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Abstract 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.

Transcription factors (TFs) play important roles in regulating multiple biological processes by binding to promoter regions and regulating the global gene transcription levels. Pseudomonas syringae is a Gram-negative phytopathogenic bacterium harbouring 301 putative TFs in its genome, approximately 50 of which are responsible for virulence-related gene and pathway regulation. Over the past decades, RNA sequencing, chromatin immunoprecipitation sequencing, high-throughput systematic evolution of ligands by exponential enrichment, and other technologies have been applied to identify the functions of master regulators and their interactions in virulence-related pathways. This review summarises the recent advances in the regulatory networks of TFs involved in the type III secretion system (T3SS) and non-T3SS virulence-associated pathways, including motility, biofilm formation, quorum sensing, nucleotide-based secondary messengers, phytotoxins, siderophore production, and oxidative stress. Moreover, this review discusses the future perspectives in terms of TF-mediated pathogenesis mechanisms and provides novel insights that will help combat P. syringae infections based on the regulatory networks of TFs.

ABSTRACT 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. IMPORTANCE Although bacterial TFs regulate transcription by binding to specific sequences at the promoter region, little is known about the mechanisms and functions of TFs binding within the CDS. In this study, we show that bacterial TFs have same binding pattern in both CDS and promoter regions, and we reveal three regulatory mechanisms of CDS-bound TF that together demonstrate the complexity of the regulatory mechanisms of bacterial TFs and the wide spread of internal cryptic promoters in CDS.