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Transcription factors (TFs) regulate transcription by binding to the specific sequences at the promoter region. However, the mechanisms and functions of TFs binding within the coding sequences (CDS) remain largely elusive in prokaryotes. To this end, we collected 409 data sets for bacterial TFs, including 104 chromatin immunoprecipitation sequencing (ChIP-seq) assays and 305 data sets from the systematic evolution of ligands by exponential enrichment (SELEX) in seven model bacteria. Interestingly, these TFs displayed the same binding capabilities for both coding and intergenic regions. Subsequent biochemical and genetic experiments demonstrated that several TFs bound to the coding regions and regulated the transcription of the binding or adjacent genes. Strand-specific RNA sequencing revealed that these CDS-binding TFs regulated the activity of the cryptic promoters, resulting in the altered transcription of the corresponding antisense RNA. TF RhpR hindered the transcriptional elongation of a subgenic transcript within a CDS. A ChIP-seq and Ribo-seq coanalysis revealed that RhpR influenced the translational efficiency of binding genes. Taken together, the present study reveals three regulatory mechanisms of CDS-bound TFs within individual genes, operons, and antisense RNAs, which demonstrate the variability of the regulatory mechanisms of TFs and expand upon the complexity of bacterial transcriptomes.

Transcription factors (TFs) play important roles in regulating multiple biological processes by binding to promoter regions and regulating the global gene transcription levels.

Background Sufficient nutrition contributes to rapid translational elongation and protein synthesis in eukaryotic cells and prokaryotic bacteria. Fast synthesis and accumulation of type III secretion system (T3SS) proteins conduce to the invasion of pathogenic bacteria into the host cells. However, the translational elongation patterns of T3SS proteins in pathogenic bacteria under T3SS-inducing conditions remain unclear. Here, we report a mechanism of translational elongation of T3SS regulators, effectors and structural protein in four model pathogenic bacteria (Pseudomonas syringae, Pseudomonas aeruginosa, Xanthomonas oryzae and Ralstonia solanacearum) and a clinical isolate (Pseudomonas aeruginosa UCBPP-PA14) under nutrient-limiting conditions. We proposed a luminescence reporter system to quantitatively determine the translational elongation rates (ERs) of T3SS regulators, effectors and structural protein under different nutrient-limiting conditions and culture durations. Results The translational ERs of T3SS regulators, effectors and structural protein in these pathogenic bacteria were negatively regulated by the nutrient concentration and culture duration. The translational ERs in 0.5× T3SS-inducing medium were the highest of all tested media. In 1× T3SS-inducing medium, the translational ERs were highest at 0 min and then rapidly decreased. The translational ERs of T3SS regulators, effectors and structural protein were inhibited by tRNA degradation and by reduced levels of elongation factors (EFs). Conclusions Rapid translational ER and synthesis of T3SS protein need adequate tRNAs and EFs in nutrient-limiting conditions. Numeric presentation of T3SS translation visually indicates the invasion of bacteria and provides new insights into T3SS expression that can be applied to other pathogenic bacteria.

The opportunistic pathogen Pseudomonas aeruginosa has evolved several systems to adapt to complex environments. The GntR family proteins play important roles in the regulation of metabolic processes and bacterial pathogenesis. In this study, we uncovered that the gene clusters of PA1513-PA1518 and PA1498-PA1502 in P. aeruginosa are required for uric acid and glyoxylate metabolism respectively. We also identified a GntR family regulator UgmR that is involved in regulation of uric acid and glyoxylate metabolism. Promoter activity measurement and biochemical assays revealed that the UgmR directly represses the transcriptional activity of PA1513-PA1518 and PA1498-PA1502, and this inhibition was relieved by the addition of uric acid. Importantly, further experiments showed that UgmR also participates in the glyoxylate shunt. Collectively, these findings contribute to a better understanding of the UgmR factor involved in uric acid and glyoxylate metabolism, which provide insights into the complex metabolic pathways in P. aeruginosa.