INTRODUCTION

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Carbon catabolite repression (CR) is a regulatory process in microorganisms, whereby the presence of a rapidly metabolizable carbon source inhibits expression of alternate catabolic functions. Although the final outcome of CR is uniform, reduced expression of certain genes and operons, the mechanisms leading to repression may be quite diverse (34). The presence of a repressing carbon source can result in lower concentrations of inducers specific for alternate routes of catabolism (35, 37), in altered activities of specific regulators (40), or in the activation of global control proteins, such as the cyclic AMP receptor protein in enteric bacteria (36) or the catabolite control protein CcpA in low-GC gram-positive bacteria (19). In many cases, genes are subject to multiple levels of control. It is therefore important to distinguish gene- or operon-specific regulatory processes from global control to be able to dissect the underlying mechanisms.

In low-GC, gram-positive bacteria, one consequence of the availability of a rapidly metabolizable carbon source is the regulation of gene expression by CcpA, the central transcriptional regulator of CR (19). CcpA, a member of the LacI/GalR family of transcription factors (29), shows a relatively weak affinity for its cognate operator sites, termed catabolite-responsive elements (cres) (20) and must therefore be activated in order to bind efficiently to cre. Several effectors, alone or in combination, have been found to enhance DNA binding of CcpA in vitro (12, 13, 16, 22, 23, 27), but only phosphorylated forms of HPr, the general phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) (31) or Crh, an HPr-like protein detected in Bacillus subtilis (14), were shown to be of significance in vivo (7, 13, 26, 44). Accordingly, loss of HPr kinase function, the enzyme responsible for regulatory phosphorylation of serine at position 46 in HPr or Crh, resulted in relief from CR (15, 33). Evidence regarding the in vivo activation of CcpA so far had been available only from B. subtilis. More recently, HPr kinase mutants, which showed a pleiotropic loss of CR, have been constructed in Staphylococcus xylosus and Lactobacillus casei (8, 21). In addition, a glucose kinase in S. xylosus has been implicated in CR (42). Both kinases appeared to modulate CcpA activity, but it was not clear whether they could act upon CcpA independently from each other.

We are interested in CR in the nonpathogenic staphylococcal species S. xylosus, which is applied in food fermentation processes. In this communication, we describe the construction and evaluation of a single-copy reporter system based on the -galactosidase gene of S. xylosus. The application of the system to study CcpA-dependent regulation is presented.

	MATERIALS AND METHODS

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Bacterial strains. The S. xylosus lactose-negative derivatives of the wild-type strain C2a (17) that were used in this study are listed in Table 1. The glucose kinase mutant strain TX60 (glkA) was constructed by introducing an internal glkA deletion into the genome of S. xylosus C2a. The resulting strain, TX60, showed the same regulatory phenotype as the original glkA::Tn917 insertion mutants (42).

View this table: [in this window] [in a new window]   TABLE 1.   S. xylosus -galactosidase-deficient strains used in this study

lacZM15

Plasmids. The lactose utilization genes of S. xylosus were obtained from plasmids pBG303 and pBG304, containing the -galactosidase gene lacH and the regulatory gene lacR, respectively (1). To construct the lac plasmids pLK1 and pLP1, the temperature-sensitive shuttle vectors pBT12 and pBT2 were used (3). Plasmid pBT12 is a derivative of pBT2, in which the multiple cloning site is replaced by the corresponding region of pBT1 (3). Plasmid pRB474, a chloramphenicol-resistant derivative of pRB374 (4), was used to isolate the B. subtilis vegII promoter and to clone the vegII(cre) promoter derivatives. Plasmid pKIN5E1 (21) was applied to inactivate the HPr kinase gene hprK in the S. xylosus chromosome.

DNA manipulations, sequencing, and transformation. DNA manipulations, plasmid DNA isolation, transformation of E. coli, and preparation of media and agar plates for E. coli were done by standard procedures (38). Sequencing was performed with a Li-Cor automated sequencer. Plasmids were introduced in S. xylosus by electroporation with glycine-treated electrocompetent cells (3). PCR was carried out with Vent DNA polymerase (New England Biolabs). S. xylosus strains were cultivated in B medium, consisting of 1% peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% KH₂PO4, supplemented with 0.5% carbohydrate as indicated. To screen for the expression of -galactosidase on agar plates, B medium agar was supplemented with 100 µg of 4-chloro-3-indolyl--D-galactopyranoside (X-Gal) per ml.

Construction of the S. xylosus lac inactivation plasmid pLK1. To use the endogenous -galactosidase gene lacH as a reporter gene, the first step was to inactivate the chromosomal copy of the gene. For that purpose, the lac deletion plasmid pLK1 (Fig. 1) was constructed. On that plasmid, the lac operon of S. xylosus (1) as outlined in Fig. 1 was modified to yield 'lacR and 'lacH truncated at their 5' ends and a complete deletion of lacP, the lactose permease gene including the lacPH promoter (Fig. 1). As the source for lac DNA, the plasmids pBG303 and -304 were used. The temperature-sensitive shuttle plasmid pBT12 served as the vector.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Genetic organization of the genomic reporter system for S. xylosus. The genetic organization of the wild-type S. xylosus lactose utilization genes lacRPH is shown in the first line. The locations of the two lac promoters are indicated by arrows. The lac deletion plasmid pLK1 harbors versions of 'lacR and 'lacH truncated at their 5' ends and lacks lacP. The promoterless -galactosidase genes lacH and 'lacR are contained on the promoter probe plasmid pLP1. The nucleotide sequence of the region preceding the reporter gene lacH is shown below pLP1. Three restriction sites, BamHI, XbaI, and SalI, are available for insertion of promoter fragments. The wild-type Shine-Dalgarno sequence (SD) of lacH (AAAGGGGT) has been changed to AAAGGAGGT on the promoter probe plasmid. The vectors used to construct pLK1 and pLP1 were the temperature-sensitive shuttle plasmids pBT12 and pBT2, respectively. After homologous recombination, the structure of the lac region in the genome of S. xylosus strains is as outlined for pLK1 and pLP1, except for the newly introduced EcoRI restriction site.

Construction of the lacH promoter probe plasmid pLP1. For the construction of the promoter probe plasmid pLP1 (Fig. 1), the intact lacH gene without promoter was restored, concomitantly changing the wild-type lacH Shine-Dalgarno sequence AGGGGT to AGGAGGT, which should allow perfect pairing with 16S rRNA. The vector for the pLP1 construction was pBT2. Promoter fragments may be inserted into the SalI, XbaI, and BamHI sites located in front of the promoterless -galactosidase gene lacH (Fig. 1).

Cloning of promoters into pLP1. S. xylosus promoters that were analyzed with the promoter probe system are shown in Fig. 2. The promoters P_malRA and P_ccpa were cloned on BamHI-SalI fragments in E. coli DH5. Successful integration of promoter fragments into pLP1 could be detected on X-Gal-containing agar plateshowever, only at 30°C. Although the -galactosidase was active at 37°C in S. xylosus, it showed no activity above 30°C in E. coli. Promoter-containing plasmids were designated pLP3 (P_malRA) and pLP4 (P_ccpa) (Fig. 2). To check the fidelity of the polymerase reactions, cloned promoters were sequenced prior to use in S. xylosus.

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Nucleotide sequence of the promoters integrated in front of the -galactosidase gene lacH. The promoter regions relevant for transcription initiation and regulation are shown. Putative RNA polymerase binding sites are underlined. Transcriptional start sites are shown in boldface. cres are underlined and shown in boldface. The elements were labeled according to the genes or operons where they originated from. Designation of the plasmids harboring the shown promoters and corresponding numbers to identify these promoters after their integration into the S. xylosus genome are shown in parentheses. Relevant restriction sites are indicated.

Construction of cre-containing vegII promoter derivatives. To convert the constitutive vegII promoter to a CcpA-regulated promoter, cre operators were placed between the HindIII and PstI restriction sites downstream of the vegII transcriptional start point (Fig. 2). To that end, overlapping pairs of oligonucleotides were designed that contained a cre in the double-stranded part and single-stranded extensions fitting into HindIII- and PstI-cut DNA. In addition, a SpeI restriction site was introduced downstream of cre to help to identify the small DNA fragment after cloning. The different cre sequences are shown in Fig. 2 in the context of the vegII(cre) promoters.

Inactivation of the lac genes in the genome of S. xylosus. The lac inactivation plasmid pLK1 (Fig. 1) was transferred by electroporation into S. xylosus wild-type C2a and the isogenic regulatory mutants listed in Table 1. Plasmid-containing colonies were selected with 20 µg of chloramphenicol per ml at 30°C. Subsequently, the transformants were patched onto B medium agar plates, supplemented with 100 µg of X-Gal per ml and 20 µg of chloramphenicol per ml and grown at 30°C for about 36 h. Although the strains appeared blue on these plates, the color was not equally distributed within the patches. Apparently, homologous recombination occurred with a high frequency introducing the lac gene deletions from plasmid pLK1 into the genome of many cells. After streaking the cells on X-Gal (100 µg/ml) agar plates without chloramphenicol and incubation at 37°C overnight, between 20 and 50% white colonies were detected. A second incubation of a white colony on agar without selective pressure efficiently cured the cells from the pLK1 plasmid, which does not replicate at 37°C. By this procedure, TX300, the -galactosidase-deficient derivative of the wild-type strain and its isogenic regulatory mutants were produced (Table 1). The chromosomal organization of the lac region in these strains was confirmed by restriction analysis of PCR fragments obtained by primers annealing outside of the cloned lac region.

lacR lacP 'lacH

ermB 'lacR lacP 'lacH

Integration of promoters in front of the chromosomal -galactosidase gene lacH. Integration of promoters in front of lacH was achieved by replacing the pLK1-generated copies of 'lacH with intact lacH from promoter-containing derivatives of pLP1. The -galactosidase-deficient strains (Table 1) were transformed with the plasmids (Fig. 2), and transformants were selected with 20 µg of chloramphenicol per ml and grown for 36 h at 30°C. After patching onto chloramphenicol- and X-Gal-containing agar plates, the colonies appeared uniformly blue, due to -galactosidase expression from plasmid. Plasmid curing on nonselective media at 37°C produced a mixture of white and blue colonies. Most of these blue colonies were chloramphenicol sensitive, indicating plasmid loss. Subsequent PCR analysis of the lac region confirmed the integration promoter-containing lacH genes into the chromosome. The resulting strains were designated according to the -galactosidase-deficient parental strains (Table 1) and the numbers that were assigned to the promoters (Fig. 2). For example, P_malRA-harboring strains (pLP3) were designated S. xylosus TX303 (wild type) and TX603 (ccpA mutant).

Integration of the promoterless -galactosidase gene into the genome of S. xylosus. To be able to determine the background level of -galactosidase expression without promoter, the modified lacH gene of pLP1 was integrated into the wild-type strain C2a and the ccpA mutant TX154 to yield S. xylosus TX301 and TX601, respectively. Both strains appeared white on X-Gal-containing agar plates, and -galactosidase activity was virtually undetectable. Therefore, the corresponding control strains with glkA, glcU, or hprK mutations were not constructed.

Determination of -galactosidase activity in cell extracts. The cells were grown at 37°C in B medium to an optical density at 578 nm (OD₅₇₈) of 1. Sugars were added to a final concentration of 25 mM, when appropriate. After 1 h of further growth, the OD₅₇₈ was determined, and the cells were harvested by centrifugation. The preparation of crude extracts and the assay of -galactosidase activity have been described previously (1). Specific -galactosidase activities are expressed in nanomoles of nitrophenol released per minute per milligram of protein. Protein concentrations were determined by the method of Bradford (2).

RNA preparation and primer extension analysis. Preparation of RNA and primer extension reactions were done as described previously (1). The primer used to map the start point of P_vegII was labeled at the 5' end with an infrared dye, IRD700. Reverse transcripts were run on 8% polyacrylamide-urea gels and detected by a Li-Cor DNA sequencer.

	RESULTS AND DISCUSSION

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Analysis of CcpA-dependent regulation of the malRA and ccpA promoters to evaluate the single-copy promoter probe system. To demonstrate that the promoter probe system is suitable for the investigation of gene regulation in S. xylosus, CcpA-mediated CR was chosen as an example. Two promoters, the promoter of the maltose utilization operon malRA (10) and one of the two ccpA promoters, had been previously shown to be subject to CcpA repression (9). In the malRA promoter region, a cre spanning positions 58 to 45 with respect to the transcriptional start point (Fig. 2) is essential for CcpA-mediated repression (9). In the ccpA promoter region, a cre overlaps the start point of the second promoter (Fig. 2) and is most likely responsible for the observed ccpA autoregulation (9). Both promoter regions were integrated in the S. xylosus genome in front of the promoterless -galactosidase gene lacH, and -galactosidase activities in the absence or presence of carbohydrates were determined in the resulting strains, TX303 ('lacR lacP P_malRA-lacH) and TX304 ('lacR lacP P_ccpA-lacH). In addition, the background level of -galactosidase expression without a cloned promoter was determined in strain TX301 ('lacR lacP lacH).

Evaluation of putative cre sequences. To define whether a cre-like sequence indeed serves as a CcpA operator, cre function must be determined in the absence of sugar-specific regulators, because alternate mechanisms of CR may mimic CcpA regulation. If specific regulators are unknown or activators are involved, it is difficult and time-consuming to obtain constitutively expressed genes. An alternative approach to analyze cre function could be to move suspected cre sequences to a constitutive promoter. Subsequently, it can be determined whether the promoter is now regulated by CcpA.

View this table: [in this window] [in a new window]   TABLE 2.   Regulation of -galactosidase expression directed by the vegII promoter and cre-containing vegII promoter derivatives in S. xylosus wild-type and mutant strains altered in catabolite repression

CcpA activity in the absence of glucose kinase GlkA and glucose uptake protein GlcU. In previous studies aimed at isolating S. xylosus mutants altered in CR, a PTS-independent glucose utilization system consisting of a glucose uptake protein, GlcU, and a glucose kinase, GlkA, have been isolated (11, 42). In glkA or glcU mutant strains, glucose-mediated CR of several enzyme activities was reduced, but not abolished. The phenotypes of these mutant strains were very similar, suggesting that both mutations affect the same process in CR. It was not clear, however, to what extent the enzymatic activities measured in these studies reflected CcpA regulation. With the CcpA-dependent promoters at hand, it was therefore of interest to determine the consequences of glcU and glkA mutation for CcpA-dependent regulation.

CcpA activity in the absence of HPr kinase. To clarify the interdependence of GlcU-GlkA and HPr kinase in CR, the HPr kinase gene, hprK (21), was inactivated in the TX300-derived wild-type strains, yielding the TX700 series of S. xylosus strains. As shown in Table 2, glucose repression of all promoters was abolished upon hprK inactivation. The slight reduction of -galactosidase activity in glucose-containing cultures is also apparent for the vegII promoter (TX702) missing a cre. Therefore, it is not due to CcpA-mediated regulation. Complete loss of CcpA regulation without a functional HPr kinase clearly shows that GlcU and GlkA do not activate CcpA independently, strongly suggesting that HPr phosphorylated at serine 46 (P-Ser-HPr) is absolutely required to trigger CcpA-dependent CR in S. xylosus. To explain the role of GlcU and GlkA in this process, it is reasonable to assume that the proteins produce a surplus of glycolytic intermediates such as glucose-6-phosphate or fructose-1,6-diphosphate (FDP). Higher levels especially of FDP could have two conceivable consequences. First, HPr kinase activity would be stimulated, resulting in elevated levels of P-Ser-HPr (15, 21, 33). Second, FDP could act, together with P-Ser-HPr, as a second signaling molecule to enhance CcpA binding to cres. The latter would be consistent with a number of in vitro experiments, in which the concomitant presence of FDP and P-Ser-HPr greatly stimulated binding of CcpA to cre (13, 23, 32). It was also shown that FDP is able to enhance specific interaction of CcpA with P-Ser-HPr (6), which could explain its stimulatory effect on CcpA binding.