Dataset Information

Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant.

ABSTRACT: The nucleotide sequence for a surface-associated protein (A. Joe, A. Yamamoto, and B. C. McBride, Infect. Immun. 61:3294-3303, 1993) of Porphyromonas gingivalis was determined. The structural gene comprises 1,338 bp and codes for a protein of 445 amino acids. The deduced molecular weight of the protein is 49,243. A data base search for homologous proteins revealed significant sequence similarity to the subunit protein of glutamate dehydrogenases (GDHs) isolated from various sources. This protein, which was previously labelled PgAg1, will now be called GDH. Recombinant GDH was purified to homogeneity, and native GDH was partially purified from P. gingivalis. Both preparations exhibited NAD-dependent GDH activity. Intact P. gingivalis and an extract of cell surface components also demonstrated NAD-dependent GDH activity. To help elucidate the role of this protein, an isogenic mutant of P. gingivalis lacking the GDH protein was generated by deletion disruption. Biological characterization of the mutant strain, P. gingivalis E51, demonstrated complete loss of GDH activity. Immunogold bead labelling of intact cells showed that GDH was no longer present on the surface of the bacterial cell. The GDH-negative mutant displayed impaired cell growth, as demonstrated by an increased generation time and an inability to grow to the same cell density as the parent.

SUBMITTER: Joe A

PROVIDER: S-EPMC186287 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

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Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant.

Joe A A Murray C S CS McBride B C BC

Infection and immunity 19940401 4

The nucleotide sequence for a surface-associated protein (A. Joe, A. Yamamoto, and B. C. McBride, Infect. Immun. 61:3294-3303, 1993) of Porphyromonas gingivalis was determined. The structural gene comprises 1,338 bp and codes for a protein of 445 amino acids. The deduced molecular weight of the protein is 49,243. A data base search for homologous proteins revealed significant sequence similarity to the subunit protein of glutamate dehydrogenases (GDHs) isolated from various sources. This protein ...[more]

PMID: 8132343

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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.

Project description:PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene encoding this proteinase, prtP, was cloned into the Escherichia coli-Bacteroides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cells, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several Bacteroides species harboring the recombinant plasmids. The proteinase was recovered from Bacteroides fragilis ATCC 25285(pFD340-prtP) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-D-Tyr-L-Pro-L-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither B. fragilis ATCC 25285(pFD340-prtP) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with knockout mutations in prtP were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other P. gingivalis proteins and describes the properties of P. gingivalis cells defective in the production of PrtP.

Dataset Information

Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant.

Publications

Nucleotide sequence of a Porphyromonas gingivalis gene encoding a surface-associated glutamate dehydrogenase and construction of a glutamate dehydrogenase-deficient isogenic mutant.

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