Oligomycins A and E, major bioactive secondary metabolites produced by Streptomyces sp. strain HG29 isolated from a Saharan soil.

An actinobacterial strain, HG29, with potent activity against pathogenic, toxigenic and phytopathogenic fungi was isolated from a Saharan soil sample of Algeria. On the basis of morphological and chemotaxonomic characteristics, the strain was classified in the genus Streptomyces. Analysis of the 16S rRNA gene sequence showed a similarity level of 99.3% with Streptomyces gancidicus NBRC 15412T. The comparison of its cultural and physiological characteristics with this species revealed significant differences. Moreover, the phylogenetic tree showed that strain HG29 forms a distinct phyletic line within the genus Streptomyces. Production of antifungal activity was investigated by following kinetics in shake broth. The highest antifungal activity was obtained after five days of fermentation, and in the dichloromethane extract. Two active compounds, NK1 and NK2, were purified by HPLC using a C18 column. Their chemical structures were identified through nuclear magnetic resonance experiments and mass spectrometry as oligomycins E and A, respectively, which have not been reported to be produced by S. gancidicus. The two bioactive compounds exhibited significant antifungal activity in vitro, showing minimal inhibitory concentrations (MICs) values between 2 and 75µg/mL.

Antifungal activity of a Saharan strain of Actinomadura sp. ACD1 against toxigenic fungi and other pathogenic microorganisms.

A new strain of actinobacteria, designated ACD1, was isolated from a Saharan soil sample in the Hoggar region (Algeria). Morphological study led to this strain being classified as a member of the Actinomadura genus. Phylogenetic analysis based on the 16S rRNA gene showed that the strain is closely related to Actinomadura sediminis DSM 45500(T) (98.5% sequence similarity). Furthermore, strain ACD1 presented a strong activity against mycotoxigenic and phytopathogenic fungi, including Aspergillus and Fusarium strains, and other pathogenic microorganisms. The kinetics of antimicrobial activity were investigated on ISP-2, Bennett and TSB media. Four solvents (n-hexane, dichloromethane, ethyl acetate and n-butanol) were used for the extraction of the produced antibiotic. The highest antimicrobial activity was obtained using the butanolic extract from the ISP-2 medium after seven days of fermentation culture. The active antibiotic was purified by reverse-phase HPLC using a C18 column. The UV-visible and mass spectra were determined. The minimum inhibitory concentrations (MIC) of this antibiotic were determined against pathogenic microorganisms.

Geodermatophilus saharensis sp. nov., isolated from sand of the Saharan desert in Chad.

A novel Gram-positive, aerobic, actinobacterial strain, CF5/5, was isolated from soil in the Sahara desert, Chad. It grew best at 20-35 °C and at pH 6.0-8.0 and with 0-4 % (w/v) NaCl, forming black-colored colonies. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G + C content was 75.9 mol%. The peptidoglycan contained meso-diaminopimelic acid; galactose and xylose were detected as diagnostic sugars. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, and phosphatidylinositol; MK-9(H(4)) was the dominant menaquinone. The major cellular fatty acids were: iso-C(16:0) and iso-C(15:0). The 16S rRNA gene showed 95.6-98.3 % sequence similarity with the other named members of the genus Geodermatophilus. Based on the polyphasic taxonomy data, the isolate is proposed to represent a novel species, Geodermatophilus saharensis with the type strain CF5/5(T) = DSM 45423 = CCUG 62813 = MTCC 11416.

Geodermatophilus arenarius sp. nov., a xerophilic actinomycete isolated from Saharan desert sand in Chad.

A novel Gram-positive, aerobic, actinobacterial strain, CF5/4(T), was isolated in 2007 during an environmental screening of arid desert soil in Ouré Cassoni, Chad. The isolate grew best in a temperature range of 28-40 °C and at pH 6.0-8.5, with 0-1 % (w/v) NaCl, forming brown-coloured and nearly circular colonies on GYM agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G + C content of the novel strain was 75.9 mol %. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diaminoacid. The main phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, diphosphatidylglycerol and a small amount of phosphatidylglycerol; MK-9(H(4)) was identified as the dominant menaquinone and galactose as diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids: iso-C(15:0) and iso-C(16:0). The 16S rRNA gene showed 96.2-98.3 % sequence identity with the three members of the genus Geodermatophilus: G. obscurus (96.2 %), G. ruber (96.5 %), and G. nigrescens (98.3 %). Based on the chemotaxonomic results, 16S rRNA gene sequence analysis and DNA-DNA hybridization with the type strain of G. nigrescens, the isolate is proposed to represent a novel species, Geodermatophilus arenarius (type strain CF5/4(T) = DSM 45418(T) = MTCC 11413(T) = CCUG 62763(T)).

Planococcus plakortidis sp. nov., isolated from the marine sponge Plakortis simplex (Schulze).

A novel coccoid-shaped strain, AS/ASP6 (II)T, was isolated from a sample taken from Plakortis simplex (Schulze), a marine sponge, collected at a depth of 30 m from the Bay of Bengal. This strain was identified by using a polyphasic taxonomic approach. The results of 16S rRNA gene sequence analysis showed that strain AS/ASP6 (II)T should be assigned to the genus Planococcus. Chemotaxonomic data (A4α-type peptidoglycan; MK-6, MK-7 and MK-8 menaquinones; mainly branched cellular fatty acids; and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as cellular phospholipids) supported taxonomic placement in the genus Planococcus. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain AS/ASP6 (II)T belonged to the genus Planococcus and was closely related to the type strains of Planococcus maritimus (99.1 %) followed by Planococcus rifietoensis (98.6 %), Planococcus maitriensis (98.5 %), Planococcus citreus (98.3 %), Planococcus salinarum (98.1 %), Planococcus columbae (97.9 %), Planococcus donghaensis (97.8 %) and Planococcus antarcticus (97.7 %); DNA-DNA hybridization values obtained were well below the threshold that is required for the proposal of a novel species. The G+C content of the genomic DNA was 51.0  mol%. The phenotypic and genotypic data showed that strain AS/ASP6 (II)T merits recognition as a representative of a novel species of the genus Planococcus, for which the name Planococcus plakortidis sp. nov. is proposed; the type strain is AS/ASP6 (II)T (=MTCC 8491T=DSM 23997T).

Auritidibacter ignavus gen. nov., sp. nov., of the family Micrococcaceae isolated from an ear swab of a man with otitis externa, transfer of the members of the family Yaniellaceae Li et al. 2008 to the family Micrococcaceae and emended description of the suborder Micrococcineae.

A Gram-reaction-positive, aerobic, catalase-positive, non-spore-forming, rod-shaped bacterium designated IMMIB L-1656(T) was isolated from an ear swab of a man and characterized using a polyphasic approach. 16S rRNA gene sequence analysis indicated that strain IMMIB L-1656(T) is related to members of the family Micrococcaceae (<95.1 % sequence similarity). Anaylsis using different phylogenetic algorithms consistently grouped strain IMMIB L-1656(T) with members of the genus Yaniella. The organism posessed a cell-wall murein based on L-lysine (variation A4α, type L-Lys-Gly-L-Glu), MK-10 as the predominant menaquinone and long-chain cellular fatty acids of straight-chain and branched-chain saturated types (with iso-C(15 : 0) and anteiso-C(17 : 0) predominating). The polar lipids included diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol in addition to unknown glycolipids. The DNA G+C content was 59.7 mol%. Based on its distinctive genotypic and phenotypic characteristics, strain IMMIB L-1656(T) represents a novel species in a novel genus, for which the name Auritidibacter ignavus gen. nov., sp. nov. is proposed. We also propose that members of the family Yaniellaceae be transferred to the family Micrococcaceae with amendments to the description of the suborder Micrococcineae. The type strain of Auritidibacter ignavus is IMMIB L-1656(T) (=DSM 45359(T) =CCUG 57943(T)).

Streptomyces youssoufiensis sp. nov., isolated from a Moroccan phosphate mine.

A novel actinomycete, strain X4(T), was isolated from a phosphate mine in Youssoufia, 100 km north of Marrakesh, Morocco. The taxonomic status of this strain was evaluated by a polyphasic approach. Strain X4(T) had white aerial mycelium with Rectiflexibiles spore chains bearing smooth-surfaced spores and did not produce diffusible pigments. Chemotaxonomic analysis showed that the cell wall of strain X4(T) contained LL-diaminopimelic acid and glycine. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence indicated that strain X4(T) belongs to the Group I streptomycetes, branching off next to Streptomyces ramulosus NRRL B-2714(T) and Streptomyces kasugaensis M338-M1(T). DNA-DNA relatedness and phenotypic data enabled strain X4(T) to be distinguished from the phylogenetically most closely related type strains. It is therefore proposed that strain X4(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces youssoufiensis sp. nov. is proposed; the type strain is X4(T) ( = CCMM B709(T)  = DSM 41920(T)).

En route to a genome-based classification of Archaea and Bacteria?

Given the considerable promise whole-genome sequencing offers for phylogeny and classification, it is surprising that microbial systematics and genomics have not yet been reconciled. This might be due to the intrinsic difficulties in inferring reasonable phylogenies from genomic sequences, particularly in the light of the significant amount of lateral gene transfer in prokaryotic genomes. However, recent studies indicate that the species tree and the hierarchical classification based on it are still meaningful concepts, and that state-of-the-art phylogenetic inference methods are able to provide reliable estimates of the species tree to the benefit of taxonomy. Conversely, we suspect that the current lack of completely sequenced genomes for many of the major lineages of prokaryotes and for most type strains is a major obstacle in progress towards a genome-based classification of microorganisms. We conclude that phylogeny-driven microbial genome sequencing projects such as the Genomic Encyclopaedia of Archaea and Bacteria (GEBA) project are likely to rectify this situation.

Emended description of the genus Actinokineospora Hasegawa 1988 and transfer of Amycolatopsis fastidiosa Henssen et al. 1987 as Actinokineospora fastidiosa comb. nov.

The species Amycolatopsis fastidiosa (ex Celmer et al. 1977) Henssen et al. 1987 was proposed, based on morphological and chemotaxonomic observations, for a strain originally described as 'Pseudonocardia fastidiosa' Celmer et al. 1977 in a US patent. In the course of a phylogenetic study of the taxa with validly published names within the suborder Pseudonocardineae based on 16S rRNA gene sequences, it became apparent that this species was misplaced in the genus Amycolatopsis. After careful evaluation of the phylogeny, morphology, chemotaxonomy and physiology of the type strain, it was concluded that this strain represents a species of the genus Actinokineospora that is unable to produce motile spores. The description of the genus Actinokineospora is therefore emended to accommodate species that do not produce motile spores, and it is proposed that Amycolatopsis fastidiosa be transferred to the genus Actinokineospora as Actinokineospora fastidiosa comb. nov. The type strain is NRRL B-16697(T) =ATCC 31181(T) =DSM 43855(T) =JCM 3276(T) =NBRC 14105(T) =VKM Ac-1419(T).

Actinomadura sputi sp. nov., isolated from the sputum of a patient with pulmonary infection.

The taxonomic position of an actinomycete, strain IMMIB L-889(T), isolated from the sputum of a 64-year-old man, was determined using a polyphasic taxonomic approach. The strain had chemical and morphological properties that were consistent with its classification in the genus Actinomadura. It formed a distinct phyletic line in the 16S rRNA gene tree of Actinomadura and was most closely related to the type strain of Actinomadura hallensis (98.4 % sequence similarity), but could be readily distinguished from the latter species using DNA-DNA relatedness and phenotypic data. The combined genotypic and phenotypic data indicate that strain IMMIB L-889(T) represents a novel species of the genus Actinomadura, for which the name Actinomadura sputi sp. nov. is proposed. The type strain is IMMIB L-889(T) =DSM 45233(T)=CCUG 56589(T). [corrected]

Streptomyces thinghirensis sp. nov., isolated from rhizosphere soil of Vitis vinifera.

A novel actinomycete, strain S10(T), was isolated from rhizosphere soil of wild Vitis vinifera in Thinghir, Ouarzazate Province, Southern Morocco. The taxonomic status of this strain was established using a polyphasic approach. Strain S10(T) had white-grey aerial mycelium with long, spiral spore chains bearing smooth surfaced spores and produced a yellow diffusible pigment. Chemotaxonomic analyses showed that the cell wall of strain S10(T) contained ll-diaminopimelic acid and glycine. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence indicated that strain S10(T) belonged to the Group I streptomycetes, branching off next to Streptomyces marokkonensis LMG 23016(T) from the Streptomyces violaceoruber group. DNA-DNA relatedness and phenotypic data distinguished strain S10(T) from the phylogenetically closest related type strains. It is therefore proposed that strain S10(T) (=CCMM B35(T)=DSM 41919(T)) represents the type strain of a novel species of the genus Streptomyces, for which the name Streptomyces thinghirensis sp. nov. is proposed.

Streptomyces marokkonensis sp. nov., isolated from rhizosphere soil of Argania spinosa L.

The novel actinomycete strain Ap1(T) was isolated from rhizosphere soil of the argan tree (Argania spinosa L.) in the south of Morocco. Strain Ap1(T) has been reported as a novel producer of the pentaene polyene macrolide isochainin, which strongly inhibits the growth of pathogenic yeasts and phytopathogenic fungi. Strain Ap1(T) shows a greyish-white aerial mycelium with chains of smooth-surfaced spores of the Spiralis type and a cell wall containing ll-diaminopimelic acid. Based on chemotaxonomy and morphological features, strain Ap1(T) was identified as a member of the genus Streptomyces. 16S rRNA gene sequence similarities based on almost-complete 16S rRNA gene sequences showed that strain Ap1(T) is closely associated with members of the Streptomyces violaceoruber species group (S. violaceoruber, S. coelescens, S. violaceorubidus, 'S. caesius', 'S. lividans', S. violaceolatus and S. humiferus) and others (Streptomyces aurantiogriseus, S. lienomycini, S. chattanoogensis, S. rubrogriseus and S. tendae). However, protein profiling, DNA-DNA hybridization and BOX-PCR fingerprinting proved a relationship above the species level. In addition, the phenotype also allowed for the differentiation of strain Ap1(T) from its closest neighbours. As a result of this polyphasic approach, we conclude that strain Ap1(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces marokkonensis sp. nov. is proposed. The type strain is Ap1(T) (=R-22003(T) =LMG 23016(T) =DSM 41918(T)).

Nocardiopsis potens sp. nov., isolated from household waste.

The taxonomic position of an actinomycete, designated strain IMMIB L-21(T), was determined using a polyphasic taxonomic approach. The organism, which had phenotypic properties consistent with its classification in the genus Nocardiopsis, formed a distinct clade in the 16S rRNA gene sequence tree together with the type strain of Nocardiopsis composta, but was readily distinguished from this species using DNA-DNA relatedness and phenotypic data. The genotypic and phenotypic data show that the organism represents a novel species of the genus Nocardiopsis, for which the name Nocardiopsis potens sp. nov. is proposed. The type strain is IMMIB L-21(T) (=DSM 45234(T)=CCUG 56587(T)).

Desmospora activa gen. nov., sp. nov., a thermoactinomycete isolated from sputum of a patient with suspected pulmonary tuberculosis, and emended description of the family Thermoactinomycetaceae Matsuo et al. 2006.

A novel Gram-positive, aerobic, catalase-positive, filamentous micro-organism, designated strain IMMIB L-1269(T), originating from sputum was characterized using phenotypic and molecular taxonomic methods. It showed cell-wall chemotype III, phospholipid type PII (with phosphatidylethanolamine as the diagnostic phospholipid) and contained an unsaturated menaquinone with seven isoprene units (MK-7) as the predominant isoprenoid quinone. It synthesized long-chain cellular fatty acids of the straight-chain saturated, monounsaturated and iso- and anteiso-branched types (with iso-C(15 : 0), C(16 : 0) and iso-C(17 : 0) predominating) and possessed a DNA G+C content of 49.3 mol%. On the basis of its morphological, biochemical and chemical characteristics, strain IMMIB L-1269(T) did not conform to any presently recognized taxon. Comparative analyses based on 16S rRNA gene sequences confirmed the distinctiveness of the isolate, as it displayed sequence-divergence values greater than 7.7 % with respect to recognized Gram-positive taxa. Phylogenetic treeing analysis served to reinforce the view that strain IMMIB L-1269(T) was distinct from recognized taxa, as it formed a relatively long subline branching within a 16S rRNA gene sequence cluster that encompassed the genera Thermoactinomyces, Laceyella, Mechercharimyces, Thermoflavimicrobium, Planifilum, Seinonella and Shimazuella of the family Thermoactinomycetaceae. On the basis of phenotypic and molecular phylogenetic evidence, strain IMMIB L-1269(T) represents a novel genus and species, for which the name Desmospora activa gen. nov., sp. nov. is proposed. The type strain of Desmospora activa is strain IMMIB L-1269(T) (=DSM 45169(T) =CCUG 55916(T)). An emended description of the family Thermoactinomycetaceae is also given.

Rapid access to genes of biotechnologically useful enzymes by partial genome sequencing: the thermoalkaliphile Anaerobranca gottschalkii.

Anaerobranca gottschalkii strain LBS3 T is an extremophile living at high temperature (up to 65 degrees C) and in alkaline environments (up to pH 10.5). An assembly of 696 DNA contigs representing about 96% of the 2.26-Mbp genome of A. gottschalkii has been generated with a low-sequence-coverage shotgun-sequencing strategy. The chosen sequencing strategy provided rapid and economical access to genes encoding key enzymes of the mono- and polysaccharide metabolism, without dilution of spare resources for extensive sequencing of genes lacking potential economical value. Five of these amylolytic enzymes of considerable commercial interest for biotechnological applications have been expressed and characterized in more detail after identification of their genes in the partial genome sequence: type I pullulanase, cyclodextrin glycosyltransferase (CGTase), two alpha-amylases (AmyA and AmyB), and an alpha-1,4-glucan-branching enzyme.

The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments.

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium that is able to grow at in situ temperatures below 0 degrees C. As abundant members of the microbial community in permanently cold marine sediments, D. psychrophila-like bacteria contribute to the global cycles of carbon and sulfur. Here, we describe the genome sequence of D. psychrophila strain LSv54, which consists of a 3 523 383 bp circular chromosome with 3118 predicted genes and two plasmids of 121 586 bp and 14 663 bp. Analysis of the genome gave insight into the metabolic properties of the organism, e.g. the presence of TRAP-T systems as a major route for the uptake of C(4)-dicarboxylates, the unexpected presence of genes from the TCA cycle, a TAT secretion system, the lack of a beta-oxidation complex and typical Desulfovibrio cytochromes, such as c(553), c(3) and ncc. D. psychrophila encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases, nine putative cold shock proteins and nine potentially cold shock-inducible proteins. A comparison of D. psychrophila's genome features with those of the only other published genome from a sulfate reducer, the hyperthermophilic archaeon Archaeoglobus fulgidus, revealed many striking differences, but only a few shared features.

Phylogenomics of hyperthermophilic Archaea and Bacteria.

The location of hyperthermophilic organisms in the tree of life has been the source of many exciting discussions during the last two decades. It inspired not only novel hypotheses for the early evolution of the organisms, but also the isolation of many new species of Archaea and Bacteria from hot environments, as well as microbial genome sequencing and phylogenomic analyses. In view of the new wealth of genetic information generated from several analysed genomes of the hyperthermophiles, we can only conclude that the question of their exact phylogenetic location and evolutionary origin is presently as open as ever before.

Identification and functional characterization of eight CYP3A4 protein variants.

The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the population studied was found to be heterozygous for one of these variants. In a bacterial heterologous expression system, two mutants, R130Q and P416L, did not result in detectable P450 holoprotein. One mutant, T363M, expressed at significantly lower levels than wild-type CYP3A4. G56D, V170I, D174H and M445T were not significantly different when compared with wild-type CYP3A4 in expression or steroid hydroxylase activity. L373F displayed a significantly altered testosterone metabolite profile and a four-fold increase in the Km value for 1'-OH midazolam formation. The results suggest a limited contribution of CYP3A4 protein variants to the inter-individual variability of CYP3A4 activity in Caucasians. Some variants may, however, play a role in the atypical response to drugs or altered sensitivity to carcinogens.

Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene.

Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.

A novel lipothrixvirus, SIFV, of the extremely thermophilic crenarchaeon Sulfolobus.

We describe a novel lipothrixvirus, SIFV, of the crenarchaeotal archaeon Sulfolobus islandicus. SIFV (S. islandicus filamentous virus) has a linear virion with a linear double-stranded DNA genome. These two features coincide in several crenarchaeotal but not in any other viruses. The SIFV core is formed by a zipper-like array of DNA-associated protein subunits and is covered by a lipid envelope containing host lipids. We sequenced approximately 96% of the virus genome excepting the DNA termini, which were modified in an unusual, yet uncharacterized, manner. Both, the 5' and the 3' DNA termini were insensitive to enzymatic degradation and labelling. Two open reading frames (ORFs) of the SIFV genome are likely to encode helicases and resemble uncharacterized ORFs from other archaea in sequence. Three ORFs showed sequence similarity with each other and each contained a glycosyl transferase motif. Another ORF of the SIFV genome showed significant sequence similarity to the ORF a291 from the well characterized, spindle-shaped Sulfolobus virus SSV1. Due to its structure, SIFV is classified as a lipothrixvirus.