Evolutionary placement of Xanthomonadales based on conserved protein signature sequences

Xanthomonadales comprises one of the largest phytopathogenic bacterial groups, and is currently classified within the gamma-proteobacteria. However, the phylogenetic placement of this group is not clearly resolved, and the results of different studies contradict one another. In this work, the evolutionary position of Xanthomonadales was determined by analyzing the presence of shared insertions and deletions (INDELs) in highly conserved proteins. Several distinctive insertions found in most of the members of the gamma-proteobacteria are absent in Xanthomonadales and groups such as Legionelalles, Chromatiales, Methylococcales, Thiotrichales and Cardiobacteriales. These INDELs were most likely introduced after the branching of Xanthomonadales from most of the gamma-proteobacteria and provide evidence for the phylogenetic placement of the early gamma-proteobacteria. Moreover, other proteins contain insertions exclusive to the Xanthomonadales order, confirming that this is a monophyletic group and provide important specific genetic markers. Thus, the data presented clearly support the Xanthomonadales group as an independent subdivision, and constitute one of the deepest branching lineage within the gamma-proteobacteria clade. (C) 2009 Elsevier Inc. All rights reserved.

Phylogenetic analysis of a near-full-length sequence of an erythrovirus genotype 3 strain isolated in Brazil

Human parvovirus B19 is the only member of the genus Erythrovirus that causes human disease. Recent findings of several strains with considerable sequence divergence from B19 have suggested a new classification for parvovirus genotypes as 1 (B19), 2 (A-6 and LaLi) and 3 (V9). In their overall DNA sequence, the three genotypes differ by similar to 10%. Here, we report the isolation of a genotype-3-related strain named BR543 during a prospective study conducted in Sao Paulo, Brazil. Analysis of the nearly full-length genome sequence of BR543 indicates that this B19 variant sequence clusters with Gh2768, a strain from Ghana belonging to subtype 3b, and showed mostly synonymous substitutions.

Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4(+) T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

Worldwide sequence conservation of transmission-blocking vaccine candidate Pvs230 in Plasmodium vivax

Pfs230, surface protein of gametocyte/gamete of the human malaria parasite, Plasmodium falciparum, is a prime candidate of malaria transmission-blocking vaccine. Plasmodium vivax has an ortholog of Pfs230 (Pvs230), however, there has been no study in any aspects on Pvs230 to date. To investigate whether Pvs230 can be a vivax malaria transmission-blocking vaccine, we performed evolutionary and population genetic analysis of the Pvs230 gene (pvs230: PVX_003905). Our analysis of Pvs230 and its orthologs in eight Plasmodium species revealed two distinctive parts: an interspecies variable part (IVP) containing species-specific oligopeptide repeats at the N-terminus and a 7.5 kb interspecies conserved part (ICP) containing 14 cysteine-rich domains. Pvs230 was closely related to its orthologs, Pks230 and Pcys230, in monkey malaria parasites. Analysis of 113 pvs230 sequences obtained from worldwide, showed that nucleotide diversity is remarkably low in the non-repeat 8-kb region of pvs230 (theta pi = 0.00118) with 77 polymorphic nucleotide sites, 40 of which results in amino acid replacements. A signature of purifying selection but not of balancing selection was seen on pvs230. Functional and/or structural constraints may limit the level of polymorphism in pvs230. The observed limited polymorphism in pvs230 should ground for utilization of Pvs230 as an effective transmission-blocking vaccine. (C) 2011 Elsevier Ltd. All rights reserved.

Brazilian coffee genome project: an EST-based genomic resource

O café é um dos principais produtos agrícolas, sendo considerado o segundo item em importância do comércio internacional de commodities. O gênero Coffea pertence à família Rubiaceae que também inclui outras plantas importantes. Este gênero contém aproximadamente 100 espécies, mas a produção comercial é baseada somente em duas espécies, Coffea arabica e Coffea canephora, que representam aproximadamente 70 % e 30 % do mercado total de café, respectivamente. O Projeto Genoma Café Brasileiro foi desenvolvido com o objetivo de disponibilizar os modernos recursos da genômica à comunidade científica e aos diferentes segmentos da cadeia produtiva do café. Para isso, foram seqüenciados 214.964 clones escolhidos aleatoriamente de 37 bibliotecas de cDNA de C. arabica, C. canephora e C. racemosa representando estádios específicos do desenvolvimento de células e de tecidos do cafeeiro, resultando em 130.792, 12.381 e 10.566 seqüências de cada espécie, respectivamente, após processo de trimagem. Os ESTs foram agrupados em 17.982 contigs e em 32.155 singletons. A comparação destas seqüências pelo programa BLAST revelou que 22 % não tiveram nenhuma similaridade significativa às seqüências no banco de dados do National Center for Biotechnology Information (de função conhecida ou desconhecida). A base de dados de ESTs do cafeeiro resultou na identificação de cerca de 33.000 unigenes diferentes. Os resultados de anotação das seqüências foram armazenados em base de dados online em http://www.lge.ibi.unicamp.br/cafe. Os recursos desenvolvidos por este projeto disponibilizam ferramentas genéticas e genômicas que podem ser decisivas para a sustentabilidade, a competitividade e a futura viabilidade da agroindústria cafeeira nos mercados interno e externo.

Whole-genome analysis for backfat thickness in a tropically adapted, composite cattle breed from Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

O objetivo deste trabalho foi desenvolver um oligonucleotídeo iniciador para reação em cadeia da polimerase (PCR) específico para as estirpes de Xylella fastidiosa que causam o mal de Pierce (PD) em videira (Vitis vinifera). Amplificações de DNA de 23 diferentes hospedeiros, usando o conjunto de oligonucleotídeos REP1-R (5'-IIIICGICGIATCCIGGC-3') e REP 2 (5'-ICGICTTATCI GGCCTAC-3') utilizando o programa: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/5 min, produziu um fragmento de 630 pb que diferenciou as estirpes de videiras dos demais. Entretanto, padrões de bandeamento REP não são considerados confiáveis para detecção devido ao par de oligonucleotídeos REP 1 e REP 2 corresponderem a seqüências repetitivas encontradas por todo o genoma bacteriano. Desse modo, o produto amplificado de 630 pb foi eluído do gel de agarose, purificado e seqüenciado. A informação da seqüência nucleotídica foi usada para identificar e sintetizar um oligonucleotídeo específico para o isolado de X. fastidiosa causadora do mal de Pierce denominado Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3'), que foi utilizado juntamente com o oligonucleotídeo REP-2 nas condições 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. Os DNAs das estirpes de X. fastidiosa de outros hospedeiros [amêndoa (Prumus amygdalus), citros (Citrus spp.), café (Coffea arabica), olmo (Ulmus americana), amora (Morus rubra), carvalho (Quercus rubra), vinca (Catharantus roseus), ameixa (Prunus salicina) e ragweed (Ambrosia artemisiifolia)] e de bactérias Gram negativas e positivas foram submetidos a amplificação com o conjunto de oligonucleotídeos Xf-1/REP 2. Um fragmento, de aproximadamente 350 pb, foi amplificado apenas com o DNA de X. fastidiosa isolada de videira.

A naturally occurring recombinant isolate of Lettuce mosaic virus

LMV-Common and LMV-Most are two seed-borne types of Lettuce mosaic virus (LMV), genus Potyvirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to lettuce by two recessive genes, mo1(1) and mo1(2). An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.

Specific detection of Lettuce mosaic virus isolates belonging to the Most type

Lettuce mosaic virus (LMV)-Most isolates can infect and are seed-borne in cultivars containing the mol gene. A reverse transcription and polymerase chain reaction (RT-PCR)-based test was developed for the specific detection of LMV-Most isolates. Based on the complete genome sequences of three LMV isolates belonging respectively to the Most type, the Common type and neither of these two types, three different assays were compared: (i) presence of a diagnostic restriction site in the region of the genome encoding the variable N-terminus of the capsid protein, in the 3' end of the genome, (ii) RT-PCR using primers designed to amplify a cDNA corresponding to a portion of the P1 coding region, in the 5' end of the genome and (iii) RT-PCR using primers designed to amplify a central region of the genome. The assays were performed against a collection of 21 isolates from different geographical origins and representing the molecular variability of LMV. RT-PCR of the central region of the genome was preferred because its results are expected to be less affected by natural recombination between LMV isolates, and it allows sensitive detection of LMV-Most in situations of single as well as mixed contamination. (C) 2004 Elsevier B.V. All rights reserved.

Further characterization of two sequiviruses infecting lettuce and development of specific RT-PCR primers

Lettuce mottle virus (LeMoV) and dandelion yellow mosaic virus (DaYMV) infect lettuce in South America and Europe, respectively. LeMoV and DaYMV possess isometric particles, occur at low concentrations in plants and have narrow host ranges. Partial genome sequences of both viruses were obtained using purified viral preparations and universal primers for members of the family Sequiviridae. DaYMV and LeMoV sequences were analyzed and showed identity with other members of the family. Universal primers that detect both viruses and specific primers for LeMoV and DaYMV were designed and used in RT-PCR-based diagnostic assays. These results provide the first molecular data on the LeMoV and DaYMV genomes and suggest that LeMoV is a member of the genus Sequivirus, probably distinct from DaYMV.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pbl 8) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pbl Ba by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Estudos ecogenômicos e bioprospectivos de Shewanella ssp

Bacteria trom Shewanella and Geobacter ganera are the most studied iron-reducing microorganisms particularly due to their electron transport systems and contribution to some industrial and environmental problems, including steel corrosion, bioenergy and bioremediation of petroleum-impacted sites. The present study was focused in two ways: the first is an in silico comparative ecogenomic study of Shewanella spp. with sequenced genomes, and the second is an experimental metagenomic work to detect iron-reducing Shewanella through PCR-DGGE of a metabolic gene. The in silico study resulted in positive correIation between copy number of 16S rDNA and genome size in Shewanella spp., with clusters of rrn near lhe origin of replication. This way, the genus is inferred as opportunist. There are no compact genomes and their sequences length varied, ranging from 4306142 nt in S. amazonensis SB2B to 5935403 nt in S. woodyi ATCC 51908, without correIation to temperature range characteristic of each specie. Intragenomic 16S rDNA sequences possess little divergence, but reasonable to resuIt in different phyIogenetic trees, depending on the sequence that is chosen to compare. For moIecuIar detection of iron-reducing Shewanella, it is proposed the mtrB gene as new biomarker. because it codes to a fundamental protein at Fe (III)-reduction. The specific primers were designed and evaluated in silico and resulted in a fragment of 360 pb. In the second study, these primers were tested in a genomic sample from S. oneidensis MR-1, amplifying the expected region. After this successfuI resuIt, the primer set was used as a tool to assess the iron-reducing communities of ShewaneIla genus under an environmental stress, i.e. crude oil contamination in mangrove sediment in Rio Grande do Norte State (Brazil). The primers presented high specificity and the reactions performed resulted in one single band of ampIification in the metagenomic samples. The fingerprinting obtained at DGGE reveaIed temporal variation of Shewanella spp. in analyzed samples. The resuIts presented show the detection of a biotechnological important group of microorganisms, the iron-reducing Shewanella spp. using a metabolic gane as target. It is concluded there are eight or more 16S rDNA sequences in Shewanella genus, with little divergence among them that affects the phylogeny; the pair of primers designed to ampIify mtrB sequences is a viable alternative to detect iron-reducing ShewanelIa in metagenomic approaches; such bacteria are present in the mangrove sediment anaIyzed, with temporal variations in the samples. This is the first experimental study that screened the iron-reducing Shewanella genus in a metagenomic experiment of mangrove sediments subjected to oil contamination through a key metabolic gene

Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST),program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genome-Wide Survey of SNP Variation Uncovers the Genetic Structure of Cattle Breeds

The imprints of domestication and breed development on the genomes of livestock likely differ from those of companion animals. A deep draft sequence assembly of shotgun reads from a single Hereford female and comparative sequences sampled from six additional breeds were used to develop probes to interrogate 37,470 single-nucleotide polymorphisms (SNPs) in 497 cattle from 19 geographically and biologically diverse breeds. These data show that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population, possibly due to bottlenecks associated with domestication, selection, and breed formation. Domestication and artificial selection appear to have left detectable signatures of selection within the cattle genome, yet the current levels of diversity within breeds are at least as great as exists within humans.