Posttranscriptional Regulation of tnaA by Protein-RNA Interaction Mediated by Ribosomal Protein L4 in Escherichia coli

ABSTRACT Escherichia coli ribosomal protein (r-protein) L4 has extraribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at nonpermissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rne temperature-sensitive [rne(Ts)] mutant strain. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and downregulates TnaA translation. We found that deletion of the 5′ or 3′ half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 to the tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary-phase bacterial growth, steady-state levels of tnaA mRNA increased but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon. IMPORTANCE Some ribosomal proteins have extraribosomal functions in addition to ribosome translation function. The extraribosomal functions of several r-proteins control operon expression by binding to own-operon transcripts. Previously, we discovered a posttranscriptional, RNase E-dependent regulatory role for r-protein L4 in the stabilization of stress-responsive transcripts. Here, we found an additional extraribosomal function for L4 in regulating the tna operon by L4-intergenic spacer mRNA interactions. L4 binds to the transcribed spacer RNA between tnaC and tnaA and alters the structural conformation of the spacer RNA, thereby reducing the translation of TnaA. Our study establishes a previously unknown L4-mediated mechanism for regulating gene expression, suggesting that bacterial cells have multiple strategies for controlling levels of tryptophanase in response to varied cell growth conditions.

MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently

ABSTRACTMvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries anmvaT-homologous gene,pmr. InPseudomonas putidaKT2440 bearing pCAR1,pmrand the chromosomally carried homologous genes,turAandturB, are transcribed at high levels, and Pmr interacts with TurA and TurBin vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome ofP. putidaKT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in theP. putidaKT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage.

2,4-Dichlorphenoxicacetic Acid at Low Concentrations Enhances Reproductive Ability and Oxidative Stress Resistance of Yeast Saccharomyces Cerevisiae

2,4-dichlorphenoxicacetic acid (2,4-D) is one of the most widely used herbicides withwell documented toxic effects on non-target organisms. In this study, the effect of lowconcentrations of 2,4-D on reproductive activity and resistance of yeast S. cerevisiae to oxidativestress was evaluated. Supplementation of the cultivation medium with 0.1-100 µM 2,4-D did notaffect the rate of yeast growth. In early stationary phase, yeast cultures grown with 0.1 and 1 µM2,4-D had higher number of reproductively active cells than control ones (without 2,4-D). Inexponential phase, S. cerevisiae cells grown in the presence of 1-100 µM 2,4-D were more resistantto hydrogen peroxide comparing to control ones. Thus, the herbicide increased reproductivepotential and cross-resistance to oxidative stress in yeast but the effective concentrations of 2,4-Dwere different for these phenomena. In summary, the results suggest possible involvement ofcertain hormetic mechanisms in the influence of 2,4-D at low concentrations on yeast

Propionibacterium-Produced Coproporphyrin III Induces Staphylococcus aureus Aggregation and Biofilm Formation

ABSTRACTThe majority of bacteria detected in the nostril microbiota of most healthy adults belong to three genera:Propionibacterium,Corynebacterium, andStaphylococcus. Among these staphylococci is the medically important bacteriumStaphylococcus aureus. Almost nothing is known about interspecies interactions among bacteria in the nostrils. We observed that crude extracts of cell-free conditioned medium fromPropionibacteriumspp. induceS. aureusaggregation in culture. Bioassay-guided fractionation implicated coproporphyrin III (CIII), the most abundant extracellular porphyrin produced by human-associatedPropionibacteriumspp., as a cause ofS. aureusaggregation. This aggregation response depended on the CIII dose and occurred during early stationary-phase growth, and a low pH (~4 to 6) was necessary but was not sufficient for its induction. Additionally, CIII induced plasma-independentS. aureusbiofilm development on an abiotic surface in multipleS. aureusstrains. In strain UAMS-1, CIII stimulation of biofilm depended onsarA, a key biofilm regulator. This study is one of the first demonstrations of a small-molecule-mediated interaction among medically relevant members of the nostril microbiota and the first description of a role for CIII in bacterial interspecies interactions. Our results indicate that CIII may be an important mediator ofS. aureusaggregation and/or biofilm formation in the nostril or other sites inhabited byPropionibacteriumspp. andS. aureus.IMPORTANCEVery little is known about interspecies interactions among the bacteria that inhabit the adult nostril, includingStaphylococcus aureus, a potential pathogen that colonizes about a quarter of adults. We demonstrated that coproporphyrin III (CIII), a diffusible small molecule excreted by nostril- and skin-associatedPropionibacteriumspp., inducesS. aureusaggregation in a manner dependent on dose, growth phase, and pH. CIII also inducesS. aureusto form a plasma-independent surface-attached biofilm. This report is the first description of a role for CIII in bacterial interspecies interactions at any human body site and a novel demonstration that nostril microbiota physiology is influenced by small-molecule-mediated interactions.

Effects of Growth Phase and Temperature onσBActivity within aListeria monocytogenesPopulation: Evidence for RsbV-Independent Activation ofσBat Refrigeration Temperatures

The alternative sigma factorσBofListeria monocytogenesis responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation ofσBwithin populations ofL.monocytogenesEGD-e wild-type, ΔsigB,and ΔrsbVthroughout growth at both 4°C and 37°C, using a reporter fusion that couples expression of EGFP to the stronglyσB-dependent promoter oflmo2230. A similarσBactivation pattern within the population was observed in wt-egfpat both temperatures, with the highest induction ofσBoccurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction ofσBactivity was heterogeneous, with only a proportion of the cells in the wt-egfppopulation being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation ofσBwas observed during growth at 4°C. This result suggests that an alternative route toσBactivation exists in the absence of RsbV, a finding that is not explained by the current model forσBregulation.