Ralstonia Species Research Articles

Interactions Between Strains of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere of Wheat

ABSTRACT Strains of fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG) are among the most effective rhizobacteria controlling diseases caused by soilborne pathogens. The genotypic diversity that exists among 2,4-DAPG producers can be exploited to improve rhizosphere competence and biocontrol activity. Knowing that D-genotype 2,4-DAPG-producing strains are enriched in some take-all decline soils and that P. fluorescens Q8r1-96, a representative D-genotype strain, as defined by whole-cell repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer, is a superior colonizer of wheat roots, we analyzed whether the exceptional rhizosphere competence of strain Q8r1-96 on wheat is characteristic of other D-genotype isolates. The rhizosphere population densities of four D-genotype strains and a K-genotype strain introduced individually into the soil were significantly greater than the densities of four strains belonging to other genotypes (A, B, and L) and remained above log 6.8 CFU/g of root over a 30-week cycling experiment in which wheat was grown for 10 successive cycles of 3 weeks each. We also explored the competitive interactions between strains of different genotypes inhabiting the same soil or rhizosphere when coinoculated into the soil. Strain Q8r1-96 became dominant in the rhizosphere and in nonrhizosphere soil during a 15-week cycling experiment when mixed in a 1:1 ratio with either strain Pf-5 (A genotype), Q2-87 (B genotype), or 1M1-96 (L genotype). Furthermore, the use of the de Wit replacement series demonstrated a competitive disadvantage for strain Q2-87 or strong antagonism by strain Q8r1-96 against Q2-87 in the wheat rhizosphere. Amplified rDNA restriction analysis and sequence analysis of 16S rDNA showed that species of Arthrobacter, Chryseobacterium, Flavobacterium, Massilia, Microbacterium, and Ralstonia also were enriched in culturable populations from the rhizosphere of wheat at the end of a 30-week cycling experiment in the presence of 2,4-DAPG producers. Identifying the interactions among 2,4-DAPG producers and with other indigenous bacteria in the wheat rhizosphere will help to elucidate the variability in biocontrol efficacy of introduced 2,4-DAPG producers and fluctuations in the robustness of take-all suppressive soils.

Burkholderiaand Emerging Pathogens in Cystic Fibrosis

Persons with cystic fibrosis (CF) are susceptible to respiratory tract infection with a variety of bacterial species. Among these are several species in the genus Burkholderia (including members of the B. cepacia complex, B. gladioli, and B. pseudomallei), Stenotrophomonas maltophilia, Achromobacter ( Alcaligenes) xylosoxidans, certain Ralstonia species, and species within the new genus Pandoraea. Enterobacteriaceae and several other rather unusual or novel species also can be found. Many of these species are closely related phylogenetically and have similar phenotypes, presenting a challenge to accurate laboratory identification. Although their role in contributing to pulmonary disease in CF is not clear, the incidence of infection by some of these species appears to be increasing. The broad-spectrum antimicrobial resistance typically exhibited by these species severely limits treatment options. Newly developed genotypic methods of identification will enable studies to better assess the epidemiology and natural history of these emerging pathogens in CF.

Development of PCR-based markers for detection of Leifsonia xyli subsp. xyli in fibrovascular fluid of infected sugarcane plants

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

Phloem-and xylem-restricted plant pathogenic bacteria

1. Introduction: an overview This review concerns plant pathogenic bacteria, which are strictly restricted either to the sieve tubes in the phloem or to the vessels in the xylem. These bacteria are endogenous as opposed to exogenous bacteria such as species of Erwinia , Pseudomonas , Ralstonia or Xanthomonas , which colonize the apoplast (intercellular spaces) of plant tissues, even though some of the exogenous bacteria, such as Xylophilus ampelina , can also induce vascular infections. Due to their vascular habitat, the endogenous bacteria have a systemic distribution throughout the plant, they are transmitted from plant to plant by graft inoculation, and most of them are vectored by insects which feed in the phloem (leafhoppers, psyllids), or the xylem (sharpshooters). Because of these virus-like properties, the diseases caused by endogenous bacteria have long been taken for virus diseases. The principal conducting cells of the phloem are the sieve tube elements [37]. These elements are joined end to end into sieve tubes, and are associated with parenchymatic, nucleated cells, the companion cells, an important role of which is to load sucrose into the sieve tubes. The sieve tube elements are living cells, which become enucleated at maturity. The sieve plates are lateral wall areas between two adjacent sieve elements. The sieve plates are clustered with pores, resembling giant plasmodesmata and interconnecting two adjacent sieve elements through their cytoplasms. The diameter of a pore ranges from a fraction of a micron (m )t o 15m and more, and is large enough to allow passage of sieve tube-restricted bacteria. The principal conducting elements of the xylem are tracheids and vessel members [37]. Both are dead cells, contain no cytoplasm, and have lignified secondary walls. The vessel elements are joined end to end into vessels, and the adjoining ends have open perforation plates. These openings allow relatively unimpeded longitudinal spread of the xylem-restricted bacteria within

Infection by Ralstonia species in cystic fibrosis patients: identification of R. pickettii and R. mannitolilytica by polymerase chain reaction.

The frequency of respiratory tract infections caused by Ralstonia species in persons with cystic fibrosis (CF) and the role of these species in CF pulmonary disease are not well documented. In part, this lack of documentation may be attributed to the difficulty in accurately identifying Ralstonia species; R. mannitolilytica and R. pickettii in particular may be misidentified as other closely related species, particularly those of the Burkholderia cepacia complex. We used polyphasic analyses to identify 42 Ralstonia isolates from sputum cultures from 38 CF patients. Several isolates that could not be identified to the species level may belong to novel Ralstonia species. We demonstrated chronic colonization by using genotyping of serial isolates recovered from the same patient. To facilitate identification of R. mannitolilytica and R. pickettii, we developed 16S ribosomal DNA-based polymerase chain reaction assays that allow sensitive and specific identification of these species.

Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend.

Thirty-one heavy-metal-resistant bacteria isolated from industrial biotopes were subjected to polyphasic characterization, including 16S rDNA sequence analysis, DNA-DNA hybridizations, biochemical tests, whole-cell protein and fatty-acid analyses. All strains were shown to belong to the Ralstonia branch of the beta-Proteobacteria. Whole-cell protein profiles and DNA-DNA hybridizations revealed two clearly distinct groups, showing low similarity to known Ralstonia species. These two groups, of 8 and 17 isolates, were assigned to two new species, for which the names Ralstonia campinensis sp. nov. and Ralstonia metallidurans sp. nov. are proposed. The type strains are WS2T (= LMG 19282T = CCUG 44526T) and CH34T (= LMG 1195T = DSM 2839T), respectively. Six isolates were allocated to Ralstonia basilensis, which presently contains only the type strain; an emendation of the latter species description is therefore proposed.

Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient.

A polyphasic taxonomic study, including 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base ratio determinations, amplified 165 rDNA restriction analysis, whole-cell protein analyses and extensive biochemical characterization, was conducted to clarify the relationships of eight isolates from root nodules of Mimosa species and one isolate from sputum of a cystic fibrosis patient. All nine isolates were classified as a novel Ralstonia species, for which the name Ralstonia taiwanensis sp. nov. is proposed. The type strain is LMG 19424T (= CCUG 44338T). R. taiwanensis effectively nodulated the Mimosa species and is the first beta-proteobacterium that is known to be capable of root nodule formation and nitrogen fixation.

Isolation and characterization of a thermotolerant bacterium Ralstonia sp. strain PHS1 that degrades benzene, toluene, ethylbenzene, and o-xylene.

A thermotolerant bacterium, designated as PHS1, was isolated from a hot spring in Pohang, Korea, on the basis of its ability to grow on benzene, toluene, ethylbenzene, and xylenes (BTEX) as a sole carbon source. Strain PHS1 is a gram-negative, rod-shaped aerobe and grows optimally at 42 degrees C and pH 7.2. According to 16 S rDNA analysis, strain PHS1 showed highest similarity to Ralstonia eutropha (previously named Alcaligenes eutrophus). Unlike its closest known Ralstonia species, however, strain PHS1 was able to utilize toluene, ethylbenzene, o-xylene, and both m- and o-cresol. The degradation of o-xylene by strain PHS1 is particularly important, since o-xylene is a compound of considerable environmental interest, owing to its recalcitrance; and very few microorganisms have been reported to utilize o-xylene as a sole carbon source. It was found that strain PHS1 transformed o-xylene to 2,3-dimethylphenol through direct oxygenation of the aromatic ring. The unique properties of strain PHS1, such as thermotolerance and the ability to degrade o-xylene, may have important implications for the treatment of BTEX-contaminated industrial effluents.

Production of catechol from benzoate by the wild strain Ralstonia species Ba-0323 and characterization of its catechol 1,2-dioxygenase.

Ralstonia sp. Ba-0323, a wild strain isolated from soil, produced catechol from benzoate and accumulated it outside the cells. The bacterium produced a maximal amount of catechol (1.6 mg/ml) from 3 mg/ml of sodium benzoate in a 20-h growing culture. The conversion rate of benzoate to catechol was 70% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 3 mg/ml of sodium benzoate reached 1.9 mg/ml at the conversion rate of 83% after 8 h of incubation. Catechol 1,2-dioxygenase, which catalyzed the ring cleavage of catechol, was purified to homogeneity from a cell extract of Ralstonia sp. Ba-0323 growing on benzoate and characterized. The specific activity of the purified enzyme was much lower than those of the dioxygenases from other microorganisms reported. The Km for catechol of the purified enzyme was much higher than those of other dioxygenases. In addition, the NH2-terminal amino acid sequence of the enzyme was less similar to the other catechol 1,2-dioxygenases than they are to each other.

Taxonomic Position of “ Pseudomonas oxalaticus” Strain Ox1 T (DSM 1105 T) (Khambata and Bhat, 1953) and its Description in the Genus Ralstonia as Ralstonia oxalatica comb. nov.

"Pseudomonas oxalaticus" strain Ox1T (= DSM 1105T), which was described as an oxalate-decomposing bacterium, was reinvestigated to clarify its taxonomic position. 16S ribosomal DNA sequence comparisons demonstrated that this species is phylogenetically related to the species of the genus Ralstonia. and represents a new species. The result of the DNA-DNA hybridization value was supported in this placement. Strain Ox1T is closely related to Ralstonia eutropha with a less than 60% DNA-DNA hybridization value. The new name Ralstonia oxalatica comb. nov. is proposed to strain Ox1T, on the basis of these results and previously published data for the G+C content of the genomic DNA and the phenotypic characters.

Staphyloferrin B, a Citrate Siderophore of Ralstonia eutropha

Abstract The structure and stereochemistry of the siderophore isolated from the culture medium of Ralstonia eutropha (formerly Alcaligenes eutrophus) could be elucidated as staphyloferrin B by spectroscopic methods and chemical degradation. The relationship of the three species of the new genus Ralstonia is reflected in the observation that all three form citrate sidero-phores.

CDC group IV c-2: a new Ralstonia species close to Ralstonia eutropha.

CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus Ralstonia, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus Ralstonia. The closest matches were obtained with Ralstonia eutropha. However, four differences in 32 biochemical tests separated R. eutropha from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus Ralstonia.

Assignment of Centers for Disease Control group IVc-2 to the genus Ralstonia as Ralstonia paucula sp. nov.

An integrated genotypic and phenotypic analysis of 12 Centers for Disease Control (CDC) group IVc-2 strains revealed that this taxon represents a novel species belonging to the genus Ralstonia. Comparative 16S rDNA sequence analysis allocated a representative CDC group IVc-2 strain to the Ralstonia branch of the beta subclass of the Proteobacteria. DNA-DNA hybridizations did not detect significant binding levels towards any presently known Ralstonia species, including Ralstonia pickettii. Its DNA base ratio is between 65 and 67 mol%. The name Ralstonia paucula sp. nov. is proposed, with strain LMG 3244 (= CDC E6793), isolated from a human respiratory tract, as the type strain. R. paucula can be differentiated from other Ralstonia species by whole-cell protein analysis, amplified rDNA restriction analysis and a variety of classical biochemical tests. Strains have been isolated from various human clinical and environmental sources.

Lipids and fatty acids of Burkholderia and Ralstonia species

Contrary to previous reports, ornithine amide lipids are produced by some strains of Ralstonia eutropha and Ralstonia pickettii under some growth conditions, thus eroding one of the characteristic differences from Burkholderia spp. The proportion of phosphatidylethanolamine containing 2-hydroxy acids also varies with growth conditions, but not significantly with growth temperature (30 or 37°C) for shake-flask cultures, unlike chemostat cultures of Burkholderia cepacia. Several fatty acid profiles were represented among the isolated lipopolysaccharides (LPS). Burkholderia spp. typically had 14:0, 3-OH-14:0 and 3-OH-16:0 as major components, but in two strains of B. cepacia the LPS was relatively rich in 12:0 (rather than 14:0) or contained 2-OH-14:0 (but little 3-OH-16:0). In two strains each of R. eutropha and R. pickettii, the LPS contained mainly 14:0 and 3-OH-14:0 with some 2-OH-14:0. In another strain of R. pickettii the LPS contained 2-OH-16:0 but not 2-OH-14:0. The data may have chemotaxonomic potential.

Evaluation of three oligonucleotide primer sets in PCR for the identification of Burkholderia cepacia and their differentiation from Burkholderia gladioli.

AIMS: To evaluate three oligonucleotide primer pairs--two specific for 16S and 23S rRNA sequences of Burkholderia cepacia, and the third specific for internal transcribed spacer region of 16S-23S sequences of...

Microbial communities of printing paper machines.

The microbial content of printing paper machines, running at a temperature of 45-50 degrees C and at pH 4.5-5, was studied. Bacteria were prevalent colonizers of the machine wet end and the raw materials. A total of 390 strains of aerobic bacteria were isolated and 86% of these were identified to genus and species by biochemical, chemotaxonomic and phylogenetic methods. The most common bacteria found at the machine wet end were Bacillus coagulans and other Bacillus species, Burkholderia cepacia, Ralstonia pickettii, and in pink slimes, accumulating in the wire area and press section, species of Deinococcus, aureobacterium and Brevibacterium. Paper-making chemicals also contained species of Aureobacterium, B. cereus, B. licheniformis, B. sphaericus, Bordetella, Hydrogenophaga, Klebsiella pneumoniae, Pantoea agglomerans, Pseudomonas stutzeri, Staphylococcus and sometimes other enteric bacteria, but these did not colonize the process water. Yeasts and moulds were not present in significant numbers. A total of 131 strains were tested for their potential to degrade paper-making raw materials; 91 strains were found to have degradative activity, mainly species of Burkholderia and Ralstonia, Sphingomonas and Bacillus, and enterobacteria produced enzymes which degraded paper-making chemicals. Stainless steel adhering strains occurred in slimes and wire water and were identified as Burkholderia cepacia, B. coagulans and Deinococcus geothermalis. Coloured slimes were formed on the machine by species of Deinococcus, Acinetobacter and Methylobacterium (pink), Aureobacterium, Pantoea and Ralstonia (yellowish) and Microbulbifer-related strains (brown). The impact of the strains and species found in the printing paper machine community on the technical quality of paper, machine operation, and as a potential biohazard (Hazard Group 2 bacteria), is discussed.