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MOLECULAR AND CELLULAR PATHOGENESIS

Effects of Enterococcus faecalis fsrGenes on Production of Gelatinase and a Serine Protease and Virulence

Xiang Qin, Kavindra V. Singh, George M. Weinstock, Barbara E. Murray
Xiang Qin
Division of Infectious Diseases, Department of Medicine,
Department of Microbiology and Molecular Genetics, and
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, Texas 77030
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Kavindra V. Singh
Division of Infectious Diseases, Department of Medicine,
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, Texas 77030
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George M. Weinstock
Department of Microbiology and Molecular Genetics, and
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, Texas 77030
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Barbara E. Murray
Division of Infectious Diseases, Department of Medicine,
Department of Microbiology and Molecular Genetics, and
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, Houston, Texas 77030
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DOI: 10.1128/IAI.68.5.2579-2586.2000
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    Fig. 1.

    Open reading frames and their transcripts ingelE flanking regions. (A) Open reading frames. The solid line represents the chromosome, and the genes and open reading frames are indicated by boxes in different shades. The orientation of the genes and open reading frames are indicated by arrows. (B) Summary of the Northern blot results using RNA from different strains and different gene probes. −, No signal in Northern blot analysis.

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    Fig. 2.

    Sequence alignment of FsrA, FsrB, and FsrC with AgrA, AgrB, and AgrC from S. aureus. Identical amino acids are indicated by a symbol as “∣”, and similar amino acids are indicated by a symbol as “:” or “.” between them. Conserved residues in H, N, and G2 blocks of histidine protein kinases and conserved Asp and Lys residues in response regulators of bacterial two-component systems (20) are boxed. The sequences fromS. aureus were as follows: AgrA, accession number M21854 (14); AgrB, accession number AF001782 (5); and AgrC, accession number AF001783 (5).

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    Fig. 3.

    Gelatinase and serine protease activities infsr and sprE mutants. These pictures are the negative images of gelatin and casein zymogram gels. (A) Gelatinase activity of mutants on gelatin zymogram gel. (B) Serine protease activity of mutants on casein zymogram gel. Lanes: o1, mutant with disruption of orf1; o, OG1RF; a, mutant with disruption offsrA; b, mutant with disruption of fsrB; c, mutant with disruption of fsrC; e, mutant with disruption ofgelE; s, mutant with disruption of sprE.

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    Fig. 4.

    Northern blot analysis of fsr andsprE mutants. (A, B, C, and D) Northern blots of mutants using fsrB, fsrC, gelE, andsprE probes, respectively. Lanes: o1, mutant with disruption of orf1; o, OG1RF; a, mutant with disruption offsrA; b, mutant with disruption of fsrB; c, mutant with disruption of fsrC; s, mutant with disruption ofsprE.

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    Fig. 5.

    Kaplan-Meier survival plots of wild-type OG1RF andfsr and sprE mutants in the mouse peritonitis model. (A) Kaplan-Meier survival plots of OG1RF, the fsrAmutant (TX5240) and an fsrC revertant. (B) OG1RF and thefsrB mutant (TX5241). (C) OG1RF and the sprEmutant (TX5243). Twelve mice were tested with each inoculum of each of the strains shown. The P value refers to mutant versus OG1RF at the same or a smaller inoculum (▴ versus ▵ or ■ versus □).

Tables

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  • Table 1.

    Bacterial strains and plasmids

    Strain or plasmidRelevant genotype or phenotypeSource or reference
    Strain
     OG1RFGelatinase and serine protease production positive/(Gel+, Spr+)10
     TX5128gelE transposon mini-γδ insertion mutant of OG1RF, Gel−, Spr−, Kanr19
     TX5240fsrA insertion mutant of OG1RF, Gel−, Spr−, KanrThis study
     TX5241fsrB insertion mutant of OG1RF, Gel−, Spr−, KanrThis study
     TX5242fsrC insertion mutant of OG1RF, Gel−, Spr−, KanrThis study
     TX5243sprE insertion mutant of OG1RF, Gel+, Spr−, KanrThis study
     TX5248orf1 insertion mutant of OG1RF, Gel+, Spr+, KanrThis study
     TX5244TX5240 harboring plasmid TEX5249, Gel+, Spr+, Kanr EmrThis study
     TX5245TX5241 harboring plasmid TEX5249, Gel+, Spr+, Kanr EmrThis study
     TX5246TX5242 harboring plasmid TEX5249, Gel+, Spr+, Kanr EmrThis study
     TX5247TX5128 harboring plasmid TEX5249, Gel−, Spr−, Kanr EmrThis study
     DH5αE. coli host strain for routine cloningStratagene
     TX5249DH5α(pTEX5249)This study
    Plasmids
     pBluescript SK(−)Cloning vector, AmprStratagene
     pTEX4577Suicide vector for single cross-over mutagenesis in E. faecalis, Kanr19
     pAT18Shuttle vector for E. coli and E. faecalis, Emr23
     pTEX5249pTEX5249 was constructed by cloning a 6-kb PstI/BglII fragment containing fsrA, fsrB, and fsrC into the shuttle vector pAT18, EmrThis study
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Effects of Enterococcus faecalis fsrGenes on Production of Gelatinase and a Serine Protease and Virulence
Xiang Qin, Kavindra V. Singh, George M. Weinstock, Barbara E. Murray
Infection and Immunity May 2000, 68 (5) 2579-2586; DOI: 10.1128/IAI.68.5.2579-2586.2000

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Effects of Enterococcus faecalis fsrGenes on Production of Gelatinase and a Serine Protease and Virulence
Xiang Qin, Kavindra V. Singh, George M. Weinstock, Barbara E. Murray
Infection and Immunity May 2000, 68 (5) 2579-2586; DOI: 10.1128/IAI.68.5.2579-2586.2000
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KEYWORDS

Bacterial Proteins
DNA-Binding Proteins
Dihydropteridine Reductase
Enterococcus faecalis
Escherichia coli Proteins
Gelatinases
Hemeproteins
NADH, NADPH Oxidoreductases
Serine Endopeptidases
Trans-Activators

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