Characterization of the divergent sacBK and sacAR operons, involved in sucrose utilization by Lactococcus lactis.

The divergently transcribed sacBK and sacAR operons, which are involved in the utilization of sucrose by Lactococcus lactis NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing sacB and sacK, a second of 3.4 kb containing sacA and sacR, and a third of 1.8 kb containing only sacR. The inactivation of the sacR gene by replacement recombination resulted in the constitutive transcription of the sacBK and sacAR operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription.

Molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr.

The Lactococcus lactis ptsH and ptsI genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene. A promoter sequence was identified upstream of the ptsHI operon, and the transcription start site was mapped by primer extension. The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb containing ptsH and a second of 2.0 kb containing both ptsH and ptsI. Disruption of the ptsH and ptsI genes in strain NZ9800 resulted in a reduced growth rate at the expense of glucose, but no growth at the expense of sucrose and fructose, confirming the dominant role of the phosphotransferase system in the uptake of these sugars in L. lactis. Complementation of the ptsH and ptsI mutants with the intact genes under the control of a regulated promoter resulted in the restoration of the wild-type phenotype. The role of HPr(Ser-P) in the recently established CcpA-mediated control of galactose metabolism as well as glycolysis was analyzed by producing an HPr mutant carrying an aspartic acid on residue 46 which mimicks a phosphorylated serine. The results of these experiments demonstrated the role of HPr(Ser-P) as corepressor in the catabolite repression of the gal operon. Furthermore, we show for the first time that HPr(Ser-P) functions as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase and L-lactate dehydrogenase genes.

Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA.

The Lactococcus lactis ccpA gene, encoding the global regulatory protein CcpA, was identified and characterized. Northern blot and primer extension analyses showed that the L. lactis ccpA gene is constitutively transcribed from a promoter that does not contain a cre sequence. Inactivation of the ccpA gene resulted in a twofold reduction in the growth rate compared with the wild type on glucose, sucrose and fructose, while growth on galactose was almost completely abolished. The observed growth defects could be complemented by the expression of either the L. lactis or the Bacillus subtilis ccpA gene. The disruption of the ccpA gene reduced the catabolite repression of the gal operon, which contains a cre site at the transcription start site and encodes enzymes involved in galactose catabolism. In contrast, CcpA activates the transcription of the cre-containing promoter of the las operon, encoding the glycolytic enzymes phosphofructokinase, pyruvate kinase and L-lactate dehydrogenase, because its transcription level was fourfold reduced in the ccpA mutant strain compared with the wild-type strain. The lower activities of pyruvate kinase and L-lactate dehydrogenase in the ccpA mutant strain resulted in the production of metabolites characteristic of a mixed-acid fermentation, whereas the fermentation pattern of the wild-type strain was essentially homolactic.

Immunological crossreactivity to the catabolite control protein CcpA Bacillus megaterium is found in many gram-positive bacteria.

The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, lactic acid bacteria, and some actinomycetes.

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli.

The celB gene encoding the cellobiose-hydrolyzing enzyme beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of beta-glucosidase that could be purified by a two-step purification procedure. The beta-glucosidase produced by E. coli had kinetic and stability properties similar to those of the beta-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of beta-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus beta-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200- or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100 degrees C.

Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction.

The post-translationally modified, antimicrobial peptide nisin is secreted by strains of Lactococcus lactis that contain the chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into the structural nisA gene (delta nisA), transcription of delta nisA is abolished. Transcription of the delta nisA gene is restored by adding subinhibitory amounts of nisin, nisin mutants, or nisin analogs to the culture medium, but not by the unmodified precursor peptide or by several other antimicrobial peptides. Upon disruption of the nisK gene, which encodes a putative sensor protein that belongs to the class of two-component regulators, transcription of delta nisA was no longer inducible by nisin. Fusion of a nisA promoter fragment to the promoterless reporter gene gusA resulted in expression of gusA in L. lactis NZ9800 (delta nisA) only upon induction with nisin species. The expression level of gusA was directly related to the amount of inducer that was added extracellularly. These results provide insight into a new mechanism of autoregulation through signal transduction in prokaryotes and demonstrate that antimicrobial peptides can exert a second function as signaling molecules.

Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus.

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105 degrees C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (Km = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100 degrees C and 13 h at 110 degrees C.