The use of bioluminescence for monitoring in planta growth dynamics of a Pseudomonas syringae plant pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Resolving the energy paradox of chaperone/usher-mediated fibre assembly

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit preassembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.

Detection and enumeration of sulphate-reducing bacteria in estuarine sediments by competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 - 5.7 × 108 cells ml-1 wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10-17 to 10-15 mol of SO42- cell-1 day-1 were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: Preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.

Bacteroides coprosuis sp nov., isolated from swine-manure storage pits

Two Gram-negative, anaerobic, non-spore-forming, rod-shaped organisms were isolated from a swine-manure storage pit. Based on morphological and biochemical criteria, the strains were tentatively identified as belonging to the genus Bacteroides but they did not appear to correspond to any recognized species of the genus. Comparative 16S rRNA gene sequencing studies showed that the strains were related closely to each other and confirmed their placement in the genus Bacteroides, but sequence divergence values of > 10% from reference Bacteroides species demonstrated that the organisms from manure represent a novel species. Based on biochemical criteria and molecular genetic evidence, it is proposed that the unknown isolates from manure be assigned to a novel species of the genus Bacteroides, as Bacteroides coprosuis sp. nov. The type strain is PC139(T) (=CCUG 50528(T)=NRRL B-41113(T)).

Uruburuella suis gen. nov., sp nov., isolated from clinical specimens of pigs

Five strains of an unusual Gram-negative, catalase-positive, oxidase-positive, coccobacillus-shaped bacterium isolated from the lungs and heart of pigs with pneumonia and pericarditis were characterized by phenotypic and molecular genetic methods. On the basis of cellular morphology and biochemical criteria, the isolates were tentatively assigned to the family Neisseriaceae, although they did not appear to correspond to any recognized genus or species. Comparative 16S rRNA gene sequencing showed that the five unidentified strains were phylogenetically highly related to each other and represent a hitherto unknown subline within the family Neisseriaceae. On the basis of both phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from pigs be classified as a novel genus and species within the family Neisseriaceae, for which the name Uruburuella suis gen. nov., sp. nov. is proposed. The type strain of U. suis is 1258/02(T) (=CCUG 47806(T) =CECT 5685(T)).

Psychrobacter pulmonis sp nov., isolated from the lungs of lambs

Unusual Gram-negative, catalase- and oxidase-positive, coccus-shaped bacteria isolated from the lungs of two lambs were characterized by phenotypic and molecular-genetic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown isolates were genealogically highly related to each other (99.8% sequence similarity) and represent a novel subline within the genus Psychrobacter. The unknown bacterium was phylogenetically closely related to, but distinct from, Psychrobacter phenylpyruvicus, Psychrobacter immobilis, Psychrobacter glacincola and Psychrobacter urativorans. The novel Psychrobacter isolates were readily distinguished from all other Psychrobacter species and other Gram-negative, oxidase-positive bacteria usually responsible for lung infections in sheep by physiological and biochemical tests. Based on molecular-genetic and phenotypic evidence, it is proposed that the unknown Psychrobacter isolates from lambs be classified as Psychrobacterpulmonis sp. nov. The type strain is strain S-606(T) (= CECT 5989(T) = CCUG 46240(T)).

Reclassification of Bacteroides putredinis (Weinberg et al., 1937) in a New Genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp nov., from human sources

During studies on the bacteriology of appendicitis in children, we often isolated from inflamed and non-inflamed tissue samples, an unusual bile-resistant pigment-producing strictly anaerobic gram-negative rod. Phenotypically this organism resembles members of Bacteroides fragilis group of species, as it is resistant to bile and exhibits a special-potency-disk pattern (resistance to vancomycin, kanamycin and colistin) typical for the B. fragilis group. However, the production of brown pigment on media containing haemolysed blood and a cellular fatty acid composition dominated by iso-C15:0, suggests that the organism most closely resembles species of the genus Porphyromonas. However, the unidentified organism differs from porphyromonads by being bile-resistant and by not producing butyrate as a metabolic end-product. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism represents a distinct sub-line, associated with but distinct from, the miss-classified species Bacteroides putredinis. The clustering of the unidentified bacterium with Bacteroides putredinis was statistically significant, but they displayed >4% sequence divergence with each other. Chromosomal DNA-DNA pairing studies further confirmed the separateness of the unidentified bacterium and Bacteroides putredinis. Based on phenotypic and phylogenetic considerations, it is proposed that Bacteroides putredinis and the unidentified bacterium from human sources be classified in a new genus Alistipes, as Alistipes putredinis comb. nov. and Alistipes finegoldii sp. nov., respectively. The type strain of Alistipes finegoldii is CCUG 46020(T) (= AHN2437(T)).

Description of Kingella potus sp nov., an organism isolated from a wound caused by an animal bite

We report the isolation and characterization of a hitherto unknown gram-negative, rod-shaped Neisseria-like organism from an infected wound resulting from a bite from a kinkajou. Based on both phenotypic and phylogenetic evidence, it is proposed that the unknown organism be classified as a new species, Kingella potus sp. nov.

Saccharospirillum impatiens gen. nov., sp nov., a novel gamma-Proteobacterium isolated from hypersaline Ekho Lake (East Antarctica)

Five Gram-negative, motile, aerobic to microaerophilic spirilla were isolated from various depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica). The strains are oxidase- and catalase-positive, metabolize a variety of sugars and carboxylic acids and have an absolute requirement for sodium ions. The predominant fatty acids of the organisms are C-16: (1)omega7c, C-16:0 and C(18:1)omega7c, with C-10:1 3-OH, C-10:0 3-OH, C-12:0 3-OH, C-14:1 3-OH, C-14:0 3-OH and C-19:1 present in smaller amounts. The main polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmonomethylamine. The DNA base composition of the strains is 54-55 mol% G + C. 16S rRNA gene sequence comparisons show that the isolates are related to the genera Oceanospirillum, Pseudospirillum, Marinospirillum, Halomonas and Chromohalobacter in the gamma-Proteobacteria. Morphological, physiological and genotypic differences from these previously described genera support the description of a novel genus and species, Saccharospirillum impatiens gen. nov., sp. nov. The type strain is EL-105(T) (= DSM 12546(T) = CECT 5721(T)).

Roseisalinus antarcticus gen. nov., sp nov., a novel aerobic bacteriochlorophyll a-producing alpha-proteobacterium isolated from hypersaline Ekho Lake, Antarctica

A Gram-negative, aerobic to microaerophilic rod was isolated from 10 m depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica). The strain was oxidase- and catalase-positive, metabolized a variety of carboxylic acids and sugars and produced lipase. Cells had an absolute requirement for artificial sea water, which could not be replaced by NaCl. A large in vivo absorption band at 870 nm indicated production of bacteriochlorophyll a. The predominant fatty acids of this organism were 16:0 and 18:1omega7c, with 3-OH 10:0, 16:1omega7c and 18:0 in lower amounts. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. Ubiquinone 10 was produced. The DNA G + C content was 67 mol%. 16S rRNA gene sequence comparisons indicated that the isolate represents a member of the Roseobacter clade within the alpha-Proteobacteria. The organism showed no particular relationship to any members of this clade but clustered on the periphery of the genera Jannaschia, Octadecabacter and 'Marinosulfonomonas' and the species Ruegeria gelatinovorans. Distinct morphological, physiological and genotypic differences to these previously described taxa supported the description of a new genus and a novel species, for which the name Roseisalinus antarcticus gen. nov., sp. nov. is proposed. The type strain is EL-88(T) (= DSM 11466(T) = CECT 7023(T)).

Subdoligranulum variabile gen. nov., sp nov from human feces

During studies on the microflora of human feces we have isolated a strictly anaerobic, non-spore-forming, Gram-negative staining organism which exhibits a somewhat variable coccus-shaped morphology. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism is phylogenetically a member of the Clostridium leptum supra-generic rRNA cluster and displays a close affinity to some rDNA clones derived from human and pig feces. The nearest named relatives of the unidentified isolate corresponded to Faecalibacterium prausnitzii (formerly Fusobacterium prausnitzii) displaying a 16S rRNA sequence divergence of approximately 9%, with Anaerofilum agile and A. pentosovorans the next closest relatives of the unidentified bacterium (sequence divergence approximately 10%). Based on phenotypic and phylogenetic considerations, it is proposed that the unusual coccoid-shaped organism be classified as a new genus and species, Subdoligranulum variabile. The type strain of S. variabile is BI 114(T) (= CCUG 47106(T) = DSM 15176(T)). (C) 2004 Elsevier Ltd. All rights reserved.

Sutterella sterconcanis sp nov., isolated from canine faeces

Morphological, biochemical and molecular genetic studies were carried out on an unknown non-spore-forming, Gram-negative, rod-shaped bacterium which was isolated from dog faeces. The bacterium grew under anaerobic conditions, was asaccharolytic, resistant to 20% (v/v) bile and was oxidase- and urease-negative. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that the unidentified bacterium clustered with Sutterella wadsworthensis, although a sequence divergence of > 5% indicated that the bacterium from dog faeces represented a previously unrecognized subline within the genus. On the basis of the presented findings, a novel species, Sutterella stercoricanis sp. nov., is described. The type strain of Sutterella stercoricanis is 5BAC4(T) ( = CCUG 47620(T) = CIP 108024(T)).

Suttonella ornithocola sp nov., from birds of the tit families, and emended description of the genus Suttonella

Phenotypic and phylogenetic studies were performed on three strains of Gram-negative, rod-shaped organisms recovered from dead birds of the tit families (blue tit, coal tit and long-tailed tit). Morphological, cultural and biochemical studies indicated that the organisms were related to the family Cardiobacteriaceae in the gamma-subclass of the Proteobacteria. Comparative 16S rRNA gene sequencing studies confirmed these findings and demonstrated that the bacterium represents a hitherto unknown subline within this family. The closest phylogenetic relative of the strains isolated from the birds was found to be Suttonella indologenes, although a sequence divergence of approximately 5 % demonstrated that the unknown bacterium represented a novel species. On the basis of the results of the phylogenetic analysis and phenotypic criteria, it is proposed that the bacteria recovered from the diseased birds represent a novel species, Suttonella ornithocola sp. nov., with strain B6/99/2(T) (=CCUG 49457(T)=NCTC 13337(T)) as the type strain.

Cetobacterium somerae sp nov from human feces and emended description of the genus Cetobacterium

Phenotypic and phylogenetic studies were performed on four isolates of an unidentified gram-negative, microaerotolerant, non-spore-forming, rod-shaped bacterium isolated from the feces of children. The unknown organism was bile resistant and produced acetic acid as the major end product of metabolism of peptides and carbohydrates. It possessed a low DNA G + C content of 31 mol %. Comparative 16S rRNA gene sequencing demonstrated that the four isolates were phylogenetically identical (100% 16S rRNA sequence similarity) and represent a hitherto unknown sub-line within the genus Cetobacterium. The novel bacterium displayed approximately 5% sequence divergence with Cetobacterium ceti, and can be readily distinguished from the latter by physiological and biochemical criteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown fecal bacterium be classified in the genus Cetobacterium, as Cetobacterium somerae sp. nov. The proposed type strain of Cetobacterium somerae is WAL 14325(T) (ATCC BAA-474(T) = CCUG 46254T).