Novel β-Lactamase Genes from Two Environmental Isolates of Vibrio harveyi

ABSTRACT Two ampicillin-resistant (Ampr) isolates ofVibrio harveyi, W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Strain W3B was isolated from marine water near a shrimp farm in North Java while HB3 was from pristine seawater in South Java. In this study, novel β-lactamase genes from W3B (bla VHW-1) and HB3 (bla VHH-1) were cloned and their nucleotide sequences were determined. An open reading frame (ORF) of 870 bp encoding a deduced protein of 290 amino acids (VHW-1) was revealed for the bla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-acid protein (VHH-1) was deduced forbla VHH-1. At the DNA level, genes for VHW-1 and VHH-1 have a 97% homology, while at the protein level they have a 91% homology of amino acid sequences. Neither gene sequence showed homology to any other β-lactamases in the databases. The deduced proteins were found to be class A β-lactamases bearing low levels of homology (<50%) to other β-lactamases of the same class. The highest level of identity was obtained with β-lactamases from Pseudomonas aeruginosa, i.e., PSE-1, PSE-4, and CARB-3, and Vibrio cholerae CARB-6. Our study showed that both strains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employingbla VHW-1 as a gene probe demonstrated that thebla gene was not located in the plasmid. A total of nine ampicillin-resistant V. harveyi strains, including W3B and HB3, were examined by pulsed-field gel electrophoresis ofNotI-digested genomic DNA. Despite a high level of intrastrain genetic diversity, thebla VHW-1 probe hybridized only to an 80- or 160-kb NotI genomic fragment in different isolates.

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The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5546-5553.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5546-5553 ◽

Cited By ~ 37

Author(s):

Kazuhiro Iwashita ◽

Tatsuya Nagahara ◽

Hitoshi Kimura ◽

Makoto Takano ◽

Hitoshi Shimoi ◽

...

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Enzyme Assays ◽

Partial Amino Acid Sequence ◽

Reading Frame ◽

Acid Protein ◽

Aspergillus Kawachii ◽

Amino Acid Protein

ABSTRACT We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA inSaccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that thebglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii.

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Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

Journal of Bacteriology ◽

10.1128/jb.187.15.5067-5074.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5067-5074 ◽

Cited By ~ 51

Author(s):

Daisuke Kasai ◽

Eiji Masai ◽

Keisuke Miyauchi ◽

Yoshihiro Katayama ◽

Masao Fukuda

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Terminal Region ◽

Ring Cleavage ◽

Sphingomonas Paucimobilis ◽

Acid Protein ◽

Demethylation Pathway ◽

Dioxygenase Gene ◽

Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

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Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus3001

Journal of Bacteriology ◽

10.1128/jb.181.21.6642-6649.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6642-6649 ◽

Cited By ~ 46

Author(s):

Jae Kweon Park ◽

Kumiko Shimono ◽

Nobuhisa Ochiai ◽

Kazutaka Shigeru ◽

Masako Kurita ◽

...

Keyword(s):

Amino Acid ◽

Fluorescent Protein ◽

Genomic Library ◽

Amino Acid Sequences ◽

Glycol Chitosan ◽

Gene Encoding ◽

Reading Frame ◽

Acid Protein ◽

Green Fluorescent ◽

Potential Inhibitors

ABSTRACT The extracellular chitosanase (34,000 M r) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40°C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag+. Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.

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The Legionella pneumophila iraAB Locus Is Required for Iron Assimilation, Intracellular Infection, and Virulence

Infection and Immunity ◽

10.1128/iai.68.3.1069-1079.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1069-1079 ◽

Cited By ~ 52

Author(s):

V. K. Viswanathan ◽

Paul H. Edelstein ◽

C. Dumais Pope ◽

Nicholas P. Cianciotto

Keyword(s):

Amino Acid ◽

Legionella Pneumophila ◽

Open Reading Frame ◽

Host Cells ◽

Reading Frame ◽

Intracellular Infection ◽

Acid Protein ◽

Iron Deficient ◽

Iron Assimilation ◽

Amino Acid Protein

ABSTRACT Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that theiraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionellaspecies. A newly derived mutant containing a targeted disruption ofiraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest thatiraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.

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Isolation and characterization of the muscle-specific isoform of creatine kinase from the zebrafish, Danio rerio

Biochemistry and Cell Biology ◽

10.1139/o01-134 ◽

2001 ◽

Vol 79 (6) ◽

pp. 779-782 ◽

Cited By ~ 6

Author(s):

Gregory Harder ◽

Ross McGowan

Keyword(s):

Creatine Kinase ◽

Amino Acid ◽

Danio Rerio ◽

Cdna Sequence ◽

Reading Frame ◽

Isolation And Characterization ◽

Acid Protein ◽

Amino Acid Levels ◽

Amino Acid Protein

We have isolated and characterized a cDNA sequence corresponding to the zebrafish muscle-specific isoform of creatine kinase. The sequence is 1552 bases in length and contains an open reading frame capable of producing a 381 amino acid protein. The sequence is very similar to muscle-specific creatine kinases isolated from other species at both the nucleotide and amino acid levels but contains some differences from a previously reported zebrafish clone.Key words: creatine kinase, muscle isoform, zebrafish, Danio rerio.

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ISCce1 and ISCce2, Two Novel Insertion Sequences in Clostridium cellulolyticum

Journal of Bacteriology ◽

10.1128/jb.185.3.714-725.2003 ◽

2003 ◽

Vol 185 (3) ◽

pp. 714-725 ◽

Cited By ~ 9

Author(s):

Hédia Maamar ◽

Pascale de Philip ◽

Jean-Pierre Bélaich ◽

Chantal Tardif

Keyword(s):

Amino Acid ◽

Insertion Site ◽

Open Reading Frame ◽

Inverted Repeats ◽

Target Sequence ◽

Insertion Sequences ◽

Reading Frame ◽

Clostridium Cellulolyticum ◽

Acid Protein ◽

Amino Acid Protein

ABSTRACT Two new insertion sequences, ISCce1 and ISCce2, were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum. In these insertional mutants, the cipC gene was disrupted either by ISCce1 alone or by both ISCce1 and ISCce2. ISCce1 is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS481 and IS3 families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce1. ISCce2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS256 family. Upon transposition, both ISCce1 and ISCce2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce1 is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce2 was found to be mostly inserted into ISCce1. In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C. cellulolyticum strain used.

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Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB fromThermotoga neapolitana

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4774-4781.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4774-4781 ◽

Cited By ~ 90

Author(s):

Jin-Duck Bok ◽

Dinesh A. Yernool ◽

Douglas E. Eveleigh

Keyword(s):

Amino Acid ◽

Molecular Mass ◽

Genomic Library ◽

Cellulose Hydrolysis ◽

Thermotoga Neapolitana ◽

Glycosyl Hydrolases ◽

Transglycosylation Activity ◽

Acid Protein ◽

High Level ◽

Amino Acid Protein

ABSTRACT Two thermostable endocellulases, CelA and CelB, were purified fromThermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high V max value and low apparentKm value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside theV max values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding thecelA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA andcelB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.

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The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2253-2262.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2253-2262

Author(s):

E M Stone ◽

M J Swanson ◽

A M Romeo ◽

J B Hicks ◽

R Sternglanz

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Gene Product ◽

Open Reading Frame ◽

Histone H4 ◽

Reading Frame ◽

Acid Protein ◽

Extragenic Suppressor ◽

Mating Type Genes ◽

Amino Acid Protein

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

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