Identification of the genes for the lactose-specific components of the phosphotransferase system in the lac operon of Staphylococcus aureus

The nucleotide and deduced amino acid sequences of the lacE and lacF genes, which code for the lactose-specific Enzyme II and Enzyme III of the Staphylococcus aureus phosphotransferase system, are presented. The primary translation products consist of a hydrophobic protein of 572 amino acids (Mr = 62,688) and a polypeptide of 103 amino acids (Mr = 11,372), respectively. The assignment of lacF as the gene for Enzyme IIIlac was based upon the known amino acid sequence of the protein. The identity of lacE as encoding Enzyme IIlac was based upon immunoreactivity of the cloned gene product with antibodies raised against purified Enzyme IIlac from S. aureus and an assay of biological function of the protein expressed in Escherichia coli. The order of the known genes of the S. aureus lac operon is lacF-lacE-lacG, the latter encoding phospho-beta-galactosidase.     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.     

Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P-GlpK dephosphorylation control Bacillus subtilis glpFK expression

The Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol-3-P and repressed by rapidly metabolizable sugars. Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK. This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co-repressors, P-Ser-HPr or P-Ser-Crh. HPr is a component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and Crh is an HPr homologue. The hprK-encoded HPr kinase phosphorylates HPr and Crh at Ser-46. But in neither ccpA nor hprK mutants was expression of a glpF'-lacZ fusion relieved from CCR, as a second, CcpA-independent CCR mechanism implying the terminator tglpFK, whose formation is prevented by the glycerol-3-P-activated antiterminator GlpP, is operative. Deletion of tglpFK led to elevated expression of the glpF'-lacZ fusion and to partial relief from CCR. CCR completely disappeared in DeltatglpFK mutants carrying a disruption of ccpA or hprK. The tglpFK-requiring CCR mechanism seems to be based on insufficient synthesis of glycerol-3-P, as CCR of glpFK was absent in ccpA mutants growing on glycerol-3-P or synthesizing H230R mutant GlpK. In cells growing on glycerol, glucose prevents the phosphorylation of GlpK by P-His-HPr. P-GlpK is much more active than GlpK, and the absence of P~GlpK formation in DeltaptsHI strains prevents glycerol metabolism. As a consequence, only small amounts of glycerol-3-P will be formed in glycerol and glucose-exposed cells (inducer exclusion). The uptake of glycerol-3-P via GlpT provides high concentrations of this metabolite in the ccpA mutant and allows the expression of the glpF'-lacZ fusion even when glucose is present. Similarly, despite the presence of glucose, large amounts of glycerol-3-P are formed in a glycerol-exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P-GlpK.     

Plasmids, loss of lactose metabolism, and appearance of partial and full lactose-fermenting revertants in Streptococcus cremoris B1.

The unstable ability to metabolize lactose (lac) via the phosphoenolpyruvate-phosphotransferase system (PTS) was examined in Streptococcus cremoris B1. The presence of functional lactose-specific PTS enzymes was correlated with the presence of a distinct plasmid species. Characterization of deoxyribonucleic acid extracted from lactose-positive (Lac+) S. cremoris B1 revealed two plasmids having molecular weights of 9 X 10(6) and 36 X 10(6). An acriflavine (BC1)-induced, lactose-negative (Lac-) mutant possessed no plasmids and was devoid of all three lac-specific PTS enzymes. A Lac- mutant (DA2) isolated by growing at elevated temperatures only possessed the 9 X 10(6)-dalton plasmid and also lacked the lac PTS enzymes. A spontaneous Lac- mutant possessed both the 9 X 10(6)-and 36 X 10(6)-dalton plasmids. This mutant displayed FIII-lac and phospho-beta-D-galactosidase (P-beta-gal) activity but was deficient in EII-lac activity. The spontaneous Lac- strain reverted to both full and partial lactose-fermenting phenotypes having FIII-lac, EII-lac, and P-beta-gal activities. BC1 and DA2 Lac- mutants reverted only to the partial lactose-fermenting phenotype having P-beta-gal activity; EII-lac and FIII-lac activities were absent. The results indicate that the genetic determinants for EII-lac, FIII-lac, and P-beta-gal are located on the 36 X 10(6)-dalton plasmid in S. cremoris B1. Evidence for a second chromosomally associated P-beta-gal gene operating in the partial lactose-fermenting revertants is also presented.     

Molecular cloning and nucleotide sequence of the factor IIIlac gene of Lactobacillus casei

The lactose-specific factor III (FIIIlac of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Lactobacillus casei and purified to homogeneity by conventional protein purification methods. Its apparent native Mr, estimated from steric exclusion chromatography (approx. 39 kDa), and subunit Mr, estimated from sodium dodecyl sulfate-polyacrylamide gels, indicated that it exists as a trimer of identical subunits of 13 kDa. The gene for FIII L. casei lac was cloned into Escherichia coli using the vector pUC18. The coding sequences were contained on an 860-bp BglII-HindIII DNA fragment of the L. casei lactose plasmid, pLZ64. A protein identical in properties to FIII L. casei lac was isolated from clones of E. coli carrying this DNA insert. The nucleotide sequence of the FIII L. casei lac gene was determined by the dideoxy chain-termination technique. The 336-bp open reading frame for FIII L. casei lac was followed by a stem-loop structure, analogous to a Rho-independent terminator. We concluded that the FIII L. casei lac was the terminal gene in what appears to be an operon comprised of the lactose-PTS-P-beta Gal-coding genes. Comparison of the deduced amino acid sequence of FIII L. caseilac with the amino acid sequence of FIII S. aureus lac (derived from peptide sequencing) demonstrated a high degree of homology (49 identical residues and 21 conservative exchanges out of 103 total aa residues). The FIII L. casei lac lacked his82, previously identified as the phosphorylation site of FIII S. aureus. lac His80 was proposed to be the site of histidyl phosphorylation of FIII L. casei lac.     

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.     

Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei. Evidence that a cysteine residue is essential for sugar phosphorylation

The gene coding for the lactose-specific Enzyme II of the Lactobacillus casei phosphoenolpyruvate-dependent phosphotransferase system, lacE, has been isolated by molecular cloning and expressed in Escherichia coli. The DNA sequence of the lacE gene and the deduced amino acid sequence are presented. The putative translation product comprises a hydrophobic protein of 577 amino acids with a calculated molecular mass of 62,350 Da. The deduced polypeptide has a high degree of sequence similarity with the corresponding lactose-specific enzymes II of Staphylococcus aureus and Lactococcus lactis. The sequence surrounding cysteine 483 was strongly conserved in the three proteins. The identity of the lacE product as the Enzyme IIlacL.casei was demonstrated by in vitro lactose phosphorylation assays using the protein expressed in E. coli. Single replacement of each of the histidine and cysteine residues by site-directed mutagenesis pointed to cysteine 483 as an amino acid residue essential for the phosphoryl group transfer reaction.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.