Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of Porphyromonas gingivalis wild type and mutant PG1432::ermF to identify genes which have their expression influenced by PG1432

ABSTRACT: The aim of this experiment is to ascertain which genes have their expression influenced by PG1432. PG1432 encodes a putative sensor histidine kinase which is part of two component signal transduction that was highly up-regulated in P. gingivalis mature biofilm cells. PG1432 was disrupted in P. gingivalis W50 by insertion of ermF cassette. The strain W50 PG1432 mutant was designated ECR222. Two independent replicates of each strain were grown for 30 days in continuous culture in chemostats using brain heart infusion media supplemented with 0.5 mg mL-1 cysteine and 5 ï¿½~Aï¿½g mL-1 hemin. Although strain W50 formed sufficient biofilm to use for total RNA harvest and microarray analysis, strain ERC222 formed only sparse biofilm showing that disruption of PG1432 impaired biofilm formation by this mutant. We were unable to collect sufficient biofilm cells of strain ECR222 therefore the microarray analysis total RNAs were harvested only from planktonic phase cells. The transcriptome of the mutant ECR222 were compared to that of strain W50.

ORGANISM(S): Porphyromonas gingivalis

SUBMITTER: Alvin Lo

PROVIDER: E-TABM-546 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

FimR and FimS: biofilm formation and gene expression in Porphyromonas gingivalis.

Lo Alvin A Seers Christine C Dashper Stuart S Butler Catherine C Walker Glenn G Walsh Katrina K Catmull Deanne D Hoffmann Brigitte B Cleal Steven S Lissel Patricia P Boyce John J Reynolds Eric E

Journal of bacteriology 20100108 5

Porphyromonas gingivalis is a late-colonizing bacterium of the subgingival dental plaque biofilm associated with periodontitis. Two P. gingivalis genes, fimR and fimS, are predicted to encode a two-component signal transduction system comprising a response regulator (FimR) and a sensor histidine kinase (FimS). In this study, we show that fimS and fimR, although contiguous on the genome, are not part of an operon. We inactivated fimR and fimS in both the afimbriated strain W50 and the fimbriated ...[more]

PMID: 20061484

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Project description:The aim of this study was to characterize the basal expression of OC, MOD and SV of newborn rats (3-5 day old) and its changes by shortâterm cultivation (24 hrs) and mild hypoxia (5 hrs). Three varieties of tissue samples were analyzed (i) Freshly prepared tissue, used as control; (ii) cultures held under normoxic conditions and (iii) cultures exposed during cultivation to 5 h hypoxia. The gene expression was analyzed using the Affymetrix GeneChip RN-U34. The functional specificity of the cochlea regions are well reflected by the gene expression pattern. For example, the basal expression of Organ of Corti is characterized by genes involved in potassium and calcium homeostasis. The MOD is characterized by genes involved in regulation of maturation of neuronal cells. The SV is characterized by genes involved in ion homeostasis and blood flow. Cultivation dramatically changes the gene expression. In contrast, mild hypoxia showed only small changes. The changes are interpreted as stress response. We used this analysis as screening approach to detect significant changes in gene expression and used the RT-PCR analyses of independent sister cultures for confirmation. Correlation analysis showed close relations of the expression levels when aliquots of the biological material were compared. However, comparisons of the expression levels received by microarray and by RT-PCR analysis of independent sister samples resulted in 70-90 % in similar predictions. This data indicate that possible discrepancies has its basis in biological sampling rather than factors of methodical errors on the level of RT-PCR. Experiment Overall Design: Pooled samples of the following cochlea preparations were studied: Experiment Overall Design: (i) Native preparations of Organ of Corti, modiolus and stria vascularis from newborn rats used as controls; (ii) Organotypic cultures of these preparations under normoxic conditions (24 h); (iii) Cultures which were exposed to 5 h hypoxia during cultivation. Samples were collected for total RNA immediately after preparation of the inner ear structures (native samples) or 24 h after onset of cultivation (normoxia, hypoxia).

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.

Dataset Information

Transcription profiling of Porphyromonas gingivalis wild type and mutant PG1432::ermF to identify genes which have their expression influenced by PG1432

Publications

FimR and FimS: biofilm formation and gene expression in Porphyromonas gingivalis.

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