Bacterial genetics and molecular pathogenesis in the age of high throughput DNA sequencing

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When Stanley Falkow introduced Molecular Koch’s Postulates (Falkow, 1988) as a conceptual framework to identify microbial factors that contributed to disease, he reaffirmed the prominent role that the basic principles of genetic analysis should play in defining genotype-phenotype associations in microbial pathogens. In classical bacterial genetics the nature of mutations is inferred through cis-trans complementation and by indirectly mapping their relative position and physical distance through recombination frequencies — all of which were made possible by the genetic tools of the day: natural transformations, conjugation and transduction. Unfortunately, many of these genetic tools are not always available to study pathogenic bacteria. The recombinant DNA revolution in the 1980s launched the field of molecular pathogenesis as genes could be treated as physical units that could be cut, spliced and transplanted from one microbe to another and thus not only ‘prove’ that an individual gene complemented a virulence defect in a mutant strain but also could impart pathogenic properties to otherwise benign microbes. The recombinant DNA revolution also enabled the generation of newer versions of genetic tools to generate mutations and engineer microbial genomes.

The last decade has ushered in next generation sequencing technologies as a new powerful tool for bacterial genetics. The routine and inexpensive sequencing of microbial genomes has increased the number and phylogenetic scope of microbes that are amenable to functional characterization and experimentation. In this review, we highlight some salient advances in this rapidly evolving area.

Section snippets

Whole genome sequencing

The options for microbial whole genome sequencing (WGS) include platforms for shorts reads, primarily Illumina, as well as ‘third generation’ DNA sequencing platforms, such as PacBio’s single molecule real-time sequencing (SMRT) [1] and Oxford Nanopore Technology (ONT) [2••]. Illumina sequencing reads can be used for de novo genome assemblies; however, its short reads can fail to assemble repetitive or transposed regions of a genome. In contrast, SMRT and ONT produce long reads that span

Transposon insertion sequencing

Genomic sequences provide insight into gene content, but gene function often relies on homologies to genes previously characterized in model organisms. A powerful tool for functional analysis of genes is transposon mutagenesis. Typically, transposon (Tn) mutants are selected or screened for a particular altered function and the location of the Tn insertion in the genome is then determined using various PCR-based methods. Tn-insertion sequencing (abbreviated TIS herein) can be used to identify

Chemical mutagenesis and whole genome sequencing

For many microbes the necessary genetic tools or DNA delivery mechanisms for Tn mutagenesis do not exist. One method to circumvent the need for molecular genetic tools is to induce mutagenesis through DNA damaging agents and mapping the resulting mutations by WGS [65]. Mutagens such as UV radiation and the DNA alkylating compounds ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) introduce point mutations randomly throughout the genome, which can inactivate or truncate a gene. As in

Large scale transcriptional profiling (RNA-seq)

High throughput sequencing has had a particularly transformative impact on the analysis of gene expression. RNA-seq has long replaced microarray as the standard method to assess global patterns of gene expression. While alternative methods such as direct sequencing of RNA molecules with ONT [38], and cDNA sequencing with SMRT [72] have recently been described, Illumina sequencing of cDNAs remains the most approach for RNA-seq. With high levels of sequencing depth, RNA-seq can to deliver

Future directions

Next generation DNA sequencing has transformed bacterial genetics and opened previously intractable organisms to genetic analysis. As technologies develop, costs decrease and more robust bioinformatic analysis tools emerge, we expect that increased applications for WGS in single bacterial cell analysis, to reveal heterogeneity in populations, and to facilitate analysis of unculturable organisms. For example, single bacteria WGS is possible [84] and technical advances in cell isolation and

Conflict of interest statement

RHV is a member of the Scientific Advisory Board of Meridian Bioscience. Meridian had no input in the writing of thus manuscript.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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