scholarly journals A luxR Homolog, aviR, in Agrobacterium vitis Is Associated with Induction of Necrosis on Grape and a Hypersensitive Response on Tobacco

2003 ◽  
Vol 16 (7) ◽  
pp. 650-658 ◽  
Author(s):  
Desen Zheng ◽  
Hongsheng Zhang ◽  
Sigrid Carle ◽  
Guixia Hao ◽  
Michele R. Holden ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Polymerase Chain Reaction Analysis ◽  
Homoserine Lactone ◽  
Wild Type ◽  
Agrobacterium Vitis ◽  
Reading Frame ◽  
Homologous Genes ◽  
Luxr Homolog ◽  
Genes Encoding ◽  
Tn5 Mutant

A Tn5 mutant of Agrobacterium vitis F2/5 (M1154) differs from the wild-type strain in that it has lost its abilities to cause necrosis on grape and a hypersensitive-like response (HR) on tobacco. The Tn5 insertion occurred in an open reading frame (ORF) aviR that is homologous to genes encoding the LuxR family of transcriptional regulators, thereby suggesting that the HR and necrosis are regulated by a quorum-sensing system. Fewer N-acyl-homoserine lactone autoinducers were detected in extracts from M1154 compared with extracts from F2/5 and from aviR-complemented M1154. The complemented mutant regained full ability to cause grape necrosis and HR. Eighteen ORFs located on a 36.6-kb insert in cosmid clone CPB221, which includes aviR, were sequenced and aligned with homologous genes from A. tumefaciens C58 and Sinorhizobium meliloti Rm1021. The order of several clustered genes is conserved among the bacteria; however, rearrangements are also apparent. Reverse transcriptase-polymerase chain reaction analysis indicated that ORF2 and ORF14 may be regulated by an aviR-encoded transcriptional regulator. Single site-directed mutations in each of the ORFs, however, had no effect on expression of HR or necrosis as compared with the wild-type parent.

A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

1998 ◽  
Vol 64 (10) ◽  
pp. 3954-3960 ◽  
Author(s):  
Donald A. Phillips ◽  
Eve S. Sande ◽  
J. A. C. Vriezen ◽  
Frans J. de Bruijn ◽  
Daniel Le Rudulier ◽  
...  
Keyword(s):  
Salt Stress ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Genetic Locus ◽  
Wild Type ◽  
Single Strain ◽  
Reading Frame ◽  
Carbon And Nitrogen ◽  
Promoter Probe ◽  
Inducible Genes

ABSTRACT Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [14C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [14C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxABfusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.

scholarly journals MucR and MucS Activate exp Genes Transcription and Galactoglucan Production in Sinorhizobium meliloti EFB1

2002 ◽  
Vol 15 (1) ◽  
pp. 54-59 ◽  
Author(s):  
Javier Lloret ◽  
Marta Martín ◽  
Roke I. Oruezabal ◽  
Ildefonso Bonilla ◽  
Rafael Rivilla
Keyword(s):  
Transcriptional Activation ◽  
Sinorhizobium Meliloti ◽  
Polymerase Chain Reaction Analysis ◽  
Wild Type ◽  
Chain Reaction ◽  
Soil Microcosms ◽  
Wild Type Level ◽  
Wild Type Phenotype ◽  
In The Wild ◽  
Polymerase Chain

When grown under standard conditions, Sinorhizobium meliloti EFB1 simultaneously produces two acidic exopolysaccharides, succinoglycan and galactoglucan, yielding very mucoid colonies. In this strain, MucR is essential for galactoglucan synthesis. A mutation in the mucS gene resulted in less mucoid colonies than in the wild-type EFB1. This mucS¯ strain was complemented to the wild-type phenotype by the cloned mucS gene, indicating that mucS is necessary for a wild-type level of galactoglucan production. Reverse transcription-polymerase chain reaction analysis of exp genes, which encode the pathway for galactoglucan production, in EFB1 and in the mutants affected in mucS, mucR, and both genes simultaneously, showed that MucS is a transcriptional activator of the exp genes but does not affect its own transcription. Furthermore, MucR is necessary for mucS transcriptional activation. As introduction of a cloned mucS gene in a mucR¯ strain yielded colonies less mucoid than the wild type, MucR could also activate exp genes transcription through other pathways. Deletion analysis of the expE promoter showed a region important for transcription and MucS activation. This region, containing a palindrome, is present in the putative expA, expC, expD, and expE promoters but not in the mucS promoter, suggesting that it is the target for MucS. A mucR¯mucS¯ mutant, which does not produce galactoglucan, was impaired in competitive nodulation of alfalfa in soil microcosms, indicating another possible role for this exopolysaccharide in symbiosis.

scholarly journals The Pseudomonas aeruginosa devB/SOL Homolog,pgl, Is a Member of the hex Regulon and Encodes 6-Phosphogluconolactonase

2000 ◽  
Vol 182 (14) ◽  
pp. 3934-3941 ◽  
Author(s):  
Paul W. Hager ◽  
M. Worth Calfee ◽  
Paul V. Phibbs
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Carbon Source ◽  
Normal Growth ◽  
Wild Type ◽  
Reading Frame ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Type Rate ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Wild Type Rate

ABSTRACT A cyclic version of the Entner-Doudoroff pathway is used byPseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form thehex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOLfamily of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5′ end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgland propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 (hexR). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl, a structural gene of the hex regulon.

scholarly journals A Burkholderia cenocepacia Orphan LuxR Homolog Is Involved in Quorum-Sensing Regulation

2009 ◽  
Vol 191 (8) ◽  
pp. 2447-2460 ◽  
Author(s):  
Rebecca J. Malott ◽  
Eoin P. O'Grady ◽  
Jessica Toller ◽  
Silja Inhülsen ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Negative Regulation ◽  
Burkholderia Cenocepacia ◽  
Acyl Homoserine Lactone ◽  
Independent Manner ◽  
Reverse Transcription Pcr ◽  
Luxr Homolog ◽  
Genes Encoding

ABSTRACT Burkholderia cenocepacia utilizes quorum sensing to control gene expression, including the expression of genes involved in virulence. In addition to CepR and CciR, a third LuxR homolog, CepR2, was found to regulate gene expression and virulence factor production. All B. cenocepacia strains examined contained this orphan LuxR homolog, which was not associated with an adjacent N-acyl-homoserine lactone synthase gene. Expression of cepR2 was negatively autoregulated and was negatively regulated by CciR in strain K56-2. Microarray analysis and quantitative reverse transcription-PCR determined that CepR2 did not influence expression of cepIR or cciIR. However, in strain K56-2, CepR2 negatively regulated expression of several known quorum-sensing-controlled genes, including genes encoding zinc metalloproteases. CepR2 exerted positive and negative regulation on genes on three chromosomes, including strong negative regulation of a gene cluster located adjacent to cepR2. In strain H111, which lacks the CciIR quorum-sensing system, CepR2 positively regulated pyochelin production by controlling transcription of one of the operons required for the biosynthesis of the siderophore in an N-acyl-homoserine lactone-independent manner. CepR2 activation of a luxI promoter was demonstrated in a heterologous Escherichia coli host, providing further evidence that CepR2 can function in the absence of signaling molecules. This study demonstrates that the orphan LuxR homolog CepR2 contributes to the quorum-sensing regulatory network in two distinct strains of B. cenocepacia.

scholarly journals Quorum Sensing Controls Exopolysaccharide Production in Sinorhizobium meliloti

2003 ◽  
Vol 185 (1) ◽  
pp. 325-331 ◽  
Author(s):  
Melanie M. Marketon ◽  
Sarah A. Glenn ◽  
Anatol Eberhard ◽  
Juan E. González
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Homoserine Lactone ◽  
Symbiotic Relationship ◽  
Wild Type ◽  
Sensing System ◽  
Quorum Sensing System

ABSTRACT Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C16:1-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.

scholarly journals Novel Mechanism for Fluoroquinolone Resistance in Acinetobacter baumannii

2012 ◽  
Vol 56 (9) ◽  
pp. 4955-4957 ◽  
Author(s):  
Sunil D. Saroj ◽  
Katy M. Clemmer ◽  
Robert A. Bonomo ◽  
Philip N. Rather
Keyword(s):  
Acinetobacter Baumannii ◽  
Fold Increase ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Content Type ◽  
Reading Frame ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Ciprofloxacin Resistance ◽  
Transposon Insertions

ABSTRACTAn EZ::TN<R6Kγori/KAN-2>Tnp transposon insertion in an open reading frame of unknown function (ncr) inAcinetobacter baumanniiresulted in an 8-fold increase in ciprofloxacin resistance (Cipr). Transposon insertions in anncrmutant that reduced Ciprback to wild type mapped to three genes encoding subunits of the RecCBD exonuclease. Thencrmutation increased transcription of therecCBDgenes, and overexpression of therecCBDgenes in a wild-type background resulted in a 4-fold increase in Cipr.

scholarly journals ExpR Coordinates the Expression of Symbiotically Important, Bundle-Forming Flp Pili with Quorum Sensing in Sinorhizobium meliloti

2014 ◽  
Vol 80 (8) ◽  
pp. 2429-2439 ◽  
Author(s):  
Hardik M. Zatakia ◽  
Cassandra E. Nelson ◽  
Umair J. Syed ◽  
Birgit E. Scharf
Keyword(s):  
Quorum Sensing ◽  
Sinorhizobium Meliloti ◽  
Consensus Sequence ◽  
Homoserine Lactone ◽  
Host Cells ◽  
Wild Type ◽  
Mobility Shift ◽  
Plant Host ◽  
Symbiotic Interaction ◽  
Content Type

ABSTRACTType IVb pili in enteropathogenic bacteria function as a host colonization factor by mediating tight adherence to host cells, but their role in bacterium-plant symbiosis is currently unknown. The genome of the symbiotic soil bacteriumSinorhizobium meliloticontains two clusters encoding proteins for type IVb pili of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the role of Flp pili in the symbiotic interaction ofS. melilotiand its host,Medicago sativa, we deletedpilA1, which encodes the putative pilin subunit in the chromosomalflp-1cluster and conducted competitive nodulation assays. ThepilA1deletion strain formed 27% fewer nodules than the wild type. Transmission electron microscopy revealed the presence of bundle-forming pili protruding from the polar and lateral region ofS. melilotiwild-type cells. The putative pilus assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral localization. Transcriptional reporter gene assays demonstrated that expression ofpilA1peaks in early stationary phase and is repressed by the quorum-sensing regulator ExpR, which also controls production of exopolysaccharides and motility. Binding of acyl homoserine lactone-activated ExpR to thepilA1promoter was confirmed with electrophoretic mobility shift assays. A 17-bp consensus sequence for ExpR binding was identified within the 28-bp protected region by DNase I footprinting analyses. Our results show that Flp pili are important for efficient symbiosis ofS. melilotiwith its plant host. The temporal inverse regulation of exopolysaccharides and pili by ExpR enablesS. melilotito achieve a coordinated expression of cellular processes during early stages of host interaction.

scholarly journals AHL Signals Induce Rubrifacine Production in a bruI Mutant of Brenneria rubrifaciens

2012 ◽  
Vol 102 (2) ◽  
pp. 195-203 ◽  
Author(s):  
Ali E. McClean ◽  
Breck A. Duerkop ◽  
E. Peter Greenberg ◽  
Daniel A. Kluepfel
Keyword(s):  
High Performance ◽  
Bacterial Species ◽  
Homoserine Lactone ◽  
Hypersensitive Reaction ◽  
Pigment Production ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Ethyl Acetate Extracts

Several members of the bacterial genus Brenneria are pathogenic on different tree species. Cell-free extracts from the bacterial phytopathogens Brenneria rubrifaciens, B. salicis, and B. nigrifluens induced production of the red pigment rubrifacine in the B. rubrifaciens bruI insertional mutant Br-212. Analysis of the bruI locus identified an adjacent open reading frame, designated bruR, with homology to luxR. High-performance liquid chromatography and mass spectrometry analysis of ethyl acetate extracts from wild-type B. rubrifaciens and Escherichia coli expressing the bruI gene identified two acyl homoserine lactone (AHL) peaks, N-(3-oxohexanoyl)-homoserine lactone (3OC6HSL) and N-hexanoyl-homoserine lactone (C6HSL). Addition of synthetic 3OC6HSL and C6HSL at 10 μM to the bruI mutant, strain Br-212, induced rubrifacine production and the ability to elicit a hypersensitive reaction (HR) in tobacco leaves. Synthetic C6HSL was less effective at inducing pigment production than 3OC6HSL at 10 μM. The bruI mutant Br-212 did not produce detectable AHLs, indicating that C6HSL and 3OC6HSL are the major AHLs produced by this species. The AHLs N-heptanoyl-DL-homoserine lactone (C7HSL), N-octanoyl-DL-homoserine lactone (C8HSL), and N-(3-oxooctanoyl)-DL-homoserine lactone (3OC8HSL) also induced pigment production in Br-212 and restored its ability to elicit an HR in tobacco, suggesting that cross-talk with other bacterial species may be possible.

scholarly journals The Chaperone GroESL Enhances the Accumulation of Soluble, Active TraR Protein, a Quorum-Sensing Transcription Factor from Agrobacterium tumefaciens

2009 ◽  
Vol 191 (11) ◽  
pp. 3706-3711 ◽  
Author(s):  
Yunrong Chai ◽  
Stephen C. Winans
Keyword(s):  
Transcription Factor ◽  
Agrobacterium Tumefaciens ◽  
Quorum Sensing ◽  
Sinorhizobium Meliloti ◽  
Transcriptional Activity ◽  
Tertiary Structure ◽  
Protein A ◽  
Homoserine Lactone ◽  
Wild Type ◽  
E Coli

ABSTRACT TraR of Agrobacterium tumefaciens is a LuxR-type quorum-sensing transcription factor that regulates genes required for replication and conjugation of the tumor-inducing (Ti) plasmid. TraR requires its cognate autoinducer N-3-oxooctanoyl-homoserine lactone (OOHL) for resistance of proteolysis in wild-type bacteria and for correct protein folding and solubility when overexpressed in E. coli. In this study, we ask whether GroESL might also play a role in TraR folding, as this molecular chaperone assists many proteins in attaining their native tertiary structure. Expression of E. coli GroESL in a strain expressing TraR increases the solubility of TraR and increases transcriptional activity of a TraR-dependent promoter. Both solubility and activity still require OOHL. We also studied the folding of TraR in the closely related bacterium Sinorhizobium meliloti. A mutation in one groEL gene slightly decreased the expression of a TraR-dependent promoter, strongly decreased the accumulation of TraR in Western immunoblot assays, and also strongly influenced the fate of pulse-labeled TraR.

scholarly journals luxR Homolog avhR in Agrobacterium vitis Affects the Development of a Grape-Specific Necrosis and a Tobacco Hypersensitive Response

2005 ◽  
Vol 187 (1) ◽  
pp. 185-192 ◽  
Author(s):  
Guixia Hao ◽  
Hongsheng Zhang ◽  
Desen Zheng ◽  
Thomas J. Burr
Keyword(s):  
Hypersensitive Response ◽  
Sinorhizobium Meliloti ◽  
Agrobacterium Vitis ◽  
Putative Gene ◽  
Dna Binding Domains ◽  
Directed Mutation ◽  
Binding Domains ◽  
Transporter Family ◽  
Luxr Homolog ◽  
A Site

ABSTRACT The luxR homolog aviR in Agrobacterium vitis strain F2/5 was recently shown to be associated with induction of a hypersensitive response (HR) on tobacco and necrosis on grape plants, indicating that the responses are regulated by quorum sensing. We now report a second luxR homolog, avhR, whose disruption (mutant M1320) results in HR-negative and reduced grape necrosis phenotypes. The deduced AvhR protein has characteristic autoinducer binding and DNA binding domains and is unique among reported functional LuxR homologs in having substitutions at highly conserved Asp70, Trp57, and Trp85 residues, which are predicted to play important roles in autoinducer binding in TraR. M1320 was fully complemented with cloned avhR. The same array of N-acylhomoserine lactones (AHL) from F2/5, M1320, and complemented M1320 were observed; however, the signal strength from extracts of 6-day-old M1320 cultures was stronger than that of F2/5. Cultures of F2/5 amended with AHL extracts from overnight and 6-day cultures of F2/5 and M1320 were not affected in ability to cause HR or necrosis. A region of about 14 kb flanking avhR was sequenced and compared with homologous regions of A. tumefaciens C58 and Sinorhizobium meliloti Rm1021 genomes. Gene order and homology are conserved between the species. A site-directed mutation in a putative gene that resides downstream of avhR and that has homology to genes belonging to the ATP-binding cassette transporter family did not affect HR or necrosis phenotypes. It was determined that avhR and aviR are expressed independently and that neither regulates the expression of a clpA homolog in F2/5.

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