Lysogenic Bacteria Research Articles

YMC-2011, a Temperate Phage of Streptococcus salivarius 57.I.

Streptococcus salivarius is an abundant isolate of the oral cavity. The genome of S. salivarius 57.I consists of a 2-Mb chromosome and a 40,758-bp circular molecule, designated YMC-2011. Annotation of YMC-2011 revealed 55 open reading frames, most of them associated with phage production, although plaque formation is not observed in S. salivarius 57.I after lytic induction using mitomycin C. Results from Southern hybridization and quantitative real-time PCR confirmed that YMC-2011 exists extrachromosomally, with an estimated copy number of 3 to 4. Phage particles were isolated from the supernatant of mitomycin C-treated S. salivarius 57.I cultures, and transmission electron microscopic examination indicated that YMC-2011 belongs to the Siphoviridae family. Phylogenetic analysis suggests that phage YMC-2011 and the cos-type phages of Streptococcus thermophilus originated from a common ancestor. An extended -10 element (p L ) and a σ70-like promoter (p R ) were mapped 5' to Ssal_phage00013 (encoding a CI-like repressor) and Ssal_phage00014 (encoding a hypothetical protein), respectively, using 5' rapid amplification of cDNA ends, indicating that YMC-2011 transcribes at least two mRNAs in opposite orientations. Studies using promoter-chloramphenicol acetyltransferase reporter gene fusions revealed that p R , but not p L , was sensitive to mitomycin C induction, suggesting that the switch from lysogenic growth to lytic growth was controlled mainly by the activity of these two promoters. In conclusion, a lysogenic state is maintained in S. salivarius 57.I, presumably by the repression of genes encoding proteins for lytic growth.IMPORTANCE The movement of mobile genetic elements such as bacteriophages and the establishment of lysogens may have profound effects on the balance of microbial ecology where lysogenic bacteria reside. The discovery of phage YMC-2011 from Streptococcus salivarius 57.I suggests that YMC-2011 and Streptococcus thermophilus-infecting phages share an ancestor. Although S. salivarius and S. thermophilus are close phylogenetically, S. salivarius is a natural inhabitant of the human mouth, whereas S. thermophilus is commonly found in the mammary mucosa of bovine species. Thus, the identification of YMC-2011 suggests that horizontal gene transfer via phage infection could take place between species from different ecological niches.

Inducible Prophage Mutant of Escherichia coli Can Lyse New Host and the Key Sites of Receptor Recognition Identification.

The use of bacteriophages as therapeutic agents is hindered by their narrow and specific host range, and by a lack of the knowledge concerning the molecular mechanism of receptor recognition. Two P2-like coliphages, named P88 and pro147, were induced from Escherichia coli strains K88 and DE147, respectively. A comparison of the genomes of these two and other P2-like coliphages obtained from GenBank showed that the tail fiber protein genes, which are the key genes for receptor recognition in other myoviridae phages, showed more diversity than the conserved lysin, replicase, and terminase genes. Firstly, replacing hypervariable region 2 (HR2: amino acids 716–746) of the tail fiber protein of P88 with that of pro147 changed the host range of P88. Then, replacing six amino acids in HR2 with the corresponding residues from pro147 altered the host range only in these mutants with changes at position 730 (leucine) and 744 (glutamic acid). Thus, we predicted that these amino acids are vital to establish the host range of P88. This study provided a vector of lysogenic bacteria that could be used to change or expand the phage host range of P88. These results illustrated that, in P2-like phage P88, the tail fiber protein determined the receptor recognition. Amino acids 716–746 and the amino acids at positions 730 and 744 were important for receptor recognition.

Identifying the lysogenic bacteria producing endolysin from Semnan soil

Identifying the lysogenic bacteria producing endolysin from Semnan soil

Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli.

Virulence of enterohemorrhagic Escherichia coli (EHEC) strains depends on production of Shiga toxins. These toxins are encoded in genomes of lambdoid bacteriophages (Shiga toxin-converting phages), present in EHEC cells as prophages. The genes coding for Shiga toxins are silent in lysogenic bacteria, and prophage induction is necessary for their efficient expression and toxin production. Under laboratory conditions, treatment with UV light or antibiotics interfering with DNA replication are commonly used to induce lambdoid prophages. Since such conditions are unlikely to occur in human intestine, various research groups searched for other factors or agents that might induce Shiga toxin-converting prophages. Among other conditions, it was reported that treatment with H2O2 caused induction of these prophages, though with efficiency significantly lower relative to UV-irradiation or mitomycin C treatment. A molecular mechanism of this phenomenon has been proposed. It appears that the oxidative stress represents natural conditions provoking induction of Shiga toxin-converting prophages as a consequence of H2O2 excretion by either neutrophils in infected humans or protist predators outside human body. Finally, the recently proposed biological role of Shiga toxin production is described in this paper, and the “bacterial altruism” and “Trojan Horse” hypotheses, which are connected to the oxidative stress, are discussed.

The Role of the Exo-Xis Region in Oxidative Stress-Mediated Induction of Shiga Toxin-Converting Prophages.

Previous studies indicated that these genetic elements could be involved in the regulation of lysogenization and prophage induction processes. The effects were dramatic in Shiga toxin-converting phage Φ24B after treatment with oxidative stress-inducing agent, hydrogen peroxide, while they were less pronounced in bacteriophage λ and in both phages irradiated with UV. The hydrogen peroxide-caused prophage induction was found to be RecA-dependent. Importantly, in hydrogen peroxide-treated E. coli cells lysogenic for either λ or Φ24B, deletion of the exo-xis region resulted in a significant decrease in the levels of expression of the S.O.S. regulon genes. Moreover, under these conditions, a dramatic decrease in the levels of expression of phage genes crucial for lytic development (particularly xis, exo, N, cro, O, Q, and R) could be observed in Φ24B-, but not in λ-bearing cells. We conclude that genes located in the exo-xis region are necessary for efficient expression of both host S.O.S regulon in lysogenic bacteria and regulatory genes of Shiga toxin-converting bacteriophage Φ24B.

Effects of antitumor derivatives of ineffective transplatin on bacterial cells: Is DNA a pharmacological target?

The effects of the two representatives of the antitumor transplatinum agents, trans-[PtCl2(methylamine)2] and trans-[PtCl2(NH3)(1-methyl-7-azaindole)] on bacterial growth were examined. The results show that these antitumor transplatinum agents can be grouped with the coordination Pt(II) compounds exhibiting antitumor activity and capable of inducing bacterial filamentation and initiate lysis in lysogenic bacteria. The results corroborate the thesis that DNA is the potential cellular target also for a class of antitumor derivatives of transplatin.

Effects of monofunctional platinum agents on bacterial growth: a retrospective study.

The effect of the novel and potent monofunctional platinum(II) agent phenanthriplatin on Escherichia coli and bacteriophage λ lysogens is reported. E. coli filamentation was observed by light microscopy when cells were grown in the presence of phenanthriplatin, cis-[Pt(NH3)2(Am)Cl](+) where Am is phenanthridine. Treatment of lysogenic bacteria with this compound resulted in lysis and the production of viral particles, as indicated by plaque formation in a bacterial lawn. The results obtained with phenanthriplatin are contextualized by comparison with those obtained using cisplatin as well as other, less active, monofunctional compounds such as [Pt(NH3)3Cl](+) and cis-[Pt(NH3)2(py)Cl](+), where py is pyridine. The ability of phenanthriplatin to induce bacterial filamentation and initiate lysis in lysogenic bacteria corroborates the hypothesis that the biological activity of this complex is mediated by its interaction with DNA.

To kill or not to kill: The balance between lytic and lysogenic viral infection is driven by trophic status

Experiments were conducted to investigate spatiotemporal patterns in lytic and lysogenic viral infection using water samples collected on the Canadian Arctic Shelf, southern Beaufort Sea. Viral production (VP) and viral‐induced mortality of bacteria (VMB) were determined using a viral reduction approach during a full seasonal cycle, while the percentage of lysogenic bacteria (PLB) in spring and summer was determined in virus‐reduced samples by induction with mitomycin C. Overall, VP (range: 0.3 × 108‐77 × 108 viruses L−1 d−1), VMB (range: 0.2 × 107‐43 × 107 bacteria L−1 d−1), and PLB (range: 4–38%) displayed marked spatiotemporal variations concomitant with changes in chlorophyll a, bacterial abundance, and production. Highest VP and VMB occurred in summer when the water was warmest, stratified, and most productive, and when viruses removed up to 29% of bacterial standing stock d−1 and released up to 4.3 µg of organic carbon L−1 d−1. In contrast, the highest PLB occurred in spring when the water was colder, well mixed, and oligotrophic. Correlative and regression analyses indicated viral lytic and lysogenic variables were significantly coupled with chlorophyll a and the abundance, production, and growth rate of bacteria, implying that viral lytic and lysogenic lifestyles were dependent on system productivity. Furthermore, lytic VP and the proportion of lysogenized bacteria were inversely related, suggesting a dynamic interplay between viral infection pathways. Lytic infection was more pronounced when system productivity was high, while lysogeny prevailed when system productivity was low. These data demonstrate the important role of viruses in bacterial mortality and carbon cycling in the Arctic Ocean, and show how their effect is influenced by trophic status.

Titanium dioxide-assisted photocatalytic induction of prophages to lytic cycle

The investigations on the kinetics of photocatalytic inactivation of bacteriophages, lactic bacteria and lysogenic lactic bacteria have shown that the rate of bacterial inactivation is ca. 10 times less than the inactivation of bacteriophages. Titania-assisted photorelease of bacteriophages from lysogenic bacteria proves that photogenerated reactive oxygen species affect the deoxyribonucleic acid (DNA) of bacteria before their deactivation. On this basis a novel photocatalytic method of a prophage induction to the lytic cycle and detection of lysogenic bacteria is proposed.

Changing phosphorus concentration and subsequent prophage induction alter composition of a freshwater viral assemblage

Summary1. The effects of nutrients on the temporal variation in viral assemblage composition, and in particular the occurrence of temperate phages, were assessed in mesotrophic Lake Erken over 5 months of the ice‐free period. The percentage of the bacterial community that contained inducible prophages (lysogenic bacteria, LB) changed over the season, being lowest in late spring and highest in early autumn. The most important variables for predicting LB were concentrations of total nitrogen (TN), total phosphorus (TP) and temperature.2. The viral assemblage composition, as determined by pulsed‐field gel electrophoresis (PFGE), also changed over the season. Prophages were induced by incubations with mitomycin C and we show, for the first time for natural communities, that the resulting temperate phages could be detected using PFGE.3. A substantial fraction (19%) of the number of detected operational taxonomic units (OTUs: defined as unique genome sizes) appeared unique to temperate phages and 41% of OTUs increased in relative abundance after treatment with mitomycin C.4. Different viral OTUs were induced at different times during the season. The most important environmental factor covarying with viral assemblage composition over the period of study, as determined by multivariate analysis, was concentration of TP. In re‐growth cultures with natural bacteria and lowered viral abundance (VA) (decreased virus to bacteria ratio), addition of PO4‐P induced prophages and resulted in subsequent production of temperate phages, as indicated by a decreased percentage of LB and increased VA. Incubations of natural bacterial communities with mitomycin C (field data) or PO4‐P (experiment) changed the viral assemblage composition at a similar rate as the observed monthly changes in the lake.

Acyl-Homoserine Lactones Can Induce Virus Production in Lysogenic Bacteria: an Alternative Paradigm for Prophage Induction

Prophage typically are induced to a lytic cycle under stressful environmental conditions or when the host's survival is threatened. However, stress-independent, spontaneous induction also occurs in nature and may be cell density dependent, but the in vivo signal(s) that can trigger induction is unknown. In the present study, we report that acyl-homoserine lactones (AHL), the essential signaling molecules of quorum sensing in many gram-negative bacteria, can trigger phage production in soil and groundwater bacteria. This phenomenon also was operative in a lambda lysogen of Escherichia coli. In model coculture systems, we monitored the real-time AHL production from Pseudomonas aeruginosa PAO1 using an AHL bioluminescent sensor and demonstrated that lambda-prophage induction in E. coli was correlated with AHL production. As a working model in E. coli, we show that the induction responses of lambda with AHL remained unaffected when recA was deleted, suggesting that this mechanism does not involve an SOS response. In the same lambda lysogen we also demonstrated that sdiA, the AHL receptor, and rcsA, a positive transcriptional regulator of exopolysaccharide synthesis, are involved in the AHL-mediated induction process. These findings relate viral reproduction to chemical signals associated with high host cell abundance, suggesting an alternative paradigm for prophage induction.

Mutant Bacteriophages, Frank Macfarlane Burnet, and the Changing Nature of “Genespeak” in the 1930s

In 1936, Frank Macfarlane Burnet published a paper entitled "Induced lysogenicity and the mutation of bacteriophage within lysogenic bacteria," in which he demonstrated that the introduction of a specific bacteriophage into a bacterial strain consistently and repeatedly imparted a specific property - namely the resistance to a different phage - to the bacterial strain that was originally susceptible to lysis by that second phage. Burnet's explanation for this change was that the first phage was causing a mutation in the bacterium which rendered it and its successive generations of offspring resistant to lysogenicity. At the time, this idea was a novel one that needed compelling evidence to be accepted. While it is difficult for us today to conceive of mutations and genes outside the context of DNA as the physico-chemical basis of genes, in the mid 1930s, when this paper was published, DNA's role as the carrier of hereditary information had not yet been discovered and genes and mutations were yet to acquire physical and chemical forms. Also, during that time genes were considered to exist only in organisms capable of sexual modes of replication and the status of bacteria and viruses as organisms capable of containing genes and manifesting mutations was still in question. Burnet's paper counts among those pieces of work that helped dispel the notion that genes, inheritance and mutations were tied to an organism's sexual status. In this paper, I analyze the implications of Burnet's paper for the understanding of various concepts - such as "mutation," and "gene," - at the time it was published, and how those understandings shaped the development of the meanings of these terms and our modern conceptions thereof.

Bacteriophages induced from lysogenic root canal isolates of Enterococcus faecalis

Bacterial viruses play crucial roles in the pathogenesis of many systemic diseases. They are known to inhabit the oral cavity, both as free virions and as prophages in lysogenic bacterial strains; however, there has been no report of bacteriophages in endodontic infections. In this study, we sought to detect, isolate, and describe temperate bacteriophages harbored by Enterococcus faecalis strains isolated from endodontic infections. Ten E. faecalis strains were isolated from root canals of teeth undergoing retreatment following unsuccessful endodontic therapy. Mitomycin C was used to induce any prophages present in the bacterial isolates. The induced phages were purified and examined using electron microscopy. The DNA extracted from one of the phage isolates was subjected to restriction endonuclease digestion and agarose electrophoresis analysis. Lysogeny was demonstrated in 4 of the 10 E. faecalis strains. Three of the lysogenic strains yielded phages exhibiting a Siphoviridae morphology, with long, non-contractile tails 130 nm in length, and spherical/icosahedral heads 41 nm in diameter. The virus induced from the fourth lysogenic E. faecalis strain had a contractile tail characteristic of Myoviridae. Restriction endonuclease analysis of NsiI and NdeI DNA fragments from one of the Siphoviridae phage isolates (phage phiEf11) indicated a genome size of approximately 41 kbp. This is the first report of lysogenic bacteria and their inducible viruses in infected root canals.

Online program ‘vipcal’ for calculating lytic viral production and lysogenic cells based on a viral reduction approach

Assessing viral production (VP) requires robust methodological settings combined with precise mathematical calculations. This contribution improves and standardizes mathematical calculations of VP and the assessment of the proportion of lysogenic cells in a sample. We present an online tool 'Viral Production Calculator' (vipcal, http://www.univie.ac.at/nuhag-php/vipcal) that calculates lytic production and the percentage of lysogenic cells based on data obtained from a viral reduction approach (VRA). The main advantage of our method lies in its universal applicability, even to different piecewise-linear curves. We demonstrate the application of our tool for calculating lytic VP and the proportion of lysogenic bacteria in an environmental sample. The program can also be used to calculate different parameters for estimating virus-induced mortality, including the percentage of lytically infected cells, lysis rate of bacteria, percentage of bacterial production lysed, proportion of bacterial loss per day, viral turnover time as well as dissolved organic carbon and nitrogen release. vipcal helps avoid differences in the calculation of VP and diverse viral parameters between studies and laboratories, which facilities interpretation of results. This tool represents a methodological step forward that can help improve our understanding of the role of viral activity in aquatic systems.

Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity

Bacteriophages and protists are major causes of bacterial mortality. Genomics suggests that phages evolved well before eukaryotic protists. Bacteria were thus initially only confronted with phage predators. When protists evolved, bacteria were caught between two types of predators. One successful antigrazing strategy of bacteria was the elaboration of toxins that would kill the grazer. The released cell content would feed bystander bacteria. I suggest here that, to fight grazing protists, bacteria teamed up with those phage predators that concluded at least a temporary truce with them in the form of lysogeny. Lysogeny was perhaps initially a resource management strategy of phages that could not maintain infection chains. Subsequently, lysogeny might have evolved into a bacterium-prophage coalition attacking protists, which became a food source for them. When protists evolved into multicellular animals, the lysogenic bacteria tracked their evolving food source. This hypothesis could explain why a frequent scheme of bacterial pathogenicity is the survival in phagocytes, why a significant fraction of bacterial pathogens have prophage-encoded virulence genes, and why some virulence factors of animal pathogens are active against unicellular eukaryotes. Bacterial pathogenicity might thus be one playing option of the stone-scissor-paper game played between phages-bacteria-protists, with humans getting into the crossfire.

Heterotrophic bacterial and viral dynamics in Arctic freshwaters: results from a field study and nutrient-temperature manipulation experiments

Heterotrophic bacterial and viral concentrations (range, 0.7 × 104 to 206.2 × 104 ml−1 and 0.05 × 106 to 128.9 × 106 ml−1, respectively) were determined in several Arctic freshwater environments, including lakes and glacial ecosystems (78.55°N, 11.56°E). Our bacteria and virus results mirrored trends seen in temperate lakes, with an average virus-to-bacteria ratio (VBR) of 13 (range, 7.3–25.2) and viral concentrations and DOC positively correlated with bacterial concentrations (R = 0.964, P < 0.01 and R = 0.813, P < 0.05, respectively). Lysogenic bacteria, determined by induction with Mitomycin C, were not detected in any of the investigated Arctic freshwater environments. Nutrient-addition experiments at in situ and at elevated temperatures were performed to elucidate the factors which influenced the bacterial growth and the virus–bacteria interactions in Arctic freshwaters. Our results suggest that multiple limiting factors interacted and constrained bacterial growth. Bacterial concentrations and doubling times increased at elevated temperatures and appeared to be co-stimulated by phosphorus and carbon. However, viral concentrations showed a lack of response to nutrient addition thus indicating an uncoupling between bacteria and viruses in the experiment.

Seasonal Viral Loop Dynamics in Two Large Ultraoligotrophic Antarctic Freshwater Lakes

The effect of viruses on the microbial loop, with particular emphasis on bacteria, was investigated over an annual cycle in 2003-2004 in Lake Druzhby and Crooked Lake, two large ultraoligotrophic freshwater lakes in the Vestfold Hills, Eastern Antarctica. Viral abundance ranged from 0.16 to 1.56 x 10(9) particles L-1 and bacterial abundances ranged from 0.10 to 0.24 x 10(9) cells L-1, with the lowest bacterial abundances noted in the winter months. Virus-to-bacteria ratios (VBR) were consistently low in both lakes throughout the season, ranging from 1.2 to 8.4. lysogenic bacteria, determined by induction with mitomycin C, were detected on three sampling occasions out of 10 in both lakes. In Lake Druzhby and Crooked Lake, lysogenic bacteria made up between 18% and 73% of the total bacteria population during the lysogenic events. Bacterial production ranged from 8.2 to 304.9 x 10(6) cells L-1 day-1 and lytic viral production ranged from 47.5 to 718.4 x 10(6) viruslike particles L-1 day-1. When only considering primary production, heterotrophic nanoflagellate (HNF) grazing and viral lysis as the major contributors to the DOC pool (i.e., autochthonous sources), we estimated a high contribution from viruses during the winter months when >60% of the carbon supplied to the DOC pool originated from viral lysis. In contrast, during the summer <20% originated from viral lysis. Our study shows that viral process in ultraoligotrophic Antarctic lakes may be of quantitative significance with respect to carbon flow especially during the dark winter period.

Virus-Bacterium Interactions in Water and Sediment of West African Inland Aquatic Systems

The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virus-bacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water.

A novel method for evaluating free radical scavenging abilities of antioxidants using ultraviolet induction of bacteriophage λ

A novel biological method used to evaluate free radical scavenging abilities of antioxidants using ultraviolet (UV) induction of bacteriophage λ in lysogenic Escherichia coli κ12 (λ +) has been developed. This method is based on the induction of bacteriophage λ from lysogenic cycle to lytic cycle by ultraviolet irradiation, and formation of free radicals during the course of induction. In the experiments, 10 8 cells/ml and 30 s (39 J/m 2) were determined as the cell density of the lysogenic bacterium and UV irradiation time, respectively. The reliability of this method was demonstrated by electron paramagnetic resonance (EPR) spectroscopy and spin trapping with DMPO. This method had good reproducibility with intra-day variations (RSD, %) of less than 4% and inter-day variations (RSD, %) of less than 5%, respectively. By this method, the free radical scavenging abilities (ID 50) of well-known antioxidants such as glutathione, superoxide dismutase (SOD), catalase (CAT) and carotenoids were determined quantitatively. The results were consistent with the ones obtained by conventional methods for evaluating free radical scavenging abilities. This developed method is reliable and uses common instruments and inexpensive, stable reagents, thus it could be utilized as a routine laboratory quantitative assay to screen a large number of substances that have potential to scavenge the free radical.

The impact of prophages on bacterial chromosomes.

Prophages were automatically localized in sequenced bacterial genomes by a simple semantic script leading to the identification of 190 prophages in 115 investigated genomes. The distribution of prophages with respect to presence or absence in a given bacterial species, the location and orientation of the prophages on the replichore was not homogeneous. In bacterial pathogens, prophages are particularly prominent. They frequently encoded virulence genes and were major contributors to the genetic individuality of the strains. However, some commensal and free-living bacteria also showed prominent prophage contributions to the bacterial genomes. Lysogens containing multiple sequence-related prophages can experience rearrangements of the bacterial genome across prophages, leading to prophages with new gene constellations. Transfer RNA genes are the preferred chromosomal integration sites, and a number of prophages also carry tRNA genes. Prophage integration into protein coding sequences can lead to either gene disruption or new proteins. The phage repressor, immunity and lysogenic conversion genes are frequently transcribed from the prophage. The expression of the latter is sometimes integrated into control circuits linking prophages, the lysogenic bacterium and its animal host. Prophages are apparently as easily acquired as they are lost from the bacterial chromosome. Fixation of prophage genes seems to be restricted to those with functions that have been co-opted by the bacterial host.