Type III protein secretion systems (T3SS) are encoded by several pathogenic or symbiotic bacteria. The central component of this nanomachine is the needle complex. Here we show in a Salmonella Typhimurium T3SS that assembly of the needle filament of this structure requires OrgC, a protein encoded within the T3SS gene cluster. Absence of OrgC results in significantly reduced number of needle substructures but does not affect needle length. We show that OrgC is secreted by the T3SS and that exogenous addition of OrgC can complement a ∆orgC mutation. We also show that OrgC interacts with the needle filament subunit PrgI and accelerates its polymerization into filaments in vitro. The structure of OrgC shows a novel fold with a shared topology with a domain from flagellar capping proteins. These findings identify a novel component of T3SS and provide new insight into the assembly of the type III secretion machine.
The 20 PDB coordinates, the assigned chemical shifts, and the restraints used in the NMR structure determination were deposited at the RCSB Protein Data Bank with the accession code PDB ID 6CJD and BMRB ID 30417. The above data were used to generate Fig. 7, Figure 6-figure supplement 1, Figure 7-figure supplement 1, 2, 3, and 4, and Supplementary File 1.
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
© 2018, Kato et al.
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Polyamines are biologically ubiquitous cations that bind to nucleic acids, ribosomes, and phospholipids and, thereby, modulate numerous processes, including surface motility in Escherichia coli. We characterized the metabolic pathways that contribute to polyamine-dependent control of surface motility in the commonly used strain W3110 and the transcriptome of a mutant lacking a putrescine synthetic pathway that was required for surface motility. Genetic analysis showed that surface motility required type 1 pili, the simultaneous presence of two independent putrescine anabolic pathways, and modulation by putrescine transport and catabolism. An immunological assay for FimA—the major pili subunit, reverse transcription quantitative PCR of fimA, and transmission electron microscopy confirmed that pili synthesis required putrescine. Comparative RNAseq analysis of a wild type and ΔspeB mutant which exhibits impaired pili synthesis showed that the latter had fewer transcripts for pili structural genes and for fimB which codes for the phase variation recombinase that orients the fim operon promoter in the ON phase, although loss of speB did not affect the promoter orientation. Results from the RNAseq analysis also suggested (a) changes in transcripts for several transcription factor genes that affect fim operon expression, (b) compensatory mechanisms for low putrescine which implies a putrescine homeostatic network, and (c) decreased transcripts of genes for oxidative energy metabolism and iron transport which a previous genetic analysis suggests may be sufficient to account for the pili defect in putrescine synthesis mutants. We conclude that pili synthesis requires putrescine and putrescine concentration is controlled by a complex homeostatic network that includes the genes of oxidative energy metabolism.
The model Gram-negative plant pathogen Pseudomonas syringae utilises hundreds of transcription factors (TFs) to regulate its functional processes, including virulence and metabolic pathways that control its ability to infect host plants. Although the molecular mechanisms of regulators have been studied for decades, a comprehensive understanding of genome-wide TFs in Psph 1448A remains limited. Here, we investigated the binding characteristics of 170 of 301 annotated TFs through chromatin immunoprecipitation sequencing (ChIP-seq). Fifty-four TFs, 62 TFs, and 147 TFs were identified in top-level, middle-level, and bottom-level, reflecting multiple higher-order network structures and direction of information flow. More than 40,000 TF pairs were classified into 13 three-node submodules which revealed the regulatory diversity of TFs in Psph 1448A regulatory network. We found that bottom-level TFs performed high co-associated scores to their target genes. Functional categories of TFs at three levels encompassed various regulatory pathways. Three and 25 master TFs were identified to involve in virulence and metabolic regulation, respectively. Evolutionary analysis and topological modularity network revealed functional variability and various conservation of TFs in P. syringae (Psph 1448A, Pst DC3000, Pss B728a, and Psa C48). Overall, our findings demonstrated a global transcriptional regulatory network of genome-wide TFs in Psph 1448A. This knowledge can advance the development of effective treatment and prevention strategies for related infectious diseases.