Ecbl and EcbR: homologs of Luxl and LuxR affecting antibiotic and exoenzyme production byErwinia carotovorasubsp.betavasculorum

1997 ◽  
Vol 43 (12) ◽  
pp. 1164-1171 ◽  
Author(s):  
José M. Costa ◽  
Joyce E. Loper
Keyword(s):  
Quorum Sensing ◽  
Antibiotic Production ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Protease Production ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
E Coli ◽  
Acylhomoserine Lactone ◽  
Lyase Activity

Erwinia carotovora subsp. betavasculorum Ecb168 causes vascular necrosis and root rot of sugar beet and produces an antibiotic(s) that is antagonistic against other Erwinia spp. EcbI−mutants of Ecb168, each containing a single transposon insertion in the ecbI gene (for Erwinia carotovora subsp. betavasculorum inducer), do not produce detectable levels of extracellular protease or antibiotic(s), and express less pectate lyase activity and virulence than the wild-type strain. A plasmid containing the cloned ecbI gene complemented the EcbI−mutants for these phenotypes. Protease production by EcbI−mutants grown on agar surfaces was restored by neighboring cells of Escherichia coli containing ecbI. Production of a diffusible N-acylhomoserine lactone autoinducer by wild-type Ecb168 was detected with indicator strains of E. coli and Agrobacterium tumefaciens. EcbI−mutant strains did not produce an autoinducer detected by the indicator strains. Antibiotic production by EcbI−mutants was restored by cell-free culture supernatants of Ecb168 or E. coli containing a cloned ecbI gene. The predicted amino acid sequence of EcbI is similar to those of CarI, ExpI, and HslI, three LuxI homologs required for production of a diffusible N-acylhomoserine lactone autoinducer in Erwinia carotovora subsp. carotovora. A luxR homolog, termed ecbR (for Erwinia carotovora subsp. betavasculorum regulator), is convergently transcribed and overlaps with ecbI by 17 bp at their 3′ ends. These results are consistent with the hypothesis that a quorum-sensing system related to the prototypic luxI–luxR gene pair controls antibiotic and exoenzyme production in Erwinia carotovora subsp. betavasculorum.Key words: quorum sensing, β-lactam, gene regulation.

scholarly journals RelA-Dependent (p)ppGpp Production Controls Exoenzyme Synthesis in Erwinia carotovora subsp. atroseptica

2007 ◽  
Vol 189 (21) ◽  
pp. 7643-7652 ◽  
Author(s):  
Jinhong Wang ◽  
Noemie Gardiol ◽  
Tom Burr ◽  
George P. C. Salmond ◽  
Martin Welch
Keyword(s):  
Quorum Sensing ◽  
Nutrient Limitation ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Competitive Inhibitor ◽  
Pcr Analysis ◽  
Wild Type ◽  
Signal Molecule ◽  
Potent Inducer

ABSTRACT In this report, we investigate the link between nutrient limitation, RelA-mediated (p)ppGpp production, and virulence in the phytopathogen Erwinia carotovora subsp. atroseptica. A relA null mutant (JWC7) was constructed by allelic exchange, and we confirmed that, unlike the wild-type progenitor, this mutant did not produce elevated levels of (p)ppGpp upon nutrient downshift. However, (p)ppGpp production could be restored in strain JWC7 during nutrient limitation by supplying relA in trans. During growth on exoenzyme-inducing minimal medium, the relA mutant showed a diminution in secreted pectate lyase and protease activities and a severe defect in motility. The relA mutant was also impaired in its ability to cause rot in potato tubers. In the presence of serine hydroxamate (a competitive inhibitor of seryl tRNA synthase and a potent inducer of the stringent response in wild-type E. carotovora subsp. atroseptica), exoenzyme production was essentially abolished in JWC7 but could be restored in the presence of plasmid-borne relA. The inhibition of exoenzyme production in JWC7 caused by serine hydroxamate could not be overcome by addition of the quorum-sensing signal molecule, N-3-oxohexanoyl-l-homoserine lactone. Quantitative reverse transcription-PCR analysis of selected RNA species confirmed that the effects of relA on secreted pectate lyase activity and motility could be attributed to a reduction in transcription of the corresponding genes. We conclude that nutrient limitation is a potent environmental cue that triggers (p)ppGpp-dependent exoenzyme production in E. carotovora subsp. atroseptica. Furthermore, our data suggest that nutrient limitation [or rather, (p)ppGpp accumulation] is a prerequisite for effective quorum-sensing-dependent activation of exoenzyme production.

scholarly journals Secretion of Anti-Plasmodium Effector Proteins from a Natural Pantoea agglomerans Isolate by Using PelB and HlyA Secretion Signals

2011 ◽  
Vol 77 (13) ◽  
pp. 4669-4675 ◽  
Author(s):  
Dawn C. Bisi ◽  
David J. Lampe
Keyword(s):  
Control Strategies ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Individual Characteristics ◽  
Pantoea Agglomerans ◽  
Effector Proteins ◽  
World Health ◽  
Content Type ◽  
E Coli ◽  
The Individual

ABSTRACTThe insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodiumspp.) in the mosquito host. We hypothesized thatPantoea agglomerans, a bacterial symbiont ofAnophelesmosquitoes, could be engineered to express and secrete anti-Plasmodiumeffector proteins, a strategy termed paratransgenesis. To this end, plasmids that include thepelBorhlyAsecretion signals from the genes of related species (pectate lyase fromErwinia carotovoraand hemolysin A fromEscherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodiumeffector proteins (SM1, anti-Pbs21, and PLA2) inP. agglomeransandE. coli.P. agglomeranssuccessfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodiumactivity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Transgenic Plants Producing the Bacterial Pheromone N-Acyl-Homoserine Lactone Exhibit Enhanced Resistance to the Bacterial Phytopathogen Erwinia carotovora

2001 ◽  
Vol 14 (9) ◽  
pp. 1035-1042 ◽  
Author(s):  
Andres Mäe ◽  
Marcos Montesano ◽  
Viia Koiv ◽  
E. Tapio Palva
Keyword(s):  
Quorum Sensing ◽  
Transgenic Tobacco ◽  
Plant Cell Wall ◽  
Ectopic Expression ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Defense Responses ◽  
Dependent Manner ◽  
Wild Type ◽  
Enhanced Resistance

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

scholarly journals Regulation of Motility in Erwinia carotovora subsp. carotovora: Quorum-Sensing Signal Controls FlhDC, the Global Regulator of Flagellar and Exoprotein Genes, by Modulating the Production of RsmA, an RNA-Binding Protein

2010 ◽  
Vol 23 (10) ◽  
pp. 1316-1323 ◽  
Author(s):  
Asita Chatterjee ◽  
Yaya Cui ◽  
Pranjib Chakrabarty ◽  
Arun K. Chatterjee
Keyword(s):  
Quorum Sensing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Erwinia Carotovora ◽  
Translational Repression ◽  
Global Regulator ◽  
Acylhomoserine Lactone ◽  
Σ Factor ◽  
Regulatory Effects

Erwinia carotovora subsp. carotovora causes soft-rotting (tissue-macerating) disease in many plants and plant organs. Although pectinases are the primary determinants of virulence, several ancillary factors that augment bacterial virulence have also been identified. One such factor is bacterial motility. Flagellum formation and bacterial movement are regulated in many enterobacteria, including E. carotovora subsp. carotovora, by FlhDC, the master regulator of flagellar genes and FliA, a flagellum-specific σ factor. We document here that motility of E. carotovora subsp. carotovora is positively regulated by the quorum-sensing signal, N-acylhomoserine lactone (AHL), and negatively regulated by RsmA, a post-transcriptional regulator. RsmA, an RNA-binding protein, causes translational repression and promotes RNA decay. Our data show that RsmA negatively regulates flhDC and fliA expression. Moreover, the chemical stabilities of transcripts of these genes are greater in an RsmA– mutant than in RsmA+ bacteria. These observations contrast with positive regulation of flhDC and motility by CsrA (= RsmA) in Escherichia coli. In the absence of AHL, the AHL receptors ExpR1/ExpR2 (= AhlR) in Erwinia carotovora subsp. carotovora negatively regulate motility and expression of flhDC and fliA by activating RsmA production. In the presence of AHL, regulatory effects of ExpR1/ExpR2 are neutralized, resulting in reduced levels of rsmA expression and enhanced motility.

scholarly journals Role of Quorum Sensing and Antimicrobial Component Production by Serratia plymuthica in Formation of Biofilms, Including Mixed Biofilms with Escherichia coli

2006 ◽  
Vol 72 (11) ◽  
pp. 7294-7300 ◽  
Author(s):  
Pieter Moons ◽  
Rob Van Houdt ◽  
Abram Aertsen ◽  
Kristof Vanoirbeek ◽  
Yves Engelborghs ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Broth Culture ◽  
Confocal Laser ◽  
Wild Type ◽  
Serratia Plymuthica ◽  
E Coli

ABSTRACT We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.

scholarly journals Elevated Temperature Enhances Virulence of Erwinia carotovora subsp. carotovora Strain EC153 to Plants and Stimulates Production of the Quorum Sensing Signal, N-Acyl Homoserine Lactone, and Extracellular Proteins

2005 ◽  
Vol 71 (8) ◽  
pp. 4655-4663 ◽  
Author(s):  
H. Hasegawa ◽  
A. Chatterjee ◽  
Y. Cui ◽  
A. K. Chatterjee
Keyword(s):  
Quorum Sensing ◽  
Extracellular Enzymes ◽  
Rna Binding ◽  
Elevated Temperatures ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Extracellular Proteins ◽  
Acyl Homoserine Lactone ◽  
Tissue Maceration

ABSTRACT Erwinia carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora produce high levels of extracellular enzymes, such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt), and the quorum-sensing signal N-acyl-homoserine lactone (AHL) at 28°C. However, the production of these enzymes and AHL by these bacteria is severely inhibited during growth at elevated temperatures (31.2°C for E. carotovora subsp. atroseptica and 34.5°C for E. carotovora subsp. betavasculorum and most E. carotovora subsp. carotovora strains). At elevated temperatures these bacteria produce high levels of RsmA, an RNA binding protein that promotes RNA decay. E. carotovora subsp. carotovora strain EC153 is an exception in that it produces higher levels of Pel, Peh, Cel, and Prt at 34.5°C than at 28°C. EC153 also causes extensive maceration of celery petioles and Chinese cabbage leaves at 34.5°C, which correlates with a higher growth rate and higher levels of rRNA and AHL. The lack of pectinase production by E. carotovora subsp. carotovora strain Ecc71 at 34.5°C limits the growth of this organism in plant tissues and consequently impairs its ability to cause tissue maceration. Comparative studies with ahlI (the gene encoding a putative AHL synthase), pel-1, and peh-1 transcripts documented that at 34.5°C the RNAs are more stable in EC153 than in Ecc71. Our data reveal that overall metabolic activity, AHL levels, and mRNA stability are responsible for the higher levels of extracellular protein production and the enhanced virulence of EC153 at 34.5°C compared to 28°C.

scholarly journals HrpW of Erwinia amylovora, a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

1998 ◽  
Vol 180 (19) ◽  
pp. 5203-5210 ◽  
Author(s):  
Jihyun F. Kim ◽  
Steven V. Beer
Keyword(s):  
Plant Cell Wall ◽  
Erwinia Amylovora ◽  
Pectate Lyase ◽  
Hypersensitive Reaction ◽  
Host Tissue ◽  
Wild Type ◽  
Pectate Lyases ◽  
Heat Stable ◽  
Lyase Activity ◽  
Type Strains

ABSTRACT Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream ofhrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwiniaspecies, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.

scholarly journals RpoS (Sigma-S) Controls Expression of rsmA, a Global Regulator of Secondary Metabolites, Harpin, and Extracellular Proteins in Erwinia carotovora

1998 ◽  
Vol 180 (14) ◽  
pp. 3629-3634 ◽  
Author(s):  
Asita Mukherjee ◽  
Yaya Cui ◽  
Weilei Ma ◽  
Yang Liu ◽  
Akira Ishihama ◽  
...  
Keyword(s):  
Sigma Factor ◽  
Parent Strain ◽  
Pectate Lyase ◽  
Extracellular Enzyme ◽  
Erwinia Carotovora ◽  
Hypersensitive Reaction ◽  
Extracellular Proteins ◽  
Global Regulator ◽  
E Coli ◽  
Multiple Copies

ABSTRACT RpoS (sigma-S or sigma-38) controls a large array of genes that are expressed during stationary phase and under various stress conditions in Escherichia coli and other bacteria. We document here that plant pathogenic and epiphytic Erwinia species, such as E. amylovora; E. carotovora subsp.atroseptica, betavasculorum, andcarotovora; E. chrysanthemi;E. herbicola; E. rhapontici; andE. stewartii, possess rpoS genes and produce the alternate sigma factor. We show that rpoStranscription in E. carotovora subsp.carotovora is driven from a major promoter which resides within the nlpD gene located upstream ofrpoS as in E. coli. RpoS− E. carotovora subsp. carotovoa strain AC5061, constructed by marker exchange, is more sensitive to hydrogen peroxide, carbon starvation, and acidic pH than its RpoS+ parent strain, AC5006. The basal levels of extracellular pectate lyase, polygalacturonase, and cellulase as well as those of transcripts of E. carotovora subsp. carotovora hrpN(hrpNEcc ), the gene for the elicitor of the hypersensitive reaction, are higher in the RpoS−strain than in the RpoS+ parent. Likewise, compared to AC5006, AC5061 causes more extensive maceration of celery petioles. Our findings with the RpoS− mutant and strains carrying multiple copies rpoS + DNA reveal thatrpoS positively controls rsmA expression. We also present evidence that supports the hypothesis that the RpoS effect on extracellular enzyme levels, hrpNEcc expression, and virulence manifests itself by the modulation ofrsmA expression.

scholarly journals Quorum-Sensing-Negative (lasR) Mutants of Pseudomonas aeruginosa Avoid Cell Lysis and Death

2005 ◽  
Vol 187 (14) ◽  
pp. 4875-4883 ◽  
Author(s):  
Karin Heurlier ◽  
Valérie Dénervaud ◽  
Marisa Haenni ◽  
Lionel Guy ◽  
Viji Krishnapillai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Point Mutations ◽  
Critical Factor ◽  
Cell Lysis ◽  
Selective Advantage ◽  
Alkaline Stress ◽  
Wild Type ◽  
Acylhomoserine Lactone ◽  
Nucleoside Hydrolase

ABSTRACT In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37°C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to ≥ 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh− phenotype, and five of these mutants, which were functionally complemented by lasR + , had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

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