Characterization of preearly genes in the terminal repetition of bacteriophage BF23 DNA by nucleotide sequencing and restriction mapping

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

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Characterization of coxsackie B virus RNA in myocardium from patients with dilated cardiomyopathy by nucleotide sequencing of reverse transcription-nested polymerase chain reaction products

Human Pathology ◽

10.1016/s0046-8177(98)80006-3 ◽

1998 ◽

Vol 29 (6) ◽

pp. 578-584 ◽

Cited By ~ 45

Author(s):

Leonard C. Archard ◽

Mahboob A. Khan ◽

Bambos A. Soteriou ◽

Hongyi Zhang ◽

Howard J.F. Why ◽

...

Keyword(s):

Nucleotide Sequencing ◽

Reaction Products ◽

Chain Reaction ◽

Nested Polymerase Chain Reaction ◽

Virus Rna ◽

B Virus ◽

Coxsackie B Virus ◽

Polymerase Chain ◽

Coxsackie B

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Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus

Journal of General Virology ◽

10.1099/vir.0.79842-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 2111-2121 ◽

Cited By ~ 92

Author(s):

Daohong Jiang ◽

Said A. Ghabrial

Keyword(s):

Molecular Characterization ◽

Penicillium Chrysogenum ◽

Sequence Similarity ◽

Minor Component ◽

Amino Acid Sequences ◽

Nucleotide Sequencing ◽

Dsrna Segment ◽

Cdna Clones

Molecular cloning and complete nucleotide sequencing of Penicillium chrysogenum virus (PcV) dsRNAs indicated that PcV virions contained four dsRNA segments with sizes of 3562, 3200, 2976 and 2902 bp. Each dsRNA segment had unique sequences and contained a single large open reading frame (ORF). In vitro translation of transcripts derived from full-length cDNA clones of PcV dsRNAs yielded single products of sizes similar to those predicted from the deduced amino acid sequences of the individual ORFs. Sequence similarity searches revealed that dsRNA1 encodes a putative RNA-dependent RNA polymerase. In this study, it was determined that dsRNA2 encodes the major capsid protein and that p4, encoded by dsRNA4, is virion-associated as a minor component. All four dsRNAs of PcV, like the genomic segments of viruses with multipartite genomes, were found to have extended regions of highly conserved terminal sequences at both ends. In addition to the strictly conserved 5′-terminal 10 nt, a second region consisting of reiteration of the sequence CAA was found immediately upstream of the AUG initiator codon. These (CAA) n repeats are reminiscent of the translational enhancer elements of tobamoviruses. The 3′-terminal 14 nt were also strictly conserved. As PcV and related viruses with four dsRNA segments (genus Chrysovirus) have not been previously characterized at the molecular level, they were provisionally classified in the family Partitiviridae, comprising viruses with bipartite genomes. This study represents the first report on molecular characterization of a chrysovirus and the results suggest the creation of a new family of mycoviruses with multipartite dsRNA genomes to accommodate PcV and related viruses.

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Characterization of a Shiga Toxin-Encoding Temperate Bacteriophage of Shigella sonnei

Infection and Immunity ◽

10.1128/iai.69.12.7588-7595.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7588-7595 ◽

Cited By ~ 65

Author(s):

Eckhard Strauch ◽

Rudi Lurz ◽

Lothar Beutin

Keyword(s):

Shiga Toxin ◽

Regulatory Region ◽

Shigella Sonnei ◽

Shigella Dysenteriae ◽

Nucleotide Sequencing ◽

Temperate Bacteriophage ◽

Cross Hybridization ◽

E Coli ◽

K 12

ABSTRACT A Shiga toxin (Stx)-encoding temperate bacteriophage ofShigella sonnei strain CB7888 was investigated for its morphology, DNA similarity, host range, and lysogenization inShigella and Escherichia coli strains. Phage 7888 formed plaques on a broad spectrum of Shigella strains belonging to different species and serotypes, including Stx-producingShigella dysenteriae type 1. With E. coli, only strains with rough lipopolysaccharide were sensitive to this phage. The phage integrated into the genome of nontoxigenic S. sonneiand laboratory E. coli K-12 strains, which became Stx positive upon lysogenization. Moreover, phage 7888 is capable of transducing chromosomal genes in E. coli K-12. The relationships of phage 7888 with the E. coli Stx1-producing phage H-19B and the E. coli Stx2-producing phage 933W were investigated by DNA cross-hybridization of phage genomes and by nucleotide sequencing of an 8,053-bp DNA region of the phage 7888 genome flanking the stx genes. By these methods, a high similarity was found between phages 7888 and 933W. Much less similarity was found between phages H-19B and 7888. As in the other Stx phages, a regulatory region involved in Q-dependent expression is found upstream of stxA and stxB (stx gene) in phage 7888. The morphology of phage 7888 was similar to that of phage 933W, which shows a hexagonal head and a short tail. Our findings demonstrate that stx genes are naturally transferable and are expressed in strains of S. sonnei, which points to the continuous evolution of human-pathogenic Shigella by horizontal gene transfer.

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Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.3830-3840.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 3830-3840 ◽

Cited By ~ 77

Author(s):

Shinichi Kawamoto ◽

Jun Shima ◽

Rumi Sato ◽

Tomoko Eguchi ◽

Sadahiro Ohmomo ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Characterization ◽

Solid Phase ◽

Exchange Chromatography ◽

Nucleotide Sequencing ◽

Lactobacillus Curvatus ◽

Amino Acid Peptide ◽

Enterococcus Mundtii

ABSTRACT Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

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Molecular Cloning and Characterization of cgs, theBrucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB andAgrobacterium tumefaciens chvB Mutants

Journal of Bacteriology ◽

10.1128/jb.180.17.4392-4400.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4392-4400 ◽

Cited By ~ 54

Author(s):

Nora Iñón de Iannino ◽

Gabriel Briones ◽

Marcelo Tolmasky ◽

Rodolfo A. Ugalde

Keyword(s):

Amino Acids ◽

Membrane Protein ◽

Rhizobium Meliloti ◽

Nodule Development ◽

Nucleotide Sequencing ◽

Insertion Mutant ◽

Reading Frame ◽

Glucan Synthesis ◽

Glucan Synthetase

ABSTRACT The animal pathogen Brucella abortus contains a gene,cgs, that complemented a Rhizobium melilotinodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved inRhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor inBrucella infection.

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Nucleotide sequence-based typing of meningococci directly from clinical samples

Journal of Medical Microbiology ◽

10.1099/jmm.0.05078-0 ◽

2003 ◽

Vol 52 (6) ◽

pp. 505-508 ◽

Cited By ~ 15

Author(s):

Mathew A. Diggle ◽

Carolyn M. Bell ◽

Stuart C. Clarke

Keyword(s):

Molecular Techniques ◽

Clinical Samples ◽

Nucleotide Sequencing ◽

Sequencing Data ◽

Conjugate Vaccines ◽

Laboratory Confirmation ◽

Highly Sensitive ◽

Culture Isolate ◽

Micro Organisms

The unpredictable characteristics of meningococcal disease (MD) make outbreaks complicated to monitor and consequently lead to high levels of public anxiety. Traditional molecular techniques have been utilized in order to understand better the epidemiology of MD, but some have disadvantages such as being highly specialized and labour-intensive, with low reproducibility. Some of these problems have been overcome by using multilocus sequence typing (MLST). This technique exploits the unambiguous nature and electronic portability of nucleotide sequencing data for the characterization of micro-organisms. The need for enhanced surveillance of MD after the introduction of serogroup C conjugate vaccines means that it is important to gain typing information from the infecting organism in the absence of a culture isolate. Here, the application of MLST for the laboratory confirmation and characterization of Neisseria meningitidis directly from clinical samples is described. This involved using a newly designed set of primers that were complementary to nucleotide sequences external to the existing MLST primers already in use for culture-based MLST of meningococci. This combination has produced a highly sensitive procedure to allow the efficient genotypic characterization of meningococci directly from clinical samples.

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