Cultivation-Based Assessment of Lysogeny Among Soil Bacteria

Lysogeny has long been proposed as an important long-term maintenance strategy for autochthonous soil bacteriophages (phages). Whole genome sequence data indicate that prophage-derived sequences pervade prokaryotic genomes, but the connection between inferred prophage sequence and an active temperate phage is tenuous. Thus, definitive evidence of phage production from lysogenic prokaryotes will be critical in determining the presence and extent of temperate phage diversity existing as prophage within bacterial genomes and within environmental contexts such as soils. This study optimized methods for systematic and definitive determination of lysogeny within a collection of autochthonous soil bacteria. Twenty bacterial isolates from a range of Delaware soil environments (five from each soil) were treated with the inducing agents mitomycin C (MC) or UV light. Six isolates (30%) carried inducible temperate phages as evidenced by an increase in virus direct counts. The magnitude of induction response was highly dependent upon specific induction conditions, and corresponding burst sizes ranged from 1 to 176. Treatment with MC for 30 min yielded the largest induction responses for three of the six lysogens. Morphological analysis revealed that four of the lysogens produced lambda-like Siphoviridae particles, whereas two produced Myoviridae particles. Additionally, pulsed-field gel electrophoresis data indicated that two of the six lysogens were polylysogens, producing more than one distinct type of phage particle. These results suggest that lysogeny is relatively common among soil bacteria.     

Prevalence of Lysogeny among Soil Bacteria and Presence of 16S rRNA and trzN Genes in Viral-Community DNA

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.     

Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage phi LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six phi LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30 degrees C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the phi LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the phi LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.     

Seasonal time bombs: dominant temperate viruses affect Southern Ocean microbial dynamics

Rapid warming in the highly productive western Antarctic Peninsula (WAP) region of the Southern Ocean has affected multiple trophic levels, yet viral influences on microbial processes and ecosystem function remain understudied in the Southern Ocean. Here we use cultivation-independent quantitative ecological and metagenomic assays, combined with new comparative bioinformatic techniques, to investigate double-stranded DNA viruses during the WAP spring–summer transition. This study demonstrates that (i) temperate viruses dominate this region, switching from lysogeny to lytic replication as bacterial production increases, and (ii) Southern Ocean viral assemblages are genetically distinct from lower-latitude assemblages, primarily driven by this temperate viral dominance. This new information suggests fundamentally different virus–host interactions in polar environments, where intense seasonal changes in bacterial production select for temperate viruses because of increased fitness imparted by the ability to switch replication strategies in response to resource availability. Further, temperate viral dominance may provide mechanisms (for example, bacterial mortality resulting from prophage induction) that help explain observed temporal delays between, and lower ratios of, bacterial and primary production in polar versus lower-latitude marine ecosystems. Together these results suggest that temperate virus–host interactions are critical to predicting changes in microbial dynamics brought on by warming in polar marine systems.     

Lysogeny and lytic viral production during a bloom of the cyanobacterium Synechococcus spp

Lytic viral production and lysogeny were investigated in cyanobacteria and heterotrophic bacteria during a bloom of Synechococcus spp. in a pristine fjord in British Columbia, Canada. Triplicate seawater samples were incubated with and without mitomycin C and the abundances of heterotrophic bacteria, cyanobacteria, total viruses and infectious cyanophage were followed over 24 h. Addition of mitomycin C led to increases in total viral abundance as well as the abundance of cyanophages infecting Synechococcus strain DC2. Given typical estimates of burst size, these increases were consistent with 80% of the heterotrophic bacteria and 0.6% of Synechococcus cells being inducible by the addition of mitomycin C. This is the highest percentage of lysogens reported for a natural microbial community and demonstrates induction in a marine Synechococcus population. It is likely that the cyanophage production following the addition of mitomycin C was much higher than that titered against a single strain of Synechococcus; hence this estimate is a minimum. In untreated seawater samples, lytic viral production was estimated to remove ca. 27% of the gross heterotrophic bacterial production, and a minimum of 1.0% of the gross cyanobacterial production. Our results demonstrate very high levels of lysogeny in the heterotrophic bacterial community, outside of an oligotrophic environment, and the presence of inducible lysogens in Synechococcus spp. during a naturally occurring bloom. These data emphasize the need for further examination of the factors influencing lytic and lysogenic viral infection in natural microbial communities.     

Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.     

Effect of petrochemical sludge concentrations on microbial communities during soil bioremediation

Qualitative and quantitative changes of microbial communities in soil microcosms during bioremediation were determined throughout one year. The soil was contaminated with 0%, 2.5%, 5%, 10% (wt/wt) of petrochemical sludge containing polynuclear aromatic hydrocarbons. We analyzed the hydrocarbon concentration in the microcosms, the number of cultivable bacteria using CFU and most probable number assays, the community structure using denaturing gradient gel electrophoresis, and the metabolic activity of soil using dehydrogenase activity and substrate-induced respiration assays. After one year of treatment, the chemical analysis suggested that the hydrocarbon elimination process was over. The biological analysis, however, showed that the contaminated microcosms suffered under long-term disturbance. The number of heterotrophic bacteria that increased after sludge addition (up to 10(8)-10(9) cells ml(-1)) has not returned to the level of the control soil (2-6 x 10(7) cells ml(-1)). The community structure in the contaminated soils differed considerably from that in the control. The substrate-induced respiration of the contaminated soils was significantly lower (approximately 10-fold) and the dehydrogenase activity was significantly higher (20-40-fold) compared to the control. Changes in the community structure of soils depended on the amount of added sludge. The species, which were predominant in the sludge community, could not be detected in the contaminated soils.     

Seasonal variation in lysogeny as depicted by prophage induction in Tampa Bay,Florida

A seasonal study of the distribution of lysogenic bacteria in Tampa Bay, Florida, was conducted over a 13-month period. Biweekly water samples were collected and either were left unaltered or had the viral population reduced by filtration (pore size, 0.2 micro m) and resuspension in filtered (pore size, 0.2 micro m) water. Virus-reduced and unaltered samples were then treated by adding mitomycin C (0.5 micro g ml(-1)) to induce prophage or were left untreated. In order to test the hypothesis that prophage induction was phosphate limited, additional induction experiments were performed in the presence and absence of phosphate. Induction was assessed as an increase in viral direct counts, relative to those obtained in controls, as detected by epifluorescence microscopy. Induction of prophage was observed in 5 of 25 (20%) unaltered samples which were obtained during or after the month of February, paralleling the results from a previous seasonal study. Induction of prophage was observed in 9 of 25 (36%) of the virus-reduced samples, primarily those obtained in the winter months, which was not observed in a prior seasonal study (P. K. Cochran and J. H. Paul, Appl. Environ. Microbiol. 64:2308-2312, 1998). Induction was noted in the months of lowest bacterial and primary production, suggesting that lysogeny was favored under conditions of poor host growth. Phosphate addition enabled prophage induction in two of nine (22%) experiments. These results indicate that prophage induction may occasionally be phosphate limited or respond to increases in phosphate concentration, suggesting that phosphate concentration may modulate the lysogenic response of natural populations.     

Disentangling the relative influence of bacterioplankton phylogeny and metabolism on lysogeny in reservoirs and lagoons

Previous studies indicate that lysogeny is preponderant when environmental conditions are challenging for the bacterial communities and when their metabolism is reduced. Furthermore, it appears that lysogeny is more frequent within certain bacterial phylogenetic groups. In this comparative study from 10 freshwater reservoirs and 10 coastal lagoons, we aim to disentangle the influence of these different factors. In eight reservoirs and four lagoons, lysogeny was detected by induction assays with mitomycin C, and induction significantly modified the bacterial community composition (BCC), whereas community composition remained constant in ecosystems in which lysogeny was not observed. Among the phylogenetic groups studied, the most abundant ones were Bacteroidetes and α-proteobacteria in lagoons, and β-proteobacteria and Bacteroidetes in reservoirs. These dominant groups comprised the highest proportions of inducible lysogens. In order to unravel the effects of bacterial metabolism from phylogeny on lysogeny, we measured bacterial community physiology and the specific activities of selected phylogenetic groups. The proportion of inducible lysogens within the α- and the β-proteobacteria decreased with increasing group-specific metabolism in lagoons and reservoirs, respectively. In contrast, this relationship was not observed for the other lysogen-containing groups. Hence, both host physiology and phylogeny are critical for the establishment of lysogeny. This study illustrates the importance of lysogeny among the most abundant phylogenetic groups, and further suggests its strong structuring impact on BCC.     

Metagenomic analysis of lysogeny in Tampa Bay: implications for prophage gene expression

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host's replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e < or =0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9 x 10(7), and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L(-1) respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L(-1). Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L(-1). In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny.     

Lysogeny in the oceans: Lessons from cultivated model systems and a reanalysis of its prevalence

In the oceans, viruses that infect bacteria (phages) influence a variety of microbially mediated processes that drive global biogeochemical cycles. The nature of their influence is dependent upon infection mode, be it lytic or lysogenic. Temperate phages are predicted to be prevalent in marine systems where they are expected to execute both types of infection modes. Understanding the range and outcomes of temperate phage–host interactions is fundamental for evaluating their ecological impact. Here, we (i) review phage-mediated rewiring of host metabolism, with a focus on marine systems, (ii) consider the range and nature of temperate phage–host interactions, and (iii) draw on studies of cultivated model systems to examine the consequences of lysogeny among several dominant marine bacterial lineages. We also readdress the prevalence of lysogeny among marine bacteria by probing a collection of 1239 publicly available bacterial genomes, representing cultured and uncultivated strains, for evidence of complete prophages. Our conservative analysis, anticipated to underestimate true prevalence, predicts 18% of the genomes examined contain at least one prophage, the majority (97%) were found within genomes of cultured isolates. These results highlight the need for cultivation of additional model systems to better capture the diversity of temperate phage–host interactions in the oceans.     

Structure of microbial communities in red ferralitic soils of Varadero National Park (Matanzas,Cuba)

Comparative study of microbial communities in red ferralitic soil, as well as tree waste and phylloplane of woody plants, of Varadero National Park (Cuba) has been performed. It is shown that the total bacterial abundance and the length of the actinomycete mycelium in the studied soil samples (A horizon) are comparable to and the length of the viable fungal mycelium is lower than the analogous parameters recorded in forest soils of the temperate zone. It is noted that the viability of bacteria is close to that in forest soils of the temperate zone. The maximum concentration of soil biota is found in the A horizon of the studied soils, in contrast to forest biogeocenoses of the temperate zone, where soil biota is concentrated in the litter. It is shown for the first time that prokaryote communities are characterized by a significant presence of filterable forms of bacteria, the content of which increases in the series: soil (A horizon) → tree waste (dry leaves) → phylloplane (green leaves).     

Comparing temperature sensitivity of bacterial growth in Antarctic marine water and soil

The western Antarctic Peninsula is an extreme low temperature environment that is warming rapidly due to global change. Little is known, however, on the temperature sensitivity of growth of microbial communities in Antarctic soils and in the surrounding oceanic waters. This is the first study that directly compares temperature adaptation of adjacent marine and terrestrial bacteria in a polar environment. The bacterial communities in the ocean were adapted to lower temperatures than those from nearby soil, with cardinal temperatures for growth in the ocean being the lowest so far reported for microbial communities. This was reflected in lower minimum (T_min) and optimum temperatures (T_opt) for growth in water (?17 and +20°C, respectively) than in soil (?11 and +27°C), with lower sensitivity to changes in temperature (Q₁₀; 0–10°C interval) in Antarctic water (2.7) than in soil (3.9). This is likely due to the more stable low temperature conditions of Antarctic waters than soils, and the fact that maximum in situ temperatures in water are lower than in soils, at least in summer. Importantly, the thermally stable environment of Antarctic marine water makes it feasible to create a single temperature response curve for bacterial communities. This would thus allow for calculations of temperature‐corrected growth rates, and thereby quantifying the influence of factors other than temperature on observed growth rates, as well as predicting the effects of future temperature increases on Antarctic marine bacteria.     

Efficiency of induction of prophage lambda mutants as a function of recA alleles.

Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.     

Seasonal Variation in Lysogeny as Depicted by Prophage Induction in Tampa Bay, Florida

A seasonal study of the distribution of lysogenic bacteria in Tampa Bay, Florida, was conducted over a 13-month period. Biweekly water samples were collected and either were left unaltered or had the viral population reduced by filtration (pore size, 0.2 μm) and resuspension in filtered (pore size, 0.2 μm) water. Virus-reduced and unaltered samples were then treated by adding mitomycin C (0.5 μg ml⁻¹) to induce prophage or were left untreated. In order to test the hypothesis that prophage induction was phosphate limited, additional induction experiments were performed in the presence and absence of phosphate. Induction was assessed as an increase in viral direct counts, relative to those obtained in controls, as detected by epifluorescence microscopy. Induction of prophage was observed in 5 of 25 (20%) unaltered samples which were obtained during or after the month of February, paralleling the results from a previous seasonal study. Induction of prophage was observed in 9 of 25 (36%) of the virus-reduced samples, primarily those obtained in the winter months, which was not observed in a prior seasonal study (P. K. Cochran and J. H. Paul, Appl. Environ. Microbiol. 64:2308-2312, 1998). Induction was noted in the months of lowest bacterial and primary production, suggesting that lysogeny was favored under conditions of poor host growth. Phosphate addition enabled prophage induction in two of nine (22%) experiments. These results indicate that prophage induction may occasionally be phosphate limited or respond to increases in phosphate concentration, suggesting that phosphate concentration may modulate the lysogenic response of natural populations.     

Bacterial Diversity of Terrestrial Crystalline Volcanic Rocks, Iceland

Bacteria inhabiting crystalline rocks from two terrestrial Icelandic volcanic lava flows of similar age and from the same geographical region, but differing in porosity and mineralogy, were characterised. Microarray (PhyloChip) and clone library analysis of 16S rRNA genes revealed the presence of a diverse assemblage of bacteria in each lava flow. Both methods suggested a more diverse community at the Dómadalshraun site (rhyolitic/andesitic lava flow) than that present at the Hnausahraun site (basaltic lava flow). Proteobacteria dominated the clone library at the Dómadalshraun site, while Acidobacteria was the most abundant phylum in the Hnausahraun site. Although analysis of similarities of denaturing gradient gel electrophoresis profiles suggested a strong correlation of community structure with mineralogy, rock porosity may also play an important role in shaping the bacterial community in crystalline volcanic rocks. Clone sequences were most similar to uncultured microorganisms, mainly from soil environments. Of these, Antarctic soils and temperate rhizosphere soils were prominent, as were clones retrieved from Hawaiian and Andean volcanic soils. The novel diversity of these Icelandic microbial communities was supported by the finding that up to 46% of clones displayed <85% sequence identities to sequences currently deposited in the RDP database.     

Phage-host interactions in soil

Abstract Phages are abundant and ubiquitous in nature, and are therefore important components of microbial communities. They can impact on host populations in several ways, including predation and alteration of host phenotype by genetic interactions. The dynamic survival of phage populations in soil requires infective interactions with host populations which must be undergoing growth. Hence survival is limited by the activity of soil bacteria, and phage populations must adopt strategies to overcome periods of inactivity. One of the most effective strategies is the lysogenic cycle of temperate phages. It is argued here that lysogeny in soil has a distinct advantage over virulence for phage and host survival, as opposed to aquatic ecosystems where virulence seems a more successful strategy for phage populations.