scholarly journals The Presence of Diverse IS Elements and an avrPphD Homologue That Acts as a Virulence Factor on the Pathogenicity Plasmid of Erwinia herbicola pv. gypsophilae

2002 ◽  
Vol 15 (7) ◽  
pp. 709-716 ◽  
Author(s):  
Ming Guo ◽  
Shulamit Manulis ◽  
Henia Mor ◽  
Isaac Barash
Keyword(s):  
Amino Acids ◽  
Pseudomonas Syringae ◽  
Upstream Region ◽  
Erwinia Herbicola ◽  
High Identity ◽  
Reading Frame ◽  
Type Iii Effectors ◽  
Is Elements ◽  
Genes Encoding ◽  
Indole 3 Acetic Acid

The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) and Erwinia herbicola pv. betae (Ehb) is dependent on a native plasmid (pPATHEhg or pPATHEhb) that harbors the hrp gene cluster, genes encoding type III effectors, phytohormones, biosynthetic genes, and several copies of IS1327. Sequence analysis of the hrp-flanking region in pPATHEhg (cosmid pLA150) revealed a cluster of four additional IS elements designated as ISEhe1, ISEhe2, ISEhe3, and ISEhe4. Two copies of another IS element (ISEhe5) were identified on the upstream region of the indole-3-acetic acid operon located on the same cosmid. Based on homology of amino acids and genetic organization, ISEhe1 belongs to the IS630 family, ISEhe2 to the IS5 family, ISEhe3 and ISEhe4 to different groups of the IS3 family, and ISEhe5 to the IS1 family. With the exception of ISEhe4, one to three copies of all the other IS elements were identified only in pathogenic strains of Erwinia herbicola pv. gypsophilae and Erwinia herbicola pv. betae whereas ISEhe4 was present in both pathogenic and nonpathogenic strains. An open reading frame that exhibited high identity (89% in amino acids) to AvrPphD of Pseudomonas syringae pv. phaseolicola was present within the cluster of IS elements. An insertional mutation in the AvrPphD Ehg reduced gall size in gypsophila by approximately 85%. In addition, remnants of known genes from four different bacteria were detected on the same cosmid.

Characterization of the indole-3-acetic acid (IAA) biosynthetic pathway in an epiphytic strain of Erwinia herbicola and IAA production in vitro

1996 ◽  
Vol 42 (6) ◽  
pp. 586-592 ◽  
Author(s):  
M. Brandi ◽  
E. M. Clark ◽  
S. E. Lindow
Keyword(s):  
Acetic Acid ◽  
Pseudomonas Syringae ◽  
Pyruvic Acid ◽  
Enzyme Assays ◽  
Erwinia Herbicola ◽  
Iaa Production ◽  
Acid Pathway ◽  
Culture Supernatants ◽  
Indole 3 Acetic Acid

An epiphytic strain of Erwinia herbicola (strain 299R) synthesized indole-3-acetic acid (IAA) from indole-3-pyruvic acid and indole-3-acetaldehyde, but not from indole-3-acetamide and other intermediates of various IAA biosynthetic pathways in enzyme assays. TLC, HPLC, and GC–MS analyses revealed the presence of indole-3-pyruvic acid, indole-3-ethanol, and IAA in culture supernatants of strain 299R. Indole-3-acetaldehyde was detected in enzyme assays. Furthermore, strain 299R genomic DNA shared no homology with the iaaM and iaaH genes from Pseudomonas syringae pv. savastanoi, even in Southern hybridizations performed under low-stringency conditions. These observations strongly suggest that unlike gall-forming bacteria which can synthesize IAA by indole-3-acetamide, the indole-3-pyruvic acid pathway is the primary route for IAA biosynthesis in this plant-associated strain. IAA synthesis in tryptophan-supplemented cultures of strain 299R was over 10-fold higher under nitrogen-limiting conditions, indicating a possible role for IAA production by bacterial epiphytes in the acquisition of nutrients during growth in their natural habitat.Key words: indole-3-acetic acid, Erwinia, tryptophan, indole-3-pyruvic acid, nitrogen.

scholarly journals Two New Polyphenol Oxidase Genes of Tea Plant (Camellia sinensis) Respond Differentially to the Regurgitant of Tea Geometrid, Ectropis obliqua

2018 ◽  
Vol 19 (8) ◽  
pp. 2414 ◽  
Author(s):  
Chen Huang ◽  
Jin Zhang ◽  
Xin Zhang ◽  
Yongchen Yu ◽  
Wenbo Bian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Camellia Sinensis ◽  
Tea Plant ◽  
High Identity ◽  
Reading Frame ◽  
Polyphenol Oxidases ◽  
Defensive Role ◽  
Molecular Masses ◽  
The Difference ◽  
Tea Plants

Polyphenol oxidases (PPOs) have been reported to play an important role in protecting plants from attacks by herbivores. Though PPO genes in other plants have been extensively studied, research on PPO genes in the tea plant (Camellia sinensis) is lacking. In particular, which members of the PPO gene family elicit the defense response of the tea plant are as yet unknown. Here, two new PPO genes, CsPPO1 and CsPPO2, both of which had high identity with PPOs from other plants, were obtained from tea leaves. The full length of CsPPO1 contained an open reading frame (ORF) of 1740 bp that encoded a protein of 579 amino acids, while CsPPO2 contained an ORF of 1788 bp that encoded a protein of 595 amino acids. The deduced CsPPO1 and CsPPO2 proteins had calculated molecular masses of 64.6 and 65.9 kDa; the isoelectric points were 6.94 and 6.48, respectively. The expression products of recombinant CsPPO1 and CsPPO2 in Escherichia coli were about 91 and 92 kDa, respectively, but the recombinant proteins existed in the form of an inclusion body. Whereas CsPPO1 is highly expressed in stems, CsPPO2 is highly expressed in roots. Further results showed that the expression of CsPPO1 and CsPPO2 was wound- and Ectropis obliqua-induced, and that regurgitant, unlike treatment with wounding plus deionized water, significantly upregulated the transcriptional expression of CsPPO2 but not of CsPPO1. The difference between regurgitant and wounding indicates that CsPPO2 may play a more meaningful defensive role against E. obliqua than CsPPO1. Meanwhile, we found the active component(s) of the regurgitant elicited by the expression of CsPPO may contain small molecules (under 3-kDa molecular weight). These conclusions advance the understanding of the biological function of two new PPO genes and show that one of these, CsPPO2, may be a promising gene for engineering tea plants that are resistant to E. obliqua.

scholarly journals The Presence of hrp Genes on the Pathogenicity-Associated Plasmid of the Tumorigenic Bacterium Erwinia herbicola pv. gypsophilae

1997 ◽  
Vol 10 (5) ◽  
pp. 677-682 ◽  
Author(s):  
Roni Nizan ◽  
Isaac Barash ◽  
Lea Valinsky ◽  
Amnon Lichter ◽  
Shulamit Manulis
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Hypersensitive Reaction ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Cytokinin Biosynthesis ◽  
Erwinia Herbicola ◽  
Hrp Genes ◽  
Genes Encoding ◽  
Bacterial Genes

The pathogenicity-associated plasmid (pPATH) of Erwinia herbicola pv. gypsophilae (Ehg), which is present only in pathogenic strains, contains a gene cluster encoding indole-3-acetic acid and cytokinin biosynthesis. The transposon-reporter Tn3-Spice was used to generate nonpathogenic mutants on two overlapping cosmids, pLA150 and pLA352, of the pPATH. A cluster of such mutations, which spanned 16 kb, mapped approximately 15 kb from the gene cluster involved in phytohormone biosynthesis. Non-pathogenic mutants also failed to elicit the hypersensitive reaction (HR) on tobacco. Pathogenicity and HR were restored concomitantly to these mutants by in trans complementation with wild-type Ehg DNA. A 3.8-kb HindIII DNA fragment that complemented the hrp mutants was sequenced and six complete and two partial open reading frames (ORFs) were identified. Comparison of the deduced amino acid sequences of the eight ORFs showed striking homology and co-linearity with hrp genes of E. amylovora as well as with other plant and mammalian pathogenic bacterial genes encoding proteins of the type III secretion system. Limited DNA sequencing at various sites on the remaining 11-kb region of pLA352 also showed high identity to Hrp proteins of E. amylovora, E. stewartii, and Pseudomonas syringae. These results suggest that hrp genes are mandatory for gall formation by E. herbicola pv. gypsophilae.

scholarly journals Characterization and In Silico Analysis of Pregnancy-Associated Glycoprotein-1 Gene of Buffalo (Bubalus bubalis)

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Jerome A. ◽  
S. K. Singh ◽  
S. K. Agarwal ◽  
Mohini Saini ◽  
Ashwin Raut
Keyword(s):  
Amino Acids ◽  
Mammalian Species ◽  
Evolutionary Relationship ◽  
In Silico Analysis ◽  
Bubalus Bubalis ◽  
Phylogenetic Study ◽  
Base Pairs ◽  
Reading Frame ◽  
Genes Encoding ◽  
Amino Acid Signal Peptide

Pregnancy-Associated Glycoproteins (PAGs) are trophoblastic proteins belonging to the Aspartic proteinase family secreted by different placental cells of many mammalian species. They play a pivotal role in placentogenesis, foetomaternal unit remodeling, and implantation. The identification of the genes encoding those proteins will be helpful to unravel the intricate embryogenomic functions during pregnancy establishment. Considering importance of these proteins, the present study was undertaken to characterize the pregnancy associated glycoprotein-1 gene of buffalo. An 1181 base pairs buffalo Pregnancy-Associated Glycoprotein PAG-1 gene was PCR amplified from the RNA obtained from the fetal cotyledons. BLAST analysis of the buffalo PAG-1 sequence retrieved a total of 20 cattle, 5 goat, and 4 sheep PAG sequences, exhibiting more than 80% similarity. Buffalo PAG-1 gene contained an uninterrupted open reading frame of 1140 base pairs encoding 380 amino acids that possess a 15 amino acid signal peptide and mature peptide of 365 amino acids. The phylogenetic study of the buffalo PAG-1 gene revealed buffalo PAG-1 is more related to cattle, goat, and sheep PAG-1 sequences. By this study characterization of buffalo PAG-1 gene and its evolutionary relationship was deduced for the first time.

scholarly journals Probing Direct Interactions between CodY and the oppD Promoter of Lactococcus lactis

2005 ◽  
Vol 187 (2) ◽  
pp. 512-521 ◽  
Author(s):  
Chris D. den Hengst ◽  
Peter Curley ◽  
Rasmus Larsen ◽  
Girbe Buist ◽  
Arjen Nauta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Nitrogen Availability ◽  
Nitrogen Sources ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Upstream Region ◽  
Binding Studies ◽  
Genes Encoding

ABSTRACT CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes include pepN, pepC, opp-pepO1, and probably prtPM, pepX, and pepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate that L. lactis CodY interacts directly with a region upstream of the promoter of its major target known so far, the opp system. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region of oppD yielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.

scholarly journals Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae

1998 ◽  
Vol 180 (4) ◽  
pp. 822-830 ◽  
Author(s):  
Xinping Wang ◽  
Craig J. Mann ◽  
Yinlin Bai ◽  
Li Ni ◽  
Henry Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Genomic Sequence ◽  
Divalent Cations ◽  
Open Reading Frame ◽  
Ethanol Metabolism ◽  
Reading Frame ◽  
Genes Encoding ◽  
Dna Fragment

ABSTRACT The full-length DNAs for two Saccharomyces cerevisiaealdehyde dehydrogenase (ALDH) genes were cloned and expressed inEscherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K+ ions. In contrast, the activity of ALDH5 increased in the presence of K+ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption Δald1 and Δald2 strains on ethanol was marginally slower than that of the parent strain. The Δald1 Δald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.

scholarly journals Global Genomic Analysis of Pseudomonas savastanoi pv. savastanoi Plasmids

2007 ◽  
Vol 190 (2) ◽  
pp. 625-635 ◽  
Author(s):  
Isabel Pérez-Martínez ◽  
Youfu Zhao ◽  
Jesús Murillo ◽  
George W. Sundin ◽  
Cayo Ramos
Keyword(s):  
Pseudomonas Syringae ◽  
Insertion Sequence ◽  
Genomic Analysis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Gene Content ◽  
Type Iv ◽  
Lack Of Information ◽  
Genes Encoding ◽  
Indole 3 Acetic Acid

ABSTRACT Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.

scholarly journals Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol

1994 ◽  
Vol 14 (4) ◽  
pp. 2298-2306
Author(s):  
M Boguta ◽  
L A Hunter ◽  
W C Shen ◽  
E C Gillman ◽  
N C Martin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Fractionation ◽  
Mitochondrial Targeting ◽  
Reading Frame ◽  
Subcellular Compartments ◽  
Initiation Of Translation ◽  
Genes Encoding ◽  
Passenger Proteins

MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.

scholarly journals Characterization of Two Newly Identified Genes, vgaD and vatG, Conferring Resistance to Streptogramin A in Enterococcus faecium

2010 ◽  
Vol 54 (11) ◽  
pp. 4744-4749 ◽  
Author(s):  
Young-Hee Jung ◽  
Eun Shim Shin ◽  
Okgene Kim ◽  
Jung Sik Yoo ◽  
Kyeong Min Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Resistance Genes ◽  
Enterococcus Faecium ◽  
Efflux Pump ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Strongly Correlated ◽  
Reading Frame ◽  
Genes Encoding ◽  
New Gene

ABSTRACT We characterized two new streptogramin A resistance genes from quinupristin-dalfopristin-resistant Enterococcus faecium JS79, which was selected from 79 E. faecium isolates lacking known genes encoding streptogramin A acetyltransferase. A 5,650-bp fragment of HindIII-digested plasmid DNA from E. faecium JS79 was cloned and sequenced. The fragment contained two open reading frames carrying resistance genes related to streptogramin A, namely, genes for an acetyltransferase and an ATP efflux pump. The first open reading frame comprised 648 bp encoding 216 amino acids with a predicted left-handed parallel β-helix domain structure; this new gene was designated vatG. The second open reading frame consisted of 1,575 bp encoding 525 amino acids with two predicted ATPase binding cassette transporters comprised of Walker A, Walker B, and LSSG motifs; this gene was designated vgaD. vgaD is located 65 bp upstream from vatG, was detected together with vatG in 12 of 179 quinupristin-dalfopristin-resistant E. faecium isolates, and was located on the same plasmid. Also, the 5.6-kb HindIII-digested fragment which was observed in JS79 was detected in nine vgaD- and vatG-containing E. faecium isolates by Southern hybridization. Therefore, it was expected that these two genes were strongly correlated with each other and that they may be composed of a transposon. Importantly, vgaD is the first identified ABC transporter conferring resistance to streptogramin A in E. faecium. Pulsed-field gel electrophoresis patterns and sequence types of vgaD- and vatG-containing E. faecium isolates differed for isolates from humans and nonhumans.

scholarly journals Identification of a Novel Pseudomonas syringae Psy61 Effector with Virulence and Avirulence Functions by a HrpL-Dependent Promoter-Trap Assay

2004 ◽  
Vol 17 (3) ◽  
pp. 254-262 ◽  
Author(s):  
L. Losada ◽  
T. Sussan ◽  
K. Pak ◽  
S. Zeyad ◽  
I. Rozenbaum ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Host Cells ◽  
Chloroplast Targeting ◽  
Trap Assay ◽  
Genomic Libraries ◽  
Reading Frame ◽  
Promoter Trap ◽  
Genes Encoding ◽  
Targeting Signal ◽  
Species Specific

The hrp pathogenicity island of Pseudomonas syringae encodes a type III secretion system (TTSS) that translocates effectors into plant cells. Most genes encoding effectors are dispersed in the P. syringae genome. Regardless of location, all are regulated coordinately by the alternative sigma factor HrpL. An HrpL-dependent promoter-trap assay was developed to screen genomic libraries of P. syringae strains for promoters whose activity in Escherichia coli is dependent on an inducible hrpL construct. Twenty-two HrpL-dependent promoter fragments were isolated from P. syringae Psy61 that included promoters for known HrpL-dependent genes. One fragment also was isolated that shared no similarity with known genes but retained a near consensus HrpL-dependent promoter. The sequence of the region revealed a 375-amino acid open reading frame encoding a 40.5-kDa product that was designated HopPsyL. HopPsyL was structurally similar to other secreted effectors and carried a putative chloroplast-targeting signal and two predicted transmembrane domains. HopPsyL′:′ AvrRpt2 fusions were translocated into host cells via the P. syringae pv. tomato DC3000 hrp TTSS. A hopPsyL∷kan mutant of Psy61 exhibited strongly reduced virulence in Phaseolus vulgaris cv. Kentucky Wonder, but did not appear to act as a defense response suppressor. The ectopically expressed gene reduced the virulence of Pseudomonas syringae DC3000 transformants in Arabidopsis thaliana Col-0. The gene was shown to be conserved in 6 of 10 P. syringae pv. syringae strains but was not detected in 35 strains of other pathovars. HopPsyL appears to be a novel TTSS-dependent effector that functions as a host-species-specific virulence factor in Psy61.

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