Characterization and Genome Study of Novel Lytic Bacteriophages against Prevailing Saprophytic Bacterial Microflora of Minimally Processed Plant-Based Food Products.

The food industry is still searching for novel solutions to effectively ensure the microbiological safety of food, especially fresh and minimally processed food products. Nowadays, the use of bacteriophages as potential biological control agents in microbiological food safety and preservation is a promising strategy. The aim of the study was the isolation and comprehensive characterization of novel bacteriophages with lytic activity against saprophytic bacterial microflora of minimally processed plant-based food products, such as mixed leaf salads. From 43 phages isolated from municipal sewage, four phages, namely Enterobacter phage KKP 3263, Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 have lytic activity against Enterobacter ludwigii KKP 3083, Citrobacter freundii KKP 3655, Enterobacter cloacae KKP 3082, and Serratia fonticola KKP 3084 bacterial strains, respectively. Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) identified Enterobacter phage KKP 3263 as an Autographiviridae, and Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 as members of the Myoviridae family. Genome sequencing revealed that these phages have linear double-stranded DNA (dsDNA) with sizes of 39,418 bp (KKP 3263), 61,608 bp (KKP 3664), 84,075 bp (KKP 3262), and 148,182 bp (KKP 3264). No antibiotic resistance genes, virulence factors, integrase, recombinase, or repressors, which are the main markers of lysogenic viruses, were annotated in phage genomes. Serratia phage KKP 3264 showed the greatest growth inhibition of Serratia fonticola KKP 3084 strain. The use of MOI 1.0 caused an almost 5-fold decrease in the value of the specific growth rate coefficient. The phages retained their lytic activity in a wide range of temperatures (from -20 °C to 50 °C) and active acidity values (pH from 4 to 11). All phages retained at least 70% of lytic activity at 60 °C. At 80 °C, no lytic activity against tested bacterial strains was observed. Serratia phage KKP 3264 was the most resistant to chemical factors, by maintaining high lytic activity across a broader range of pH from 3 to 11. The results indicated that these phages could be a potential biological control agent against saprophytic bacterial microflora of minimally processed plant-based food products.

The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity.

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

Understanding the mechanism of asymmetric gene regulation determined by the VqmA of vibriophage.

The quorum-sensing (QS) system between the phages and their hosts is important for the phage lysis-lysogeny decision. In Vibrio cholerae, the QS system consists of a LuxR-type receptor VqmA (VqmAVc) and an autoinducer molecule 3,5-dimethylpyrazin-2-ol (DPO). A VqmA homolog encoded by vibriophage VP882 (VqmAPhage) can intervene the host QS system via binding to both the host-produced DPO and its cognate promoter (Pqtip) to induce the phage lysogeny-to-lysis transition, whereas VqmAVc cannot influence the VqmAPhage-induced pathway, suggesting an asymmetry regulation. In this study, we report the crystal structure of VqmAPhage-DPO complex at 2.65 Å and reveal that the mechanism of DPO recognition is conserved in VqmA homologs. Besides, we identify a non-classical palindrome sequence in Pqtip, which can be effectively recognized by VqmAPhage but not VqmAVc. The sequence contains an interval longer than that in the vqmR promoter recognized by VqmAVc. In addition, the two DBD regions in the VqmAPhage dimer exhibit more relaxed architecture than that of the reported VqmAVc, which is likely to be in the conformation that may easily bind to target promoter containing a longer interval. In summary, our findings provide a structural and biochemical basis for the DBD-dependent DNA recognition in different promoter regions in the phage lysogeny-to-lysis decision communication system, and provide clues for developing phage therapies against Vibrio cholerae infection.

[Jules Bordet, a man of conviction. Centenary of his Nobel Prize]. / Jules Bordet, un homme de conviction - Centenaire de l'attribution de son prix Nobel.

Jules Bordet came to the Institut Pasteur soon after his MD graduation at the Université libre de Bruxelles, thanks to a grant from the Belgian government. He joined there the laboratory of Elie Metchnikoff, the father of phagocytes and cellular immunity. Amazingly, he will decipher there some of the key mechanisms of humoral immunity initially discovered by the German school against which his mentor was fighting. He described the mechanisms that govern bacteriolysis and hemolysis, following the action of immune sera. Even if he favored the term alexin coined by Hans Buchner, he is indeed one of the founding fathers of the complement system (term coined by Paul Ehrlich). It is for these works that he was awarded in October 1920 the 1919 Nobel Prize. Back in Belgium, he became the director of Institut Pasteur du Brabant and made another landmark discovery, namely the identification of the bacillus of whooping cough, now named Bordetella pertussis.

TITLE: Jules Bordet, un homme de conviction - Centenaire de l'attribution de son prix Nobel. ABSTRACT: Docteur en médecine, bénéficiant d'une bourse du gouvernement belge, Jules Bordet vint se former au sein du laboratoire du père de l'immunité cellulaire, Elie Metchnikoff, à l'Institut Pasteur. Paradoxalement, il va y déchiffrer certains des mécanismes clés de l'immunité humorale, initialement découverte par l'école allemande. Il y décrit notamment les mécanismes qui aboutissent à la bactériolyse et l'hémolyse par l'action d'immunsérums. Même s'il favorisa le terme d'alexine, créé par Hans Buchner, c'est bien le système du complément (terme inventé par Paul Ehrlich) dont il est un des pères fondateurs. C'est pour ces travaux qu'il se verra attribué en octobre 1920 le prix Nobel de physiologie ou médecine millésimé 1919. Il identifia aussi le bacille de la coqueluche, qui porte son nom Bordetella pertussis.

The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by ß-lactam antibiotics or inhibitors of WTA biosynthesis.

ß-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of ß-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of ß-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with ß-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by ß-lactams or by inhibiting an early step in WTA biosynthesis. The finding that ß-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that ß-lactams do not kill S. aureus simply by weakening the cell wall.

Phage Lysis: Multiple Genes for Multiple Barriers.

The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.

The "hole" story of predatory outer-membrane vesicles.

All Gram-negative bacteria release membrane vesicles. These vesicles contain a cargo of proteins and enzymes that include one or more autolysins. Autolysins are a group of enzymes with specificity for the different linkages within peptidoglycan sacculi that if uncontrolled cause bacteriolysis. This minireview, written in honor and memory of Terry Beveridge, presents an overview of autolytic activity and focuses on Beveridge's important original observations regarding predatory membrane vesicles and their associated autolysin cargo.

Modeling the probability distribution of the bacterial burst size via a game-theoretic approach.

Based on previous studies, empirical distribution of the bacterial burst size varies even in a population of isogenic bacteria. Since bacteriophage progenies increase linearly with time, it is the lysis time variation that results in the bacterial burst size variations. Here, the burst size variation is computationally modeled by considering the lysis time decisions as a game. Each player in the game is a bacteriophage that has initially infected and lysed its host bacterium. Also, the payoff of each burst size strategy is the average number of bacteria that are solely infected by the bacteriophage progenies after lysis. For calculating the payoffs, a new version of ball and bin model with time dependent occupation probabilities (TDOP) is proposed. We show that Nash equilibrium occurs for a range of mixed burst size strategies that are chosen and played by bacteriophages, stochastically. Moreover, it is concluded that the burst size variations arise from choosing mixed lysis strategies by each player. By choosing the lysis time and also the burst size stochastically, the released bacteriophage progenies infect a portion of host bacteria in environment and avoid extinction. The probability distribution of the mixed burst size strategies is also identified.

Potential use of soilborne lytic Podoviridae phage as a biocontrol agent against Ralstonia solanacearum.

A new podovirus RsPod1EGY Ralstonia phage (GenBank accession no MG711516) with a specific action against R. solanacearum phylotype IIa, sequevar I (race 3, biovar 2) was isolated from Egyptian soil. The potential efficacy of the isolated phage to be used as biocontrol agent was evaluated in vitro and under greenhouse conditions. The podovirus phage produced a plaque size of 3.0-4.0 mm in diameter and completed its infection cycle in 180 min after infection with a burst size of ∼27 virions per infected cell. On the basis of restriction endonuclease analysis, the genome size of the phage was about 41 kb of double-stranded DNA. In vitro studies showed that RsPod1EGY is stable at higher temperatures (up to 60 °C), and at a wide pH range (5-9). SDS-PAGE analysis indicated the major structural protein to be approximately 32 kDa. Bacteriolytic activity of RsPod1EGY against R. solanacearum was detected at different multiplicity of infection (MOI). RsPod1EGY proved to be effective in reduction and prevention of formation of surface polysaccharides of R. solanacearum, during the exponential growth phase of the latter. Interestingly, RsPod1EGY was effective in suppression of R. solanacearum under greenhouse conditions. All Phage-treated tomato plants showed no wilt symptoms or any latent infection during the experimental period, whereas all untreated plants have wilted by 10 days post-infection. The lytic stability of RsPod1EGY phage at higher temperature as well as its effective suppression of wilting symptoms under greenhouse conditions would contribute to biocontrol the bacterial wilt disease in Egypt under field conditions.

Isolation and characterization of bacteriophages from India, with lytic activity against Mycobacterium tuberculosis.

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages - PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.

The applications of bacteriophages and their lysins as biocontrol agents against the foodborne pathogens Listeria monocytogenes and Campylobacter: An updated look.

Listeria monocytogenes and Campylobacter spp. are foodborne pathogens responsible for outbreaks and disease in humans. The emerging problem of bacterial antibiotic resistance and the persistence of pathogens in the environment, especially where foods are processed, are some of the reasons that have led to a re­emerging interest in bacteriophages and their lysins as potential candidates for bio­control. This review focuses on the use of bacteriophages and their lysins as alternative strategies for controlling the foodborne pathogens L. monocytogenes and Campylobacter spp. In addition, the application of bacteriophages and their lysins in food safety and animal health, as well as phage­resistance development, legislation, and future prospects were discussed.

A novel autolysis system controlled by magnesium and its application to poly (3-hydroxypropionate) production in engineered Escherichia coli.

The release of intracellular products, especially polyhydroxyalkanoates, is still a great challenge in industry. To solve this bottleneck, a novel autolysis system strictly controlled with magnesium was constructed and applied to poly(3-hydroxypropionate) production in engineered Escherichia coli. The autolysis system was constructed by inserting the 5'untranslated region (5'UTR) behind promoter PmgtA with lysis genes (S, R, and Rz, from E. coli) overexpressed. The autolysis system functioned well (lysis efficiency of more than 90%) in the P3HP producer with double plasmids containing lysis genes and P3HP biosynthesis genes, whereas the P3HP production was reduced due to plasmid losses. After the autolysis genes and P3HP biosynthesis genes were integrated into one plasmid, the P3HP content of 72.7% (2.4 times of the control) and the plasmid stability of 79.8 ± 3.1% were achieved in strain Q2646 with promoter PmgtA-UTR. However, the strain Q2647 with promoter PmgtA could not accumulate P3HP because of rapid cell lysis. The novel autolysis system activated in Mg2+-depleted conditions proves to be feasible for polyhydroxyalkanoates production, which may have great application potential for other intracellular products.

Overexpression of the fratricide immunity protein ComM leads to growth inhibition and morphological abnormalities in Streptococcus pneumoniae.

The important human pathogen Streptococcus pneumoniae is a naturally transformable species. When developing the competent state, it expresses proteins involved in DNA uptake, DNA processing and homologous recombination. In addition to the proteins required for the transformation process, competent pneumococci express proteins involved in a predatory DNA acquisition mechanism termed fratricide. This is a mechanism by which the competent pneumococci secrete a muralytic fratricin termed CbpD, which lyses susceptible sister cells or closely related streptococcal species. The released DNA can then be taken up by the competent pneumococci and integrated into their genomes. To avoid committing suicide, competent pneumococci produce an integral membrane protein, ComM, which protects them against CbpD by an unknown mechanism. In the present study, we show that overexpression of ComM results in growth inhibition and development of severe morphological abnormalities, such as cell elongation, misplacement of the septum and inhibition of septal cross-wall synthesis. The toxic effect of ComM is tolerated during competence because it is not allowed to accumulate in the competent cells. We provide evidence that an intra-membrane protease called RseP is involved in the process of controlling the ComM levels, since △rseP mutants produce higher amounts of ComM compared to wild-type cells. The data presented here indicate that ComM mediates immunity against CbpD by a mechanism that is detrimental to the pneumococcus if exaggerated.

Isolation of Bdellovibrio sp. from soil samples in Mexico and their potential applications in control of pathogens.

In this study, two strains of Bdellovibrio were isolated from soil samples using the culture-dependent technique and two members of the family Enterobacteriaceae (Klebsiella sp. and Salmonella sp.) as prey. The Bdellovibrio strains were bacteriolytic, plaque-forming, and highly motile gram-negative bacteria. We identified and confirmed the Bdellovibrio strains using microscopy, PCR amplification, and sequencing of the 16S rRNA gene. They were observed to be different strains based on hit locus and prey range analyses. Here, the first report on Bdellovibrio strains isolated from soil in Mexico corroborates earlier report indicating that populations of Bdellovibrio found in soil are heterogeneous thereby the need to identify the various strains.

More than a hole: the holin lethal function may be required to fully sensitize bacteria to the lytic action of canonical endolysins.

Double-strand DNA bacteriophages employ the holin-endolysin dyad as core components of different strategies to lyse bacterial hosts. In the so-called canonical model the holin holes play an essential role in lysis as they provide a conduit for passage of the cytoplasm-accumulated endolysin to the cell wall (CW), where it degrades the peptidoglycan. It is considered that once synthesized canonical endolysins immediately acquire their fully active conformation, having thus the capacity to efficiently cleave the peptidoglycan if contact to the CW is allowed. We show here however that holin-mediated cell death may be required to fully sensitize cells to the lytic action of canonical endolysins, a role that is obviously masked by the key function of the holin in endolysin release. We demonstrate that in certain conditions Bacillus subtilis cells are capable of counteracting the activity of the phage SPP1 endolysin attacking the CW either from within or from without. This capacity is lost after holin action or in presence of agents that mimic its membrane-depolarizing role. We have observed a similar relationship between lytic activity and membrane proton motive force for a staphylococcal endolysin. The possible implications of these findings in the exploitation of endolysins as enzybiotics are discussed.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc.

Bacterial killing via a type IV secretion system.

Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and protein-DNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.