Erwinia Species Research Articles

Erwinia alni, a New Species Causing Bark Cankers of Alder (Alnus Miller) Species

The causal agent of an undetermined disease of black alder (Alnus glutinosa) and Italian alder (Alnus cordata) was identified as an Erwinia species. This alder bacterium induces dark brown necrotic cankers on alder plants; these cankers are often longitudinally elongated and occur in the bark of the trunks and also in the bark of branches, twigs, and suckers. A dark watery liquid often exudes from small cracks in the cankers and stains the bark surface. Disease symptoms were produced on the trunks of 2-year-old black and Italian alder trees after artificial inoculation of selected strains of bacteria obtained from typical bark cankers (G. Surico and L. Mugnai, Inf. tore Fitopatol. 12:41-43, 1992). Morphological examination of the pathogen by electron microscopy showed that it is a motile rod-shaped organism. All of the strains examined were gram negative, oxidase negative, and facultatively anaerobic with fermentative metabolism and had the general characteristics and fatty acids of members of the family Enterobacteriaceae. Strains PVFi 20T (T = type strain), PVFi 23, PVFi 25, and PVFi 27 were chosen for further characterization. These four strains exhibited 96 to 100% DNA homology in hybridization experiments performed at 40 and 50°C. They were most closely related to Erwinia nigrifluens (levels of homology 40°C, 49 to 65%). Phenotypic differentiation from E. nigrifluens, which induces a similar disease on Persian walnut but is nonpathogenic on alder, is based on positive reactions by the alder strains for acetoin (Voges-Proskauer reaction), endoglucanase activity, and acid production from maltose and negative reactions for esculin hydrolysis and acid production from raffinose, melibiose, sorbitol, and inositol. The fatty acid profiles of the alder strains were quantitatively different from those of all previously validly named plant-pathogenic species belonging to the Enterobacteriaceae. On the basis of data described above, the name Erwinia alni is proposed for the new organism.

Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway.

The type II or Sec-dependent secretion system is used by diverse Gram-negative bacteria for secretion of extracellular proteins. Of the 12-15 proteins involved in secretion, the requirement for many has not been demonstrated and little is known about their functions in the secretion process. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete extra-cellular pectate lyases (Pels) using the type II or Out pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed genes from the other species, suggesting the presence of species-specific recognition factors in the Out systems of the two Erwinia species. We previously reported the isolation of a cosmid clone, pCPP2OO6, from E. chrysanthemi EC16, which enables Escherichia coil to secrete heterologously expressed E. chrysanthemi Pels. Sequencing in a region required for secretion revealed the presence of 12 genes, outC-M and outO. We report here the construction of functionally non-polar mutations in each gene in the outC-M operon and outS and outB using a polA(ts) strain of E. coli to facilitate homologous recombination between out genes carrying deletions and their wild-type copies on pCPP2006. By testing for complementation of each deletion with wild-type out genes from E. chrysanthemi EC16 and E. carotovora SCRI193 we have demonstrated that: (i) each out gene is required for secretion of E. chrysanthemi PelE from E. coli with the exception of outH; (ii) each mutation can be complemented by its homologue from E. carotovora, except for outC and outD; (iii) outC and outD from E. carotovora do not confer secretion of Pel1 on the E. chrysanthemi Out system; and (iv) Pel1 secretion can be conferred on the E. chrysanthemi Out system by the presence of outC-M, S and B from E. carotovora. The data suggest that OutC and OutD are gatekeepers of the Out system involved in recognition of Pels targeted for secretion but that OutC and OutD from E. carotovora cannot be successfully assembled into the E. chrysanthemi Out system.

Genetics and molecular biology of carotenoid pigment biosynthesis

The crucial roles of carotenoids and their metabolites in photooxidative protection and photosynthesis, not to mention nutrition, vision, and cellular differentiation, make them an important and complex class of biological pigments. Significant advances within the last few years have enhanced our understanding of the genetics and molecular biology of carotenoid biosynthesis in bacteria, fungi, algae, and plants. All of the genes involved in carotenoid biosynthesis from Rhodobacter capsulatus, an anoxygenic photosynthetic bacterium, and from several species of Erwinia, nonphotosynthetic bacteria, have been molecularly characterized. Recent studies have revealed that two early enzymes of carotenoid biosynthesis, geranylgeranyl pyrophosphate synthase and phytoene synthase, are structurally and functionally related in all carotenogenic organisms. In contrast, the subsequent conversion of phytoene, the first C(40) carotenoid, to beta-carotene requires two desaturases and one cyclase in oxygenic photosynthetic organisms (cyanobacteria, algae, and higher plants) but only one structurally distinct desaturase and a structurally distinct cyclase in other carotenogenic bacteria and in fungi. Studies of the enzymes that introduce oxygen-containing functional groups into carotenes to produce xanthophylls, the vast majority of all carotenoids, are still in their infancy. This review summarizes the most recent developments in carotenoid biosynthesis from a molecular genetic standpoint.

A Rapid and Sensitive PCR-Based Assay for Concurrent Detection of Bacteria Causing Common and Halo Blights in Bean Seed

A quick polymerase chain reaction (PCR)-based procedure was developed for concurrent detection of the causal agents of bean common blight, Xanthomonas campestris pv. phaseoli, and bean halo blight, Pseudomonas syringae pv. phaseolicola, in bean seed. A rapid DNA extraction procedure, consisting of briefly wetting intact or crushed seeds with a solution of sodium hydroxide, was used to extract bacterial DNA from externally and internally blight-contaminated seeds. G+C-rich oligonucleotide primers were designed from the phaseolotoxin gene cluster of P syringae pv. phaseolicola and tested under high-stringency conditions in PCR assays. The HB14 primers specifically directed the amplification of a 1.4-kb fragment from DNA of 19 P syringae pv. phaseolicola isolates, whereas templates from 62 other bacterial strains, including the bean pathogens P syringae pv. syringae and X. campestris pv. phaseoli, and plant-pathogenic species of Agrobacterium, Clavibacter, Erwinia, and Xanthomonas did not produce any discrete bands upon amplification. The HB14 primers for P. syringae pv. phaseolicola, and the previously reported G+C-rich X4 primers, specific for X. campestris pv. phaseoli, amplified discrete DNA fragments by soaking extracts of white bean seeds contaminated with halo and common blights, respectively. DNA extracts from blight-infested colored seeds were recalcitrant to PCR-amplification unless polyvinylpyrrolidone was added to the extraction buffer. In combination, X4 and HB14 primers successfully detected individual and mixed infections of bean common and halo blights and yielded distinctive DNA fragments from batches containing as few as 1 infected seed in 10,000 seeds.

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction

Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease‐coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato‐derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 103 culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100‐fold excess of DNA from both related and unrelated bacteria. This PCR‐based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.

Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.

A Chlorella Virus Gene Promoter Functions as a Strong Promoter Both in Plants and Bacteria

An upstream region from an algal virus methyltransferase gene was tested for promoter function in transgenic plants, electroporated monocot protoplasts and bacteria. Fusion of the 851 bp upstream region to a reporter gene significantly enhanced the reporter gene expression in transgenic Arabidopsis and potato plants and in electroporated maize and Sorghum cells relative to the cauliflower mosaic virus 35S promoter. The promoter was also functional in several Escherichia coli strains and in three species of phytopathogenic bacteria, Erwinia, Pseudomonas, and Xanthomonas. These findings indicate that the upstream region is a strong promoter uniquely functional in both eukaryotes and prokaryotes and capable of using both eukaryotic RNA polymerase II and bacterial RNA polymerases.

Nitrogen fertilization and head rot in broccoli

An increase in the amount of N supplied (from 0 to 196 kg N/ha) to broccoli (Brassica oleracea var. italica) cv. Emperor led to an increased incidence of head rot (probably caused by a number of Pseudomonas and Erwinia species), which resulted in a decrease of the amount of marketable produce. Indications were found that head weight and head DM content are, probably indirectly, involved in limiting head rot at low N levels. The harvest index was not influenced by N treatment. Identification of the mechanisms which resulted in limited head rot at low N levels, could help to identify breeding characteristics for screening in agronomic evaluation programmes for broccoli cultivars under normal N levels.

EXTRACELLULAR ENZYMES AND PATHOGENESIS OF SOFT-ROT ERWINIA

Historically the genus Erwinia has served as a repository for plant-pathogenic or plant-associated bacteria (25, 96, 128). Consequently, diverse bacteria that occupy different habitats on or within plants and plant remains have been lumped into this genus. Their diversity is also reflected in the range of symp­ toms, i.e. necrosis, wilts, galls, and rots elicited in a wide range of plants. This diversity notwithstanding, these bacteria are related genetically to other en­ terobacteria such as Escherichia coli and Salmonella typhimurium that have served well as model systems for genetic and physiological studies. Microbi­ ologists and plant pathologists alike were initially attracted to Erwinia because of the potential for the use of genetic tools developed for the E. coli system. Thus, several Erwinia species were selected as model systems for the analysis

Colonization of Apple Wounds by Naturally Occurring Microflora and Introduced Candida oleophila and Their Effect on Infection by Botrytis cinerea during Storage

The colonization of apple wounds by natural microflora and an introduced antagonist, Candida oleophila, was followed at 4 and 18°C. Fresh wounds had initial populations of yeasts, filamentous fungi, and bacteria ranging between 102 to 103 cfu/wound. Colonization at 18°C by the natural microflora was rapid with fungal and bacterial populations increasing to about 104 to 105 and 106 cfu/wound, respectively, within the first 2 to 4 days and changing little afterwards. Aureobasidium pullulans and yeasts (mainly Sporobolomyces roseus) were the dominant fungal wound invaders, while species of Erwinia, Gluconobacter, and Pseudomonas were the most common bacteria isolated. At 4°C, little difference was noted in the composition of the microflora in wounds except for the absence of Gluconobacter sp. and lower populations of A. pullulans. The colonization process was more gradual at this temperature, reaching populations of about 5 × 104 and 105 cfu/wound by day 20 for fungi and bacteria, respectively. Populations of introduced C. oleophila applied at levels of 105 or 106 cfu/wound increased by about one log within 1 day at 18°C and 4 days at 4°C and stabilized thereafter. Introduced C. oleophila outnumbered the natural colonizers, forming a film of cells on the wound surface. Some of the main components of the natural microflora (Erwinia sp., A. pullulans, and S. roseus) coinoculated into wounds with C. oleophila in equal proportions did not have any effect on the establishment of C. oleophila on wounds. However, their introduction to wounds reduced the incidence of gray mold rot at 4°C after 23 days but not 30 days. Erwinia sp. and S. roseus coinoculated on wounds with C. oleophila did not affect the performance of C. oleophila in the biocontrol of gray mold rot, but A. pullulans improved it. Thus, the presence of the naturally occurring microflora of apple wounds does not interfere with the biocontrol of storage rot by C. oleophila and, in some cases, may even be beneficial.

Identification of Erwinia stewartii by a ligase chain reaction assay.

A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed such that only E. stewartii DNA gave a product in the LCR assay. The ligated product was separated from the radioactively labelled primers by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Twenty-four different Erwinia species and strains were tested by PCR-coupled LCR to verify the specificity of the assay, and only E. stewartii strains gave a positive reaction. In addition, infected and healthy plant material was also assayed. E. stewartii was detected in infected plant material, even when large populations of epiphytic bacteria were present. No enrichment was necessary for detection of the pathogen in corn leaves. This assay has potential as a diagnostic technique for the detection of E. stewartii in infected plant and vector material.

Differentiation of Erwinia Species in the “Amylovorap” Group by Class Analysis of Cellular Fatty Acids

Cellular fatty acids of Erwinia species in the Amylovora group (E. amylovora, E. nigrifluens, E. quercina, E. rubrifaciens, E. salicis and E. tracheiphila), grown on King's medium B agar for 1,3, and 6 days at 28°C, were analyzed by gas‐liquid chromatography, grouped by chemical class, and compared in order to differentiate the species. For the group in general, the average percentage in 1‐day‐old cells ranged 36.4–41.5% for the saturated straight‐chain even‐carbon fatty acids, (Class A), 0.3–0.7% for saturated straight‐chain odd‐carbons (Class B), 43.4–53.3% for unsaturated acids (Class C), 4.5–7.1% for hydroxy‐substituted acids (Class D), 0.2–2.0% for branched‐chain acids (Class E), and 0–7.5% for cyclopropane fatty acids (Class F). Species could be differentiated in 4 sequential steps based on: 1) percentage of the 12:0 fatty acid (E. amylovora >3.6%, E. quercina (1%);2) sum of Classes E + F (E. tracheiphila (0.3%);3) ratio Class A/(E + F) (E. salicis <8); and percentage of 18:1 (E. rubrifaciens >14%, E. nigrifluens < 14%). Physiological age significantly affected relative percentages of Classes B, C and F in E. amylovora and E. salicis; of Classes A, C and F in E. quercina; of Class A in E. tracheiphila; and of Class C in E. nigrifluens.

Differentiation of Erwinia Species in the "Herbicola" Group by Class Analysis of Cellular Fatty Acids

Differentiation of Erwinia Species in the "Herbicola" Group by Class Analysis of Cellular Fatty Acids

An anionic galactomannan polysaccharide gum from a newly-isolated lactose-utilizing bacterium. I. Strain description and gum characterization.

As part of an effort to obtain microorganisms able to produce polysaccharide gums from whey and whey permeate, soil samples from farm fields regularly treated with whey were screened for bacteria able to produce gums from lactose. The most promising organism isolated (ATCC 55046) is a facultative anaerobe, tentatively identified as a new Erwinia species on the basis of biochemical and morphological tests. The organism produces a polysaccharide gum from lactose and other sugars (herein named lactan gum) composed of mannose, galactose, and galacturonic acid with an approximate molar ratio of 5:3:2 and containing no organic acid modifying groups. The weight average molecular weight of the gum is approximately 7 x 10(6). Aqueous solutions of lactan gum exhibit shear-thinning and elastic flow behavior with an estimated power law model flow index of 0.26 at 1% (w/w) gum. The viscosity of aqueous 1% (w/w) lactan gum solutions is stable over a pH range of 2-11, being particularly stable in alkaline environments. Aqueous 1% (w/w) gum solutions at pH 5-11 show excellent thermostability, retaining at least 80% of the original viscosity after being heated to 121 degrees C for 15 min. These flow properties indicate potential industrial applications in food and nonfood products requiring a moderate degree of thickening, wet-end additives and coating agents for paper products, ceramics, detergents, and binders for building materials.

Bacterial Interference with in Vitro Assays of Tall Fescue Seeds for Acremonium Coenophialum

In vitro embryo‐derived callus culture is an efficient and useful technique for determining viable fungal endophyte (Acremonium coenophialum Morgan‐Jones & Gams) levels in tall fescue (Festuca arundinacea Schreb.) seed lots; however, the presence of a yellow‐pigmented bacterium occasionally interferes with detection of the fungus. The objectives of this study were to control and identify this bacterium. Excised embryos from 26 seed lots were plated on Schenk and Hildebrandt medium amended with 20 μM dicamba (2‐methoxy‐3,6‐dichlorobenzoic acid); bacteria‐infested seeds were found in 22 lots. Infestation levels ranged from 1 to 100%. Medium supplemented with 50 mg L−1 streptomycin eliminated bacterial growth without interfering with the growth of A. coenophialum. Based on microscopy, biochemical tests, and fatty acid analyses, the bacterium was identified as an Erwinia species with characteristics of the herbicola group.

Calcium Deficiency of Artichoke Buds in Relation to Salinity

Globe artichokes (Cynara scolymus L.) were grown for 2 years in artificially salinized field plots in the irrigated desert area near Brawley, Calif. Saline treatments were imposed by irrigation with waters that contained equal weights of NaCl and CaCl,. Increased incidence and severity of Ca deficiency in the inner bracts of artichoke buds were directly related to increased levels of salinity. The Ca-deficient bracts were subject to infection by species of Botrytis and Erwinia. The number of marketable artichokes was reduced 20% or more when irrigation water salinity exceeded 2.0 dS·m–1, and up to 50% at 10 dS·m–1. Calcium deficiency in the artichoke bud is believed to be the result of disparate Ca distribution among the tissues, which is caused by high transpiration rates in the desert environment and the reduction in root pressure by soil salinity. Elemental analysis of leaf tissues can not be used to predict Ca deficiency within the artichoke buds.

Phosphatidylinositol, a phospholipid of ice-nucleating bacteria

The nature of the phospholipids of the various bacteria that have ice nucleation activity in supercooled water has been determined. The seven bacteria studied included Pseudomonas syringae, Erwinia herbicola, three Escherichia coli K-12 strains that are phenotypically Ice+ because they contain plasmids with different amounts of either P. syringae or E. herbicola cloned DNA, and two E. coli K-12 strains without cloned ice gene DNA. All five Ice+ bacterial strains contained small amounts (0.1 to 1.0% of the total phospholipids) of phosphatidylinositol (PI), a phospholipid not previously detected in E. coli, Pseudomonas, or Erwinia species. The Ice- E. coli strains also contained trace level of PI that amounted to 2 to 30% of the level found in the Ice+ E. coli strains. Extracts of Ice+ strains contained low but measurable activities of PI synthase, while the activities in Ice- strains amounted to only 8 to 12% or less of that found in extracts of Ice+ bacteria. The functioning of the ice gene apparently increased both the PI synthase activity and the PI content of Ice+ strains from low endogenous levels. The relative ice nucleation activity at -4 degrees C or above (class A nucleation activity) of all Ice+ strains was found to be proportional to their PI content. The addition of myo-inositol (5 x 10(-4) M) to synthetic culture media increased the class A nucleation activity of both Ice+ E. coli strains and P. syringae up to sevenfold but had no stimulating effect on ice nucleation at lower temperatures (class B and class C nucleation activities). If these cells after fusion with PI vesicles were incubated with an energy source, the class A nucleation activity increased 70-fold over that present before fusion. These results indicate that PI plays an important role in ice nucleation at warm temperatures and is a likely precursor or component of the class A structure.

Nucleotide sequence and molecular characterization of pnlA, the structural gene for damage-inducible pectin lyase of Erwinia carotovora subsp. carotovora 71

In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N-terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins.

ChemInform Abstract: A Chiral Synthesis of trans‐Carbapenam‐3‐carboxylic Acid and the Assignment of (3S,5S)‐Configuration to the Corresponding Product from Serratia and Erwinia Species.

AbstractThe oxazolidonepropionic acid (Ia), synthesized from the corresponding (R)‐glutamic acid and paraformaldehyde, is converted into the trans‐alkene (III) by reduction and olefination.

Bacteriophage λ-mediated transposon mutagenesis of phytopathogenic and epiphytic Erwinia species is strain dependent

Using transformation and conjugal mobilization, plasmids carrying the lamB gene of Escherichia coli were transferred to a range of Erwinia strains. The resultant strains were infected with lambda 467, and kanamycin resistant transductants were screened for various mutant phenotypes including auxotrophy and altered extracellular enzyme activities. Reversion analysis suggested that most mutant phenotypes were due to Tn5 insertion. The applicability of the techniques was highly strain dependent. However a rapid and simple route to mutant isolation was obtained, which could allow the use of other lambda-related genetic techniques in several important species which, to date, have not been genetically manipulated.