Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae.

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae

Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.     

Diversity of antifungal and plant-associated Serratia plymuthica strains

A total of 21 plant-associated Serratia plymuthica strains were characterized phenotypically by their nutritional patterns, susceptibility to antibiotics, antifungal and haemolytic properties, and genotypically by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA, PCR fingerprints using BOX primers (BOX-PCR) and pulsed-field gel electrophoresis (PFGE) after digestion with SpeI. All of the investigated strains demonstrated antifungal activity in vitro against fungal pathogens while only six strains produced the antifungal antibiotic prodigiosin. Haemolytic activity and antibiotic resistance patterns were investigated to assess the risk associated with the use of isolates in plant protection. The strains were haemolytic at human-relevant temperatures. The level of resistance to antibiotics was low. This work shows that BOX-PCR and PFGE are useful fingerprinting methods to characterize Ser. plymuthica strains, although the discriminatory effect between the two methods differed. Computer-assisted analysis of phenotypic and genotypic features demonstrated relationships between the origin of isolation, the production of prodigiosin and the molecular fingerprint.     

Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China

To investigate the distribution and diversity of the pathogens associated with Fusarium crown rot in the Huanghuai wheat-growing region (HHWGR) of China, we collected wheat samples with symptomatic stem bases from seven provinces in the HHWGR between 2013 and 2016. A total of 1196 isolates obtained from 222 locations were identified as 9 Fusarium species based on morphological and molecular identification. Of these pathogen species, F. pseudograminearum was the dominant species. Furthermore, F. sinensis was isolated from the disease specimens and tested for virulence to wheat. The result of the pathogenicity revealed that an intraspecific differentiation existed in F. pseudograminearum; sequence analysis of the EF-1α gene showed that 194 F. pseudograminearum isolates were differentiated into two distinct clades which closed to the strains from Australia and China respectively, but neither pathogenicity nor EF-1α sequence was related to the geographic origins of these isolates. However, universal rice primers-polymerase chain reaction showed a correlation with the geographical origins of the 194 isolates, which were divided into eight subclusters, the level of genetic diversity was higher within a geographical population than among the different populations. The results of these analyses can be directly used to facilitate disease monitoring and development of control strategies.     

Cadmium resistance in some members of Enterobacteriaceae

Strains of members of Enterobacteriaceae, namely Escherichia coli (18), Klebsiella aerogenes (16), and Serratia marcescens (16) were screened for Cd resistance or sensitivity. Only one strain each of these was resistant to high levels (25 n moles/0.05 ml) CdCl2. The Minimal inhibitory concentration (MIC) of sensitive strains ranged from 0.8-5 micrograms/ml. All the resistant strains were simultaneously resistant to a number of antibiotics. Treatment with sodium dodecyl sulfate eliminated resistance to Cd and to some antibiotics.     

Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction

A multiplex polymerase chain reaction (PCR) assay for simultaneous, fast and reliable detection of the main soft rot and blackleg potato pathogens in Europe has been developed. It utilises three pairs of primers and enables detection of three groups of pectinolytic bacteria frequently found in potato, namely: Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum together with Pectobacterium wasabiae and Dickeya spp. in a multiplex PCR assay. In studies with axenic cultures of bacteria, the multiplex assay was specific as it gave positive results only with strains of the target species and negative results with 18 non‐target species of bacteria that can possibly coexist with pectinolytic bacteria in a potato ecosystem. The developed assay could detect as little as 0.01 ng µL^–1 of Dickeya sp. genomic DNA, and down to 0.1 ng µL^–1 of P. atrosepticum and P. carotovorum subsp. carotovorum genomic DNA in vitro. In the presence of competitor genomic DNA, isolated from Pseudomonas fluorescens cells, the sensitivity of the multiplex PCR decreased tenfold for P. atrosepticum and Dickeya sp., while no change was observed for P. carotovorum subsp. carotovorum and P. wasabiae. In spiked potato haulm and tuber samples, the threshold level for target bacteria was 10¹ cfu mL^–1 plant extract (10² cfu g^–1 plant tissue), 10² cfu mL^–1 plant extract (10³ cfu g^–1 plant tissue), 10³ cfu mL^–1 plant extract (10⁴ cfu g^–1 plant tissue), for Dickeya spp., P. atrosepticum and P. carotovorum subsp. carotovorum/P. wasabiae, respectively. Most of all, this assay allowed reliable detection and identification of soft rot and blackleg pathogens in naturally infected symptomatic and asymptomatic potato stem and progeny tuber samples collected from potato fields all over Poland.     

Lipids of antibiotic-resistant and -susceptible members of the Enterobacteriaceae.

Lipids of antibiotic-resistant and related -susceptible strains of the Enterobacteriaceae were extracted with chloroform-methanol and characterized by thin-layer chromatography, densitometry, and fatty acid analysis using gas chromatography. Quantitative differences which correlated with antibiotic resistance existed among the phospholipids and fatty acids. A relatively higher concentration of a ninhydrin-positive phospholipid concentration with a lower amount of phosphatidylethanolamine was observed in antibiotic-resistant strains of serratia marcescens. Bacterial strains which harbored R-factor 222 had a higher ratio of phosphatidylglycerol to diphosphatidylglycerol than their respective parent strains while those strains which were resistant to the polymyxins had a lower ratio of these phospholipids. Differences in the relative amounts of certain unsaturated and cyclopropane fatty acids were observed between susceptible and resistant strains. Such differences, however, were dependent upon a particular genus and species.     

Multiplex detection and identification of bacterial pathogens causing potato blackleg and soft rot in Europe,using padlock probes

The objective of this study was to develop a multiplex detection and identification protocol for bacterial soft rot coliforms, namely Pectobacterium wasabiae (Pw), Pectobacterium atrosepticum (Pba) and Dickeya spp., responsible for potato blackleg and tuber soft rot. The procedures were derived from the phylogenetic relationships of these and other Enterobacteriaceae based on recA sequences. The group of Pw strains was highly homogeneous and could be distinguished from the other species. A ligation‐based method for detection of Pw was developed. Five padlock probes (PLPs) were designed, targeting recA sequences to identify the Pw, Pba or Dickeya spp., whereas a sixth probe recognised recA sequences of all soft rot coliforms including Pectobacterium carotovorum subsp. carotovorum (Pcc). Two PLP‐based applications were developed: one using real‐time PCR and one using universal microarrays. Assay sensitivity and specificity were demonstrated using 71 strains of Pw, Pcc, Pba and Dickeya spp. Both multiplex methods can be potentially used for seed testing and in ecological studies, but further validation is required.     

Asperpyrone D and other metabolites of the plant-associated fungal strain Aspergillus tubingensis

Bioactivity-guided fractionation of a cytotoxic extract of Aspergillus tubingensis, a fungal strain occurring in the rhizosphere of the Sonoran desert plant, Fallugia paradoxa, afforded a dimeric naphtho-gamma-pyrone asperpyrone D, nine known naphtho-gamma-pyrones, funalenone, and the cytotoxic cyclic penta-peptide, malformin A1.     

Haemagglutinins and fimbriae of soft rot Erwinias

A. WALLACE AND M.C.M. PÉROMBELON. 1992. Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the β-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA⁺ bacteria of E.c. carotovora and E.c. atroseptica.     

Autoinducer-2 of the fire blight pathogen Erwinia amylovora and other plant-associated bacteria

Autoinducers are important for cellular communication of bacteria. The luxS gene has a central role in the synthesis of autoinducer-2 (AI-2). The gene was identified in a shotgun library of Erwinia amylovora and primers designed for PCR amplification from bacterial DNA. Supernatants of several Erwinia amylovora strains were assayed for AI-2 activity with a Vibrio harveyi mutant and were positive. Many other plant-associated bacteria also showed AI-2 activity such as Erwinia pyrifoliae and Erwinia tasmaniensis. The luxS genes of several bacteria were cloned, sequenced, and complemented Escherichia coli strain DH5alpha and a Salmonella typhimurium mutant, both defective in luxS, for synthesis of AI-2. Assays to detect AI-2 activity in culture supernatants of several Pseudomonas syringae pathovars failed, which may indicate the absence of AI-2 or synthesis of another type. Several reporter strains did not detect synthesis of an acyl homoserine lactone (AHL, AI-1) by Erwinia amylovora, but confirmed AHL-synthesis for Erwinia carotovora ssp. atroseptica and Pantoea stewartii.     

Pseudomonads associated with midrib rot and soft rot of butterhead lettuce and endive

During the past ten years, bacterial soft rot and midrib rot of glasshouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) and field-grown endive (Cichorium endivia L.) has become increasingly common in the region of Flanders, Belgium. Severe losses and reduced market quality caused by bacterial rot represent an important economical threat for the production sector. Symptoms of midrib rot are a brownish rot along the midrib of one or more inner leaves, often accompanied by soft rot of the leaf blade. Twenty-five symptomatic lettuce and endive samples were collected from commercial growers at different locations in Flanders. Isolations of dominant bacterial colony types on dilution plates from macerated diseased tissue extracts yielded 282 isolates. All isolates were characterized by colony morphology and fluorescence on pseudomonas agar F medium, oxidase reaction, and soft rot ability on detached chicory leaves. Whole-cell fatty acid methyl esters profile analyses identified the majority of isolates (85%) as belonging to the Gammaproteobacteria, which included members of the family Enterobacteriaceae (14%) and of the genera Pseudomonas (73%), Stenotrophomonas (9%), and Acinetobacter (3%). Predominant bacteria were a diverse group of fluorescent Pseudomonas species. They were further differentiated based on the non-host hypersensitive reaction on tobacco and the ability to rot potato slices into 4 phenotypic groups: HR-/P- (57 isolates), HR-/P+ (54 isolates), HR+/P (16 isolates) and HR+/P+ (35 isolates). Artificial inoculation of suspensions of HR-, pectolytic fluorescent pseudomonads in the leaf midrib of lettuce plants produced various symptoms of soft rot, but they did not readily cause symptoms upon spray inoculation. Fluorescent pseudomonads with phenotype HR+ were consistently isolated from typical dark midrib rot symptoms, and selected isolates reproduced the typical midrib rot symptoms when spray-inoculated onto healthy lettuce plants.     

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus

Background

Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling.

Results

Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster.

Conclusions

Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1763-2) contains supplementary material, which is available to authorized users.     

Dulcitol-malonate-phenylalanine agar for the identification of Salmonella and other Enterobacteriaceae

An agar medium combining dulcitol fermentation, malonate utilization, and phenylalanine deamination was evaluated with 229 isolates representing 19 genera. All reactions agreed with those obtained on conventional media.