Phylogenetic identification of bacterial MazF toxin protein motifs among probiotic strains and foodborne pathogens and potential implications of engineered probiotic intervention in food

  • Xianghe Yan1Email author,

    Affiliated with

    • Joshua B Gurtler1,

      Affiliated with

      • Pina M Fratamico1,

        Affiliated with

        • Jing Hu2 and

          Affiliated with

          • Vijay K Juneja1

            Affiliated with

            Cell & Bioscience20122:39

            DOI: 10.1186/2045-3701-2-39

            Received: 14 May 2012

            Accepted: 10 October 2012

            Published: 27 November 2012

            Abstract

            Background

            Toxin-antitoxin (TA) systems are commonly found in bacteria and Archaea, and it is the most common mechanism involved in bacterial programmed cell death or apoptosis. Recently, MazF, the toxin component of the toxin-antitoxin module, has been categorized as an endoribonuclease, or it may have a function similar to that of a RNA interference enzyme.

            Results

            In this paper, with comparative data and phylogenetic analyses, we are able to identify several potential MazF-conserved motifs in limited subsets of foodborne pathogens and probiotic strains and further provide a molecular basis for the development of engineered/synthetic probiotic strains for the mitigation of foodborne illnesses. Our findings also show that some probiotic strains, as fit as many bacterial foodborne pathogens, can be genetically categorized into three major groups based on phylogenetic analysis of MazF. In each group, potential functional motifs are conserved in phylogenetically distant species, including foodborne pathogens and probiotic strains.

            Conclusion

            These data provide important knowledge for the identification and computational prediction of functional motifs related to programmed cell death. Potential implications of these findings include the use of engineered probiotic interventions in food or use of a natural probiotic cocktail with specificity for controlling targeted foodborne pathogens.

            Keywords

            toxin-antitoxin module probiotic cocktail engineered probiotics foodborne pathogens

            Background

            Foodborne illnesses continue to be an important public health concern in developing, as well as in developed countries, thus prevention of foodborne illness outbreaks through effective and novel interventions should be given priority. The U.S. Public Health Service has identified ten important foodborne pathogens causing human illnesses, including pathogenic strains of Escherichia coli, Salmonella, Listeria, Clostridium botulinum, Shigella, and Campylobacter, which are associated with more than 250 known foodborne diseases (http://​www3.​niaid.​nih.​gov/​topics/​foodborne/​default.​htm).

            In addition, according to the World Health Organization (WHO), antibiotic overuse in food animal production is a major contributor to the emergence of antibiotic resistant foodborne pathogens [1]. The use of antibiotics in food animals for growth promotion and treatment disrupts the normal beneficial commensal bacterial microflora in the animal intestinal tract [26]. Recently, the human and chicken gut microbiome projects [712] have shed new light on the existence of a bacterial ‘phylogenetic core’ [13] consisting of a wide diversity of gastrointestinal bacteria by using new technologies such as next generation sequencing, 16S rRNA screens, metagenomics, and metaproteomics. Healthful, commensal bacteria found in the GI tract might be key members of known or potential probiotic strains revealed in this ‘phylogenetic core’, which may include Bacillus clausii, Bacillus pumilus, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus rhamnosus GG (ATCC 53103), Bifidobacterium infantis, Saccharomyces boulardii, Lactobacillus ruminis, Lactobacillus johnsonii str. NCC 533, and many others. These known probiotic strains have been used as dietary supplements, as treatments for illnesses caused by foodborne pathogens, and for disease prevention. Use of probiotic strains not only reduce invasion by bacterial pathogens, but also restore and maintain an optimal balance of healthy commensal bacteria in the human gut via production of antimicrobials [1423].

            One of the major mechanisms recognized as being responsible for apoptosis, or programmed cell death, and production of toxic metabolites in bacteria is through the regulation of a wide variety of bacterial toxin-antitoxin modules [2426], such as MazE/MazF, a chromosomal toxin-antitoxin module [2730], plasmid-encoded par D [3133], chpBIK[26, 34], relBE[35, 36], and the PhoQ-PhoP system [3740]. MazEF is one of the most well-studied toxin–antitoxin (TA) systems and has been found on the chromosomes of many bacteria. The MazF protein has been recently categorized as an endoribonuclease [41, 42] or as a type of RNA interference enzyme [43]. The link between this TA system and quorum sensing has also been explored recently through a small pentapeptide (NNWNN) called the Extracellular Death Factor (EDF) [44]. This small peptide motif (such as NNWNN) is known to be an extra-cellular death factor in E. coli and other bacterial species. The necessity of an “extracellular death factor” (EDF) or “cell death factor” (CDF) via MazEF-mediated cell death is a population phenomenon requiring the activation of quorum-sensing (cell-to-cell signaling) peptides in bacteria. High cell density was found to be associated with elevated concentrations of EDF, and the presence of EDF resulted in MazF-induced cell death [44]. From a food safety and public health perspective, use of EDF or a similar strategy may be used in place of antibiotics, resulting in less usage of antibiotics. We also noticed in one very interesting study that the induction of toxin MazF and the use of antibiotic share a similar mechanism by inhibiting the transcription and/or translation of the MazE antitoxin [45].

            It has been theorized that there is “one toxin for one antitoxin” and interestingly MazF, in some bacteria, exhibits a selective inhibition of ribosomes and mRNAs [43, 46]. Numerous strains of probiotic bacteria, such as Lactobacillus spp., have been reported to produce antimicrobial agents [47], such as bacteriocins, that inhibit or kill closely-related species, or even different strains of the same species through the inhibition of transcription and translation by receptor binding. The antimicrobial activities of bacteriocins are due to a heterologous subgroup of ribosomally synthesized cationic peptides [48]. Nisin, a polycyclic antibacterial peptide 34 amino acid residues long, is one of the most studied bacteriocins and is produced by many strains of Lactococcus lactis. It has been approved by the FDA for use as a food preservative, and certain probiotic Lactobacillus strains have been thoroughly studied and evaluated in vitro and in vivo. For example, L. reuteri controls diarrhea in children and suppresses the growth and pathogenicity of harmful foodborne pathogens such as Salmonella, E. coli, Staphylococcus, and Listeria[49, 50]. L. casei GG has been used to treat Clostridium difficile infections and to reduce intestinal permeability [5154]. L. reuteri is known to produce a broad-spectrum antimicrobial agent, reuterin, composed of the natural metabolic compound 3-hydroxypropionaldehyde, which has been used on the surface of sausages to inhibit growth of harmful bacteria and fungi [15, 16]. However, the molecular mechanisms underlying the effectiveness of individual probiotic strains have not been systematically studied and characterized. Several potential mechanisms of action, including their ability to generate diverse natural toxic metabolites, lactic acid, and other organic acids, enzymes, vitamins, and hydrogen peroxide, as well as antimicrobial peptides such as nisin, have been well-described [20].

            The work reported herein explores the experimental antimicrobial possibilities and/or procedures for (1) expression of an engineered, stress-induced recombinant secreted fusion gene encoded by MazF and a small extracellular cell death factor (CDF) on the surface or extracellular space of recombinant probiotic bacteria or (2) for potential application of a cocktail of natural probiotic strains via experimental in vitro selection to control foodborne pathogens for improving the safety and quality of foods, as well as improving human health. The use of engineered probiotic strains or the natural probiotic cocktail consisting of mixed probiotic strain populations for targeting foodborne pathogens will potentially be able to selectively inactivate these pathogens, even in complex food matrices. Through gene/motif reshuffling [55, 56] and computational molecular modeling, this engineered and secreted bacterial fusion protein, MazF-CDF, which contains an enterokinase (EK) cleavage site [5760] for releasing active CDF and MazF after protein secretion, should have a narrow range of applicability, limited to inactivating only specific foodborne pathogens such as E. coli O157, Salmonella, Listeria, Campylobacter, and potentially other human pathogens. This pathogen specificity is due to the fact that the genetically-engineered MazF-CDF fusion protein could be modified by the inclusion of specific genomic DNA sequences from various commensal, as well as pathogenic bacterial strains, identified through DNA/protein structure and functional comparisons. Additionally, this engineered antimicrobial polypeptide and MazF will be non-toxic to beneficial (healthful/probiotic) bacteria, as well as to its host that expresses the protein/peptide. Moreover, these engineered and secreted CDFs and MazF proteins/peptides can easily pass through infectious foodborne pathogen cell barriers mediating cell death, and thus could potentially reduce the use of deleterious compounds such as antibiotics or other harmful chemicals in the feed and food industry.

            Results and discussion

            General genetic analysis of probiotics and foodborne pathogen genomes

            The genomic information from selected probiotic strains and foodborne pathogens is shown in Table 1. It revealed little diversity in genomic GC-content in the Bifidobacterium genus, while showing an astonishing diversity in the GC-content among Lactobacillus species, ranging from 33 to 51.5%. It has been demonstrated that genomic GC-content is correlated with a number of factors [61], including genome size [62] from species such as Lactobacillus, which ranges from 1.8 to 3.4 Mb in length. This demonstrates that the GC-content and genome size of Lactobacillus genomes may have implications related to the biological complexity and adaptation of this genus, and could be due to the rate of recombination that has been extensively studied in the E. coli genome [63]. Knowledge of genetic diversity is fundamental to development of synthetic probiotic genomes and/or nucleic acid sequence reshuffling strategies. In this paper, we demonstrate that the MazF protein is a suitable candidate for the determination of genetic relationships within sets of MazF proteins in combination with with functional motif analysis.
            Table 1

            Genomic information of selected microorganisms considered as potential probiotic strains and some major food-borne pathogens used in this study

            Organism

            Strain

            Pathotype /or others

            GenBank Accession /Assembly

            GC (%)

            Genome Size (~ Mb)

            Campylobacter upsaliensis

            JV21

            pathogenic

            ASM18534v1

            NA

            1.6

            Lactobacillus coleohominis

            101-4-CHN

            probiotic

            ASM16193v1

            41.3

            1.7

            Campylobacter jejuni

            RM1221

            pathogenic

            NC_003912.7

            30.3

            1.8

            Lactobacillus johnsonii

            FI9785

            probiotic

            NC_013504.1

            34.4

            1.8

            Pediococcus pentosaceus

            ATCC 25745

            probiotic

            CP000422.1

            37.4

            1.8

            Streptococcus thermophilus LMG

            LMG 18311

            probiotic

            NC_006448.1

            39.1

            1.8

            Lactobacillus gasseri

            ATCC 33323

            probiotic

            NC_008530.1

            35.3

            1.9

            Lactobacillus sakei subsp. sakei

            23K

            probiotic

            NC_007576.1

            41.3

            1.9

            Lactobacillus delbrueckii subsp. bulgaricus

            ATCC 11842

            probiotic

            NC_008054.1

            49.7

            1.9

            Lactobacillus delbrueckii subsp. bulgaricus

            ATCC BAA-365

            probiotic

            NC_008529.1

            49.7

            1.9

            Leuconostoc citreum

            KM20

            probiotic

            ASM2640v1

            38.9

            1.9

            Bifidobacterium animalis subsp. Lactis

            Bl-04

            probiotic

            NC_012814.1

            60.5

            1.9

            Bifidobacterium animalis subsp. Lactis

            DSM 10140

            probiotic

            NC_012815.1

            60.5

            1.9

            Bifidobacterium animalis subsp. Lactis

            AD011

            probiotic

            NC_011835.1

            60.5

            1.9

            Lactobacillus amylovorus

            GRL1118

            probiotics

            ASM19411v1

            38.0

            2.0

            Lactobacillus johnsonii

            NCC 533

            probiotic

            NC_005362.1

            34.6

            2.0

            Lactobacillus acidophilus

            NCFM

            probiotic

            NC_006814.3

            34.7

            2.0

            Lactobacillus reuteri

            DSM 20016

            probiotic

            NC_009513.1

            38.9

            2.0

            Lactobacillus reuteri

            JCM 1112

            probiotic

            NC_010609.1

            38.9

            2.0

            Listeria monocytogenes

            FSL J1-208

            pathogenic

            ASM16843v1

            37.7

            2.0

            Lactobacillus salivarius

            UCC118

            probiotic

            NC_007929.1

            33

            2.1

            Lactobacillus helveticus

            DPC 4571

            probiotic

            NC_010080.1

            37.1

            2.1

            Leuconostoc mesenteroides subsp. mesenteroides

            ATCC 8293

            probiotic

            NC_008531.1

            37.7

            2.1

            Campylobacter concisus

            13826

            pathogenic

            NC_009802.1

            39.3

            2.1

            Lactobacillus fermentum

            IFO 3956

            probiotic

            NC_010610.1

            51.5

            2.1

            Bifidobacterium adolescentis

            ATCC 15703

            probiotic

            NC_008618.1

            59.2

            2.1

            Bifidobacterium bifidum

            PRL2010

            probiotic

            NC_014638.1

            Na

            2.2

            Bifidobacterium bifidum

            S17

            probiotic

            NC_014616.1

            Na

            2.2

            Lactobacillus amylovorus

            GRL 1112

            probiotic

            NC_014724.1

            Na

            2.2

            Lactobacillus brevis

            ATCC 367

            probiotic

            NC_008497.1

            46.1

            2.3

            Lactobacillus crispatus

            CTV-05

            probiotic

            ASM16588v1

            37.1

            2.3

            Bifidobacterium longum

            NCC2705

            probiotic

            NC_004307.2

            60.1

            2.3

            Bifidobacterium longum subsp. longum

            BBMN68

            probiotic

            NC_014656.1

            Na

            2.3

            Bifidobacterium longum subsp. infantis

            157F

            probiotic

            NC_015052.1

            Na

            2.4

            Bifidobacterium longum subsp. longum

            JCM 1217

            probiotic

            NC_015067.1

            Na

            2.4

            Lactococcus lactis subsp. lactis

            Il1403

            probiotic

            NC_002662.1

            35.3

            2.4

            Bifidobacterium longum

            DJO10A

            probiotic

            NC_010816.1

            60.2

            2.4

            Bifidobacterium longum subsp. longum

            JDM301

            probiotic

            NC_014169.1

            Na

            2.5

            Lactococcus lactis subsp. cremoris

            MG1363

            probiotic

            NC_009004.1

            35.7

            2.5

            Listeria grayi

            DSM 20601

            pathogenic

            ASM14899v1*

            41.6

            2.6

            Propionibacterium freudenreichii subsp. shermanii

            CIRM-BIA1

            probiotic

            NC_014215.1

            Na

            2.6

            Lactococcus lactis subsp. lactis

            KF147

            probiotic

            NC_013656.1

            34.9

            2.6

            Lactococcus lactis subsp. cremoris

            SK11

            probiotic

            NC_008527.1

            35.8

            2.6

            Bifidobacterium dentium

            Bd1

            probiotic

            NC_013714.1

            58.5

            2.6

            Bifidobacterium longum subsp. infantis

            ATCC 15697

            probiotic

            NC_011593.1

            59.9

            2.8

            Listeria monocytogenes

            EGD-e

            pathogenic

            NC_003210.1

            38

            2.9

            Lactobacillus casei str.

            Zhang

            probiotic

            NC_014334.1

            40.1

            2.9

            Lactobacillus casei

            ATCC 334

            probiotic

            NC_008526.1

            46.6

            2.9

            Lactobacillus paracasei subsp. paracasei

            ATCC 25302

            probiotic

            ASM15949v1

            46.5

            2.9

            Lactobacillus rhamnosus

            GG

            probiotic

            NC_013198.1

            46.7

            3.0

            Lactobacillus rhamnosus

            Lc 705

            probiotic

            NC_013199.1

            46.7

            3.0

            Lactobacillus casei

            BL23

            probiotic

            NC_010999.1

            46.3

            3.1

            Lactobacillus plantarum

            JDM1

            probiotic

            NC_012984.1

            44.7

            3.2

            Enterococcus faecalis

            V583

            probiotic

            NC_004668.1

            37.4

            3.3

            Lactobacillus plantarum

            WCFS1

            probiotic

            NC_004567.1

            44.4

            3.3

            Lactobacillus plantarum subsp. plantarum

            ST-III

            probiotic

            NC_014554.1

            Na

            3.4

            Bacillus selenitireducens

            MLS10

            Bio agent

            ASM9308v1

            48.7

            3.6

            Bacillus pumilus

            SAFR-032

            probiotic

            NC_009848.1

            41.3

            3.7

            Coprobacillus sp.

            29_1

            Non-pathogenic

            Coprobacillus_sp_29_1_V1

            NA

            3.8

            Vibrio cholerae

            M66-2

            pathogenic

            NC_012578.1

            47.6

            3.9

            Bacillus halodurans

            C-125

            Non-pathogenic

            ASM1114v1

            43.7

            4.2

            Bacillus amyloliquefaciens

            Y2

            probiotic

            ASM28439v1

            45.9

            4.2

            Clostridium difficile

            630

            pathogenic

            NC_009089.1

            29.1

            4.3

            Bacillus clausii

            KSM-K16

            probiotic

            NC_006582.1

            44.8

            4.3

            Shigella dysenteriae

            Sd197

            pathogenic

            ASM1200v1]

            50.9

            4.6

            Shigella flexneri

            8401

            pathogenic

            NC_008258.1

            50.9

            4.6

            Salmonella enterica subsp. arizonae serovar

            RSK2980

            pathogenic

            NC_010067.1

            51.4

            4.6

            Enterobacter sp.

            638

            pathogenic

            NC_009436.1

            52.9

            4.7

            Vibrio vulnificus

            CMCP6

            pathogenic

            NC_004459.2

            46.7

            5.1

            Escherichia coli

            E24377A

            pathogenic

            NC_009801.1

            50.6

            5.2

            Citrobacter rodentium

            ICC168

            pathogenic

            NC_013716.1

            54.6

            5.4

            Klebsiella variicola

            At-22

            pathogenic

            NC_013850.1

            57.6

            5.5

            Bacillus megaterium

            QM B1551

            probiotic

            ASM2582v1

            38.0

            5.5

            Escherichia coli O157:H7

            Sakai

            pathogenic

            NC_002695.1

            50.5

            5.6

            Bacillus thuringiensis serovar israelensis

            ATCC 35646

            Bio agents

            ASM16769v1

            35.0

            5.9

            Lachnospiraceae bacterium

            3_1_57FAA_CT1

            Non-pathogenic

            Lach_bact_3_1_57FAA_CT1_V1

            NA

            7.7

            *: assembly accession.

            Ubiquitous existence of MazE/toxin, MazF

            MazEF is a toxin-antitoxin (TA) module widely distributed among many bacterial species, including both foodborne pathogens and probiotic strains (Table 1, 2), such as Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus rhamnosus GG (ATCC 53103), Escherichia coli, Selenomonas sputigena, Enterobacter spp., Campylobacter spp., Citrobacter spp., Thermoanaerobacter tengcongensis, Pelotomaculum thermopropionicum, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus buchneri, Bifidobacterium longum subsp. infantis, Clostridium botulinum, Clostridium difficile, Vibrio spp., Listeria spp., Bacillus spp., Klebsiella variicola, and Salmonella enterica. Recent literature and computational analyses have shown the presence of many different types of TA modules in various localizations, e.g. some TA modules have been found within prophages, prophage-like elements, and other mobile genetic elements, such as plasmids [3133]. Due to the existence of varying types of toxin-antitoxin modules and possible gene duplication events, in this paper we present the above one-to-one best matches of chromosomal-encoded mazF orthologs and homologs among 75 publically available genome sequences of foodborne pathogens and probiotic strains (Table 1), and the publically-available databases such as NCBI and the Uniprot database (http://​www.​uniprot.​org/​) in Table 2.
            Table 2

            The genetic characterization of the transcriptional modulator MazF , a chromosomal cell death factor from potential probiotic strains and some major food-borne pathogens used in this study

            Organism

            Type of organisms

            Strains

            Protein accession (GenBank)

            Gene annotation

            E. coli

            Pathogen

            EDL933

            NP_289336.1

            toxin ChpA

             

            Pathogen

            Sakai

            NP_311669.1

             
             

            Antibiotic resistance

            B185

            ZP_06660634.1*

             

            Enterobacter sp.

            pathogen

            638

            ABP60743.1*

            transcriptional modulator of MazE/toxin, MazF

            Shigella dysenteriae

            pathogen

            Sd197

            YP_405833.1*

            toxin ChpB

            Citrobacter rodentium

            pathogen

            ICC168

            YP_003366767.1*

            Toxin component of the ChpB-ChpS toxin-antitoxin system

            Vibrio vulnificus

            pathogen

            CECT4999=R99

            YP_001393091.1*

            growth inhibitor

            Listeria welshimeri serovar 6b str.

            pathogen

            SLCC5334

            YP_849071.1

            PemK family transcriptional regulator

              

            FSL J1-208

            ZP_05296226.1*

            PemK family transcriptional regulator

            Listeria monocytogenes

             

            SLCC3954

            YP_003464028.1

            transcriptional regulator, PemK family

              

            FSL S4-120

            ZP_07870171.1

            toxin-antitoxin system, antitoxin component, MazF family

            Listeria seeligeri serovar 1/2b str.

                

            Listeria marthii

                

            Listeria grayi

            pathogen

            DSM 20601

            ZP_07054243.1*

            MazF family toxin-antitoxin system protein

            Clostridium difficile

            pathogen

            630

            YP_001089981.1*

            putative regulator of cell growth

              

            QCD-66c26

            ZP_05273557.1

            putative regulator of cell growth

              

            CIP 107932

            ZP_05323891.1

            putative regulator of cell growth

            Klebsiella variicola

            pathogen

            At-22

            YP_003438577.1*

            transcriptional modulator of MazE/toxin, MazF pemK; protein PemK

              

            1_1_55

            ZP_06548086.1

             

            Salmonella enterica subsp. arizonae serovar

            pathogen

            RSK2980

            YP_001573441.1*

            hypothetical protein SARI_04525

            Campylobacter showae

            pathogen

            RM3277

            ZP_05364729.1

            addiction module antitoxin, RelB/DinJ family

            Campylobacter concisus

            pathogen

            13826

            YP_001466677.1

            addiction module antitoxin

            Campylobacter jejuni subsp.

            pathogen

            IA3902

            ADC28395.1

            prevent-host-death family protein

            Campylobacter upsaliensis

            pathogen

            JV21

            ZP_07894578.1*

            ChpA/MazF transcriptional modulator

            Shigella flexneri 2a str.

            pathogen

            301

            NP_709188.1*

            PemK protein

            Shigella flexneri 5 str.

             

            8401

            YP_690766.1

            growth inhibitor, PemK-like, autoregulated

            Cronobacter sakazakii

            pathogen

            ATCC BAA-894

            YP_001436394.1

            bifunctional antitoxin/transcriptional repressor RelB

            Lactobacillus coleohominis

            probiotic

            101-4-CHN

            ZP_05553821.1*

            regulatory protein

            Bacillus megaterium

            probiotic

            QM B1551

            YP_003560763.1*

            endoribonuclease EndoA

              

            DSM 319

            YP_003595503.1

             

            Bacillus thuringiensis serovar israelensis

            probiotic

            ATCC 35646

            ZP_00740711.1*

            MazF protein

              

            KBAB4

            YP_001643128.1

            MazF protein

            Bacillus weihenstephanensis

             

            AH1134

            ZP_03233130.1

            PemK family

            Bacillus cereus

                

            Coprobacillus sp.

            probiotic

            29_1

            ZP_08009764.1*

            PemK family transcriptional regulator

            Enterococcus faecalis

            probiotic

            V583

            NP_814592.1

            PemK family transcriptional regulator

              

            TX0102

            EFQ11602.1*

             
                

            Toxin-antitoxin system, toxin compoment, MazF

              

            TX2134

            ZP_07557645.1*

             
                

            Toxin-antitoxin system, toxin compoment, MazF

              

            HH22

            ZP_03985009.1

             
                

            PemK family transcriptional regulator

            Lactobacillus plantarum

            probiotic

            WCFS1

            NP_786238.1*

            cell growth regulatory protein

            Lactobacillus rhamnosus

            probiotic

            LMS2-1

            ZP_04441477.1*

            cell growth regulatory protein

              

            Lc 705

            YP_003175134.1

            transcriptional modulator of MazE/toxin, MazF

            Lactobacillus johnsonii

            probiotic

            ATCC 33200

            ZP_04007131.1*

            PemK family growth inhibitor

            Lactobacillus crispatus

            probiotics

            CTV-05

            ZP_07789240.1*

            ppGpp-regulated growth inhibitor

            Lactobacillus gasseri

            probiotic

            ATCC 33323

            YP_815460.1*

            toxin-antitoxin system, toxin component, MazF family

              

            224-1

            ZP_06262236.1

             
              

            MV-22

            ZP_07711551.1

             

            Lactobacillus casei

            probiotic

            BL23

            YP_001986080.1

            Cell growth regulatory protein

            Lactobacillus amylovorus

            probiotic

            GRL1118

            YP_005854914.1*

            transcriptional modulator of MazE/toxin MazF

            Vibrio cholerae

            probiotic

            1587

            ZP_01950611.1*

            transcriptional modulator of MazE/toxin, MazF

              

            NCTC 8457

            ZP_01969676.1

             

            Pediococcus pentosaceus

            probiotic

            ATCC 25745

            YP_805020.1*

            toxin-antitoxin addiction module toxin component MazF (an endoRNAse)

            Bacillus amyloliquefaciens

            probiotic

            Y2

            YP_006327269.1*

            Endoribonuclease

            Bacillus pumilus

            probiotic

            SAFR-032

            YP_001485694.1*

            PemK family growth inhibitor endoribonuclease EndoA

              

            ATCC 7061

            ZP_03054587.1

             

            Lactobacillus sakei subsp. sakai

            probiotic

            Sakei 23K

            YP_396224.1

            DNA-binding protein PemK family

            Leuconostoc mesenteroides subsp. mesenteroides

            probiotic

            ATCC 8293

            YP_819271.1*

            toxin-antitoxin addiction module toxin component MazF

              

            ATCC 19254

            ZP_03913232.1

            PemK family growth inhibitor

            Leuconostoc mesenteroides subsp. cremoris

                

            Enterococcus faecalis

            probiotic

            TX0102

            EFQ11602.1*

            toxin-antitoxin system, toxin component, MazF family

              

            TX0031

            EFT96737.1

             

            Bifidobacterium animalis subsp. lactis

            probiotic

            HN019

            ZP_02964075.1*

            transcriptional modulator of MazE/toxin, MazF

            Bifidobacterium bifidum

            probiotic

            NCIMB 41171

            ZP_03645723.1

            RelB antitoxin

            Bifidobacterium longum subsp. longum

            probiotic

            JDM301

            YP_003660624.1*

            RelB antitoxin

              

            DSM 20213

            ZP_06596513.1

            toxin-antitoxin system protein

            Bifidobacterium breve

                

            Bifidobacterium bifidum

            probiotic

            S17

            YP_003938101.1

            hypothetical protein with RelB antitoxin domain

            Lactobacillus casei

            Probiotic

            ATCC 334

            YP_807723.1

            toxin-antitoxin addiction module toxin component MazF (an endoRNAse)

            Lactobacillus casei

            Probiotic

            BL23

            YP_001988635.1

            Growth inhibitor

            Lactobacillus paracasei subsp. paracasei

            Probiotic

            ATCC 25302

            ZP_03963082.1*

            PemK family transcriptional regulator

            Lactobacillus casei str.

            probiotic

            Zhang

            YP_003789562.1

            toxin-antitoxin addiction module toxin component MazF

            Lachnospiraceae bacterium

            probiotic

            3_1_57FAA_CT1

            ZP_08609193.1*

            hypothetical protein HMPREF0994_05199

            Leuconostoc citreum

            Probiotic

            KM20

            YP_001727503.1*

            growth inhibitor

            Lactobacillus reuteri

            probiotic

            DSM 20016

            YP_001270863.1*

            transcriptional modulator of MazE/toxin, MazF

              

            JCM 1112

            YP_001841242.1

            hypothetical protein LAR_0246

              

            MM2-3

            ZP_03847756.1

            PemK family growth inhibitor

            Streptococcus sp. oral taxon 071 str.

            probiotic

            73H25AP

            ZP_07458552.1*

            ChpA/MazF transcriptional modulator

            Bacillus selenitireducens

            probiotic

            MLS10

            YP_003700699.1*

            transcriptional modulator of MazE/toxin, MazF

            Bacillus halodurans

            probiotic

            C-125

            NP_244588.1*

            ppGpp-regulated growth inhibitor (ChpA/MazF)

            Enterococcus faecium

            probiotic

            DO

            ZP_05714797.1*

            PemK family protein

              

            TX0133a04

            ZP_07845579.1

            toxin component, MazF family

              

            TX0133C

            ZP_07850444.1

            toxin component, MazF family

            *: data are presented in Figure 1.

            Phylogenetic analyses and cluster analysis of MazF/antitoxin protein, a growth inhibitor

            The phylogenetic tree in Figure 1 displays the phylogentic relationships of many well-recognized genera within Enterobacteriaceae, including several important foodborne pathogens such as pathogenic E. coli, Salmonella, Listeria, and Campylobacter, as well as some major probiotic strains. To build a phylogenetic tree from the data in Table 2, the amino acid sequences of all the MazF or growth inhibitor proteins were analyzed using the Geneious software package v5.5.7 with Neighbor-joining (NJ) method by applying ClustalW for sequence alignment (Figure 1). Three main clusters (viz., groups 1, 2, and 3) are given in Figure 1. At least two representatives of potential probiotic strains are listed for each group, depending on the complexity of groups. In group 1, the potential probiotic, non-pathogenic strains, such as Lactobacillus amylovorus, Lactobacillus crispatus, Streptococcus, Enterococcus faecium, Enterococcus, Lactobacillus plantarum, and Lactobacillus rhamnosus, are grouped with the foodborne pathogens E. coli, Vibrio vulnificus, and Vibrio cholerae. In group 2, the foodborne pathogens Enterobacter, Campylobacter upsaliensis, Klebsiella variicola, Salmonella enterica, Shigella flexneri, Shigella dysenteriae, and Citrobacter rodentium were shown to be genetically closer to Bacillus selenitireducens, Bacillus halodurans, and Enterococcus faecalis In group 3, some probiotic strains, such as the Bacillus, Lactobacillus, Leuconostoc, and the Bifidobacterium genera are categorized along with other major foodborne pathogens, such as Clostridium difficile, Listeria monocytogenes, Listeria grayi, and an emerging foodborne pathogen Pediococcus pentosaceus.
            http://static-content.springer.com/image/art%3A10.1186%2F2045-3701-2-39/MediaObjects/13578_2012_Article_78_Fig1_HTML.jpg
            Figure 1

            Phylogenetic analyses of Enterobacteriaceae based on MazF (toxin), a growth inhibitor. The NCBI accession numbers are shown after each taxon name.

            Phylogenetic identification of bacterial toxin MazF protein motifs and the relationship between gene structure and phylogenetic classification

            In Figure 2, it is shown that the number of candidate motifs are slightly different in each group, but with a high degree of amino acid sequence variability within conserved “hot spots” between groups 1, 2, and 3. To determine which motifs are the best candidates for the engineered MazF construction (discussed in next section) in each individual group, recent studies [43] suggest that the N-terminal (the first 80 amino acids in Figure 2) of the MazF protein are the most suitable motifs. The other motifs in the C-terminal might be referred to as ‘incorrect’ motifs. There are three criteria for evaluating the remaining motifs as “incorrect and without biological significance”, and this is related to: the mean hydrophobicity, identity, and the gene structure. In Figure 2, in the analysis for the phylogenetic identification of bacterial Toxin MazF protein motifs, the mean hydrophobicity sequence and identity were computed and compared for each sequence; it is interesting to find that all the compared MazF proteins have a higher degree of mean hydrophobicity, identity, and more conserved gene structure among several conserved amino acid regions, particular in the N-terminal. The selection of potential functional MazF motifs is discussed in the next section. The functional importance of the mean hydrophobicity has also been discussed to involve mRNA and protein degradation and slower translation of mRNA for disordered proteins (http://​employees.​csbsju.​edu/​hjakubowski/​classes/​ch331/​protstructure/​olprotfold.​html).
            http://static-content.springer.com/image/art%3A10.1186%2F2045-3701-2-39/MediaObjects/13578_2012_Article_78_Fig2_HTML.jpg
            Figure 2

            ClustalW output (default settings) for the aligned amino acid sequences of MazF. The pdf images of the alignments were generated using Geneious v5.5.7. Conserved amino acids are highlighted in colors; the numbers (1 to 39) in this Figure correspond to the taxon names in Figure 1.

            Bacterial probiotic cocktail strains

            It is important to note that the benefits of probiotics are strain-specific [64, 65]. A commercial product, VSL#3, developed by Sigma-Tau Pharmaceuticals, Inc., provides a mixture of probiotic bacteria (B. breve, B. infantis, B. longum, L. acidophilus, L. bulgaricus, L. casei, L. plantarum, and Streptococcus thermophilus) to help protect GI tract integrity [66]. In this study, the bacterial probiotic cocktail strains we propose would be comprised of all representatives of groups 1, 2, and 3 shown in Figure 1. The principal basis behind the composition of these probiotic cocktail strains is the assumption that a combination of organisms might be more effective than the application of a single strain, which potentially could suppress many foodborne pathogens, such as the E. coli and vibros in Group 1, several foodborne pathogens, such as Enterobacter, Klebsiella variicola, Salmonella enterica, Shigella flexneri, Shigella dysenteriae, Citrobacter rodentium, and Campylobacter upsaliensis, (one of the most common Campylobacter strain found in people with diarrhea in Group 2), Clostridium difficile, Listeria monocytogenes, and Listeria grayi, in Group 3 of Figure 1. Table 1 shows an astonishing diversity in the genomic GC-content and genome size among Lactobacillus species and their diverse distribution in all groups within Figure 1, which indicates a potential to further identify other closely-related Lactobacillus species (not listed in Table 1) into the three previously-described groups. The hypothesis underlying our approach is that probiotic strains found within the same group with foodborne pathogens will have a reasonable degree of genetic and molecular phylogenetic compatibility and could bridge a relationship similar to a “symbiosis” of entities, including exchanging toxin/antitoxin molecules among the probiotic and pathogenic strains. Lactobacillus species are known to produce antimicrobial substances, including bacteriocins, lactic acid, and hydrogen peroxide. The MazEF toxin component may provide a basis for bacterial growth inhibition within the same group (Figure 1 and 2). Therefore, this toxin-antitoxin module may have great potential to inhibit the growth of potentially-pathogenic bacteria through a possible competitive exclusion due to selective inhibition [46]. Figures 1 and 2 list all possible cocktails of these probiotic strains. In reality, a foodborne outbreak is more likely to be associated with one particular foodborne pathogen in particular foods. For example, there is a low incidence of Campylobacter in ground beef and pork, while Campylobacter is the major foodborne pathogens associated with poultry, therefore, a single food-borne pathogen with the application of mixed probiotics will be considered initially.

            Molecular recombination techniques: construction of genetically engineered synthetic probiotic strains

            Figure 3 details an engineered probiotic strain bearing a recombinant plasmid containing a stress-induced promoter, followed by an in-frame gene fusion accomplished by fusing an appropriate signal peptide, a functional cell death peptide/factor (CDF), an enterokinase (EK) binding site, and a genetically-modified engineered MazF gene, which will be constructed and transformed into the probiotic bacteria. In this recombinant probiotic strain, environmental stress, relevant to food environments will trigger gene expression of this fusion protein under an environmental stress-inducible promoter. The signal peptide directs the encoded fusion protein to the extracellular space of the engineered probiotic strains. The signal peptide depleted fusion proteins will be cleaved by a biliary tract enterokinase directly into the digestive system to release the functionally-active CDF and MazF. This event will occur based on the ability of probiotic strains to form stable, non-infectious biofilm-type aggregates, which may attach with other bacteria, including foodborne pathogens, in the urogenital and intestinal tracts [22]. These active species-specific CDF and MazF proteins will bind and pass through “unfriendly” bacterial cell surface barriers into the cytoplasm. These processed CDFs and MazF proteins will selectively inactivate and/or inhibit proteins involved in cell survival and induce the synthesis of more cell death-related proteins with activity against foodborne pathogens, eventually controlling, inhibiting, or inactivating “unfriendly” cells. Although antitoxin MazE could reverse the bacteriocidal effect of the overexpressed MazF, MazE cannot impede the downstream cascade already initiated by MazF at early stages of the MazF-mediated cascade [42].
            http://static-content.springer.com/image/art%3A10.1186%2F2045-3701-2-39/MediaObjects/13578_2012_Article_78_Fig3_HTML.jpg
            Figure 3

            A Schematic representation of the engineered, secreted CDF and MazF -mediated cell death during food processing and storage. SP: promoter, CDF: cell death factor/peptide, EKB: enterokinase binding site.

            The overall hypothesis for this experiment is graphically presented in Figure 3. The engineered MazF gene sequence will be designed based on the genomic sequences of all publically-available foodborne pathogenic strains by using reasoned random gene biosynthesis and/or genuine gene reshuffling to rapidly combine functions and properties of parental genes for the development of improved gene specificity and generality, molecular modeling (transcription factor binding site identification, etc.), and systems biology technologies/tools.

            Conclusions

            Survival of foodborne pathogens in cultures or in animal GI tracts may be very genus- or species-specific. Data presented in this paper can be explored to develop effective intervention strategies applied directly during food processing and preparation, as well as in the animal feed supply, which may lead to an overall reduction in use of antibiotic growth promoters (AGP) throughout the world. Recent research in molecular biology and genomics has provided potential applications of probiotic strains as dietary supplements, which could replace AGP in animal diets or as biotherapeutic agents in cases of antibiotic-associated diarrhea in travelers and in childhood diarrhea and other bacterial gastrointestinal illnesses. Experiments relating to this potential probiotic application may reveal a further greater range of potential benefits. For many of these potential benefits, current research is limited, and only preliminary results are available. All effects can only be attributed to the individual strain(s) tested. Testing of a specific supplement may not be extrapolated to benefits from any other strain of the same species, and testing results do not imply that comparable benefits will be imparted from other LAB (or other probiotics). In this study, we have computationally explored several potential intervention strategies to control foodborne pathogens, either by using a cocktail of probiotic strains or an engineered probiotic strain.

            The inhibition of pathogens by probiotic strains is mainly due to the production of antibacterial peptides [67], the release of short-chain fatty acids, or reduction of the pH within the lumen [68, 69] by the production of organic acids or by decreasing pathogen adherence to intestinal epithelial cells [70]. Therefore, the benefits of probiotics could be very strain- or species-specific, and probiotic strains may rely on different mechanisms to suppress growth, attachment, or other metabolic processes, inherent to pathogenic bacteria. Moreover, the optimal effects of probiotic strains may involve the simultaneous use of more than one strain. Our contention in this paper is not experimental proof but, rather, a clear scientifically-backed hypothesis in the form of a detailed accompanying method that multi-probiotic strain composites with diverse genetic backgrounds may complement [71] one another as vectors of competitive exclusion and, therefore, could maximize the potential to inhibit an array of common foodborne pathogens [72] in the gastrointestinal tract of humans or livestock, as well as in foods and animal feed.

            The use of probiotic bacteria with the ability to produce CDFs and engineered MazF to selectively inactivate pathogens is a novel approach to controlling pathogens in foods, and possibly treating human infections. A number of studies suggest that this project could have practical significance and be a potentially new approach for the development of novel and cost-effective food safety intervention technologies for the control of foodborne pathogens and improving public health [7375].

            The approaches described above represent a first attempt to describe a systematic approach or method to test the hypothesis that “friendly” bacteria can be used to inactivate or inhibit pathogens in food based on expression of MazF. There are many specific cell death factors that may be associated with bacterial programmed cell death and multi-cellular behavior mechanisms in foodborne pathogens. Through computational modeling, remodeling, genetic recombination, or further gene reshuffling, and exploring experimental approaches, it may be possible to evaluate and elucidate more effective CDFs and MazF to be used for controlling foodborne pathogens, which will ultimately result in a reduction in the use of antimicrobial compounds in humans and animals, as well as during food processing and storage.

            Materials and methods

            Genomic sequences

            All of the genome and gene sequences examined in this study (Table 1 & 2) are available in GenBank (http://​www.​ncbi.​nlm.​nih.​gov/​genbank/​GenbankOverview.​html).

            Identification and analysis of bacterial toxin-antitoxin modules, MazE/MazF

            The prediction accuracy of the best chromosomal-encoded MazF orthologs among relatively distinct genome strains is critical for the performance of molecular phylogenetic analysis. We used a sequential BLAST workflow based on pairwise comparison by applying either an E. coli programmed cell death toxin MazF (Genbank accession: ZP_06660634.1), an endoribonuclease MazF, a MazF protein from Vibrio cholerae (ZP_01950611.1), or a PemK family transcriptional regulator protein (ZP_05296226.1) from Listeria monocytogenes to perform a BLASTP homology search of these combined protein sequences with all 75 publically available foodborne pathogen and probiotic strain genomic sequences (presented in Table 1) publically available databases such as NCBI and Uniprot database (http://​www.​uniprot.​org/​). BLAST search results were used to list MazF or MazF-like proteins from 75 different strains being represented as 39 different species (Table 2).

            Phylogenetic analysis and computational identification of phylogenetic motifs of the MazF protein

            In order to identify potential functional motifs of MazF proteins, phylogentic analyses of MazF proteins were conducted by applying the embedded multiple sequence alignment ClustalW program in the Geneious software package v 5.5.7 [76, 77] with the Neighbor Joining method. ClustalW output for the aligned amino acid sequences and the pdf images of the alignments were generated using the Geneious software package v 5.5.7.

            Declarations

            Acknowledgements

            We express our appreciation to Dr. James Smith for his critical review of the manuscript. The mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

            Authors’ Affiliations

            (1)
            Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture
            (2)
            Department of Mathematics and Computer Science, Franklin & Marshall College

            References

            1. Expert workshop on non-human antimicrobial usage and antimicrobial resistance. 2003,http://​www.​who.​int/​foodsafety/​publications/​micro/​en/​amr.​pdf, Geneva
            2. Are antibacterial-containing products (soaps, household cleaners, etc.) better for preventing the spread of infection? Does their use add to the problem of resistance?. 2009, Antibiotic Resistance Questions & Answers, Centers for Disease Control and Prevention, Atlanta
            3. Ferber D: Antibiotic resistance. Livestock feed ban preserves drugs’ power. Science. 2002, 295: 27-28.PubMed
            4. Goossens H, Ferech M, Vander Stichele R, Elseviers M: Outpatient antibiotic use in Europe and association with resistance: a cross-national database study. Lancet. 2005, 365: 579-587.View ArticlePubMed
            5. Mathew AG, Cissell R, Liamthong S: Antibiotic resistance in bacteria associated with food animals: a United States perspective of livestock production. Foodborne Pathog Dis. 2007, 4: 115-133. 10.1089/fpd.2006.0066View ArticlePubMed
            6. WHO: Use of antimicrobials outside human medicine and resultant antimicrobial resistance in humans [R].:World Health Organization; 2002.http://​www.​who.​int/​mediacentre/​factsheets/​fs268/​en/​index.​html.
            7. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI: Obesity alters gut microbial ecology. Proc Natl Acad Sci U S A. 2005, 102: 11070-11075. 10.1073/pnas.0504978102PubMed CentralView ArticlePubMed
            8. Possemiers S, Grootaert C, Vermeiren J, Gross G, Marzorati M, Verstraete W, Van de Wiele T: The intestinal environment in health and disease - recent insights on the potential of intestinal bacteria to influence human health. Curr Pharm Des. 2009, 15: 2051-2065. 10.2174/138161209788489159View ArticlePubMed
            9. De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet JB, Massart S, Collini S, Pieraccini G, Lionetti P: Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci U S A. 2010, 107: 14691-14696. 10.1073/pnas.1005963107PubMed CentralView ArticlePubMed
            10. Qu A, Brulc JM, Wilson MK, Law BF, Theoret JR, Joens LA, Konkel ME, Angly F, Dinsdale EA, Edwards RA, Nelson KE, White BA: Comparative metagenomics reveals host specific metavirulomes and horizontal gene transfer elements in the chicken cecum microbiome. PLoS One. 2008, 3: e2945. 10.1371/journal.pone.0002945PubMed CentralView ArticlePubMed
            11. Torok VA, Ophel-Keller K, Loo M, Hughes RJ: Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism. Appl Environ Microbiol. 2008, 74: 783-791. 10.1128/AEM.01384-07PubMed CentralView ArticlePubMed
            12. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, Sogin ML, Jones WJ, Roe BA, Affourtit JP, Egholm M, Henrissat B, Heath AC, Knight R, Gordon JI: A core gut microbiome in obese and lean twins. Nature. 2009, 457: 480-484. 10.1038/nature07540PubMed CentralView ArticlePubMed
            13. Tap J, Mondot S, Levenez F, Pelletier E, Caron C, Furet JP, Ugarte E, Munoz-Tamayo R, Paslier DL, Nalin R, Dore J, Leclerc M: Towards the human intestinal microbiota phylogenetic core. Environ Microbiol. 2009, 11: 2574-2584. 10.1111/j.1462-2920.2009.01982.xView ArticlePubMed
            14. Cabana MD, Shane AL, Chao C, Oliva-Hemker M: Probiotics in primary care pediatrics. Clin Pediatr (Phila). 2006, 45: 405-410. 10.1177/0009922806289614. 10.1177/0009922806289614View Article
            15. Cadieux P, Wind A, Sommer P, Schaefer L, Crowley K, Britton RA, Reid G: Evaluation of reuterin production in urogenital probiotic Lactobacillus reuteri RC-14. Appl Environ Microbiol. 2008, 74: 4645-4649. 10.1128/AEM.00139-08PubMed CentralView ArticlePubMed
            16. Cadieux PA, Burton J, Devillard E, Reid G: Lactobacillus by-products inhibit the growth and virulence of uropathogenic Escherichia coli. J Physiol Pharmacol. 2009, 60 (Suppl 6): 13-18.PubMed
            17. Canny GO, McCormick BA: Bacteria in the intestine, helpful residents or enemies from within?. Infect Immun. 2008, 76: 3360-3373. 10.1128/IAI.00187-08PubMed CentralView ArticlePubMed
            18. Gillor O, Etzion A, Riley MA: The dual role of bacteriocins as anti- and probiotics. Appl Microbiol Biotechnol. 2008, 81: 591-606. 10.1007/s00253-008-1726-5PubMed CentralView ArticlePubMed
            19. Lidbeck A, Edlund C, Gustafsson JA, Kager L, Nord CE: Impact of Lactobacillus acidophilus on the normal intestinal microflora after administration of two antimicrobial agents. Infection. 1988, 16: 329-336. 10.1007/BF01644541View ArticlePubMed
            20. Ng SC, Hart AL, Kamm MA, Stagg AJ, Knight SC: Mechanisms of action of probiotics: recent advances. Inflamm Bowel Dis. 2009, 15: 300-310. 10.1002/ibd.20602View ArticlePubMed
            21. Reid G, Bruce AW: Urogenital infections in women: can probiotics help?. Postgrad Med J. 2003, 79: 428-432. 10.1136/pmj.79.934.428PubMed CentralView ArticlePubMed
            22. Reid G, Jass J, Sebulsky MT, McCormick JK: Potential uses of probiotics in clinical practice. Clin Microbiol Rev. 2003, 16: 658-672. 10.1128/CMR.16.4.658-672.2003PubMed CentralView ArticlePubMed
            23. Reid G, Sanders ME, Gaskins HR, Gibson GR, Mercenier A, Rastall R, Roberfroid M, Rowland I, Cherbut C, Klaenhammer TR: New scientific paradigms for probiotics and prebiotics. J Clin Gastroenterol. 2003, 37: 105-118. 10.1097/00004836-200308000-00004View ArticlePubMed
            24. Grady R, Hayes F: Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium. Mol Microbiol. 2003, 47: 1419-1432. 10.1046/j.1365-2958.2003.03387.xView ArticlePubMed
            25. Masuda Y, Miyakawa K, Nishimura Y, Ohtsubo E: chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100. J Bacteriol. 1993, 175: 6850-6856.PubMed CentralPubMed
            26. Masuda Y, Ohtsubo E: Mapping and disruption of the chpB locus in Escherichia coli. J Bacteriol. 1994, 176: 5861-5863.PubMed CentralPubMed
            27. Gerdes K, Christensen SK, Lobner-Olesen A: Prokaryotic toxin-antitoxin stress response loci. Nat Rev Microbiol. 2005, 3: 371-382. 10.1038/nrmicro1147View ArticlePubMed
            28. Kolodkin-Gal I, Verdiger R, Shlosberg-Fedida A, Engelberg-Kulka H: A differential effect of E. coli toxin-antitoxin systems on cell death in liquid media and biofilm formation. PLoS One. 2009, 4: e6785. 10.1371/journal.pone.0006785PubMed CentralView ArticlePubMed
            29. Mittenhuber G: Occurrence of mazEF-like antitoxin/toxin systems in bacteria. J Mol Microbiol Biotechnol. 1999, 1: 295-302.PubMed
            30. Sat B, Hazan R, Fisher T, Khaner H, Glaser G, Engelberg-Kulka H: Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality. J Bacteriol. 2001, 183: 2041-2045. 10.1128/JB.183.6.2041-2045.2001PubMed CentralView ArticlePubMed
            31. Johnson EP, Strom AR, Helinski DR: Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein. J Bacteriol. 1996, 178: 1420-1429.PubMed CentralPubMed
            32. Santos-Sierra S, Pardo-Abarrio C, Giraldo R, Diaz-Orejas R: Genetic identification of two functional regions in the antitoxin of the parD killer system of plasmid R1. FEMS Microbiol Lett. 2002, 206: 115-119. 10.1111/j.1574-6968.2002.tb10995.xView ArticlePubMed
            33. Sobecky PA, Easter CL, Bear PD, Helinski DR: Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2. J Bacteriol. 1996, 178: 2086-2093.PubMed CentralPubMed
            34. Zhang Y, Zhu L, Zhang J, Inouye M: Characterization of ChpBK, an mRNA interferase from Escherichia coli. J Biol Chem. 2005, 280: 26080-26088. 10.1074/jbc.M502050200View ArticlePubMed
            35. Bech FW, Jorgensen ST, Diderichsen B, Karlstrom OH: Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene. EMBO J. 1985, 4: 1059-1066.PubMed CentralPubMed
            36. Gotfredsen M, Gerdes K: The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Mol Microbiol. 1998, 29: 1065-1076. 10.1046/j.1365-2958.1998.00993.xView ArticlePubMed
            37. Gunn JS, Miller SI: PhoP-PhoQ activates transcription of pmrAB, encoding a two-component regulatory system involved in Salmonella typhimurium antimicrobial peptide resistance. J Bacteriol. 1996, 178: 6857-6864.PubMed CentralPubMed
            38. Lippa AM, Goulian M: Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide. PLoS Genet. 2009, 5: e1000788. 10.1371/journal.pgen.1000788PubMed CentralView ArticlePubMed
            39. Monsieurs P, De Keersmaecker S, Navarre WW, Bader MW, De Smet F, McClelland M, Fang FC, De Moor B, Vanderleyden J, Marchal K: Comparison of the PhoPQ regulon in Escherichia coli and Salmonella typhimurium. J Mol Evol. 2005, 60: 462-474. 10.1007/s00239-004-0212-7View ArticlePubMed
            40. Zwir I, Shin D, Kato A, Nishino K, Latifi T, Solomon F, Hare JM, Huang H, Groisman EA: Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica. Proc Natl Acad Sci U S A. 2005, 102: 2862-2867. 10.1073/pnas.0408238102PubMed CentralView ArticlePubMed
            41. Amitai S, Kolodkin-Gal I, Hananya-Meltabashi M, Sacher A, Engelberg-Kulka H: Escherichia coli MazF leads to the simultaneous selective synthesis of both “death proteins” and “survival proteins”. PLoS Genet. 2009, 5 (3): e1000390. 10.1371/journal.pgen.1000390PubMed CentralView ArticlePubMed
            42. Amitai S, Yassin Y, Engelberg-Kulka H: MazF-mediated cell death in Escherichia coli: a point of no return. J Bacteriol. 2004, 186: 8295-8300. 10.1128/JB.186.24.8295-8300.2004PubMed CentralView ArticlePubMed
            43. Park JH, Yamaguchi Y, Inouye M: Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases. Appl Environ Microbiol. 2012, 78 (11): 3794-3799. 10.1128/AEM.00364-12PubMed CentralView ArticlePubMed
            44. Belitsky M, Avshalom H, Erental A, Yelin I, Kumar S, London N, Sperber M, Schueler-Furman O, Engelberg-Kulka H: The Escherichia coli extracellular death factor EDF induces the endoribonucleolytic activities of the toxins MazF and ChpBK. Mol Cell. 2012, 41 (6): 625-635.View Article
            45. Engelberg-Kulka H, Sat B, Reches M, Amitai S, Hazan R: Bacterial programmed cell death systems as targets for antibiotics. Trends Microbiol. 2004, 12 (2): 66-71. 10.1016/j.tim.2003.12.008View ArticlePubMed
            46. Rothenbacher FP, Suzuki M, Hurley JM, Montville TJ, Kirn TJ, Ouyang M, Woychik NA: Clostridium difficile MazF toxin exhibits selective, not global, mRNA cleavage. J Bacteriol. 2012, 194 (13): 3464-3474. 10.1128/JB.00217-12PubMed CentralView ArticlePubMed
            47. Juneja VK, Dwivedi HP, Yan X: Novel natural food antimicrobials. Annu Rev Food Sci Technol. 2012, 3: 381-403. 10.1146/annurev-food-022811-101241View ArticlePubMed
            48. de Vuyst L, Vandamme E: Antimicrobial potential of lactic acid bacteria. In Bacteriocins of Lactic Acid Bacteria. Microbiology, Genetics and Applications. Edited by de Vuyst L, Vandamme EJ. London: Blackie Acad Prof; 1994:91–142.View Article
            49. Shornikova AV, Casas IA, Isolauri E, Mykkanen H, Vesikari T: Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children. J Pediatr Gastroenterol Nutr. 1997, 24: 399-404. 10.1097/00005176-199704000-00008View ArticlePubMed
            50. Williams MD, Ha CY, Ciorba MA: Probiotics as therapy in gastroenterology: a study of physician opinions and recommendations. J Clin Gastroenterol. 2010, 44: 631-636.PubMed CentralPubMed
            51. Goldin BR, Gorbach SL, Saxelin M, Barakat S, Gualtieri L, Salminen S: Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. Dig Dis Sci. 1992, 37: 121-128. 10.1007/BF01308354View ArticlePubMed
            52. Plummer S, Weaver MA, Harris JC, Dee P, Hunter J: Clostridium difficile pilot study: effects of probiotic supplementation on the incidence of C. difficile diarrhoea. Int Microbiol. 2004, 7: 59-62.PubMed
            53. Vanderhoof JA, Whitney DB, Antonson DL, Hanner TL, Lupo JV, Young RJ: Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children. J Pediatr. 1999, 135: 564-568. 10.1016/S0022-3476(99)70053-3View ArticlePubMed
            54. Vanderhoof JA, Young RJ: Current and potential uses of probiotics. Ann Allergy Asthma Immunol. 2004, 93: S33-S37. 10.1016/S1081-1206(10)61730-9View ArticlePubMed
            55. Arber W: Genetic variation: molecular mechanisms and impact on microbial evolution. FEMS Microbiol Rev. 2000, 24: 1-7. 10.1111/j.1574-6976.2000.tb00529.xView ArticlePubMed
            56. Saito H, Kashida S, Inoue T, Shiba K: The role of peptide motifs in the evolution of a protein network. Nucleic Acids Res. 2007, 35: 6357-6366. 10.1093/nar/gkm692PubMed CentralView ArticlePubMed
            57. Hadorn B, Steiner N, Sumida C, Peters TJ: Intestinal enterokinase. Mechanisms of tts “secretion” into the lumen of the small intestine. Lancet. 1971, 1: 165-166.View ArticlePubMed
            58. Kitamoto Y, Yuan X, Wu Q, McCourt DW, Sadler JE: Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains. Proc Natl Acad Sci U S A. 1994, 91: 7588-7592. 10.1073/pnas.91.16.7588PubMed CentralView ArticlePubMed
            59. Song HW, Choi SI, Seong BL: Engineered recombinant enteropeptidase catalytic subunit: effect of N-terminal modification. Arch Biochem Biophys. 2002, 400: 1-6. 10.1006/abbi.2001.2737View ArticlePubMed
            60. Zhang Y, Vankemmelbeke MN, Holland LE, Walker DC, James R, Penfold CN: Investigating early events in receptor binding and translocation of colicin E9 using synchronized cell killing and proteolytic cleavage. J Bacteriol. 2008, 190: 4342-4350. 10.1128/JB.00047-08PubMed CentralView ArticlePubMed
            61. Hildebrand F, Meyer A, Eyre-Walker A: Evidence of Selection upon Genomic GC-Content in Bacteria. PLoS Genet. 2010, 6 (9): e1001107. 10.1371/journal.pgen.1001107PubMed CentralView ArticlePubMed
            62. Bentley SD, Parkhill J: Comparative genomic structure of prokaryotes. Annu Rev Genet. 2004, 38: 771-792. 10.1146/annurev.genet.38.072902.094318View ArticlePubMed
            63. Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier C, Bouvet O, Calteau A, Chiapello H, Clermont O, Cruveiller S, Danchin A, Diard M, Dossat C, Karoui ME, Frapy E, Garry L, Ghigo JM, Gilles AM, Johnson J, Le Bouguenec C, Lescat M, Mangenot S, Martinez-Jehanne V, Matic I, Nassif X, Oztas S, et al: Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet. 2009, 5: e1000344. 10.1371/journal.pgen.1000344PubMed CentralView ArticlePubMed
            64. Canani RB, Cirillo P, Terrin G, Cesarano L, Spagnuolo MI, De Vincenzo A, Albano F, Passariello A, De Marco G, Manguso F, Guarino A: Probiotics for treatment of acute diarrhoea in children: randomised clinical trial of five different preparations. BMJ. 2007, 335: 340. 10.1136/bmj.39272.581736.55PubMed CentralView ArticlePubMed
            65. Weizman Z, Asli G, Alsheikh A: Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents. Pediatrics. 2005, 115: 5-9.PubMed
            66. Quigley EMM, Sanders ME: Probiotic foods for gastrointestinal health. Gastroenterology & Endoscopy News Special Edition. 2009, 7: 27-33.
            67. Corr SC, Gahan CG, Hill C: Impact of selected Lactobacillus and Bifidobacterium species on Listeria monocytogenes infection and the mucosal immune response. FEMS Immunol Med Microbiol. 2007, 50: 380-388. 10.1111/j.1574-695X.2007.00264.xView ArticlePubMed
            68. Marteau P, Seksik P: Tolerance of probiotics and prebiotics. J Clin Gastroenterol. 2004, 38: S67-S69. 10.1097/01.mcg.0000128929.37156.a7View ArticlePubMed
            69. Marteau P, Seksik P, Lepage P, Dore J: Cellular and physiological effects of probiotics and prebiotics. Mini Rev Med Chem. 2004, 4: 889-896. 10.2174/1389557043403369View ArticlePubMed
            70. Mack DR, Michail S, Wei S, McDougall L, Hollingsworth MA: Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression. Am J Physiol. 1999, 276: G941-G950.PubMed
            71. Klaenhammer TR, Kullen MJ: Selection and design of probiotics. Int J Food Microbiol. 1999, 50: 45-57. 10.1016/S0168-1605(99)00076-8View ArticlePubMed
            72. Cursino L, Smajs D, Smarda J, Nardi RM, Nicoli JR, Chartone-Souza E, Nascimento AM: Exoproducts of the Escherichia coli strain H22 inhibiting some enteric pathogens both in vitro and in vivo. J Appl Microbiol. 2006, 100: 821-829. 10.1111/j.1365-2672.2006.02834.xView ArticlePubMed
            73. Iacono A, Raso GM, Canani RB, Calignano A, Meli R: Probiotics as an emerging therapeutic strategy to treat NAFLD: focus on molecular and biochemical mechanisms. J Nutr Biochem. 2010, 22: 699-711.View Article
            74. Lu TK, Collins JJ: Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy. Proc Natl Acad Sci U S A. 2009, 106: 4629-4634. 10.1073/pnas.0800442106PubMed CentralView ArticlePubMed
            75. Westwater C, Kasman LM, Schofield DA, Werner PA, Dolan JW, Schmidt MG, Norris JS: Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections. Antimicrob Agents Chemother. 2003, 47: 1301-1307. 10.1128/AAC.47.4.1301-1307.2003PubMed CentralView ArticlePubMed
            76. Drummond A, Ashton B, Buxton S: Geneious v5.4. 2011, http://​www.​geneious.​com,
            77. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, et al: Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012, 28 (12): 1647-1649. 10.1093/bioinformatics/bts199PubMed CentralView ArticlePubMed

            Copyright

            © Yan et al.; licensee BioMed Central Ltd. 2012

            This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

            Advertisement