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T-even bacteriophages are known to employ lysis inhibition (LIN), where the lysis of an infected host is delayed in response to secondary adsorptions. Upon the eventual burst of the host, significantly more phage progenies are released. Here, we analysed the competitive advantage of LIN using a mathematical model. In batch culture, LIN provides a bigger phage yield at the end of the growth where all the hosts are infected due to an exceeding number of phage particles and, in addition, gives a competitive advantage against LIN mutants with rapid lysis by letting them adsorb to already infected hosts in the LIN state. By simulating plaque formation in a spatially structured environment, we show that, while LIN phages will produce a smaller zone of clearance, the area over which the phages spread is actually comparable to those without LIN. The analysis suggests that LIN induced by secondary adsorption is favourable in terms of competition, both in spatially homogeneous and inhomogeneous environments., Competing Interests: The authors have declared that no competing interests exists., (Copyright: © 2024 Hvid, Mitarai. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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.

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 (VqmA Vc ) and an autoinducer molecule 3,5-dimethylpyrazin-2-ol (DPO). A VqmA homolog encoded by vibriophage VP882 (VqmA Phage ) 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 VqmA Vc cannot influence the VqmA Phage -induced pathway, suggesting an asymmetry regulation. In this study, we report the crystal structure of VqmA Phage -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 VqmA Phage but not VqmA Vc . The sequence contains an interval longer than that in the vqmR promoter recognized by VqmA Vc . In addition, the two DBD regions in the VqmA Phage dimer exhibit more relaxed architecture than that of the reported VqmA Vc , 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2020 médecine/sciences – Inserm.)

β-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 β-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 β-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 β-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 β-lactams or by inhibiting an early step in WTA biosynthesis. The finding that β-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 β-lactams do not kill S. aureus simply by weakening the cell wall., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2019 Elsevier Inc. All rights reserved.)

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 re‑emerging interest in bacteriophages and their lysins as potential candidates for bio‑control. 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 phage‑resistance development, legislation, and future prospects were discussed.

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.

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.

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., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 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 Mg 2+ -depleted conditions proves to be feasible for polyhydroxyalkanoates production, which may have great application potential for other intracellular products.

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.

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., (© 2016 Wiley Periodicals, Inc.)

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., (© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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., (© 2016 John Wiley & Sons Ltd.)

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.

Although the mechanisms underlying the cytotoxic effect of NK cells on tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell-surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection., (© 2014 Society for Leukocyte Biology.)

Polyamines are small cationic molecules that have far-reaching roles in biology. In the case of pathogenic bacteria, these functions include those central to their pathogenesis. Streptococcus pneumoniae is a major bacterial pathogen, causing a diverse range of diseases that account for significant morbidity and mortality worldwide. In this work, we characterize the polyamine biosynthetic pathway of S. pneumoniae, demonstrating that this organism produces spermidine from arginine. The synthesis of spermidine was found to be nonessential for growth in a polyamine-free chemically defined medium. However, mutant strains lacking the ability to synthesize or transport spermidine displayed a significant delay in the onset of autolysis. We provide evidence for a model in which spermidine modulates the activity of the major autolysin LytA in the pneumococcal cell wall compartment via interactions with negatively charged molecules, such as teichoic acids., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We report the detection by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry analyses of the secreted 2,5-diketopiperazines (DKPs) cyclo(-Ala-Pro), cyclo(-Gly-Pro), cyclo(-Val-Pro), cyclo(-Ile-Pro), cyclo(-Leu-Pro), cyclo(-Pro-Pro), cyclo(-HyP-Pro), cyclo(-Met-Pro), and cyclo(-Phe-Pro) produced by Bacillus pumilus. The study focuses on a marine isolate and a laboratory test strain of B. pumilus with capabilities to lyse pregrown living cell lawns of different bacterial species, among them Arthrobacter citreus. Chromatographic methods were used to analyze induced bioactive compounds. At least 13 different DKPs are produced by B. pumilus. Both strains respond with an increased production of the DKPs cyclo(-Gly-Pro), cyclo(-Ala-Pro), and cyclo(-Val-Pro) to the presence of pasteurized A. citreus cells after 4 h in a nutrient-poor liquid medium. In agar diffusion assays, these DKPs did not cause lysis zones in living cell lawns, but they did inhibit further growth of several pregrown test bacteria in microplates even at concentrations as low as 1 μg ml(-1). Antibiotic substances produced by B. pumilus after 20 h of cultivation in a special lysis medium showed lytic activity in cell-free extracts of B. pumilus culture supernatants.

t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an N(in)-C(out) topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N(out)-C(in)) of the coliphage lambda and S68 (2 TMDs; N(in)-C(in)) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Streptococcus pneumoniae (SP) and nontypeable Haemophilus influenzae (NTHi) are common commensals of the human airway and major bacterial pathogens of otitis media (OM) and other upper airway infections. The interaction between them may play an important role in the pathogenesis of polymicrobial infections. Although previous studies suggested NTHi could promote pneumococcal survival and biofilm formation, how NTHi affects pneumococcal activities has not been defined. Our data in the present studies indicated that the outcome of the interaction between SP and NTHi was in a cell-density-dependent manner and the enhancement of pneumococcal survival happened at the later stages of culturing. Using quantitative PCR, we found that the expression of pneumococcal genes regulating autolysis and fratricide, lytA and cbpD, were significantly down-regulated in co-culture with NTHi. We further observed that influence of NTHi was not on direct cell-to-cell contact, but that this contact may contribute to the interaction between these two microorganisms. These results suggest that pneumococcal survival and biofilm formation can be enhanced by down-regulating pneumococcal cell wall hydrolase production thereby inhibiting pneumococcal autolysis and fratricide in the presence of NTHi., (Copyright © 2013. Published by Elsevier Masson SAS.)

Vibrio tubiashii has been linked to disease outbreaks in molluscan species, including oysters, geoducks, and clams, and shellfish hatcheries in the Pacific Northwest have been plagued by intermittent vibriosis outbreaks since 2006. Like V. tubiashii, Vibrio coralliilyticus has recently been described as an oyster pathogen in addition to its role in coral disease. Here, we describe an autolysis phenotype in V. tubiashii and its close relative V. coralliilyticus and characterize the effects of environmental conditions on this phenotype. We also explored whether the survivors of autolysis were resistant to the phenotype and if material from the autolysed culture would either regrow or have a population of viable cells. Ultimately, this work contributes to the larger understanding of bacterial population dynamics as it relates to aquaculture pathogens.

CspA of the Lyme disease spirochete Borrelia burgdorferi represents a key molecule in immune evasion, protecting borrelial cells from complement-mediated killing. As previous studies focused almost exclusively on CspA of B. burgdorferi, here we investigate the different binding capacities of CspA orthologs of Borrelia burgdorferi, B. afzelii, and B. spielmanii for complement regulator factor H and plasminogen and their ability to inhibit complement activation by either binding these host-derived plasma proteins or independently by direct interaction with components involved in formation of the lethal, pore-like terminal complement complex. To further examine their function in serum resistance in vivo, a serum-sensitive B. garinii strain was used to generate spirochetes, ectopically producing functional CspA orthologs. Irrespective of their species origin, all three CspA orthologs impart resistance to complement-mediated killing when produced in a serum-sensitive B. garinii surrogate strain. To analyze the inhibitory effect on complement activation and to assess the potential to inactivate C3b by binding of factor H and plasminogen, recombinant CspA orthologs were also investigated. All three CspA orthologs simultaneously bound factor H and plasminogen but differed in regard to their capacity to inactivate C3b via bound plasmin(ogen) and inhibit formation of the terminal complement complex. CspA of B. afzelii binds plasmin(ogen) and inhibits the terminal complement complex more efficiently than CspA of B. burgdorferi and B. spielmanii. Taken together, CspA orthologs of serum-resistant Lyme disease spirochetes act as multifunctional evasion molecules that inhibit complement on two central activation levels, C3b generation and assembly of the terminal complement complex.

Some clinical isolates of Pseudomonas aeruginosa stored in our culture collection did not grow or grew poorly and showed lysis on the culture plates when removed from the collection and inoculated on MacConkey agar. One hypothesis was that bacteriophages had infected and killed those clinical isolates. To check the best storage conditions to maintain viable P. aeruginosa for a longer time, clinical isolates were stored at various temperatures and were grown monthly. We investigated the presence of phage in 10 clinical isolates of P. aeruginosa stored in our culture collection. Four strains of P. aeruginosa were infected by phages that were characterized by electron microscopy and isolated to assess their ability to infect. The best condition to maintain the viability of the strains during storage was in water at room temperature. Three Siphoviridae and two Myoviridae phages were visualized and characterized by morphology. We confirmed the presence of bacteriophages infecting clinical isolates, and their ability to infect and lyse alternative hosts. Strain PAO1, however, did not show lysis to any phage. Mucoid and multidrug resistant strains of P. aeruginosa showed lysis to 50% of the phages tested.

Bacteriophages have developed multiple host cell lysis strategies to promote release of descendant virions from infected bacteria. This review is focused on the lysis mechanisms employed by tailed double-stranded DNA bacteriophages, where new developments have recently emerged. These phages seem to use a least common denominator to induce lysis, the so-called holin-endolysin dyad. Endolysins are cell wall-degrading enzymes whereas holins form 'holes' in the cytoplasmic membrane at a precise scheduled time. The latter function was long viewed as essential to provide a pathway for endolysin escape to the cell wall. However, recent studies have shown that phages can also exploit the host cell secretion machinery to deliver endolysins to their target and subvert the bacterial autolytic arsenal to effectively accomplish lysis. In these systems the membrane-depolarizing holin function still seems to be essential to activate secreted endolysins. New lysis players have also been uncovered that promote degradation of particular bacterial cell envelopes, such as that of mycobacteria., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Lambdoid phage 21 uses a pinholin-signal anchor release endolysin strategy to effect temporally regulated host lysis. In this strategy, the pinholin S(21)68 accumulates harmlessly in the bilayer until suddenly triggering to form lethal membrane lesions, consisting of S(21)68 heptamers with central pores <2 nm in diameter. The membrane depolarization caused by these pores activates the muralytic endolysin, R(21), leading immediately to peptidoglycan degradation. The lethal S(21)68 complexes have been designated as pinholes to distinguish from the micrometer-scale holes formed by canonical holins. Here, we used GFP fusions of WT and mutant forms of S(21)68 to show that the holin accumulates uniformly throughout the membrane until the time of triggering, when it suddenly redistributes into numerous small foci (rafts). Raft formation correlates with the depletion of the proton motive force, which is indicated by the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol. By contrast, GFP fusions of either antiholin variant irsS(21)68, which only forms inactive dimers, or nonlethal mutant S(21)68(S44C), which is blocked at an activated dimer stage of the pinhole formation pathway, were both blocked in a state of uniform distribution. In addition, fluorescence recovery after photobleaching revealed that, although the antiholin irsS(21)68-GFP fusion was highly mobile in the membrane (even when the proton motive force was depleted), more than one-half of the S(21)68-GFP molecules were immobile, and the rest were in mobile states with a much lower diffusion rate than the rate of irsS(21)68-GFP. These results suggest a model in which, after transiting into an oligomeric state, S(21)68 migrates into rafts with heterogeneous sizes, within which the final pinholes form.

Bacterial ghost is intact envelope of Gram-negative bacteria, which is produced by the function of the lysis gene E from bacteriophage PhiX174. The expression of the lysis gene E is usually controlled by the thermosensitive lambdapL/pR-cI857 promoter. In this study, we described a mutation (T --> C) at the ninth nucleotide of the OR2 in the lambdapR promoter of the lambdapL/pR-cI857 system by overlap PCR. The bacteriolytic assay showed that the mutation in the lambdapL/pR-cI857 system enhanced the temperature of repressing the expression of gene E up to 37 degrees C. The lysis efficiency of altered lambdapR promoter in Escherichia coli DH5a and avian pathogenic E. coli DE17 was up to 99.9%. The expanded range of temperature will benefit for the production of bacterial ghost.

The aim of this study was to investigate the molecular characteristics of induced vancomycin resistance in Staphylococcus haemolyticus. Autolytic properties and phenotypic characteristics of passage-selected vancomycin-resistant S. haemolyticus strains were examined. In addition, expression of autolysis-related genes (atl, lrgAB, sarA and lytS) was investigated using the RNase protection assay (RPA). The RPA results indicated that only the expression of the atl gene was significantly upregulated (2.5- to 6-fold increase) in vancomycin-intermediate and vancomycin-resistant strains. The vancomycin-resistant strains exhibited lower expression of murein hydrolase proteins and reduced autolytic activity compared with the parent strain. In addition, a reduced growth rate, cell wall thickening and higher survival rate in the presence of lysostaphin were observed in vancomycin-intermediate and vancomycin-resistant induced strains compared with the parent strain. In conclusion, altered autolytic properties, in particular upregulation of the atl gene, may contribute to vancomycin resistance in S. haemolyticus.

The fmtA gene is a member of the Staphylococcus aureus core cell wall stimulon. The FmtA protein interacts with β-lactams through formation of covalent species. Here, we show that FmtA has weak D-Ala-D-Ala-carboxypeptidase activity and is capable of covalently incorporating C14-Gly into cell walls. The fluorescence microscopy study showed that the protein is localized to the cell division septum. Furthermore, we show that wall teichoic acids interact specifically with FmtA and mediate recruitment of FmtA to the S. aureus cell wall. Subjection of S. aureus to FmtA concentrations of 0.1 μM or less induces autolysis and biofilm production. This effect requires the presence of wall teichoic acids. At FmtA concentrations greater than 0.2 μM, autolysis and biofilm formation in S. aureus are repressed and growth is enhanced. Our findings indicate dual roles of FmtA in S. aureus growth, whereby at low concentrations, FmtA may modulate the activity of the major autolysin (AtlA) of S. aureus and, at high concentrations, may participate in synthesis of cell wall peptidoglycan. These two roles of FmtA may reflect dual functions of FmtA in the absence and presence of cell wall stress, respectively.

Biofilms are currently recognised as the predominant bacterial life-style and it has been suggested that biofilm development is influenced by a number of different processes such as adhesion, detachment, mass transport, quorum sensing, cell death and active dispersal. One of the least understood processes and its effects on biofilm development is cell death. However, experimental studies suggest that bacterial death is an important process during biofilm development and many studies show a relationship between cell death and dispersal in microbial biofilms. We present a model of the process of cell death during biofilm development, with a particular focus on the spatial localisation of cell death or cell damage. Three rules governing cell death or cell damage were evaluated which compared the effects of starvation, damage accumulation, and viability during biofilm development and were also used to design laboratory based experiments to test the model. Results from model simulations show that actively growing biofilms develop steep nutrient gradients within the interior of the biofilm that affect neighbouring microcolonies resulting in cell death and detachment. Two of the rules indicated that high substrate concentrations lead to accelerated cell death, in contrast to the third rule, based on the accumulation of damage, which predicted earlier cell death for biofilms grown with low substrate concentrations. Comparison of the modelling results with experimental results suggests that cell death is favoured under low nutrient conditions and that the accumulation of damage may be the main cause of cell death during biofilm development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Acinetobacter baumannii is known for its ability to develop resistance to the major groups of antibiotics, form biofilms, and survive for long periods in hospital environments. The prevalence of infections caused by multidrug-resistant A. baumannii is a significant problem for the modern health care system, and application of lytic bacteriophages for controlling this pathogen may become a solution., Methodology/principal Findings: In this study, using atomic force microscopy (AFM) and microbiological assessment we have investigated A. baumannii bacteriophage AP22, which has been recently described. AFM has revealed the morphology of bacteriophage AP22, adsorbed on the surfaces of mica, graphite and host bacterial cells. Besides, morphological changes of bacteriophage AP22-infected A. baumannii cells were characterized at different stages of the lytic cycle, from phage adsorption to the cell lysis. The phage latent period, estimated from AFM was in good agreement with that obtained by microbiological methods (40 min). Bacteriophage AP22, whose head diameter is 62±1 nm and tail length is 88±9 nm, was shown to disperse A. baumannii aggregates and adsorb to the bacterial surface right from the first minute of their mutual incubation at 37°C., Conclusions/significance: High rate of bacteriophage AP22 specific adsorption and its ability to disperse bacterial aggregates make this phage very promising for biomedical antimicrobial applications. Complementing microbiological results with AFM data, we demonstrate an effective approach, which allows not only comparing independently obtained characteristics of the lytic cycle but also visualizing the infection process.

Transcription initiation is a critical step in bacterial gene regulation and is often controlled by transcription regulators. The alternate sigma factor (σ(54)) is one such regulator that facilitates activator-dependent transcription initiation and thus modulates the expression of a variety of genes involved in metabolism and pathogenesis in bacteria. This study describes the role of σ(54) in the nosocomial pathogen Enterococcus faecalis. Biofilm formation is one of the important pathogenic mechanisms of E. faecalis, as it elevates the organism's potential to cause surgical site and urinary tract infections. Lysis of bacterial cells within the population contributes to biofilm formation by providing extracellular DNA (eDNA) as a key component of the biofilm matrix. Deletion of rpoN rendered E. faecalis resistant to autolysis, which in turn impaired eDNA release. Despite the significant reduction in eDNA levels compared to the parental strain, the rpoN mutant formed more robust biofilms as observed using laser scanning confocal microscopy and Comstat analysis, indicating and emphasizing the presence of other matrix components. Initial adherence to a polystyrene surface was also enhanced in the mutant. Proteinase K treatment at early stages of biofilm development significantly reduced the accumulation of biofilm by the rpoN mutant. In conclusion, our data indicate that other factors in addition to eDNA might contribute to the overall composition of the enterococcal biofilm and that the regulatory role of σ(54) governs the nature and composition of the biofilm matrix.

Bacteriophage λ, rediscovered in the early 1950s, has served as a model in molecular biology studies for decades. Although currently more complex organisms and more complicated biological systems can be studied, this phage is still an excellent model to investigate principles of biological processes occurring at the molecular level. In fact, very few other biological models provide possibilities to examine regulations of biological mechanisms as detailed as performed with λ. In this chapter, recent advances in our understanding of mechanisms of bacteriophage λ development are summarized and discussed. Particularly, studies on (i) phage DNA injection, (ii) molecular bases of the lysis-versus-lysogenization decision and the lysogenization process itself, (iii) prophage maintenance and induction, (iv), λ DNA replication, (v) phage-encoded recombination systems, (vi) transcription antitermination, (vii) formation of the virion structure, and (viii) lysis of the host cell, as published during several past years, will be presented., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway.

A bactericidal mechanism mediated by human serum was investigated by a field emission scanning electron microscope and a strain of drug-resistant Pseudomonas aeruginosa. When the bacteria were treated with meropenem, a carbapenem antibiotic, spheroplasts and bulges (spheroidization) appeared after 1-3 h. When 40% serum was added to the bacteria, the bacteria agglutinated within 2 min and then lysed after 5-30 min. Immunoelectron micrographic analyses showed dispositions of complement component C9 molecules on the cell surface of lysed bacteria by the serum treatment that might suggest formation of a membrane attack complex. Immunoglobulin G (IgG) depletion from the serum diminished the lytic activity and adding human intravenous immunoglobulin (IVIG) restored it, suggesting that lysis was induced by specific IgG binding to the bacteria. IVIG may help patients with less IgG against bacteria to overcome severe infection.

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.

Introduction: Dental caries remains one of the most common chronic infectious diseases throughout the world. The formation of dental plaque is one of the caries risk factors. As a consequence, the removal of plaque may reduce the incidence of caries development. We identified an autolysin produced by Streptococcus mutans named auto-mutanolysin (Aml). Aml selectively lyses S. mutans and Streptococcus sobrinus. The specificity towards these cariogenic bacteria suggests that Aml may be used to prevent dental caries. Here, with the aim towards therapeutic application, we investigated the lytic activity of Aml against clinical isolates of S. mutans and S. sobrinus using planktonic cells and biofilms., Methods: Planktonic cell suspensions and biofilms of clinically isolated streptococci were treated with Aml in the absence or the presence of Triton X-100. The lytic activity of Aml was monitored as the change in turbidity. The disruption of biofilms was evaluated by detecting the released DNA by polymerase chain reaction and observing the alteration of optical density of treated biofilms., Results: Triton X-100 enhances the lytic ability of Aml. Using planktonic cells, Aml had various lysis levels against clinical strains. Repeated Aml treatment showed disruption of the biofilm using the representative clinical strains., Conclusion: Our study demonstrates that Aml has an ability to lyse planktonic and biofilm cells of clinically isolated mutans streptococci in the presence of Triton X-100. These results suggest the possibility of using Aml as an alternative or additional approach for caries prevention.

Perhaps the simplest of biological timing systems, bacteriophage holins accumulate during the phage morphogenesis period and then trigger to permeabilize the cytoplasmic membrane with lethal holes; thus, terminating the infection cycle. Canonical holins form very large holes that allow nonspecific release of fully-folded proteins, but a recently discovered class of holins, the pinholins, make much smaller holes, or pinholes, that serve only to depolarize the membrane. Here, we interrogate the structure of the prototype pinholin by negative-stain transmission electron-microscopy, cysteine-accessibility, and chemical cross-linking, as well as by computational approaches. Together, the results suggest that the pinholin forms symmetric heptameric structures with the hydrophilic surface of one transmembrane domain lining the surface of a central channel approximately 15 A in diameter. The structural model also suggests a rationale for the prehole state of the pinholin, the persistence of which defines the duration of the viral latent period, and for the sensitivity of the holin timing system to the energized state of the membrane.

Two classes of bacteriophages, the single-stranded DNA Microviridae and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing "protein antibiotics", or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91-amino acid membrane protein encoded by the prototype microvirus, varphiX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to overexpression because of its lethality to Escherichia coli. Moreover, using a fluorescently labeled analogue of the sugar-nucleotide substrate, we demonstrate that E acts as a noncompetitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential heteromultimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E resistance mutations map and the fact that the result of this binding is a conformational change that inactivates the enzyme.

The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales. IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.

The potential of laser tweezers Raman spectroscopy (LTRS) to study complex and dynamic cellular processes was investigated on the model of single E. coli cells lysed (1) from "outside" with egg white lysozyme and (2) from "within" by temperature-induced temperate bacteriophage lambdacI857. The two lysis processes differed in the final outcome (incomplete vs complete cell lysis) as revealed by the dynamic laser light scattering and exhibited distinctive dynamic Raman spectra changes. The technique enabled for the first time at the cellular level to observe and quantify real time interaction of lysozyme with E. coli cells, "visualize" a side effect of the process due to the presence of EDTA, and correlate the process of cell wall disruption, as evidenced by the onset and development of asymmetric speckle scattering patterns, with release/escape of intracellular material (ribosomes, nucleic acids, proteins, etc.) quantified by the intensity changes of Raman signatures. Raman spectra changes observed during the lysis from "within" suggest alleged production of heat shock proteins are consistent with the occurring synthesis of phage-related proteins and are in good agreement with the calculated potential contribution of the above proteins to the Raman spectra. It was also established and validated that the contribution of cellular DNA to the Raman spectra of bacterial cells is negligible compared to RNA. The results open new venues for LTRS research and strongly suggest that LTRS has a great potential especially in investigation of real-time processes.

Aims: Frequency of lysogeny in Lactobacillus delbrueckii strains (from commercial and natural starters) and preliminary characterization of temperate bacteriophages isolated from them., Methods and Results: Induction of strains (a total of 16) was made using mitomycin C (MC) (0.5 mug ml(-1)). For 37% of the MC-treated supernatants, it was possible to detect phage particles or presence of killing activity, but only two active bacteriophages were isolated. The two temperate phages isolated were prolate-headed phages which belonged to group c of Lact. delbrueckii bacteriophages classification. Different DNA restriction patterns were obtained for each phage, while the structural protein profiles and packaging sites were identical. Distinctive one-step growth curves were exhibited by each phage. An influence of calcium ions was observed for their lysis in broth but not on the adsorption levels., Conclusions: Our study showed that lysogeny is also present in Lact. delbrueckii strains, including commercial strains., Significance and Impact of the Study: Commercial strains could be lysogenic and this fact has a great practical importance since they could contribute to the dissemination of active-phage particles in industrial environments.

We have devised and constructed a biological containment system designed to cause programmed bacterial cell lysis with no survivors. We have validated this system, using Salmonella enterica serovar Typhimurium vaccines for antigen delivery after colonization of host lymphoid tissues. The system is composed of two parts. The first component is Salmonella typhimurium strain chi8937, with deletions of asdA and arabinose-regulated expression of murA, two genes required for peptidoglycan synthesis and additional mutations to enhance complete lysis and antigen delivery. The second component is plasmid pYA3681, which encodes arabinose-regulated murA and asdA expression and C2-regulated synthesis of antisense asdA and murA mRNA transcribed from the P22 P(R) promoter. An arabinose-regulated c2 gene is present in the chromosome. chi8937(pYA3681) exhibits arabinose-dependent growth. Upon invasion of host tissues, an arabinose-free environment, transcription of asdA, murA, and c2 ceases, and concentrations of their gene products decrease because of cell division. The drop in C2 concentration results in activation of P(R), driving synthesis of antisense mRNA to block translation of any residual asdA and murA mRNA. A highly antigenic alpha-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with chi8937(pYA3685) developed antibody responses to PspA and Salmonella outer membrane proteins. No viable vaccine strain cells were detected in host tissues after 21 days. This system has potential applications with other Gram-negative bacteria in which biological containment would be desirable.

The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.