Yersinia enterocolitica ClpB affects levels of invasin and motility

Abstract

Expression of the Yersinia enterocolitica inv gene is dependent on growth phase and temperature. inv is maximally expressed at 23 degrees C in late-exponential- to early-stationary-phase cultures. We previously reported the isolation of a Y. enterocolitica mutant (JB1A8v) that shows a decrease in invasin levels yet is hypermotile when grown at 23 degrees C. JB1A8v has a transposon insertion within uvrC. Described here is the isolation and characterization of a clone that suppresses these mutant phenotypes of the uvrC mutant JB1A8v. This suppressing clone encodes ClpB (a Clp ATPase homologue). The Y. enterocolitica ClpB homologue is 30 to 40% identical to the ClpB proteins from various bacteria but is 80% identical to one of the two ClpB homologues of Yersinia pestis. A clpB::TnMax2 insertion mutant (JB69Qv) was constructed and determined to be deficient in invasin production and nonmotile when grown at 23 degrees C. Analysis of inv and fleB (flagellin gene) transcript levels in JB69Qv suggested that ClpB has both transcriptional and posttranscriptional effects. In contrast, a clpB null mutant, BY1v, had no effect on invasin levels or motility. A model accounting for these observations is presented.

FIG. 1

Physical maps of various clones used this study. (A) Restriction endonuclease sites: H, HindIII; R, EcoRI; S, SalI; Sa, SacII; P, PstI; N, NarI. (B) Schematic of strain lineages for strains used and/or constructed in this study.

FIG. 2

Suppression of JB1A8v invasin motility phenotypes by pJB69. (A) Western analysis of invasin expression by JB41v(pTM100), JB1A8v(pTM100), and JB1A8v(pJB69). Whole-cell extracts were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a rabbit polyclonal anti-invasin antibody as described in Materials and Methods. The arrow points to the band representing full-length invasin. It is typical to see full-length invasin along with several breakdown products. (B) Invasion phenotype and AP activity exhibited by JB41v(pTM100), JB1A8v(pTM100), and JB1A8v(pJB69). AP activities were assayed in duplicate and are represented as means ± ranges. Invasion assays with HEp-2 cells were performed in duplicate, and data represent means ± ranges as described in Materials and Methods. (C) Motility assays were performed with JB41v(pTM100), JB1A8v(pTM100), and JB1A8v(pJB69) in 0.3% motility soft agar plates incubated at 23°C for 16 h. (D) Western analysis of flagellin preparations probed with monoclonal antibody 15D8 as described in Materials and Methods. Data shown are from a single experiment and are representative of several experiments performed with similar results.

FIG. 3

Comparison of the ClpB proteins of E. coli and Y. enterocolitica. The predicted amino acid sequence of the Y. enterocolitica (Ye) ClpB protein was aligned to that of the E. coli (Ec) ClpB protein (GenBank accession no. PO3815) using the ClustalW alignment software (MacVector) and the identity alignment matrix. Similar residues are boxed; identical residues are boxed and shaded. The Walker A and B boxes are underlined with dotted lines, and the spacer region is solid underlined. The asterisk indicates the position of an amino acid translated from a GTG codon that initiates the synthesis of a truncated ClpB protein in E. coli. Arrowheads point to the positions of the pBY38 and TnMax2-Q insertions (giving mutants BY1v and JB69Qv, respectively).

FIG. 4

Invasin and motility phenotypes of the clpB mutant JB69Qv. (A) Whole-cell extracts of indicated strains grown at 23°C were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a rabbit polyclonal anti-invasin antibody as described in Materials and Methods. The arrow points to the band representing full-length invasin. (B) Motility assays were performed with JB580v and JB69Qv in 0.3% motility soft agar plates incubated at 23°C for 16 h. (C) Western analysis of flagellin preparations probed with monoclonal antibody 15D8 as described in Materials and Methods. Arrows point to the three flagellin subunits in Y. enterocolitica. Data shown are from a single experiment and are representative of several experiments performed with similar results.

FIG. 5

Complementation of the clpB mutant JB69Qv and the uvrC mutant JB1A8v. (A) Western analysis of whole-cell extracts of indicated strains grown at 23°C using an antibody to invasin. The numbers listed at the bottom (motility) indicate the percentage of the size of the motility zone in soft agar seen with the wild type (i.e., zone for JB580v pCR2.1 = 100). The “v” indicates the presence of vector pCR2.1, and “+” indicates the presence of the clpB plasmid pJB302. (B) (Top) Western analysis of whole-cell extracts of indicated strains grown at 23°C using an antibody to invasin. (Bottom) Western analysis of flagellin preparations from the same strains probed with monoclonal antibody 15D8 as described in Materials and Methods. The “v” indicates the presence of vector pTM100, and “+” indicates the presence of the clpB plasmid pJB305. Data shown are from a single experiment and are representative of several experiments performed with similar results.

FIG. 6

Analysis of transcript levels and summary of phenotypes of the various mutants. Transcription of inv, fleB, and fliA was measured by RNA slot blot analysis. RNA was prepared from late-log- to early-stationary-phase cells grown at 23°C. Wild-type transcript levels were set at 100%, and relative amounts of specific mRNA from other strains were determined. For mutant JB1A8v, transcript levels are shown relative to the parental strain JB41v (wild-type inv and inv::phoA). Transcript levels for JB69Qv are shown relative to the parental strain JB580v. Data presented represent the mean ± the range from an individual representative experiment containing duplicate samples and reflect the results from several experiments using independently prepared RNA samples.