scholarly journals Identification and Characterization of a Repeat-in-Toxin Gene Cluster in Vibrio anguillarum

2008 ◽  
Vol 76 (6) ◽  
pp. 2620-2632 ◽  
Author(s):  
Ling Li ◽  
Jessica L. Rock ◽  
David R. Nelson
Keyword(s):  
Atlantic Salmon ◽  
Gene Cluster ◽  
Vibrio Anguillarum ◽  
Hypothetical Protein ◽  
Pcr Analysis ◽  
Toxin Gene ◽  
Reverse Transcription Pcr ◽  
Hemolysin Gene ◽  
Negative Mutant

ABSTRACT Vibrio anguillarum is the causative agent of vibriosis in fish. Hemolysins of V. anguillarum have been considered virulence factors during infection. One hemolysin gene, vah1, has been previously identified but does not account for all hemolytic activity. The mini-Tn10Km mutagenesis performed with a vah1 mutant resulted in a hemolysin-negative mutant. The region surrounding the mutation was cloned and sequenced, revealing a putative rtx operon with six genes (rtxACHBDE), where rtxA encodes an exotoxin, rtxC encodes an RtxA activator, rtxH encodes a conserved hypothetical protein, and rtxBDE encode the ABC transporters. Single mutations in rtx genes did not result in a hemolysin-negative phenotype. However, strains containing a mutation in vah1 and a mutation in an rtx gene resulted in a hemolysin-negative mutant, demonstrating that the rtx operon is a second hemolysin gene cluster in V. anguillarum M93Sm. Reverse transcription-PCR analysis revealed that the rtxC and rtxA genes are cotranscribed, as are the rtxBDE genes. Additionally, Vah1 and RtxA each have cytotoxic activity against Atlantic salmon kidney (ASK) cells. Single mutations in vah1 or rtxA attenuate the cytotoxicity of V. anguillarum M93Sm. A vah1 rtxA double mutant is no longer cytotoxic. Moreover, Vah1 and RtxA each have a distinct cytotoxic effect on ASK cells, Vah1 causes cell vacuolation, and RtxA causes cell rounding. Finally, wild-type and mutant strains were tested for virulence in juvenile Atlantic salmon. Only strains containing an rtxA mutation had reduced virulence, suggesting that RtxA is a major virulence factor for V. anguillarum.

scholarly journals Identification of Nitric Oxide Responsive Genes in the Rudimentary Leaves of Litchi chinensis

2018 ◽  
Vol 46 (2) ◽  
pp. 608-614
Author(s):  
Xingyu LU ◽  
Houbin CHEN ◽  
Zhiqun HU ◽  
Biyan ZHOU
Keyword(s):  
Nitric Oxide ◽  
Hot Spring ◽  
Subtractive Hybridization ◽  
Hypothetical Protein ◽  
Pcr Analysis ◽  
Litchi Chinensis ◽  
Tropical Regions ◽  
Reverse Transcription Pcr ◽  
Protein Encoding ◽  
Floral Differentiation

Litchi (Litchi chinensis Sonn.) is an evergreen woody fruit tree widely cultivated in subtropical and tropical regions. Warm winter and hot spring often leads to abnormal floral differentiation in litchi. Under this condition, the rudimentary leaves in the floral buds expand and the inflorescences will stop developing. Thus, how to promote abortion of rudimentary leaves in litchi inflorescence are important for floral development. Previous study indicated that nitric oxide (NO) produced by sodium nitroprusside (SNP) promoted flowering and abortion of rudimentary leaves in litchi. In the present study, a suppression subtractive hybridization (SSH) was used to identify NO responsive genes. As a result, 16 high homologous ESTs were obtained from the SSH library of the SNP treated rudimentary leaves. The ESTs were classified into three groups. They are disease/defensive, protein destination and storage, and protein synthesis. Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that 6 out of the 7 randomly selected ESTs’expression showed an increasing trend from 0 h to 10 h of SNP treatment. It is suggested that the litchi homologs 18S ribosomal RNA gene, cytochrome P450 like TBP, and the senescence-associated protein, chaperone protein, and a hypothetical protein encoding genes may be involved in the NO-induced senescence in litchi rudimentary leaves. LcERD15-like may be a key gene involved in this process.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

scholarly journals A gene cluster involved in the biosynthesis of vanchrobactin, a chromosome-encoded siderophore produced by Vibrio anguillarum

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3517-3528 ◽  
Author(s):  
Miguel Balado ◽  
Carlos R. Osorio ◽  
Manuel L. Lemos
Keyword(s):  
Gene Cluster ◽  
Vibrio Anguillarum ◽  
No Production ◽  
Genetic Determinants ◽  
Dihydroxybenzoic Acid ◽  
Peptide Synthetase ◽  
Genes Encoding ◽  
In Trans ◽  
Culture Supernatants

Vibrio anguillarum serotype O2 strains produce a catechol siderophore named vanchrobactin, which has been identified as N-[N′-(2,3-dihydroxybenzoyl)-arginyl]-serine. This work describes a chromosomal region that harbours the genetic determinants necessary for the biosynthesis of vanchrobactin. The authors have identified the genes involved in 2,3-dihydroxybenzoic acid (DHBA) biosynthesis (vabA, vabB and vabC) and activation (vabE), and a gene (vabF) encoding a non-ribosomal peptide synthetase, which is putatively involved in the assembly of the siderophore components. Also described are the identification and characterization of genes encoding a putative vanchrobactin exporter (vabS) and a siderophore esterase (vabH). In-frame deletion mutants in vabA, vabB, vabC, vabE, vabF and vabH were impaired for growth under conditions of iron limitation, and the analysis of culture supernatants by chrome azurol-S and cross-feeding assays showed almost no production of siderophores in any of the vabABCEF mutants. In addition, deletion mutations of vabA, vabB and vabC abolished production of DHBA, as assessed by chemical and biological analyses. Complementation of each mutant with the corresponding gene provided in trans confirmed the involvement of this gene cluster in the biosynthesis of DHBA and vanchrobactin in V. anguillarum strain RV22. Based on chemical and genetic data, and on published models for other catechol siderophores, a model for vanchrobactin biosynthesis is proposed.

Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽  
2011 ◽  
Vol 138 (14) ◽  
pp. 1832-1842 ◽  
Author(s):  
V. RISCO-CASTILLO ◽  
V. MARUGÁN-HERNÁNDEZ ◽  
A. FERNÁNDEZ-GARCÍA ◽  
A. AGUADO-MARTÍNEZ ◽  
E. JIMÉNEZ-RUIZ ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Cluster ◽  
Neospora Caninum ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Initial Characterization ◽  
Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

Molecular characterization of the PR-toxin gene cluster in Penicillium roqueforti and Penicillium chrysogenum: Cross talk of secondary metabolite pathways

2014 ◽  
Vol 62 ◽  
pp. 11-24 ◽  
Author(s):  
Pedro I. Hidalgo ◽  
Ricardo V. Ullán ◽  
Silvia M. Albillos ◽  
Olimpio Montero ◽  
María Ángeles Fernández-Bodega ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Molecular Characterization ◽  
Secondary Metabolite ◽  
Cross Talk ◽  
Penicillium Chrysogenum ◽  
Toxin Gene ◽  
Penicillium Roqueforti

scholarly journals Isolation and molecular characterization of a urease-negative Actinobacillus pleuropneumoniae mutant

2017 ◽  
Vol 30 (1) ◽  
pp. 172-174 ◽  
Author(s):  
Hiroya Ito ◽  
Sayaka Takahashi ◽  
Tetsuo Asai ◽  
Yutaka Tamura ◽  
Koshi Yamamoto
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Gene Cluster ◽  
Molecular Characterization ◽  
Dna Sequence ◽  
Actinobacillus Pleuropneumoniae ◽  
Nucleotide Sequence Analysis ◽  
Urease Gene ◽  
Negative Mutant

An atypical urease-negative mutant of Actinobacillus pleuropneumoniae serovar 2 was isolated in Japan. Nucleotide sequence analysis of the urease gene cluster revealed that the insertion of a short DNA sequence into the cbiM gene was responsible for the urease-negative activity of the mutant. Veterinary diagnostic laboratories should be watchful for the presence of aberrant urease-negative A. pleuropneumoniae isolates.

scholarly journals Identification and Molecular Characterization of the Chromosomal Exopolysaccharide Biosynthesis Gene Cluster from Lactococcus lactis subsp. cremoris SMQ-461

2005 ◽  
Vol 71 (11) ◽  
pp. 7414-7425 ◽  
Author(s):  
N. Dabour ◽  
G. LaPointe
Keyword(s):  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Repeat Unit ◽  
Raw Milk ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Biosynthesis Gene Cluster ◽  
Glycosyltransferase Gene ◽  
Reverse Transcription Pcr ◽  
Exopolysaccharide Biosynthesis

ABSTRACT The exopolysaccharide (EPS) capsule-forming strain SMQ-461 of Lactococcus lactis subsp. cremoris, isolated from raw milk, produces EPS with an apparent molecular mass of >1.6 × 106 Da. The EPS biosynthetic genes are located on the chromosome in a 13.2-kb region consisting of 15 open reading frames. This region is flanked by three IS1077-related tnp genes (L. lactis) at the 5′ end and orfY, along with an IS981-related tnp gene, at the 3′ end. The eps genes are organized in specific regions involved in regulation, chain length determination, biosynthesis of the repeat unit, polymerization, and export. Three (epsGIK) of the six predicted glycosyltransferase gene products showed low amino acid similarity with known glycosyltransferases. The structure of the repeat unit could thus be different from those known to date for Lactococcus. Reverse transcription-PCR analysis revealed that the eps locus is transcribed as a single mRNA. The function of the eps gene cluster was confirmed by disrupting the priming glycosyltransferase gene (epsD) in Lactococcus cremoris SMQ-461, generating non-EPS-producing reversible mutants. This is the first report of a chromosomal location for EPS genetic elements in Lactococcus cremoris, with novel glycosyltransferases not encountered before in lactic acid bacteria.

scholarly journals Evolution and Characterization of Tetraonine Endogenous Retrovirus: a New Virus Related to Avian Sarcoma and Leukosis Viruses

2001 ◽  
Vol 75 (4) ◽  
pp. 2002-2009 ◽  
Author(s):  
Derek E. Dimcheff ◽  
Mallika Krishnan ◽  
David P. Mindell
Keyword(s):  
Southern Blotting ◽  
Phylogenetic Analyses ◽  
Endogenous Retrovirus ◽  
Pcr Analysis ◽  
Bonasa Umbellus ◽  
Long Terminal Repeats ◽  
Proviral Sequence ◽  
Reverse Transcription Pcr ◽  
Avian Sarcoma

ABSTRACT In a previous study, we found avian sarcoma and leukosis virus (ASLV) gag genes in 19 species of birds in the order Galliformes including all grouse and ptarmigan (Tetraoninae) surveyed. Our data suggested that retroviruses had been transmitted horizontally among some host species. To further investigate these elements, we sequenced a replication-defective retrovirus, here named tetraonine endogenous retrovirus (TERV), from Bonasa umbellus (ruffed grouse). This is the first report of a complete, replication-defective ASLV provirus sequence from any bird other than the domestic chicken. We found a replication-defective proviral sequence consisting of putative Gag and Env proteins flanked by long terminal repeats. Reverse transcription-PCR analysis showed that retroviral gagsequences closely related to TERV are transcribed, supporting the hypothesis that TERV is an active endogenous retrovirus. Phylogenetic analyses suggest that TERV may have arisen via recombination between different retroviral lineages infecting birds. Southern blotting usinggag probes showed that TERV occurs in tetraonines but not in chickens or ducks, suggesting that integration occurred after the earliest phasianid divergences but prior to the radiation of tetraonine birds.

scholarly journals Characterization of the Protocatechuate 4,5-Cleavage Pathway Operon in Comamonas sp. Strain E6 and Discovery of a Novel Pathway Gene

2010 ◽  
Vol 76 (24) ◽  
pp. 8093-8101 ◽  
Author(s):  
Naofumi Kamimura ◽  
Taichi Aoyama ◽  
Rieko Yoshida ◽  
Kenji Takahashi ◽  
Daisuke Kasai ◽  
...  
Keyword(s):  
Growth Defect ◽  
Pcr Analysis ◽  
Aromatic Acids ◽  
Transcription Start ◽  
Reverse Transcription Pcr ◽  
Degrading Bacterium ◽  
Pathway Gene ◽  
Insertional Inactivation ◽  
Dioxygenase Gene

ABSTRACT The protocatechuate (PCA) 4,5-cleavage (PCA45) pathway is the essential catabolic route for the degradation of various aromatic acids in the genus Comamonas. All of the PCA45 pathway genes, orf1-pmdKEFDABC, as well as another PCA 4,5-dioxygenase gene, pmdA II B II, were isolated from a phthalate-degrading bacterium, Comamonas sp. strain E6. Disruption of pmdB and pmdD in E6, which code for the β subunit of PCA 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate (PDC) hydrolase, respectively, resulted in a growth defect on PCA, indicating that these genes are essential for the growth of E6 on PCA. On the other hand, inactivation of pmdB II did not affect the growth of E6 on PCA. Disruption of pmdK, which is related to a 4-hydroxybenzoate/PCA transporter of Pseudomonas putida, resulted in growth retardation on PCA. The insertional inactivation of orf1 in E6, whose deduced amino acid sequence has no similarity with proteins of known function, led to the complete loss of growth on PCA and the accumulation of PDC and 4-oxalomesaconate (OMA) from PCA. These results indicated the involvement of orf1 in the PCA45 pathway, and this gene, designated pmdU, was suggested to code for OMA tautomerase. Reverse transcription-PCR analysis suggested that the pmdUKEFDABC genes constitute an operon. The transcription start site of the pmd operon was mapped at 167 nucleotides upstream of the initiation codon of pmdU. The pmd promoter activity was enhanced 20-fold when the cells were grown in the presence of PCA. Inducers of the pmd operon were found to be PCA and PDC, but PDC was the more effective inducer.

scholarly journals Characterization of the anthranilate degradation pathway in Geobacillus thermodenitrificans NG80-2

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Xueqian Liu ◽  
Yangpeng Dong ◽  
Xiaomin Li ◽  
Yi Ren ◽  
Yanxia Li ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Degradation Pathway ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Geobacillus Thermodenitrificans ◽  
Reservoir Conditions ◽  
Important Intermediate ◽  
First Time

Anthranilate is an important intermediate of tryptophan metabolism. In this study, a hydroxylase system consisting of an FADH2-utilizing monooxygenase (GTNG_3160) and an FAD reductase (GTNG_3158), as well as a bifunctional riboflavin kinase/FMN adenylyltransferase (GTNG_3159), encoded in the anthranilate degradation gene cluster in Geobacillus thermodenitrificans NG80-2 were functionally characterized in vitro. GTNG_3159 produces FAD to be reduced by GTNG_3158 and the reduced FAD (FADH2) is utilized by GTNG_3160 to convert anthranilate to 3-hydroxyanthranilate (3-HAA), which is further degraded to acetyl-CoA through a meta-cleavage pathway also encoded in the gene cluster. Utilization of this pathway for the degradation of anthranilate and tryptophan by NG80-2 under physiological conditions was confirmed by real-time RT-PCR analysis of representative genes. This is believed to be the first time that the degradation pathway of anthranilate via 3-HAA has been characterized in a bacterium. This pathway is likely to play an important role in the survival of G. thermodenitrificans in the oil reservoir conditions from which strain NG80-2 was isolated.

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