Open Access

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

Contributed equally
Cell & Bioscience20111:17

DOI: 10.1186/2045-3701-1-17

Received: 25 February 2011

Accepted: 19 April 2011

Published: 19 April 2011

Abstract

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.

Results

For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.

Conclusions

The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.

Background

In 2001, Laribacter hongkongensis, a novel genus and species that belongs to the Neisseriaceae family of β-subclass of the Proteobacteria, was discovered from the blood and empyema pus of a patient with underlying alcoholic cirrhosis [1]. Subsequently, it was observed that L. hongkongensis was associated with freshwater fish borne community-acquired gastroenteritis and traveler's diarrhea in human [27]. The clinical syndrome of associated gastroenteritis is similar to those of Salmonella or Campylobacter gastroenteritis. About 80% and 20% of the patients have watery and bloody diarrhea respectively, one third of them have systemic symptoms and another one third have vomiting [4]. Pulsed-field gel electrophoresis of Spe I digested chromosomal DNA and multilocus sequence typing using seven housekeeping gene loci independently showed that the L. hongkongensis isolates recovered from freshwater fish and patients fell into separate clusters. These suggested that some L. hongkongensis clones could be more virulent or adapted to human than others [8, 9].

For a gastrointestinal tract pathogen to cause infection, after transmission through the oral route, the bacterium has to be able to survive the hostile acidic environment of the stomach, resist the action of bile in the small intestine, colonize the gastrointestinal tract epithelium through binding of adhesins of the bacterium to receptors on epithelial cells, evade host immune defense mechanisms before causing diarrhea and/or invading the gastrointestinal tract and cause systemic infections, as in the case of bacteremia and empyema thoracis [1]. Moreover, the possession of drug resistance determinants and phages also enhance the potential capability of the bacterium to resist to killing by antimicrobials and causing diseases. In this article, we present an overview of the genes and gene cassettes of the L. hongkongensis genome related to these various steps in the infective process, as well as drug resistance and phages. The phylogeny of these genes, most of them were thought to be acquired through horizontal gene transfer, was also analyzed.

Results and discussion

Resistance to acid

Urease

Similar to other gastrointestinal tract pathogens, L. hongkongensis has to face the highly hostile and acidic environment of the stomach before reaching the intestine. L. hongkongensis possesses a urease, that is able to hydrolyze the limited amount of urea available in the stomach to generate carbon dioxide and ammonia, which increases the pH. In the L. hongkongensis genome, a complete urease cassette, that occupies a 7,556 bp region, is observed. The cassette includes eight CDSs, which encodes three urease structural proteins (UreA, UreB and UreC) and five accessory proteins (UreE, UreF, UreG, UreD and UreI) [10]. Similar to the urease of other bacteria, the urease of L. hongkongensis is presumably a nickel containing enzyme [11]. The histidine residues at the carboxyl terminal of UreE are supposed to bind to the nickel ions that are transported into L. hongkongensis through a nickel transporter, and donate the nickel ions to UreC during urease activation. Most of the eight genes in the urease cassette of L. hongkongensis are most closely related to their homologues in bacteria of α- and γ-proteobacteria, rather than those in other bacteria of β-proteobacteria [1216].

Arginine deiminase

Two arc gene clusters were encoded in the L. hongkongensis genome. Each cluster consists of four genes, arcA, arcB, arcC and arcD. arcA, arcB and arcC encode the three enzymes, arginine deiminase, ornithine carbamoyltransferase and carbamate kinase, of the arginine deiminase pathway, whereas arcD encodes a membrane bound arginine-ornithine antiporter. The arginine deiminase pathway converts L-arginine to carbon dioxide, ATP, and ammonia, which increases the pH. It has been shown in various bacteria, such as Streptococcus sanguis, Streptococcus rattus, Streptococcus suis, Streptococcus pyogenes, Enterococcus faecium and Pseudomonas aeruginosa that this gene cluster is useful for bacterial survival in acidic environment [1719]. In S. pyogenes, it has also been shown that this pathway facilitates cell invasion and inhibits proliferation of human peripheral blood mononuclear cells [20, 21]. Phylogenetically, these four genes of the arc gene cluster in L. hongkongensis are most closely related to the corresponding homologues in Chromobacterium violaceum (Figure 1, 2, 3, and 4), whereas the gene cluster is absent in Neiserria meningitidis and Neisseria gonorrhoeae. Among all bacteria with complete genomes sequenced, L. hongkongensis is the only one that contains two adjacent arc gene clusters (Figure 5).
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Figure 1

Phylogenetic analysis of ArcA encoded in the arc gene cluster in L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Four hundred and nine and 409 amino acid positions in ArcA1 and ArcA2, respectively, were included in the analysis. The scale bars indicate the estimated number of substitutions per 10 amino acids. All names and accession numbers are given as cited in the GenBank database.

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Figure 2

Phylogenetic analysis of ArcB proteins encoded in the arc gene cluster in L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Three hundred and thirty-four and 335 amino acid positions in ArcB1 and ArcB2, respectively, were included in the analysis. The scale bar indicates the estimated number of substitutions per 20 amino acids. All names and accession numbers are given as cited in the GenBank database.

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Figure 3

Phylogenetic analysis of ArcC proteins encoded in the arc gene cluster in L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Two hundred and ninety-one and 314 amino acid positions in ArcC1 and ArcC2, respectively, were included in the analysis. The scale bars indicate the estimated number of substitutions per 10 amino acids. All names and accession numbers are given as cited in the GenBank database.

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Figure 4

Phylogenetic analysis of ArcD encoded in the arc gene cluster in L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Four hundred and ninety-two, 478 and 478 amino acid positions in ArcD1, ArcD2 and ArcD3, respectively, were included in the analysis. The scale bars indicate the estimated number of substitutions per 10 amino acids. All names and accession numbers are given as cited in the GenBank database.

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Figure 5

Genetic organization of ADI clusters in L. hongkongensis and other representative microbial genomes. The arrow boxes represent the CDSs. The relative positions of each gene are assigned as predicted by nucleotide sequence analysis.

Bile resistance

Efflux pumps

Efflux of bile salts from bacteria is mediated through a number of efflux systems. These efflux systems pump a variety of compounds, including antibiotics, oxidative stress agents, organic solvents and bile salts, out of the bacterial cytoplasm. Among these efflux systems, the best studied one is encoded by acrAB-tolC of the resistance nodulation division (RND) family. This system has been shown to be present in the genomes of a variety of pathogenic and non-pathogenic bacteria of the human gastrointestinal tract, such as Escherichia coli and Salmonella Typhimurium [22, 23]. In the L. hongkongensis genome, three complete copies of acrAB-tolC, of which AcrB is located in the inner membrane and contains the conserved ACR_tran domain, AcrA is located in the periplasmic space and contains the conserved HlyD domain and TolC as the outer membrane channel protein, are present. A recent bioinformatics analysis on bile resistance mechanisms in Campylobacterales also found that one complete copy of acrAB-tolC is present in the C. jejuni genome [24]. In addition to efflux pumps encoded by acrAB-tolC, the genome of L. hongkongensis also contains two copies of emrAB-tolC of the major facilitator superfamily, one copy of acrAD-tolC of the RND family (AcrD is also an inner membrane protein and contains the conserved ACR_tran domain similar to AcrB), one copy of mdtABC-tolC of the RND family and one copy of ydgFE/mdtJI of the small multidrug resistance family. These four gene cassettes were also found to be encoding efflux pumps related to bile resistance in E. coli[22, 2527]. In addition, acrAD-tolC and mdtABC-tolC have been documented to be related to bile salt resistance in S. Typhimurium [28].

Lipopolysaccharide (LPS) and Tol proteins

In addition to the efflux pumps, the integrity of the outer membrane is also important in resistance against bile. The O-antigen has been shown to be related to bile resistance in S. Typhimurium [29, 30]. Tol proteins, which are cytoplasmic and periplasmic proteins encoded by a gene cluster that consists of five genes (tolQ, tolR, tolA, tolB and pal), are also important in maintaining the integrity of the outer membrane and bile resistance, as shown in E. coli, S. Typhimurium and Erwinia chrysanthemi[3133]. In the genomes of L. hongkongensis and C. violaceum, tolQ was not clustered with tolR, tolA, tolB and pal, although all five genes are present in their genomes.

Colonization of intestinal mucosa

The first step of infection is adhesion to host cells. In the L. hongkongensis genome, a putative adhesin, with 27-30% amino acid identity to the adhesins of diffusely adherent E. coli (DAEC) [3436] and enterotoxigenic E. coli (ETEC) [3740], encoded by aidA and tibA respectively, was observed (Figure 6). It has been shown that aidA deletion mutants of DAEC lost the ability to adhere to HeLa cells and tibA deletion mutants of ETEC lost the ability to adhere to human intestine epithelial cells [37, 41, 42]; and E. coli HB101 transformed with tib loci was able to adhere to HCT 8 cells [37, 42]. aidA and tibA encode proteins of the autotransporter family, type V protein secretion system of Gram-negative bacteria [43]. Proteins of this family possess three domains, an N-terminal signal sequence, a passenger or α-domain and a translocation or β-domain, which enable the proteins to transport themselves to cell surfaces. These three domains are all present in the putative adhesin in L. hongkongensis. Amino acid residues 1-36 is the putative signal sequence (predicted by SignalP). As in the passenger domains of other autotransporters, no cysteine residues, which were thought to interfere with transport of the proteins to cell surfaces because of formation of disulphide bonds, were present in the putative passenger domain (amino acid residues 37-756) of the putative adhesin in L. hongkongensis[41]. In the passenger domains of AIDA in DAEC, multiple copies of the consensus sequence VXNSGG, acceptor sites for heptose, addition of which catalyzed by AAH heptosyltransferase, encoded by aah located upstream to aidA, are present [44]. The addition of heptose was shown to be essential for the adhesion properties in the tibA adhesin in ETEC [45]. In the putative passenger domain of the putative adhesin in L. hongkongensis, nine copies of VXSGG, but not VXNSGG, were present; and a putative heptosyltransferase, with 52% amino acid identity to the TibC heptosyltransferase of ETEC, was present upstream to the putative adhesin gene in the L. hongkongensis genome. Interestingly, in the putative passenger domain of tibA adhesin in ETEC, 11 copies of VXSGG, but not VXNSGG, were present, but whether VXSGG is the acceptor sites for heptose has not been documented. In addition to their roles for adhesion, the passenger domains may also possess virulence functions, such as autoaggregation, biofilm formation, invasion and cytotoxicity. In the putative translocation domain, the consensus motif (Y/V/I/F/W)-X-(F/W) at the extreme carboxyl terminus of other autotransporter proteins, predicted to play a role in outer membrane localization and/or stability of these proteins, was present [41].
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Figure 6

Phylogenetic analysis of the putative adhesin of L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Six hundred and eight amino acid positions of the passenger domain were included in the analysis. The scale bar indicates the estimated number of substitutions per 20 amino acids. All names and accession numbers are given as cited in the GenBank database.

Evasion of host defense

To protect from the active oxygen species (superoxide and hydrogen peroxide) released from phagocytic cells, the genome of L. hongkongensis encodes superoxide dismutase and catalases, in line with its catalase-positive phenotype. The putative superoxide dismutase of L. hongkongensis, which decomposes superoxide to hydrogen peroxide and oxygen, is most closely related to those of C. violaceum, N. meningitidis and N. gonorrhoeae. There are three putative catalases in the L. hongkongensis genome, encoded by a katE (encoding hydroperoxidase II) and two katG (encoding hydroperoxidase I with catalase-peroxidase activity). These decompose hydrogen peroxide to water and oxygen. katE in L. hongkongensis is most closely related to the homologues in Ralstonia eutropha, whereas the two katG were most closely related to those in Shewanella amazonensis and Vibrio cholerae respectively. In addition to protection against the active oxygen species, some efflux pumps may export host-derived antimicrobial agents in addition to antibiotics, bile and other substances, hence protecting from such naturally produced molecules of the host.

Virulence factors

Lipopolysaccharide

LPS consists of three parts: lipid A, core oligosaccharide, and polysaccharide side chains. In E. coli, the minimal LPS required for growth include lipid A and two keto-deoxyoctulonate (Kdo) units of the core oligosaccharide. The LPS of wild type strains of E. coli consist of additional core sugars and polysaccharide side chains. The polysaccharide side chains are also known as the O-antigen, which varies among different species of Gram-negative bacteria and different strains of the same species. These sugars enhance survival during environmental stress, and help the bacteria evade the host immune system by modification of the structure. Lipid A, also known as the endotoxin, is the hydrophobic anchor of LPS. It is a glucosamine based phospholipid inserted into the outer membranes of most Gram-negative bacteria. Most Gram-negative bacteria synthesize lipid A by pathways similar to the one in E. coli. Through binding to Toll-like receptor 4 and CD14, lipid A of Gram-negative bacteria trigger the synthesis and secretion of pro-inflammatory cytokines. The actions of these cytokines lead to local and systemic inflammatory responses, which result in various clinical manifestations, and even deaths, of patients.

The same set of genes that encode enzymes in the biosynthetic pathways of lipid A, the two Kdo units and the heptose units are present in the L. hongkongensis, C. violaceum, N. meningitidis, N. gonorrhoeae and E. coli genomes. In contrast to E. coli, the lipid A of C. violaceum, N. meningitidis and N. gonorrhoeae had a symmetrical acylation pattern [46]. Both the reducing and terminal N-acetyl-glucosamine residues in these bacteria carry three acyl groups. The sequential addition of the last 12-carbon acyl group to the reducing and terminal N-acetyl-glucosamine residues are catalyzed by enzymes encoded by the htrB and msbB genes, respectively. It was found that msbB deletion mutants of N. meningitidis and N. gonorrhoeae had lower abilities to activate human macrophages to produce pro-inflammatory cytokines [4749]. Phylogenetic analysis of the experimentally confirmed htrB and msbB genes in N. meningitidis and N. gonorrhoeae and the putative htrB and msbB genes in L. hongkongensis and C. violaceum showed that the four htrB genes and the four msbB genes fell into two separate clusters, with very high bootstrap values (Figure 7). Therefore, we speculate that the htrB and msbB genes in L. hongkongensis and C. violaceum serve similar functions as those in N. meningitidis and N. gonorrhoeae and that the lipid A of L. hongkongensis also had a symmetrical acylation pattern.
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Figure 7

Phylogenetic analysis of confirmed/putative HtrB and MsbB of L. hongkongensis , C. violaceum , N. meningitidis and N. gonorrhoeae. The tree was constructed by neighbor joining method using Kimura's correction and was rooted using HtrB of Rickettsia typhi (YP_067645). Two hundred and eighty-four amino acid positions were included in the analysis. The scale bar indicates the estimated number of substitutions per 20 amino acids. Numbers at nodes indicated levels of bootstrap support calculated from 1000 trees. All names and accession numbers are given as cited in the GenBank database.

The genes that are responsible for the synthesis of α-chain L1, α-chain L2, β-chain and γ-chain in the core oligosaccharide in N. meningitidis and N. gonorrhoeae (lgtA, lgtB, lgtC, lgtD, lgtE, lgtF, lgtG, rfaK) and those for the addition of sialic acids to these chains (lst) are absent in the genomes of L. hongkongensis and C. violaceum[50]. On the other hand, nine genes which encode putative enzymes for biosynthesis of the polysaccharide side chains are present in the L. hongkongensis genome. Four of these genes (rfbA, rfbB, rfbC and rfbD) are also present in the genomes of C. violaceum, N. meningitidis and N. gonorrhoeae. The enzymes encoded by these four genes catalyzed reactions for the synthesis of dTDP-rhamnose, although mutations of them in N. meningitidis and N. gonorrhoeae did not result in any change in their phenotypes [51, 52]. The other five genes (wbmF, wbmG, wbmH, wbmI and wbmK), which encode putative nucleotide sugar epimerases/dehydratases and amidotransferase, are not present in the C. violaceum, N. meningitidis and N. gonorrhoeae genomes, but are most closely related to the corresponding genes for the biosynthesis of the O-antigens in Bordetella parapertussis and Bordetella bronchoseptica[53]. Although the structures of the LPS of L. hongkongensis and C. violaceum remain to be determined, these imply that the structures of the LPS of L. hongkongensis and C. violaceum are probably quite different from those of the lipooligosaccharides of N. meningitidis and N. gonorrhoeae.

Recently, a number of genes that encode proteins for the assembly and transport of LPS in E. coli have been discovered [54]. All these genes were also present in the genomes of L. hongkongensis, C. violaceum, N. meningitidis and N. gonorrhoeae (Table 1). The exact functions of these proteins have not been fully elucidated.
Table 1

Genes for assembly and transport of LPS

Protein

Gene

E. coli str. K-12 substr. MG1655

L. hongkongensis

C. violaceum

N. gonorrhoeae FA1090

N. meningitidis MC58

Periplasmic LPS-binding protein

lptA

b3200

LHK_02023

CV3330

NGO1606

NMB0355

Lipopolysaccharide export, IM-tethered periplasmic protein of LptBFGC export complex

lptC

b3199

LHK_02022

CV3329

NGO1607

NMB0354

Lipopolysaccharide export ABC transporter ATP-binding protein of LptBFGC export complex

lptB

b3201

LHK_02024

CV3331

NGO1605

NMB0356

Lipopolysaccharide export ABC permease of LptBFGC export complex

lptF

b4261

LHK_01413

CV2915

NGO1228

NMB1570

Lipopolysaccharide export ABC permease of LptBFGC export complex

lptG

b4262

LHK_01412

CV2916

NGO1229

NMB1571

LPS assembly OM complex LptDE, beta-barrel component

lptD

b0054

LHK_03193

CV4229

NGO1715

NMB0280

LPS assembly OM complex LptDE, lipoprotein component

lptE

b0641

LHK_00118

CV0506

NGO0282

NMB0707

Cytotoxins

The L. hongkongensis genome contains a number of CDSs that encode putative cytotoxins. These include cell surface acting cytotoxins, such as RTX toxin and hemolysins; and intracellular cytotoxins such as patatin-like proteins.

RTX toxins
RTX toxins, originally discovered in E. coli (α-hemolysin) [55, 56], are most commonly found in bacteria of the Pasteurellaceae family. Most RTX toxins are hemolysins or leukotoxins [57, 58]. The L. hongkongensis genome contains an RTX gene cluster (tolC-rtxA1-rtxD-rtxB) and an isolated rtxA2 gene. In the RTX gene cluster (Figure 8), tolC encodes the outer membrane component of the type I secretion apparatus, rtxA1 encodes the structural toxin, rtxD encodes the adaptor protein anchored to the inner membrane and rtxB encodes the inner membrane ATPase. TolC, RtxD and RtxB form the secretion apparatus for exporting RtxA. Similar to RtxA of other bacteria, RtxA1 and RtxA2 of L. hongkongensis possess tandem arrays of glycine-rich nonapeptide repeats (GGXGXDX[L/I/V/W/Y/F]X, where X is any amino acid) for binding of calcium ions (Figure 8). There are five nonapeptide repeats in RtxA1 and nine nonapeptide repeats in RtxA2. Unlike most other bacteria which contain rtxC genes, the RTX gene cluster of L. hongkongensis does not possess this gene. Instead, it contains a gene of putative adhesive function, located between rtxA1 and rtxD. Domain search using InterProScan showed that this gene contains nine repeats of 22 amino acids (TDNGTVTNVTLSSVTNGQTVAE) with parallel beta-helix structures. Each repeat is separated from the adjacent one by 82 amino acids (Figure 8). Although the genomes of L. hongkongensis, C. violaceum and N. meningitidis all contain RTX toxin, RtxA1 and RtxA2 of L. hongkongensis do not show clustering with the homologues in C. violaceum and N. meningitidis. This is in contrast to the other genes (tolC, rtxD and rtxB) in the RTX gene cluster, which are all most closely related to the corresponding homologues in C. violaceum and other species of β-proteobacteria [59, 60] (Figure 9, 10, 11, and 12). Moreover, the amino acid identities between TolC, RtxD and RtxB and their homologues in C. violaceum are much higher than those between RtxA1 or RtxA2 and their homologues in any other bacteria (Figure 9, 10, 11, and 12). These suggest that rtxA1 and rtxA2 have evolved much faster than tolC, rtxD and rtxB, so that the toxins can bind to their corresponding host cells more efficiently. Interestingly, similar to rtxA2 of L. hongkongensis, the structural toxin genes (frpC and frpA) in N. meningitidis are not linked to genes of the type I secretion system. However, it has been shown that FrpC and FrpA can be secreted by E. coli harboring hlyBD genes, indicating that they are probably secreted by secretion systems unlinked to their corresponding genes [61].
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Figure 8

Genetic organization of the RTX gene cluster ( tolC-rtxA1-rtxD-rtxB ) in L. hongkongensis. The boxes represent the CDSs. The number of amino acid residues of each gene is indicated above the boxes. The basic functional activities of the corresponding gene products are given on the top. Five copies of glycine-rich nonapeptide repeats (GGXGXDX[L/I/V/W/Y/F]X, where X is any amino acid) of rtxA1 are underlined. An CDS of unknown function, located between rtxA1 and rtxD, are also depicted, where nine repeats of 22 amino acids are highlighted. The relative positions of each gene are assigned as predicted by nucleotide sequence analysis.

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Figure 9

Phylogenetic analysis of TolC in the RTX gene cluster of L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Four hundred and forty-two amino acid positions were included in the analysis. The scale bars indicate the estimated number of substitutions per 20 amino acids. All names and accession numbers are given as cited in the GenBank database.

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Figure 10

Phylogenetic analysis of RtxA1 in the RTX gene cluster of L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. One thousand and eighty-seven amino acid positions were included in the analysis. The scale bars indicate the estimated number of substitutions per 20 amino acids. All names and accession numbers are given as cited in the GenBank database.

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Figure 11

Phylogenetic analysis of RtxD in the RTX gene cluster of L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Four hundred and fifty-two amino acid positions were included in the analysis. The scale bars indicate the estimated number of substitutions per 20 amino acids. All names and accession numbers are given as cited in the GenBank database.

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Figure 12

Phylogenetic analysis of RtxB in the RTX gene cluster of L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Seven hundred and twenty amino acid positions were included in the analysis. The scale bars indicate the estimated number of substitutions per 20 amino acids. All names and accession numbers are given as cited in the GenBank database.

Hemolysins

In the L. hongkongensis genome, there are two gene loci that encode putative hemolysins. The first putative hemolysin contains three domains, the first one of the DUF21 superfamily, the second one of the CBS_pair superfamily and the third one of the CorC_HlyC superfamily. Among the five most closely related protein sequences, three of them were putative hemolysins of three different Yersinia species, and the other two were hypothetical proteins. The second putative hemolysin belongs to the HlyIII superfamily, which contains seven transmembrane domains with conserved amino acid residues present. It is most closely related to the hemolysin III of C. violaceum.

Patatin-like protein
Patatin, originally described in plants such as potatoes, has diverse functions such as storage glycoproteins [62], signal transduction [63] and defense against parasites [64]. In 2003, it was found that toxin ExoU of P. aeruginosa, delivered to eukaryotic cells via a type III secretion system, possessed the catalytic domains of patatin, iPLA(2) and cPLA(2) [65]. Direct injection of ExoU in mammalian cells resulted in irreversible damage to cellular membranes and rapid necrotic death [66]. Similar to patatin, ExoU of P. aeruginosa possessed phopholipase A2 activity. P. aeruginosa mutants with mutations at the active sites of the patatin-like protein were less virulent than wild type P. aeruginosa in a mouse model [67]. Subsequently, genes that encode putative patatin-like proteins were observed in many bacterial genomes, although none of them was characterized phenotypically [68]. It was also observed that the average copy number of genes that encode patatin-like proteins is higher in plant/animal bacterial pathogens than in non-pathogens [68]. In some pathogens, up to eight copies of genes that encode putative patatin-like proteins can be found. Similar to P. aeruginosa, the genome of L. hongkongensis also contains three copies of genes that encode putative patatin-like proteins. The lengths of the genes that encode putative patatin-like proteins in the genomes of L. hongkongensis, C. violaceum (7 copies), N. meningitidis (1 copy) and N. gonorrhoeae (1 copy) varied from 894 to 2,337 bp. The three copies in the L. hongkongensis genome are 951, 963 and 2,232 bp respectively. All three copies contain all the four domains that can be found in bacterial patatin-like proteins, including a putative oxyanion hole, a serine hydrolase G-X-S-X-G domain, a potential serine-containing phosphorylation site and an aspartate-containing active site domain (Figure 13). The serine in the hydrolase domain and the aspartate made up a patatin-specific catalytic dyad that has not been described in any other known proteins [68].
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Figure 13

Multiple alignments of the four conserved domains in the putative patatin-like proteins in the genomes of L. hongkongensis , C. violaceum , N. meningitidis and N. gonorrhoeae. The two arrows indicate the Ser-Asp catalytic dyad. Conserved amino acids in the four domains are in bold. ω, number of amino acids before and after the conserved domains.

Enzymes

Outer membrane phospholipase A
It has been shown that outer membrane phospholipase A (OMPLA) is a virulence factor in a number of bacteria, including Helicobacter pylori and C. coli. Located on the outer membrane of bacteria, OMPLA lyses the outer membrane, leading to release of other virulence factors, such as urease and VacA in H. pylori. In the L. hongkongensis genome, a gene that encodes a putative OMPLA is observed. This OMPLA possesses a complete and highly specific consensus sequence motif (YTQ-Xn-G-X2-H-X-SNG) found in OMPLA of other bacteria. Phylogenetically, it is most closely related to the OMPLA of Methylibium petroleiphilum, a methyl tert-butyl ether-degrading methylotroph of β-proteobacteria (Figure 14) [69].
https://static-content.springer.com/image/art%3A10.1186%2F2045-3701-1-17/MediaObjects/13578_2011_Article_16_Fig14_HTML.jpg
Figure 14

Phylogenetic analysis of outer membrane phospholipase A of L. hongkongensis. The tree was constructed by neighbor joining method using Kimura's correction and bootstrap values calculated from 1000 trees. Three hundred and seventy-seven amino acid positions were included in the analysis. The scale bar indicates the estimated number of substitutions per 10 amino acids. All names and accession numbers are given as cited in the GenBank database.

Drug resistance

A genome-wide analysis using similarity searches revealed the presence of a large number of antibiotic resistance-related genes in L. hongkongensis strain HLHK9. They are related to β-lactam (Table 2), multidrug efflux (Table 3) and other resistance genes (Table 4).
Table 2

CDSs related to beta-lactam antibiotics in L. hongkongensis

CDS

Gene

Product

Organism with the closest matching sequences

E-value

Identities

Remarksa

LHK_00876

 

β-lactamase domain protein

Thauera sp.

6e-77

135/204 (66%)

 

LHK_00878

gloB

Hydroxyacylglutathione hydrolase

Rickettsiella grylli

6e-64

126/259 (48%)

 

LHK_00975

dacC

D-alanyl-D-alanine-carboxypeptidase

C. violaceum

e-140

254/379 (67%)

PBP6a

LHK_02726

pbpG

D-alanyl-D-alanine-endopeptidase

C. violaceum

4e-94

183/288 (63%)

PBP7

LHK_02764

prc

Carboxy-terminal processing protease

C. violaceum

1e-173

315/480 (65%)

PBP3 processing protease

LHK_02836

dacB

Serine-type D-Ala-D-Ala carboxypeptidase

C. violaceum

3e-81

207/427 (48%)

PBP4a

LHK_02959

mrcA

Peptidoglycan glycosyltransferase

C. violaceum

0

512/795 (64%)

PBP1a

LHK_03028

ampC

β-lactamase

C. violaceum

7e-91

189/381 (49%)

 

LHK_03062

ftsI

Penicillin-binding protein 3 precursor

C. violaceum

0

349/586 (59%)

PBP3

LHK_03073

mrdA

Penicillin-binding protein 2

C. violaceum

0

404/583 (69%)

PBP2

aPBP, penicillin-binding protein

Table 3

CDSs related to multidrug resistance in L. hongkongensis

CDS

Gene

Product

Organism with the closest matching sequence

E-value

Identities

No. of TMSa

Remarksb

LHK_00138

tolC

TolC family type I secretion outer membrane protein

Polaromonas naphthalenivorans

2e-72

203/455 (44%)

--

OMP

LHK_00140

acrB

Acriflavin resistance protein

P. naphthalenivorans

0

723/1066 (67%)

13

RND

LHK_00141

acrA

Efflux transporter, RND family, MFP subunit

P. naphthalenivorans

2e-51

153/355 (43%)

--

MFP

LHK_00142

arsR

Transcription regulator ArsR

Bordetella parapertussis

2e-26

62/99 (62%)

--

TR

LHK_00221

 

RND efflux system outer membrane lipoprotein

Pelobacter propionicus

1e-108

207/424 (48%)

--

OMP

LHK_00222

macB

Macrolide-specific ABC-type efflux carrier

Bordetella avium

0

429/655 (65%)

4

ABC

LHK_00223

macA

Efflux transporter, RND family, MFP subunit

Lutiella nitroferrum

4e-127

252/384 (65%)

--

MFP

LHK_00466

 

Hypothetical protein

Dorea longicatena

6e-45

131/439 (29%)

12

MATE

LHK_00743

mdfA

Probable multidrug translocase protein

C. violaceum

e-139

253/394 (64%)

12

MFS

LHK_01214

 

Probable multiple antibiotic resistance protein MarC

C. violaceum

7e-58

118/208 (56%)

--

MarC

LHK_01285

mdtA

Probable membrane protein

C. violaceum

3e-94

188/340 (55%)

--

MFP

LHK_01286_01287

mdtB

Drug efflux pump transmembrane protein

C. violaceum

0

700/1018 (68%)

12

RND

LHK_01288

mdtC

Drug efflux pump transmembrane protein

C. violaceum

0

678/994 (68%)

10

RND

LHK_01289

tolC

Putative outer membrane protein precursor

Acinetobacter sp.

4e-79

189/433 (43%)

--

OMP

LHK_01373

emrB

Multidrug resistance protein

C. violaceum

0

323/490 (65%)

14

MFS

LHK_01374

emrA

Multidrug efflux membrane fusion protein

Ralstonia eutropha

e-101

194/370 (52%)

--

MFP

LHK_01375

mdtP

Outer membrane efflux protein

Yersinia enterocolitica

1e-38

142/469 (30%)

--

OMP

LHK_01376

emrR

MarR family transcriptional regulator

C. violaceum

4e-32

69/156 (44%)

--

TR

LHK_01383

 

Probable multiple antibiotic resistance protein MarC

C. violaceum

4e-80

149/232 (64%)

--

MarC

LHK_01384

mdtJ

Multidrug efflux system protein MdtJ

Klebsiella pneumoniae

3e-20

52/119 (43%)

3

SMR

LHK_01385

mdtI

Multidrug efflux system protein MdtI

Salmonella enterica

6e-21

63/109 (57%)

4

SMR

LHK_01424

 

RND efflux system, outer membrane lipoprotein, NodT family

Syntrophobacter fumaroxidans

7e-108

223/446 (50%)

--

OMP

LHK_01425

 

Transporter, hydrophobe/amphiphile efflux-1 (HAE1) family

Pelobacter propionicus

0

553/1036 (53%)

12

RND

LHK_01426

 

Efflux transporter, RND family, MFP subunit

S. fumaroxidans

7e-95

186/364 (51%)

--

MFP

LHK_01870

 

Putative multidrug resistance protein

R. eutropha

8e-47

153/483 (31%)

14

MFS

LHK_01934

 

Probable multiple antibiotic resistance protein MarC

C. violaceum

3e-80

152/205 (74%)

--

MarC

LHK_01967

 

ABC transporter, transmembrane region:ABC transporter related

R. eutropha

0

550/732 (75%)

6

ABC

LHK_02051

 

Lipoprotein releasing system, ATP-binding protein

Pseudomonas stutzeri

4e-58

134/227 (59%)

--

ABC

LHK_02129

mexA

Multidrug resistance protein

Xanthomonas campestris

3e-104

223/375 (59%)

--

MFP

LHK_02130

acrB

AcrB/AcrD/AcrF family protein

Cellvibrio japonicus

0

768/1034 (74%)

14

RND

LHK_02131

nodT

RND efflux system, outer membrane lipoprotein, NodT

Geobacter metallireducens

8e-142

266/466 (57%)

--

OMP

LHK_02132

 

Transcriptional regulator, TetR/AcrR family

Cellvibrio japonicus

2e-46

93/187 (49%)

--

TR

LHK_02173

 

Probable MFS transporter

C. violaceum

1e-82

195/370 (52%)

12

MFS

LHK_02235

 

Putative integral membrane efflux protein

Yersinia pestis

0

379/505 (75%)

13

abgT family protein

LHK_02238

 

ABC transporter

Azoarcus sp.

e-157

281/371 (75%)

7

ABC

LHK_02239

yhiH

ABC transporter related

Thauera sp.

0

699/954 (73%)

6

ABC

LHK_02240

 

Conserved hypothetical protein, predicted secretion protein HlyD family

Azoarcus sp.

6e-96

232/339 (68%)

--

MFP

LHK_02241

oprM3

Outer membrane efflux protein

B. avium

1e-100

241/453 (53%)

--

OMP

LHK_02292

 

Probable multiple antibiotic resistance protein MarC

C. violaceum

2e-60

116/218 (53%)

--

MarC

LHK_02533

 

Multidrug efflux protein NorA

C. violaceum

e-122

230/447 (51%)

12

MATE

LHK_02539

 

EmrB/QacA family drug resistance transporter

P. stutzeri

2e-147

277/481 (57%)

13

MFS

LHK_02783

 

Hypothetical protein

multiple antibiotic resistance (MarC)-related protein

C. violaceum

5e-74

139/200 (69%)

--

MarC

LHK_02825

natC

Periplasmic type I secretion system

C. violaceum

4e-82

190/439 (43%)

--

OMP

LHK_02826

acrB

Probable transmembrane drug efflux protein

C. violaceum

0

693/1019 (68%)

12

RND

LHK_02827

acrA

Probable transport/efflux transmembrane protein

C. violaceum

3e-83

174/351 (49%)

--

MFP

LHK_02828

acrR

TetR/AcrR family transcriptional regulator

C. violaceum

6e-44

92/183 (50%)

--

TR

LHK_02929

acrA

Probable multidrug efflux membrane permease

C. violaceum

1e-89

203/372 (54%)

--

MFP

LHK_02930

acrD

Acriflavin resistance protein D

C. violaceum

0

717/1036 (69%)

12

RND

LHK_02931

oprM

Outer membrane efflux protein

C. violaceum

e-136

252/467 (53%)

--

OMP

LHK_02949

msbA

Transport ATP-binding protein MsbA

C. violaceum

0

344/554 (62%)

5

ABC

LHK_02975

bcr

Probable MFS transporter

C. violaceum

e-147

269/388 (69%)

12

MFS

LHK_03132

emrB

Probable multidrug resistance protein

C. violaceum

0

303/492 (61%)

14

MFS

LHK_03133

emrA

Multidrug resistance protein

Burkholderia thailandensis

1e-108

202/377 (53%)

--

MFP

LHK_03134

tolC

Outer membrane efflux protein

R. eutropha

6e-45

153/453 (33%)

--

OMP

a TMS, transmembrane segment domain

bRND, resistance-nodulation-division family; MFS, major facilitator superfamily; ABC, ATP-binding cassette transporter superfamily; SMR, small multidrug resistance family; MATE, multidrug and toxic compound extrusion; MFP, membrane fusion protein; OMP, outer membrane (channel) protein; TR, transcription regulator; MarC, MarC-like protein.

Table 4

Miscellaneous resistance genes in L. hongkongensis

CDS

Gene

Product

Organism with the closest matching sequence

E-value

Identities

LHK_00025

ksgA

Dimethyladenosine transferase

C. violaceum

1e-94

178/260 (68%)

LHK_00913

 

Arsenical-resistance protein

Burkholderia oklahomensis

2e-65

153/183 (83%)

LHK_01038

crcB

Camphor resistance protein CrcB

Brucella abortus

2e-33

88/129 (68%)

LHK_01039

crcB

Camphor resistance protein CrcB

Y. pestis

3e-34

78/123 (63%)

LHK_01350

rarD

RarD protein, chloamphenicol sensitive

C. violaceum

2e-81

172/285 (60%)

LHK_02940

bacA

Undecaprenol kinase, putative bacitracin resistance protein

Burkholderia graminis

3e-83

174/278 (62%)

β-lactam resistance-related genes

A total of 10 CDSs related to β-lactam resistance were identified in the L. hongkongensis genome. Genes that exhibit similarity to penicillin-binding proteins (PBPs) (6 CDSs) of other bacterial species were found (Table 2). The PBPs identified in L. hongkongensis include PBP1a, PBP2, PBP3, PBP4a, PBP6a, and PBP7, which are essential proteins that are involved in biosynthesis of murein and peptidoglycan, and are targets for inhibition by β-lactams [70, 71]. Although the presence of PBPs per se does not confer resistance, chromosomal mutations in PBPs may render the bacteria resistant to β-lactams [7275].

Apart from the ampC gene (LHK_03028) that encodes the previously characterized class C β-lactamase [76], there are two other putative β-lactamases (LHK_00876 and LHK_00878) observed in the L. hongkongensis genome. They are both putative metallo-β-lactamases containing a metallo-β-lactamase superfamily domain which included two zinc ligand-binding sites essential for its hydrolytic function on the β-lactam ring (Figure 15) [7779]. However, these zinc ligand-binding sites were also present in most proteins of the metallo-β-lactamase superfamily, the function of which is not limited to β-lactam hydrolysis [7981]. Therefore, in vitro experiments are required to confirm the actual function of these two putative metallo-β-lactamases.
https://static-content.springer.com/image/art%3A10.1186%2F2045-3701-1-17/MediaObjects/13578_2011_Article_16_Fig15_HTML.jpg
Figure 15

Multiple alignment of the partial amino acid sequences of the two putative metallo-β-lactamases in L. hongkongensis and those of known metallo-β-lactamases showing the conserved zinc-ligand binding sites. Amino acid residues high-lighted in yellow and blue representing two independent putative zinc-ligand binding sites of class B3 metallo-β-lactamase His116-His118-His196 and Asp120-His121-His263. Numbers in parentheses indicate the corresponding positions in the amino acid sequences. L1, Stenotrophomonas maltophilia IID1275 (accession no. CAA52968); FEZ-1, Legionella gormanii ATCC33297 (accession no. CAB96921); CAU-1, Caulobacter vibrioides DSM 4727 (accession no. CAC87665); BJP-1, Bradyrhizobium japonicum USDA 110 (accession no. NP_772870)

Multidrug resistance genes

A total of 54 CDSs related to multidrug efflux were identified in L. hongkongensis genome (Table 3). The five major families of drug extrusion translocases were all present, including the Major Facilitator Superfamily (MFS) (7 CDSs), Small Multidrug Resistance (SMR) family (2 CDSs), RND family (7 CDSs), Multidrug and Toxic compound Extrusion (MATE) family (2 CDSs), and ATP-Binding Cassette (ABC) superfamily (6 CDSs).

Resistance-Nodulation-cell Division (RND) family proteins

For Gram-negative bacteria, the efflux pumps that are associated with most clinically significant resistance to antibiotics are those of the RND family. In this family, three gene loci homologous to acrRAB-tolC (LHK_00138, LHK_00140-00142; LHK_02129-02132; LHK_02825-02828) and one gene locus homologous to acrAD-tolC (LHK_02929-02931) of Escherichia coli were identified in the genome of L. hongkongensis. These three AcrRAB-TolC and the AcrAD-TolC multidrug efflux systems shared typical tripartite structure with other multidrug efflux systems in the RND family [82]. AcrB and AcrD are membrane transporter proteins, AcrA is membrane fusion protein and TolC is outer membrane channel protein. acrR is a transcription regulator gene located upstream of the acrAB-tolC loci. As a multidrug efflux system with broad-substrate spectrum, AcrAB-TolC confers resistance to chloramphenicol, tetracyclines, erythromycin, trimethoprim, β-lactams, and other organic and inorganic antiseptic agents in E. coli[83, 84]. AcrAD-TolC is less commonly reported compared to AcrAB-TolC system, where AcrD is a close homolog of AcrB. AcrAD-TolC multidrug efflux system is capable of exporting antibiotics of the aminoglycoside class including amikacin, gentamicin, neomycin, kanamycin, tobramycin, and streptomycin in E. coli[85, 86]. Another putative multidrug efflux system of the RND family identified in the genome of L. hongkongensis is homologous to MdtABC-TolC system (LHK_01285, LHK_01286, LHK_01288, LHK_01289). MdtABC-TolC system in E. coli confers at least novobiocin and bile salt resistance in the bacterium. A uniqueness of this system is that MdtB and MdtC will form a heterodimer as a membrane efflux component in cooperation with membrane fusion protein MdtA and outer membrane channel protein TolC. [27, 87] Moreover, one RND family multidrug efflux system with homology to hydrophobe/amphiphile efflux-1 subfamily was also discovered (LHK_01424-01426).

Major Facilitator Superfamily (MFS)

Two loci (LHK_01373-01376; LHK_03132-03134) homologous to emrAB-tolC system of E. coli belonging to MFS were found in the genome of L. hongkongensis. One of them had an additional transcription regulator emrR gene (LHK_01376) in its upstream sequence. EmrAB-TolC system in E. coli confers nalidixic acid and other toxic novobiocin substances resistance to bacterium [88]. Moreover, mutation of the emrR gene has been shown to lead to over-expression of the EmrAB pump and increased resistance to antimicrobial agents [89]. However, the substrate specificity of these EmrAB-TolC homologs identified in the genome of L. hongkongensis is yet to be investigated. There are five other multidrug efflux proteins belonging to MFS (LHK_00743; LHK_01870; LHK_02173; LHK_02539; LHK_02975) in the L. hongkongensis genome. One of them (LHK_00743) is a homolog to mdfA gene while another (LHK_02975) has high identities to bcr gene. mdfA encodes an MF-related protein, MdfA, which results in resistance to a diverse group of cationic and zwitterionic lipophilic compounds and antibiotics such as chloramphenicol and erythromycin when over-expressed in E. coli[90]. bcr gene codes for an efflux protein which is associated with bicyclomycin resistance in E. coli[91].

Small Multidrug Resistance (SMR) family

Two adjacently located multidrug efflux genes (LHK_01384 and LHK_01385) of the SMR family were identified in the genome of L. hongkongensis. They are homologous to mdtJI (also named ydgEF) genes in E. coli which confers resistance to spermidine and, deoxycholate and sodium dodecyl sulfate at low level [92, 93]. mdtJI have to be co-expressed for functionality and it is suggested that MdtJI may function as a heterodimer or heterooligomer [9294].

Multidrug and Toxic compound Extrusion (MATE) family

Two multidrug efflux genes of the MATE family (LHK_00466 and LHK_02533) were also discovered in the genome of L. hongkongensis. One of them (LHK_02533) is a homolog of multidrug efflux protein NorA from Staphylococcus aureus, which confers resistance to antibiotics of the quinolone class and various organic compounds [95, 96]. Mutation of the norA gene in S. aureus has resulted in 5- to 30-fold increase in susceptibility to norfloxacin [96].

ATP-Binding Cassette (ABC) superfamily

Six CDSs of the ABC transporter family related to multidrug resistance were identified in the L. hongkongensis genome. A tripartite multidrug efflux system of the ABC transporter family composed of membrane transporter (LHK_02239), MFP (LHK_02240), and OMP (LHK_02241) was identified in the genome of L. hongkongensis. This system of proteins probably functions as a complex with composition resembling to that of RND family. Five other standalone putative ABC transporter genes (LHK_00222; LHK_01967; LHK_02051; LHK_02238; LHK_02949) coding for multidrug efflux proteins were scattered over the L. hongkongensis genome. One (LHK_02949) of them possessed homology to msbA from E. coli, which is responsible for mediating the transport of the lipid A core of LPS to the outer membrane [97, 98]. Interestingly, expression of E. coli MsbA in Lactococcus lactis which lacks LPS has been shown to significantly increase resistance to erythromycin [98].

In addition to these five major families, the L. hongkongensis genome also encodes a number of other possible multidrug resistance-related genes. Among these, there are five marC-like genes (LHK_01214; LHK_01383; LHK_01934; LHK_02292; LHK_02783), the expression of which was once believed to be associated with multidrug efflux system MarRAB in E. coli[99]. However, a recent report has shown that mutation in marC did not increase antibiotic susceptibility on E. coli[100]. Therefore, the actual function of MarC is still not identified yet. One CDS (LHK_02235) coding for a protein with 75% amino acid identities to putative integral membrane efflux protein of Yersinia pestis and possessing an AbgT family domain was also identified in the genome of L. hongkongensis. AbgT protein family includes two transporter members, AbgT protein of E. coli and MtrF of N. gonorrhoeae[101, 102]. MtrF, as an inner membrane protein, which enhances the activity of multidrug efflux system MtrCDE of the RND family, conferring higher level of resistance to hydrophobic antibiotics such as penicillin and erythromycin etc. [102, 103]. Since no mtrCDE gene homologs were found in the genome of L. hongkongensis, the role and function of the AbgT family protein in L. hongkongensis remains to be elucidated.

Miscellaneous resistance genes

Six other CDSs with homologies to other drug resistance genes were identified in the L. hongkongensis genome (Table 4). A putative dimethyladenosine transferase, encoded by ksgA gene (LHK_00025) was found. Kasugamycin and streptomycin resistance as a result of mutations in ksgA have been documented [104106]. A bacA gene (LHK_02940) encoding putative bacitracin resistance protein BacA was also identified. BacA protein confers bacitracin resistance to E. coli by catalyzing the dephosphorylation of undecaprenyl diphosphate (C55-PP) into C55-P, which is important in peptidoglycan synthesis. The conversion of C55-PP into C55-P is normally catalyzed by a specific phosphatase which is inhibited by bacitracin leading to halted peptidoglycan synthesis [107]. The other four CDSs encode putative arsenical-resistance protein (LHK_00913), two camphor resistance proteins CrcB (LHK_01038 and LHK_01039), and chloramphenicol sensitive protein RarD (LHK_01350). Overexpression of CrcB in E. coli has been shown to protect the bacteria against chromosome decondensation by camphor [108]. The presence of two crcB genes in L. hongkongensis genome, but only one copy in the closely related bacterium, C. violaceum, and none in N. gonorrheae or N. meningitidis genomes suggested that this is an important defense mechanism in L. hongkongensis. Since the L. hongkongensis strain, HLHK9, used for genome sequencing is susceptible to tetracycline (MIC = 0.5 μg/ml), the tetA gene previously identified in L. hongkongensis strains resistant to tetracycline is not found in the present genome [109]. Recently, class 1 integrons carrying multiple antimicrobial resistance genes were identified in 6.5% of L. hongkongensis isolates from aquatic products in Guangzhou city, China [110]. However, such integron is not present in the genome of strain HLHK9.

Bacteriophages

The L. hongkongensis genome (genome size 3.16 Mbp) contains a total of eight putative prophages named LhP1 to LhP8, the positions of which are shown in Figure 16 and Table 5. This high number of prophages, compared to 3 prophages in C. violaceum (genome size 4.75 Mbp) (GenBank accession no. AE016825), 1 to 3 in N. meningitidis (genome size 2.14 to 2.27 Mbp) (GenBank accession no. CP000381, FM999788, AM421808, AE002098, AL157959, AM889136, CP001561) and 6 in N. gonorrhoeae (genome size 2.15 to 2.23 Mbp) (GenBank accession no. AE004969, CP001050) using the same parameters for prophage prediction by Prophage Finder, suggested that this is an important mechanism for acquisition and exchange of genetic materials in L. hongkongensis. While N. meningitides and C. violaceum cause mainly meningitis and invasive infections respectively that can lead to fatal septicemia, N. gonorrheae and L. hongkongensis were mainly isolated from human genital and gastrointestinal tract respectively. Interestingly, the presence of apparently high number of prophages also in N. gonorrhoeae is in line with our previous observation that horizontal gene transfer was particularly frequent among bacteria residing in human gastrointestinal and probably genital tract [111], suggesting that these anatomical sites may be an excellent incubator for bacterial gene transfer.
https://static-content.springer.com/image/art%3A10.1186%2F2045-3701-1-17/MediaObjects/13578_2011_Article_16_Fig16_HTML.jpg
Figure 16

Position of the LhP prophages and the CDSs coding for transposases in the L. hongkongensis genome. LhP1 to LhP8: L. hongkongensis prophages 1 to 8.

Table 5

Prophages of L. hongkongensis HLHK9

Prophage

Nucleotide Positions

Size (bp)

GC (%)

No. of CDS

Total No. of Phage related CDS

Phage related CDS (No.)

LhP1

356028-387345

31,318

63.07

47

34

P2-like (29), unclassified Myoviridae (2), unclassified Siphoviridae (2), Mu-like (1)

LhP2

1109928-1136068

26,141

64.81

32

25

Mu-like (10), P2-like (5), lambda-like (3), unclassified phage (3), epsilon15-like (1), unclassified Myoviridae (1), unclassified Podoviridae (1), unclassified Siphoviridae (1)

LhP3

1454673-1465841

11,169

58.70

19

14

BPP-1-like (3), lambda-like (3), epsilon15-like (2), unclassified Podoviridae (2), P1-like viruses (1), unclassified Myoviridae (1), unclassified Siphoviridae (1), unclassifed phage (1)

LhP4

1477589-1511963

34,375

58.78

36

23

BPP-1-like (14), P4-like (4), P2-like (1), P22-like (1), epsilon15-like (1), unclassified Siphoviridae (1), unclassified Myoviridae (1)

LhP5

1568789-1612785

43,997

59.00

64

32

Mu-like (9), lambda-like (7), unclassified Podoviridae (5), unclassified phage (4), unclassified Myoviridae (3), unclassified Siphoviridae (2), P2-like (1), P22-like (1)

LhP6

1671244-1693161

21,918

62.04

31

25

unclassified Siphoviridae (12), lambda-like (5), unclassified phage (3), T1-like (2), unclassified Myoviridae (2), unclassified Podoviridae (1)

LhP7

1888197-1908188

19,992

55.59

31

18

unclassified phage (7), Mu-like (4), unclassified Myoviridae (2), lambda-like (1), P22-like (1), unclassified Podoviridae (1), unclassified Siphoviridae (1), BPP-1-like (1)

LhP8

2462791-2496581

33,791

63.87

48

37

P2-like (30), Myoviridae (2), unclassified Siphoviridae (2), unclassified phage (2), Mu-like (1)

LhP1

Bacteriophage LhP1 is composed of 47 CDSs, accounting for 31,318 bp with G+C content 63.07%, close to the G+C content of the L. hongkongensis genome. LhP1 contains 34 phage-related CDSs. Analysis of these CDSs indicated that LhP1 is likely a P2-like phage, as 29 of its 34 phage-related CDSs were most similar to CDSs in P2-like prophages (Figure 17). A P2-like phage typically possesses an icosahedral head with a diameter of about 60 nm, containing a linear double-stranded DNA molecule of about 30-35 kb with cohesive ends and a straight tail with a contractile sheath [112]. Based on their morphology, P2-like phages are classified as members of the Myoviridae family (phages with contractile tails) in the order Caudovirales (tailed phages) [113]. Other CDSs exhibit similarity to other genes of phages such as Mu-like phages and unclassified phages under Myoviridae and Siphoviridae (phages with long non-contractile tails).
https://static-content.springer.com/image/art%3A10.1186%2F2045-3701-1-17/MediaObjects/13578_2011_Article_16_Fig17_HTML.jpg
Figure 17

Dot-plot analysis for LhP1, LhP8 and E. coli phage P2. (A) Dot-plot alignment of LhP8 sequences (vertical axis) versus LhP1 sequences (horizontal axis). (B) Dot-plot alignment of LhP1 sequences (vertical axis) versus Enterobacteria phage P2 sequences (horizontal axis). (C) Dot-plot alignment of LhP8 sequences (vertical axis) versus Enterobacteria phage P2 sequences (horizontal axis).

LhP2

Bacteriophage LhP2 is composed of 32 CDSs, accounting for 26,141 bp with G+C content 64.81%. Analysis of its CDSs indicated that LhP2 is likely a Mu-like phage, with 10 of the 25 phage-related CDSs most similar to CDSs in Mu-like phages of C. violaceum (CvP1), Haemophilus influenzae and N. meningitidis. There are also other CDSs similar to other phage genes of lambda- and P2-like phages.

LhP3

Bacteriophage LhP3 is the smallest prophage in the L. hongkongensis genome. It is composed of 19 CDSs, accounting for 11,169 bp with G+C content 58.70%, lower than that of the host genome (62.35%), reflecting its heterologous origin. Of the 19 CDSs, 14 were phage-related CDSs with similarity to genes of BPP-1-, lambda- and epsilon15-like phages and other unclassified phages, indicating its genetic complexity. Further studies are required if this relatively small prophage is a functional tailed phage.

LhP4

Bacteriophage LhP4 is composed of 36 CDSs, accounting for 34,375 bp with G+C content 58.78%, also lower than that of the host genome, indicating its heterologous origin. Of the 23 phage-related CDSs, 14 possessed similarity to genes of Bordetella phage BPP-1. Other phage related genes resemble those of P4-, P2-, P22- and episolon15-like phages and unclassified phages of Siphoviridae and Myoviridae.

LhP5

Bacteriophage LhP5 is the largest prophage identified in the L. hongkongensis genome. Composed of 64 CDSs, it accounts for 43,997 bp with G+C content 59%, lower than that of the host genome. Of the 32 phage-related CDSs, 9 possessed homologies to genes of Mu-like phages, 7 even possessed homologies to genes of lambda-like phages. The other phage-related CDSs are most closely related to those of various phages including those belonging to Podoviridae (phages with short tails), Myoriviridae and Siphoviridae.

LhP6

Bacteriophage LhP6 is composed of 31 CDSs, accounting for 21,918 bp with G+C content of 62.04%. The 25 phage-related CDSs exhibit similarity to phage genes of Bordetella bronchiseptica and Bordetella avium. Of these 25 CDSs, 12 possessed homologies to genes of unclassified phages belonging to Siphoviridae and 5 to lambda-like phages.

LhP7

Bacteriophage LhP7 is composed of 31 CDSs, accounting for 19,992 bp with the lowest G+C content of 55.59% among the eight prophages, suggesting a heterologous origin. Of the 18 phage-related CDSs, 4 exhibits similarity to phage genes of N. meningitidis, Burkholderia, and C. violaceum genes of Mu-like phages, and others to those of unclassified phages, lambda-, P22-, and BPP-1-like phages.

LhP8

Similar to LhP1, bacteriophage LhP8 is also a P2-like phage (Figure 17). It is composed of 48 CDSs, accounting for 33,791 bp with G+C content of 63.87%, similar to that of the host genome. It contains the highest number of phage-related CDSs (n = 37) among the eight phages. Of the 37 phage-related CDSs, 30 were most similar to genes of P2-like phages and others to phages of Myoviridae, Siphoviridae and Mu-like phages. In fact, LhP1 and LhP8 are highly similar with the exception of a few CDSs, with most of their CDSs exhibiting similarity to phage proteins found in other gram-negative bacteria including Salmonella, Burkholderia, Yersinia, and Shigella species. Their gene organizations are also highly similar to P2 phage (Table 6) (Figure 17).
Table 6

CDSs of LhP1 and LhP8 from the L. hongkongensis HLHK9, and comparison of genome structures of LhP1 (reverse complement), LhP8 and E. coli P2 phage.

P2

LhP1

LhP8

Function

Q

LHK_00420

LHK_02579

Capsid portal protein

P

LHK_00419

LHK_02580/LHK_02582

Large terminase subunit

O

LHK_00418

LHK_02581/LHK_02583

Capsid scaffold

N

LHK_00417

LHK_02584

Major capsid precursor

M

LHK_00416

LHK_02585

Small terminase subunit

L

LHK_00415

LHK_02586

Capsid completion

X

LHK_00414

LHK_02587

Tail

Y

-

-

Lysis - holin

K

-

-

Lysis - endolysin

-

LHK_00413

LHK_02588

Phage-related transmembrane protein

-

LHK_00412

LHK_02589

Hypothetical protein

-

LHK_00411

LHK_02590

Putative phage-related protein (hydrolase)

lysA

-

-

Timing of lysis

lysB

LHK_00410

LHK_02591

Timing of lysis

lysC

-

-

Regulation of lysis

-

LHK_00409

LHK_02592

Hypothetical protein

R

LHK_00408

LHK_02593

Tail completion

S

LHK_00407

LHK_02594

Tail completion

V

LHK_00406/LHK_00405

LHK_02595

Baseplate assembly

W

LHK_00404

LHK_02596

Baseplate assembly

J

LHK_00403

LHK_02597

Baseplate assembly

I

LHK_00402

LHK_02598

Baseplate assembly

H

LHK_00401

LHK_02599

Tail fiber

-

LHK_00400

LHK_02600

Hypothetical protein

-

LHK_00399

LHK_02601

Mu-like prophage protein Com

-

LHK_00398

LHK_02602

DNA adenine methylase

-

LHK_00397

LHK_02603

Hypothetical protein

G

-

-

Tail fiber assembly

Z/fun

-

-

Blocks phage T5

FI

LHK_00396

LHK_02604

Tail sheath

FII

LHK_00395

LHK_02605

Tail tube

E+E'

LHK_00394

LHK_02606

Tail

E

LHK_00393

LHK_02607

Tail

T

LHK_00392

LHK_02608

Tail

U

LHK_00391

LHK_02609

Tail

D

LHK_00390

LHK_02610

Tail

-

LHK_00389

-

Hypothetical protein

-

LHK_00388

-

Hypothetical protein

-

LHK_00387

-

Hypothetical protein

-

LHK_00386

-

Hypothetical protein

-

LHK_00385

-

Hypothetical protein

-

-

LHK_02611

Anthranilate synthase component I

-

-

LHK_02612

Hypothetical protein

-

-

LHK_02613

Hypothetical protein

-

-

LHK_02614

Hypothetical protein

-

LHK_00384

LHK_02615

Hypothetical protein

Ogr

LHK_00383

LHK_02616

Late promoter activator

-

LHK_00382

LHK_02617

Hypothetical protein

-

LHK_00381

-

Hypothetical protein

-

LHK_00380

LHK_02618

Hypothetical protein

-

-

LHK_02619

Hypothetical protein

-

LHK_00379

LHK_02620

Cro/CI family transcriptional regulator

-

LHK_00378

LHK_02621

Hypothetical protein

Int

-

-

Integrase

C

-

-

Immunity repressor

Cox

-

-

Inhibits integration

B

-

-

DNA replication

A

LHK_00377

LHK_02622

DNA replication

-

LHK_00376

-

Hypothetical protein

-

LHK_00375

-

Hypothetical protein

-

-

LHK_02623

Hypothetical protein

-

-

LHK_02624

DNA binding protein, excisionase family

tin

-

-

Blocks growth of T-even phages

old

-

-

Blocks growth of phage lambda

-

LHK_00374

LHK_02625

Integrase

Remnant phages

Among the eight putative prophages, LhP1 and LhP8 are most likely to represent intact prophages, while the remaining six prophages encode a diversity of prophage elements of phage-related structural and non-structural proteins. In addition to these putative prophages, 17 other phage-related CDSs were found scattered in the L. hongkongensis genome. However, these CDSs are either not flanked by other phage-related genes or that the region of these phage-related gene clusters was too short for confident prediction as prophages. Further studies are required to ascertain if the present putative prophages and phage-related gene clusters are intact or remnant phages.

Transposases and insertion sequences

There are 26 CDSs coding for transposases in the L. hongkongensis genome (Table 7). Fourteen of these 26 transposases possessed homologies to transposases of IS3 family, nine to those of IS5 family and three to those of IS481 family. The presence of transposases of IS481 family is unique in L. hongkongensis, as they are absent in other members of the Neisseriaceae family such as the pathogenic Neisseria species and C. violaceum[114]. The transposases of L. hongkongensis are most closely related to those of other members of β-proteobacteria, especially of the order Burkholderiales, with seven most closely related to those of Comamonas testosteroni, seven to those of Janthinobacterium sp., and four to those of Polaromonas sp. However, only two pairs of these transposases carry short imperfect inverted repeats at their ends that form insertion sequences most closely related to the IS3 family. Other transposases are likely remnant insertion sequences and lack associated inverted repeats. The first insertion sequence, of 1,183 bp, contains two ORFs, LHK_01280 (ORFb) and LHK_01281 (ORFa), with 38-bp inverted repeats with six mismatches. The second insertion sequence is relatively short in length, with 603 bp containing two ORFs, LHK_02311 and LHK_02312 (ORFa) and 50-bp inverted repeats with ten mismatches. The G+C content of both putative insertion sequences are lower (57.4% and 54.89% respectively) than that of the L. hongkongensis genome, suggestive of heterologous origin.
Table 7

Transposases identified in the genome of L. hongkongensis HLHK9

CDS

IS name

IS family

IS group

Origin

Identity (%)

E-value

Size (bp)

LHK_00816

ISCte3

IS3

IS407

Comamonas testosteroni

79.76

2e-38

294

LHK_00817

ISCte3

IS3

IS407

Comamonas testosteroni

77.01

9e-36

264

LHK_00911

ISAisp3

IS481

-

Acidovorax sp.

62.5

7e-54

588

LHK_01023

ISJsp2

IS5

IS903

Janthinobacterium sp.

47.92

6e-50

822

LHK_01024

ISPosp5

IS3

IS3

Polaromonas sp.

71.21

2e-24

339

LHK_01025

ISPosp5

IS3

IS3

Polaromonas sp.

65.98

1e-33

336

LHK_01171

ISPosp5

IS3

IS3

Polaromonas sp.

71.21

2e-24

342

LHK_01172

ISPosp5

IS3

IS3

Polaromonas sp.

65.98

2e-33

336

LHK_01280

ISKpn10

IS3

IS407

Klebsiella pneumoniae

68.29

2e-50

360

LHK_01281

ISKpn10

IS3

IS407

Klebsiella pneumoniae

84.09

2e-39

267

LHK_01366

ISJsp2

IS5

IS903

Janthinobacterium sp.

60.68

4e-36

744

LHK_01998

ISJsp2

IS5

IS903

Janthinobacterium sp.

75.86

2e-09

186

LHK_01999

ISPpa4

IS5

IS903

Paracoccus pantotrophus

69

2e-17

219

LHK_02013

ISRme14

IS481

-

Ralstonia metallidurans

59.46

2e-22

249

LHK_02014

ISAisp3

IS481

-

Acidovorax sp.

71.26

8e-27

276

LHK_02015

ISJsp2

IS5

IS903

Janthinobacterium sp.

60.53

7e-35

540

LHK_02176

ISJsp2

IS5

IS903

Janthinobacterium sp.

58.02

7e-23

273

LHK_02311

ISCte3

IS3

IS407

Comamonas testosteroni

88.89

6e-09

141

LHK_02312

ISCte3

IS3

IS407

Comamonas testosteroni

78.05

8e-14

126

LHK_02314

ISCte3

IS3

IS407

Comamonas testosteroni

96

3e-20

399

LHK_02540

ISJsp2

IS5

IS903

Janthinobacterium sp.

59.54

9e-40

648

LHK_02711

IS476

IS3

IS407

Xanthomonas campestris pv. vesicatoria 81-23 race 2

63.64

7e-42

387

LHK_02712

IS1421

IS5

IS427

Ralstonia solanacearum

57.38

8e-33

357

LHK_02720

ISCte3

IS3

IS407

Comamonas testosteroni

72.73

4e-14

627

LHK_02721

ISCte3

IS3

IS407

Comamonas testosteroni

77.01

4e-12

264

LHK_03256

ISJsp2

IS5

IS903

Janthinobacterium sp.

62.79

3e-27

477

Conclusions

The L. hongkongensis genome possessed genes and gene cassettes for acid and bile resistance, colonization of the intestinal mucosa, evasion of host defense and cytotoxicity and invasion. In addition, a broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, together with other phage-related CDSs and CDSs coding for transposases, were also identified.

Methods

CDSs identified in the L. hongkongensis genome were annotated as described in our previous publication and classified functionally according to the Clusters of Orthologous Groups (COG) methodology [10]. CDSs belonging to COG clusters potentially associated with virulence (such as intracellular trafficking, secretion and vesicular transport) were selected for further examination, whereas those associated with housekeeping functions (such as chromatin structure and dynamics) were removed. The CDSs were then examined by comparison with the latest release of the reference Virulence Factor Database (VFDB) [115] and keyword searching using the following words and their variants: virulence, toxin, hemolysin/hemolysis, pathogenicity, adherence, invasion, secretion, phagocytosis, phase variation, stress, iron uptake, siderophore, resistance, efflux pump, damaging and regulation. For drug resistance, CDSs that were classified to COG V (defense mechanisms), COG Q (secondary metabolites biosynthesis, transport and catabolism), and COQ M (cell wall/membrane/envelope biogenesis) were manually annotated for identification of antibiotic resistance-related genes. CDSs from other COGs were searched for additional genes using keywords: resistance antibiotic, efflux, multi etc. Prophages were identified by Prophage finder http://bioinformatics.uwp.edu/~phage/ searches [116]. The genome was run under the parameters with an e-value of 0.01, hits per prophage of 7, and hit spacing of 5000. Transposases were identified by performing BlastP analyses for all CDSs identified in the genome of L. hongkongensis HLHK9 against the ISfinder database http://www-is.biotoul.fr/is.html[117] and inverted repeats by einverted (EMBOSS package) [118]. Manual confirmation of the assigned function was performed by sequence similarity search using BLAST against the NCBI nr database, and assisted by conserved domain search (CD-search), identification of signature sequence motifs and sequence analysis using InterProScan. Localization patterns of putative virulence factors were predicted using PSORTb where appropriate [119].

Notes

List of abbreviations

ABC: 

ATP-Binding Cassette

ATP: 

Adenosine triphosphate

BLAST: 

Basic Local Alignment Search Tool

bp: 

Base pair

C55-P: 

Undecaprenyl pyrophosphate

C55-PP: 

Undecaprenyl diphosphate

CD14: 

Cluster of differentiation 14

CDS(s): 

Coding sequence(s)

COG(s): 

Clusters of orthologous group(s)

CvP: 

Chromobacterium violaceum phage

DAEC: 

Diffusely adherent Escherichia coli

DNA: 

Deoxyribonucleic acid

dTDP: 

Deoxythymidine diphosphate

EMBOSS: 

European Molecular Biology Open Software Suite

ETEC: 

Enterotoxigenic Escherichia coli

IS: 

Insertion sequence

Kdo: 

Keto-deoxyoctulonate

LhP: 

Laribacter hongkongensis prophage

LPS: 

Lipopolysaccharide

MATE: 

Multidrug and Toxic compound Extrusion

Mbp: 

Mega base pairs

MF: 

Major facilitator

MFP: 

Membrane fusion protein

MFS: 

Major Facilitator Superfamily

MIC: 

Minimum inhibitory concentration

OMP: 

Outer membrane (channel) protein

OMPLA: 

Outer membrane phospholipase A

ORF(s): 

Open reading frame(s)

PBP(s): 

Penicillin-binding protein(s)

RND: 

Resistance nodulation division

RTX: 

Repeats in toxin

SMR: 

Small Multidrug Resistance family.

Declarations

Acknowledgements

This work is partly supported by the Research Grant Council Grant, Committee for Research and Conference Grant and University Development Fund, The University of Hong Kong; the HKSAR Research Fund for the Control of Infectious Diseases of the Health, Welfare and Food Bureau. We are grateful to support from the Genome Research Centre, The University of Hong Kong, and the generous support of Mrs. Carol Yu, Professor Richard Yu, Mr. Hui Hoy and Mr. Hui Ming in the genomic sequencing platform.

Authors’ Affiliations

(1)
State Key Laboratory of Emerging Infectious Diseases
(2)
Research Centre of Infection and Immunology, The University of Hong Kong
(3)
Carol Yu Centre of Infection, The University of Hong Kong
(4)
Department of Microbiology, The University of Hong Kong

References

  1. Yuen KY, Woo PC, Teng JL, Leung KW, Wong MK, Lau SK: Laribacter hongkongensis gen. nov., sp. nov., a novel gram-negative bacterium isolated from a cirrhotic patient with bacteremia and empyema. J Clin Microbiol. 2001, 39: 4227-4232. 10.1128/JCM.39.12.4227-4232.2001PubMed CentralPubMedGoogle Scholar
  2. Lau SK, Woo PC, Hui WT, Li MW, Teng JL, Que TL, Luk WK, Lai RW, Yung RW, Yuen KY: Use of cefoperazone MacConkey agar for selective isolation of Laribacter hongkongensis. J Clin Microbiol. 2003, 41: 4839-4841. 10.1128/JCM.41.10.4839-4841.2003PubMed CentralPubMedGoogle Scholar
  3. Woo PC, Kuhnert P, Burnens AP, Teng JL, Lau SK, Que TL, Yau HH, Yuen KY: Laribacter hongkongensis: a potential cause of infectious diarrhea. Diagn Microbiol Infect Dis. 2003, 47: 551-556. 10.1016/S0732-8893(03)00161-5PubMedGoogle Scholar
  4. Woo PC, Lau SK, Teng JL, Que TL, Yung RW, Luk WK, Lai RW, Hui WT, Wong SS, Yau HH, Yuen KY: Association of Laribacter hongkongensis in community-acquired gastroenteritis with travel and eating fish: a multicentre case-control study. Lancet. 2004, 363: 1941-1947. 10.1016/S0140-6736(04)16407-6PubMedGoogle Scholar
  5. Woo PC, Lau SK, Teng JL, Yuen KY: Current status and future directions of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis and traveller's diarrhoea. Curr Opin Infect Dis. 2005, 18: 413-419. 10.1097/01.qco.0000180162.76648.c9PubMedGoogle Scholar
  6. Lau SK, Woo PC, Fan RY, Lee RC, Teng JL, Yuen KY: Seasonal and tissue distribution of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, in retail freshwater fish in Hong Kong. Int J Food Microbiol. 2007, 113: 62-66. 10.1016/j.ijfoodmicro.2006.07.017PubMedGoogle Scholar
  7. Lau SK, Woo PC, Fan RY, Ma SS, Hui WT, Au SY, Chan LL, Chan JY, Lau AT, Leung KY, Pun TC, She HH, Wong CY, Wong LL, Yuen KY: Isolation of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, from drinking water reservoirs in Hong Kong. J Appl Microbiol. 2007, 103: 507-515. 10.1111/j.1365-2672.2006.03263.xPubMedGoogle Scholar
  8. Teng JL, Woo PC, Ma SS, Sit TH, Ng LT, Hui WT, Lau SK, Yuen KY: Ecoepidemiology of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis. J Clin Microbiol. 2005, 43: 919-922. 10.1128/JCM.43.2.919-922.2005PubMed CentralPubMedGoogle Scholar
  9. Woo PC, Teng JL, Tsang AK, Tse H, Tsang VY, Chan KM, Lee EK, Chan JK, Ma SS, Tam DM, Chung LM, Lau SK, Yuen KY: Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea. BMC Microbiol. 2009, 9: 21. 10.1186/1471-2180-9-21PubMed CentralPubMedGoogle Scholar
  10. Woo PC, Lau SK, Tse H, Teng JL, Curreem SO, Tsang AK, Fan RY, Wong GK, Huang Y, Loman NJ, Snyder LA, Cai JJ, Huang JD, Mak W, Pallen MJ, Lok S, Yuen KY: The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats. PLoS Genet. 2009, 5: e1000416. 10.1371/journal.pgen.1000416PubMed CentralPubMedGoogle Scholar
  11. Mobley HL, Island MD, Hausinger RP: Molecular biology of microbial ureases. Microbiol Rev. 1995, 59: 451-480.PubMed CentralPubMedGoogle Scholar
  12. Bandara AB, Contreras A, Contreras-Rodriguez A, Martins AM, Dobrean V, Poff-Reichow S, Rajasekaran P, Sriranganathan N, Schurig GG, Boyle SM: Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice. BMC Microbiol. 2007, 7: 57. 10.1186/1471-2180-7-57PubMed CentralPubMedGoogle Scholar
  13. Sangari FJ, Seoane A, Rodríguez MC, Agüero J, García Lobo JM: Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium. Infect Immun. 2007, 75: 774-780. 10.1128/IAI.01244-06PubMed CentralPubMedGoogle Scholar
  14. de Koning-Ward TF, Ward AC, Robins-Browne RM: Characterisation of the urease-encoding gene complex of Yersinia enterocolitica. Gene. 1994, 145: 25-32. 10.1016/0378-1119(94)90318-2PubMedGoogle Scholar
  15. Riot B, Berche P, Simonet M: Urease is not involved in the virulence of Yersinia pseudotuberculosis in mice. Infect Immun. 1997, 65: 1985-1990.PubMed CentralPubMedGoogle Scholar
  16. Sebbane F, Devalckenaere A, Foulon J, Carniel E, Simonet M: Silencing and reactivation of urease in Yersinia pestis is determined by one G residue at a specific position in the ureD gene. Infect Immun. 2001, 69: 170-176. 10.1128/IAI.69.1.170-176.2001PubMed CentralPubMedGoogle Scholar
  17. Marquis RE, Bender GR, Murray DR, Wong A: Arginine deiminase system and bacterial adaptation to acid environments. Appl Environ Microbiol. 1987, 53: 198-200.PubMed CentralPubMedGoogle Scholar
  18. Gruening P, Fulde M, Valentin-Weigand P, Goethe R: Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis. J Bacteriol. 2006, 188: 361-369. 10.1128/JB.188.2.361-369.2006PubMed CentralPubMedGoogle Scholar
  19. Degnan BA, Fontaine MC, Doebereiner AH, Lee JJ, Mastroeni P, Dougan G, Goodacre JA, Kehoe MA: Characterization of an isogenic mutant of Streptococcus pyogenes Manfredo lacking the ability to make streptococcal acid glycoprotein. Infect Immun. 2000, 68: 2441-2448. 10.1128/IAI.68.5.2441-2448.2000PubMed CentralPubMedGoogle Scholar
  20. Degnan BA, Kehoe MA, Goodacre JA: Analysis of human T cell responses to group A streptococci using fractionated Streptococcus pyogenes proteins. FEMS Immunol Med Microbiol. 1997, 17: 161-170. 10.1111/j.1574-695X.1997.tb01009.xPubMedGoogle Scholar
  21. Degnan BA, Palmer JM, Robson T, Jones CE, Fischer M, Glanville M, Mellor GD, Diamond AG, Kehoe MA, Goodacre JA: Inhibition of human peripheral blood mononuclear cell proliferation by Streptococcus pyogenes cell extract is associated with arginine deiminase activity. Infect Immun. 1998, 66: 3050-3058.PubMed CentralPubMedGoogle Scholar
  22. Thanassi DG, Cheng LW, Nikaido H: Active efflux of bile salts by Escherichia coli. J Bacteriol. 1997, 179: 2512-2518.PubMed CentralPubMedGoogle Scholar
  23. Prouty AM, Brodsky IE, Manos J, Belas R, Falkow S, Gunn JS: Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile. FEMS Immunol Med Microbiol. 2004, 41: 177-185. 10.1016/j.femsim.2004.03.002PubMedGoogle Scholar
  24. Okoli AS, Wadstrom T, Mendz GL: MiniReview: bioinformatic study of bile responses in Campylobacterales. FEMS Immunol Med Microbiol. 2007, 49: 101-123. 10.1111/j.1574-695X.2006.00194.xPubMedGoogle Scholar
  25. Elkins CA, Nikaido H: Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops. J Bacteriol. 2002, 184: 6490-6498. 10.1128/JB.184.23.6490-6499.2002PubMed CentralPubMedGoogle Scholar
  26. Nishino K, Yamaguchi A: Analysis of a complete library of putative drug transporter genes in Escherichia coli. J Bacteriol. 2001, 183: 5803-5812. 10.1128/JB.183.20.5803-5812.2001PubMed CentralPubMedGoogle Scholar
  27. Nagakubo S, Nishino K, Hirata T, Yamaguchi A: The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system, MdtABC. J Bacteriol. 2002, 184: 4161-4167. 10.1128/JB.184.15.4161-4167.2002PubMed CentralPubMedGoogle Scholar
  28. Nishino K, Latifi T, Groisman EA: Virulence and drug resistance roles of multidrug efflux systems of Salmonella enterica serovar Typhimurium. Mol Microbiol. 2006, 59: 126-141. 10.1111/j.1365-2958.2005.04940.xPubMedGoogle Scholar
  29. Picken RN, Beacham IR: Bacteriophage-resistant mutants of Escherichia coli K12. Location of receptors within the lipopolysaccharide. J Gen Microbiol. 1977, 102: 305-318.PubMedGoogle Scholar
  30. Gunn JS: Mechanisms of bacterial resistance and response to bile. Microbes Infect. 2000, 2: 907-913. 10.1016/S1286-4579(00)00392-0PubMedGoogle Scholar
  31. Ray MC, Germon P, Vianney A, Portalier R, Lazzaroni JC: Identification by genetic suppression of Escherichia coli TolB residues important for TolB-Pal interaction. J Bacteriol. 2000, 182: 821-824. 10.1128/JB.182.3.821-824.2000PubMed CentralPubMedGoogle Scholar
  32. Prouty AM, Van Velkinburgh JC, Gunn JS: Salmonella enterica serovar Typhimurium resistance to bile: identification and characterization of the tolQRA cluster. J Bacteriol. 2002, 184: 1270-1276. 10.1128/JB.184.5.1270-1276.2002PubMed CentralPubMedGoogle Scholar
  33. Dubuisson JF, Vianney A, Hugouvieux-Cotte-Pattat N, Lazzaroni JC: Tol-Pal proteins are critical cell envelope components of Erwinia chrysanthemi affecting cell morphology and virulence. Microbiology. 2005, 151: 3337-3347. 10.1099/mic.0.28237-0PubMedGoogle Scholar
  34. Benz I, Schmidt MA: AIDA-I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule. Mol Microbiol. 1992, 6: 1539-1546. 10.1111/j.1365-2958.1992.tb00875.xPubMedGoogle Scholar
  35. Benz I, Schmidt MA: Isolation and serologic characterization of AIDA-I, the adhesin mediating the diffuse adherence phenotype of the diarrhea-associated Escherichia coli strain 2787 (O126:H27). Infect Immun. 1992, 60: 13-18.PubMed CentralPubMedGoogle Scholar
  36. Sherlock O, Schembri MA, Reisner A, Klemm P: Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation. J Bacteriol. 2004, 186: 8058-8065. 10.1128/JB.186.23.8058-8065.2004PubMed CentralPubMedGoogle Scholar
  37. Elsinghorst EA, Weitz JA: Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein. Infect Immun. 1994, 62: 3463-3471.PubMed CentralPubMedGoogle Scholar
  38. Sherlock O, Vejborg RM, Klemm P: The TibA adhesin/invasin from enterotoxigenic Escherichia coli is self recognizing and induces bacterial aggregation and biofilm formation. Infect Immun. 2005, 73: 1954-1963. 10.1128/IAI.73.4.1954-1963.2005PubMed CentralPubMedGoogle Scholar
  39. Lindenthal C, Elsinghorst EA: Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells. Infect Immun. 2001, 69: 52-57. 10.1128/IAI.69.1.52-57.2001PubMed CentralPubMedGoogle Scholar
  40. Lindenthal C, Elsinghorst EA: Identification of a glycoprotein produced by enterotoxigenic Escherichia coli. Infect Immun. 1999, 67: 4084-4091.PubMed CentralPubMedGoogle Scholar
  41. Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala'Aldeen D: Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev. 2004, 68: 692-744. 10.1128/MMBR.68.4.692-744.2004PubMed CentralPubMedGoogle Scholar
  42. Henderson IR, Nataro JP: Virulence functions of autotransporter proteins. Infect Immun. 2001, 69: 1231-1243. 10.1128/IAI.69.3.1231-1243.2001PubMed CentralPubMedGoogle Scholar
  43. Dautin N, Bernstein HD: Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu Rev Microbiol. 2007, 61: 89-112. 10.1146/annurev.micro.61.080706.093233PubMedGoogle Scholar
  44. Benz I, Schmidt MA: Glycosylation with heptose residues mediated by the aah gene product is essential for adherence of the AIDA-I adhesin. Mol Microbiol. 2001, 40: 1403-1413. 10.1046/j.1365-2958.2001.02487.xPubMedGoogle Scholar
  45. Moormann C, Benz I, Schmidt MA: Functional substitution of the TibC protein of enterotoxigenic Escherichia coli strains for the autotransporter adhesin heptosyltransferase of the AIDA system. Infect Immun. 2002, 70: 2264-2270. 10.1128/IAI.70.5.2264-2270.2002PubMed CentralPubMedGoogle Scholar
  46. Kulshin VA, Zähringer U, Lindner B, Frasch CE, Tsai CM, Dmitriev BA, Rietschel ET: Structural characterization of the lipid A component of pathogenic Neisseria meningitidis. J Bacteriol. 1992, 174: 1793-1800.PubMed CentralPubMedGoogle Scholar
  47. van der Ley P, Steeghs L, Hamstra HJ, ten Hove J, Zomer B, van Alphen L: Modification of lipid A biosynthesis in Neisseria meningitidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, and adjuvant activity. Infect Immun. 2001, 69: 5981-5990. 10.1128/IAI.69.10.5981-5990.2001PubMed CentralPubMedGoogle Scholar
  48. Harvey HA, Post DM, Apicella MA: Immortalization of human urethral epithelial cells: a model for the study of the pathogenesis of and the inflammatory cytokine response to Neisseria gonorrhoeae infection. Infect Immun. 2002, 70: 5808-5815. 10.1128/IAI.70.10.5808-5815.2002PubMed CentralPubMedGoogle Scholar
  49. Teghanemt A, Zhang D, Levis EN, Weiss JP, Gioannini TL: Molecular basis of reduced potency of underacylated endotoxins. J Immunol. 2005, 175: 4669-4676.PubMedGoogle Scholar
  50. Arking D, Tong Y, Stein DC: Analysis of lipooligosaccharide biosynthesis in the Neisseriaceae. J Bacteriol. 2001, 183: 934-941. 10.1128/JB.183.3.934-941.2001PubMed CentralPubMedGoogle Scholar
  51. Hammerschmidt S, Birkholz C, Zähringer U, Robertson BD, van Putten J, Ebeling O, Frosch M: Contribution of genes from the capsule gene complex (cps) to lipooligosaccharide biosynthesis and serum resistance in Neisseria meningitidis. Mol Microbiol. 1994, 11: 885-896. 10.1111/j.1365-2958.1994.tb00367.xPubMedGoogle Scholar
  52. Robertson BD, Frosch M, van Putten JP: The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis. J Bacteriol. 1994, 176: 6915-6920.PubMed CentralPubMedGoogle Scholar
  53. Preston A, Allen AG, Cadisch J, Thomas R, Stevens K, Churcher CM, Badcock KL, Parkhill J, Barrell B, Maskell DJ: Genetic basis for lipopolysaccharide O-antigen biosynthesis in bordetellae. Infect Immun. 1999, 67: 3763-3767.PubMed CentralPubMedGoogle Scholar
  54. Sperandeo P, Lau FK, Carpentieri A, De Castro C, Molinaro A, Dehò G, Silhavy TJ, Polissi A: Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli. J Bacteriol. 2008, 190: 4460-4469. 10.1128/JB.00270-08PubMed CentralPubMedGoogle Scholar
  55. Felmlee T, Pellett S, Welch RA: Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol. 1985, 163: 94-105.PubMed CentralPubMedGoogle Scholar
  56. Cavalieri SJ, Bohach GA, Snyder IS: Escherichia coli alpha-hemolysin: characteristics and probable role in pathogenicity. Microbiol Rev. 1984, 48: 326-343.PubMed CentralPubMedGoogle Scholar
  57. Lally ET, Hill RB, Kieba IR, Korostoff J: The interaction between RTX toxins and target cells. Trends Microbiol. 1999, 7: 356-361. 10.1016/S0966-842X(99)01530-9PubMedGoogle Scholar
  58. Coote JG: Structural and functional relationships among the RTX toxin determinants of gram-negative bacteria. FEMS Microbiol Rev. 1992, 8: 137-161.PubMedGoogle Scholar
  59. Thompson SA, Wang LL, West A, Sparling PF: Neisseria meningitidis produces iron-regulated proteins related to the RTX family of exoproteins. J Bacteriol. 1993, 175: 811-818.PubMed CentralPubMedGoogle Scholar
  60. Thompson SA, Wang LL, Sparling PF: Cloning and nucleotide sequence of frpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins. Mol Microbiol. 1993, 9: 85-96. 10.1111/j.1365-2958.1993.tb01671.xPubMedGoogle Scholar
  61. Thompson SA, Sparling PF: The RTX cytotoxin-related FrpA protein of Neisseria meningitidis is secreted extracellularly by meningococci and by HlyBD+ Escherichia coli. Infect Immun. 1993, 61: 2906-2911.PubMed CentralPubMedGoogle Scholar
  62. Andrews DL, Beames B, Summers MD, Park WD: Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector. Biochem J. 1988, 252: 199-206.PubMed CentralPubMedGoogle Scholar
  63. Holk A, Rietz S, Zahn M, Quader H, Scherer GF: Molecular identification of cytosolic, patatin-related phospholipases A from Arabidopsis with potential functions in plant signal transduction. Plant Physiol. 2002, 130: 90-101. 10.1104/pp.006288PubMed CentralPubMedGoogle Scholar
  64. Strickland JA, Orr GL, Walsh TA: Inhibition of Diabrotica Larval Growth by Patatin, the Lipid Acyl Hydrolase from Potato Tubers. Plant Physiol. 1995, 109: 667-674.PubMed CentralPubMedGoogle Scholar
  65. Sato H, Frank DW, Hillard CJ, Feix JB, Pankhaniya RR, Moriyama K, Finck-Barbançon V, Buchaklian A, Lei M, Long RM, Wiener-Kronish J, Sawa T: The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU. EMBO J. 2003, 22: 2959-2969. 10.1093/emboj/cdg290PubMed CentralPubMedGoogle Scholar
  66. Sato H, Frank DW: ExoU is a potent intracellular phospholipase. Mol Microbiol. 2004, 53: 1279-1290. 10.1111/j.1365-2958.2004.04194.xPubMedGoogle Scholar
  67. Pankhaniya RR, Tamura M, Allmond LR, Moriyama K, Ajayi T, Wiener-Kronish JP, Sawa T: Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU. Crit Care Med. 2004, 32: 2293-2299.PubMedGoogle Scholar
  68. Banerji S, Flieger A: Patatin-like proteins: a new family of lipolytic enzymes present in bacteria?. Microbiology. 2004, 150: 522-525. 10.1099/mic.0.26957-0PubMedGoogle Scholar
  69. Nakatsu CH, Hristova K, Hanada S, Meng XY, Hanson JR, Scow KM, Kamagata Y: Methylibium petroleiphilum gen. nov., sp. nov., a novel methyl tert-butyl ether-degrading methylotroph of the Betaproteobacteria. Int J Syst Evol Microbiol. 2006, 56: 983-989. 10.1099/ijs.0.63524-0PubMedGoogle Scholar
  70. Massova I, Mobashery S: Kinship and diversification of bacterial penicillin-binding proteins and beta-lactamases. Antimicrob Agents Chemother. 1998, 42: 1-17. 10.1093/jac/42.1.1PubMed CentralPubMedGoogle Scholar
  71. Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P: The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev. 2008, 32: 234-258. 10.1111/j.1574-6976.2008.00105.xPubMedGoogle Scholar
  72. Sibold C, Henrichsen J, Konig A, Martin C, Chalkley L, Hakenbeck R: Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis. Mol Microbiol. 1994, 12: 1013-1023. 10.1111/j.1365-2958.1994.tb01089.xPubMedGoogle Scholar
  73. Dabernat H, Delmas C, Seguy M, Pelissier R, Faucon G, Bennamani S, Pasquier C: Diversity of beta-lactam resistance-conferring amino acid substitutions in penicillin-binding protein 3 of Haemophilus influenzae. Antimicrob Agents Chemother. 2002, 46: 2208-2218. 10.1128/AAC.46.7.2208-2218.2002PubMed CentralPubMedGoogle Scholar
  74. Gerrits MM, Schuijffel D, van Zwet AA, Kuipers EJ, Vandenbroucke-Grauls CM, Kusters JG: Alterations in penicillin-binding protein 1A confer resistance to beta-lactam antibiotics in Helicobacter pylori. Antimicrob Agents Chemother. 2002, 46: 2229-2233. 10.1128/AAC.46.7.2229-2233.2002PubMed CentralPubMedGoogle Scholar
  75. Antignac A, Boneca IG, Rousselle JC, Namane A, Carlier JP, Vazquez JA, Fox A, Alonso JM, Taha MK: Correlation between alterations of the penicillin-binding protein 2 and modifications of the peptidoglycan structure in Neisseria meningitidis with reduced susceptibility to penicillin G. J Biol Chem. 2003, 278: 31529-31535. 10.1074/jbc.M304607200PubMedGoogle Scholar
  76. Lau SK, Ho PL, Li MW, Tsoi HW, Yung RW, Woo PC, Yuen KY: Cloning and characterization of a chromosomal class C beta-lactamase and its regulatory gene in Laribacter hongkongensis. Antimicrob Agents Chemother. 2005, 49: 1957-1964. 10.1128/AAC.49.5.1957-1964.2005PubMed CentralPubMedGoogle Scholar
  77. Wang Z, Fast W, Valentine AM, Benkovic SJ: Metallo-beta-lactamase: structure and mechanism. Curr Opin Chem Biol. 1999, 3: 614-622. 10.1016/S1367-5931(99)00017-4PubMedGoogle Scholar
  78. Garau G, Garcia-Saez I, Bebrone C, Anne C, Mercuri P, Galleni M, Frere JM, Dideberg O: Update of the standard numbering scheme for class B beta-lactamases. Antimicrob Agents Chemother. 2004, 48: 2347-2349. 10.1128/AAC.48.7.2347-2349.2004PubMed CentralPubMedGoogle Scholar
  79. Bebrone C: Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol. 2007, 74: 1686-1701. 10.1016/j.bcp.2007.05.021PubMedGoogle Scholar
  80. Carfi A, Pares S, Duee E, Galleni M, Duez C, Frere JM, Dideberg O: The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold. Embo J. 1995, 14: 4914-4921.PubMed CentralPubMedGoogle Scholar
  81. Daiyasu H, Osaka K, Ishino Y, Toh H: Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold. FEBS Lett. 2001, 503: 1-6. 10.1016/S0014-5793(01)02686-2PubMedGoogle Scholar
  82. Paulsen IT, Brown MH, Skurray RA: Proton-dependent multidrug efflux systems. Microbiol Rev. 1996, 60: 575-608.PubMed CentralPubMedGoogle Scholar
  83. Ma D, Cook DN, Alberti M, Pon NG, Nikaido H, Hearst JE: Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol. 1995, 16: 45-55. 10.1111/j.1365-2958.1995.tb02390.xPubMedGoogle Scholar
  84. George AM: Multiple antimicrobial resistance. Frontiers in antimicrobial resistance: a tribute to Stuart B Levy. Edited by: White DG, Alekshun MN, McDermott PF, Levy SB. 2005, 151-164. Washington, DC, ASM Press.Google Scholar
  85. Rosenberg EY, Ma D, Nikaido H: AcrD of Escherichia coli is an aminoglycoside efflux pump. J Bacteriol. 2000, 182: 1754-1756. 10.1128/JB.182.6.1754-1756.2000PubMed CentralPubMedGoogle Scholar
  86. Aires JR, Nikaido H: Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli. J Bacteriol. 2005, 187: 1923-1929. 10.1128/JB.187.6.1923-1929.2005PubMed CentralPubMedGoogle Scholar
  87. Baranova N, Nikaido H: The BaeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate. J Bacteriol. 2002, 184: 4168-4176. 10.1128/JB.184.15.4168-4176.2002PubMed CentralPubMedGoogle Scholar
  88. Lomovskaya O, Lewis K: Emr, an Escherichia coli locus for multidrug resistance. Proc Natl Acad Sci USA. 1992, 89: 8938-8942. 10.1073/pnas.89.19.8938PubMed CentralPubMedGoogle Scholar
  89. Lomovskaya O, Lewis K, Matin A: EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB. J Bacteriol. 1995, 177: 2328-2334.PubMed CentralPubMedGoogle Scholar
  90. Edgar R, Bibi E: A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA. Embo J. 1999, 18: 822-832. 10.1093/emboj/18.4.822PubMed CentralPubMedGoogle Scholar
  91. Bentley J, Hyatt LS, Ainley K, Parish JH, Herbert RB, White GR: Cloning and sequence analysis of an Escherichia coli gene conferring bicyclomycin resistance. Gene. 1993, 127: 117-120. 10.1016/0378-1119(93)90625-DPubMedGoogle Scholar
  92. Kobayashi A, Hirakawa H, Hirata T, Nishino K, Yamaguchi A: Growth phase-dependent expression of drug exporters in Escherichia coli and its contribution to drug tolerance. J Bacteriol. 2006, 188: 5693-5703. 10.1128/JB.00217-06PubMed CentralPubMedGoogle Scholar
  93. Higashi K, Ishigure H, Demizu R, Uemura T, Nishino K, Yamaguchi A, Kashiwagi K, Igarashi K: Identification of a spermidine excretion protein complex (MdtJI) in Escherichia coli. J Bacteriol. 2008, 190: 872-878. 10.1128/JB.01505-07PubMed CentralPubMedGoogle Scholar
  94. Jack DL, Storms ML, Tchieu JH, Paulsen IT, Saier MH: A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins. J Bacteriol. 2000, 182: 2311-2313. 10.1128/JB.182.8.2311-2313.2000PubMed CentralPubMedGoogle Scholar
  95. Kaatz GW, Seo SM: Inducible NorA-mediated multidrug resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1995, 39: 2650-2655.PubMed CentralPubMedGoogle Scholar
  96. Hsieh PC, Siegel SA, Rogers B, Davis D, Lewis K: Bacteria lacking a multidrug pump: a sensitive tool for drug discovery. Proc Natl Acad Sci USA. 1998, 95: 6602-6606. 10.1073/pnas.95.12.6602PubMed CentralPubMedGoogle Scholar
  97. Reuter G, Janvilisri T, Venter H, Shahi S, Balakrishnan L, van Veen HW: The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities. J Biol Chem. 2003, 278: 35193-35198. 10.1074/jbc.M306226200PubMedGoogle Scholar
  98. Woebking B, Reuter G, Shilling RA, Velamakanni S, Shahi S, Venter H, Balakrishnan L, van Veen HW: Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA. J Bacteriol. 2005, 187: 6363-6369. 10.1128/JB.187.18.6363-6369.2005PubMed CentralPubMedGoogle Scholar
  99. Miller PF, Gambino LF, Sulavik MC, Gracheck SJ: Genetic relationship between soxRS and mar loci in promoting multiple antibiotic resistance in Escherichia coli. Antimicrob Agents Chemother. 1994, 38: 1773-1779.PubMed CentralPubMedGoogle Scholar
  100. McDermott PF, McMurry LM, Podglajen I, Dzink-Fox JL, Schneiders T, Draper MP, Levy SB: The marC gene of Escherichia coli is not involved in multiple antibiotic resistance. Antimicrob Agents Chemother. 2008, 52: 382-383. 10.1128/AAC.00930-07PubMed CentralPubMedGoogle Scholar
  101. Hussein MJ, Green JM, Nichols BP: Characterization of mutations that allow p-aminobenzoyl-glutamate utilization by Escherichia coli. J Bacteriol. 1998, 180: 6260-6268.PubMed CentralPubMedGoogle Scholar
  102. Veal WL, Shafer WM: Identification of a cell envelope protein (MtrF) involved in hydrophobic antimicrobial resistance in Neisseria gonorrhoeae. J Antimicrob Chemother. 2003, 51: 27-37. 10.1093/jac/dkg031PubMedGoogle Scholar
  103. Folster JP, Shafer WM: Regulation of mtrF expression in Neisseria gonorrhoeae and its role in high-level antimicrobial resistance. J Bacteriol. 2005, 187: 3713-3720. 10.1128/JB.187.11.3713-3720.2005PubMed CentralPubMedGoogle Scholar
  104. Inoue K, Basu S, Inouye M: Dissection of 16S rRNA methyltransferase (KsgA) function in Escherichia coli. J Bacteriol. 2007, 189: 8510-8518. 10.1128/JB.01259-07PubMed CentralPubMedGoogle Scholar
  105. Duffin PM, Seifert HS: ksgA mutations confer resistance to kasugamycin in Neisseria gonorrhoeae. Int J Antimicrob Agents. 2009, 33: 321-327. 10.1016/j.ijantimicag.2008.08.030PubMed CentralPubMedGoogle Scholar
  106. Ochi K, Kim JY, Tanaka Y, Wang G, Masuda K, Nanamiya H, Okamoto S, Tokuyama S, Adachi Y, Kawamura F: Inactivation of KsgA, a 16S rRNA methyltransferase, causes vigorous emergence of mutants with high-level kasugamycin resistance. Antimicrob Agents Chemother. 2009, 53: 193-201. 10.1128/AAC.00873-08PubMed CentralPubMedGoogle Scholar
  107. Cain BD, Norton PJ, Eubanks W, Nick HS, Allen CM: Amplification of the bacA gene confers bacitracin resistance to Escherichia coli. J Bacteriol. 1993, 175: 3784-3789.PubMed CentralPubMedGoogle Scholar
  108. Hu KH, Liu E, Dean K, Gingras M, DeGraff W, Trun NJ: Overproduction of three genes leads to camphor resistance and chromosome condensation in Escherichia coli. Genetics. 1996, 143: 1521-1532.PubMed CentralPubMedGoogle Scholar
  109. Lau SK, Wong GK, Li MW, Woo PC, Yuen KY: Distribution and molecular characterization of tetracycline resistance in Laribacter hongkongensis. J Antimicrob Chemother. 2008, 61: 488-497. 10.1093/jac/dkm539PubMedGoogle Scholar
  110. Feng JL, Yan H, Chowdhury N, Neogi SB, Yamasaki S, Shi L, Hu J, Chen Q: Identification and characterization of integron-associated antibiotic resistant Laribacter hongkongensis isolated from aquatic products in China. Int J Food Microbiol. 2011, 144: 337-341. 10.1016/j.ijfoodmicro.2010.10.014PubMedGoogle Scholar
  111. Lau SK, Woo PC, To AP, Lau AT, Yuen KY: Lack of evidence that DNA in antibiotic preparations is a source of antibiotic resistance genes in bacteria from animal or human sources. Antimicrob Agents Chemother. 2004, 48: 3141-3146. 10.1128/AAC.48.8.3141-3146.2004PubMed CentralPubMedGoogle Scholar
  112. Richard C: The Bacteriophages. 2006, Oxford University Press, 2.
  113. Ackermann HW: Tailed bacteriophages: the order caudovirales. Adv Virus Res. 1998, 51: 135-201. full_textPubMedGoogle Scholar
  114. Siguier P, Filée J, Chandler M: Insertion sequences in prokaryotic genomes. Curr Opin Microbiol. 2006, 9: 526-531. 10.1016/j.mib.2006.08.005PubMedGoogle Scholar
  115. Yang J, Chen L, Sun L, Yu J, Jin Q: VFDB 2008 release: an enhanced web-based resource for comparative pathogenomics. Nucleic Acids Res. 2008, 36: D539-542. 10.1093/nar/gkm951PubMed CentralPubMedGoogle Scholar
  116. Bose M, Barber RD: Prophage Finder: a prophage loci prediction tool for prokaryotic genome sequences. In Silico Biol. 2006, 6: 223-227.PubMedGoogle Scholar
  117. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M: ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res. 2006, D32-D36. 34 Database.
  118. Rice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000, 16: 276-277. 10.1016/S0168-9525(00)02024-2PubMedGoogle Scholar
  119. Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FS: PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics. 2010, 26: 1608-1615. 10.1093/bioinformatics/btq249PubMed CentralPubMedGoogle Scholar

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