Transductomics: sequencing-based detection and analysis of transduced DNA in pure cultures and microbial communities.

BACKGROUND: Horizontal gene transfer (HGT) plays a central role in microbial evolution. Our understanding of the mechanisms, frequency, and taxonomic range of HGT in polymicrobial environments is limited, as we currently rely on historical HGT events inferred from genome sequencing and studies involving cultured microorganisms. We lack approaches to observe ongoing HGT in microbial communities. RESULTS: To address this knowledge gap, we developed a DNA sequencing-based "transductomics" approach that detects and characterizes microbial DNA transferred via transduction. We validated our approach using model systems representing a range of transduction modes and show that we can detect numerous classes of transducing DNA. Additionally, we show that we can use this methodology to obtain insights into DNA transduction among all major taxonomic groups of the intestinal microbiome. CONCLUSIONS: The transductomics approach that we present here allows for the detection and characterization of genes that are potentially transferred between microbes in complex microbial communities at the time of measurement and thus provides insights into real-time ongoing horizontal gene transfer. This work extends the genomic toolkit for the broader study of mobile DNA within microbial communities and could be used to understand how phenotypes spread within microbiomes. Video Abstract.

CTAG-containing cleavage site profiling to delineate Salmonella into natural clusters.

BACKGROUND: The bacterial genus Salmonella contains thousands of serotypes that infect humans or other hosts, causing mild gastroenteritis to potentially fatal systemic infections in humans. Pathogenically distinct Salmonella serotypes have been classified as individual species or as serological variants of merely one or two species, causing considerable confusion in both research and clinical settings. This situation reflects a long unanswered question regarding whether the Salmonella serotypes exist as discrete genetic clusters (natural species) of organisms or as phenotypic (e.g. pathogenic) variants of a single (or two) natural species with a continuous spectrum of genetic divergence among them. Our recent work, based on genomic sequence divergence analysis, has demonstrated that genetic boundaries exist among Salmonella serotypes, circumscribing them into clear-cut genetic clusters of bacteria. METHODOLOGIES/PRINCIPAL FINDINGS: To further test the genetic boundary concept for delineating Salmonella into clearly defined natural lineages (e.g., species), we sampled a small subset of conserved genomic DNA sequences, i.e., the endonuclease cleavage sites that contain the highly conserved CTAG sequence such as TCTAGA for XbaI. We found that the CTAG-containing cleavage sequence profiles could be used to resolve the genetic boundaries as reliably and efficiently as whole genome sequence comparisons but with enormously reduced requirements for time and resources. CONCLUSIONS: Profiling of CTAG sequence subsets reflects genetic boundaries among Salmonella lineages and can delineate these bacteria into discrete natural clusters.

Genomic comparison of Salmonella typhimurium DT104 with non-DT104 strains.

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.

A genome map of Salmonella enterica serovar Agona: numerous insertions and deletions reflecting the evolutionary history of a human pathogen.

Salmonella enterica serovar Agona is an important zoonotic pathogen, causing serious human illness worldwide, but knowledge about its genetics and evolution, especially regarding the genomic events that might have contributed to the formation of S. Agona as an important pathogen, is lacking. As a first step toward understanding this pathogen and characterizing its genomic differences with other salmonellae, we constructed a physical map of S. Agona in strain SARB1 using I-CeuI, XbaI, AvrII and Tn10 insertions with pulsed-field gel electrophoresis techniques. On the 4815-kb genomic map, we located 82 genes, revealed one inversion of about 1000 kb and resolved seven deletions and seven insertions ranging from 10 to 67 kb relative to the genome of Salmonella typhimurium LT2. These genomic features clearly distinguish S. Agona from other previously analyzed salmonellae and provide clues to the molecular basis for its genomic divergence. Additionally, these kinds of physical maps, combined with emerging high-speed sequencing technologies, such as the Solexa or SOLiD techniques, which require a pre-existing high-resolution physical map such as the S. Agona map reported here, will play important roles in genomic comparative studies of bacteria involving large numbers of strains.

High-throughput genotyping of Salmonella enterica serovar Typhi allowing geographical assignment of haplotypes and pathotypes within an urban District of Jakarta, Indonesia.

High-throughput epidemiological typing systems that provide phylogenetic and genotypic information are beneficial for tracking bacterial pathogens in the field. The incidence of Salmonella enterica serovar Typhi infection in Indonesia is high and is associated with atypical phenotypic traits such as expression of the j and the z66 flagellum antigens. Utilizing a high-throughput genotyping platform to investigate known nucleotide polymorphisms dispersed around the genome, we determined the haplotypes of 140 serovar Typhi isolates associated with Indonesia. We identified nine distinct serovar Typhi haplotypes circulating in Indonesia for more than 30 years, with eight of these present in a single Jakarta suburb within a 2-year period. One dominant haplotype, H59, is associated with j and z66 flagellum expression, representing a potential pathotype unique to Indonesia. Phylogenetic analysis suggests that H59 z66(+), j(+) isolates emerged relatively recently in terms of the origin of serovar Typhi and are geographically restricted. These data demonstrate the potential of high-throughput genotyping platforms for analyzing serovar Typhi populations in the field. The study also provides insight into the evolution of serovar Typhi and demonstrates the value of a molecular epidemiological technique that is exchangeable, that is internet friendly, and that has global utility.

A novel linear plasmid mediates flagellar variation in Salmonella Typhi.

Unlike the majority of Salmonella enterica serovars, Salmonella Typhi (S. Typhi), the etiological agent of human typhoid, is monophasic. S. Typhi normally harbours only the phase 1 flagellin gene (fliC), which encodes the H:d antigen. However, some S. Typhi strains found in Indonesia express an additional flagellin antigen termed H:z66. Molecular analysis of H:z66+ S. Typhi revealed that the H:z66 flagellin structural gene (fljB(z66)) is encoded on a linear plasmid that we have named pBSSB1. The DNA sequence of pBSSB1 was determined to be just over 27 kbp, and was predicted to encode 33 coding sequences. To our knowledge, pBSSB1 is the first non-bacteriophage-related linear plasmid to be described in the Enterobacteriaceae.

Role of N-acetylglucosamine within core lipopolysaccharide of several species of gram-negative bacteria in targeting the DC-SIGN (CD209).

Our recent studies have shown that the dendritic cell-specific ICAM nonintegrin CD209 (DC-SIGN) specifically binds to the core LPS of Escherichia coli K12 (E. coli), promoting bacterial adherence and phagocytosis. In this current study, we attempted to map the sites within the core LPS that are directly involved in LPS-DC-SIGN interaction. We took advantage of four sets of well-defined core LPS mutants, which are derived from E. coli, Salmonella enterica serovar Typhimurium, Neisseria gonorrhoeae, and Haemophilus ducreyi and determined interaction of each of these four sets with DC-SIGN. Our results demonstrated that N-acetylglucosamine (GlcNAc) sugar residues within the core LPS in these bacteria play an essential role in targeting the DC-SIGN receptor. Our results also imply that DC-SIGN is an innate immune receptor and the interaction of bacterial core LPS and DC-SIGN may represent a primeval interaction between Gram-negative bacteria and host phagocytic cells.

Genome plasticity and ori-ter rebalancing in Salmonella typhi.

Genome plasticity resulting from frequent rearrangement of the bacterial genome is a fascinating but poorly understood phenomenon. First reported in Salmonella typhi, it has been observed only in a small number of Salmonella serovars, although the over 2,500 known Salmonella serovars are all very closely related. To gain insights into this phenomenon and elucidate its roles in bacterial evolution, especially those involved in the formation of particular pathogens, we systematically analyzed the genomes of 127 wild-type S. typhi strains isolated from many places of the world and compared them with the two sequenced strains, Ty2 and CT18, attempting to find possible associations between genome rearrangement and other significant genomic features. Like other host-adapted Salmonella serovars, S. typhi contained large genome insertions, including the 134 kb Salmonella pathogenicity island, SPI7. Our analyses showed that SPI7 disrupted the physical balance of the bacterial genome between the replication origin (ori) and terminus (ter) when this DNA segment was inserted into the genome, and rearrangement in individual strains further changed the genome balance status, with a general tendency toward a better balanced genome structure. In a given S. typhi strain, genome diversification occurred and resulted in different structures among cells in the culture. Under a stressed condition, bacterial cells with better balanced genome structures were selected to greatly increase in proportion; in such cases, bacteria with better balanced genomes formed larger colonies and grew with shorter generation times. Our results support the hypothesis that genome plasticity as a result of frequent rearrangement provides the opportunity for the bacterial genome to adopt a better balanced structure and thus eventually stabilizes the genome during evolution.

The genome of Salmonella enterica serovar gallinarum: distinct insertions/deletions and rare rearrangements.

Salmonella enterica serovar Gallinarum is a fowl-adapted pathogen, causing typhoid fever in chickens. It has the same antigenic formula (1,9,12:--:--) as S. enterica serovar Pullorum, which is also adapted to fowl but causes pullorum disease (diarrhea). The close relatedness but distinct pathogeneses make this pair of fowl pathogens good models for studies of bacterial genomic evolution and the way these organisms acquired pathogenicity. To locate and characterize the genomic differences between serovar Gallinarum and other salmonellae, we constructed a physical map of serovar Gallinarum strain SARB21 by using I-CeuI, XbaI, and AvrII with pulsed-field gel electrophoresis techniques. In the 4,740-kb genome, we located two insertions and six deletions relative to the genome of S. enterica serovar Typhimurium LT2, which we used as a reference Salmonella genome. Four of the genomic regions with reduced lengths corresponded to the four prophages in the genome of serovar Typhimurium LT2, and the others contained several smaller deletions relative to serovar Typhimurium LT2, including regions containing srfJ, std, and stj and gene clusters encoding a type I restriction system in serovar Typhimurium LT2. The map also revealed some rare rearrangements, including two inversions and several translocations. Further characterization of these insertions, deletions, and rearrangements will provide new insights into the molecular basis for the specific host-pathogen interactions and mechanisms of genomic evolution to create a new pathogen.

Diversity of genome structure in Salmonella enterica serovar Typhi populations.

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136 wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonuclease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangements due to homologous recombination between the rrn operons leading to inversions and translocations. Recombination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S. enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. enterica serovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need for genome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types 1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectional replication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb off balance), indicating that the survival of the best-balanced strains was greater. In addition, the need for appropriate gene dosage apparently selected against rearrangements which moved genes from their accustomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S. enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved in rearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmonella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a prophage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appeared to be independent of the origins of the genome types.

Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid.

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their approximately 4,400 protein coding sequences: 173 in Paratyphi A and approximately 210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).

Salmonella enterica serovar Typhi strains from which SPI7, a 134-kilobase island with genes for Vi exopolysaccharide and other functions, has been deleted.

Salmonella enterica serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between pheU and 'pheU (truncated), two genes for tRNA(Phe). SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage. Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion. In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative. We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons. Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single pheU gene; microarrays show that all SPI7 genes are deleted. Two of the strains produce amplicons which have A derived from pheU at bp 27, while five have C derived from 'pheU at this position; thus, the position of the crossover which results in the deletion can be inferred. The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the viaB operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained. We propose that these deletions occurred during storage following isolation.

Genomic diversification among archival strains of Salmonella enterica serovar typhimurium LT7.

To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at -70 degrees C or in serovar Typhimurium LT2 stocked either at -70 degrees C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at -70 degrees C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi.

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.

The evolving genome of Salmonella enterica serovar Pullorum.

Salmonella enterica serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make S. enterica serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of S. enterica serovar Pullorum RKS5078, using I-CeuI, XbaI, AvrII, and SpeI and Tn10 insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas. However, the genome of S. enterica serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most S. enterica serovar Pullorum strains examined, with different structures in a minority of S. enterica serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with S. enterica serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among S. enterica serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the S. enterica serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.