Leal-Morales, A. | bioRxiv Science

bioRxiv2023

The regulation of polar flagella assembly in Pseudomonas putida

The Gram-negative bacterium Pseudomonas putida bears a tuft of flagella at a single cell pole. New flagella must be assembled de novo every cell cycle to secure motility of both daughter cells. Here we show that the coordinated action of FimV, FlhF and FleN sets the location, timing and number of flagella assembled. The polar landmark proteins FimV and FlhF are independently targeted to the nascent new pole during or shortly after cell division, but FimV stabilizes FlhF association with the cell poles. FlhF determines the polar position of the flagella by targeting early flagellar components to the cell pole and preventing their nucleation at non-polar sites. FlhF also promotes efficient flagellar assembly and indirectly stimulates Class III flagellar promoter activation by promoting secretion of the anti-FliA anti-{sigma} factor FlgM. The MinD-like ATPase FleN partitions between the cell poles and the cytoplasm. Cytoplasmic FleN regulates flagellar number by preventing excessive accumulation of FlhF at the cell poles that may otherwise lead to hyperflagellation, likely by antagonizing FleQ-dependent transcriptional activation. FimV is essential to FleN polar location. FimV and FleN temporally regulate the onset of flagellar assembly by preventing premature polar targeting of FlhF and the ensuing premature targeting of additional flagellar components. Our results shed new light on the mechanisms that ensure the timely assembly of the appropriate number of flagella at the correct polar location in polarly flagellated bacteria. HIGHLIGHTSO_LIFimV, FlhF and FleN determine the position, number and timing of flagellar assembly C_LIO_LIFimV is essential to the normal intracellular distribution of FlhF and FleN C_LIO_LIFlhF restricts flagellar location to the cell poles and promotes efficient assembly C_LIO_LISoluble, cytoplasmic FleN prevents polar FlhF accumulation and hyperflagellation C_LIO_LIPole-bound FimV and FleN prevent premature FlhF recruitment and flagellar assembly C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/571843v5_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f57b6dorg.highwire.dtl.DTLVardef@7a38c7org.highwire.dtl.DTLVardef@259c58org.highwire.dtl.DTLVardef@13b6f20_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Transcriptional organization and regulation of the Pseudomonas putida flagellar system

A single region of the Pseudomonas putida genome, designated the flagellar cluster, includes 59 genes potentially involved in the biogenesis and function of the flagellar system. Here we combine bioinformatics and in vivo gene expression analyses to clarify the transcriptional organization and regulation of the flagellar genes in the cluster. We have identified eleven flagellar operons and characterized twenty-two primary and internal promoter regions. Our results indicate that synthesis of the flagellar apparatus and core chemotaxis machinery is regulated by a three-tier cascade in which fleQ is a Class I gene, standing at the top of the transcriptional hierarchy. FleQ- and{sigma} 54-dependent Class II genes encode most components of the flagellar structure, part of the chemotaxis machinery and multiple regulatory elements, including the flagellar{sigma} factor FliA. FliA activation of Class III genes enables synthesis of the filament, one stator complex and completion of the chemotaxis apparatus. Accessory regulatory proteins and an intricate operon architecture add complexity to the regulation by providing feedback and feed-forward loops to the main circuit. Because of the high conservation of the gene arrangement and promoter motifs, we believe that the regulatory circuit presented here may also apply to other environmental pseudomonads. ORIGINALITY-SIGNIFICANCE STATEMENTThis is the first integrative study of the flagellar transcriptional cascade in Pseudomonas putida. Our results provide a new transcriptional organization featuring several operons with a nested architecture, detailed regulatory characterization of twenty-two flagellar promoters and a novel hierarchy for the regulatory circuit of the flagellar transcriptional cascade. The results presented represent a significant departure from previous models for other related bacteria. High conservation of flagellar gene organization and promoter sequences suggest that our observations may be relevant to other pseudomonads.

microbiology↗