RESUMO
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.
Assuntos
Serina Endopeptidases/genética , Staphylococcus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Staphylococcus/enzimologia , Especificidade por SubstratoRESUMO
The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).
Assuntos
Bacteriófagos/genética , Proteínas de Ligação a DNA/metabolismo , Lactobacillus/virologia , Proteínas Repressoras/isolamento & purificação , Proteínas não Estruturais Virais/isolamento & purificação , Sequência de Bases , Sítios de Ligação/genética , Reagentes de Ligações Cruzadas , Pegada de DNA , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I , Dimerização , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS-PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.
Assuntos
Acetiltransferases , Aciltransferases/isolamento & purificação , Escherichia coli/enzimologia , Acetilcoenzima A/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Cinética , Plasmídeos , ortoaminobenzoatos/metabolismoRESUMO
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.
Assuntos
Bacteriófagos/genética , Regulação Viral da Expressão Gênica , Lactobacillus/virologia , Sequência de Aminoácidos , Ligação Competitiva , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA/metabolismo , Escherichia coli/genética , Genes Virais/genética , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Carcinógenos/toxicidade , Dinitrobenzenos/toxicidade , Salmonella typhimurium/genética , Acetiltransferases/química , Carcinógenos/metabolismo , Dinitrobenzenos/metabolismo , Testes de Mutagenicidade , Nitrorredutases/química , Oxirredução , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/enzimologiaRESUMO
The Lactobacillus plantarum phage og1e (42
Assuntos
Bacteriófagos/genética , Proteínas de Ligação a DNA , Genes Virais , Lactobacillus/virologia , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genética , Bacteriófagos/metabolismo , Sequência de Bases , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/biossíntese , Clonagem Molecular , Escherichia coli/genética , Genótipo , Lactobacillus/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/metabolismo , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
Lactobacillus plantarum phage phi gle has two consecutive cell lysis genes hol-lys (Oki et al., 1996b). In the present study, functional and structural properties of the hol protein (Hol) were characterized in Escherichia coli. Electron microscopic examinations showed that hol under plac in E. coli XL1-Blue injured the inner membrane to yield empty ghost cells with the bulk of the cell wall undisturbed. Northern blot analysis indicated that hol-lys genes under plac were co-transcribed, although the amount of hol transcript was larger than that of lys, ceasing via an apparently rho-independent terminator just downstream of hol. However, deletion and/or fusion experiments suggested that: (1) the N-terminal half of phi gle Hol composed of three putative transmembrane domains may be responsible for interaction with membrane; (2) the N-terminal end (five amino acids) seems nonessential; and (3) the C-terminal half containing charged amino acids appears to be involved in proper hol function. These results suggest that phi gle Hol is a member of the lambdoid holin family, but divergent in several properties from lambda holin.
Assuntos
Bacteriófagos/genética , Escherichia coli/virologia , Lactobacillus/virologia , Proteínas de Membrana/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Bacteriólise , Sequência de Bases , Membrana Celular/ultraestrutura , DNA Viral/química , Genes Virais/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Viral/análise , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Proteínas Virais/fisiologia , Proteínas Estruturais Virais/genéticaRESUMO
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.
Assuntos
Bacteriófagos/genética , DNA Viral/química , Genoma Viral , Lactobacillus/virologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Capsídeo/genética , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Sequências Hélice-Volta-Hélice , Mitomicina/farmacologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribossomos/metabolismo , Análise de Sequência , Homologia de Sequência de Aminoácidos , Raios Ultravioleta , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
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.
Assuntos
Bacteriófagos/genética , Clonagem Molecular , DNA Nucleotidiltransferases/genética , Regulação Bacteriana da Expressão Gênica , Integrases/genética , Lactobacillus/virologia , Lisogenia/genética , Proteínas Virais , Sequência de Aminoácidos , Sítios de Ligação Microbiológicos/genética , Bacteriófago lambda/genética , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , DNA Nucleotidiltransferases/fisiologia , Escherichia coli/genética , Escherichia coli/virologia , Integrases/fisiologia , Lactococcus/virologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Staphylococcus/virologiaRESUMO
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.
Assuntos
Bacteriófagos/enzimologia , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Integrases/metabolismo , Bacteriófagos/genética , Sequência de Bases , Pegada de DNA , DNA Viral , Proteínas de Ligação a DNA/genética , Integrases/genética , Lactobacillus/virologia , Dados de Sequência Molecular , Plasmídeos , Ligação ProteicaRESUMO
Bacteriophage phi gle was induced from a lysogenic Lactobacillus strain Gle. phi gle genome is double-stranded DNA of approximately 42.5 kilo-base (kb) pairs. SDS poly-acrylamide gel electrophoresis demonstrated that the phage particles contain 4 major structural (capsid) proteins, gpB, gpG, gpO, and gpP, whose molecular weights (MW) are estimated to be 64, 43, 29 and 26 kilodaltons (kDa), respectively. More than 16 minor proteins ranging from 113 to 9.6 kDa were also detected. The genes for the major capsid proteins were cloned and each DNA sequence was determined. N-terminal amino acid alignments determined by protein sequencing completely coincided with those deduced from the nucleotide sequences.
Assuntos
Bacteriófagos/química , Capsídeo/genética , Genes Virais/genética , Lactobacillus/virologia , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Bacteriófagos/ultraestrutura , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Microscopia Eletrônica , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
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.
Assuntos
Bacteriófagos/genética , Lactobacillus/virologia , Análise de Sequência de DNA , Proteínas Virais/genética , Sequência de Aminoácidos , Bacteriófagos/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Viral , Expressão Gênica , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The microvirid phage phi K, specific for Escherichia coli K12, contains a circular single-stranded (SS) DNA in the icosahedral virion, which comprises four phage gene products, F (capsid), G (major spike), H (minor spike), and J (core). phi KhT, a host-range mutant of phi K, can grow on E. coli C and B, besides K12, and is more thermosensitive than the parental phage phi K. Sequencing analysis revealed that the genome of phi K and phi KhT consists of 6,089 nucleotides (nt), and codes for eleven genes, whose sequences are similar to those of alpha 3, phi X174, and G4 infective to strain C. In phi KhT, two nt had changed: one is in the gene G, resulting in replacement of the 75th codon Ala with Ser, and the other is at 67th codon of the gene H: Val to Ala. Chemically synthesized gene J protein composed of 23 amino acids (aa) binds to phi K SS DNA more tightly than and preferentially over the host E. coli SS-DNA-binding protein (SSB). These results indicate that the two spike proteins G and H are involved in the determination of phi K host-range, and support a model in which the gene J protein functions in packaging the viral SS DNA into the virion vesicle.