Sequence and Structure Analysis of Cryptic Plasmid pN30 from Oil-Oxidizing Strain <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> 30

Nucleotide sequence of cryptic plasmid pN30 from a Rhodococcus erythropolis 30 soil isolate was determined. Plasmid DNA consists of 5403 nucleotide pairs and contains about 62% GC pairs, which is typical of Rhodococcus DNA. No significant homology was determined between the pN30 DNA sequence and those of known plasmids. Computer-aided analysis of pN30 sequence revealed open reading frames that encode proteins strongly homologous to replicative proteins encoded by small cryptic plasmids of different actinomycetes.     

Primary Structure of NM.<Emphasis Type="Italic">Bst</Emphasis>SEI Operon from <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>, the Producer of N.<Emphasis Type="Italic">Bst</Emphasis>SEI Site-Specific Nicking Endonuclease

The nucleotide sequence of Bacillus stearothermophilus SE-589 DNA fragment including an operon for the site-specific nicking-modification (NM) system with a gene for BstSEI nicking endonuclease (nickase) has been determined. An analysis of the regions adjacent to the nickase gene has revealed two genes encoding DNA methyltransferases belonging to different classes. Three genes that form the system operon are separated by short open reading frames (ORFs). An analysis of these ORFs has shown that the polypeptides they encode are homologous to different parts of BstSEI nickase, NatB protein, and arginase. A difference in the GC content of the beginning and ending regions of the cloned DNA fragment and the presence of short ORFs similar to genes for known proteins indicate that the NM.BstSEI system operon has probably evolved by horizontal DNA transfer.     

The complete genome sequence and phylogenetic analysis of the mitochondrial DNA of the wood-decaying fungus <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>

The complete mitochondrial genome sequence of the wood-decaying fungus Fomitopsis palustris (Basidiomycete, Agaricomycotina) was determined by next-generation sequencing technology. The complete sequence of the circular mitochondrial DNA of F. palustris was 63,479 bp in length with a 75.98% AT content. The mitochondrial genome encoded 14 conserved proteins, 2 ribosomal RNAs, 26 transfer RNAs, and 19 additional open reading frames. The coxI and cob genes contained six and one group I introns, respectively, and encoded eight open reading frames, including seven intron-encoded endonucleases. The complete mitochondrial genome of F. palustris presented herein represents the first such report for brown rot basidiomycetes. In addition, the BLAST score ratio and phylogenetic analysis may open new avenues to understanding the evolutionary status of this fungus .     

Nucleotide sequence and structural analysis of cryptic plasmid pBL90 from <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis>

The nucleotide sequence of cryptic plasmid (designated as pBL90) detected in the cells of Brevibacterium lactofermentum DSM 1412 was determined. The length of plasmid DNA is 67826 bp. Comparison of the nucleotide sequence of pBL90 with known plasmid sequences showed no long regions of significant homology. Computer analysis of the plasmid DNA revealed 29 open reading frames (ORFs). The amino acid sequences of 15 ORFs (approximately 25% of plasmid length) have a high (>70%) level of identity to proteins from different plasmids of Corynebacterium representatives, including replicative proteins. Unusual in pBL90 is the presence of replicative genes from two different families and types of replication.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Transposition mechanism,molecular characterization and evolution of IS<Emphasis Type="Italic">6110</Emphasis>, the specific evolutionary marker of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex

The mycobacterial insertion sequence IS6110 proved crucial in deciphering tuberculosis (TB) transmission dynamics. This sequence was also shown to play an important role in the pathogenicity (transmission ability and/or virulence) of Mycobacterium tuberculosis, the main causative agent of TB in humans. In this study, we explored the usefulness of IS6110 and its potential as a phylogenetic/typing marker. We also analyzed the genetic polymorphism and evolutionary trends (selective pressure) of its transposase-encoding open reading frames (ORFs), A and B, using the maximum likelihood method. Both ORFs evolved chronologically through random single nucleotide polymorphisms. They were subjected to strict purifying selection more tight on orfA, with no evidence of significant recombination events. OrfA proved to have a crucial role in regulating the transpositional process. Several analyses showed that IS6110 acquisition antedated the emergence of the Mycobacterium tuberculosis complex. This original copy of IS6110 element was functionally optimal. In conclusion, this study not only demonstrated the usefulness of IS6110 in terms of phylogenetic and typing purposes and its transpositional mechanism, but also informed the scientific community on its evolutionary history.     

Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.     

The complete chloroplast genome of the green algae <Emphasis Type="Italic">Hariotina reticulata</Emphasis> (Scenedesmaceae,Sphaeropleales, Chlorophyta)

In this study, the chloroplast genome of Hariotina reticulata was fully sequenced and compared to other Sphaeropleales chloroplast genomes. It is 210,757 bp larger than most Sphaeropleales cpDNAs. It presents a traditional chloroplast structure, and contains 103 genes, including 68 protein-coding genes, six rRNA genes and 29 tRNA genes. The coding region constitutes of 43% of the whole cpDNA. Eighteen introns are found in 11 genes and six introns are unique for Hariotina. 11 open reading frames are identified among these introns. The synteny between Hariotina and Acutodesmus cpDNAs is in general identical, while within Sphaeropleales order, high variability in cpDNA architecture is indicated by general high DCJ distances. Ankyra judayi exhibits the greatest dissimilarity in gene synteny to the others and share some unique gene clusters with Treubaria triappendiculata. The phylogenomic analyses show that A. judayi is clustered with Treubariaceae species and sister to Chlorophyceae incertae sedis and other Sphaeropleales species. The monophyly of Sphaeropleales is rejected.     

Analysis of two large functionally uncharacterized regions in the <Emphasis Type="Italic">Methanopyrus kandleri</Emphasis> AV19 genome

Background

For most sequenced prokaryotic genomes, about a third of the protein coding genes annotated are "orphan proteins", that is, they lack homology to known proteins. These hypothetical genes are typically short and randomly scattered throughout the genome. This trend is seen for most of the bacterial and archaeal genomes published to date.

Results

In contrast we have found that a large fraction of the genes coding for such orphan proteins in the Methanopyrus kandleri AV19 genome occur within two large regions. These genes have no known homologs except from other M. kandleri genes. However, analysis of their lengths, codon usage, and Ribosomal Binding Site (RBS) sequences shows that they are most likely true protein coding genes and not random open reading frames.

Conclusions

Although these regions can be considered as candidates for massive lateral gene transfer, our bioinformatics analysis suggests that this is not the case. We predict many of the organism specific proteins to be transmembrane and belong to protein families that are non-randomly distributed between the regions. Consistent with this, we suggest that the two regions are most likely unrelated, and that they may be integrated plasmids.

     

Start codon in the <Emphasis Type="Italic">Bacillus intermedius</Emphasis> gene for serine proteinase

The translation initiation site was determined for Bacillus intermedius aprBi (AN AY754946), coding for extracellular subtilisin-like serine proteinase secreted at the stationary growth phase. Analysis of the aprBi open reading frame revealed three potential translation start sites (TTG, GTG, and ATG). Using the SignalP online freeware program, the probabilities of their functional activity were evaluated. To identify the translation start, modified subtilisin-like protease genes with nucleotide replacements in putative start codons were obtained by oligonucleotide-directed mutagenesis. The expression of these genetic constructs was investigated in protease-deficient strain B. subtilis AJ73. The results indicated that aprBi translation starts from the alternative GTG codon.     

Genetic characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> from Great Salt Lake brine flies

Wolbachia are intracellular prokaryotic endosymbionts associated with a wide distribution of arthropod and nematode hosts. Their association ranges from parasitism to mutualism, and there is growing evidence that Wolbachia can have dramatic effects on host reproduction, physiology, and immunity. Although all Wolbachia are currently considered as single species, W. pipientis, phylogenetic studies reveal about a dozen monophyletic groups, each designated as a supergroup. This study uses 16S rRNA gene sequences to examine the genetic diversity of Wolbachia present in three species of Great Salt Lake brine flies, Cirrula hians, Ephydra gracilis, and Mosillus bidentatus. The brine fly Wolbachia sequences are highly similar, with an average nucleotide sequence divergence among the three species of 0.00174. The brine fly Wolbachia form a monophyletic group that is affiliated with a subset of supergroup B, indicating that this supergroup may be more diverse than previously thought. These findings expand the phylogenetic diversity of Wolbachia and extend their host range to taxa adapted to a hypersaline environment.     

The single-stranded DNA-binding protein of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background

Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.

Results

A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSB_Dr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.

Conclusions

The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

     

Subcellular localization and detection of <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> ORF6 protein by immunoelectron microscopy

Members of the genus Tobamovirus represent one of the best-characterized groups of plant positive, single stranded RNA viruses. Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. The amino acid sequences of ORF6 proteins encoded by different tobamoviruses are highly divergent. The potential role of ORF6 proteins in replication of tobamoviruses still needs to be elucidated. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses. For example, in the genomes of other members of the tobamovirus subgroup 1, including Rehmannia mosaic virus, Paprika mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Tomato mottle mosaic virus, and Nigerian tobacco latent virus, sequence comparisons revealed the existence of a similar open reading frame like ORF6 of TMV.     

Characterization of a <Emphasis Type="Italic">Salmonella</Emphasis> Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.     

Comparative study on mitogenomes of green tide algae

Since 2007, the annual green tide disaster in the Yellow Sea has brought serious economic losses to China. There is no research on the genetic similarities of four constituent species of green tide algae at the genomic level. We previously determined the mitochondrial genomes of Ulva prolifera, Ulva linza and Ulva flexuosa. In the present work, the mitochondrial genome of another green tide (Ulva compressa) was sequenced and analyzed. With the length of 62,311 bp, it contained 29 encoding genes, 26 tRNAs and 10 open reading frames. By comparing these four mitochondrial genomes, we found that U. compressa was quite different from the other three types of Ulva species. However, there were similarities between U. prolifera and U. linza in the number, distribution and homology of open reading frames, evolutionary and codon variation of tRNA, evolutionary relationship and selection pressure of coding genes. Repetitive sequence analysis of simple sequence repeats, tandem repeat and forward repeats further supposed that they have evolved from the same origin. In addition, we directly analyzed gene homologies and translocation of four green tide algae by Mauve alignment. There were gene order rearrangements among them. With fast-evolving genomes, these four green algal mitochondria have both conservatism and variation, thus opening another window for the understanding of origin and evolution of Ulva.     

Single nucleotide polymorphisms in <Emphasis Type="Italic">TaER</Emphasis> genes and their association with carbon isotope discrimination in wheat genotypes under drought

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. The ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.