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Pathogenic multidrug-resistant bacteria with hybrid chromosomes have emerged as a significant global healthcare threat. These include the pandemic Escherichia coli ST1193, the product of homologous recombination events involving two phylogenetically distant strains of E. coli, in which mutant alleles of the widely separated genes, gyrA and parC, generating high-level fluoroquinolone resistance were acquired. The mechanisms and frequency of hybrid formation are poorly understood. We developed a robust hybrid selection procedure and applied it to 118 clinical UTI isolates of E. coli mixed with suitable recipient strains. Hybrids were selected from 39% of isolates. All hybrids were recombinants of donor and recipient chromosomal DNA (median length of donor DNA 367 kb), with 90% also acquiring conjugative mobile genetic elements (MGE) from the donor. We showed that individual conjugative plasmids, and integrative conjugative elements (ICE), from donors were sufficient to drive hybrid formation. These observations strongly support conjugative chromosomal DNA transfer as the major mechanism underlying hybrid formation. ICE are genome-integrated and passively propagated but when transferring to recipients they normally do so by excising and producing their own conjugation machinery. We found that ICE were responsible for the highest frequencies of hybrid chromosome formation. They could mobilize DNA around the full length of the chromosome, including the simultaneous acquisition of mutant variants of gyrA and parC, separated by ∼826 kb, generating highly fluoroquinolone-resistant bacteria in a single event. Bacterial hybrid chromosome formation driven by conjugative MGE may be an important and widespread mechanism in the emergence and evolution of high-risk bacterial pathogens.

Horizontal gene transfer (HGT) between bacterial species is an important contributor to the development of multidrug-resistant (MDR) clinical pathogens. One example is the transfer of MDR conjugative plasmids from Klebsiella pneumoniae into Escherichia coli occurring within co-infected human hosts. Here, we asked whether, in mixed-species bacterial populations, we could also detect the transfer of chromosomal DNA. To address this, we screened 50 K. pneumoniae clinical isolates for their ability to generate chromosomal hybrids with E. coli as recipient. We found that 7/50 (14%) generated chromosomal hybrids at frequencies above our limit of detection (~10-11) up to a frequency of 5x10-10. Genome sequence analysis of 28 hybrids revealed multiple underlying mechanisms of DNA transfer and recombination. These included two different mechanisms by which a region of the E. coli chromosome was replaced with K. pneumoniae chromosomal DNA (Hfr-like mechanisms, with up to 586 kb of K. pneumoniae DNA acquired), and three different mechanisms by which F’-like plasmids carrying K. pneumoniae chromosomal DNA were created and transferred into E. coli. Accordingly, clinical isolates of K. pneumoniae have the ability to transfer, not just plasmids, but also intrinsically non-mobile chromosomal DNA into E. coli to create novel hybrid strains. Several of the isolated hybrids acquired genes related to virulence or antibiotic resistance. Chromosomal hybrids were stable after evolution in rich media and had a 70-95% fitness compared to MG1655. The results provide insights into the mechanisms involved in creating bacteria with novel hybrid chromosomes, which may have important implications for bacterial evolution, and the spread of antibiotic resistance and virulence genes.

Integrated conjugative elements can drive the transfer of chromosomal DNA from one Escherichia coli strain into another, generating strains with hybrid chromosomes. Several pandemic virulent multidrug-resistant strains of E. coli are chromosomal hybrids. Here we show that the frequency of hybrid formation can differ by orders of magnitude depending on the parental combination. However, the donor-recipient characteristics affecting hybridization efficiency are poorly understood. The laboratory workhorse strain E. coli MG1655 is significantly more proficient as a recipient in hybrid generation than E. coli ATCC25922, the strain used for quality control in antibiotic susceptibility testing. Focusing on these two strains we tested several hypotheses to identify barriers to hybrid formation. Among these, we found that relative conjugation efficiency correlated with hybrid formation frequency. To explore this, we used transposon mutagenesis to identify genes in ATCC25922 whose inactivation relieves a conjugation barrier. Among the first 1500 mutants tested we isolated and identified one that was 500-fold more conjugation-permissive than the parental strain. Whole genome sequencing identified this candidate ‘barrier’ gene as a protein-coding sequence on plasmid 3. Understanding the barrier mechanism will require further study. We are currently testing whether inactivation of this gene also increases chromosomal hybrid formation. The importance of this work is that it could significantly deepen our understanding of preferred pathways of HGT, something that will have broad application in both clinical and evolutionary biology.

Escherichia coli is a prototroph and can synthesize all twenty proteinogenic amino acids when required to grow in minimal medium. There are approximately sixty protein-coding genes individually essential for amino acid synthesis. This is a large mutational target for the accumulation of detrimental mutations. E. coli can rewire biosynthetic pathways in response to mutational damage but the limits of this capacity are poorly understood. Here, to address evolutionary robustness, we asked whether and how the phenotypes of irreversible mutations causing auxotrophy could be suppressed or bypassed in the absence of horizontal gene transfer (HGT). Spontaneous suppressors could be selected for only ten of fifty-nine mutants tested (detection limit ~7x10^-11). Mechanisms of suppression included: regional amplifications; mutations increasing gene or operon expression; mutations relaxing enzyme specificity; and mutations causing biochemical pathway diversions. Overall, the data show that spontaneous suppression of auxotrophy caused by an irreversible mutation is an evolutionary survival mechanism relevant only to a minority of the genes essential for amino acid synthesis. As a consequence, the essential genetic foundations for amino acid prototrophy are expected to be degraded over time by mutations (Muller’s ratchet) and metabolic rewiring alone will be insufficient to counteract this effect. This implies that maintaining phenotypes, including prototrophy in E. coli, and potentially other bacterial species, is likely to be reliant on HGT of housekeeping genes to counteract the effects of inevitable mutational inactivation. Accordingly, chromosomal HGT in bacteria may be critical for survival across diverse environmental niches.