Biology subjects

Govantes, F.

Publications and source records attributed to Govantes, F..

5 recordsLinked to original sources

bioRxiv2025

FimV, ParC and ParP coordinate polar location of the chemosensory arrays in Pseudomonas putida

Motile bacteria rely on chemotaxis to promote directional motion in response to specific environmental signals. The bacterial chemotaxis machinery is typically arranged in highly ordered arrays containing multiple chemoreceptors and signal transduction proteins. Pseudomonas putida is a polarly flagellated soil bacterium displaying chemotaxis towards numerous organic compounds. Here we demonstrate the involvement of the polar landmark proteins FimV, ParC and ParP in the assembly of the flagella-associated chemosensory arrays at the cell poles in P. putida. Our results show that FimV, ParC and ParP are sequentially recruited to the new cell pole during the cell cycle. FimV is located at the new cell pole concurrently with cell division, and is further required for ParC recruitment. FimV and ParC subsequently stimulate polar localization of ParP. Bacterial two-hybrid assays and structural modeling suggest the involvement of direct protein-protein interactions between the three proteins. We also show that ParC and ParP are decisive to the assembly of fully formed polar chemosensory arrays, by recruiting chemosensory proteins to the cell pole, promoting the stable association of the histidine kinase CheA with the cell pole and enabling the assembly of the functional array structure. Polar targeting of both flagella and chemosensory arrays to the new pole in the timespan of a cell cycle is a means to secure correct segregation of the motility apparatus to both daughter cells during cell division. HIGHLIGHTSO_LIFimV, ParC and ParP promote chemosensory array assembly at the new cell pole C_LIO_LIFimV is essential to the normal intracellular distribution of ParC and ParP C_LIO_LIParC and ParP recruit and stabilize the array components at the cell poles C_LIO_LIParP and CheA promote polar clustering of the chemoreceptors C_LIO_LIAssembly in the timespan of the cell cycle endows chemotaxis to both daughter cells C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=120 HEIGHT=200 SRC="FIGDIR/small/684771v3_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@143bbe3org.highwire.dtl.DTLVardef@1e46d26org.highwire.dtl.DTLVardef@1b9fea9org.highwire.dtl.DTLVardef@1fe3d6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

HsbA represses stationary phase biofilm formation in Pseudomonas putida

Pseudomonas putida biofilm growth is associated to nutrient-sufficient conditions and biofilm dispersal is induced by nutrient starvation, signaled by the stringent response-associated nucleotide alarmone (p)ppGpp. We have used transcriptomic analysis to show that (p)ppGpp regulates the hsbAR-hptB gene cluster, encoding components of a phosphorelay pathway and an anti-{sigma} factor antagonist, and cfcR, encoding a response regulator with diguanylate cyclase (DGC) activity. Transcription of hsbAR-hptB and cfcR is RpoS-dependent and induced by stationary phase and the stringent response. A {Delta}hsbA mutant resumed biofilm formation after dispersal in late stationary phase and displayed increased pellicle formation at the medium-air interphase and Congo Red adsorption. All these phenotypes were traced down to increased c-di-GMP levels in stationary phase, dependent on the activity of CfcR and its cognate sensor kinase, CfcA. HsbA was reversibly phosphorylated by the combined action of HptB and HsbR. HsbA phosphorylation conditioned its interaction with CfcR and CfcA and the subcellular distribution of the three proteins. In spite of this, HsbA retained its ability to prevent biofilm formation regardless of its phosphorylation state. Our results support a model in which HsbA forms a complex with CfcR to inhibit its DGC activity regardless of its phosphorylation state. Upon HsbA dephosphorylation, this complex is recruited to the cell membrane by CfcA to strengthen the inhibitory effect. While this pathway contributes to biofilm dispersal by denying de novo c-di-GMP synthesis during nutrient starvation, it may also enable quick restoration of the biofilm phenotype to colonize new sites or during biofilm maturation. HIGHLIGHTSO_LITranscription of hsbAR-hptB is activated by the stringent response and RpoS. C_LIO_LIHptB and HsbR control the phosphorylation state of HsbA. C_LIO_LIHsbA prevents biofilm formation in stationary phase. C_LIO_LIHsbA inhibits the DGC activity of CfcR regardless of its phosphorylation state. C_LIO_LIUnphosphorylated HsbA elicits formation of a membrane-bound HsbA-CfcR-CfcA complex. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/625174v3_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1ca16dorg.highwire.dtl.DTLVardef@1aa17e2org.highwire.dtl.DTLVardef@6277forg.highwire.dtl.DTLVardef@b0f2b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

microbiology↗

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↗

bioRxiv2022

Exploring generality of experimental conformational changes with AlphaFold predictions

Structural predictions have matched the accuracy of experimental structures in the case of close homologues, outperformed docking methods for multimeric complexes and helped sampling the conformational landscape of transporters and receptors. Such successes prompt the question whether predictions can be used to relate experimental structures in the context of available knowledge. LysR-type transcriptional regulators (LTTR) constitute the most common family of bacterial regulators. Intriguingly, their experimental structures are remarkably diverse. The active species, composed of flexible monomers dimerizing through their N- and C-terminal domains in a circular arrangement, differ across LTTR, due to intrinsic sequence differences or because crystals stabilize diverse snapshots of a common dynamic mechanism. We have used AlphaFold2 (AF) to interrogate the experimental AtzR structure in the context of predictions guided towards the different hetero-multimeric conformations known for other LTTR. Our approach drives AF prediction with the structure-based selection of the information input through sequence alignment and template conformation, linked to examination of the energy with PISA and interactions with ALEPH.

biophysics↗

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↗