Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.15 (pectinase)
 2,440 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.
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PMID:Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production. 149 88

A set of gene fusions was constructed between the pehA gene encoding the secreted endopolygalacturonase (PehA) and the bla gene coding for a normally periplasmic beta-lactamase (Bla). The resulting hybrid proteins were specifically and actively routed out of the cells via the Out-terminal branch of the general secretory pathway (GSP) in Erwinia carotovora subsp. carotovora (Ecc), provided that no more than the last two amino acids (aa) of the PehA domain were excluded from the fusion. However, both PehA-Bla hybrid proteins and PehA variants lacking at least four aa from the C-terminus of the PehA accumulated in the periplasm. Also, overexpression of the gene fusions prevented extracellular targeting of the hybrid proteins. Site-directed mutagenesis of the codons -4 and -3 (encoding Asn-373 and Val-374, respectively) from the end of the pehA gene and analysis of the protein products suggested that the Val-374 was important both for the structure and secretion of PehA, while the Asn-373 proved to be insignificant. We conclude that: (i) the GSP of Ecc is capable of secreting heterologous proteins; (ii) as the PehA protein can accommodate C-terminal extensions, secretion can occur with no part of the proposed targeting signal lying within the C-terminal extremity of a secreted molecule; and (iii) residues within the C-terminus of PehA play a role in secretion, possibly through stabilization of a structure needed for proper exposition of the proposed targeting motif.
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PMID:The extreme C-terminus is required for secretion of both the native polygalacturonase (PehA) and PehA-Bla hybrid proteins in Erwinia carotovora subsp. carotovora. 855 64

Abstract A gene resembling enterobacterial ampD was identified in the bacterial wilt pathogen, Ralstonia solanacearum. The gene lies 13 bp 3' of pehSR, a two-component positive regulator of virulence factors such as plant cell wall-degrading polygalacturonases and bacterial motility. AmpD, an N-acetylmuramyl-l-alanine amidase, degrades and recycles bacterial cell wall components and also plays a role in the induction of beta-lactamase, which confers ampicillin resistance. AmpD is probably not involved in beta-lactamase regulation in R. solanacearum, because the species produces no detectable beta-lactamase activity and is not ampicillin resistant. However, the R. solanacearum ampD gene restores inducible beta-lactamase activity to an Escherichia coli ampD mutant, demonstrating that the gene encodes an AmpD protein that can function in a heterologous background. An R. solanacearumampD chromosomal mutant was motile, produced wild-type levels of polygalacturonase activity and had wild-type cell and colony morphology. This mutant also grew normally in minimal medium and in plant tissue. Nonetheless, the ampD mutant was significantly reduced in bacterial wilt virulence on eggplant and tomato, suggesting a previously unsuspected role for N-acetylmuramyl-l-alanine amidase in plant pathogenesis.
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PMID:Ralstonia solanacearum AmpD is required for wild-type bacterial wilt virulence. 2057 64