Effect of hmuY and hmuR or husA and husB genes deletion on Porphyromonas gingivalis gene expression
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ABSTRACT:
ORGANISM(S): Porphyromonas gingivalis
PROVIDER: GSE275439 | GEO | 2024/12/31
REPOSITORIES: GEO
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Project description:Porphyromonas gingivalis is one of the main etiological agents responsible for initiating and progressing periodontal diseases in humans. P. gingivalis does not synthesize heme, and it uses host hemoproteins as a source of this molecule. The main heme uptake mechanism utilized by P. gingivalis is the Hmu system composed of six proteins where HmuY (hemophore-like protein) and HmuR (TonB-dependant outer membrane heme receptor) play a crucial role in heme supply. The second heme uptake mechanism is the Hus system composed of four proteins, with HusA (hemophore-like protein) and HusB (TonB-dependant outer membrane receptor), having a supporting role in heme uptake. Heme and iron starvation influence the P. gingivalis phenotype, therefore the aim was to determine the influence of deletion of HmuY and HmuR (ΔhmuYR strain) or HusA and HusB (ΔhusAB strain) encoding genes on P. gingivalis phenotype including the impact on protein production in response to the analyzed deletion. The mutant strains were constructed in wild-type A7436 P. gingivalis strain. Bacteria were grown in basal medium (BM) supplemented with 7.7 µM heme. After analysis of bacteria lysates with SDS-PAGE and CBB G-250 staining, in ΔhmuYR a protein (~45 kDa) was detected produced in higher amounts, than in the wild-type and the ΔhusAB strains. The gel band of interest, as well as its corresponding sections in the WT lanes were excised for bottom-up LC-MS analysis, and the protein was identified as FimA.
2024-12-28 | PXD054703 | Pride
Project description:Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane. In this work we determined the C. crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur are those encoding a putative high affinity ABC uptake system (znuCBA), three TonB-dependent receptors (znuD, znuE and znuF) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by β-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuD and znuE mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems.
2014-04-29 | GSE57136 | GEO
Project description:Porphyromonas gingivalis is a major pathogen associated with the microbial biofilm-mediated disease chronic periodontitis. P. gingivalis has an obligate requirement for iron and protoporphyrin IX which it satisfies by transporting heme and iron liberated from the human host. The level of cellular iron in P. gingivalis affects the expression of a distinct iron-associated regulon of 64 genes and low iron invokes an iron sparing response. Iron homeostasis is usually mediated in Gram-negative bacteria at the transcriptional level by the Ferric Uptake Regulator (Fur). There is a single predicted P. gingivalis Fur superfamily orthologue named Har (heme associated regulator) that lacks the conserved metal binding residues found in other Fur orthologues. We show that Har binds both heme and ferrous iron resulting in a conformational change in the protein. Har was unable to complement the Escherichia coli H1780 fur mutant and there was no change in cellular metal content in a P. gingivalis Har mutant compared with the wild-type. The Har regulon of 44 genes is not predicted to play a role in iron homeostasis. Together these data indicated that Har does not regulate iron homeostasis in P. gingivalis. However, Har was required for heme-responsive biofilm development and its regulon overlapped P. gingivalis regulons previously identified after growth in heme limitation or as a homotypic biofilm. P. gingivalis is unique as an iron-dependent Gram-negative bacterium with a single heme-binding Fur superfamily orthologue, Har, that does not regulate iron homeostasis.
2015-04-01 | GSE37099 | GEO
Project description:Porphyromonas gingivalis is an anaerobic, Gram-negative oral pathogen associated with chronic periodontitis. P. gingivalis has an obligate requirement for heme which it obtains from the host. Heme availability has been linked to disease initiation and progression. In this study we used continuous culture of the bacterium to determine the effect of heme limitation and excess on the P. gingivalis proteome. Four biological replicates of whole cell lysate (WCL) and outer membrane vesicle (OMV) samples were digested with trypsin and analysed by tandem mass spectrometry and MaxQuant label-free quantification. In total, 1211 proteins were quantified with 108 and 49 proteins significantly changing in abundance more than 1.5-fold (p<0.05) in the WCLs and OMVs respectively. The proteins most upregulated in response to heme limitation were those involved in binding and transporting heme while the four proteins most upregulated in the heme excess condition constitute a putative heme efflux system. In general, the protein abundance ratios obtained for OMVs and WCLs agreed, indicating that changes to the OM protein composition are passed on to OMVs, however 16 proteins were preferentially packaged into OMVs in one condition more than the other. In particular, moonlighting cytoplasmic proteins were preferentially associated with OMVs under heme excess.
2018-05-18 | PXD009730 | Pride
Project description:Porphyromonas gingivalis is a major pathogen associated with the microbial biofilm-mediated disease chronic periodontitis. P. gingivalis has an obligate requirement for iron and protoporphyrin IX which it satisfies by transporting heme and iron liberated from the human host. The level of cellular iron in P. gingivalis affects the expression of a distinct iron-associated regulon of 64 genes and low iron invokes an iron sparing response. Iron homeostasis is usually mediated in Gram-negative bacteria at the transcriptional level by the Ferric Uptake Regulator (Fur). There is a single predicted P. gingivalis Fur superfamily orthologue named Har (heme associated regulator) that lacks the conserved metal binding residues found in other Fur orthologues. We show that Har binds both heme and ferrous iron resulting in a conformational change in the protein. Har was unable to complement the Escherichia coli H1780 fur mutant and there was no change in cellular metal content in a P. gingivalis Har mutant compared with the wild-type. The Har regulon of 44 genes is not predicted to play a role in iron homeostasis. Together these data indicated that Har does not regulate iron homeostasis in P. gingivalis. However, Har was required for heme-responsive biofilm development and its regulon overlapped P. gingivalis regulons previously identified after growth in heme limitation or as a homotypic biofilm. P. gingivalis is unique as an iron-dependent Gram-negative bacterium with a single heme-binding Fur superfamily orthologue, Har, that does not regulate iron homeostasis. Paired samples were compared on the same microarray using a two-colour system. A total of 6 paired microarray hybridizations were performed representing 6 biological replicates, where a balanced dye design was used, with the overall analysis including three microarrays where P. gingivalis 33277 samples were labeled with Cy3 and the paired ECR455 samples were labeled with Cy5 and three other microarrays where samples were labeled with the opposite combination of fluorophores.
2015-04-01 | E-GEOD-37099 | biostudies-arrayexpress
Project description:Cell-surface signaling is a sophisticated regulatory mechanism used by gram-negative bacteria to sense signals from outside the cell and transmit them into the cytoplasm. This regulatory system consists of an outer membrane-localized TonB-dependent receptor (TonB-dependent transducer), a cytoplasmic membrane-localized anti-sigma factor and an extracytoplasmic function (ECF) sigma factor. By microarray analysis we have identified the regulons of four novel P. aeruginosa signaling systems. For that, the ECF sigmas PA0149, PA2050, PA2093 and PA4896 have been overexpressed in P. aeruginosa and their target gene candidates have been identified using DNA microarray. Keywords: Overexpression of ECF sigma factors
2007-11-24 | GSE9657 | GEO
Project description:Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane. In this work we determined the C. crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur are those encoding a putative high affinity ABC uptake system (znuCBA), three TonB-dependent receptors (znuD, znuE and znuF) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by β-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuD and znuE mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems. The DNA microarray experiments were performed as described in (da Silva Neto et al., 2013). Briefly, cDNA was generated from 12.5 µg of total RNA and labeled with either Cy3 or Cy5 fluorescent dyes (FairPlay III Microarray Labeling System, Stratagene). Labeled cDNA samples were hybridized to a Caulobacter DNA oligo microarray (Agilent), and the arrays were scanned with an Agilent High Resolution Microarray Scanner. RNA from three independent biological cultures was used for DNA microarray analysis. We considered as differentially expressed genes those showing a minimum of 2-fold change relative to the control, considering at least three out of the four last probes for each gene (that are downstream of the translational start site) in at least two of three biological replicates. The values for the relative expression of each gene were obtained as the average of the four last probes.
2014-04-29 | E-GEOD-57136 | biostudies-arrayexpress
Project description:Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS senses signals from outside the cell and transmits them into the cytoplasm. It consists of a TonB-dependent outer membrane receptor, a cytoplasmic membrane-localized sigma factor regulator (or anti-sigma factor), and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa ECF sigma factors are involved in the regulation of iron uptake, we have identified a novel ECF sigma factor (PA0675) involved in the regulation of virulence. By microarray analysis of cells overexpressing PA0675 from the pMUM3 plasmid we have identified the genes regulated by this sigma factor. Two different samples are analyzed namely P. aeruginosa (pMMB67EH) (control/reference sample) and P. aeruginosa (pMUM3) (overexpressing the PA0675 ECF sigma factor). Two different replicates per sample are included.
2009-04-21 | E-GEOD-15697 | biostudies-arrayexpress
Project description:Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS senses signals from outside the cell and transmits them into the cytoplasm. It consists of a TonB-dependent outer membrane receptor, a cytoplasmic membrane-localized sigma factor regulator (or anti-sigma factor), and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa ECF sigma factors are involved in the regulation of iron uptake, we have identified a novel ECF sigma factor (PA0675) involved in the regulation of virulence. By microarray analysis of cells overexpressing PA0675 from the pMUM3 plasmid we have identified the genes regulated by this sigma factor.
2009-04-21 | GSE15697 | GEO
Project description:Transcription profiling of P. gingivalis W50 grown in continuous culture under conditions of heme-excess and heme-limitation.
2008-11-19 | GSE13375 | GEO