Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage phig1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ' gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1-7) MKLKNKL, mature-Lys (27-33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide.These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli.Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of phig1e Lys are essential for the lytic activity.     

The genetic switch for the regulatory pathway of Lactobacillus plantarum phage (phi)g1e: characterization of the promoter P(L), the repressor gene cpg, and the cpg-encoded protein Cpg in Escherichia coli

The structural and functional features of the approximately 530 bp P(L)/Gb5-Gb6-cpg-Gb7 region (P(L) overlaps Gb5) for the lysogenic pathway of L. plantarum phage (phi)gle were investigated using the cat gene of E. coli plasmid pKK232-8 as a reporter. In E. coli XL1-Blue, a recombinant plasmid pKPL2 (cat under P(L)/Gb5-Gb6) exhibited distinct CAT activity, whereas the activity of pKPLCP1 (cat under P(L)/Gb5-Gb6-cpg) was only marginal. When pKPL2 was coexistent with a compatible derivative of plasmid pACYC177 carrying P(L)/Gb5-Gb6-cpg, the CAT activity was declined to the level of pKPLCP1. On the other hand, the cpg-encoded protein Cpg was overproduced in E. coli under P(T7). The molecular mass of the purified Cpg (14.5 kDa on a SDS gel) corresponded well with that (15.1 kDa) predicted from the DNA sequence. Gel-shift and footprinting assays demonstrated that Cpg selectively binds to about 25 bp bases centered around the GATAC-box (from 1 to 7). Moreover, protein crosslinking experiments using glutaraldehyde showed that Cpg most likely functions as a dimeric form. Thus, the present results indicate that Cpg probably represses P(L) through binding to the operator GATAC-box(es), and the P(L)/cpg region might participate in the lysogenic pathway.     

Purification and characterization of repressor of temperate S. aureus phage phi11

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.     

Functionally important correlated motions in the single-stranded DNA-binding protein encoded by filamentous phage Pf3

To elucidate the interplay between different parts of dimeric single-stranded DNA-binding proteins we have studied the correlated motions in the protein encoded by filamentous phage Pf3 via the combined use of 15N-NMR relaxation experiments, molecular dynamics simulations and essential dynamics calculations. These studies provide insight into the mechanism underlying the protein-DNA binding reaction. The most important motions can be described by a few essential modes. Most outstanding is the correlated symmetric motion of the DNA-binding wings, which are far apart in the structure. This motion determines the access of DNA to the DNA-binding domain. A correlation between the motion of the DNA-binding wing and the complex loop is indicated to play a role in the cooperative binding of the protein to DNA. These motions are in the nanosecond regime in correspondence with the 15N-NMR relaxation experiments.     

Purification and properties of phage P22 c2 repressor.

The c2 repressor of phage P22 has been purified to homogeneity. It specifically binds to lambdaimm21 and P22 DNA. Its affinity for the presumed operator mutant P22 virB is reduced. The initial dissociation rates of the complex between c2 repressor and lambdaimm21 DNA are 0.02 min-1 at 0 degrees C, 0.08 min-1 at 20 degrees C and 0.17 min-1 at 32 degrees C. The dissociation rates of complexes formed between the c2 repressor and the lambdaimm21 operators OR, OL and OR vira were measured and compared to the corresponding rates obtained with 21 cI repressor.     

Mutational and biochemical analyses of the endolysin Lys(gaY) encoded by the Lactobacillus gasseri JCM 1131T phage phi gaY

The Lys(gaY) of Lactobacillus gasseri JCM 1131(T) phage phigaY endolysin was purified to homogeneity using the Escherichia coli/His.Tag system. Zymographic and spectrophotometric assays showed that Lys(gaY) lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37 degrees C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys(gaY) were involved in cell-lytic activity: in the beta/alpha(gaY) domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b(gaY) domain, Y272 and W284. In addition, deletion analysis demonstrated that the beta/alpha(gaY) domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys(gaY). These mutational experiments suggested that beta/alpha(gaY) (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b(gaY) promotes beta/alpha(gaY) possibly through cell-wall binding function. The purified His-tagged SH3b(gaY) domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys(gaY), (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131(T), L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys(gaY), and (iv) impeded autolysis of L. gasseri JCM 1131(T) in buffer systems. A truncated protein HDeltaSH3b(gaY) lacking in N-terminal 21 residues (from S217 to E237) of SH3b(gaY) and an amino-acid substituted protein HSH3b(gaY)G (W284G) lost the activities of HSH3b(gaY), showing that the N-terminal region and W284 probably play important roles in the SH3b(gaY) function(s).     

Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum

Pyruvate oxidase (EC 1.2.3.3) was isolated and characterized from Lactobacillus plantarum. The enzyme catalyzes the oxidative decarboxylation of pyruvate in the presence of phosphate and oxygen, yielding acetyl phosphate, carbon dioxide, and hydrogen peroxide. This pyruvate oxidase is a flavoprotein, with the relatively tightly bound cofactors flavin adenine dinucleotide, thiamine pyrophosphate, and a divalent metal ion, with Mn2+ being the most effective. The enzyme is only slightly inhibited by EDTA, implying that the enzyme-bound metal ion is poorly accessible to EDTA. Only under relatively drastic conditions, such as acid ammonium sulfate precipitation, could a colorless and entirely inactive apoenzyme be obtained. A partial reactivation of the enzyme was only possible by the combined addition of flavin adenine dinucleotide, thiamine pyrophosphate, and MnSO4. The enzyme has a molecular weight of ca. 260,000 and consists of four subunits with apparently identical molecular weights of 68,000. For catalytic activity the optimum pH is 5.7, and the optimum temperature is 30 degrees C. The Km values for pyruvate, phosphate, and arsenate are 0.4, 2.3, and 1.2 mM, respectively. The substrate specificity revealed that the enzyme reacts also with certain aldehydes and that phosphate can be replaced by arsenate. In addition to oxygen, several artificial compounds can function as electron acceptors.     

Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21).

In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.     

Purification and properties of the DNA-binding protein HPB12 from Bacillus subtilis nucleoid

We report the purification to homogeneity of a 12 KDa protein (HPB12) present in the nucleoids of Bacillus subtilis. From the purification data the abundance of the protein was estimated to about 20,000 monomers per cell. The HPB12 protein is heat-stable and acid-soluble and binds preferentially to supercoiled and linearized double-stranded DNAs. The binding of the protein to the supercoiled DNA occurs very rapidly and appears to be cooperative. Moreover, the complexes are extremely stable and do not dissociate after 90 min. These properties are consistent with a role of the HPB12 protein in the structure of the B. subtilis chromosome.     

Interaction of the Bacillus subtilis phage phi 105 repressor DNA: a genetic analysis.

The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14-bp operator sequence which does not exhibit 2-fold rotational symmetry. To facilitate a genetic analysis of this sequence-dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor-operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N-terminal part (positions 1-43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N-terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln-18 to Arg-37, which we propose to be the DNA-binding alpha-helix--beta-turn--alpha-helix motif, common to all lambda Cro-like repressors. The second ('recognition') helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half-site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's 'recognition helix'.