Bacterial chromosomal mobility via lateral transduction exceeds that of classical mobile genetic elements

It is commonly assumed that the horizontal transfer of most bacterial chromosomal genes is limited, in contrast to the frequent transfer observed for typical mobile genetic elements. However, this view has been recently challenged by the discovery of lateral transduction in Staphylococcus aureus, where temperate phages can drive the transfer of large chromosomal regions at extremely high frequencies. Here, we analyse previously published as well as new datasets to compare horizontal gene transfer rates mediated by different mechanisms in S. aureus and Salmonella enterica. We find that the horizontal transfer of core chromosomal genes via lateral transduction can be more efficient than the transfer of classical mobile genetic elements via conjugation or generalized transduction. These results raise questions about our definition of mobile genetic elements, and the potential roles played by lateral transduction in bacterial evolution.

Effects of the Newly Isolated T4-Like Phage on Transmission of Plasmid-Borne Antibiotic Resistance Genes via Generalized Transduction

Bacteriophages are the most abundant biological entities on earth and may play an important role in the transmission of antibiotic resistance genes (ARG) from host bacteria. Although the specialized transduction mediated by the temperate phage targeting a specific insertion site is widely explored, the carrying characteristics of “transducing particles” for different ARG subtypes in the process of generalized transduction remains largely unclear. Here, we isolated a new T4-like lytic phage targeting transconjugant Escherichia coli C600 that contained plasmid pHNAH67 (KX246266) and encoded 11 different ARG subtypes. We found that phage AH67C600_Q9 can misload plasmid-borne ARGs and package host DNA randomly. Moreover, for any specific ARG subtype, the carrying frequency was negatively correlated with the multiplicity of infection (MOI). Further, whole genome sequencing (WGS) identified that only 0.338% (4/1183) of the contigs of an entire purified phage population contained ARG sequences; these were floR, sul2, aph(4)-Ia, and fosA. The low coverage indicated that long-read sequencing methods are needed to explore the mechanism of ARG transmission during generalized transduction.

Evaluation of Potential ARG Packaging by Two Environmental T7-Like Phage during Phage-Host Interaction

The increase in antimicrobial resistance is a threat to both human and animal health. The transfer of antibiotic resistance genes (ARG) via plasmids has been studied in detail whereas the contribution of bacteriophage-mediated ARG transmission is relatively little explored. We isolated and characterized two T7-like lytic bacteriophages that infected multidrug-resistant Escherichia coli hosts. The morphology and genomic analysis indicated that both phage HZP2 and HZ2R8 were evolutionarily related and their genomes did not encode ARGs. However, ARG-like raw reads were detected in offspring sequencing data with a different abundance level implying that potential ARG packaging had occurred. PCR results demonstrated that six fragments of genes (qnrS, cmlA, tetM, blaTEM, sul3, mcr-1) were potentially packaged by phage HZP2 and four (qnrS, cmlA, blaTEM, mcr-1) by phage HZ2R8. Further quantitative results showed that ARG abundance hierarchies were similar. The gene blaTEM was the most abundant (up to 1.38 × 107 copies/mL) whereas cmlA and qnrS were the least. Moreover, the clinically important mcr-1 gene was the second most abundant ARG indicating a possibility for spread through generalized transduction. Together, our results indicated that these structurally similar phage possessed similar characteristics and potential packaging during phage-host interaction displayed an ARG preference rather than occurring randomly.

Genome Sequences of vB_RleM_RL38JI and vB_RleM_RL2RES, Two Virulent Rhizobium leguminosarum Transducing Phages

Phages vB_RleM_RL38JI and vB_RleM_RL2RES are known to mediate generalized transduction in Rhizobium leguminosarum. The RL38JI genome consists of 158,577 nucleotides and 270 predicted genes, whereas RL2RES has a 156,878-bp genome with 262 predicted genes. The two genomes are similar, with 82.88% nucleotide identity to each other.

Molecular Piracy: Redirection of Bacteriophage Capsid Assembly by Mobile Genetic Elements

Horizontal transfer of mobile genetic elements (MGEs) is a key aspect of the evolution of bacterial pathogens. Transduction by bacteriophages is especially important in this process. Bacteriophages—which assemble a machinery for efficient encapsidation and transfer of genetic material—often transfer MGEs and other chromosomal DNA in a more-or-less nonspecific low-frequency process known as generalized transduction. However, some MGEs have evolved highly specific mechanisms to take advantage of bacteriophages for their own propagation and high-frequency transfer while strongly interfering with phage production—“molecular piracy”. These mechanisms include the ability to sense the presence of a phage entering lytic growth, specific recognition and packaging of MGE genomes into phage capsids, and the redirection of the phage assembly pathway to form capsids with a size more appropriate for the size of the MGE. This review focuses on the process of assembly redirection, which has evolved convergently in many different MGEs from across the bacterial universe. The diverse mechanisms that exist suggest that size redirection is an evolutionarily advantageous strategy for many MGEs.

Phages rarely encode antibiotic resistance genes: a cautionary tale for virome analyses

Antibiotic resistance genes (ARG) are pervasive in gut microbiota, but it remains unclear how often ARG are transferred, particularly to pathogens. Traditionally, ARG spread is attributed to horizontal transfer mediated either by DNA transformation, bacterial conjugation or generalized transduction. However, recent viral metagenome (virome) analyses suggest that ARG are frequently carried by phages, which is inconsistent with the traditional view that phage genomes rarely encode ARG. Here we used exploratory and conservative bioinformatic strategies found in the literature to detect ARG in phage genomes, and experimentally assessed a subset of ARG predicted using exploratory thresholds. ARG abundances in 1,181 phage genomes were vastly over-estimated using exploratory thresholds (421 predicted vs 2 known), due to low similarities and matches to protein unrelated to antibiotic resistance. Consistent with this, 4 ARG predicted using exploratory thresholds were experimentally evaluated and failed to confer antibiotic resistance in Escherichia coli. Re-analysis of available human-or mouse-associated viromes for ARG and their genomic context suggested that bona fide ARG attributed to phages in viromes were previously over-estimated. These findings provide guidance for documentation of ARG in viromes, and re-assert that ARG are rarely encoded in phages.

Recombinant transfer in the basic genome ofEscherichia coli

An approximation to the ∼4-Mbp basic genome shared by 32 strains ofEscherichia colirepresenting six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.

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

ABSTRACTAnimal-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 commensalEscherichia coliin 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 entericE. colibacteria 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 inE. coliby 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 8E. colibacteria/hour within the population of 1.47 × 108E. colibacteria/liter luminal contents. In comparison, the plasmidicblaCMY-2gene carried by ∼2% of entericE. coliwas transferred by conjugation at a rate at least 1.4 × 103times 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 entericE. coli.