High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model–based analyses of transposon-insertion sequencing data

Abstract

The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data.

Cell Separation in Vibrio cholerae Is Mediated by a Single Amidase Whose Action Is Modulated by Two Nonredundant Activators

“…V. cholerae AmiB also contains three putative LysM PG-binding domains at its C terminus that are not present in the E. coli PG amidases. Analysis of previously reported transposon insertions within VC0344 indicates that disruption of this C-terminal region is less detrimental than disruption of the remainder of the gene (25), suggesting that the LysM domains are not critical to the function of AmiB.…”

“…Transposon insertion sequencing (TnSeq) was performed as described previously (25). In brief, 200,000 to 300,000 transposon mutants were generated for each strain by transformation with transposon delivery vector pSC189, and pooled genomic DNA fragments were analyzed on an Illumina MiSeq benchtop sequencer (Illumina, San Diego, CA).…”

“…In brief, 200,000 to 300,000 transposon mutants were generated for each strain by transformation with transposon delivery vector pSC189, and pooled genomic DNA fragments were analyzed on an Illumina MiSeq benchtop sequencer (Illumina, San Diego, CA). Insertion sites were identified as described previously (25). Results were visualized using Artemis (26).…”

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum

“…This has implications for the development of strain-independent therapeutics and underscores the importance of functional studies in pathogenic strains of interest. more sophisticated methods using hidden Markov models have incorporated mutant abundance data successfully but do not consider biological variability (18,19).…”

Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

“…Genes that contain fewer than expected transposon insertions have a growth disadvantage (here termed "essential") whereas those with larger numbers of insertions have an advantage ("enriched"). Comparisons of the transposon insertion profiles (21,22) of the wild-type and mutant strains revealed both essential and enriched genes-two types of genetic interaction-in each strain (Fig. 2).…”