Ecc Strain Research Articles

Regulation of corA, the Magnesium, Nickel, Cobalt Transporter, and Its Role in the Virulence of the Soft Rot Pathogen, Pectobacterium versatile Strain Ecc71.

Pectobacterium versatile (formally P. carotovorum) causes disease on diverse plant species by synthesizing and secreting copious amount of plant-cell-wall-degrading exoenzymes including pectate lyases, polygalacturonases, cellulases, and proteases. Exoenzyme production and virulence are controlled by many factors of bacterial, host, and environmental origin. The ion channel forming the magnesium, nickel, and cobalt transporter CorA is required for exoenzyme production and full virulence in strain Ecc71. We investigated CorA's role as a virulence factor and its expression in P. versatile. Inhibiting the transport function of CorA by growing a CorA+ strain in the presence of specific CorA inhibitor, cobalt (III) hexaammine (Co (III)Hex), has no effect on exoenzyme production. Transcription of pel-1, encoding a pectate lyase isozyme, is decreased in the absence of CorA, suggesting that CorA influences exoenzyme production at the transcriptional level, although apparently not through its transport function. CorA- and CorA+ strains grown in the presence of Co (III)Hex transcriptionally express corA at higher levels than CorA+ strains in the absence of an inhibitor, suggesting the transport role of corA contributes to autorepression. The expression of corA is about four-fold lower in HrpL- strains lacking the hrp-specific extracytoplasmic sigma factor. The corA promoter region contains a sequence with a high similarity to the consensus Hrp box, suggesting that corA is part of Hrp regulon. Our data suggest a complex role, possibly requiring the physical presence of the CorA protein in the virulence of the Pectobacterium versatile strain Ecc71.

Integrated assessment of left ventricular electrical activation and myocardial strain mapping in heart failure patients

Objectives This study sought to test the accuracy of strain measurements based on anatomo-electromechanical mapping (AEMM) measurements compared with magnetic resonance imaging (MRI) tagging, to evaluate the diagnostic value of AEMM-based strain measurements in the assessment of myocardial viability, and the additional value of AEMM over peak-to-peak local voltages. Background The in vivo identification of viable tissue, evaluation of mechanical contraction, and simultaneous left ventricular activation is currently achieved using multiple complementary techniques. Methods In 33 patients, AEMM maps (NOGA XP, Biologic Delivery Systems, Division of Biosense Webster, a Johnson & Johnson Company, Irwindale, California) and MRI images (Siemens 3T, Siemens Healthcare, Erlangen, Germany) were obtained within 1 month. MRI tagging was used to determine circumferential strain (Ecc) and delayed enhancement to obtain local scar extent (%). Custom software was used to measure Ecc and local area strain (LAS) from the motion field of the AEMM catheter tip. Results Intertechnique agreement for Ecc was good (R2 = 0.80), with nonsignificant bias (0.01 strain units) and narrow limits of agreement (−0.03 to 0.06). Scar segments showed lower absolute strain amplitudes compared with nonscar segments: Ecc (median [first to third quartile]: nonscar −0.10 [−0.15 to −0.06] vs. scar −0.04 [−0.06 to −0.02]) and LAS (−0.20 [−0.27 to −0.14] vs. −0.09 [−0.14 to −0.06]). AEMM strains accurately discriminated between scar and nonscar segments, in particular LAS (area under the curve: 0.84, accuracy = 0.76), which was superior to peak-to-peak voltages (nonscar 9.5 [6.5 to 13.3] mV vs. scar 5.6 [3.4 to 8.3] mV; area under the curve: 0.75). Combination of LAS and peak-to-peak voltages resulted in 86% accuracy. Conclusions An integrated AEMM approach can accurately determine local deformation and correlates with the scar extent. This approach has potential immediate application in the diagnosis, delivery of intracardiac therapies, and their intraprocedural evaluation.

Phenomenological interpretation of the shear behavior of reinforced Engineered Cementitious Composite beams

This paper describes an experimental investigation of the shear behavior of beams consisting of steel Reinforced Engineered Cementitious Composites (R/ECC). This study investigates and quantifies the effect of ECC's strain hardening and multiple cracking behavior on the shear capacity of beams loaded in shear. The experimental program consists of R/ECC beams with short (8 mm) randomly distributed Polyvinyl Alcohol (PVA) fiber and conventional Reinforced Concrete (R/C) counterparts for comparison with varying shear reinforcement arrangements. Beams were loaded until failure while a Digital Image Correlation (DIC) measurement technique was used to measure surface displacements and crack formation. The shear crack mechanisms of R/ECC are described in detail based on findings of DIC measurements and can be characterized by an opening and sliding of the cracks. Multiple micro-cracks developed in a diagonal arrangement between the load and support points due to the strain-hardening response of ECC in tension. The strain-hardening response strongly influenced the shear response of the beam specimen.

9 Sex differences in circumferential and longitudinal strain assessed using strain encoded cardiac magnetic resonance at 3.0T: Abstract 9 Table 1

<h3>Background</h3> Displacement encoding with stimulated echoes (DENSE) encodes myocardial tissue displacement into the phase of the MRI image, thus allowing direct quantification of myocardial displacement at multiple cardiac phases. Strain-encoded CMR with DENSE has high spatial (3.2 mm × 3.2 mm × 8 mm) and temporal resolution (TR= 27.34 ms). We aimed to measure myocardial strain values with DENSE in healthy adults across a broad age range at 3.0 Tesla. <h3>Methods</h3> Healthy volunteers with no prior medical history or treatment were enrolled and underwent CMR at 3.0T (Magnetom Verio, Siemens healthcare, Erlangen, Germany). Mid-left ventricular short axis and horizontal long axis DENSE sequences were acquired and analysed using CIM_DENSE2D software (University of Auckland, New Zealand and Siemens Healthcare). Segmental and global myocardial circumferential (Ecc) and longitudinal (Ell) strain were obtained. <h3>Results</h3> Ecc and Ell were analysed in 77 volunteers (mean age 45 ± 18 years, 49% male) (Table 1). Mean Ecc strain was greater in women than in men. These sex-differences were mainly related to strain values in the anterior and antero-lateral LV segments. There is no difference for Ell. <h3>Conclusion</h3> Left ventricular circumferential contractility differs between men and women, and the differences were regionalised to the antero-lateral myocardial regions where LV displacement is greatest. <h3>Funding</h3> Medical Research Scotland project grant. Professor Berry was supported by a Senior Clinical Fellowship from the Scottish Funding Council.

Prospective comparison of circumferential and longitudinal strain in asymptomatic children with single left ventricle, single right ventricle and normal hearts

Background Ventricular dysfunction in patients with a single right ventricle (SRV) or as ingle left ventricle (SLV) is a known risk factor for morbidity and mortality. However, the differences in SRV and SLV function remain poorly understood, with only a few studies performed1-3. In this study, we measure the strain using cardiac MRI and perform comprehensive comparison of the global and regional strain in both the circumferential (ecc) and longitudinal (eL) dimension. Purpose: In normal subjects and asymptomatic patients with SLV and SRV after total cavopulmonary connection (TCPC), compare: 1) Global ecc and eL strain, 2) Regional circumferential and longitudinal strains at free wall (ecc-free, eL-free) and septum (ecc-sept, eL-sept), 3) ecc and eL across the ventricle from apex to base. Methods We performed a prospective analysis of 18 subjects (7 normal age: 11.8 +/- 3; 6 SRV age: 11.4 +/- 2.3; 5 SLV age: 12.7 +/- 4.2). Acquisition Protocol: Strain information was acquired at three short axis slices at basal, mid-cavity, and apical locations in all 18 subjects in a 1.5T MRI scanner (Philips Acheiva) using: a) Complementary Spatial Modulation of Magnetization (CSPAMM)4 images: Used for generating ecc; and b) Fast-Strain Encoded (fSENC)5 images: Used for generating eL. Data Analysis: ecc and eL across all cardiac phases and slices were calculated from SAX slices using DiagnosoftTM. The ventricular regions at each slice were assigned based upon the AHA 16 segment model. ecc-sept, eL-sept, ecc-free, and eL-free were also calculated for each slice and compared. Results

Salvage assessment with cardiac MRI following acute myocardial infarction underestimates potential for recovery of systolic strain

Our aim was to evaluate the relationship between the degree of salvage following acute ST elevation myocardial infarction (STEMI) and subsequent reversible contractile dysfunction using cardiac magnetic resonance (CMR) imaging. Thirty-four patients underwent CMR examination 1-7 days after primary percutaneous coronary intervention (PPCI) for acute STEMI with follow-up at 1 year. The ischaemic area-at-risk (AAR) was assessed with T2-weighted imaging and myocardial necrosis with late gadolinium enhancement. Myocardial strain was quantified with complementary spatial modulation of magnetisation (CSPAMM) tagging. Ischaemic segments with poor (<25 %) or intermediate (26-50 %) salvage index were associated with worse Eulerian circumferential (Ecc) strain immediately post-PPCI (-9.1 % ± 0.6, P = 0.033 and -11.8 % ± 1.3, P = 0.003, respectively) than those with a high (51-100 %) salvage index (-14.4 % ± 1.3). Mean strain in ischaemic myocardium improved between baseline and follow-up (-10.1 % ± 0.5 vs. -16.2 % ± 0.5 %, P < 0.0001). Segments with poor salvage also showed an improvement in strain by 1 year (-9.1 % ± 0.6 vs. -15.3 % ± 0.6, P = 0.033) although they remained the most functionally impaired. Partial recovery of peak systolic strain following PPCI is observed even when apparent salvage is less than 25 %. Late gadolinium enhancement (LGE) may not equate to irreversibly injured myocardium and salvage assessment performed within the first week of revascularisation may underestimate the potential for functional recovery. • MRI can measure how much myocardium is damaged after a heart attack. • Heart muscle that appears initially non-viable may sometimes partially recover. • Enhancement around the edges of infarcts may resolve over time. • Evaluating new cardio-protective treatments with MRI requires appreciation of its limitations.

HIV infection and abnormal regional ventricular function

To examine the effect of HIV infection on regional left ventricular dysfunction in cardiovascularly asymptomatic individuals. Nineteen HIV-negative and 27 HIV-positive cardiovascularly asymptomatic study participants in Baltimore, Maryland were selected and underwent tagged cardiac magnetic resonance imaging. Regional left ventricular myocardial mid-wall peak systolic circumferential strain (Ecc) and early diastolic strain rate (SRE) of the left ventricle were assessed with the use of the harmonic phase analysis. The average Ecc and SRE measurements were compared between HIV-negative and HIV-positive individuals. Compared with the HIV-negatives, the HIV-positives had lower average Ecc and SRE measurements in 90% of the 16 standard left ventricular segments. Of the 14 segments with decreased Ecc strain, 3 were statistically significant and of 14 with decreased strain rate (SRE), 6 were statistically significant. HIV infection may be associated with subclinical regional left ventricular systolic and diastolic dysfunction in individuals free of overt cardiovascular disease.

Subclinical Atherosclerosis and Incipient Regional Myocardial Dysfunction in Asymptomatic Individuals: The Multi-Ethnic Study of Atherosclerosis (MESA)

This study sought to determine whether increased carotid intima-media thickness (IMT) is related to reduced regional myocardial function in participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Carotid artery IMT is an established index of subclinical atherosclerosis, and tagged magnetic resonance imaging (MRI) can detect incipient alterations of segmental function that precede overt myocardial failure. The MESA study is a prospective observational study including four ethnic groups free from clinical cardiovascular disease. Peak midwall systolic circumferential strain (ECC) and regional strain rates were calculated by harmonic phase from tagged MRI data of 500 participants. Systolic ECC and diastolic strain rate were regressed on IMT of the common carotid artery defined by ultrasound, with adjustments for body mass index, blood pressure, cholesterol, diabetes, smoking, left ventricular hypertrophy, C-reactive protein, age, and gender. The mean participant age was 66 +/- 10 years (mean +/- SD). Among the 58 participants, 4% were male and the interquartile (25th to 75th percentile) range for IMT was 0.25 mm. Multiple linear regression analyses showed that increased IMT was related to reduced systolic regional function (less shortening ECC) in all myocardial regions (p < 0.05), except in the inferior wall. The analyses also showed that greater IMT was associated with a lower diastolic strain rate (diastolic reduced function) in all regions (p < 0.01), except in the anterior wall. Greater carotid IMT is associated with alterations of myocardial strain parameters reflecting reduced systolic and diastolic myocardial function. These observations indicate a relationship between subclinical atherosclerosis and incipient myocardial dysfunction in a population free of clinical heart disease.

Cooperation of two distinct ExpR regulators controls quorum sensing specificity and virulence in the plant pathogen Erwinia carotovora

Quorum sensing, the population density-dependent regulation mediated by N-acylhomoserine lactones (AHSL), is essential for the control of virulence in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc). In Erwinia carotovora ssp. the AHSL signal with an acyl chain of either 6 or 8 carbons is generated by an AHSL synthase, the expI gene product. This work demonstrates that the AHSL receptor, ExpR1, of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. We have also identified a second AHSL receptor (ExpR2) and demonstrate a novel quorum sensing mechanism, where ExpR2 acts synergistically with the previously described ExpR1 to repress virulence gene expression in Ecc. We show that this repression is released by addition of AHSLs and appears to be largely mediated via the negative regulator RsmA. Additionally we show that ExpR2 has the novel property to sense AHSLs with different acyl chain lengths. The expI expR1 double mutant is able to act in response to a number of different AHSLs, while the expI expR2 double mutant can only respond to the cognate signal of Ecc strain SCC3193. These results suggest that Ecc is able to react both to the cognate AHSL signal and the signals produced by other bacterial species.

Identification of regulators of hrp/hop genes of Erwinia carotovora ssp. carotovora and characterization of HrpLEcc (SigmaLEcc), an alternative sigma factor

Summary Erwinia carotovora ssp. carotovora (Ecc) possesses hrpN(Ecc)[hrp = gene for hypersensitive reaction (HR) and pathogenicity], the structural gene for Harpin(Ecc), the inducer of the HR-like response and genes for the type III secretion system. In Ecc, RsmA, an RNA-binding protein responsible for the accelerated decay of RNA species, tightly controls the expression of the Hrp regulon. We document here that Ecc strain 71 possesses several presumed Hrp regulators: HrpX(Ecc), HrpY(Ecc), HrpS(Ecc), and HrpL(Ecc). Nucleotide sequence data indicate that the regulatory genes occur as a cluster. The data also suggest that HrpX(Ecc) is a putative sensor kinase, HrpY(Ecc) is the cognate response regulator, and HrpS(Ecc) is an NtrC-like regulator. hrpL(Ecc) consists of a 543-bp open reading frame (ORF), which encodes a c. 21-kDa protein product. This protein shares significant homology with members of the extracytoplasmic function (ECF) subfamily of alternative sigma factors, including SigE. To examine the regulatory role of hrpL(Ecc), we constructed by marker exchange an HrpL(Ecc) (-) derivative of an RsmA(-) strain. The levels of transcripts of hrcC(Ecc), a gene for the type III secretion system, and hrpN(Ecc) were lower in the RsmA(-)HrpL(Ecc) (-) mutant compared to its RsmA(-)HrpL(Ecc) (+) parent. The RsmA(-)HrpL(Ecc) (-) strain, like the RsmA(-)HrpL(Ecc) (+) parent, caused maceration of celery petioles and produced extracellular pectinases, cellulase, and protease, indicating that the alternative sigma factor is not required for the type I and type II secretion systems, for tissue maceration, or for the production of proteins secreted by these pathways. However, the RsmA(-)HrpL(Ecc) (-) strain, unlike the RsmA(-)HrpL(Ecc) (+) strain, did not elicit the HR in tobacco leaves. In addition, the RsmA(-)HrpL(-) strain failed to cause symptoms in Arabidopsis thaliana. Genetic and biochemical data revealed that Harpin(Ecc) is required for symptom production in Arabidopsis, but is not sufficient by itself to cause necrosis. These observations raise the possibility that another 'effector' protein of Ecc, secreted by the type III system, acts in conjunction with Harpin(Ecc) to trigger responses resulting in cell death in Arabidopsis.

Effects of the two-component system comprising GacA and GacS of Erwinia carotovora subsp. carotovora on the production of global regulatory rsmB RNA, extracellular enzymes, and harpinEcc.

Posttranscriptional regulation mediated by the regulator of secondary metabolites (RSM) RsmA-rsmB pair is the most important factor in the expression of genes for extracellular enzymes and HarpinEcc in Erwinia carotovora subsp. carotovora. RsmA is a small RNA-binding protein, which acts by lowering the half-life of a mRNA species. rsmB specifies an untranslated regulatory RNA and neutralizes the RsmA effect. It has been speculated that GacA-GacS, members of a two-component system, may affect gene expression via RsmA. Because expA, a gacA homolog, and expS (or rpfA), a gacS homolog, have been identified in E. carotovora subsp. carotovora, we examined the effects of these gacA and gacS homologs on the expression of rsmA, rsmB, and an assortment of exoprotein genes. The gacA gene of E. carotovora subsp. carotovora strain 71 stimulated transcription of genes for several extracellular enzymes (pel-1, a pectate lyase gene; peh-1, a polygalacturonase gene; and celV, a cellulase gene), hrpNEcc (an E. carotovora subsp. carotovora gene specifying the elicitor of hypersensitive reaction), and rsmB in GacA+ and GacS+ E. carotovora subsp. carotovora strains. Similarly, the E. carotovora subsp. carotovora gacA gene stimulated csrB (rsmB) transcription in Escherichia coli. A GacS- mutant of E. carotovora subsp. carotovora strain AH2 and a GacA- mutant of E. carotovora subsp. carotovora strain Ecc71 compared with their parent strains produced very low levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts but produced similar levels of rsmA RNA and RsmA protein as well as transcripts of hyperproduction of extracellular enzymes (Hex) hexA, kdgR (repressor of genes for uronate and pectate catabolism), rsmC, and rpoS (gene for Sigma-S, an alternate Sigma factor). The levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts as well as production of pectate lyase, polygalacturonase, cellulase, protease, and HarpinEcc proteins were stimulated in GacS- and GacA- mutants by GacS+ or GacA+ plasmids, respectively. The GacA effect on exoenzyme genes and hrpNEcc was abrogated in E. carotovora subsp. carotovora mutants deficient in RsmA and RsmC or RsmA, RsmC, and rsmB RNA. The expression of lacZ transcriptional fusions of rsmB of Erwinia amylovora and Erwinia herbicola pv. gypsophilae was markedly reduced in a GacA- and a GacS- mutant of Pseudomonas syringae pv. syringae. Southern blot hybridization revealed the presence of gacA and gacS homologs in all tested strains of soft-rotting Erwinia spp. and several nonsoft-rotting Erwinia species such as E. amylovora, E. rhapontici, E. herbicola, E. stewartii, and E. herbicola pv. gypsophilae. These findings establish that the GacA-GacS system controls transcription of rsmB of E. carotovora subsp. carotovora, E. amylovora, and E. herbicola pv. gypsophilae and support the hypothesis that the effects of this two-component system on extracellular protein production in E. carotovora subsp. carotovora is mediated, at least in part, via the levels of rsmB transcripts.

Quorum sensing controls the synthesis of virulence factors by modulating rsmA gene expression in Erwinia carotovora subsp. carotovora.

The plant-pathogenic bacterium Erwinia carotovora subsp. carotovora (Ecc) causes disease mainly by means of a number of extracellular plant cell wall-degrading enzymes (PCWDEs), also referred to as virulence factors. The production of PCWDEs is coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone (HSL) in a population density-dependent manner ("quorum sensing"). ExpI is the enzyme responsible for the synthesis of HSL. The Rsm system negatively regulates the production of PCWDEs. It includes three components: RsmA is an RNA-binding protein which promotes mRNA decay; rsmB is a unique regulator RNA, and RsmC regulates expression of rsmA positively and of rsmB negatively. We report here that in an expI knockout mutant of Ecc strain SCC3193, the levels of rsmA and rsmB RNA are remarkably enhanced in comparison to the wild-type strain, while the level of the rsmC transcript is not affected. The increase in transcription of rsmA in the expI strain represses production of PCWDEs, which in turn leads to the avirulent phenotype of this mutant. In the expI- mutant, addition of exogenous HSL caused repression of rsmA and rsmB transcription to the wild-type level, whereas the expression of rsmC was not affected. Taken together, these data suggest that HSL affects the expression of rsmA, and that this effect is not mediated by RsmC. This specific effect and the previous demonstration that HSL is required for PCWDE production in Ecc support the hypothesis that regulation by quorum sensing in Ecc, in contrast to most other systems already described, requires HSL to repress rsmA transcription, which in turn leads to the activation of PCWDE production. A model is presented that explains how HSL controls the production of PCWDEs by modulating the expression of rsmA.

Cryptic carbapenem antibiotic production genes are widespread in Erwinia carotovora: facile trans activation by the carR transcriptional regulator.

Few strains of Erwinia carotovora subsp. carotovora (Ecc) make carbapenem antibiotics. Strain GS101 makes the basic carbapenem molecule, 1-carbapen-2-em-3-carboxylic acid (Car). The production of this antibiotic has been shown to be cell density dependent, requiring the accumulation of the small diffusible molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) in the growth medium. When the concentration of this inducer rises above a threshold level, OHHL is proposed to interact with the transcriptional activator of the carbapenem cluster (CarR) and induce carbapenem biosynthesis. The introduction of the GS101 carR gene into an Ecc strain (SCRI 193) which is naturally carbapenem-negative resulted in the production of Car. This suggested that strain SCRI 193 contained functional cryptic carbapenem biosynthetic genes, but lacked a functional carR homologue. The distribution of trans-activatable antibiotic genes was assayed in Erwinia strains from a culture collection and was found to be common in a large proportion of Ecc strains. Significantly, amongst the Ecc strains identified, a larger proportion contained trans-activatable cryptic genes than produced antibiotics constitutively. Southern hybridization of the chromosomal DNA of cryptic Ecc strains confirmed the presence of both the car biosynthetic cluster and the regulatory genes. Identification of homologues of the transcriptional activator carR suggests that the cause of the silencing of the carbapenem biosynthetic cluster in these strains is not the deletion of carR. In an attempt to identify the cause of the silencing in the Ecc strain SCRI 193 the carR homologue from this strain was cloned and sequenced. The SCRI 193 CarR homologue was 94% identical to the GS101 CarR and contained 14 amino acid substitutions. Both homologues could be expressed from their native promoters and ribosome-binding sites using an in vitro prokaryotic transcription and translation assay, and when the SCRI 193 carR homologue was cloned in multicopy plasmids and reintroduced into SCRI 193, antibiotic production was observed. This suggested that the mutation causing the silencing of the biosynthetic cluster in SCRI 193 was leaky and the cryptic Car phenotype could be suppressed by multiple copies of the apparently mutant transcriptional activator.

Genetic and physiological evidence for the production of N-acyl homoserine lactones by Pseudomonas syringae pv. syringae and other fluorescent plant pathogenic Pseudomonas species

N-acyl homoserine lactones (AHLs) function as cell density (quorum) sensing signals and regulate diverse metabolic processes in several gram negative bacteria. We report that strains of Pseudomonas syringae pvs. syringae (Pss), tabaci and tomato as well as P. corrugata and P. savastanoi produce difussible AHLs that activate the lux operons of Vibrio fischeri or the tra::lacZ fusion of Agrobacterium tumefaciens. In Pss strain B3A, AHL production occurs in cell density dependent manner. Nucleotide sequence and genetic complementation data revealed the presence of ahlIPss, a luxI homolog within the Ahl+ DNA of Pss strain B3A. The \(ahlI_{Pss}^ + \) DNA expresses in AHL-deficient strains of P. fluorescens and E. carotovora subsp. carotovora (Ecc), and restores extracellular enzyme production and pathogenicity in the Ecc strain. The derivatives of Pss strains B3A and 301D carrying chromosomal ahlI::lacZ do not produce AHL, but like their wild type parents, produce extracellular protease and the phytotoxin syringomycin as well as elicit the hypersensitive reaction in tobacco leaves. While these strains also produce a basal level of β-galactosidase activity, the expression of ahlI::lacZ is substantially stimulated in the presence of multiple copies of the \(ahlI_{Pss}^ + \) DNA or by the addition of cell-free spent cultures containing AHL. The activation of β-galactosidase production occurs with spent cultures of some, but not all Pseudomonas strains which produce AHL as indicated by the Lux and tra::lacZ assays. Pss strains deficient in the global regulatory genes, gacA or lemA, produce very low levels of AHL. Since inactivation of ahlIPss eliminates AHL production and since Ahl+ Pseudomonas strains carry the homolog of ahlIPss, we conclude that ahlIPss specifies a key step in AHL biosynthesis and it has been conserved in many plant pathogenic pseudomonads.

Characterization of a novel regulatory gene aepA that controls extracellular enzyme production in the phytopathogenic bacterium Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora strain Ecc71 produces an array of extracellular enzymes including pectate lyase (Pel), polygalacturonase, cellulase, and protease. In strain Ecc71, these enzymes are coregulated by aepA, which encodes an activator of extracellular protein production (H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, and A. K. Chatterjee, Mol. Plant-Microbe Interact, 4:239-246, 1991). The nucleotide sequence of a 2.7-kb aepA+ DNA segment revealed an open reading frame (ORF) of 1,395 bp which matches with the size of the aepA transcript determined by Northern blot analysis. aepA is predicted to encode a protein of 465 amino acid residues with a molecular mass of approximately 51 kDa and a pI of 6.52. The occurrence of a putative signal sequence and several hydrophobic domains suggest membrane localization of AepA. An aepA-lacZ operon fusion was constitutively expressed in E. coli (DH5 alpha) but inducible by pectate and celery extract in E. c. subsp. carotovora (AC5006). These findings suggest that aepA expression may be negatively regulated in E. c. subsp. carotovora. By assaying for the transcript of pel-1, which species a major secreted Pel species in strain Ecc71, and by following the expression of a pel1-lacZ operon fusion we determined that AepA activates pel-1 transcription. The characteristics of aepA including the lack of homology with other prokaryotic regulatory genes indicate that aepA encodes a novel regulatory protein required for extracellular protein production. Whereas homologs of Ecc71 aepA occur in E. c. subsp. carotovora and E. c. subsp. atroseptica strains, activation of exoenzyme production is markedly stimulated by aepA in E. c. subsp. carotovora than in E. c. subsp. atroseptica.

Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production.

In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.

Potato Protoplast‐derived Callus Tissue Challenged with Erwinia carotovora subsp. carotovora: Survival, Growth and Identification of Resistant Callus Lines*

Abstract Potato (cv. Crystal) protoplast‐derived callus tissue was evaluated for survival and growth when exposed to Erwinia carotovora subsp. carotovora (strain Ecc71). Calli were either directly exposed to the pathogen by inoculation or to metabolites produced by the pathogen via a bilayer medium. Individual calli were inoculated with 0.5 μl of bacterial suspensions at 104, 105, 106, 107, 108 and 109 cfu/ml. The bilayer consistedof 10 ml of callus proliferation medium supplemented with pectin (2 g/l) and contained bacteria at 102, 103, 104, 105 and 106 cfu/ml. This medium was overlaid with 10 ml of bacteria free callus induction medium. Mean callus diameter of the inoculated treatments increased for 24 h, then declined. Over 90% of the inoculated calli were killed within 5 days but some survived as long as 14 days. Calli grown on the bilayer medium containing 106, 105 and 104 cfu/ml also decreased in size. Most were killed within 9 days but some survived 20 days. Calli exposed to 103 and 102 cfu/ml experienced limited growth with 20% and 7%, respectively, surviving after 27 days. Reactions to the pathogen varied considerably within the callus populations and individual calli with extended survival were identified in both experiments.

Serological characteristics of Erwinia carotovora isolated from Japan and Thailand.

Seventy-nine strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various soft rot vegetables in Japan and Thailand were compared in serological properties by Ouch-terlony double diffusion method. Seven antisera produced against representative strains of different Ecc biovars (B, C and D) and one antiserum against E. c. subsp. atroseptica (Eca) were used. All antigens were heat-treated before use. All Ecc strains did not react with anti-Eca-serum and all Eca strains did not react with all of the anti-Ecc-sera. None of the strains of Ecc belonging to biovar DG (typical Ecc strains) gave specific precipitin band with antisera to strains belonging to biovar B and C (intermediate strains) which had some properties of Eca. With antisera to the typical Ecc strain (N7129 of biovar D), none of the intermediate strains gave specific precipitin band. These results indicated that the intermediate strains could be serologically distinguished from both typical Ecc and Eca.

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora.

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.

Enumeration of two competing Erwinia carotovora populations in potato tubers by a membrane filter‐immunofluorescence procedure

Relative increases in cell populations of specific Erwinia carotovora strains injected into potato tubers either singly or in combination of two strains were determined by depositing tissue extracts on membrane filters and staining cells of individual strains by immunofluorescence. The population increase of an Erw. carotovora subsp. atroseptica (Eca) strain was, in general, not affected by the presence of an Erw. carotovora subsp. carotovora (Ecc) strain. However, the increase of an Ecc strain was inhibited by the presence of an Eca strain, especially at incubation temperatures of 5° and 15°C but not at 26°C. One Ecc strain consistently increased in population over another Ecc strain at a greater rate when they were inoculated together at the same loci in comparison to when they were inoculated separately at different loci.