scholarly journals Mutations in Bartonella bacilliformis gyrB Confer Resistance to Coumermycin A1

1998 ◽  
Vol 42 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
James M. Battisti ◽  
Laura S. Smitherman ◽  
D. Scott Samuels ◽  
Michael F. Minnick
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Extract ◽  
Natural Resistance ◽  
Temperature Sensitive ◽  
Bartonella Bacilliformis ◽  
Reading Frame ◽  
E Coli ◽  
Isolation And Characterization ◽  
Spontaneous Mutants

ABSTRACT This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of ∼77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants ofB. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124→Ser), Arg184→Gln, and Thr214→Ala or Thr214→Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes fromBorrelia burgdorferi, E. coli,Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.

scholarly journals Molecular Identification of Family 38 α-Mannosidase of Bacillus sp. Strain GL1, Responsible for Complete Depolymerization of Xanthan

2002 ◽  
Vol 68 (6) ◽  
pp. 2731-2736 ◽  
Author(s):  
Hirokazu Nankai ◽  
Wataru Hashimoto ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Extract ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Sequence Information ◽  
Reading Frame ◽  
A Cell ◽  
Terminal Amino ◽  
Amino Acid Sequence Information

ABSTRACT When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, α-mannosidase exhibiting activity toward p-nitrophenyl-α-d-mannopyranoside (pNP-α-d-Man) was produced intracellularly. The 350-kDa α-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-α-d-Man (Km = 0.49 mM) and d-mannosyl-(α-1,3)-d-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for α-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of α-mannosidases belonging to glycoside hydrolase family 38.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

scholarly journals Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli

1992 ◽  
Vol 288 (3) ◽  
pp. 1037-1044 ◽  
Author(s):  
R E Milner ◽  
J Busaan ◽  
M Michalak
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Fusion Proteins ◽  
Cytoskeletal Proteins ◽  
Terminal Region ◽  
Soluble Form ◽  
E Coli ◽  
Terminal Domain ◽  
Isolation And Characterization

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

scholarly journals The yiaE Gene, Located at 80.1 Minutes on the Escherichia coli Chromosome, Encodes a 2-Ketoaldonate Reductase

1998 ◽  
Vol 180 (22) ◽  
pp. 5984-5988 ◽  
Author(s):  
Do-Young Yum ◽  
Bong-Yong Lee ◽  
Dae-Hyum Hahm ◽  
Jae-Gu Pan
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Metal Chelate ◽  
Reading Frame ◽  
Sequencing Project ◽  
E Coli ◽  
Dehydrogenase Gene ◽  
Translational Start

ABSTRACT An open reading frame located in the bisC-cspAintergenic region, or at 80.1 min on the Escherichia colichromosome, encodes a hypothetical 2-hydroxyacid dehydrogenase, which was identified as a result of the E. coli Genome Sequencing Project. We report here that the product of the gene (yiaE) is a 2-ketoaldonate reductase (2KR). The gene was cloned and expressed with a C-terminal His tag in E. coli, and the protein was purified by metal-chelate affinity chromatography. The determination of the NH2-terminal amino acid sequence of the protein defined the translational start site of this gene. The enzyme was found to be a 2KR catalyzing the reduction of 2,5-diketo-d-gluconate to 5-keto-d-gluconate, 2-keto-d-gluconate (2KDG) to d-gluconate, 2-keto-l-gulonate tol-idonate. The reductase was optimally active at pH 7.5, with NADPH as a preferred electron donor. The deduced amino acid sequence showed 69.4% identity with that of 2KR from Erwinia herbicola. Disruption of this gene on the chromosome resulted in the loss of 2KR activity in E. coli. E. coli W3110 was found to grow on 2KDG, whereas the mutant deficient in 2KR activity was unable to grow on 2KDG as the carbon source, suggesting that 2KR is responsible for the catabolism of 2KDG in E. coli and the diminishment of produced 2KDG from d-gluconate in the cultivation of E. coli harboring a cloned gluconate dehydrogenase gene.

scholarly journals A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate

2004 ◽  
Vol 186 (9) ◽  
pp. 2757-2765 ◽  
Author(s):  
Eiji Masai ◽  
Miyuki Sasaki ◽  
Yasunori Minakawa ◽  
Tomokuni Abe ◽  
Tomonori Sonoki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Extract ◽  
Growth Defect ◽  
Sphingomonas Paucimobilis ◽  
Reading Frame ◽  
E Coli ◽  
Growth Deficiency ◽  
Glycine Cleavage ◽  
Derivative Strain

ABSTRACT Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H4folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H4folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H4folate, forming 5-methyl-H4folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H4folate. The possible role of ligH is discussed.

scholarly journals d-alanine: d-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme

1992 ◽  
Vol 282 (3) ◽  
pp. 747-752 ◽  
Author(s):  
O A M al-Bar ◽  
C D O'Connor ◽  
I G Giles ◽  
M Akhtar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Phosphinic Acid ◽  
Mature Protein ◽  
Bamhi Fragment ◽  
E Coli ◽  
Escherichia Coli Expression ◽  
Slow Binding Inhibitor

A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis

1991 ◽  
Vol 11 (8) ◽  
pp. 4022-4035
Author(s):  
A R Kubelik ◽  
B Turcq ◽  
A M Lambowitz
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Temperature Sensitivity ◽  
Temperature Sensitive ◽  
Cellular Transport ◽  
Missense Mutations ◽  
Reading Frame ◽  
Trna Synthetases ◽  
Cytosolic Protein

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.

scholarly journals Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

1996 ◽  
Vol 40 (3) ◽  
pp. 616-620 ◽  
Author(s):  
A Bauernfeind ◽  
I Stemplinger ◽  
R Jungwirth ◽  
P Mangold ◽  
S Amann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Beta Lactamase ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
The Family ◽  
Extended Spectrum Beta Lactamases ◽  
Lactamase Gene

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

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