Characterization of the ccpA gene of Enterococcus faecalis: identification of starvation-inducible proteins regulated by ccpA

Abstract

Inactivation of ccpA in Enterococcus faecalis leads to reduction of the growth rate, derepression of the galKETR operon in the presence of a mixture of glucose and galactose, and reduction of transcription of ldh in the presence of glucose. Moreover, the E. faecalis ccpA gene fully complements a Bacillus subtilis ccpA mutant, arguing for similar functions of these two homologous proteins. Protein comparison on two-dimensional gels from the wild-type cells and the ccpA mutant cells revealed a pleiotropic effect of the mutation on gene expression. The HPr protein of the carbohydrate-phosphotransferase system was identified by microsequencing, and a modification of its phosphorylation state was observed between the wild-type and the mutant strains. Moreover, at least 16 polypeptides are overexpressed in the mutant, and 6 are repressed. Interestingly, 13 of the 16 polypeptides whose synthesis is enhanced in the mutant were also identified as glucose starvation proteins. The N-terminal amino acid sequences of four of them match sequences deduced from genes coding for L-serine dehydratase, dihydroxyacetone kinase (two genes), and a protein of unknown function from Deinococcus radiodurans.

FIG. 1

Determination of the 5′ end of the ccpA transcript by primer extension. A DNA-sequencing preparation was run in parallel using the same primer. The arrowhead corresponds to the point within the sequence representing the apparent 5′ end.

FIG. 2

β-Galactosidase activities of B. subtilis strains QB7144 and QB7147 containing different plasmids. The specific activities of β-galactosidase were determined in extracts prepared from exponentially growing cells (OD₆₀₀ = 0.5). The mean values of three independent experiments are presented. Cells were grown in CSK medium supplemented with 0.2% xylose (solid bars) or with 0.2% xylose and 1% glucose (open bars). The method of Miller (32) was used for the determination of β-galactosidase activity.

FIG. 3

(A) Northern blot analysis of the expression of the galK gene in the E. faecalis strains JH2-2 (lanes 1, 2, and 3) and CL14 (lanes 4, 5, and 6) grown with 0.15% glucose (lanes 1 and 4), 0.15% glucose plus 0.15% galactose (lanes 2 and 5), or 0.15% galactose (lanes 3 and 6). (B) Northern blot analysis of the expression of the pfk gene in E. faecalis strains JH2-2 (lane 1) and CL14 (lane 2) grown with glucose. (C) Northern blot analysis of the expression of the ldh gene in E. faecalis strains JH2-2 (lane 1) and CL14 (lane 2) grown with glucose.

FIG. 4

The 2-D electrophoresis protein pattern of E. faecalis strains JH2-2 (A) and CL14 (B) grown on glucose and harvested in the exponential growth phase. The arrows indicate proteins showing modified expression in the ccpA mutant strain. The spots indicated by a G and a number and those indicated only by numbers are polypeptides under negative control by CcpA, whereas proteins indicated by letters are under positive control by CcpA. The majority of the G proteins have been identified in a previous study (13) as inducible upon glucose starvation (Gls proteins). Three additional Gls proteins (G43, G44, and G45) have been identified since that time (unpublished results). The proteins indicated by numbers are specific to the ccpA mutant. N-terminal microsequencing of proteins isolated from two spots showed that they correspond to the HPr protein of E. faecalis. The more acidic spot seems to be the form of this protein phosphorylated on serine 46. For more details, see the text.

FIG. 5

(A) Western blot analysis of HPr of E. faecalis strains JH2-2 (lanes 1 and 2) and CL14 (lanes 3 and 4). The extracts in lanes 1 and 3 correspond to native proteins, and the extracts in lanes 2 and 4 were boiled for 5 min before loading, leading to the dephosphorylation of histidine. The positions of the different forms of HPr are indicated by arrowheads. (B) Percentages of the different HPr forms in E. faecalis wild-type and ccpA mutant strains were deduced from the results of the Western blot analysis after scanning and densitometry analysis by OptiQuant image analysis Software.

FIG. 6

Northern blot analysis of the E. faecalis gls27 and gls17 genes. Total RNA was isolated from strains JH2-2 (lanes 1 and 3) and CL14 (lane 2) exponentially grown on glucose (lanes 1 and 2) or at the onset of glucose starvation (lane 3). Hybond N+ membranes were hybridized with a gls27-specific probe (A) or a gls17-specific probe (B).