Mihaela Pruteanu

Bacterial metabolism plays a fundamental role in gut microbiota ecology and host–microbiome interactions. Yet the metabolic capabilities of most gut bacteria have remained unknown. Here we report growth characteristics of 96 phylogenetically diverse gut bacterial strains across 4 rich and 15 defined media. The vast majority of strains (76) grow in at least one defined medium, enabling accurate assessment of their biosynthetic capabilities. These do not necessarily match phylogenetic similarity, thus indicating a complex evolution of nutritional preferences. We identify mucin utilizers and species inhibited by amino acids and short-chain fatty acids. Our analysis also uncovers media for in vitro studies wherein growth capacity correlates well with in vivo abundance. Further value of the underlying resource is demonstrated by correcting pathway gaps in available genome-scale metabolic models of gut microorganisms. Together, the media resource and the extracted knowledge on growth abilities widen experimental and computational access to the gut microbiota.

The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease, but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens (C. perfringens), have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin, and junctional adhesion molecule 1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin, and we demonstrate that this process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of E-cadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins, which is likely to enable access to the extracellular matrix components by the paracellular pathway.

The aim of this study was to investigate the diversity and composition of the intestinal microbiota of elderly subjects using a combination of culture-dependent techniques and 16S rRNA gene amplicon sequencing. The study was performed as part of the ELDERMET project, in which 368 faecal samples were assessed for viable numbers of Bifidobacterium spp., Lactobacillus spp. and Enterobacteriaceae on selective agar. However, the Bifidobacterium selective medium used also supported the growth of Clostridium perfringens, which appeared as distinct colonies and were subsequently characterized phenotypically and genotypically. All the isolates were confirmed as toxin biotype A producers. In addition, three isolates tested also had the genetic determinants for the β2 toxin. Of the 368 faecal samples assessed, C. perfringens was detected in 28 samples (7.6 %). Moreover, C. perfringens was observed in samples from subjects in all the residence locations assessed but was most prevalent in subjects from long-stay residential care, with 71.4 % of the samples (63.2 % of the subjects) being from this residence location, and with a shedding level in excess of 106 c.f.u. (g faeces)−1. Microbiota profiling revealed some significant compositional changes across both the family and genus taxonomic levels between the C. perfringens-positive and -negative datasets. Levels of culturable Bifidobacterium spp. and Lactobacillus spp. were significantly (P<0.05) lower in the C. perfringens-positive samples. Sequence-based methods also confirmed a significant difference in the Bifidobacterium spp. level (P106 c.f.u. (g faeces)−1] of C. perfringens in stool samples may be indicative of a less healthy microbiota in the intestine of elderly people in long-stay residential care.

Summary: RprA is a small regulatory RNA known to weakly affect the translation of σ^S (RpoS) in Escherichia coli. Here we demonstrate that csgD, which encodes a stationary phase‐induced biofilm regulator, as well as ydaM, which encodes a diguanylate cyclase involved in activating csgD transcription, are novel negatively controlled RprA targets. As shown by extensive mutational analysis, direct binding of RprA to the 5′‐untranslated and translational initiation regions of csgD mRNA inhibits translation and reduces csgD mRNA levels. In the case of ydaM mRNA, RprA base‐pairs directly downstream of the translational start codon. In a feedforward loop, RprA can thus downregulate > 30 YdaM/CsgD‐activated genes including those for adhesive curli fimbriae. However, during early stationary phase, when csgD transcription is strongly activated, the synthesis of csgD mRNA exceeds that of RprA, which allows the accumulation of CsgD protein. This situation is reversed when csgD transcription is shut off – for instance, later in stationary phase or during biofilm formation – or by conditions that further activate RprA expression via the Rcs two‐component system. Thus, antagonistic regulation of csgD and RprA at the mRNA level integrates cell envelope stress signals with global gene expression during stationary phase and biofilm formation.

Background. Proteolytic degradation of the extracellular matrix, a feature of mucosal homeostasis and tissue renewal, also contributes to the complications of

Summary: UV irradiation damages DNA and activates expression of genes encoding proteins helpful for survival under DNA stress. These proteins are often deleterious in the absence of DNA damage. Here, we investigate mechanisms used to regulate the levels of DNA‐repair proteins during recovery by studying control of the nucleotide excision repair (NER) protein UvrA. We show that UvrA is induced after UV irradiation and reaches maximum levels between ∼20 and 120 min post UV. During post‐UV recovery, UvrA levels decrease principally as a result of ClpXP‐dependent protein degradation. The rate of UvrA degradation depends on the amount of unrepaired pyrimidine dimers present; this degradation rate is initially slow shortly after UV, but increases as damage is repaired. This increase in UvrA degradation as repair progresses is also influenced by protein–protein interactions. Genetic and in vitro experiments support the conclusion that UvrA–UvrB interactions antagonize degradation. In contrast, Mfd appears to act as an enhancer of UvrA turnover. Thus, our results reveal that a complex network of interactions contribute to tuning the level of UvrA in the cell in response to the extent of DNA damage and nicely mirror findings with excision repair proteins from eukaryotes, which are controlled by proteolysis in a similar manner.

Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment.Escherichia coliZntR, a zinc-responsive transcriptional regulator, was identified by proteomic experiments as a likely ClpXP substrate, suggesting that protein turnover may play a role in regulation of zinc homeostasis. When intracellular zinc levels are high, ZntR activates expression of ZntA, an ATPase essential for zinc export. We find that ZntR is degraded in vivo in a manner dependent on both the ClpXP and Lon proteases. However, ZntR degradation decreases in the presence of high zinc concentrations, the level of ZntR rises, and transcription of thezntAexporter is increased. Mutagenesis experiments reveal that zinc binding does not appear to be solely responsible for the zinc-induced protection from proteolysis. Therefore, we tested whether DNA binding was important in the zinc-induced stabilization of ZntR by mutagenesis of the DNA binding helices. Replacement of a conserved arginine (R19A) in the DNA binding domain both enhances ZntR degradation and abolishes zinc-induced transcriptional activation ofzntA. Biochemical and physical analysis of ZntR^R19Ademonstrates that it is structurally similar to, and binds zinc as well as does, the wild-type protein but is severely defective in binding DNA. Thus, we conclude that two different ligands—zinc and DNA—function together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.