Predicted ATP-binding cassette systems in the phytopathogenic mollicute <Emphasis Type="Italic">Spiroplasma kunkelii</Emphasis>

Spiroplasma kunkelii is a cell wall-free, helical, and motile mycoplasma-like organism that causes corn stunt disease in maize. The bacterium has a compact genome with a gene set approaching the minimal complement necessary for cellular life and pathogenesis. A set of 21 ATP-binding cassette (ABC) domains was identified during the annotation of a draft S. kunkelii genome sequence. These 21 ABC domains are present in 18 predicted proteins, and are components of 16 functional systems, which account for 5% of the protein coding capacity of the S. kunkelii genome. Of the 16 systems, 11 are membrane-bound transporters, and two are cytosolic systems involved in DNA repair and the oxidative stress response; the genes for the remaining three hypothetical systems harbor nonsense and/or frameshift mutations, so their functional status is doubtful. Assembly of the 11 multicomponent transporters, and comparisons with other known systems permitted functional predictions for the S. kunkelii ABC transporter systems. These transporters convey a wide variety of substrates, and are critical for nutrient uptake, multidrug resistance, and perhaps virulence. Our findings provide a framework for functional characterization of the ABC systems in S. kunkelii.Communicated by W. Goebel     

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize ( Zea mays L.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.Communicated by W. Goebel     

Biodegradation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether by newly identified soil microorganisms in a simple mineral solution

Methyl tert-butyl ether (MTBE) is a widely used fuel ether, which has become a soil and water contaminant. In this study, 12 microbial strains were isolated from gasoline-contaminated soils and selected because of their capacity to grow in MTBE. The strains were identified by 16S/ITS rDNA gene sequencing and screened for their ability to consume MTBE aerobically in a simple mineral solution. Solid phase microoextraction and gas chromatography were used to detect MTBE degradation. High levels of MTBE biodegradation were obtained using resting cells of the bacteria Achromobacter xylosoxidans MCM1/1 (78%), Enterobacter cloacae MCM2/1 (50%), and Ochrobactrum anthropi MCM5/1 (52%) and the fungus Exophiala dermatitidis MCM3/4 (14%). Our phylogenetic analysis clearly shows that bacterial MTBE biodegraders belong to the clade of Proteobacteria. For further insight, MTBE-degrader strains were profiled by denaturing gel gradient electrophoresis (DGGE) of PCR-amplified 16S rRNA gene sequences. This approach could be used to analyse microbial community dynamics in bioremediation processes.     

FACIL: Fast and Accurate Genetic Code Inference and Logo

MOTIVATION: The intensification of DNA sequencing will increasingly unveil uncharacterized species with potential alternative genetic codes. A total of 0.65% of the DNA sequences currently in Genbank encode their proteins with a variant genetic code, and these exceptions occur in many unrelated taxa. RESULTS: We introduce FACIL (Fast and Accurate genetic Code Inference and Logo), a fast and reliable tool to evaluate nucleic acid sequences for their genetic code that detects alternative codes even in species distantly related to known organisms. To illustrate this, we apply FACIL to a set of mitochondrial genomic contigs of Globobulimina pseudospinescens. This foraminifer does not have any sequenced close relative in the databases, yet we infer its alternative genetic code with high confidence values. Results are intuitively visualized in a Genetic Code Logo. Availability and implementation: FACIL is available as a web-based service at http://www.cmbi.ru.nl/FACIL/ and as a stand-alone program.     

Cryptic plasmid pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an adhesin and components of a type IV translocation-related conjugation system

A cryptic plasmid of the wall-less plant pathogenic mollicute, Spiroplasma kunkelii CR2-3X, was cloned and its sequence analyzed. The 14,615 bp plasmid, designated pSKU146, has a nucleotide content of 28 mol% G + C, and contains 18 potential protein-coding regions (open reading frames, ORFs), of which six encode proteins that exhibit similarity to virulence-associated proteins involved in cell-to-cell adhesion or conjugal DNA transfer. One ORF encodes a 96 kDa protein, SkARP1, that is highly similar to SARP1 adhesin involved in attachment of Spiroplasma citri to insect vector gut membrane. Five ORFs encode proteins similar to TraE and Mob in walled bacteria, and to ORFs found in the integrative, conjugative element (ICEF) of Mycoplasma fermentans, respectively. Presence of domains similar to proteins of the Type IV secretion system in pathogenic bacteria suggests that spiroplasma possesses a related translocation system. Plasmid pSKU146 also contains two identical oriT regions each containing a nick sequence characteristic of the IncP conjugative plasmid family, as well as a 58 bp palindromic sequence, palSK1. Features in pSKU146 suggest that the plasmid functions as a mobile genetic element in conjugative transmission of spiroplasma pathogenicity-related genes.     

Lineage-specific decay of folate biosynthesis genes suggests ongoing host adaptation in phytoplasmas

Phytoplasmas are nonculturable cell wall-less, obligate intracellular pathogens of plants and insect vectors. In their descent from walled bacterial ancestors, phytoplasmas underwent massive genome reduction, resulting in some of the smallest cellular genomes known in nonsymbiotic bacteria. While requirements for in vitro culture of phytoplasmas remain unknown, two opposing reports have appeared concerning genes encoding the ability of phytoplasmas to synthesize folates de novo. One study found pseudogene homologs of folP and folK, obviating folate synthesis in "Candidatus Phytoplasma asteris"-related strain CPh, whereas, a separate study found intact genes encoding a complete folate biosynthesis pathway in "Ca. Phytoplasma asteris"-related strain OY. To resolve the apparent conflict, we hypothesized that evolutionary adaptation to the availability of folate and/or other metabolites in host cells is an ongoing process in the phytoplasma clade that is reflected in part by differences among phytoplasmas in the status of genes of the folate biosynthesis pathway. By studying folP and folK loci in 11 closely related phytoplasmas, we determined that these essential folate biosynthesis genes are intact in some phytoplasmas but are deteriorating in closely related strains. We suggest that the status of the folate biosynthesis pathway and the course of gene decay are lineage-specific, predicting the eventual, lineage-related loss of recognizable folP and folK homologs in phytoplasma genomes.     

Characterization of Thermophilic Proteolytic Spore-Forming Bacteria from a Geothermal Site in Lithuania Based on 16S rDNA RFLP and ITS-PCR Analyses1

Forty-two strains of gram-positive, aerobic, heterotrophic, obligately thermophilic, spore-forming bacteria were isolated from a geothermal site near the Baltic Sea in Lithuania. All of the strains were able to hydrolyze collagen and/or casein. Since characteristics of proteolytic activity are correlated with taxonomic positions of bacteria, the strains were grouped on the basis of molecular biological analyses. On the basis of RFLP patterns of 16S rDNA and 16S–23S rDNA ITS-PCR analysis, the strains were subdivided into nine groups.