Lindsay I. Sly

Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria.

OBJECTIVES:Mucosa-associated bacteria are increased in inflammatory bowel disease (IBD), which suggests the possibility of an increased source of digestible endogenous mucus substrate. We hypothesized that mucolytic bacteria are increased in IBD, providing increased substrate to sustain nonmucolytic mucosa-associated bacteria.METHODS:Mucolytic bacteria were characterized by the ability to degrade human secretory mucin (MUC2) in pure and mixed anaerobic cultures. Real-time PCR was used to enumerate mucosa-associated mucolytic bacteria in 46 IBD and 20 control patients. Bacterial mucolytic activity was tested in vitro using purified human MUC2.RESULTS:We confirm increased total mucosa-associated bacteria 16S rRNA gene in macroscopically and histologically normal intestinal epithelium of both Crohns disease (CD) (mean 1.9-fold) and ulcerative colitis (UC) (mean 1.3-fold). We found a disproportionate increase in some mucolytic bacteria. Mean Ruminococcus gnavus were increased >4-fold and Ruminococcus torques ∼100-fold in macroscopically and histologically normal intestinal epithelium of both CD and UC. The most abundantly detected mucolytic bacterium in controls, Akkermansia muciniphila, was reduced many fold in CD and in UC. Coculture of A. muciniphila with MUC2 as the sole carbon source led to reduction in its abundance while it augmented growth of other bacteria.CONCLUSIONS:Mucolytic bacteria are present in healthy humans, where they are an integral part of the mucosa-associated bacterial consortium. The disproportionate increase in R. gnavus and R. torques could explain increased total mucosa-associated bacteria in IBD.

A New Chemolithoautotrophic Arsenite-Oxidizing Bacterium Isolated from a Gold Mine: Phylogenetic, Physiological, and Preliminary Biochemical Studies

ABSTRACT 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 theAgrobacterium/Rhizobium branch of the α-Proteobacteria and may represent a new species. This recently discovered organism is the most rapidly growing chemolithoautotrophic arsenite oxidizer known.

Revised Taxonomy of the Methanotrophs: Description of Methylobacter gen. nov., Emendation of Methylococcus, Validation of Methylosinus and Methylocystis Species, and a Proposal that the Family Methylococcaceae Includes Only the Group I Methanotrophs

Numerical taxonomic, DNA-DNA hybridization, and phospholipid fatty acid composition analyses were performed on an extensive range of methanotrophic strains, including reference strains and environmental isolates obtained from sites throughout eastern Australia. When the results of these studies were related to the results of a study based on genomic physicochemical properties, they clarified group I and II methanotroph genus and species interrelationships. The group I methanotrophs were found to be made up of three broadly phenotypically and genotypically homologous clusters of species. The first group I methanotroph cluster included the carotenoid-containing species Methylomonas methanica, Methylomonas fodinarum, and Methylomonas aurantiaca. These species represent the true members of the genus Methylomonas. The second group I methanotroph cluster was made up of two subclusters of strains. One subcluster included species not capable of producing resting cells and consisted of the species “Methylomonas agile,” “Methylomonas alba,” and Methylomonas pelagica. The other subcluster included species capable of forming desiccation-resistant cysts and included Methylococcus luteus, marine Methylomonas-like strains, and Methylococcus whittenburyi. Strains designated “Methylococcus ucrainicus” and Methylococcus vinelandii were found to be synonyms of Methylococcus whittenburyi, while Methylococcus bovis was a synonym of Methylococcus luteus. It is proposed that these subclusters represent a new genus, Methylobacter gen. nov. The species in the new genus are type species Methylobacter luteus comb. nov., Methylobacter agilis sp. nov., Methylobacter albus sp. nov., nom. rev., Methylobacter marinus sp. nov., Methylobacter pelagicus comb. nov., and Methylobacter whittenburyi comb. nov. The remaining group I methanotrophs included the moderately thermophilic species Methylococcus capsulatus and Methylococcus thermophilus and a group of unnamed strains closely related to Methylococcus capsulatus. It is proposed that these species represent the true members of the genus Methylococcus. The group II methanotrophs consisted of two closely related groups. The first group included budding, exospore-producing strains, while the second group included nonmotile, cyst-forming strains. These groups represent the genera Methylosinus and Methyocystis, which are revived here. The genus Methylosinus gen. nov., nom. rev. includes the species Methylosinus trichosporium sp. nov., nom. rev. and Methylosinus sporium sp. nov., nom. rev., while the genus Methylocystis gen. nov., nom. rev. includes the species Methylocystis parvus sp. nov., nom. rev. and Methylocystis echinoides sp. nov., nom. rev.

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

Abstract 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.

Thauera selenatis gen. nov., sp. nov., a member of the beta subclass of Proteobacteria with a novel type of anaerobic respiration.

A recently isolated, selenate-respiring microorganism (strain AXT [T = type strain]) was classified by using a polyphasic approach in which both genotypic and phenotypic characteristics were determined. Strain AXT is a motile, gram-negative, rod-shaped organism with a single polar flagellum. On the basis of phenotypic characteristics, this organism can be classified as a Pseudomonas sp. However, a comparison of the 16S rRNA sequence of strain AXT with the sequences of other organisms indicated that strain AXT is most similar to members of the beta subclass (level of similarity, 86.8%) rather than to members of the gamma subclass (level of similarity, 80.2%) of the Proteobacteria. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) excluded strain AXT from the authentic genus Pseudomonas and allowed placement in the beta subclass of the Proteobacteria. Within the beta subclass, strain AXT is related to Iodobacter fluvatile. The phylogenetic distance (level of similarity, less than 90%), as well as a lack of common phenotypic characteristics between these organisms, prevents classification of strain AXT as a member of the genus Iodobacter. In addition, strain AXT possesses a unique mechanism for anaerobic respiration, which allows it to utilize selenate as an electron acceptor without interference by nitrate. Therefore, we propose that strain AXT should be the first member of a new genus and species, Thauera selenatis.

The use of second derivative plots for the determination of mol% guanine plus cytosine of DNA by the thermal denaturation method

Procedures for the estimation of thermal denaturation temperatures for the determination of the mol% G+C base composition of bacterial DNA have been examined with a view to simplifying the procedure and taking advantage of developments in microprocessor controlled spectrophotometers. Thermal denaturation profiles of DNA from 40 strains with base compositions ranging from 35 to 70 mol% G+C were studied. Thermal denaturation temperatures were determined by the conventional Tm method and by using second derivative plots to determine the temperature of inflection (Ti). The results showed that there is a close linear correlation between mol% G+C values calculated using Tm or Ti as the thermal denaturation temperature, provided the thermal denaturation temperature of the internal reference DNA is estimated in the same way. Actual Ti values were approximately 0.3°C higher than corresponding Tm values but the increase was consistent across the range of DNA samples studied. The results also showed that the error introduced by disregarding correction for the effect of thermal expansion was not consistent across the range of DNA samples studied. However, the error was small and in practical terms could be neglected.

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.

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.

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.

Streptococcus gallolyticus sp. nov. ; gallate degrading organisms formerly assigned to Streptococcus bovis

Summary Phenotypic charcteristics including the abilites two hydrolyze tannin and to decarboxylate gallic acid in 31 strains of S. bovis and 3 strains of S. equinus were compared with their dexoyribonucleic acid (DNA) relatedness. It was found that the strains capable of decarboxylating gallic acid all belong to a single DNA homology group which does not include either the type strain of S. bovis or the type strain of S. equinus . Thus it was concluded that the gallate degrading strains of S. bovis should be re-assigned to a new species, for which the name Streptococcus gallolyticus sp. nov. is proposed.