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Role and function of Class III LitR, a photosensor homolog from Burkholderia multivorans ATCC 17616 in the regulation of folate production.

ABSTRACT: The LitR/CarH protein family is a adenosyl B12 (AdoB12)-dependent photoreceptor with DNA-binding activity and its homologs are widely distributed in the genome of diverged bacterial genera. Here, we studied a role and function of a LitR homolog from a Gram-negative soil bacteria Burkholderia multivorans, which has no AdoB12-binding domain. Transcriptome analysis revealed the existence of 19 light-induced genes (folE, cfa, litS, a Photolyase gene phrB2, cryB2) located in the flanking region of litR. Gene disruption of litR caused a constitutive expression of all light-inducible genes, while the mutation of the light-dependent expressed sigma factor litS abolished the transcription in cryB2, phrB2 operon and cfa operon, indicating that LitR and LitS play a central role in the light-inducible transcription. Gel-shift assay showed that a recombinant protein LitR specifically binds to the promoter region of litR and folE2 operon, and its ability was lowered by UV-A illumination. LitR absorbs maximally near 340 nm, and exhibited a photocyclic response and light-dependent subunit dissociation. Furthermore, the litR mutant produced a 20-fold high intracellular folate compared to the wild-type strain. Overall, the line of evidence suggest that Class III LitR light-dependently regulates the transcription of litR itself and folE2-litS operon, leading the production of folate, and then expressed RNA polymerase-LitS directs the transcription for phrB2 operon and cfa operon. Light-dependent characteristics suggests that Class III LitR in complex with a UV-A absorbing molecule has a novel light-sensing mechanism.

ORGANISM(S): Burkholderia multivorans ATCC 17616 Burkholderia multivorans

PROVIDER: GSE112472 | GEO | 2018/10/09

REPOSITORIES: GEO

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Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12M-BM-:C. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR. V. salmonicida wild type strain LFI1238 (control) and the isogenic DlitR mutant were grown as statical biofilm in SWT medium, at 4M-BM-0C and harvested after 72 hours. Biological replicates for each sample: 4 wild type, 4 DlitR mutant (including one dye swap), independently grown and harvested. One replicate per array.

Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12M-BM-:C. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR. V. salmonicida wild type strain LFI1238 (control) and the isogenic DlitR deletion mutant were grown in suspension in SWT medium at 8M-BM-0C with 200 rpm and harvested at OD=0.8. Biological replicates for each sample: 4 wild type, 4 DlitR mutant (including one dye swap), independently grown and harvested. One replicate per array.

Project description:The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the V. cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production. Importance: TcpP homologs are widespread throughout the Vibrio genus; however, the only protein in this family described thus far is a V. cholerae virulence regulator. Here we show that HbtR, the TcpP homolog in V. fischeri, has both a biological role and regulatory pathway completely unlike that in V. cholerae. Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of the E. scolopes light organ. While LitR becomes activated within the crypts, and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion of V. fischeri cells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival of V. fischeri outside of its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.

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Role and function of Class III LitR, a photosensor homolog from Burkholderia multivorans ATCC 17616 in the regulation of folate production.

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