Catenulispora acidiphila gen. nov., sp. nov., a novel, mycelium-forming actinomycete, and proposal of Catenulisporaceae fam. nov.

A novel, Gram-positive bacterial strain was isolated from forest soil. Among species with validly published names, the 16S rRNA gene sequence is related most closely (approx. 93 % similarity) to that of Sporichthya polymorpha DSM 43042(T). However, differently from this species, it forms both vegetative and aerial mycelia. The aerial hyphae are straight to slightly flexuous, starting to septate to form chains of more than 20 cylindrical spores with a rugose surface. The strain is acidophilic, with a pH range for robust growth between 4.3 and 6.8 and an optimum around 6.0. The peptidoglycan type is A3gamma ll-Dpm-Gly. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and two unknown phospholipids. Predominant menaquinones are MK-9(H(6)) and -9(H(4)), and iso- and anteiso-branched C(16 : 0) and C(17 : 0) are the main cellular fatty acids. The DNA G+C content is 71.9 mol%. The distinct phylogenetic position and the unusual combination of chemotaxonomic characteristics justify the proposal of Catenulispora gen. nov., with the type species Catenulispora acidiphila sp. nov. (type strain, ID139908(T) =DSM 44928(T)=NRRL B-24433(T)). Catenulisporaceae fam. nov. is also proposed.

Actinospica robiniae gen. nov., sp. nov. and Actinospica acidiphila sp. nov.: proposal for Actinospicaceae fam. nov. and Catenulisporinae subord. nov. in the order Actinomycetales.

Two novel Gram-positive, acidophilic bacterial strains were isolated from forest soil. According to their 16S rRNA gene sequences, these strains are related closely to each other and form a distinct cluster within the order Actinomycetales. They show the typical features of filamentous actinomycetes, with branched vegetative hyphae and production of aerial hyphae. The distinct phylogenetic positions and the combination of chemotaxonomic characteristics of these strains justify the proposal of Actinospica gen. nov. Both strains display 3-hydroxydiaminopimelic acid plus traces of meso-diaminopimelic acid, the phospholipids diphosphatidylglycerol, phosphatidylethanolamine, methylphosphatidylethanolamine and phosphatidylinositol, the predominant cellular fatty acids i-C(15 : 0), i-C(16 : 0) and ai-C(15 : 0) and the whole-cell sugars mannose and rhamnose. They differ in the fatty acid profiles, in the quantitative ratios of the major menaquinones MK-9(H(4)), MK-9(H(6)) and MK-9(H(8)) and in the occurrence of additional whole-cell sugars (arabinose and xylose in strain GE134766(T) and galactose in strain GE134769(T)). Differences in the phenotypic characteristics and in the 16S rRNA gene sequences suggest the description of two species, Actinospica robiniae gen. nov., sp. nov. (the type species) and Actinospica acidiphila sp. nov., with the type strains GE134769(T) (=DSM 44927(T)=NRRL B-24432(T)) and GE134766(T) (=DSM 44926(T)=NRRL B-24431(T)), respectively. The DNA G+C contents of strains GE134769(T) and GE134766(T) are 70.8 and 69.2 mol%, respectively. Due to the large phylogenetic distance from known actinomycete genera, it is proposed to accommodate Actinospica gen. nov. in Actinospicaceae fam. nov. In addition, Catenulisporineae subord. nov. is proposed to harbour Actinospicaceae fam. nov. and the newly proposed family Catenulisporaceae, described in the accompanying paper.

Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes.

The discovery of new antibiotics and other bioactive microbial metabolites continues to be an important objective in new drug research. Since extensive screening has led to the discovery of thousands of bioactive microbial molecules, new approaches must be taken in order to reduce the probability of rediscovering known compounds. The authors have recently isolated slow-growing acidophiles belonging to the novel genera Catenulispora and Actinospica within the order Actinomycetales. These strains, which likely belong to a new suborder, grow as filamentous mycelia, have a genome size around 8 Mb, and produce antimicrobial activities. In addition, a single strain harbours simultaneously genes encoding type I and type II polyeketide synthases, as well as non-ribosomal peptide synthetases. The metabolite produced by one strain was identified as a previously reported dimeric isochromanequinone. In addition, at least the Catenulispora strains appear globally distributed, since a PCR-specific signal could be detected in a significant fraction of acidic soils from different continents, and similar strains have been independently isolated from an Australian soil (Jospeh et al., Appl Environ Microbiol 69, 7210-7215, 2003). Thus, these previously uncultured actinomycetes share several features with Streptomyces and related antibiotic-producing genera, and represent a promising source of novel antibiotics.

Detection of HLA polymorphisms by ligase detection reaction and a universal array format: a pilot study for low resolution genotyping.

We present our results in the identification of polymorphic sites within the second exon of the human leukocyte antigen A (HLA-A) region using the DNA microarray technology. Allele specific detection was performed by polymerase chain reaction followed by ligase detection reaction (LDR) in combination with a universal array, a powerful method for high throughput DNA sequence analysis. By this approach we confirmed 32 human samples previously characterized by direct DNA sequencing, thus demonstrating the interest of this approach.

Two efficient polymeric chemical platforms for oligonucleotide microarray preparation.

In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step with appropriate silanes (gamma-aminopropyltriethoxysilane-APTES or 3-glycidoxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/ stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction).

BACKGROUND: PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. RESULTS: Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. CONCLUSIONS: The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria.

Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem-loop structured probes.

Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem-loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.

Mutations in two independent genes lead to suppression of the shoot apical meristem in maize.

The shoot apical meristem (SAM), initially formed during embryogenesis, gives rise to the aboveground portion of the maize (Zea mays) plant. The shootless phenotype (sml) described here is caused by disruption of SAM formation due to the synergistic interaction of mutations at two genetic loci. Seedlings must be homozygous for both sml (shootmeristemless), and the unlinked dgr (distorted growth) loci for a SAM-less phenotype to occur. Seedlings mutant only for sml are impaired in their morphogenesis to different extents, whereas the dgr mutation alone does not have a recognisable phenotype. Thus, dgr can be envisaged as being a dominant modifier of sml and the 12 (normal):3 (distorted growth):1 (shoot meristemless) segregation observed in the F(2) of the double heterozygote is the result of the interaction between the sml and dgr genes. Other segregation patterns were also observed in the F(2), suggesting instability of the dgr gene. Efforts to rescue mutant embryos by growth on media enriched with hormones have been unsuccessful so far. However, mutant roots grow normally on medium supplemented with kinetin at a concentration that suppresses wild-type root elongation, suggesting possible involvement of the mutant in the reception or transduction of the kinetin signal or transport of the hormone. The shootless mutant appears to be a valuable tool with which to investigate the organization of the shoot meristem in monocots as well as a means to assay the origins and relationships between organs such as the scutellum, the coleoptile, and leaves that are initiated during the embryogenic process.