Reiner M. Kroppenstedt

The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov.

The genus Nocardiopsis was shown to be phylogenetically coherent and to represent a distinct lineage within the radiation of the order Actinomycetales. The closest relatives of the genus Nocardiopsis are members of the genera Actinomadura, Thermomonospora, Streptosporangium, and Microtetraspora. The intrageneric structure of the genus Nocardiopsis is shown to consist of a highly related species group containing Nocardiopsis dassonvillei, Nocardiopsis alborubida, and Nocardiopsis antarctica and a second group of less highly related species comprising Nocardiopsis alba subsp. alba, Nocardiopsis alba subsp. prasina, and Nocardiopsis listeri. Nocardiopsis lucentensis occupies a position intermediate between the two species groups. The results of a 16S ribosomal DNA sequence analysis are generally consistent with the available chemotaxonomic, phenotypic, and DNA-DNA hybridization data. The phylogenetic position and the morpho- and chemotaxonomic properties of Nocardiopsis species support the creation of a family for the genus Nocardiopsis, Nocardiopsaceae fam. nov.

Effects of T4 Lysozyme Release from Transgenic Potato Roots on Bacterial Rhizosphere Communities Are Negligible Relative to Natural Factors

ABSTRACT Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or α- or β-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.

Phylogeny of the Genus Nocardia Based on Reassessed 16S rRNA Gene Sequences Reveals Underspeciation and Division of Strains Classified as Nocardia asteroides into Three Established Species and Two Unnamed Taxons

ABSTRACT Conventional identification of Nocardia in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified as belonging to the species Nocardia asteroides, which comprises the type strain and isolates with drug pattern types II and VI. The 16S rRNA gene of 74 representative strains of the genus Nocardia, encompassing 25 established species, was sequenced in order to provide a molecular basis for accurate species identification and with the aim of reassessing the phylogeny of taxons assigned to the species N. asteroides. The result of this phylogenetic analysis confirms that the interspecies heterogeneity of closely related nocardial species can be considerably low (a sequence divergence of only 0.5% was found between N. paucivorans and N. brevicatena). We observed a sequence microheterogeneity (sequence divergence of fewer than five bases) in 8 of 11 species of which more than one strain in the species was studied. At least 10 taxons were found that merit description as new species. Strains previously classified as N. asteroides fell into five distinct phylogenetic groups: the type strain cluster (N. asteroides sensu strictu), N. abscessus, N. cyriacigeorgica, and two clusters closely related to N. carnea or N. flavorosea. The strains within the latter two groups probably represent new species, pending further genetic and phenotypic evaluation. Restricted phenotypic data revealed that N. abscessus, N. cyriacigeorgica, and the two Nocardia species taxons are equivalent to drug patterns I, VI, and II, respectively. In the future, these data will help in finding species-specific markers after adoption of a more precise nomenclature for isolates closely related to N. asteroides and unravel confusing phenotypic data obtained in the past for unresolved groups of strains that definitely belong to separate taxons from a phylogenetic point of view.

A numerical classification of the genera Streptomyces and STreptoverticillium using miniaturized physiological tests

Eight hundred and twenty-one strains of the genera Streptomyces and Streptoverticillium were physiologically characterized using a total of 329 miniaturized tests. Overall similarities of all strains were determined by numerical taxonomic techniques using the UPGMA algorithm and the SSM and the S J coefficients as measures of similarity. Test error was within acceptable limits. Comparison of photometric and visual test reading revealed overall differences of 7.45%. A total of 15 major clusters (six or more strains), 34 minor clusters (less than six strains) and 40 single-member clusters were defined at the 81.5% similarity level (S SM). Two clusters containing physiologically, and in some cases morphologically and genetically, different groups could be further subdivided at the 84.0% similarity level (S SM). Generally, similar groupings were obtained with the Jaccard coefficient at similarity levels ranging from 59.6% to 64.6% similarity (S J), with changes in the definition of clusters and subclusters. The cophenetic correlation coefficients r CS for the UPGMA/S J and the UPGMA/S sm analysis were 0.6929 and 0.8239, respectively. Several phena showed significant overlap with others, indicating the physiological variability within the species. The phenetic data in most cases confirm the major phena of the study of Williams et al. (1983), Journal of General Microbiology 129, 1743-1813 (although the cluster-groups defined in that study could only be detected in part) and the results indicate that the genus Streptomyces is still overspeciated. Some of the major groupings obtained were very much in line with chemotaxonomic and genetical data. However, several clusters containing only a few strains should be regarded as preliminary ‘species’ until further information is available. The majority of Streptoverticillium strains presently assigned to different species formed a homogeneous subcluster defined at the 84.0% similarity level (S SM). Thus, on the basis of numerical phenetic and (published) molecular genetic and chemotaxonomic data, our study supports the suggestion that members of the genus Streptoverticillium be reclassified into the genus Streptomyces.

Dietzia, a new genus including Dietzia maris comb. nov., formerly Rhodococcus maris.

Sequencing of the 16S ribosomal DNAs (rDNA) of two strains of Rhodococcus maris was performed to determine the relationship of this species to other mycolic acid-containing actinomycetes. For this purpose we also determined the 16S rDNA sequences for the type species of the genus Rhodococcus, Rhodococcus rhodochrous, and for Mycobacterium chlorophenolicum (formerly Rhodococcus chlorophenolicus), Rhodococcus erythropolis, Gordona bronchialis, and Gordona terrae, for which only partial sequence data have been available previously. The sequences of the two strains of R. maris were identical. The results of a distance matrix analysis indicated that R. maris is not a member of the genus Rhodococcus but is located between members of the genus Corynebacterium and members of the Rhodococcus-Nocardia-Mycobacterium-Gordona-Tsukamurella cluster. The finding that R. maris is phylogenetically isolated is supported by the presence of N-acetyl residues in the glycan moiety of the peptidoglycan and the lack of phosphatidylinositol and phosphatidylinositol mannosides, characteristics which distinguish this taxon from related taxa. On the basis of our results and previous findings, we propose that R. maris should be reclassified in a new genus, Dietzia. The type species is Dietzia maris comb. nov.

Cellular fatty acid composition of Desulfovibrio species and its use in classification of sulfate-reducing bacteria

Summary Forty strains of non-sporeforming sulfate-reducing bacteria including eighteen type strains of the genus Desulfovibrio and six type strains of other genera have been analysed for their cellular fatty acid (FA) composition. Iso-branched 17:1 FA, regarded as a biomarker FA for the genus Desulfovibrio, and, in many cases, also iso-15:0 FA predominated in most Desulfovibrio species, in Desulfomicrobium and Desulfomonas, but their proportions were significantly lower (1.6–9.5%) in D. alcoholovorans, D. carbinolicus, D. fructosovorans, D. giganteus, D. gigas DSM 496 and D. sulfodismutans. The latter species had anteiso-15:0 FA (30–54%) as the major FA. Iso-17:l FA was absent in D. baarsii, D. gigas DSM 1382T, D. sapovorans and D. thermophilus. The overall FA composition of D. baarsii, D. thermophilus, Desulfobacter curvatus, Desulfobacterium autotrophicum, Desulfuromonas acetoxidans and Thermodesulfobacterium commune was very distinct from those of what may be called “true” desulfovibrios. Surprisingly, Desulfobacterium autotrophicum did not contain iso or anteiso FA, but contained 10-methyl 16:0 FA which is reported for the first time in a member of this genus. The structure of the dendrogram produced by numerical cluster analysis of the FA composition of all strains studied was in good agreement with the phylogenetic relationships of the organisms. Most desulfo vibrios clustered in two separate groups reflecting the different distribution of iso-17:l FA. Some desulfovibrios, e.g. D. gigas DSM 496, and salt requiring strains D. desulfuricans El Agheila Z and D. vulgaris DSM 1744, did not cluster with the respective type strain indicating that they might have been incorrectly classified. On the other hand, transfer of D. baculatus to Desulfomicrobium and of D. thermophilus to Thermodesulfobacterium, as recently suggested, is clearly supported by our FA data.

Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India

Neptunia natans is a unique aquatic legume indigenous to tropical and sub-tropical regions and is nodulated symbiotically by rhizobia using an unusual infection process unlike any previously described. Previously, isolates of neptunia-nodulating rhizobia from Senegal were characterized as Allorhizobium undicola. Here we report on a different group of neptunia-nodulating rhizobia isolated from India. Sequencing of the 16S rDNA gene from two of these Indian isolates (strains J1T and J2) show that they belong in the genus Devosia rather than Allorhizobium. Currently, the only described Devosia species is D. riboflavina (family Hyphomicrobiaceae, order Rhizobiales). The complete 16S rDNA sequences of strains J1T and J2 are 95.9% homologous to the type strain, D. riboflavina LMG 2277T, suggesting that these neptunia-nodulating strains from India belong to a new Devosia species. This hypothesis was confirmed by further studies of polyphasic taxonomy (DNA-DNA hybridisation, TP-RAPD patterns, SDS-PAGE of cellular proteins, 16S rDNA RFLP patterns, carbon source utilisation, cellular fatty acid analysis and other phenotypic characterisations), all of which support the proposal that these neptunia-nodulating strains constitute a new Devosia species, which we name Devosia neptuniae sp. nov. These gram negative, strictly aerobic short rods are motile by a subpolar flagellum, positive for catalase, oxidase, urease and beta-galactosidase, can utilise several carbohydrates (but not organic acids) as carbon sources and contain C18:0 3-OH, cis-7 C18:1 11-methyl and cis-7 C18:1 as their major cellular fatty acids. Unlike D. riboflavina, the longer-chain C24:1 3-OH and C26:1 3-OH hydroxy fatty acids are not detected. The type strain of D. neptuniae is LMG 21357T (CECT 5650T). Assignment of this new taxon represents the fourth example in the literature of a non-rhizobial genus of bacteria capable of forming a bonafide dinitrogen-fixing root-nodule symbiosis with legume plants.

Biodegradation of cis-1,4-Polyisoprene Rubbers by Distinct Actinomycetes: Microbial Strategies and Detailed Surface Analysis

ABSTRACT Several actinomycetes isolated from nature were able to use both natural rubber (NR) and synthetic cis-1,4-polyisoprene rubber (IR) as a sole source of carbon. According to their degradation behavior, they were divided into two groups. Representatives of the first group grew only in direct contact to the rubber substrate and led to considerable disintegration of the material during cultivation. The second group consisted of weaker rubber decomposers that did not grow adhesively, as indicated by the formation of clear zones (translucent halos) around bacterial colonies after cultivation on NR dispersed in mineral agar. Taxonomic analysis of four selected strains based on 16S rRNA similarity examinations revealed two Gordonia sp. strains, VH2 and Kb2, and one Mycobacterium fortuitumstrain, NF4, belonging to the first group as well as oneMicromonospora aurantiaca strain, W2b, belonging to the second group. Schiffs reagent staining tests performed for each of the strains indicated colonization of the rubber surface, formation of a bacterial biofilm, and occurrence of compounds containing aldehyde groups during cultivation with NR latex gloves. Detailed analysis by means of scanning electron microscopy yielded further evidence for the two different microbial strategies and clarified the colonization efficiency. Thereby, strains VH2, Kb2, and NF4 directly adhered to and merged into the rubber material, while strain W2b produced mycelial corridors, especially on the surface of IR. Fourier transform infrared spectroscopy comprising the attenuated total reflectance technique was applied on NR latex gloves overgrown by cells of theGordonia strains, which were the strongest rubber decomposers. Spectra demonstrated the decrease in number ofcis-1,4 double bonds, the formation of carbonyl groups, and the change of the overall chemical environment, indicating that an oxidative attack at the double bond is the first metabolic step of the biodegradation process.

Phylogenetic analysis of the genera Rhodococcus and Nocardia and evidence for the evolutionary origin of the genus Nocardia from within the radiation of Rhodococcus species

SUMMARY: 16S rDNA analysis was performed on 32 strains of 26 species of the genera Rhodococcus and Nocardia in order to investigate the phylogenetic structure of these genera within the radiation of other mycolic-acid-containing genera such as Corynebacterium, Dietzia, Gordona, Mycobacterium and Tsukamurella. The genus Rhodococcus shows a complex structure, consisting of six phylogenetically equidistant lineages. The genus Nocardia does not appear to be a sister taxon of Rhodococcus but branches off from within the radiation of Rhodococcus; thus its species can be considered to be derived from a Rhodococcus ancestor. The main known phenotypic feature that separates Nocardia species from those of Rhodococcus appears to be the presence of a cyclic component in menaquinone of the MK-8(H4) type.

Taxonomic Revision of the Actinomycete Genera Actinomadura and Microtetraspora

Summary The type strains of 33 Actinomadura , 2 Excellospora , and 4 Microtetraspora species, together with those of Microbispora echinospora and Nocardiopsis africana , were assigned to two aggregate groups that merited generic status given chemical, molecular and numerical taxonomic evidence. The aggregate taxa corresponded with the genera Actinomadura and Microtetraspora as; emended. The emended genus Actinomadura showed its closest affinty to Thermomonospora and Microtetraspora to the genera Microbispora, Planobispora, Planomonospora and Streptosporangium . It is proposed that Actinomadura coeruleoviolacea be transferred to the genus Saccharothrix as Saccharothrix coeruleoviolacea comb. nov. Further studies are required to establish the taxonomic status of Actinomadura aurantiaca and Actinomadura carminata . The revised genus Actinomadura encompasses Actinomadura atramentaria, Actinomadura citrea, Actinomadura coerulea, Actinomadura cremea, Actinomadura fibrosa, Actinomadura fulvescens, Actinomadura kijianata, Actinomadura libanotica, Actinomadura livida, Actinomadura luteofluorescens, Actinomadura macra, Actinomadura madurae (the type species), Actinomadura oligospora, Actinomadura pelletieri, Actinomadura spadix, Actinomadura umbrina, Actinomadura verrucosospora, Actinomadura vinacea, Actinomadura viridis (including Actinomadura malachitica ) and Actinomadura yumaensis . It is proposed that Microbispora echinospora and Excellospora rubrobrunea nom. rev. be transferred to the emended genus Actinomadura . It is also proposed that Actinomadura fastidiosa, Actinomadura ferruginea, Actinomadura flexuosa, Actinomadura helvata, Actinomadura polychroma, Actinomadura pusilla, Actinomadura recticatena, Actinomadura roseola, Actinomadura roseoviolacea, Actinomadura rubra, Actinomadura salmonea, Actinomadura spiralis and Actinomadura turkmeniaca, Micropolyspora angiospora and Nocardiopsis africana be transferred to the revised genus Microtetraspora .