Addressing the sublime scale of the microbial world: reconciling an appreciation of microbial diversity with the need to describe species.

There are fewer than 20,000 prokaryotic species with validly published names, meaning >99% of a reasonable estimate of microbial diversity remains formally unnamed. Here we explore the damaging consequences of the current practice in which each new species is described in a standardized publication, most typically a 'single strain species description'. This approach is both an impediment to scaling up progress in naming the microbial world and also a significant factor in the poor reputation of the discipline of microbial taxonomy. We conclude that significant changes in author habits are needed and make constructive suggestions as to how author practice should adapt.

Genomic analysis of a novel nontoxigenic Corynebacterium diphtheriae strain isolated from a cancer patient.

The genome of a novel nontoxigenic Corynebacterium diphtheriae, strain 5015, isolated from a patient with adenoid cystic carcinoma was sequenced and compared with 117 publically available genomes. This strain is phylogenetically distinct and lacks virulence genes encoding the toxin, BigA and Sdr-like adhesins. Strain 5015 possesses spaD-type and spaH-type pilus gene clusters with a loss of some gene functions, and 31 unique genes that need molecular characterization to understand their potential role in virulence characteristics.

Genomic analyses reveal two distinct lineages of Corynebacterium ulcerans strains.

Corynebacteriumulcerans is an important zoonotic pathogen which is causing diphtheria-like disease in humans globally. In this study, the genomes of three recently isolated C. ulcerans strains, 4940, 2590 and BR-AD 2649, respectively from an asymptomatic carrier, a patient with pharyngitis and a canine host, were sequenced to investigate their virulence potential. A comparative analysis was performed including the published genome sequences of 16 other C. ulcerans isolates. C. ulcerans strains belong to two lineages; 13 strains are grouped together in lineage 1, and six strains comprise lineage 2. Consistent with the zoonotic nature of C. ulcerans infections, isolates from both the human and canine hosts clustered in both the lineages. Most of the strains possessed spaDEF and spaBC gene clusters along with the virulence genes cpp, pld, cwlH, nanH, rpfI, tspA and vsp1. The gene encoding Shiga-like toxin was only present in one strain, and 11 strains carried the tox gene encoding the diphtheria-like toxin. However, none of strains 4940, 2590 and BR-AD 2649 carried any toxin genes. These strains varied in the number of prophages in their genomes, which suggests that they play an important role in introducing diversity in C. ulcerans. The pan-genomic analyses revealed a variation in the number of membrane-associated and secreted proteins that may contribute to the variation in pathogenicity among different strains.

Phenotypic variation in Streptomyces sp. DSM 40537, a lipoteichoic acid producing actinomycete.

AIMS: To reinvestigate the production of lipoteichoic acid (LTA) by the actinomycete strain Streptomyces sp. DSM 40537 (=ATCC 3351). METHODS AND RESULTS: LTA was extracted and purified from strain Streptomyces sp. DSM 40537. The identification of the LTA was confirmed by Western blotting with a monoclonal antibody. During these studies, two stable phenotypic variants of DSM 40537 were obtained, one of which released a distinctive orange pigment. 16S rRNA gene sequencing of each variant yielded identical sequences and allowed phylogenetic analysis to be performed. CONCLUSIONS: Streptomyces sp. DSM 40537 was shown to exhibit stable morphological variation. The strain was confirmed to be a LTA-producing actinomycete and to belong to the Streptomyces albidoflavus cluster within the genus Streptomyces. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide important support for the hypothesis that the distribution of LTA is linked to that of wall teichoic acids and emphasizes the need to reinvestigate LTA distribution in actinomycetes.

Reclassification of [Pseudomonas] doudoroffii (Baumann et al. 1983) into the genus Oceanomonas gen. nov. as Oceanomonas doudoroffii comb. nov., and description of a phenol-degrading bacterium from estuarine water as Oceanomonas baumannii sp. nov.

A bacterium, isolate GB6T, capable of degrading phenol in the presence of elevated salinity was isolated from the estuary of the River Wear, UK. The bacterium was subjected to biochemical and molecular analysis to determine its taxonomic status. These studies indicated that the bacterium was a distinct species closely related to [Pseudomonas] doudoroffii. However, the phylogenetic analysis indicated that [Pseudomonas] doudoroffii was misclassified, as noted previously. Analysis of the characteristics of isolate GB6T and the type strain of [Pseudomonas] doudoroffii confirmed that these bacteria belonged to the same novel genus, which we have named Oceanomonas gen. nov. The type strain of Oceanomonas doudoroffii (Baumann et al. 1983) comb. nov. is ATCC 27123T (= DSM 7028T. The DNA-DNA homology between isolate GB6T and [Pseudomonas] doudoroffii is low and phenotypic differences between the two organisms are evident. Isolate GB6T (= ATCC 700832T = NCIMB 13685T) is therefore proposed as the type strain of a new species, Oceanomonas baumannii sp. nov.

Surface immunolocalisation of HPr in the equine pathogen Streptococcus equi.

We have investigated the surface localisation of the phosphotransferase system protein HPr in the equine pathogen Streptococcus equi subsp. equi using immunogold localisation and transmission electron microscopy. Like the LppC acid phosphatase lipoprotein, a reference surface antigen, the S. equi HPR could be clearly detected on the surfaces of intact cells. This study is consistent with previous reports that some streptococcal HPr is cell surface associated and suggests that the extracytoplasmic mobilisation and transfer of phosphate groups by streptococci warrant further investigation.

Characterization of acid phosphatase activities in the equine pathogen Streptococcus equi.

Acid phosphatases hydrolyse phosphomonoesters at acidic pH in a variety of physiological contexts. The recently defined class C family of acid phosphatases includes the 32 kDa LppC lipoprotein of Streptococcus equisimilis. To define further the distribution of acid phosphatases in the genus Streptococcus we have examined the equine pathogens Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Whole cell assays indicated that these organisms possess two acid phosphatases with activity optima at pH 5.0 and pH 6.0-6.5 and that only the former of these was, like LppC, resistant to EDTA. Western blotting with a polyclonal anti-LppC antiserum revealed the presence of a cross-reactive 32 kDa protein in both organisms. The cross-reactive protein in S. equi was shown to be a surface accessible lipoprotein as its processing was inhibited by the antibiotic globomycin and it was released from whole cells by treatment with trypsin. The presence of DNA sequences homologous to the S. equisimilis lppC gene were confirmed by PCR. These data strongly suggest that Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus produce a lipoprotein acid phosphatase homologous to LppC of S. equisimilis.

Identification of methionine-processed HPr in the equine pathogen Streptococcus equi.

Using preparative electrophoresis, a low molecular weight protein has been partially purified from a cell extract of the equine pathogen Streptococcus equi susp. equi. N-terminal sequence analysis and Western blotting revealed the protein to be HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Interestingly, the only form of the HPr protein detected in S. equi was one with the amino-terminal methionine removed, a modification that has previously been associated with surface localization of streptococcal HPr proteins.

Identification of lipoprotein homologues of pneumococcal PsaA in the equine pathogens Streptococcus equi and Streptococcus zooepidemicus.

Streptococcus equi and Streptococcus zooepidemicus are major etiological agents of upper and lower airway disease in horses. Despite the considerable animal suffering and economic burden associated with these diseases, the factors that contribute to the virulence of these equine pathogens have not been extensively investigated. Here we demonstrate the presence of a homologue of the Streptococcus pneumoniae PsaA protein in both of these equine pathogens. Inhibition of signal peptide processing by the antibiotic globomycin confirmed the lipoprotein nature of the mature proteins, and surface exposure was confirmed by their release from intact cells by mild trypsinolysis.

The modification of the membrane of Oceanomonas baumannii when subjected to both osmotic and organic solvent stress.

Oceanomonas baumanniioff a novel halotolerant bacterium which was isolated from the estuary of the river Wear (Sunderland, UK). When grown in tryptone soya broth it can tolerate high levels of phenol, which is not utilised as a carbon source in this medium. However, the level of tolerance was reduced from 35 mM to 3 mM phenol as salinity increased from 1% to 12% NaCl (w/v). Increasing salinity up to 12% NaCl also decreased the growth rate 8-fold and caused modifications to the cytoplasmic membrane particularly anionic phosphatidylglycerol levels, which doubled at the expense of zwitterionic phosphatidylethanolamine. In addition, changes in the phospholipid fatty acid composition were noted, cis-vaccenic acid decreased significantly at higher salinities. Intracellular solute levels also increased with increasing salinity and there was an accumulation of the compatible solutes ectoine, glycine betaine and glutamate. The addition of phenol to osmotically compromised cultures led to a further modification of the cytoplasmic membrane phospholipid composition, in particular, that the decrease in zwitterionic phosphatidylethanolamine and the increase of anionic phospholipid species was much less pronounced. A further decrease in unsaturation, particularly in the proportion of cis-vaccenic acid, and the mean chain length of the fatty acids suggested that this response was important in maintaining membrane integrity in the presence of phenol.

Characterisation of a lipomannan lipoglycan from the mycolic acid containing actinomycete Dietzia maris.

Lipoglycans such as the mycobacterial lipoarabinomannans (LAM) are important cell envelope components of actinomycetes. To further our understanding of the diversity of these enigmatic macromolecules the lipoglycan composition of Dietzia maris has been investigated. Phenol-water extraction and hydrophobic interaction chromatography were used to purify a lipoglycan which was unusually small and predominantly lipomannan in nature. The presence of minor levels of arabinose along with components consistent with the presence of a phosphatidylinositol anchor suggest that this lipoglycan is a novel representative of the lipomannan/LAM structural archetype. This was further supported by the observed cross-reaction of the D. maris lipoglycan with an antiserum raised against LAM from Mycobacterium tuberculosis. These findings reveal a previously unsuspected diversity in the lipoglycan composition of the mycolic acid containing actinomycetes and are further discussed in relation to the apparent absence of phosphatidylinositolmannoside glycolipids in D. maris.

Identification of a lipoarabinomannan-like lipoglycan in Gordonia rubropertincta.

Lipoarabinomannans are well characterised lipoglycans present in the cell envelopes of mycobacteria and closely related bacteria. To define further the distribution of these lipoglycans we have investigated the mycolic acid-containing bacterium Gordonia rubropertincta and, by analysis of carbohydrate composition and antigenic cross-reactivity, demonstrated the presence of a lipoarabinomannan-like lipoglycan. A fraction that appeared to contain higher phosphatidylinositolmannosides was also isolated.

Cell envelope composition and organisation in the genus Rhodococcus.

A knowledge of the organisation of the rhodococcal cell envelope is of fundamental importance if the environmental and biotechnological significance of these bacteria are to be understood and successfully exploited. The genus Rhodococcus belongs to a distinctive suprageneric taxon, the mycolata, which includes among others the genera Corynebacterium, Mycobacterium and Nocardia. Members of this taxon exhibit an unusual complexity in their cell envelope composition and organisation compared to other Gram-positive bacteria. Models that describe the architecture of the mycobacterial cell envelope are extrapolated here to provide a model of the rhodococcal cell envelope. The rhodococcal cell envelope is dominated by the presence of an arabinogalactan cell wall polysaccharide and large 2-alkyl 3-hydroxy branched-chain fatty acids, the mycolic acids, which are covalently assembled into a peptidoglycan-arabinogalactan-mycolic acid matrix. This review further emphasises that the mycolic acids in this complex form the basis of an outer lipid permeability barrier. The localisation and roles of other cell envelope components, notably complex free lipids, lipoglycans, proteins and lipoproteins are also considered.

Macroamphiphilic cell envelope components of Rhodococcus equi and closely related bacteria.

Recent progress towards an understanding of the architecture of the mycobacterial cell envelope (P.J. Brennan and H. Nikaido, Annual Review of Biochemistry 64 (1995) 29-63) provides a model with features more generally applicable to cell envelope organisation in other mycolic acid-containing bacteria. Using this archetype, a model for the organisation of the rhodococcal cell envelope is presented here, with particular reference to cell envelope composition in Rhodococcus equi. The likelihood that mycolic acids bound to the cell wall arabinogalactan contribute to the formation of a distinct outer lipid layer is emphasised. Furthermore, the model incorporates recent work which has characterised rhodococcal macroamphiphiles (lipoglycans and lipoproteins), including the VapA virulence-associated lipoproteins of R. equi.

An investigation of the intra-generic structure of Rothia by pyrolysis mass spectrometry.

Previous studies of the genus Rothia have indicated that members of the only species, Rothia dentocariosa, are heterogeneous and may form more than one species. To study the intrageneric taxonomic structure of the Rothia taxon eighteen strains identified as R. dentocariosa, including reference organisms from culture collections (3 strains), isolates from healthy subjects (5 strains) and from clinical sources (10 strains) were examined using pyrolysis mass spectrometry. The ordination plots of the pyrolysis data indicated that all the strains clustered in a closely related group, with the exception of three strains which out-grouped. Phenotypic testing and fatty acid data indicated that the latter three strains are probably misclassified in the genus Rothia. Reanalysis of the PyMS data including only the fifteen authentic Rothia strains indicated that ten of these organisms formed a group which included the type strain, R. dentocariosa NCTC 10917. Four out of the remaining five organisms formed a diffuse group; the remaining strain was recovered as a single member cluster. These data indicate that R. dentocariosa is heterogeneous though at present there are no suitable criteria for assigning members of this taxon to more than one species.

Organization and nucleotide sequence of the Streptococcus mutans galactose operon.

The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The Leloir pathway was induced by the presence of galactose in the growth medium or following the release of intracellular galactose after uptake and cleavage of alpha-galactosides by the multiple sugar metabolism system. Analysis of the mechanism of galactose transport confirmed the absence of a galactose-specific phosphotransferase system and suggested the presence of an inducible galactose permease. Evidence is presented that galactose transport is independent of the proton motive force and may be ATP-dependent.

A chemotaxonomic study of the lipoglycans of Rhodococcus rhodnii N445 (NCIMB 11279).

Rhodococcus rhodnii N445 was investigated for the presence of macroamphiphilic lipoglycan. Purification of a hot phenol-water extract by hydrophobic interaction chromatography allowed the resolution of three lipoglycan fractions. The two main preparations contained lipoglycans with carbohydrate compositions consistent with the presence of lipoarabinomannan and lipomannan, whilst the minor fraction appeared to contain a mixture of these two lipoglycans. The fatty acid composition of the lipoglycans resembled that of the whole cells except that the relative proportion of unsaturated fatty acids was decreased. Although lipoarabinomannan and structurally-related lipomannan lipoglycans from representatives of the genus Mycobacterium have been extensively studied, this is the first report of the lipoglycan composition of a representative of the genus Rhodococcus as presently defined. These findings provide further chemotaxonomic evidence that lipoarabinomannan-type lipoglycans are widely distributed throughout the mycolic acid-containing actinomycetes.

Identification of a lipoarabinomannan-like lipoglycan in Corynebacterium matruchotii.

The oral organism Corynebacterium matruchotii was investigated for the presence of lipoteichoic acid, as this common polyanionic macroamphiphilic component of Gram-positive bacteria has been implicated in phenomena related to calcium binding. Phenol-water extraction followed by a small-scale, hydrophobic-interaction chromatography step yielded carbohydrate-containing preparations that were distinguished from lipoteichoic acid by their low phosphorus content. Subsequently, large-scale phenol-water extracts from each of three strains of C. matruchotii were purified by hydrophobic-interaction chromatography and shown to contain a heterogeneous lipoglycan fraction. The major fatty acids present were the same as for the whole-cell fatty acid profiles but differed in their relative amounts. Qualitative analysis of the lipoglycan fractions revealed similarities of carbohydrate composition with a previously characterized lipoglycan fraction from C. diphtheriae and with the lipoarabinomannan/lipomannans found in the genus Mycobacterium. The carbohydrate composition and the low phosphorus content indicated that lipoteichoic acid was absent from C. matruchotii. The calcium-binding properties of C. matruchotii therefore cannot be attributed to lipoteichoic acid.