Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis - PubMed (original) (raw)

Comparative Study

Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis

A Rincé et al. J Bacteriol. 2001 Feb.

Abstract

The Enterococcus faecalis general stress protein Gsp65 has been purified from two-dimensional gel electrophoresis. Determination of its N-terminal sequence and characterization of the corresponding gene revealed that the gsp65 product is a 133-amino-acid protein sharing homologies with organic hydroperoxide resistance (Ohr) proteins. Transcriptional analysis of gsp65 gave evidence for a monocistronic mRNA initiated 52 nucleotides upstream of the ATG start codon and for an induction in response to hydrogen peroxide, heat shock, acid pH, detergents, ethanol, sodium chloride, and tert-butylhydroperoxide (tBOOH). A gsp65 mutant showed increased sensitivity to the organic hydroperoxide tBOOH and to ethanol.

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Figures

FIG. 1

Amino acid sequence comparison of Gsp65 with Ohr proteins from B. subtilis (YkzA Bs and YklA Bs) (accession numbers F69870 and D69857) (30), X. campestris (Ohr Xc) (accession number AF036166) (21), D. radiodurans (Ohr Dr) (accession number AE002025.1) (31), and E. coli OsmC (OsmC Ec) (accession number P23929) (14). Complete amino acid sequence identity in three out of the six proteins is indicated in boldface. Asterisks and dots indicate positions where, respectively, identical and functionally related (H, K, and R; F, Y, and W; L, I, M, and V; G and A; S and T; D and E; N and Q; C and P) amino acid are found in the six proteins. The consensus sequence is given with lowercase and capital letters, which represent identity in all and in five out of the six proteins, respectively.

FIG. 2

(A) Schematic representation of the genetic organization of the gsp65 chromosomal region. Large arrows represent the ORFs, and their orientation shows the transcriptional direction. The nucleotide sequences of the gsp65 promoter region (Pr) and of a putative Rho-independent terminator (T1) located 2 nt downstream of the gsp65 stop codon are shown. The transcriptional initiation nucleotide (+1) and the putative −35 and −10 motifs are boxed. Primer positions are indicated by small arrows. (B) Primer extension experiment. Lane 1, primer extension signal obtained with the primer Pext and RNA extracted from E. faecalis JH2-2 cells incubated for 10 min with 0.08% bile salts. Lanes C, A, T, and G, products of the sequencing reactions performed with the transcribed DNA strand used for standardization. The arrowhead indicates the primer extension signal which corresponds to the transcriptional start site. (C) Electropherogram obtained from 5′ RACE PCR experiment. The sequence in the electropherogram was obtained using the primer Pext and cDNA from 5′ A-tailed RNA, using the 3′/5′ RACE kit (Roche Molecular Biochemicals). The last base (C) upstream from the 16-n A tail corresponded to the first nucleotide transcribed. The corresponding G on the reverse complement strand is indicated (+1).

FIG. 3

(A) Northern blot hybridization of E. faecalis JH2-2 RNA extracted from exponentially growing cells (lane 1) and from cells incubated for 10, 20, and 30 min (lanes 2 to 4, respectively) with 0.08% bile salts. Hybridization was performed with a single-stranded DNA probe corresponding to the DNA region located between primers P4 and P5. The size of the transcript determined with RNA molecular size markers (Amersham) is indicated on the right. (B) Dot blot hybridization of total RNA (1 μg) from E. faecalis JH2-2 cells harvested in exponential growth phase (control) and from exponentially growing cells incubated for 10 min under the indicated conditions with the single-stranded DNA probe corresponding to the DNA region located between primers P4 and P5. (C) Northern blot hybridization of E. faecalis JH2-2 RNA extracted from exponentially growing cells (lane 1) and from cells incubated for 10 min with 0.3, 0.6, 0.9, and 1.2 M NaCl (lanes 2 to 5, respectively) with the single-stranded DNA probe corresponding to the DNA region located between primers P4 and P5. The size of the transcript determined with RNA molecular size markers (0.56- to 0.94-kb RNA ladder; Amersham) is indicated on the right.

FIG. 4

(A) Effect of tBOOH on wild-type E. faecalis JH2-2 and gsp65 mutant growth. Cultures grown in BHI medium to an OD at 600 nm of 0.2 were divided and either treated with 0.15 mM tBOOH or not. Squares, JH2-2; circles, gsp65 mutant; closed symbols, treated; open symbol; untreated. The values are the averages of results obtained in three independent experiments. (B and C) Effect of tBOOH (B) and ethanol (C) on wild-type E. faecalis JH2-2 and gsp65 mutant survival. Bacteria grown in BHI medium to an OD at 600 nm of 0.5 were harvested and resuspended in BHI medium (control) or BHI medium containing 20 mM tBOOH or 22% ethanol (challenges). The percent viability represents the ratio between the number of cells surviving after 30 min of challenge and the number surviving prior challenge.

FIG. 5

Autoradiogram of 2-D-labeled protein gels of E. faecalis JH2-2 (A) and the gsp65 mutant (B). The pictures represent parts of the autoradiograms obtained after electrophoresis of labeled proteins during the 30-min treatment with 2 mM tBOOH. The arrow shows the position of the spot corresponding to Gsp65.

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Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis - PubMed (original) (raw)