scholarly journals Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Katarzyna Hupert-Kocurek ◽  
Urszula Guzik ◽  
Danuta Wojcieszyńska
Keyword(s):  
Kinetic Studies ◽  
High Reactivity ◽  
Homology Search ◽  
Nucleotide Sequencing ◽  
Positive Bacterium ◽  
Gene Encoding ◽  
Genes Encoding ◽  
High Phenol Concentration ◽  
Dioxygenase Activity

This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.

scholarly journals Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Sakuko Ueshima ◽  
Hisashi Muramatsu ◽  
Takanori Nakajima ◽  
Hiroaki Yamamoto ◽  
Shin-ichiro Kato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Homology Search ◽  
Hydroxyl Group ◽  
Enzymatic Reactions ◽  
Reaction Products ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Single Operon

The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

scholarly journals Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

1998 ◽  
Vol 36 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Claire Poyart ◽  
Gilles Quesne ◽  
Stephane Coulon ◽  
Patrick Berche ◽  
Patrick Trieu-Cuot
Keyword(s):  
Superoxide Dismutase ◽  
Pcr Assay ◽  
Degenerate Primers ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Type Strains ◽  
Internal Fragment ◽  
Rrna Sequences ◽  
16S Rrna Sequences

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

scholarly journals Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

2007 ◽  
Vol 73 (17) ◽  
pp. 5411-5420 ◽  
Author(s):  
Yu-Sin Jang ◽  
Young Ryul Jung ◽  
Sang Yup Lee ◽  
Ji Mahn Kim ◽  
Jeong Wook Lee ◽  
...  
Keyword(s):  
Succinic Acid ◽  
Shuttle Vector ◽  
Green Fluorescence Protein ◽  
Expression Vectors ◽  
Rumen Bacteria ◽  
Gene Encoding ◽  
Shuttle Vectors ◽  
Genes Encoding ◽  
Fluorescence Protein

ABSTRACT Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 106 and 7.1 × 106 transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM)

Parasitology ◽  
1998 ◽  
Vol 117 (4) ◽  
pp. 321-330 ◽  
Author(s):  
R. A. SKILTON ◽  
R. P. BISHOP ◽  
C. W. WELLS ◽  
P. R. SPOONER ◽  
E. GOBRIGHT ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Signal Sequence ◽  
Peptide Sequence ◽  
Theileria Parva ◽  
Repeat Region ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Immunodominant Antigens ◽  
Proline Rich Region

To identify the genes encoding novel immunodominant antigens of Theileria parva a λgt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host–sporozoite interaction.

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 Biochemical and Mutational Analysis of Glutamine Synthetase Type III from the Rumen Anaerobe Ruminococcus albus 8

2005 ◽  
Vol 187 (21) ◽  
pp. 7481-7491 ◽  
Author(s):  
Kensey R. Amaya ◽  
Svetlana A. Kocherginskaya ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Glutamine Synthetase ◽  
Mutational Analysis ◽  
Sufficient Conditions ◽  
Kinetic Studies ◽  
Type I ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Subunit Organization ◽  
Glutamyl Transferase ◽  
First Time

ABSTRACT Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the γ-glutamyl transferase (γ-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.

scholarly journals Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

2010 ◽  
Vol 192 (6) ◽  
pp. 1624-1633 ◽  
Author(s):  
Chantal Fernandes ◽  
Vitor Mendes ◽  
Joana Costa ◽  
Nuno Empadinhas ◽  
Carla Jorge ◽  
...  
Keyword(s):  
Sequence Homology ◽  
Thermotoga Maritima ◽  
Functional Characterization ◽  
Compatible Solute ◽  
Homologous Gene ◽  
Gene Encoding ◽  
Alternative Pathways ◽  
Cell Extracts ◽  
Genes Encoding

ABSTRACT The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.

scholarly journals Generation of Markerless Deletions in the Nosocomial PathogenClostridium difficileby Induction of DNA Double-Strand Breaks

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Elena-Stella Theophilou ◽  
Prerna Vohra ◽  
Maurice P. Gallagher ◽  
Ian R. Poxton ◽  
Garry W. Blakely
Keyword(s):  
Clostridium Difficile ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Strand Breaks ◽  
Allelic Exchange ◽  
Genes Encoding

ABSTRACTClostridium difficileis an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions inC. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddABmutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of thefliCgene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCEMost sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution inClostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions inC. difficileand expands the toolbox available for genetic analysis of this important anaerobic pathogen.

scholarly journals Characterization of Staphylococcus pseudintermedius isolated from diseased dogs in Lithuania

2016 ◽  
Vol 19 (1) ◽  
pp. 7-14 ◽  
Author(s):  
M. Ruzauskas ◽  
N. Couto ◽  
A. Pavilonis ◽  
I. Klimiene ◽  
R. Siugzdiniene ◽  
...  
Keyword(s):  
Companion Animals ◽  
Penicillin G ◽  
Special Focus ◽  
Reproductive Disorders ◽  
Gene Encoding ◽  
Staphylococcus Pseudintermedius ◽  
Genes Encoding ◽  
Species Specific ◽  
Staphylococcus Spp

AbstractThe aim of this study was to characterize Staphylococcus pseudintermedius for its antimicrobial resistance and virulence factors with a special focus on methicillin-resistant (MRSP) strains isolated from sick dogs in Lithuania. Clinically sick adult dogs suffering from infections (n=214) and bitches with reproductive disorders (n=36) from kennels were selected for the study. Samples (n=192) from the 250 tested (76.8%) dogs were positive for Staphylococcus spp. Molecular profiling using the species-specific nuc gene identified 51 isolates as S. pseudintermedius (26.6% from a total number of isolated staphylococci) of which 15 isolates were identified as MRSP. Ten MRSP isolates were isolated from bitches with reproductive disorders from two large breeding kennels. Data on susceptibility of S. pseudintermedius to different antimicrobials revealed that all isolates were susceptible to vancomycin, daptomycin and linezolid. Two isolates (3.9%) were resistant to rifampicin. A high resistance was seen towards penicillin G (94.1%), tetracycline (64.7%) and macrolides (68.7%). Resistance to fluoroquinolones ranged from 25.5% (gatifloxacin) to 31.4% (ciprofloxacin). The most prevalent genes encoding resistance included blaZ, aac(6’)-Ie-aph(2’’)-Ia, mecA, and tet(M). The Luk-I gene encoding a leukotoxin was detected in 29% of the isolates, whereas the siet gene encoding exfoliative toxin was detected in 69% of the S. pseudintermedius isolates. This report of MRSP in companion animals represents a major challenge for veterinarians in terms of antibiotic therapy and is a concern for both animal and public health.

Increased or Decreased Levels of Caenorhabditis elegans lon-3, a Gene Encoding a Collagen, Cause Reciprocal Changes in Body Length

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 83-97 ◽  
Author(s):  
Josefin Nyström ◽  
Zai-Zhong Shen ◽  
Margareta Aili ◽  
Anthony J Flemming ◽  
Armand Leroi ◽  
...  
Keyword(s):  
Body Length ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Loss Of Function ◽  
Gene Encoding ◽  
C Elegans ◽  
Morphometric Analyses ◽  
Genes Encoding ◽  
New Mutations

Abstract Body length in C. elegans is regulated by a member of the TGFβ family, DBL-1. Loss-of-function mutations in dbl-1, or in genes encoding components of the signaling pathway it activates, cause worms to be shorter than wild type and slightly thinner (Sma). Overexpression of dbl-1 confers the Lon phenotype characterized by an increase in body length. We show here that loss-of-function mutations in dbl-1 and lon-1, respectively, cause a decrease or increase in the ploidy of nuclei in the hypodermal syncytial cell, hyp7. To learn more about the regulation of body length in C. elegans we carried out a genetic screen for new mutations causing a Lon phenotype. We report here the cloning and characterization of lon-3. lon-3 is shown to encode a putative cuticle collagen that is expressed in hypodermal cells. We show that, whereas putative null mutations in lon-3 (or reduction of lon-3 activity by RNAi) causes a Lon phenotype, increasing lon-3 gene copy number causes a marked reduction in body length. Morphometric analyses indicate that the lon-3 loss-of-function phenotype resembles that caused by overexpression of dbl-1. Furthermore, phenotypes caused by defects in dbl-1 or lon-3 expression are in both cases suppressed by a null mutation in sqt-1, a second cuticle collagen gene. However, whereas loss of dbl-1 activity causes a reduction in hypodermal endoreduplication, the reduction in body length associated with overexpression of lon-3 occurs in the absence of defects in hypodermal ploidy.

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