Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

ABSTRACT We cloned the rpoN (ntrA andglnF) gene encoding ς54 from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the ς54 protein encoded by thePseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation,rpoN::Kmr. In contrast to wild-type ES4326, ES4326 rpoN::Kmr was nonmotile and could not utilize nitrate, urea, C4-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources.rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326rpoN::Kmr did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thalianaleaves, did not elicit the accumulation of severalArabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Kmrcarrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326rpoN::Kmr partially restored defense-related mRNA accumulation, showing a direct role for thehrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression ofhrpL in ES4326 rpoN::Kmrdid not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.

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Genetic and Functional Investigation of Zn 2 Cys 6 Transcription Factors RSE2 and RSE3 in Podospora anserina

Eukaryotic Cell ◽

10.1128/ec.00172-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 53-65 ◽

Cited By ~ 10

Author(s):

Elodie Bovier ◽

Carole H. Sellem ◽

Adeline Humbert ◽

Annie Sainsard-Chanet

Keyword(s):

Transcription Factors ◽

Phosphoenolpyruvate Carboxykinase ◽

Constitutive Expression ◽

Podospora Anserina ◽

Loss Of Function ◽

Gene Encoding ◽

Content Type ◽

Zinc Cluster ◽

Wide Range ◽

Genes Encoding

ABSTRACT In Podospora anserina , the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase ( aox ) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase ( pck ) and fructose-1,6-biphosphatase ( fbp ). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3 . Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3 . It showed that in addition to aox , fbp , and pck , RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn 2 Cys 6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

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Differential Expression of Genes Encoding Arabidopsis Phospholipases After Challenge with Virulent or Avirulent Pseudomonas Isolates

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.8.808 ◽

2002 ◽

Vol 15 (8) ◽

pp. 808-816 ◽

Cited By ~ 59

Author(s):

Marta de Torres Zabela ◽

Isabelle Fernandez-Delmond ◽

Totte Niittyla ◽

Pedro Sanchez ◽

Murray Grant

Keyword(s):

Pseudomonas Syringae ◽

Defense Responses ◽

Gene Interactions ◽

Gene Encoding ◽

Cellular Processes ◽

Expression Of Genes ◽

Pathogen Challenge ◽

Genes Encoding ◽

Plant Pathogen Interactions ◽

Gene For Gene

Phospholipase D (PLD; EC 3.1.4.4) has been linked to a number of cellular processes, including Tran membrane signaling and membrane degradation. Four PLD genes (α, β, γ1, and γ2) have been cloned from Arabidopsis thalami. They encode isoforms with distinct regulatory and catalytic properties but little is known about their physiological roles. Using cDNA amplified fragment length polymorphism display and RNA blot analysis, we identified Arabidopsis PLDγ1 and a gene encoding a lysophospholipase (EC 3.1.1.5), lysoPL1, to be differentially expressed during host response to virulent and avirulent pathogen challenge. Examination of the expression pattern of phospholipase genes induced in response to pathogen challenge was undertaken using the lysoPL1 and gene-specific probes corresponding to the PLD isoforms α, β, and γ1. Each mRNA class exhibited different temporal patterns of expression after infiltration of leaves with Pseudomonas syringae pv. tomato with or without avrRpm1. PLDα was rapidly induced and remained constitutively elevated regardless of treatment. PLDβ was transiently induced upon pathogen challenge. However, mRNA for the lysoPL1 and PLDγ1 genes showed enhanced and sustained elevation during an incompatible interaction, in both ndr1 and overexpressing NahG genetic backgrounds. Further evidence for differential engagement of these PLD mRNA during defense responses, other than gene-for-gene interactions, was demonstrated by their response to salicylic acid treatment or wounding. Our results indicate that genes encoding lysoPL1, PLDγ1, and PLDβ are induced during early responses to pathogen challenge and, additionally, PLDγ1 and lysoPL1 are specifically upregulated during gene-for-gene interactions, leading to the hypersensitive response. We discuss the possible role of these genes in plant-pathogen interactions.

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GadY, a Small-RNA Regulator of Acid Response Genes in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.186.20.6698-6705.2004 ◽

2004 ◽

Vol 186 (20) ◽

pp. 6698-6705 ◽

Cited By ~ 227

Author(s):

Jason A. Opdyke ◽

Ju-Gyeong Kang ◽

Gisela Storz

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Small Rna ◽

Acid Resistance ◽

Base Pairs ◽

Gene Encoding ◽

Mrna Accumulation ◽

Long Form ◽

Genes Encoding ◽

Opposite Strand

ABSTRACT A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor σS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abolished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3′ end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3′-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

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Expression of avrPphB, an Avirulence Gene from Pseudomonas syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.2.247 ◽

1997 ◽

Vol 10 (2) ◽

pp. 247-256 ◽

Cited By ~ 66

Author(s):

Nakul Puri ◽

Carol Jenner ◽

Mark Bennett ◽

Ruth Stewart ◽

John Mansfield ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Induction Time ◽

Phenotypic Expression ◽

Constitutive Expression ◽

Hypersensitive Reaction ◽

Full Length ◽

Avirulence Gene ◽

Over Expression ◽

E Coli ◽

Induction Kinetics

Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY - strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.

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Characterization of LgnR, an IclR family transcriptional regulator involved in the regulation of l-gluconate catabolic genes in Paracoccus sp. 43P

Microbiology ◽

10.1099/mic.0.074286-0 ◽

2014 ◽

Vol 160 (3) ◽

pp. 623-634 ◽

Cited By ~ 7

Author(s):

Tetsu Shimizu ◽

Akira Nakamura

Keyword(s):

Constitutive Expression ◽

Transcriptional Regulator ◽

Pcr Analysis ◽

Mobility Shift ◽

Gene Encoding ◽

Reverse Transcription Pcr ◽

Dnase I Footprinting ◽

Catabolic Genes ◽

Genes Encoding ◽

Mobility Shift Assay

Five genes encoding enzymes required for l-gluconate catabolism, together with genes encoding components of putative ABC transporters, are located in a cluster in the genome of Paracoccus sp. 43P. A gene encoding a transcriptional regulator in the IclR family, lgnR, is located in front of the cluster in the opposite direction. Reverse transcription PCR analysis indicated that the cluster was transcribed as an operon, termed the lgn operon. Two promoters, P lgnA and P lgnR , are divergently located in the intergenic region, and transcription from these promoters was induced by addition of l-gluconate or d-idonate, a catabolite of l-gluconate. Deletion of lgnR resulted in constitutive expression of lgnA, lgnH and lgnR, indicating that lgnR encodes a repressor protein for the expression of the lgn operon and lgnR itself. Electrophoretic mobility shift assay and DNase I footprinting analyses revealed that recombinant LgnR binds to both P lgnA and P lgnR , indicating that LgnR represses transcription from these promoters by competing with RNA polymerase for binding to these sequences. d-Idonate was identified as a candidate effector molecule for dissociation of LgnR from these promoters. Phylogenetic analysis revealed that LgnR formed a cluster with putative proteins from other genome sequences, which is distinct from those proteins of known regulatory functions, in the IclR family of transcriptional regulators. Additionally, the phylogeny suggests an evolutionary linkage between the l-gluconate catabolic pathway and d-galactonate catabolic pathways distributed in Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria.

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A Physical Map of the Syringomycin and Syringopeptin Gene Clusters Localized to an Approximately 145-kb DNA Region of Pseudomonas syringae pv. syringae Strain B301D

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.12.1426 ◽

2001 ◽

Vol 14 (12) ◽

pp. 1426-1435 ◽

Cited By ~ 36

Author(s):

Brenda K. Scholz-Schroeder ◽

Jonathan D. Soule ◽

Shi-En Lu ◽

Ingeborg Grgurina ◽

Dennis C. Gross

Keyword(s):

Gene Cluster ◽

Pseudomonas Syringae ◽

Physical Map ◽

Regulatory Element ◽

Gene Clusters ◽

Gene Encoding ◽

Peptide Synthetases ◽

Peptide Synthetase ◽

Genes Encoding ◽

Size Estimates

Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts.

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

Enzyme Research ◽

10.4061/2010/597010 ◽

2010 ◽

Vol 2010 ◽

pp. 1-10 ◽

Cited By ~ 2

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.

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Use of Tn5-gusA5to investigate environmental and nutritional effects on gene expression in the coronatine biosynthetic gene cluster ofPseudomonas syringaepv.glycinea

Canadian Journal of Microbiology ◽

10.1139/m97-074 ◽

1997 ◽

Vol 43 (6) ◽

pp. 517-525 ◽

Cited By ~ 13

Author(s):

David A. Palmer ◽

Carol L. Bender ◽

Shashi B. Sharma

Keyword(s):

Gene Expression ◽

Amino Acid ◽

Gene Cluster ◽

Pseudomonas Syringae ◽

Nitrogen Sources ◽

Biosynthetic Gene Cluster ◽

Amide Bond ◽

Transcriptional Level ◽

Coronafacic Acid ◽

Genes Encoding

Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafacic acid (CFA) coupled via an amide bond to the ethylcyclopropyl amino acid coronamic acid (CMA). Both CFA and CMA function as intermediates in the pathway to coronatine, and genes encoding their synthesis have been localized; however, the precise factors that regulate the production of COR and its precursors remain unclear. In the present study, a λ delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster. Selected carbon (fructose and xylose) and amino acid (isoleucine and valine) sources significantly decreased COR biosynthesis at the transcriptional level. Transcriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18–24 °C and significantly less expression at 14 and 30 °C. Interestingly, changes in osmolarity and the addition of complex carbon and nitrogen sources to the growth medium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced. These results indicate that multiple factors impact COR production and only some of these directly affect transcription in the COR gene cluster.Key words: transcriptional fusion, glucuronidase, gene expression, reporter gene.

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Lack of Control of Nitrite Assimilation by Ammonium in an Oceanic Picocyanobacterium, Synechococcus sp. Strain WH 8103

Applied and Environmental Microbiology ◽

10.1128/aem.02606-06 ◽

2007 ◽

Vol 73 (9) ◽

pp. 3028-3033 ◽

Cited By ~ 12

Author(s):

Michael Wyman ◽

Clare Bird

Keyword(s):

Surface Waters ◽

Nitrogen Sources ◽

Ammonium Transporter ◽

Marine Cyanobacteria ◽

Gene Encoding ◽

Expression Of Genes ◽

Oceanic Surface ◽

Lack Of Control ◽

Genes Encoding ◽

Synechococcus Sp

ABSTRACT In cyanobacteria, the transcriptional activator NtcA is involved in global nitrogen control and, in the absence of ammonium, regulates the expression of genes involved in the assimilation of alternative nitrogen sources. The oceanic picocyanobacterium Synechococcus sp. strain WH 8103 harbors a copy of ntcA, but in the present study, we show that unlike other marine cyanobacteria that have been investigated, this strain is capable of coassimilating nitrite when grown in the presence of ammonium. Transcript levels for the genes encoding the nitrate/nitrite-bispecific permease NrtP and nitrate reductase (NarB) were substantially down-regulated by ammonium, whereas the abundances of nitrite reductase (NirA) transcripts were similar in nitrite- and ammonium-grown cells. The growth of Synechococcus sp. strain WH 8103 in medium containing both ammonium and nitrite resulted in only minor changes in the expression profile in comparison to that of nitrite-grown cells with the exception that the gene encoding the high-affinity ammonium transporter Amt1 was down-regulated to the levels seen in ammonium-grown cells. Whereas the expression of nrtP, narB, and amt1 appears to be NtcA dependent in this marine cyanobacterium, the transcription and expression of nirA appear not to be. The ability to coassimilate nitrite and reduced-nitrogen sources like ammonium may be an adaptive trait that enables oceanic strains like Synechococcus sp. strain WH 8103 to exploit the low nitrite concentrations found in oceanic surface waters that are not available to their principal and more numerous competitor, Prochlorococcus.

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Cyclin-Dependent Kinase-Associated Proteins Cks1 and Cks2 Are Essential during Early Embryogenesis and for Cell Cycle Progression in Somatic Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01833-07 ◽

2008 ◽

Vol 28 (18) ◽

pp. 5698-5709 ◽

Cited By ~ 62

Author(s):

Hanna-Stina Martinsson-Ahlzén ◽

Vasco Liberal ◽

Björn Grünenfelder ◽

Susana R. Chaves ◽

Charles H. Spruck ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Cycle Progression ◽

Cyclin B1 ◽

Open Reading Frames ◽

Promoter Regions ◽

Gene Encoding ◽

Direct Role ◽

Cycle Arrest ◽

Genes Encoding

ABSTRACT Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G2, followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription.

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