Determinants of extracellular protein secretion in gram-negative bacteria

Lory, Stephen

doi:10.1128/jb.174.11.3423-3428.1992

Cited by 102 publications

(67 citation statements)

References 59 publications

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“…Hydropathy analysis (33) indicated that the predicted protein is very hydrophilic, with no likely membrane-spanning domains. Furthermore, the predicted protein contains no similarity to N-terminal signal peptides or C-terminal hemolysin-like secretion signals (36). A search of the Protein Information Resource (PIR), EMBL, and translated GenBank data bases revealed no proteins with significant sequence similarities.…”

Section: Resultsmentioning

confidence: 99%

Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes

et al. 1993

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The avrRpt2 locus from Pseudomonas syringae pv. tomato causes virulent strains ofP. syringae to be avirulent on some, but not all, lines ofAabidopsis thaliana and Glycine mar (soybean). We determined the DNA sequence of the avrRpt2 locus and identified the avrRpt2 gene as a 768-bp open reading frame encoding a putative 28.2-kDa protein. Deletion analysis and transcription studies provided further evidence that this open reading frame encodes AvrRpt2. We found that the avrRpt2 gene also has avirulence activity in P. syringae pathogens of Phaseous vulgaris (common bean), suggesting that disease resistance genes specific to avrRpt2 are functionally conserved among diverse plant species. The predicted AvrRpt2 protein is hydrophilic and contains no obvious membrane-spanning domains or export signal sequences, and there was no significant similarity of AvrRpt2 to sequences in the GenBank, EMBL, or Swiss PIR data bases. A comparison of the avrRpt2 DNA sequence to nine other P. syringac avinlence genes revealed a highly conserved sequence, GGAACCNA-N14-CCACNNA, upstream of the translation initiation codon. This motif is located 6 to 8 nucleotides upstream of the transcription start site in all four P. syringae avirulence genes for which a transcription start site has been determined, suggesting a role as a binding site for a novel form of RNA polymerase. Regulation ofavrRpt2 was similar to other P. syrmigae avirulence genes; expression was high in minimal medium and low in rich medium and depended on the hrpRS locus and an additional locus at the opposite end of the hrp cluster of P. syringae pv. tomato.

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Section: Resultsmentioning

confidence: 99%

Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes

et al. 1993

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“…Considerable effort has been devoted to understanding the mechanism of protein translocation in gram-negative bacteria (30,32,40). However, the precise events leading to the actual liberation of protein from the bacterial surface to the outside environment are not yet fully understood.…”

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confidence: 99%

Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion

1995

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Pseudomonas aeruginosa blebs-off membrane vesicles (MVs) into culture medium during normal growth. Release of these vesicles increased approximately threefold after exposure of the organism to four times the MIC of gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) were isolated by filtration and differential centrifugation, and several of their biological activities were characterized. Electron microscopy of both n-MVs and g-MVs revealed that they were spherical bilayer MVs with a diameter of 50 to 150 nm. Immunoelectron microscopy and Western blot (immunoblot) analysis of the vesicles demonstrated the presence of B-band lipopolysaccharide (LPS), with a slightly higher proportion of B-band LPS in g-MVs than in n-MVs. A-band LPS was occasionally detected in g-MVs but not in n-MVs. In addition to LPS, several enzymes, such as phospholipase C, protease, hemolysin, and alkaline phosphatase, which are known to contribute to the pathogenicity of Pseudomonas infections were found to be present in both vesicle types. Both types of vesicles contained DNA, with a significantly higher content in g-MVs. These vesicles could thus play an important role in genetic transformation and disease by serving as a transport vehicle for DNA and virulence factors and are presumably involved in septic shock.

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“…In some species, several of the genes involved in biogenesis of type IV fimbriae are clustered together close to the pilin structural gene. Those genes identified often include the peptidase responsible for amino-terminal processing of the prepilin and a cytoplasmic protein with highly conserved nucleoside triphosphate binding sites (25,30,48). The cleavage site for the type IV prepilin is usually between glycine and phenylalanine residues (methionine in TcpA of V. cholerae) in the sequence Gly-Phe(Met)-Thr-Leu-Ile(Leu)-Glu, which is highly conserved among members of the type IV fimbria family (44).…”

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confidence: 99%

A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli

1994

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Enteropathogenic Escherichia coli, a leading agent of infantile diarrhea worldwide, adheres to tissue culture cells in a pattern called "localized adherence." Localized adherence is associated with bundle-forming pili encoded by the plasmid bfpA gene, the product of which is homologous with the major structural subunit proteins of type IV fimbriae in other bacteria. Several of these proteins have been shown to be processed from a precursor by a specific prepilin peptidase. We cloned restriction fragments downstream of the bfpA gene into an E. coli-Pseudomonas aeruginosa shuttle vector and mobilized them into a P. aeruginosa prepilin peptidase (pilD) mutant. A plasmid containing a 1.3-kb PstI-BamHI fragment was able to complement the pilD mutation, as demonstrated by restoration of sensitivity to the pilus-specific bacteriophage PO4. The DNA sequence of this fragment revealed an open reading frame, designated bfpP, the predicted product of which is homologous to other prepilin peptidases, including TcpJ of Vibrio cholerae (30% identical amino acids), PulO of Klebsiella oxytoca (29%), and PilD of P. aeruginosa (28%). A bfpA::TnphoA mutant complemented with a bfpA-containing DNA fragment only partially processes the BfpA protein. When complemented with a larger fragment containing bfpP as well as bfpA, the mutant expresses the fully processed BfpA protein. P. aeruginosa PAK, but not a pilD mutant of PAK, expresses mature BfpA protein when the bfpA gene is mobilized into this strain. Thus, as in other type IV fimbria systems, enteropathogenic E. coli utilizes a specific prepilin peptidase to process the major subunit of the bundle-forming pilus. This prepilin petidase contains sequence and reciprocal functional homologies with the PilD protein of P. aeruginosa.

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Determinants of extracellular protein secretion in gram-negative bacteria

Cited by 102 publications

References 59 publications

Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes

Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes

Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion

A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli

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