1 Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Polígono de la Coma s/n, Apartado de Correos (PO Box) 73, 46100-Burjassot, Valencia, Spain
2 Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany
Correspondence
Gaspar Pérez-Martínez
gaspar.perez{at}iata.csic.es
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ABSTRACT |
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INTRODUCTION |
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CcpA inactivation pleiotropically affects the expression of approximately 8 % of the genes in Bacillus subtilis (Moreno et al., 2001). Genes of very important metabolic pathways are regulated by CcpA, such as citB and citZ contributing to the Krebs cycle (Kim et al., 2002
), gltAB for ammonium assimilation (Faires et al., 1999
) or the ilvleu operon for branched-chain amino-acid biosynthesis (Ludwig et al., 2002
). Other studies revealed that CcpA in Gram-positive bacteria also regulates carbon catabolite activation (CCA) of acetoin secretion, acetate biosynthesis genes in B. subtilis (Turinsky et al., 2000
) and glycolytic genes in B. subtilis, Lactococcus lactis and Enterococcus faecalis (Luesink et al., 1998
; Leboeuf et al., 2000
; Ludwig et al., 2001
). However, the number of genes subject to CCA could be significantly lower than those under CCR (Leboeuf et al., 2000
; Moreno et al., 2001
; Titgemeyer & Hillen, 2002
).
However, CcpA has also been found to regulate other processes; for example, biofilm formation is subject to CcpA-mediated CCR in B. subtilis, while intact CcpA was required for formation of a full biofilm in Streptococcus mutans (Wen & Burne, 2002; Stanley et al., 2003
). Also, transcription of the capsular polysaccharide biosynthesis locus (cps) was significantly reduced by mutation of regM (ccpA homologue) in Streptococcus pneumoniae (Giammarinaro & Paton, 2002
). Its involvement in such diverse processes made researchers in the field consider CcpA as a global regulator. As a consequence, inactivation of ccpA is known to have a strong effect on growth rate (Miwa et al., 1994
; Hueck et al., 1995
; Monedero et al., 1997
; Leboeuf et al., 2000
). This growth defect in B. subtilis has been proposed to be related to lack of expression of the above-mentioned gltAB operon, encoding glutamate synthase, necessary for ammonium assimilation (Faires et al., 1999
), and the ilvleu operon (Ludwig et al., 2002
).
In Bacillus megaterium, four single mutations in CcpA were described that showed independent effects on growth and CCR (Küster et al., 1999a): three mutations showed no CCR but normal growth and one was solely defective in growth. All of these mutations are located within the N-terminal DNA-binding domain (DBD) of CcpA. Five additional mutations showed glucose-independent CCR and were located in the co-repressor-binding domain (CBD) (Küster et al., 1999b
).
The process leading to growth depression has not been studied in lactobacilli up until now. Therefore, during this work, mutations equivalent to those in B. megaterium were obtained in L. casei CcpA to elucidate if there was similarity in the role of these residues/domains between these species.
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METHODS |
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Site-directed mutagenesis.
Construction of ccpA mutant alleles was carried out by site-directed mutagenesis following the protocol of Landt et al. (1990). Oligonucleotides for site-specific mutagenesis of ccpA (5'-CACGCATAAATGCTAATTGTTTGC-3', 5'-CTGCATTCGGATGATAATCAAGC-3', 5'-GAGCAACTGCGCTCGGCCGAT-3', 5'-CAAGCTTGAGCAGAACATGTT-3', 5'-CACGAGCCAAGAGTGAGAAGAAC-3', 5'-CTGACGGAAGTGACTCGG-3', 5'-CTTGCTGATCAAGATGATG-3'; introduced oligonucleotide exchanges are underlined) were used in combination with either Universal or Reverse Primer to construct mutants T7S, R50H, N52S, F78C, S80L, M283V and T307I, respectively, using pUCCPA (1 ng) as template. Amplification products were restricted with SacI and PstI, and cloned in pUCCPA digested with the same endonucleases, yielding the corresponding pUCCPA derivatives. All constructs were verified by DNA sequencing using an ABI PRISM 310 Genetic Analyser (Applied Biosystems). Mutant genes were subcloned into pGAL9 following the same procedure described above for the wild-type gene to obtain the series of vectors named pGCCPA-T7S to pGCCPA-T307I (Table 1
).
Construction of pccpA.
pCCPA2.6 containing a 2·6 kb insert carrying the L. casei ccpA gene and surrounding chromosomal DNA (Monedero et al., 1997) was amplified by reverse PCR using oligonucleotides
CCPA1 (5'-GAAGATCTCCGCCTTTTTCAGAAAGCC-3') and
CCPA2 (5'-GAAGATCTCGAATTGTCAAACTAAGTGC-3') introducing BglII restriction sites (underlined). The PCR product was digested with BglII, ligated and used to transform E. coli. The derived vector contained a DNA fragment that excluded ccpA joining the regions upstream and downstream of ccpA (700 bp each). This fragment was isolated by restriction with SalI/XbaI and ligated to SalI/XbaI-digested pUCm1 (Monedero et al., 1997
), yielding plasmid p
ccpA (Fig. 1
).
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Enzymic assays.
Measurements of N-acetylglucosaminidase activity of L. casei cells grown on MRS basal medium supplemented with glucose or ribose were carried out as described previously (Monedero et al., 1997).
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RESULTS |
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The deletion of ccpA required a process that involved two steps of a Campbell-like recombination followed by selection of the appropriate recombinants. For this purpose, L. casei BL71, carrying an inactive ccpA gene disrupted by an erythromycin-resistance cassette, was transformed with the integrative plasmid pccpA. One erythromycin-/chloramphenicol-resistant transformant that had undertaken recombination upstream of ccpA was selected; as revealed by Southern blot analysis (not shown). This transformant was grown in antibiotic-free medium for 50 generations. Strains that underwent a second recombination event downstream of ccpA were selected as erythromycin-/chloramphenicol-sensitive colonies on MRS-agar replica plates. The genetic structure of one of these colonies (strain BL190) was analysed by PCR and dot-blot to ensure a complete deletion of the ccpA gene (data not shown and Fig. 1
). BL190 is the first food-grade strain of Lactobacillus with a deletion of the ccpA gene.
Mutagenesis of ccpA and expression of CcpA variants in BL190
To gain molecular information on the CcpA protein of L. casei and to possibly dissect CCR from growth function, mutations leading to single amino acid mutants were introduced in L. casei ccpA as described in Methods (Table 1). Positions for mutagenesis were chosen following detailed studies on CcpA of B. megaterium, where amino acid residues affecting either CCR and/or growth had been identified previously (Küster et al., 1999a
, b
). The ccpA-deleted strain, BL190, was transformed by electroporation with pGAL9 derivatives containing the wild-type and the collection of ccpA mutants obtained. The resultant set of BL190 derivatives expressing ccpA (wild-type and mutants) was used to study the effect of CcpA mutations on CCR (N-acetylglucosaminidase activity) and growth rate. To ensure that the differences found were due to the mutations in CcpA and not merely to different expression levels or stability of the mutant proteins, a Western blot analysis of the different transformants was performed. As depicted in Fig. 2
, a band corresponding to L. casei CcpA could be clearly detected, although other bands appeared as unspecific reaction of the polyclonal antibodies used (Küster et al., 1996
). The amount of CcpA protein in all mutants was very similar and also similar to the amount of wild-type CcpA expressed from pGCCPA. This result was therefore considered a valid control to analyse the phenotype of the mutants in further experiments. Expression of CcpA from pGAL9 derivatives was slightly higher than expression from the chromosomal copy (Fig. 2
).
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Growth effect of ccpA mutations
In addition to the effect on CCR, deletion of ccpA also resulted in a reduced growth rate. To study the influence of CcpA mutations on this phenotype, the doubling times of strains BL23, BL190 and BL190 carrying the plasmids expressing mutant CcpA proteins (T7S to T307I) and the wild-type protein were determined on MRS basal medium plus 0·5 % glucose (Table 3). The doubling time of BL23 was 93 min, and when ccpA was deleted (BL190) the doubling time increased up to 118 min. However, the generation time of the wild-type was not restored when BL190 was transformed with pGCCPA. This growth restriction may be due to the expression of CcpA, to the presence of pGCCPA, or derivatives, or to the use of erythromycin in the culture medium. The determination of the growth rate of L. casei carrying a similar plasmid but without ccpA, such as pGAL9, could help to identify the cause of the growth defect. The doubling time of BL23(pGAL9) was 126 min, very similar to that of BL190(pGCCPA), suggesting that the presence of ccpA in multicopy might not be the factor affecting growth. Therefore, the doubling time of BL190(pGCCPA) should be taken as a reference. All the mutants grew slower than the wild-type; however, differences in doubling times were always below 13·5 % [BL190(pGCCPA-F78C)]. Thus, the growth defect was not linked to the CCR phenotype as was observed in the respective B. megaterium CcpA mutants.
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DISCUSSION |
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Interestingly, none of the CBD mutations in L. casei CcpA had the same effect as the corresponding mutations in B. megaterium CcpA, for the reporters assayed. From these results, it can be inferred that, although CCR signal response is efficiently fulfilled by both bacteria, the residues involved and molecular changes in CcpA caused by interactions with the cofactor(s) could be different in both species. In a previous work, remarkable structural differences between B. megaterium and L. casei CcpA were suggested by the inefficient interaction of L. casei CcpA with the B. megaterium P-Ser-HPr, as determined by surface plasmon resonance and lack of CCR complementation by L. casei ccpA in a B. megaterium ccpA strain (Mahr et al., 2002
). However, it has been described that in B. subtilis there are a large number of genes under CCR which show a different response to the inactivation of ccpA (Moreno et al., 2001
); therefore, it could be conceivable that CcpA mutations, also in L. casei, could have a different effect on other promoters.
The growth effect of specific mutations was shown to be completely independent from the CCR of N-acetylglucosaminidase activity. Mutation F78C showed the highest increase in doubling time and it was not affected in CCR. The two hyper-repressing mutations, S80L and T307I, had a slightly different behaviour with respect to growth. S80L showed almost normal growth, while T307I had a small growth defect (6 % increase in doubling time). The three mutations in the DBD subdomain, two of them with a de-repressed phenotype, differed in their growth capabilities, following the increasing order: N52S<R50H<T7S.
In summary, results obtained in this work suggest that CCR and growth defect phenotypes of CcpA mutants might not be linked in L. casei as they possibly are in B. megaterium and that conserved amino acid residues in equivalent positions may not be playing the same role in B. megaterium and L. casei. Two mutations leading to a CCR hyper-repressing phenotype were identified. Up until now, such a phenotype had never been reported, but it represents an excellent starting point for further studies. In particular, future works should analyse the effect of these mutations on CCR/CCA on a global basis or, at least, of a larger number of genes.
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ACKNOWLEDGEMENTS |
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Received 22 July 2003;
revised 16 October 2003;
accepted 30 October 2003.
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