scholarly journals CAU-1, a Subclass B3 Metallo-β-Lactamase of Low Substrate Affinity Encoded by an Ortholog Present in the Caulobacter crescentus Chromosome

2002 ◽  
Vol 46 (6) ◽  
pp. 1823-1830 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Fabrizio Pantanella ◽  
Francesco Giuliani ◽  
Maria Cristina Thaller ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Hypothetical Protein ◽  
Exchange Chromatography ◽  
Substrate Affinity ◽  
Gene Encoding ◽  
Native Form ◽  
Significant Similarity ◽  
Expression Studies ◽  
Genes Encoding ◽  
Isoelectric Ph

ABSTRACT The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Km values were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, the bla CAU determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the class Proteobacteria.

scholarly journals The Bartonella vinsonii subsp. arupensis Immunodominant Surface Antigen BrpA Gene, Encoding a 382-Kilodalton Protein Composed of Repetitive Sequences, Is a Member of a Multigene Family Conserved among Bartonella Species

2005 ◽  
Vol 73 (5) ◽  
pp. 3128-3136 ◽  
Author(s):  
Robert D. Gilmore ◽  
Travis M. Bellville ◽  
Steven L. Sviat ◽  
Michael Frace
Keyword(s):  
Bartonella Henselae ◽  
Repetitive Sequences ◽  
Expression Library ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Bartonella Quintana ◽  
Genomic Expression ◽  
Genes Encoding ◽  
Multiple Regions ◽  
Adhesin Protein

ABSTRACT Bartonella proteins that elicit an antibody response during an infection are poorly defined; therefore, to characterize antigens recognized by the host, a Bartonella genomic expression library was screened with serum from an infected mouse. This process led to the discovery of a Bartonella vinsonii subsp. arupensis gene encoding a 382-kDa protein, part of a gene family encoding large proteins, each containing multiple regions of repetitive segments. The genes were termed brpA to -C (bartonella repeat protein) and bore significant similarity to genes encoding the BadA adhesin protein and members of the variably expressed outer membrane protein family of proteins from Bartonella henselae and Bartonella quintana, respectively.

scholarly journals Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

2015 ◽  
Vol 81 (24) ◽  
pp. 8294-8306 ◽  
Author(s):  
Heba Khairy ◽  
Jan Hendrik Wübbeler ◽  
Alexander Steinbüchel
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Rhodococcus Erythropolis ◽  
Model Organism ◽  
Hypothetical Protein ◽  
Gene Encoding ◽  
Content Type ◽  
Gene Coding ◽  
Initial Cleavage ◽  
Genes Encoding

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

fur-independent regulation of the Pasteurella multocida hbpA gene encoding a haemin-binding protein

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2273-2281 ◽  
Author(s):  
M. Elena Garrido ◽  
Montserrat Bosch ◽  
Ricardo Medina ◽  
Anna Bigas ◽  
Montserrat Llagostera ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Pasteurella Multocida ◽  
Outer Membrane Proteins ◽  
Protein Composition ◽  
Electrophoretic Analysis ◽  
Gene Encoding ◽  
Expression Studies ◽  
Genes Encoding ◽  
Fur Protein ◽  
Iron Manganese

Treatment of bacterial cultures with chelating agents such as 2,2′-dipyridyl (DPD) induces expression of iron-regulated genes. It is known that in the γ-Proteobacteria, the Fur protein is the major regulator of genes encoding haem- or haemoglobin-binding proteins. Electrophoretic analysis of outer-membrane proteins of the γ-proteobacterium Pasteurella multocida has revealed the induction of two proteins of 60 and 40 kDa in DPD-treated cultures in both wild-type and fur-defective strains. These two proteins have the same N-terminal amino acid sequence, which identifies this protein as the product of the PM0592 ORF. Analysis of the sequence of this ORF, which encodes a protein of 60 kDa, revealed the presence of a hexanucleotide (AAAAAA) at which a programmed translational frameshift can occur giving rise to a 40 kDa protein. Analyses conducted in Escherichia coli, using the complete PM0592 ORF and a derivative truncated at the hexanucleotide position, have shown that both polypeptides bind haemin. For this reason, the PM0592 ORF product has been designated HbpA (for haemin-binding protein). Expression studies using both RT-PCR and lacZ fusions, as well as electrophoretic profiles of outer-membrane protein composition, have demonstrated that the hbpA gene is negatively regulated by iron, manganese and haemin through a fur-independent pathway. Despite the fact that serum of mice infected with P. multocida contained antibodies that reacted with both the 60 and 40 kDa products of the hbpA gene, these proteins did not offer protection when used in immunization assays against this micro-organism.

scholarly journals Thioredoxin Is Involved in U(VI) and Cr(VI) Reduction in Desulfovibrio desulfuricans G20

2009 ◽  
Vol 191 (15) ◽  
pp. 4924-4933 ◽  
Author(s):  
Xiangkai Li ◽  
Lee R. Krumholz
Keyword(s):  
Specific Inhibitor ◽  
Thioredoxin Reductase ◽  
Desulfovibrio Desulfuricans ◽  
Receptor Protein ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Transposon Insertion ◽  
Expression Studies ◽  
Genes Encoding ◽  
Camp Receptor

ABSTRACT A transposon insertion mutant has been identified in a Desulfovibrio desulfuricans G20 mutant library that does not grow in the presence of 2 mM U(VI) in lactate-sulfate medium. This mutant has also been shown to be deficient in the ability to grow with 100 μM Cr(VI) and 20 mM As(V). Experiments with washed cells showed that this mutant had lost the ability to reduce U(VI) or Cr(VI), providing an explanation for the lower tolerance. A gene encoding a cyclic AMP (cAMP) receptor protein (CRP) was identified as the site of the transposon insertion. The remainder of the mre operon (metal reduction) contains genes encoding a thioredoxin, thioredoxin reductase, and an additional oxidoreductase whose substrate has not been predicted. Expression studies showed that in the mutant, the entire operon is downregulated, suggesting that the CRP may be involved in regulating expression of the whole operon. Exposure of the cells to U(VI) resulted in upregulation of the entire operon. CdCl2, a specific inhibitor of thioredoxin activity, inhibits U(VI) reduction by washed cells and inhibits growth of cells in culture when U(VI) is present, confirming a role for thioredoxin in U(VI) reduction. The entire mre operon was cloned into Escherichia coli JM109 and the transformant developed increased U(VI) resistance and the ability to reduce U(VI) to U(IV). The oxidoreductase protein (MreG) from this operon was expressed and purified from E. coli. In the presence of thioredoxin, thioredoxin reductase, and NADPH, this protein was shown to reduce both U(VI) and Cr(VI), providing a mechanism for the cytoplasmic reduction of these metals.

scholarly journals CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

2010 ◽  
Vol 192 (20) ◽  
pp. 5480-5488 ◽  
Author(s):  
Vânia S. Braz ◽  
José F. da Silva Neto ◽  
Valéria C. S. Italiani ◽  
Marilis V. Marques
Keyword(s):  
Stationary Phase ◽  
Mutant Strain ◽  
Caulobacter Crescentus ◽  
Transcriptional Start Site ◽  
Hypothetical Protein ◽  
Zinc Homeostasis ◽  
Gene Encoding ◽  
Transmembrane Segments ◽  
Zinc Depletion

ABSTRACT Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. β-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 999-1007
Author(s):  
R G Gregerson ◽  
L Cameron ◽  
M McLean ◽  
P Dennis ◽  
J Strommer
Keyword(s):  
Chromosomal Location ◽  
Higher Plants ◽  
Gene Encoding ◽  
Genomic Libraries ◽  
Regulatory Strategy ◽  
Adh1 Gene ◽  
Adh Genes ◽  
Genes Encoding ◽  
Adh Gene ◽  
Polymerase Chain

Abstract In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

scholarly journals Non-Syndromic Dentinogenesis Imperfecta Caused by Mild Mutations in COL1A2

2021 ◽  
Vol 11 (6) ◽  
pp. 526
Author(s):  
Yejin Lee ◽  
Youn Jung Kim ◽  
Hong-Keun Hyun ◽  
Jae-Cheoun Lee ◽  
Zang Hee Lee ◽  
...  
Keyword(s):  
Collagen Type ◽  
Molecular Genetic ◽  
Collagen Type I ◽  
Type I ◽  
Dentinogenesis Imperfecta ◽  
Gene Encoding ◽  
Korean Families ◽  
Whole Exome ◽  
Genes Encoding ◽  
Candidate Gene Sequencing

Hereditary dentin defects can be categorized as a syndromic form predominantly related to osteogenesis imperfecta (OI) or isolated forms without other non-oral phenotypes. Mutations in the gene encoding dentin sialophosphoprotein (DSPP) have been identified to cause dentinogenesis imperfecta (DGI) Types II and III and dentin dysplasia (DD) Type II. While DGI Type I is an OI-related syndromic phenotype caused mostly by monoallelic mutations in the genes encoding collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 2 chain (COL1A2). In this study, we recruited families with non-syndromic dentin defects and performed candidate gene sequencing for DSPP exons and exon/intron boundaries. Three unrelated Korean families were further analyzed by whole-exome sequencing due to the lack of the DSPP mutation, and heterozygous COL1A2 mutations were identified: c.3233G>A, p.(Gly1078Asp) in Family 1 and c.1171G>A, p.(Gly391Ser) in Family 2 and 3. Haplotype analysis revealed different disease alleles in Families 2 and 3, suggesting a mutational hotspot. We suggest expanding the molecular genetic etiology to include COL1A2 for isolated dentin defects in addition to DSPP.

scholarly journals Essential and nonessential histone H2A variants in Tetrahymena thermophila.

1996 ◽  
Vol 16 (8) ◽  
pp. 4305-4311 ◽  
Author(s):  
X Liu ◽  
B Li ◽  
GorovskyMA
Keyword(s):  
Tetrahymena Thermophila ◽  
Essential Gene ◽  
Histone Variants ◽  
Gene Replacement ◽  
Histone H2a ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Simple Mass ◽  
Essential Function ◽  
High Degree

Although variants have been identified for every class of histone, their functions remain unknown. We have been studying the histone H2A variant hv1 in the ciliated protozoan Tetrahymena thermophila. Sequence analysis indicates that hv1 belongs to the H2A.F/Z type of histone variants. On the basis of the high degree of evolutionary conservation of this class of histones, they are proposed to have one or more distinct and essential functions that cannot be performed by their major H2A counterparts. Considerable evidence supports the hypothesis that the hv1 protein in T. thermophila and hv1-like proteins in other eukaryotes are associated with active chromatin. In T. thermophila, simple mass transformation and gene replacement techniques have recently become available. In this report, we demonstrate that either the HTA1 gene or the HTA2 gene, encoding the major H2As, can be completely replaced by disrupted genes in the polyploid, transcriptionally active macronucleus, indicating that neither of the two genes is essential. However, only some of the HTA3 genes encoding hv1 can be replaced by disrupted genes, indicating that the H2A.F/Z type variants have an essential function that cannot be performed by the major H2A genes. Thus, an essential gene in T. thermophila can be defined by the fact that it can be partially, but not completely, eliminated from the polyploid macronucleus. To our knowledge, this study represents the first use of gene disruption technology to study core histone gene function in any organism other than yeast and the first demonstration of an essential gene in T. thermophila using these methods. When a rescuing plasmid carrying a wild-type HTA3 gene was introduced into the T. thermophila cells, the endogenous chromosomal HTA3 could be completely replaced, defining a gene replacement strategy that can be used to analyze the function of essential genes.

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