Prophage Research Articles

Induction of the Stringent Response Underlies the Antimicrobial Action of Aliphatic Isothiocyanates.

Bacterial resistance to known antibiotics comprises a serious threat to public health. Propagation of multidrug-resistant pathogenic strains is a reason for undertaking a search for new therapeutic strategies, based on newly developed chemical compounds and the agents present in nature. Moreover, antibiotic treatment of infections caused by enterotoxin toxin-bearing strain—enterohemorrhagic Escherichia coli (EHEC) is considered hazardous and controversial due to the possibility of induction of bacteriophage-encoded toxin production by the antibiotic-mediated stress. The important source of potentially beneficial compounds are secondary plant metabolites, isothiocyanates (ITC), and phytoncides from the Brassicaceae family. We reported previously that sulforaphane and phenethyl isothiocyanate, already known for their chemopreventive and anticancer features, exhibit significant antibacterial effects against various pathogenic bacteria. The mechanism of their action is based on the induction of the stringent response and accumulation of its alarmones, the guanosine penta- and tetraphosphate. In this process, the amino acid starvation path is employed via the RelA protein, however, the precise mechanism of amino acid limitation in the presence of ITCs is yet unknown. In this work, we asked whether ITCs could act synergistically with each other to increase the antibacterial effect. A set of aliphatic ITCs, such as iberin, iberverin, alyssin, erucin, sulforaphen, erysolin, and cheirolin was tested in combination with sulforaphane against E. coli. Our experiments show that all tested ITCs exhibit strong antimicrobial effect individually, and this effect involves the stringent response caused by induction of the amino acid starvation. Interestingly, excess of specific amino acids reversed the antimicrobial effects of ITCs, where the common amino acid for all tested compounds was glycine. The synergistic action observed for iberin, iberverin, and alyssin also led to accumulation of (p)ppGpp, and the minimal inhibitory concentration necessary for the antibacterial effect was four- to eightfold lower than for individual ITCs. Moreover, the unique mode of ITC action is responsible for inhibition of prophage induction and toxin production, in addition to growth inhibition of EHEC strains. Thus, the antimicrobial effect of plant secondary metabolites by the stringent response induction could be employed in potential therapeutic strategies.

Fitness benefits to bacteria of carrying prophages and prophage-encoded antibiotic-resistance genes peak in different environments.

Understanding the role of horizontal gene transfer (HGT) in adaptation is a key challenge in evolutionary biology. In microbes, an important mechanism of HGT is prophage acquisition (phage genomes integrated into bacterial chromosomes). Prophages can influence bacterial fitness via the transfer of beneficial genes (including antibiotic‐resistance genes, ARGs), protection from superinfecting phages, or switching to a lytic lifecycle that releases free phages infectious to competitors. We expect these effects to depend on environmental conditions because of, for example, environment‐dependent induction of the lytic lifecycle. However, it remains unclear how costs/benefits of prophages vary across environments. Here, studying prophages with/without ARGs in Escherichia coli, we disentangled the effects of prophages alone and adaptive genes they carry. In competition with prophage‐free strains, benefits from prophages and ARGs peaked in different environments. Prophages were most beneficial when induction of the lytic lifecycle was common, whereas ARGs were more beneficial upon antibiotic exposure and with reduced prophage induction. Acquisition of prophage‐encoded ARGs by competing strains was most common when prophage induction, and therefore free phages, were common. Thus, selection on prophages and adaptive genes they carry varies independently across environments, which is important for predicting the spread of mobile/integrating genetic elements and their role in evolution.

Characterization of Heterogeneity and Dynamics of Lysis of Single Bacillus subtilis Cells upon Prophage Induction During Spore Germination, Outgrowth, and Vegetative Growth Using Raman Tweezers and Live-Cell Phase-Contrast Microscopy.

A prophage comprises a bacteriophage genome that has integrated into a host bacterium's DNA, which generally permits the cell to grow and divide normally. However, the prophage can be induced by various stresses, or induction can occur spontaneously. After prophage induction, viral replication and production of endolysins begin until the cell lyses and phage particles are released. However, the heterogeneity of prophage induction and lysis of individual cells in a population and the dynamics of a cell undergoing lysis by prophage induction have not been fully characterized. Here, we used Raman tweezers and live-cell phase-contrast microscopy to characterize the Raman spectral and cell length changes that occur during the lysis of individual Bacillus subtilis cells from spores that carry PBSX prophage during spores' germination, outgrowth, and then vegetative growth. Major findings of this work are as follows: (i) After addition of xylose to trigger prophage induction, the intensities of Raman spectral bands associated with nucleic acids of single cells in induced cultures gradually fell to zero, in contrast to the much smaller changes in protein band intensities and no changes in nucleic acid bands in uninduced cultures; (ii) the nucleic acid band intensities from an individual induced cell exhibited a rapid decrease, following a long lag period; (iii) after the addition of nutrient-rich medium with xylose, single spores underwent a long period (228 ± 41.4 min) for germination, outgrowth, and vegetative growth, followed by a short period of cell burst in 1.5 ± 0.8 min at a cell length of 8.2 ± 5.5 μm; (iv) the latent time (Tlatent) between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (ΔTburst) from the latent time to the completion of cell lysis was quite small; (v) in a poor medium with l-alanine alone, addition of xylose caused prophage induction following spore germination but with longer Tlatent and ΔTburst times and without cell elongation; (vi) spontaneous prophage induction and lysis of individual cells from spores in a minimal nutrient medium were observed without xylose addition, and cell length prior to cell lysis was ∼4.1 μm, but spontaneous prophage induction was not observed in a rich medium; (vii) in a rich medium, addition of xylose at a time well after spore germination and outgrowth significantly shortened the average Tlatent time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual B. subtilis cells derived from spores upon prophage induction.

Differential induction of Shiga toxin in environmental Escherichia coli O145:H28 strains carrying the same genotype as the outbreak strains

Shiga toxin-producing Escherichia coli (STEC) O145 is a major serotype associated with severe human disease. Production of Shiga toxins (Stxs), especially Stx2a, is thought to be correlated with STEC virulence. Since stx genes are located in prophages genomes, induction of prophages is required for effective Stxs production. Here, we investigated the production of Stxs in 12 environmental STEC O145:H28 strains under stresses STEC encounter in natural habitats and performed comparative analysis with two O145:H28 clinical strains, one linked to a 2010 U.S. lettuce-associated outbreak (RM13514) and the other linked to a 2007 Belgium ice cream-associated outbreak (RM13516). Similar to the outbreak strains, all environmental strains belong to Sequence Type (ST)-78 using the EcMLST typing scheme. Although all Stx1a-prophages were grouped together, variations in Stx1a production were observed prior to or following the inductions. Among all stx2a positive environmental strains, only the Stx2a-prophage in cattle isolate RM9154-C1 was clustered with the Stx2a-prophages in RM13514, the Stx2a-phage induced from a STEC O104:H4 strain linked to the 2011 outbreak of enterohemorrhagic infection in Germany, and the Stx2a-prophage in STEC O157:H7 strain EDL933, a prototype of enterohemorrhagic E. coli. Furthermore, the Stx2a-prophage in RM9154-C1 shared the same chromosomal insertion site and carried the same antiterminator Q gene and the late promoter PR' as the Stx2a-prophage in RM13514. Following mitomycin C or enrofloxacin treatment, the production of Stx2a in RM9154-C1 was the highest among all environmental strains tested. In contrast, following acid challenge and recovery, the production of Stx2a in RM9154-C1 was the lowest among all the environmental strains tested, at a level comparable to the clinical strains. A significant increase in Stx2a production was detected in all strains when exposed to H2O2, although the induction fold was much lower than those by other inducers. This low-efficiency induction of Stx-prophages by H2O2, a natural inducer of Stx-prophages, supports the hypothesis of bacterial altruism in controlling Stxs production, a strategy that assures the survival of the STEC population as a whole by sacrificing a small fraction of cells for Stxs production and release. Differential induction of Stxs among strains carrying nearly identical Stx-prophages suggests a role of host bacteria in regulating Stxs production. Our study revealed diverse Stx-prophages in STEC O145:H28 strains that were genotypically indistinguishable. Identification of a cattle isolate harboring a Stx2a-prophage associated with high virulence supports the premise that cattle, a natural reservoir of STEC, serve as a source of hypervirulent STEC strains.

Microbial single-cell RNA sequencing by split-pool barcoding.

Single-cell RNA sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes, but current methods are incompatible with bacteria. Here, we introduce microSPLiT (microbial split-pool ligation transcriptomics), a high-throughput scRNA-seq method for Gram-negative and Gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction and also identified unexpected gene expression states, including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities that are otherwise not amenable to single-cell analysis, such as natural microbiota.

A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression.

Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

Articles of Significant Interest in This Issue

Shiga toxins, the major virulence factors of enterohemorrhagic Escherichia coli (EHEC) strains, are encoded within temperate bacteriophage genomes. Their expression is coupled to prophage induction, which can be stimulated by interactions with other microbes in the intestinal environment. Nawrocki et al. (e00509-20) review the effects of microbial communities on Shiga toxin production, focusing on phage induction by DNA-damaging molecules and phage replication in susceptible host strains. These influences may contribute to severe systemic complications by increasing Shiga toxin levels in EHEC infections.

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186.

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.

Long inverted repeats around the chromosome replication terminus in the model strain Bacillus thuringiensis serovar israelensis BGSC 4Q7

Bacillus thuringiensis serovar israelensis is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain, B . thuringiensis serovar israelensis BGSC 4Q7 (4Q7), has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element, pBtic235, which has been shown to be an inducible prophage, although three putative chromosomal prophages have been lost. Moreover, a 492 kb region, potentially including the standard replication terminus, has also been deleted in the 4Q7 strain, indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain, and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area, close to the 492 kb deletion, was found to be duplicated. This duplication presumably restored the equal sizes of the replichores, and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome, and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins, potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235, but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural B. thuringiensis serovar israelensis strains, suggesting a duplication or over-replication in 4Q7. Thus, the 4Q7 strain is not a pure plasmid-less offshoot, but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence, genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model, but they also raise interesting fundamental questions about the functioning of the Bacillus genome.

Genotoxicity and antimicrobial activity of Myrtus communis L., Ziziphus spina-christi (L.) Willd and Cassia angustifolia Vahl extracts

Genotoxicity and antimicrobial activity of the leaf extract of Myrtus communis, Ziziphus spina-christi and Cassia angustifolia plants were evaluated. The prophage induction assay was used to test the genotoxicity. The results showed that, no fold increase in Pfu/ml over the spontaneous release for F116 and R3 phages, so these results indicated that Myrtus communis, Ziziphus spina-christi and Cassia angustifolia leaves extract did not have genotoxic effect, so they can be used as antimutagen. On the other hand, Myrtus communis and Ziziphus spina-christi showed strong antimicrobial activity against all tested microbes. The antimicrobial effect increased by the increase of the concentration. While Cassia angustifolia leaves extract showed moderate antimicrobial activity against some strains. The results of antimicrobial activity suggest that these plant extracts have cytotoxic effect.

Mimicking prophage induction in the body: induction in the lab with pH gradients.

The majority of bacteria within the human body are lysogens, often harboring multiple bacteriophage sequences (prophages) within their genomes. While several different types of environmental stresses can trigger or induce prophages to enter into the lytic cycle, they have yet to be fully explored and understood in the human microbiota. In the laboratory, the most common induction method is the DNA damaging chemical Mitomycin C. Although pH has been listed in the literature as an induction method, it is not widely used. Here, we detail a protocol for prophage induction by culture under different pH conditions. We explored the effects of pH on prophage induction in bacterial isolates from the bladder, where the pH is well documented to vary significantly between individuals as well as between healthy individuals and individuals with urinary tract symptoms or disease. Using this protocol, we successfully induced phages from seven bladder E. coli strains. Testing conditions and stressors appropriate to the environment from which a lysogen is isolated may provide insight into community dynamics of the human microbiota.

Impact of Viral Lysis on the Composition of Bacterial Communities and Dissolved Organic Matter in Deep-Sea Sediments.

Viral lysis is a main mortality factor for bacteria in deep-sea sediments, leading to changing microbial community structures and the release of cellular components to the environment. Nature and fate of these compounds and the role of viruses for microbial diversity is largely unknown. We investigated the effect of viruses on the composition of bacterial communities and the pool of dissolved organic matter (DOM) by setting up virus-induction experiments using mitomycin C with sediments from the seafloor of the Bering Sea. At the sediment surface, no substantial prophage induction was detected, while incubations from 20 cm below seafloor showed a doubling of the virus-to-cell ratio. Ultra-high resolution mass spectrometry revealed an imprint of cell lysis on the molecular composition of DOM, showing an increase of molecular formulas typical for common biomolecules. More than 50% of these compounds were removed or transformed during incubation. The remaining material potentially contributed to the pool of refractory DOM. Next generation sequencing of the bacterial communities from the induction experiment showed a stable composition over time. In contrast, in the non-treated controls the abundance of dominant taxa (e.g., Gammaproteobacteria) increased at the expense of less abundant phyla. Thus, we conclude that viral lysis was an important driver in sustaining bacterial diversity, consistent with the “killing the winner” model.

Bifurcation analysis of a phage-bacteria interaction model with prophage induction.

A predator-prey model is used to investigate the interactions between phages and bacteria by considering the lytic and lysogenic life cycles of phages and the prophage induction. We provide answers to the following conflictual research questions: (1) what are conditions under which the presence of phages can purify a bacterial infected environment? (2) Can the presence of phages triggers virulent bacterial outbreaks? We derive the basic offspring number $\mathcal N_0$ that serves as a threshold and the bifurcation parameter to study the dynamics and bifurcation of the system. The model exhibits three equilibria: an unstable environment-free equilibrium, a globally asymptotically stable (GAS) phage-free equilibrium (PFE) whenever $\mathcal N_0<1$, and a locally asymptotically stable environment-persistent equilibrium (EPE) when $\mathcal N_0>1$. The Lyapunov-LaSalle techniques are used to prove the GAS of the PFE and estimate the EPE basin of attraction. Through the center manifold approximation, topological types of the PFE are precised. Existence of transcritical and Hopf bifurcations are established. Precisely, when $\mathcal N_0>1$, the EPE loses its stability and periodic solutions arise. Furthermore, increasing $\mathcal N_0$ can purify an environment where bacteriophages are introduced. Purposely, we prove that for large values of $\mathcal N_0$, the overall bacterial population asymptotically approaches zero, while the phage population sustains. Ecologically, our results show that for small values of $\mathcal N_0$, the existence of periodic solutions could explain the occurrence of repetitive bacteria-borne disease outbreaks, while large value of $\mathcal N_0$ clears bacteria from the environment. Numerical simulations support our theoretical results.

Studies on the gene regulation involved in the lytic-lysogenic switch in Staphylococcus aureus temperate bacteriophage Phi11.

Antirepressor proteins of bacteriophages are chiefly involved in interfering with the function of the repressor protein and forcing the bacteriophage to adopt the lytic cycle. The genome of Staphylococcus aureus phage, Phi11 has already been sequenced; from the genome sequence, we amplified gp07 gene and analysed its involvement in the developmental pathway of Phi11. Our results indicate that Gp07 functions as a novel antirepressor and regulates the developmental pathway of Phi11 by enhancing the binding of the Cro repressor protein to its cognate operator. We also report our finding that the CI repressor protein of Phi11 binds to the putative operator of Gp07 and regulates its expression. We further report that S.aureus transcriptional repressor LexA and coprotease RecA play a crucial role in the lytic-lysogenic switching in Phi11. We also identified that the N-terminal domain (Bro-N) of Gp07 is actually responsible for enhancing the binding of Cro repressor to its cognate operator. Our results suggest that Phi11 prophage induction is different from other bacteriophages. This study furnishes a first-hand report regarding the regulation involved in the developmental pathway of Phi11.

Temporal Dynamics of Soil Virus and Bacterial Populations in Agricultural and Early Plant Successional Soils.

As reported in many aquatic environments, recent studies in terrestrial ecosystems implicate a role for viruses in shaping the structure, function, and evolution of prokaryotic soil communities. However, given the heterogeneity of soil and the physical constraints (i.e., pore-scale hydrology and solid-phase adsorption of phage and host cells) on the mobility of viruses and bacteria, phage-host interactions likely differ from those in aquatic systems. In this study, temporal changes in the population dynamics of viruses and bacteria in soils under different land management practices were examined. The results showed that bacterial abundance was significantly and positively correlated to both virus and inducible prophage abundance. Bacterial and viral abundance were also correlated with soil organic carbon and nitrogen content as well as with C:N ratio. The seasonal variability in viral abundance increased with soil organic carbon content. The prokaryotic community structure was influenced more by land use than by seasonal variation though considerable variation was evident in the early plant successional and grassland sites. The free extracellular viral communities were also separated by land use, and the forest soil viral assemblage exhibiting the most seasonal variability was more distinct from the other sites. Viral assemblages from the agricultural soils exhibited the least seasonal variability. Similar patterns were observed for inducible prophage viral assemblages. Seasonal variability of viral assemblages was greater in mitomycin-C (mitC) induced prophages than in extracellular viruses irrespective of land use and management. Taken together, the data suggest that soil viral production and decay are likely balanced but there was clear evidence that the structure of viral assemblages is influenced by land use and by season.

Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori, which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori. Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

Quorum Sensing Signals Alter in vitro Soil Virus Abundance and Bacterial Community Composition.

Cell-density dependent quorum sensing (QS) is fundamental for many coordinated behaviors among bacteria. Most recently several studies have revealed a role for bacterial QS communication in bacteriophage (phage) reproductive decisions. However, QS based phage-host interactions remain largely unknown, with the mechanistic details revealed for only a few phage-host pairs and a dearth of information available at the microbial community level. Here we report on the specific action of eight different individual QS signals (acyl-homoserine lactones; AHLs varying in acyl-chain length from four to 14 carbon atoms) on prophage induction in soil microbial communities. We show QS autoinducers, triggered prophage induction in soil bacteria and the response was significant enough to alter bacterial community composition in vitro. AHL treatment significantly decreased the bacterial diversity (Shannon Index) but did not significantly impact species richness. Exposure to short chain-length AHLs resulted in a decrease in the abundance of different taxa than exposure to higher molecular weight AHLs. Each AHL targeted a different subset of bacterial taxa. Our observations indicate that individual AHLs may trigger prophage induction in different bacterial taxa leading to changes in microbial community structure. The findings also have implications for the role of phage-host interactions in ecologically significant processes such as biogeochemical cycles, and phage mediated transfer of host genes, e.g., photosynthesis and heavy metal/antibiotic resistance.

Release mechanisms and molecular interactions of Pseudomonas aeruginosa extracellular DNA.

Pseudomonas aeruginosa infection is a significant threat for clinicians. Increasing incidents of resistant biofilm infection result in high mortality rates worldwide. There is a considerable current interest in the field of extracellular DNA (eDNA)-mediated P. aeruginosa biofilm formation. eDNA acts as a glue to make biofilm more stable. This review focuses on the diverse mechanisms and factors, which enhance the eDNA release into the extracellular milieu. Furthermore, eDNA-mediated molecular interactions within the biofilm are emphasized. In addition, drug resistance mechanisms due to the versatility of eDNA are discussed. Spatial physiological diversity is expected due to different metabolic activity of bacterial subpopulation present in P. aeruginosa biofilm layers. In P. aeruginosa, eDNA release is accomplished by cell lysis and OMVs (outer membrane vesicles). eDNA release is a spontaneous and multifactorial process, which may be accomplished by PQS, pyocyanin, and lambda prophage induction. Hydrogen peroxide and pyocin trigger cell death, which may facilitate eDNA release. Lung mucosa of cystic fibrosis patients is enriched with eDNA, which acidifies biofilm and develops P. aeruginosa resistance to aminoglycosides. Further studies on spatial and molecular characterization of bacterial subpopulation in biofilm will shed light on eDNA-biofilm interaction more precisely.Key Points• Extracellular DNA (eDNA) is a key component of Pseudomonas aeruginosa biofilm.• P. aeruginosa eDNA acts as a glue to make biofilm more stronger.• Bacterial cell death or lysis may be the potential way to release P. aeruginosa eDNA into extracellular milieu.• P. aeruginosa eDNA contributes to develop resistance to antimicrobials.

Bacteriophage induction versus vaginal homeostasis: role of H(2)O(2) in the selection of Lactobacillus defective prophages.

Vaginal disorders associated with systemic chemotherapy arise by direct inhibition of the resident microbiota (dominated by lactobacilli) or, possibly, by induction of prophages harbored in their genomes, leading to cell lysis. In the present study, proficient Lactobacillus phages could not be isolated from vaginal exudates. However, lysogeny appeared to be widespread, although about half of the strains harbored prophage sequences that were not responsive to SOS activation. In other cases, prophage induction was achieved, but viable phages were not generated, despite the fact that the induced supernatants of some strains were bactericidal. In one case, this activity was accompanied by the production of a bacteriophage subsequently identified as a member of the family Siphoviridae (isometric capsid and long non-contractile tail). Most of the lactobacilli tested generated hydrogen peroxide, which acted as an inducer of the SOS response, suggesting that H2O2 selects for strains that harbor SOS-insensitive, defective prophages, which are thus unable to promote vaginal lactobacilli phage-induced lysis.

A Novel Inducible Prophage from Burkholderia Vietnamiensis G4 is Widely Distributed across the Species and Has Lytic Activity against Pathogenic Burkholderia.

Burkholderia species have environmental, industrial and medical significance, and are important opportunistic pathogens in individuals with cystic fibrosis (CF). Using a combination of existing and newly determined genome sequences, this study investigated prophage carriage across the species B. vietnamiensis, and also isolated spontaneously inducible prophages from a reference strain, G4. Eighty-one B. vietnamiensis genomes were bioinformatically screened for prophages using PHASTER (Phage Search Tool Enhanced Release) and prophage regions were found to comprise up to 3.4% of total genetic material. Overall, 115 intact prophages were identified and there was evidence of polylysogeny in 32 strains. A novel, inducible Mu-like phage (vB_BvM-G4P1) was isolated from B. vietnamiensis G4 that had lytic activity against strains of five Burkholderia species prevalent in CF infections, including the Boston epidemic B. dolosa strain SLC6. The cognate prophage to vB_BvM-G4P1 was identified in the lysogen genome and was almost identical (>93.5% tblastx identity) to prophages found in 13 other B. vietnamiensis strains (17% of the strain collection). Phylogenomic analysis determined that the G4P1-like prophages were widely distributed across the population structure of B. vietnamiensis. This study highlights how genomic characterization of Burkholderia prophages can lead to the discovery of novel bacteriophages with potential therapeutic or biotechnological applications.