Method for Host-Independent Detection of Generalized Transducing Bacteriophages in Natural Habitats

Despite an increasing interest in horizontal gene transfer in bacteria, the role of generalized transduction in this process has not been well investigated yet. Certainly one of the reasons is that only a small fraction of general transducing bacteriophages have been characterized, because many bacterial hosts needed for propagation and identification are not culturable or are simply unknown. A method for host-independent detection of transducing bacteriophages was developed. Phage-encapsulated DNA was used as a template for PCR amplification of 16S ribosomal DNA using primers specific for the 16S rRNA genes of most eubacteria. Sequencing of the cloned amplification products permits the identification of the host bacteria. The Salmonella phage P22 was used as an example. Applying this method to a sample of the supernatant of the mixed liquor in the aeration tank of an activated sludge treatment works revealed the presence of transducing phages infecting several bacterial species for which such phages have not yet been described. This method is suitable for estimating the contribution of generalized transduction to horizontal gene transfer in different habitats.     

A broad-host-range, generalized transducing phage (SN-T) acquires 16S rRNA genes from different genera of bacteria

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 x 10(-9) transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.     

A Broad-Host-Range, Generalized Transducing Phage (SN-T) Acquires 16S rRNA Genes from Different Genera of Bacteria

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 × 10⁻⁹ transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.     

Bacteriophages and genetic mobilization in sewage and faecally polluted environments

Bacteriophages are one of the most abundant entities on the planet and are present in high concentrations within humans and animals, mostly in the gut. Phages that infect intestinal bacteria are released by defecation and remain free in extra‐intestinal environments, where they usually persist for longer than their bacterial hosts. Recent studies indicate that a large amount of the genetic information in bacterial genomes and in natural environments is of phage origin. In addition, metagenomic analysis reveals that a substantial number of bacterial genes are present in viral DNA in different environments. These facts support the belief that phages can play a significant role in horizontal gene transfer between bacteria. Bacteriophages are known to transfer genes by generalized and specialized transduction and indeed there are some examples of phages found in the environment carrying and transducing genes of bacterial origin. A successful transduction in the environment requires certain conditions, e.g. phage and bacterial numbers need to exceed certain threshold concentrations, the bacteria need to exist in an infection‐competent physiological state, and lastly, the physical conditions in the environment (pH, temperature, etc. of the supporting matrix) have to be suitable for phage infection. All three factors are reviewed here, and the available information suggests: (i) that the number of intestinal bacteria and phages in faecally contaminated environments guarantees bacteria–phage encounters, (ii) that transduction to intestinal bacteria in the environment is probable, and (iii) that transduction is more frequent than previously thought. Therefore, we suggest that phage‐mediated horizontal transfer between intestinal bacteria, or between intestinal and autochthonous bacteria in extra‐intestinal environments, might take place and that its relevance for the emergence of new bacterial strains and potential pathogens should not be ignored.     

Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences

Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences.     

Bacteriophage ?MAM1, a Viunalikevirus,Is a Broad-Host-Range,High-Efficiency Generalized Transducer That Infects Environmental and Clinical Isolates of the Enterobacterial Genera Serratia and Kluyvera

Members of the enterobacterial genus Serratia are ecologically widespread, and some strains are opportunistic human pathogens. Bacteriophage ϕMAM1 was isolated on Serratia plymuthica A153, a biocontrol rhizosphere strain that produces the potently bioactive antifungal and anticancer haterumalide oocydin A. The ϕMAM1 phage is a generalized transducing phage that infects multiple environmental and clinical isolates of Serratia spp. and a rhizosphere strain of Kluyvera cryocrescens. Electron microscopy allowed classification of ϕMAM1 in the family Myoviridae. Bacteriophage ϕMAM1 is virulent, uses capsular polysaccharides as a receptor, and can transduce chromosomal markers at frequencies of up to 7 × 10⁻⁶ transductants per PFU. We also demonstrated transduction of the complete 77-kb oocydin A gene cluster and heterogeneric transduction of a plasmid carrying a type III toxin-antitoxin system. These results support the notion of the potential ecological importance of transducing phages in the acquisition of genes by horizontal gene transfer. Phylogenetic analyses grouped ϕMAM1 within the ViI-like bacteriophages, and genomic analyses revealed that the major differences between ϕMAM1 and other ViI-like phages arise in a region encoding the host recognition determinants. Our results predict that the wider genus of ViI-like phages could be efficient transducing phages, and this possibility has obvious implications for the ecology of horizontal gene transfer, bacterial functional genomics, and synthetic biology.     

Isolation of generalized transducing bacteriophages for uropathogenic strains of Escherichia coli

The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.     

Application of DNA probes to analysis of bacteriophage distribution patterns in the environment.

Radiolabeled bacteriophage DNA probes have been used in this study to determine the distribution of Pseudomonas aeruginosa-infecting bacteriophages in natural samples of lake water, sediment, soil, and sewage. The sensitivity of detection of bacteriophage with the DNA probes was between 10(3) and 10(4) PFU and 10(6) to 10(7) CFU of lysogenized bacteria detectable with a homologous phage DNA probe. Analyses of environmental samples suggest that up to 40% of P. aeruginosa in natural ecosystems contain DNA sequences homologous to phage genomes. By using different bacteriophage DNA probes, the diversity of the bacteriophage population in sewage was estimated to be higher than that in other natural samples. The indication that transducing phages and prophages are widely distributed in the Pseudomonas populations investigated has considerable implications for the frequency of natural gene transfer by transduction and of lysogenic conversion of host bacteria in natural ecosystems.     

Application of DNA probes to analysis of bacteriophage distribution patterns in the environment.

Radiolabeled bacteriophage DNA probes have been used in this study to determine the distribution of Pseudomonas aeruginosa-infecting bacteriophages in natural samples of lake water, sediment, soil, and sewage. The sensitivity of detection of bacteriophage with the DNA probes was between 10(3) and 10(4) PFU and 10(6) to 10(7) CFU of lysogenized bacteria detectable with a homologous phage DNA probe. Analyses of environmental samples suggest that up to 40% of P. aeruginosa in natural ecosystems contain DNA sequences homologous to phage genomes. By using different bacteriophage DNA probes, the diversity of the bacteriophage population in sewage was estimated to be higher than that in other natural samples. The indication that transducing phages and prophages are widely distributed in the Pseudomonas populations investigated has considerable implications for the frequency of natural gene transfer by transduction and of lysogenic conversion of host bacteria in natural ecosystems.     

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.     

Bacteriophages Isolated from Chicken Meat and the Horizontal Transfer of Antimicrobial Resistance Genes

Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.     

Factors that affect transduction in microorganisms]

Data from literature concerning general and specialized transduction in microorganisms are given in the paper. The process of exogenic DNA penetration to the cells of bacteria and participation of protein products of separate phage genes in this process are described. The so-called E-proteins in a set with DNA penetrate through a cell membrane. In phage P22 they are protein products of phage genes 7, 16, 20. In P22 mutants with an altered transducing frequencies (HFT and LFT) the due functions are also coded by the phage genes. It is shown that the process of DNA packing in phages P22, phi 80, lambda and others is genetically determined. The gene transfer frequency depends on UV radiation and the very nature of transducing phages itself. In virulent phages the UV radiation up to inactivation level 95-99% evokes a decrease of their "killer" ability, which is accompanied by an increase of survivability of the formed transductants and, as a result, by enhancement of the transduction transfer frequency. An important role of the transduction analysis for fine mapping of a genome of microorganisms and its significance for practice are shown. A mathematical analysis of the data on cotransduction of linkage markers is presented as such that may be used when determining the value of transduced fragment of a chromosome.     

Modeling the Infection Dynamics of Bacteriophages in Enteric Escherichia coli: Estimating the Contribution of Transduction to Antimicrobial Gene Spread

Animal-associated bacterial communities are infected by bacteriophages, although the dynamics of these infections are poorly understood. Transduction by bacteriophages may contribute to transfer of antimicrobial resistance genes, but the relative importance of transduction among other gene transfer mechanisms is unknown. We therefore developed a candidate deterministic mathematical model of the infection dynamics of enteric coliphages in commensal Escherichia coli in the large intestine of cattle. We assumed the phages were associated with the intestine and were predominantly temperate. Model simulations demonstrated how, given the bacterial ecology and infection dynamics, most (>90%) commensal enteric E. coli bacteria may become lysogens of enteric coliphages during intestinal transit. Using the model and the most liberal assumptions about transduction efficiency and resistance gene frequency, we approximated the upper numerical limits (“worst-case scenario”) of gene transfer through specialized and generalized transduction in E. coli by enteric coliphages when the transduced genetic segment is picked at random. The estimates were consistent with a relatively small contribution of transduction to lateral gene spread; for example, generalized transduction delivered the chromosomal resistance gene to up to 8 E. coli bacteria/hour within the population of 1.47 × 10⁸ E. coli bacteria/liter luminal contents. In comparison, the plasmidic bla_CMY-2 gene carried by ∼2% of enteric E. coli was transferred by conjugation at a rate at least 1.4 × 10³ times greater than our generalized transduction estimate. The estimated numbers of transductants varied nonlinearly depending on the ecology of bacteria available for phages to infect, that is, on the assumed rates of turnover and replication of enteric E. coli.     

Prophage-like gene transfer agents-novel mechanisms of gene exchange for Methanococcus, Desulfovibrio, Brachyspira, and Rhodobacter species

Gene transfer agents (GTAs) are novel mechanisms for bacterial gene transfer. They resemble small, tailed bacteriophages in ultrastructure and act like generalized transducing prophages. In contrast to functional prophages, GTAs package random fragments of bacterial genomes and incomplete copies of their own genomes. The packaged DNA content is characteristic of the GTA and ranges in size from 4.4 to 13.6kb. GTAs have been reported in species of Brachyspira, Methanococcus, Desulfovibrio, and Rhodobacter. The best studied GTAs are VSH-1 of the anaerobic, pathogenic spirochete Brachyspira hyodysenteriae and RcGTA of the nonsulfur, purple, photosynthetic bacterium Rhodobacter capsulatus. VSH-1 and RcGTA have likely contributed to the ecology and evolution of these bacteria. The existence of GTAs in phylogenetically diverse bacteria suggests GTAs may be more common in nature than is now appreciated.     

The mechanisms of horizontal gene transfer: the role of bacteriophages and integrons in the evolution of pathogenic bacteria

The role of bacteriophages, e.g. filamentous phages, whose single-stranded DNA comprise the coding virulence factors, as well as of certain suspected bacteriophage derivatives like constin-elements, integrons and chromosomal super-integron, played by them in the horizontal gene transfer and in the evolution of bacteria is under discussion.     

Transduction in Bacillus stearothermophilus.

Temperate and virulent bacteriophages isolated from soil were shown to carry out generalized transduction of Bacillus stearothermophilus NUB36. A transducing frequency of 1 X 10(-5) to 7 X 10(-4) was obtained for temperate phages TP-42 and TP-56. The transducing frequency for virulent phage TP-68 was two to three orders of magnitude lower. Cotransfer analysis with the three phages showed that hom-1 is linked to thr-1 and that gly-1 is linked to his-1.     

Role of horizontal gene transfer by bacteriophages in the origin of pathogenic bacteria

The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent phi KZ-like phages) of conditional pathogen Pseudomonas aeruginosa. Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches.     

Rapid differentiation of bacterial species by high resolution melting curve analysis

Molecular based differentiation of various bacterial species is important in phylogenetic studies, diagnostics and epidemiological surveillance, particularly where unusual phenotype makes the classical phenotypic identification of bacteria difficult. Molecular approach based on the sequence of 16S ribosomal RNA gene analysis can achieve fast and reliable identification of bacteria. High resolution melting (HRM) curve analysis has been developed as an attractive novel technique for DNA sequence discrimination but it’s application for bacteria differentiation has not been well studied yet. We have developed HRM assay for differentiation of sixteen pathogenic or opportunistic bacterial species. Amplified partial 16S ribosomal RNA gene region between 968 and 1401 positions (E. coli reference numbering) was subsequently used in high resolution melting curve analysis of PCR products for bacterial species differentiation. Sixteen bacterial species were simultaneously discerned by difference plot of normalized and temperatures shifted melting curves, without need for spiking of DNA, hetero-duplexing experiments or application of several primer pairs. High resolution melting curve analysis of duplex DNA is simple, fast and reliable tool for bacterial species differentiation and may efficiently complement phenotypic identification of bacteria.     

Effects of Suspended Particulates on the Frequency of Transduction among Pseudomonas aeruginosa in a Freshwater Environment

Transduction has been shown to play a significant role in the transfer of plasmid and chromosomal DNA in aquatic ecosystems. Such ecosystems contain a multitude of environmental factors, any one of which may influence the transduction process. It was the purpose of this study to show how one of these factors, particulate matter, affects the frequency of transduction. In situ transduction rates were measured in lake water microcosms containing either high or low concentrations of particulate matter. The microcosms were incubated in a freshwater lake in central Oklahoma. Transduction frequencies were found to be enhanced as much as 100-fold in the presence of particulates. Our results suggest that aggregations of bacteriophages and bacterial cells are stimulated by the presence of these suspended particulates. This aggregation increases the probability of progeny phages and transducing particles finding and infecting new host cells. Consequently, both phage production and transduction frequencies increase in the presence of particulate matter.