Molecular Microbiology最新文献

Salmonella enterica serovar Typhimurium (S. Tm) is a major cause of foodborne diarrhea. However, in healthy individuals, the microbiota typically restricts the growth of incoming pathogens, a protective mechanism termed colonization resistance (CR). To circumvent CR, Salmonella strains can utilize private nutrients that remain untapped by the resident microbiota. However, the metabolic pathways and environmental niches promoting pathogen growth are still not completely understood. Here, we investigate the significance of the gfr operon in gut colonization of S. Tm, which is essential for the utilization of fructoselysine (FL) and glucoselysine (GL). These Amadori compounds are present in heated foods with high protein and carbohydrate contents. We detected FL in both mouse chow and the intestinal tract of mice and showed that gfr mutants are attenuated during the initial phase of colonization in the murine model. Experiments in gnotobiotic mice and competition experiments with Escherichia coli suggest that gfr-dependent fitness advantage is context-dependent. We conclude that dietary Amadori products like FL can support S. Tm gut colonization, depending on the metabolic capacities of the microbiota.

The insertion sequence IS200 is widely distributed in Eubacteria. Despite its prevalence, IS200 does not appear to be mobile and as such is considered an ancestral component of bacterial genomes. Previous work in Salmonella enterica revealed that the IS200 tnpA transcript is processed to form a small, highly structured RNA (5'tnpA) that participates in the posttranscriptional control of invF expression, encoding a key transcription factor in this enteropathogen's invasion regulon. To further examine the scope of 5'tnpA transcript integration into Salmonella gene expression networks, we performed comparative RNA-seq, revealing the differential expression of over 200 genes in a Salmonella SL1344 5'tnpA disruption strain. This includes the genes for the master regulators of both invasion and flagellar regulons (HilD and FlhDC, respectively), plus genes involved in cysteine biosynthesis and an operon (phsABC) encoding a thiosulfate reductase complex. These expression changes were accompanied by an 80-fold increase in Salmonella invasion of HeLa cells. Follow-up experimentation suggested an additional direct target of 5'tnpA to be the small RNA PinT, which has previously been shown to be a negative regulator of invasion genes through its inhibitory action on key transcription factors governing the Salmonella pathogenicity island 1 regulon. This study provides a powerful new example of bacterial transposon domestication that is based not on the production/use of a regulatory protein or regulatory DNA sequences, but on the function of a transposon-derived small RNA.

Streptococcus agalactiae (group B Streptococcus; GBS) is a leading cause of neonatal sepsis and meningitis. Despite advances in molecular microbiology, GBS genome engineering remains laborious due to inefficient mutagenesis protocols. Here, we report a versatile and rapid Cas12a-based toolkit for GBS genetic manipulation. We developed two shuttle plasmids-pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference-derived from an Enterococcus faecium system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template-free mutagenesis via alternative end-joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand-specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off-target activity, as confirmed by whole-genome sequencing. In benchmarking, template-less Cas12a mutagenesis yielded sequence-confirmed mutants in ~7 days and homology-directed edits in ~7-14 days; aTC-resistant colonies arose at ~10^-4 of uninduced CFU, and 27%-65% of resistant clones carried the intended homology-directed edit depending on locus and homology arm length (e.g., ~27% markerless deletion; ~35% insertion; 65% with 1 kb arms). These workflows provide a rapid alternative to temperature-sensitive plasmid mutagenesis protocols that typically require ≥ 4 weeks. This Cas12a-based platform offers an efficient, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.