Development of a colorimetric colony-screening assay for detection of defluorination by micro-organisms.

AIMS: To develop a colorimetric colony-screening assay to facilitate the isolation of micro-organisms capable of defluorination. METHODS AND RESULTS: A metal-dye chelate, zirconium-xylenol orange was used to detect fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1:2. Using depolymerised zirconium and xylenol orange (150 and 300 nmol l(-1) respectively), the assay could detect a fluoride application spot (5 mmol l(-1)) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions needed to be restricted to 0.1 g l(-1) and some proteins digest to between 1 and 5 g l(-1). A microbial enrichment culture growing on solidified medium containing 20 mmol l(-1) fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the genus Burkholderia isolated. CONCLUSIONS: A method was developed that is sensitive, rapid and reliable for detecting defluorination by micro-organisms growing on solidified medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This method can be used to facilitate the isolation of micro-organisms capable of defluorination.

Development of a Bio-PCR Protocol for the Detection of Xanthomonas arboricola pv. pruni.

A real-time SYBR Green I assay was developed and evaluated as a biological and enzymatic polymerase chain reaction (Bio-PCR) protocol for the detection of Xanthomonas arboricola pv. pruni. Suppression subtractive hybridization was used to generate a X. arboricola pv. pruni-specific subtracted DNA library, using X. arboricola pv. corylina as the driver strain. Primer pair 29F/R, designed from cloned sequence, showed no homology to GenBank sequences and amplified a 344-bp product in all X. arboricola pv. pruni isolates. Compared with other published X. arboricola pv. pruni primers, this primer pair was shown to be the only one capable of differentiating X. arboricola pv. pruni from all other X. arboricola pathovars. A real-time assay was developed and shown to be capable of detecting less than 10 CFU and 0.1 pg of DNA. Epiphytic bacteria isolated from plum tissue was used to further evaluate the specificity of the assay. A Bio-PCR protocol, developed for field evaluation, confirmed X. arboricola pv. pruni isolation from asymptomatic and symptomatic plum tissue over a 9-week period between host flowering and the first appearance of leaf and fruit symptoms in an orchard. Dilution plating enabled X. arboricola pv. pruni numbers to be quantified, providing supportive evidence for the usefulness of the Bio-PCR protocol in plant pathology and quarantine surveillance.

A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies.

A previously unknown chemolithoautotrophic arsenite-oxidizing bacterium has been isolated from a gold mine in the Northern Territory of Australia. The organism, designated NT-26, was found to be a gram-negative motile rod with two subterminal flagella. In a minimal medium containing only arsenite as the electron donor (5 mM), oxygen as the electron acceptor, and carbon dioxide-bicarbonate as the carbon source, the doubling time for chemolithoautotrophic growth was 7.6 h. Arsenite oxidation was found to be catalyzed by a periplasmic arsenite oxidase (optimum pH, 5.5). Based upon 16S rDNA phylogenetic sequence analysis, NT-26 belongs to the Agrobacterium/Rhizobium branch of the alpha-Proteobacteria and may represent a new species. This recently discovered organism is the most rapidly growing chemolithoautotrophic arsenite oxidizer known.

Two new arsenate/sulfate-reducing bacteria: mechanisms of arsenate reduction.

Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system.

Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug.

A new species of the genus Gluconacetobacter, for which the name Gluconacetobacter sacchari sp. nov. is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia. The nearest phylogenetic relatives in the alpha-subclass of the Proteobacteria are Gluconacetobacter liquefaciens and Gluconacetobacter diazotrophicus, which have 98.8-99.3% and 97.9-98.5% 16S rDNA sequence similarity, respectively, to members of Gluconacetobacter sacchari. On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q10 ubiquinone, this new species was assigned to the genus Gluconacetobacter. No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0.01% malachite green, growth on 30% glucose, an inability to fix nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source. The type strain of this species is strain SRI 1794T (= DSM 12717T).

Phylogenetic position of Chitinophaga pinensis in the Flexibacter-Bacteroides-Cytophaga phylum.

Comparison of the 16S rRNA gene sequence determined for Chitinophaga pinensis showed that this species is most closely related to Flexibacter filiformis in the Flexibacter-Bacteroides-Cytophaga phylum. These two chitinolytic bacteria, which are characterized by transformation into spherical bodies on ageing, belong to a strongly supported lineage that also includes Cytophaga arvensicola, Flavobacterium ferrugineum and Flexibacter sancti. The lineage is distinct from the microcyst-forming species Sporocytophaga myxococcoides.

The phylogenetic relationships of Caulobacter, Asticcacaulis and Brevundimonas species and their taxonomic implications.

The phylogenetic relationships among the species of Caulobacter, Asticcacaulis and Brevundimonas were studied by comparison of their 16S rDNA sequences. The analysis of almost complete sequences confirmed the early evolutionary divergence of the freshwater and marine species of Caulobacter reported previously [Stahl, D. A., Key, R., Flesher, B. & Smit, J. (1992). J Bacteriol 174, 2193-2198]. The freshwater species formed two distinct clusters. One cluster contained the species Caulobacter bacteroides, Caulobacter crescentus, Caulobacter fusiformis and Caulobacter henricii. C. bacteroides and C. fusiformis are very closely related (sequence identity 99.8%). The second cluster was not exclusive and contained the specis Caulobacter intermedius, Caulobacter subvibrioides and Caulobacter variabilis, as well as Brevundimonas diminuta and Brevundimonas vesicularis. The marine species Caulobacter halobacteroides and Caulobacter maris were very closely related, with a sequence identity of 99.7%. These two species were most closely but distantly related to the marine hyphal/budding bacteria Hyphomonas jannaschiana and Hirschia baltica, which formed a deep phylogenetic line with Rhodobacter sphaeroides and Rhodobacter capsulatus. Caulobacter leidyia is unrelated to the other species of Caulobacter and belongs to the alpha-4 subclass of the Proteobacteria, forming a distinct cluster with Asticcacaulis excentricus and Asticcacaulis biprosthecium. The taxonomic implications of the polyphyletic nature of the genus Caulobacter and the absence of a type culture for the type species of the genus Caulobacter vibrioides, are discussed.

Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov.

The phylogenetic relationships among members of the family Comamonadaceae and several unclassified strains were studied by direct sequencing of their PCR-amplified 16S rRNA genes. Based on the 16S rRNA gene sequence analysis, members of the family formed a coherent group. The closest relatives are species of the Rubrivivax sub-group: Leptothrix discophora, Ideonella dechloratans and Rubrivivax gelatinosus. The genus Hydrogenophaga formed two subclusters, as did the species of Acidovorax, whereas the five species of the genus [Aquaspirillum] were polyphyletic. Comamonas acidovorans was phylogenetically distant from the type species of Comamonas, Comamonas terrigena. On the basis of this work and previous studies, Comamonas acidovorans is removed from the genus Comamonas and renamed as Delftia acidovorans gen. nov., comb. nov. Descriptions of the new genus Delftia and of the type species Delftia acidovorans, for which the type strain is ATCC 15668T, are presented.

Phylogenetic heterogeneity within the genus Herpetosiphon: transfer of the marine species Herpetosiphon cohaerens, Herpetosiphon nigricans and Herpetosiphon persicus to the genus Lewinella gen. nov. in the Flexibacter-Bacteroides-Cytophaga phylum.

Analysis of the 16S rDNA sequences of species currently assigned to the genus Herpetosiphon revealed intrageneric phylogenetic heterogeneity. The thermotolerant freshwater species Herpetosiphon geysericola is most closely related to the type species Herpetosiphon aurantiacus in the Chloroflexus subdivision of the green non-sulfur bacteria. The marine species Herpetosiphon cohaerens, Herpetosiphon nigricans and Herpetosiphon persicus, on the other hand, were found to form a cluster with sheathed bacterium Haliscomenobacter hydrossis in the Saprospira group of the Flexibacter-Bacteroides-Cytophaga (FBC) phylum. A proposal is made to transfer these marine species to the genus Lewinella gen. nov. as Lewinella cohaerens comb. nov., Lewinella nigricans comb. nov. and Lewinella persica comb. nov. The marine sheathed gliding bacterium Flexithrix dorotheae was also found to be a member of the FBC phylum but on a separate phylogenetic line to the marine herpetosiphons now assigned to the genus Lewinella.

Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus.

The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers. The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense. The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences. Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet. diazotrophicus from environmental samples with nifH gene-based primers.

The tannin-degrading species Streptococcus gallolyticus and Streptococcus caprinus are subjective synonyms.

The tannin-degrading species Streptococcus gallolyticus and Streptococcus caprinus have been shown to be subjective synonyms on the basis of their levels of 16S rRNA sequence similarity (98.3%) and DNA-DNA homology (> 70%) and the phenotypes of their type strains. S. gallolyticus has nomenclatural priority according to Rule 24b(2) of the International Code of Nomenclature of Bacteria.

PCR-mediated detection of acidophilic, bioleaching-associated bacteria.

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg.

Isolation of Lautropia mirabilis from sputa of a cystic fibrosis patient.

An aggregate-forming coccus, isolated twice as the predominant microorganism in sputa from a cystic fibrosis patient on consecutive days, was shown to belong to the species Lautropia mirabilis on the bases of similarities of 16S rRNA gene sequences and phenotype. These isolates of L. mirabilis appear to be the first reported from a patient with cystic fibrosis and outside of Denmark.

Phylogenetic relationships and uncertain taxonomy of Pedomicrobium species.

The phylogenetic relationships among the species of the genus Pedomicrobium were studied by comparing their 16S rRNA sequences. The Pedomicrobium species form a coherent phylogenetic cluster within the genera of the hyphal budding bacteria in the alpha-Proteobacteria. The sequences of two strains of Pedomicrobium australicum were obtained from DNAs extracted from nonviable freeze-dried cells, which are the only source of material available, and were found to be almost identical (level of similarity, 99.9%). Overall, the Pedomicrobium species are closely related, with sequence similarities ranging from 96.2 to 99.9%. Pedomicrobium manganicum is phylogenetically the most distantly related species and exhibits the lowest similarity (96.2%) with Pedomicrobium americanum. Australian isolate Pedomicrobium sp. strain ACM 3067, P. americanum, and P. australicum are all very highly related, with similarities greater than 99%. Pedomicrobium sp. strain ACM 3067 is most closely related to P. australicum (level of similarity, 99.6%) and P. americanum (99.4%). These manganese-oxidizing species are more closely related to the iron-oxidizing species Pedomicrobium ferrugineum than to the other manganese-oxidizing species, P. manganicum. Taxonomic uncertainties resulting from the loss of the type culture of P. australicum are discussed.

Transfer of Blastobacter natatorius (Sly 1985) to the genus Blastomonas gen. nov. as Blastomonas natatoria comb. nov.

The budding bacterium Blastobacter natatorius belongs to the alpha-4 group of the Proteobacteria and clusters phylogenetically on a deep branch with Sphingomonas capsulata, with which it shares 93.9% 16S rRNA sequence similarity. On phylogenetic, phenotypic, and chemotaxonomic grounds a proposal is made to transfer B. natatorius to the genus Blastomonas gen. nov. as Blastomonas natatoria comb. nov.

Reassessment of the phylogenetic position of Caulobacter subvibrioides.

Determination of the 16S rRNA gene sequence of Caulobacter subvibrioides ATCC 15264T (T = type strain) confirmed that this species is a member of the alpha subclass of the Proteobacteria and showed that it is phylogenetically most closely related to the Caulobacter group comprising the species Caulobacter bacteroides, Caulobacter crescentus, and Brevundimonas (Pseudomonas) diminuta, for which 16S rRNA sequences of the type strains are currently available. The closest known relative of strain ATCC 15264T among these species is B. diminuta (level of direct pairwise sequence similarity, 95%). On the basis of its previously determined 16S rRNA sequence (accession number M83797), C. subvibrioides is most closely related to Sphingomonas adhaesiva in the alpha-4 subgroup (level of similarity, 97.7%). Analysis of the hydroxy fatty acids of C. subvibrioides ATCC 15264T showed that the 2-hydroxymyristic acid which is characteristic of the genus Sphingomonas was absent.

Chrysiogenes arsenatis gen. nov., sp. nov., a new arsenate-respiring bacterium isolated from gold mine wastewater.

A new strictly anaerobic bacterium (strain BAL-1T) has been isolated from a reed bed at Ballarat Goldfields in Australia. The organism grew by reducing arsenate [As(V)] to arsenite [As(III)], using acetate as the electron donor and carbon source; acetate alone did not support growth. When BAL-1T was grown with arsenate as the terminal electron acceptor, acetate could be replaced by pyruvate, L- and D-lactate, succinate, malate, and fumarate but not by H2, formate, citrate, glutamate, other amino acids, sugars, or benzoate. When acetate was the electron donor, arsenate could be replaced by nitrate or nitrite but not by sulfate, thiosulfate, or iron oxide. Nitrate was reduced to ammonia via nitrite. The doubling time for growth on acetate (5 mM) plus arsenate (5 mM) or nitrate (5 mM) was 4 h. The G+C content of the DNA is 49 mol%. The 16S rRNA sequence data for the organism support the hypothesis that this organism is phylogenetically unique and at present is the first representative of a new deeply branching lineage of the Bacteria. This organism is described as Chrysiogenes arsenatis gen. nov., sp. nov.

Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii, and the blood disease bacterium of banana based on 16S rRNA gene sequences.

We determined nearly complete 16S rRNA gene sequences for 19 isolates of Burkholderia solanacearum, three isolates of the blood disease bacterium of bananas, and two isolates of Pseudomonas syzygii, the cause of Sumatra disease of cloves. The dendrogram produced by comparing all of these sequences revealed that there were two divisions, which corresponded to the results obtained previously in a restriction fragment length polymorphism analysis (D. Cook, E. Barlow, and L. Sequeira, Mol. Plant Microbe Interact. 2:113-121, 1989) and a total 16S ribosomal DNA (rDNA) sequence analysis of four isolates representing four biovars of B. solanacearum (X. Li, M. Dorsch, T. Del Dot, L. I. Sly, E. Stackebrandt, and A. C. Hayward, J. Appl. Bacteriol. 74:324-329, 1993). Division 1 comprised biovars 3, 4, and 5 and an aberrant biovar 2 isolate (strain ACH0732), and division 2 included biovars 1, 2, and N2, the blood disease bacterium, and P. syzygii. Specific nucleotides at positions 458 to 460 (UUC) and 474 (A) characterized division 2, whereas in division 1 the nucleotides at these positions were ACU and U, respectively. However, strain ACH0732 had a U at position 458, as did division 2 isolates, and G instead of U at position 474. Division 2 consisted of two subdivisions; one subdivision contained two B. solanacearum isolates that originated from Indonesia, P. syzygii strains, and blood disease bacterium strains, and the other subdivision contained all of the other division 2 isolates. Within division 1, the level of 16S rDNA sequence similarity ranged from 99.8 to 100%, and within division 2, the levels of 16S rDNA sequence similarity ranged from 99.1 to 100%. The division 1 isolates exhibited an average level of 16S rDNA sequence similarity to division 2 isolates of 99.3% (range, 99.1 to 99.5%). The occurrence of consistent polymorphisms in the 16S rDNA sequences of B. solanacearum strains, in particular unique 16S rDNA sequence differences in aberrant biovar 2 isolate ACH0732, and the occurrence of the Indonesian subdivision of division 2 suggest that this group is a rapidly evolving (tachytelic) group.

The phylogenetic position of the family Methylococcaceae.

The 16S ribosomal DNA-based phylogenetic positions of various members of the Methylococcaceae (group I methanotrophs) were investigated. The Methylococcaceae as a whole formed a distinct branch in the gamma subdivision of the Proteobacteria, and this branch had five distinct subbranches. On the basis of a number of phenotypic traits, phospholipid fatty acid patterns, and the results of a 16S ribosomal DNA analysis, we determined that the species belonging to one subbranch, Methylobacter albus, Methylobacter agilis, and Methylobacter pelagicus, formed a distinct group that could be differentiated from other members of the genus Methylobacter, which grouped in an adjacent subbranch. We propose that these species belong to a new taxon, Methylomicrobium gen. nov.

The phylogenetic position of Helicobacter nemestrinae.

Comparison of the Helicobacter nemestrinae 16S ribosomal DNA with published homologous sequences from members of the genera Helicobacter, Wolinella, and Campylobacter reveals a close relationship between H. nemestrinae, H. pylori, and H. acinonyx. This finding is unexpected since these species differ significantly in their DNA guanine-plus-cytosine contents (24 to 38 mol%).