Lopez-Sanchez, A. | bioRxiv Science

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↗