Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination

Abstract

Background: Shigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches. Results: We have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new hybrid strategy to prepare gapless, highly accurate genome sequences, which also cover AT-rich tracks or repetitive sequences that are transcribed. Furthermore, we have performed genome-wide analysis of transcriptional start sites (TSS) and determined the length of 5′ untranslated regions (5′-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6723 primary TSS (pTSS) and 7328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam. mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) databases to use their analysis tools in the S. flexneri 5a M90T genome. Conclusions: We provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. Further, we present a new hybrid strategy to prepare gapless, highly accurate genome sequences. Unlike currently used hybrid strategies combining long-and short-read DNA sequencing technologies to maximize accuracy, our workflow using long-read DNA sequencing and short-read RNA sequencing provides the added value of using non-redundant technologies, which yield distinct, exploitable datasets.

The T3SS of Shigella: Expression, Structure, Function, and Role in Vacuole Escape

“…We will refer generically to the various proteins forming the T3SA as its parts and use their Shigella names throughout, but at the first occurrence where their name in the unified nomenclature is mentioned [5]. Shigella harbors a ~232 kbp virulence plasmid (VP) that is maintained by an intricate combination of toxin-antitoxin systems [8] and harbors most T3SS genes [9]. The parts of the T3SA in Shigella, as in other species [4], are encoded on a single DNA fragment of approximately 30 kbp named the entry region and composed of the mxi/spa and ipa operons [10,11].…”

CRISPR-Cas guided mutagenesis of chromosome and virulence plasmid inShigella flexneriby cytosine base editing

“…Generating transposon libraries, on the other hand, is fast and easy but it still requires verification of phenotypes by generating individual loss-of-function mutants (42). Within this context, a number of chromosomal genes in Shigella are transcribed with no function assigned to them and no high-throughput effort, to the best of our knowledge, has been undertaken to characterize these genes (7). We sought to bridge this gap by adopting a recently improved CRISPR-Cas base editing method, shown to work in C. glutamicum , for generating targeted loss-of-function mutants with higher throughput, in Shigella (21).…”

“…W e recently resequenced the genome of S. flexneri serotype 5a M90T, a widely used lab reference strain, and mapped global transcription start sites (7). Unsurprisingly, many transcription start sites led to identification of genes that await functional characterization to reveal new and interesting information about Shigella physiology.…”

Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri

“…The peak lists were searched against the sequences of 34 target proteins and 34 decoy proteins. The target proteins are 34 proteins that are encoded by the “entry region” of the Shigella virulence plasmid (Buchrieser et al, 2000; Cervantes‐Rivera et al, 2020). The decoy proteins were constructed with random amino acid sequences; however, they have the same length as the target proteins and in the sequences, trypsin cleavage amino acids (lysing and arginine) are at the identical position to the target proteins.…”