Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M.

A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.

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.

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains.

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes<2,4-DNT<2,2'-dimethyl-5, 5'-dinitroazoxybenzene (2,2'-DM-5, 5'-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)<<4, 4'-dimethyl-3,3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB), and aminonitrotoluenes (2A4AT, 4A2NT)<2,4-DNT<4HA2NT4,4'-dimethyl-3, 3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2, 6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2'-dimethyl-3, 3'-dinitroazoxybenzene (2,2'-DM-3,3'-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.

Promoter/repressor system of Lactobacillus plantarum phage og1e: characterization of the promoters pR49-pR-pL and overproduction of the cro-like protein cng in Escherichia coli.

The Lactobacillus plantarum phage og1e (42259bp) has two repressor-like genes cng and cpg oriented oppositely, accompanied by three potential promoters pR, pL and pR49, and seven operator-like sequences (GATAC-boxes) (Kodaira et al., 1997). In this study, the og1e putative promoters were introduced into the Escherichia coli promoter-detecting plasmid pKK232-8. In E. coli CK111, pR (pKPR1), pL (pKPL1) and pR49 (pKPR49) exhibited distinct CAT activities. When pKPR1 or pKPL1 was coexistent with a compatible plasmid pACYC184 carrying pR-cng (pA4PRCN1), the CAT activity was decreased significantly. On the other hand, cng directed a protein (Cng) of 10.1 kDa in E. coli under the control of T7 promoter. Gel mobility-shift assays demonstrated that Cng binds specifically to a DNA region containing the GATAC-boxes. In addition, primer extension analyses demonstrated that the two sequences pR and pL act as a promoter in L. plantarum as well as in E. coli. These results suggested that the potential promoters pR and pL probably function for the lytic and lysogenic pathways, respectively, and Cng may act as a repressor presumably through the GATAC-boxes as operators.

Genome structure of the Lactobacillus temperate phage phi g1e: the whole genome sequence and the putative promoter/repressor system.

The complete genome sequence of a Lactobacillus temperate phage phi g1e was established. The double-stranded DNA is composed of 42,259 bp, and encodes for sixty-two possible open reading frames (ORF) as well as several potential regulatory sequences. Based on comparative analysis with other related proteins of the Lactobacillus and Lactococcus phages as well as the Escherichia coli phages (such as lambda), functions were putatively assigned to several phi g1e ORFs: cng and cpg (encoding for repressors), hel (helicase), ntp (NTPase), and several ORFs (e.g., minor capsid proteins). An about 1000-bp DNA region of phi g1e containing cpg and cng was inferred to function as a promoter/repressor system for the phi g1e lysogenic and lytic pathway.

Characterization of the genes encoding integrative and excisive functions of Lactobacillus phage øg1e: cloning, sequence analysis, and expression in Escherichia coli.

øg1e is a temperate phage of the Lactobacillus strain G1e. The phage-host junctions attR and attL cloned from the lysogen have a 24-bp common (core) sequence implicated in recombination. DNA sequencing analysis of a 5.2-kbp SacI fragment of the øg1e phage genome (42.5 kbp) revealed two possible open reading frames (ORF), xis and int, and the phage attachment (recombination) site (attP), whose 24-bp sequence is identical to the core sequence detected in attR and attL. The deduced int product (Int) is a basic protein of 391 amino acids with an estimated pI of 9.70, and significantly resembles other presumed integrases encoded by the Lactobacillus and Lactococcus phages including øadh and øLC3, as well as the Escherichia coli phages such as lambda. The predicted øg1e xis protein (Xis) is small and very acidic (66 amino acids; pI 4.55), and shows a resemblance (32% overall identity) with a putative excisionase encoded by the Staphylococcus phage ø11. The øg1e Int with a deduced molecular mass of 45.5 kDa was overproduced in E. coli cells, and electrophoretically analyzed.

Purification and DNA-binding properties of the integrase protein Int encoded by Lactobacillus plantarum phage.

The Lactobacillus plantarum temperate phage phi g1e (42,259 bp) encodes an integrase gene int linked to a phage attachment site attP (Kakikawa et al., 1997). To investigate phi g1e recombination, the integrase protein Int was overproduced in Escherichia coli under the T7 promoter, and purified. The Int protein had an apparent molecular mass of 42.0 kDa, corresponding well with that (45.5 kDa) predicted from the DNA sequence. Amino-acid sequencing revealed that the N-terminal 20 amino-acids of the purified Int protein completely coincided with those deduced from the DNA sequence, although deficient in the first methionine. Gel mobility-shift assays demonstrated that Int bound specifically to the attP region. In addition, footprinting analysis showed that Int protected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interaction of Int with the attP site, the reaction prerequisite for integration and excision of the phi g1e genome into and/or out of the host chromosome.

Cloning, sequence analysis, and expression of the genes encoding lytic functions of Bacteriophage phi g1e.

The lysis genes of a Lactobacillus phage phi g1e were cloned, sequenced, and expressed in Escherichia coli. Nucleotide sequencing of a 3813-bp phi g1e DNA revealed five successive open reading frames (ORF), Rorf50, Rorf118, hol, and lys and Rorf175, in the same DNA strand. By comparative analysis of the DNA sequence, the putative hol product (holin) has an estimated molecular weight is 14.2 kDa, and contains two potential transmembrane helices and highly charged N- and C-termini, resembling predicted holins (which are thought to be a cytoplasmic membrane-disrupting protein) encoded by other phages such as mv1 from Lactobacillus bulgaricus, phi adh from Lactobacillus gasseri, as well as monocins from Listeria. On the other hand, the putative phi g1e lys product (lysin) of 48.4 kDa shows significant similarity with presumed muramidase, known as a cell wall peptidoglycandegrading enzyme, encoded by the Lactobacillus phage mv1 and phi adh, the Lactococcus lactis phage phi LC3, and the Streptococcus pneumoniae phages Cp-1, Cp-7 and Cp-9. When expressed in E. coli, the phi g1e lysin and/or holin decreased the cell turbidity significantly, suggesting that the phi g1e hol-lys system is involved in cytolytic process.

The unc-18 gene encodes a novel protein affecting the kinetics of acetylcholine metabolism in the nematode Caenorhabditis elegans.

Genes affecting acetylcholine (ACh) levels without influencing choline acetyltransferase activity have been identified in Caenorhabditis elegans. We have examined one such gene, unc-18. We isolated a transposon-insertion allele for unc-18 and used it to clone a genomic region containing the unc-18 locus. The unc-18 location within this region was determined by rescuing the unc-18 mutant phenotype in a germ-line transformation experiment and identifying transcripts affected by four independent unc-18 mutations. A single-sized poly(A)+ RNA was synthesized from the gene. Expression of the transcript appears to be stage specific: The transcript is found in abundance at the early larval stage but in decreased amounts at the fourth larval and the adult stages. These results show that the unc-18 gene plays a role in development as well as in the kinetics of ACh metabolism.

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.

Synthesis of alpha3 phage DNA in replication mutants of Escherichia coli.

Host functions for DNA replication of bacteriophage alpha3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna+ cells, conversion of phage alpha3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30--150 microgram/ml of chloramphenicol, 200 microgram/ml of rifampicin, 50 microgram/ml of nalidixic acid, or 200 microgram/ml of novobiocin. At 43 degrees C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage alpha3 progeny RF was prevented by 50 microgram/ml of mitomycin C (in hcr+ bacteria), 50 microgram/ml of nalidixic acid or 200 microgram/ml of novoviocin, but neither by 30 microgram/ml of chloramphenicol nor by 200 microgram/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for progeny RF synthesis. Host factor dependence of alpha3 was relatively simple and, in contrast with phages phiX174 and G4, alpha3 did not require dnaB and dnaC(D) activities.

Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.