scholarly journals Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.

1996 ◽  
Vol 40 (10) ◽  
pp. 2350-2355 ◽  
Author(s):  
J A Aínsa ◽  
C Martin ◽  
B Gicquel ◽  
R Gomez-Lus
Keyword(s):  
Dna Hybridization ◽  
Mycobacterium Smegmatis ◽  
Open Reading Frame ◽  
Mycobacterium Fortuitum ◽  
Aminoglycoside Resistance ◽  
Gene Encoding ◽  
Reading Frame ◽  
Putative Protein ◽  
Providencia Stuartii

A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.

scholarly journals Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

2000 ◽  
Vol 66 (12) ◽  
pp. 5480-5483 ◽  
Author(s):  
Sean S. Dineen ◽  
Marite Bradshaw ◽  
Eric A. Johnson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Clostridium Botulinum ◽  
Shuttle Vector ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Heat Stable ◽  
Group I

ABSTRACT Boticin B is a heat-stable bacteriocin produced byClostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene,btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing theHindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.

scholarly journals Characterization of IS18, an Element Capable of Activating the Silent aac(6′)-Ij Gene ofAcinetobacter sp. 13 Strain BM2716 by Transposition

1998 ◽  
Vol 42 (10) ◽  
pp. 2759-2761 ◽  
Author(s):  
Eric Rudant ◽  
Patrice Courvalin ◽  
Thierry Lambert
Keyword(s):  
Resistance Gene ◽  
Insertion Sequence ◽  
Resistant Mutant ◽  
Open Reading Frame ◽  
Inverted Repeats ◽  
Aminoglycoside Resistance ◽  
Reading Frame ◽  
Terminal Inverted Repeats ◽  
Hybrid Promoter

ABSTRACT Insertion sequence IS18 was detected by analysis of the spontaneous aminoglycoside resistant mutant Acinetobactersp. 13 strain BM2716-1. Insertion of the element upstream from the silent acetyltransferase gene aac(6′)-Ij created a hybrid promoter that putatively accounts for the expression of the aminoglycoside resistance gene. The 1,074-bp IS18 element contained partially matched (20 out of 26 bases) terminal inverted repeats, one of which overlapped the 3′ end of a 935-bp open reading frame potentially encoding a protein related to the transposases of the IS30 family. IS18 was found in 6 out of 29 strains of Acinetobacter sp. 13 but not in 10 strains each of A. baumannii and A. haemolyticus.

Sequence of a Rhodococcus gene cluster encoding the subunits of ethanolamine ammonia-lyase and an APC-like permease

1994 ◽  
Vol 40 (5) ◽  
pp. 403-407 ◽  
Author(s):  
René De Mot ◽  
Istvan Nagy ◽  
Geert Schoofs ◽  
Jos Vanderleyden
Keyword(s):  
Open Reading Frame ◽  
Genomic Fragment ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gene Encoding ◽  
Reading Frame ◽  
Transporter Family ◽  
Rhodococcus Species ◽  
Rhodococcus Sp

Sequence analysis of a 5173-bp genomic fragment from the nocardioform actinomycete Rhodococcus sp. strain NI86/21 revealed the presence of two genes, eutB and eutC, encoding the putative homologues of the large and small subunits of the ethanolamine ammonia-lyase, respectively, from Salmonella typhimurium. This is the first report of the characterization of these genes in a Gram-positive species. Immediately upstream of eutB, a gene encoding a putative permease of the APC (amino acids, polyamines, choline) transporter family was located. At present, no other Gram-positive members of this permease family are known. The translational coupling of these eut genes suggests an operon-like organization of the ethanolamine genes in Rhodococcus species. A truncated open reading frame downstream of eutC contained an N-terminal motif characteristic of membrane-anchored lipoproteins.Key words: nocardioform actinomycete, cobalamin, APC transporter, membrane-anchored lipoprotein, Gram-positive bacterium.

scholarly journals Cloning and Expression of the algL Gene, Encoding the Azotobacter chroococcum Alginate Lyase: Purification and Characterization of the Enzyme

1999 ◽  
Vol 181 (5) ◽  
pp. 1409-1414 ◽  
Author(s):  
Ana Peciña ◽  
Alberto Pascual ◽  
Antonio Paneque
Keyword(s):  
Gene Sequence ◽  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Azotobacter Chroococcum ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Reading Frame ◽  
Encoding Gene

ABSTRACT The alginate lyase-encoding gene (algL) ofAzotobacter chroococcum was localized to a 3.1-kbEcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

scholarly journals Loss of intrinsic aminoglycoside resistance in Acinetobacter haemolyticus as a result of three distinct types of alterations in the aac(6')-Ig gene, including insertion of IS17.

1997 ◽  
Vol 41 (12) ◽  
pp. 2646-2651 ◽  
Author(s):  
E Rudant ◽  
P Courvalin ◽  
T Lambert
Keyword(s):  
Point Mutation ◽  
Dna Hybridization ◽  
Aminoglycoside Resistance ◽  
Gene Encoding ◽  
Reading Frame ◽  
Loss Of Resistance ◽  
Acinetobacter Haemolyticus

The distribution of the aac(6')-Ig gene, encoding aminoglycoside 6'-N-acetyltransferase-Ig [AAC(6')-Ig], was studied in 96 Acinetobacter haemolyticus strains and 12 proteolytic Acinetobacter strains, including Acinetobacter genomospecies 6, 13, and 14 and 3 unnamed species assigned to this genomic group by DNA-DNA hybridization. This gene was detected by DNA-DNA hybridization in all 96 A. haemolyticus strains and by PCR in 95 strains but was not detected in strains of other species, indicating that it may be used to identify A. haemolyticus. Three A. haemolyticus strains were susceptible to tobramycin and did not produce an aminoglycoside 6'-N-acetylating activity, although they contained aac(6')-Ig-related sequences. An analysis of three susceptible A. haemolyticus strains indicated that aminoglycoside resistance was abolished by the following three distinct mechanisms: (i) a point mutation in aac(6')-Ig that led to a Met56-->Arg substitution, which was shown by analysis of a revertant to be responsible for the loss of resistance; (ii) a polythymine insertion that altered the reading frame; and (iii) insertion of IS17, a new member of the IS903 family. These observations indicated that AAC(6')-Ig is not essential for the viability of A. haemolyticus, although the aac(6')-Ig gene was detected in all members of this species.

scholarly journals Functional Characterization of Alanine Racemase fromSchizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

2001 ◽  
Vol 183 (7) ◽  
pp. 2226-2233 ◽  
Author(s):  
Takuma Uo ◽  
Tohru Yoshimura ◽  
Naotaka Tanaka ◽  
Kaoru Takegawa ◽  
Nobuyoshi Esaki
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Source ◽  
Functional Characterization ◽  
Open Reading Frame ◽  
Alanine Racemase ◽  
Sole Nitrogen Source ◽  
Recombinant Yeast ◽  
Reading Frame ◽  
Putative Protein

ABSTRACT Schizosaccharomyces pombe has an open reading frame, which we named alr1 +, encoding a putative protein similar to bacterial alanine racemase. We cloned thealr1 + gene in Escherichia coli and purified the gene product (Alr1p), with an M rof 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparentKm and V max values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that ofl-alanine, respectively. S. pombe usesd-alanine as a sole nitrogen source, but deletion of thealr1 + gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway forl-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but notd-alanine as a sole nitrogen source. Moreover,d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1 + gene enabled S. cerevisiae to grow efficiently ond-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine.

scholarly journals Knot Formation Caused by Pseudomonas syringae subsp. savastanoi on Olive Plants Is hrp-Dependent

2004 ◽  
Vol 94 (5) ◽  
pp. 484-489 ◽  
Author(s):  
A. Sisto ◽  
M. G. Cipriani ◽  
M. Morea
Keyword(s):  
Sequence Analysis ◽  
Pseudomonas Syringae ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Phytopathogenic Bacteria ◽  
Reading Frame ◽  
Nonhost Plant ◽  
Induced Mutant

The virulence of Pseudomonas syringae subsp. savastanoi, which causes hyperplastic symptoms (knots) on olive plants, is associated with secreted phytohormones. We identified a Tn5-induced mutant of P. syringae subsp. savastanoi that did not cause disease symptoms on olive plants although it was still able to produce phytohormones. In addition, the mutant failed to elicit a hypersensitive response in a nonhost plant. Molecular characterization of the mutant revealed that a single Tn5 insertion occurred within an open reading frame encoding a protein 92% identical to the HrcC protein of P. syringae pv. syringae. Moreover, sequence analysis revealed that the gene encoding the HrcC protein in P. syringae subsp. savastanoi was part of an operon that included five genes arranged as in other phytopathogenic bacteria. These results imply that hrp/hrc genes are functional in P. syringae subsp. savastanoi and that they play a key role in the pathogenicity of this plant pathogen.

scholarly journals Cloning and Characterization of a Sulfonate/α-Ketoglutarate Dioxygenase from Saccharomyces cerevisiae

1999 ◽  
Vol 181 (18) ◽  
pp. 5876-5879 ◽  
Author(s):  
Deborah A. Hogan ◽  
Thomas A. Auchtung ◽  
Robert P. Hausinger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Sequence Identity ◽  
Ralstonia Eutropha ◽  
Open Reading Frame ◽  
Yeast Protein ◽  
Gene Encoding ◽  
Reading Frame ◽  
Sequence Identity ◽  
Ketoglutarate Dioxygenase

ABSTRACT The Saccharomyces cerevisiae open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to Escherichia coli taurine/α-ketoglutarate dioxygenase and 27% identity to Ralstonia eutropha TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/α-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being isethionate and taurocholate. Disruption of the gene encoding this enzyme negatively affects the use of isethionate and taurine as sulfur sources byS. cerevisiae, providing strong evidence that YLL057c plays a role in sulfonate catabolism.

scholarly journals Identification of a Sterol Δ7 Reductase Gene Involved in Desmosterol Biosynthesis in Mortierella alpina 1S-4

2007 ◽  
Vol 73 (6) ◽  
pp. 1736-1741 ◽  
Author(s):  
Shuo Zhang ◽  
Eiji Sakuradani ◽  
Sakayu Shimizu
Keyword(s):  
Mortierella Alpina ◽  
Open Reading Frame ◽  
Predicted Amino Acid Sequence ◽  
Control Strain ◽  
Gene Encoding ◽  
Reading Frame ◽  
African Clawed Frog ◽  
Reductase Gene ◽  
Clawed Frog

ABSTRACT Molecular cloning of the gene encoding sterol Δ7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoΔ7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoΔ7SR showed highest homology of 51% with that of sterol Δ7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoΔ7SR gene in yeast Saccharomyces cerevisiae revealed that MoΔ7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Δ7 reductase. In addition, with gene silencing of MoΔ7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoΔ7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Δ7 reductase from a microorganism.

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