scholarly journals Characterization of the argA Gene Required for Arginine Biosynthesis and Syringomycin Production by Pseudomonas syringae pv. syringae

2003 ◽  
Vol 69 (12) ◽  
pp. 7273-7280 ◽  
Author(s):  
Shi-En Lu ◽  
Jonathan D. Soule ◽  
Dennis C. Gross
Keyword(s):  
Pseudomonas Syringae ◽  
Culture Media ◽  
Toxin Production ◽  
Arginine Biosynthesis ◽  
Reading Frame ◽  
High Conservation ◽  
Plant Pathogenicity ◽  
Arginine Deficiency ◽  
Dna Fragment

ABSTRACT Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.

scholarly journals Knot Formation Caused by Pseudomonas syringae subsp. savastanoi on Olive Plants Is hrp-Dependent

2004 ◽  
Vol 94 (5) ◽  
pp. 484-489 ◽  
Author(s):  
A. Sisto ◽  
M. G. Cipriani ◽  
M. Morea
Keyword(s):  
Sequence Analysis ◽  
Pseudomonas Syringae ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Phytopathogenic Bacteria ◽  
Reading Frame ◽  
Nonhost Plant ◽  
Induced Mutant

The virulence of Pseudomonas syringae subsp. savastanoi, which causes hyperplastic symptoms (knots) on olive plants, is associated with secreted phytohormones. We identified a Tn5-induced mutant of P. syringae subsp. savastanoi that did not cause disease symptoms on olive plants although it was still able to produce phytohormones. In addition, the mutant failed to elicit a hypersensitive response in a nonhost plant. Molecular characterization of the mutant revealed that a single Tn5 insertion occurred within an open reading frame encoding a protein 92% identical to the HrcC protein of P. syringae pv. syringae. Moreover, sequence analysis revealed that the gene encoding the HrcC protein in P. syringae subsp. savastanoi was part of an operon that included five genes arranged as in other phytopathogenic bacteria. These results imply that hrp/hrc genes are functional in P. syringae subsp. savastanoi and that they play a key role in the pathogenicity of this plant pathogen.

scholarly journals Phylogeny and Characterization of Three nifH-Homologous Genes from Paenibacillus azotofixans

2003 ◽  
Vol 69 (6) ◽  
pp. 3658-3662 ◽  
Author(s):  
Quok-Cheong Choo ◽  
Mohd-Razip Samian ◽  
Nazalan Najimudin
Keyword(s):  
Phylogenetic Analysis ◽  
Methanogenic Archaea ◽  
Gene Organization ◽  
Open Reading Frame ◽  
The Other ◽  
Sequencing Analysis ◽  
Reading Frame ◽  
Homologous Genes ◽  
Dna Fragment

ABSTRACT In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.

scholarly journals Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

2008 ◽  
Vol 191 (5) ◽  
pp. 1565-1573 ◽  
Author(s):  
Sang-Hwal Yoon ◽  
Tae Seok Moon ◽  
Pooya Iranpour ◽  
Amanda M. Lanza ◽  
Kristala Jones Prather
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Pseudomonas Syringae ◽  
Glucuronic Acid ◽  
Glucaric Acid ◽  
Michaelis Constant ◽  
Turnover Number ◽  
Reading Frame ◽  
E Coli ◽  
Uronate Dehydrogenase

ABSTRACT Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K m ) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k cat = 1.9 × 102 s−1 on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

scholarly journals The Mangotoxin Biosynthetic Operon (mbo) Is Specifically Distributed within Pseudomonas syringae Genomospecies 1 and Was Acquired Only Once during Evolution

2012 ◽  
Vol 79 (3) ◽  
pp. 756-767 ◽  
Author(s):  
Víctor J. Carrión ◽  
José A. Gutiérrez-Barranquero ◽  
Eva Arrebola ◽  
Leire Bardaji ◽  
Juan C. Codina ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Phylogenetic Analyses ◽  
Housekeeping Genes ◽  
Phenotypic Characterization ◽  
Content Type ◽  
Group Iii ◽  
Genomic Location ◽  
High Sequence Conservation ◽  
Dna Fragment

ABSTRACTMangotoxin production was first described inPseudomonas syringaepv. syringae strains. A phenotypic characterization of 94P. syringaestrains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair specific for thembooperon to examine its distribution within theP. syringaecomplex. These primers amplified a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor thembooperon, whereas 35 non-mangotoxin-producing strains did not yield any amplification. This, together with the analysis of draft genomes, allowed the identification of thembooperon in five pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of thembogenes in theP. syringaecomplex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing thembooperon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. Thembooperon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that thembooperon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within theP. syringaecomplex.

scholarly journals Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene from Ralstonia eutropha H16 and Characterization of the Gene Product

2001 ◽  
Vol 183 (1) ◽  
pp. 94-100 ◽  
Author(s):  
Haruhisa Saegusa ◽  
Mari Shiraki ◽  
Chie Kanai ◽  
Terumi Saito
Keyword(s):  
Gene Product ◽  
Ralstonia Eutropha ◽  
Nucleotide Sequence Analysis ◽  
Rich Medium ◽  
Wild Type ◽  
Reading Frame ◽  
Phb Depolymerase ◽  
Freeze Dried ◽  
Dna Fragment

ABSTRACT An intracellular poly[d(−)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned fromRalstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric d(−)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutrophawhose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.

scholarly journals SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (1) ◽  
pp. 202-212 ◽  
Author(s):  
F Damak ◽  
E Boy-Marcotte ◽  
D Le-Roscouet ◽  
R Guilbaud ◽  
M Jacquet
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Open Reading Frame ◽  
Gene Products ◽  
Terminal Part ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Dna Fragment ◽  
Degree Of Similarity ◽  
Entire Gene

In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.

SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae

1991 ◽  
Vol 11 (1) ◽  
pp. 202-212 ◽  
Author(s):  
F Damak ◽  
E Boy-Marcotte ◽  
D Le-Roscouet ◽  
R Guilbaud ◽  
M Jacquet
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Open Reading Frame ◽  
Gene Products ◽  
Terminal Part ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Dna Fragment ◽  
Degree Of Similarity ◽  
Entire Gene

In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.

scholarly journals Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster

1998 ◽  
Vol 331 (3) ◽  
pp. 897-904 ◽  
Author(s):  
Francisca REYES ◽  
Mónica GAVIRA ◽  
Francisco CASTILLO ◽  
Conrado MORENO-VIVIÁN
Keyword(s):  
Nitrate Reduction ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Transcriptional Start Site ◽  
Azorhizobium Caulinodans ◽  
Reading Frame ◽  
Phototrophic Bacterium ◽  
Mutant Strains ◽  
Dna Fragment

The phototrophic bacterium Rhodobacter sphaeroidesDSM 158 is able to reduce nitrate to nitrite by means of a periplasmic nitrate reductase which is induced by nitrate and is not repressed by ammonium or oxygen. Recently, a 6.8 kb PstI DNA fragment carrying the napABCgenes coding for this periplasmic nitrate-reducing system was cloned [Reyes, Roldán, Klipp, Castillo and Moreno-Vivián (1996) Mol. Microbiol. 19, 1307–1318]. Further sequence and genetic analyses of the DNA region upstream from the napABCgenes reveal the presence of four additional napgenes. All these R. sphaeroidesgenes seem to be organized into a napKEFDABCtranscriptional unit. In addition, a partial open reading frame similar to the Azorhizobium caulinodans yntCgene and the Escherichia coli yjcCand yhjKgenes is present upstream from this napgene cluster. The R. sphaeroides napKgene codes for a putative 6.3 kDa transmembrane protein which is not similar to known proteins and the napEgene codes for a 6.7 kDa transmembrane protein similar to the Thiosphaera pantotrophaNapE. The R. sphaeroides napFgene product is a 16.4 kDa protein with four cysteine clusters that probably bind four [4Fe-4S] centres. This iron–sulphur protein shows similarity to the NapF and NapG proteins of E. coliand Haemophilus influenzae.Finally, the napDgene product is a 9.4 kDa soluble protein which is also found in E. coliand T. pantotropha. The 5´ end of the naptranscript has been determined by primer extension, and a δ70-like promoter has been identified upstream from the napKgene. The same transcriptional start site is found for cells growing aerobically or anaerobically with nitrate. Different mutant strains carrying defined polar and non-polar insertions in each napgene were constructed. Characterization of these mutant strains demonstrates the participation of the napgene products in the periplasmic nitrate reduction in R. sphaeroides.

scholarly journals Characterization of the Escherichia coli gene encoding a new member of the short-chain dehydrogenase/reductase (SDR) family.

1997 ◽  
Vol 44 (1) ◽  
pp. 153-157 ◽  
Author(s):  
A Sirko ◽  
A Wegleńska ◽  
M Hryniewicz ◽  
D M Hulanicka
Keyword(s):  
Escherichia Coli ◽  
Short Chain ◽  
Chromosomal Dna ◽  
Large Protein ◽  
Gene Encoding ◽  
Reading Frame ◽  
Large Protein Family ◽  
Dna Fragment

The nucleotide sequence of a chromosomal DNA fragment located upstream from the cysPTWAM operon of Escherichia coli was established. Sequence analysis indicates the presence of an open reading frame which has been designated ucpA (upstream cys P). The potential protein products exhibits strong sequence homology to the members of a large protein family, short-chain dehydrogenases/reductases. Involvement of Crp, FruR and IHF in the regulation of ucpA transcription in vivo was demonstrated.

scholarly journals Novel Virulence Gene of Pseudomonas syringae pv. tomato Strain DC3000

2005 ◽  
Vol 187 (22) ◽  
pp. 7805-7814 ◽  
Author(s):  
Karen Preiter ◽  
David M. Brooks ◽  
Alejandro Penaloza-Vazquez ◽  
Aswathy Sreedharan ◽  
Carol L. Bender ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Virulence Gene ◽  
Transcriptional Regulators ◽  
Disease Symptoms ◽  
Tomato Plants ◽  
Reading Frame ◽  
Type Iii ◽  
Mutant Strains

ABSTRACT Previously, we conducted a mutant screen of Pseudomonas syringae pv. tomato strain DC3000 to identify genes that contribute to virulence on Arabidopsis thaliana plants. Here we describe the characterization of one mutant strain, DB4H2, which contains a single Tn5 insertion in PSPTO3576, an open reading frame that is predicted to encode a protein belonging to the TetR family of transcriptional regulators. We demonstrate that PSPTO3576 is necessary for virulence in DC3000 and designate the encoded protein TvrR (TetR-like virulence regulator). TvrR, like many other TetR-like transcriptional regulators, negatively regulates its own expression. Despite the presence of a putative HrpL binding site in the tvrR promoter region, tvrR is not regulated by HrpL, an alternative sigma factor that regulates the expression of many known DC3000 virulence genes. tvrR mutant strains grow comparably to wild-type DC3000 in culture and possess an intact type III secretion system. However, tvrR mutants do not cause disease symptoms on inoculated A. thaliana and tomato plants, and their growth within plant tissue is significantly impaired. We demonstrate that tvrR mutant strains are able to synthesize coronatine (COR), a phytotoxin required for virulence of DC3000 on A. thaliana. Given that tvrR mutant strains are not defective for type III secretion or COR production, tvrR appears to be a novel virulence factor required for a previously unexplored process that is necessary for pathogenesis.

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