ABSTRACT
Low-abundance members of microbial communities are difficult to study in their native habitats. This includes Escherichia coli, a minor, but common inhabitant of the gastrointestinal tract and opportunistic pathogen, including of the urinary tract, where it is the primary pathogen. While multi-omic analyses have detailed critical interactions between uropathogenic Escherichia coli (UPEC) and the bladder that mediate UTI outcome, comparatively little is known about UPEC in its pre-infection reservoir, partly due to its low abundance there (<1% relative abundance). To accurately and sensitively explore the genomes and transcriptomes of diverse E. coli in gastrointestinal communities, we developed E. coli PanSelect which uses a set of probes designed to specifically recognize and capture E. coli's broad pangenome from sequencing libraries. We demonstrated the ability of E. coli PanSelect to enrich, by orders of magnitude, sequencing data from diverse E. coli using a mock community and a set of human stool samples collected as part of a cohort study investigating drivers of recurrent urinary tract infections (rUTI). Comparisons of genomes and transcriptomes between E. coli residing in the gastrointestinal tracts of women with and without a history of rUTI suggest that rUTI gut E. coli are responding to increased levels of oxygen and nitrate, suggestive of mucosal inflammation, which may have implications for recurrent disease. E. coli PanSelect is well suited for investigations of native in vivo biology of E. coli in other environments where it is at low relative abundance, and the framework described here has broad applicability to other highly diverse, low abundance organisms.
ABSTRACT
The bacterial FtsZ-ring initiates division by recruiting a large repertoire of proteins (the divisome; Z-ring) needed for septation and separation of cells. Although FtsZ is essential and its role as the main orchestrator of cell division is conserved in most eubacteria, the regulators of Z-ring presence and positioning are not universal. This study characterizes factors that regulate divisome presence and placement in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn), focusing on FtsZ, EzrA, SepF, ZapA, and ZapJ, which is reported here as a partner of ZapA. Epi-fluorescence microscopy (EFm) and high-resolution microscopy experiments showed that FtsZ and EzrA co-localize during the entire Spn cell cycle, whereas ZapA and ZapJ are late-arriving divisome proteins. Depletion and conditional mutants demonstrate that EzrA is essential in Spn and required for normal cell growth, size, shape homeostasis, and chromosome segregation. Moreover, EzrA(Spn) is required for midcell placement of FtsZ-rings and PG synthesis. Notably, overexpression of EzrA leads to the appearance of extra Z-rings in Spn. Together, these observations support a role for EzrA as a positive regulator of FtsZ-ring formation in Spn. Conversely, FtsZ is required for EzrA recruitment to equatorial rings and for the organization of PG synthesis. In contrast to EzrA depletion, which causes a bacteriostatic phenotype in Spn, depletion of FtsZ results in enlarged spherical cells that are subject to LytA-dependent autolysis. Co-immunoprecipitation and bacterial two-hybrid assays show that EzrA(Spn) is in complexes with FtsZ, Z-ring regulators (FtsA, SepF, ZapA, MapZ), division proteins (FtsK, StkP), and proteins that mediate peptidoglycan synthesis (GpsB, aPBP1a), consistent with a role for EzrA at the interface of cell division and PG synthesis. In contrast to the essentiality of FtsZ and EzrA, ZapA and SepF have accessory roles in regulating pneumococcal physiology. We further show that ZapA interacts with a non-ZapB homolog, named here as ZapJ, which is conserved in Streptococcus species. The absence of the accessory proteins, ZapA, ZapJ, and SepF, exacerbates growth defects when EzrA is depleted or MapZ is deleted. Taken together, these results provide new information about the spatially and temporally distinct proteins that regulate FtsZ-ring organization and cell division in Spn.
