Sequence analysis of theGluconobacter oxydansRecA protein and construction of arecA-deficient mutant

1999 ◽  
Vol 45 (4) ◽  
pp. 347-351 ◽  
Author(s):  
Yu-Tien Liu ◽  
Chia-Geun Chen ◽  
Der-Chiang Chao ◽  
Fan Lee ◽  
Ching-Len Liao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Uv Irradiation ◽  
Reca Protein ◽  
Kanamycin Resistance ◽  
Reca Gene ◽  
Amino Acid Residues ◽  
Deficient Mutant ◽  
Wild Type ◽  
E Coli ◽  
Nucleotide Sequence Alignment

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, andPseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.Key words: Gluconobacter orphans, recA gene, DNA repair, recA mutant, SOS box.

Molecular characterization ofGluconobacter oxydans recAgene and its inhibitory effect on the function of the host wild-typerecAgene

1998 ◽  
Vol 44 (2) ◽  
pp. 149-156 ◽  
Author(s):  
Yu-Tien Liu ◽  
Der-Chiang Chao ◽  
Fan Lee ◽  
Chia-Geun Chen ◽  
Dar-Der Ji
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Reca Protein ◽  
Gluconobacter Oxydans ◽  
Inhibitory Effect ◽  
Reca Gene ◽  
Wild Type ◽  
E Coli

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-typerecA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.Key words: Gluconobacter oxydans, recA gene, recombination, SOS function, interference.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Sensitization of Human Aortic Endothelial Cells to Lipopolysaccharide via Regulation of Toll-Like Receptor 4 by Bacterial Fimbria-Dependent Invasion

2005 ◽  
Vol 73 (12) ◽  
pp. 8050-8059 ◽  
Author(s):  
Hiromichi Yumoto ◽  
Hsin-Hua Chou ◽  
Yusuke Takahashi ◽  
Michael Davey ◽  
Frank C. Gibson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Periodontal Disease ◽  
Deficient Mutant ◽  
Wild Type ◽  
Microbial Infection ◽  
Monocyte Chemoattractant Protein 1 ◽  
Aortic Endothelial Cells ◽  
E Coli ◽  
Human Aortic Endothelial Cells ◽  
Aortic Endothelial

ABSTRACT Toll-like receptors (TLRs) are differentially up-regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Epidemiological data support the idea that periodontal disease may be a risk factor for acceleration of atherosclerosis. Porphyromonas gingivalis, the etiological agent of periodontal disease, invades endothelium, has been detected in human atheromatous tissue, and accelerates atheroma formation in apolipoprotein E−/− mice with concurrent induction of TLRs in the aorta. As endothelial cells can present antigen via TLRs and play an important role in the development of atherosclerosis, we examined TLR expression in human aortic endothelial cells (HAEC) cultured with wild-type P. gingivalis, a fimbria-deficient mutant, and purified antigens. We observed increased TLR expression in HAEC infected with wild-type P. gingivalis by fluorescence-activated cell sorter, but not with noninvasive, fimbria-deficient mutant or purified P. gingivalis antigens. Following a wild-type P. gingivalis challenge, functional TLR2 and TLR4 activation was assessed by subsequent stimulation with TLR agonists Staphylococcus aureus lipoteichoic acid (SLTA; TLR2 ligand) and Escherichia coli lipopolysaccharide (LPS; TLR4 ligand). Unchallenged HAEC failed to elicit monocyte chemoattractant protein 1 (MCP-1) in response to LPS or SLTA but did so when cultured with wild-type P. gingivalis. P. gingivalis-induced TLR2 and -4 expression on HAEC functionally reacted to SLTA and E. coli LPS as measured by a further increase in MCP-1 production. Furthermore, MCP-1 expression elicited by E. coli LPS was inhibitable with TLR4-specific antibody and polymyxin B. These results indicate that invasive P. gingivalis stimulates TLR expression on the surface of endothelium and these primed cells respond to defined TLR-specific ligands.

scholarly journals Identification of a Gene Involved in Assembly ofActinomyces naeslundii T14V Type 2 Fimbriae

1998 ◽  
Vol 66 (4) ◽  
pp. 1482-1491 ◽  
Author(s):  
Maria K. Yeung ◽  
Jacob A. Donkersloot ◽  
John O. Cisar ◽  
Pamela A. Ragsdale
Keyword(s):  
Amino Acid ◽  
Kanamycin Resistance ◽  
Amino Acid Residues ◽  
Subunit Protein ◽  
Actinomyces Naeslundii ◽  
Cell Extracts ◽  
Acid Protein ◽  
Structural Subunit ◽  
Antipeptide Antibody

ABSTRACT The nucleotide sequence of the Actinomyces naeslundiiT14V type 2 fimbrial structural subunit gene, fimA, and the 3′ flanking DNA region was determined. The fimA gene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal cell wall sorting sequences. A second gene, designated orf365, that encoded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3′ to fimA. Mutants in which either fimA or orf365 was replaced with a kanamycin resistance gene did not participate in type 2 fimbriae-mediated coaggregation with Streptococcus oralis34. Type 2 fimbrial antigen was not detected in cell extracts of thefimA mutant by Western blotting with anti-A. naeslundii type 2 fimbrial antibody, but the subunit protein was detected in extracts of the orf365 mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide representing the C-terminal 20 amino acid residues of the predicted FimA. The antipeptide antibody reacted with FimA isolated from the recombinant Escherichia coliclone containing fimA but did not react with purified type 2 fimbriae in extracts of the wild-type strain. These results indicate that synthesis of type 2 fimbriae in A. naeslundii T14V may involve posttranslational cleavage of both the N-terminal and C-terminal peptides of the precursor subunit and also the expression oforf365.

Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1763-1770 ◽  
Author(s):  
Ryszard Zielke ◽  
Aleksandra Sikora ◽  
Rafał Dutkiewicz ◽  
Grzegorz Wegrzyn ◽  
Agata Czyż
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Repair ◽  
Gene Product ◽  
Uv Irradiation ◽  
Vibrio Harveyi ◽  
Bacterial Species ◽  
Wild Type ◽  
E Coli ◽  
Stimulation Of

CgtA is a member of the Obg/Gtp1 subfamily of small GTP-binding proteins. CgtA homologues have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Nevertheless, despite the fact that cgtA is an essential gene in most bacterial species, its function in the regulation of cellular processes is largely unknown. Here it has been demonstrated that in two bacterial species, Escherichia coli and Vibrio harveyi, the cgtA gene product enhances survival of cells after UV irradiation. Expression of the cgtA gene was found to be enhanced after UV irradiation of both E. coli and V. harveyi. Moderate overexpression of cgtA resulted in higher UV resistance of E. coli wild-type and dnaQ strains, but not in uvrA, uvrB, umuC and recA mutant hosts. Overexpression of the E. coli recA gene in the V. harveyi cgtA mutant, which is very sensitive to UV light, restored the level of survival of UV-irradiated cells to the levels observed for wild-type bacteria. Moreover, the basal level of the RecA protein was lower in a temperature-sensitive cgtA mutant of E. coli than in the cgtA + strain, and contrary to wild-type bacteria, no significant increase in recA gene expression was observed after UV irradiation of this cgtA mutant. Finally, stimulation of uvrB gene transcription under these conditions was impaired in the V. harveyi cgtA mutant. All these results strongly suggest that the cgtA gene product is involved in DNA repair processes, most probably by stimulation of recA gene expression and resultant activation of RecA-dependent DNA repair pathways.

bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans

1991 ◽  
Vol 99 (4) ◽  
pp. 711-719
Author(s):  
K.L. O'Donnell ◽  
A.H. Osmani ◽  
S.A. Osmani ◽  
N.R. Morris
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Essential Gene ◽  
Tetratricopeptide Repeat ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reading Frame ◽  
Integrative Transformation

The recessive, temperature-sensitive bimA1 mutation of Aspergillus nidulans blocks nuclei in metaphase at restrictive temperature. To determine whether the bimA product is essential, integrative transformation was used to create a mutation in the bimA gene. The mutation was maintained in a heterokaryon and the phenotype of spores produced by the heterokaryon was analyzed. Molecular disruption of the wild-type bimA gene is recessive in the heterokaryon and causes a metaphase block, demonstrating that bimA is an essential gene for mitosis. bimA was cloned by DNA-mediated complementation of its mutant phenotype at restrictive temperature, and the nucleotide sequence of a full-length cDNA was determined. A single large open reading frame was identified in the cDNA sequence, which predicts a protein containing 806 amino acid residues that is related (30.4% identity) to the Schizosaccharomyces pombe nuc2+ gene product, which also is required for completion of mitosis. The sequence of the bimA gene indicates that it is a member of a family of mostly nuclear proteins that contain a degenerate 34 amino acid repeat, the TPR (tetratricopeptide repeat) gene family.

Homologous recombination involving a large heterology in Escherichia coli.

Genetics ◽  
1988 ◽  
Vol 119 (4) ◽  
pp. 759-769
Author(s):  
K Yamamoto ◽  
N Takahashi ◽  
H Yoshikura ◽  
I Kobayashi
Keyword(s):  
Escherichia Coli ◽  
Gene Conversion ◽  
Kanamycin Resistance ◽  
Coli Strain ◽  
The Other ◽  
Reca Gene ◽  
Inverted Repeats ◽  
Crossing Over ◽  
Wild Type ◽  
Parental Allele

Abstract Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain. The two homologous regions were in an inverted orientation on the same plasmid molecule. Kanamycin-resistant plasmids were selected and analyzed. The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene. It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form. Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats. These results were interpreted as follows. Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other. The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis. In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate. In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E. coli strain.

Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

1990 ◽  
Vol 45 (5) ◽  
pp. 538-543 ◽  
Author(s):  
D. Friedberg ◽  
J. Seijffers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Amino Acid Level ◽  
Acetolactate Synthase ◽  
Mutant Gene ◽  
Wild Type ◽  
E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

scholarly journals PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2702-2707 ◽  
Author(s):  
Sujoy Kumar Sarkar ◽  
Mouparna Dutta ◽  
Chiranjit Chowdhury ◽  
Akash Kumar ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Parent Strain ◽  
Amino Acid Identity ◽  
Enzymic Activity ◽  
Exponential Phase ◽  
Wild Type ◽  
Acid Identity ◽  
Penicillin Binding Proteins ◽  
E Coli

Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic β-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared β-lactam sensitivity of the mutants and the parent strain. β-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for β-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter β-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored β-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic β-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.

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