A Xanthomonas citri subsp citri hypothetical protein related to virulence contains a non-functional HD domain and is implicated in flagellar motility.

Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp citri (Xac), severely affects most economically important citrus varieties worldwide. A previous study showed that disruption of the ORF XAC1201 from the Xac 306 strain by transposon Tn5 decreased bacterium virulence in the Rangpur lime host (Citrus limonia L. Osbeck). However, little is known regarding the possible function of the hypothetical protein XAC1201 and how it affects the virulence of Xac 306. Here, we confirmed that disruption of ORF XAC1201 reduces Xac 306 virulence in two different hosts, delaying the onset of typical symptoms. In silico analysis suggested that XAC1201 interacts with the flagellar proteins FliM and FliL, known to be an important factor for virulence. In fact, motility assays revealed that the XAC1201 mutant has a significant difference in motility compared to the wild-type Xac 306. Also, a 3-D structure model revealed modified cofactor binding sites and suggested that XAC1201 has a non-functional HD domain. This hypothesis was confirmed by enzymatic assays performed in purified, XAC1201 recombinant protein expressed in Escherichia coli, which revealed no significant activities previously associated with HD domains for the tested substrates. Thus, the role of the XAC1201 protein in Xac 306 virulence seems to be related to flagellar motility, although a non-classic role for the HD domain cannot be dismissed.

Mapping and validation of Xanthomonas citri subsp citri genes regulated by putative plant-inducible promoter box (PIP-box).

Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp citri (Xac), is a major disease affecting citriculture worldwide, because of the susceptibility of the host and the lack of efficient control methods. Previous studies have reported that some genes of phytopathogenic bacteria possess a consensus nucleotide sequence (TTCGC...N15...TTCGC) designated the "plant-inducible-promoter box" (PIP box) located in the promoter region, which is responsible for activating the expression of pathogenicity and virulence factors when the pathogen is in contact with the host plant. In this study, we mapped and investigated the expression of 104 Xac genes associated with the PIP box sequences using a macroarray analysis. Xac gene expression was observed during in vitro (Xac grown for 12 or 20 h in XAM1 induction medium) or in vivo (bacteria grown in orange leaves for 3 to 5 days) infection conditions. Xac grown in non-induction NB liquid medium was used as the control. cDNA was isolated from bacteria grown under the different conditions and hybridized to the macroarray, and 32 genes differentially expressed during the infection period (in vitro or in vivo induction) were identified. The macroarray results were validated for some of the genes through semi-quantitative RT-PCR, and the functionality of the PIP box-containing promoter was demonstrated by activating b-glucuronidase reporter gene activity by the PIP box-containing promoter region during Xac-citrus host interaction.

Differential gene expression in drought-tolerant sugarcane roots.

Drought is one of the most frequent abiotic stresses limiting the productivity and geographical distribution of sugarcane culture. The use of drought-tolerant genotypes is one approach for overcoming the effects of water stress. We conducted a comparative study to identify gene expression profiles under water stress in tolerant sugarcane roots. Two different cultivars, 1 drought tolerant (RB867515) and 1 drought susceptible (SP86-155), were evaluated at 4 sampling time points (1, 3, 5, and 10 days) using the cDNA-amplified fragment length polymorphism technique. A total of 173 fragments were found to be differentially expressed in response to water stress in the tolerant cultivar. Seventy of these were cloned, sequenced, and categorized. Similarity analysis using BLAST revealed that 64% of the fragments differentially expressed code proteins classified as no hits (23%), hypothetical (21%), or involved in stress response (20%), with others were involved in communication pathways and signal transduction, bioenergetics, secondary metabolism, and growth and development. Four genes were analyzed and validated using real-time quantitative polymerase chain reaction to determine their expression and showed consistency with the cDNA-amplified fragment length polymorphism analyses. Our results contribute insight into the molecular responses to water stress in sugarcane and possibility to the development of cultivars with improved tolerance to drought.

Genetic Diversity of a Brazilian Strain Collection of Xanthomonas citri subsp. citri Based on the Type III Effector Protein Genes.

Exclusion and eradication or management based on an integrated approach with less susceptible varieties, copper-based bactericides, and windbreaks are the two main strategies used to prevent or control citrus canker. Field tolerance or resistance to citrus canker is not found in the most important commercial sweet orange cultivars, and pathogen-derived resistance has been developed and applied in different crops to obtain resistant genotypes to plant pathogens. We describe the development of DNA primers and probes based on the type III effector genes avrXacE1, avrXacE2, avrXacE3, avrBs2, pthA4, hpaF, and XAC3090 (leucine rich protein), and their application in the evaluation of the genetic diversity of the pathogen. A total of 49 haplotypes were identified in 157 strains by Southern blot analysis. No genetic polymorphism was detected by BOX elements - and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis, nor with the genes avrBs2, XAC3090, and hpaF. Nei's genetic diversity indexes varied from 0.65 to 0.96 for subcollections of the pathogen. One or few haplotypes were most frequent in the strain collection, but several haplotypes were represented by solely one or few strains. The PthA4 probe resulted in the higher number of haplotypes identified in the Brazilian subcollections. Greater variation in the frequency of haplotypes occurred within subcollections (93.7%) than among subcollections. Only some haplotypes were genetically distant from all others, especially those originated from Rio Grande do Sul and Santa Catarina states. These bacterial effectors are widely spread in the collections and are useful for a better understanding of the host-pathogen interaction and the search for resistance genes in host and nonhost plants.

Identification and characterization of polymorphisms within the 5' flanking region, first exon and part of first intron of bovine GH gene.

The aim of the present study was to identify and characterize polymorphisms within the 5' flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental x Nelore (n = 30), Canchim x Nelore (n = 30) and Angus x Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648, AY662649 and AY662650, respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652, respectively). The allele sequences were compared with GenBank sequences (M57764, AF007750 and AH009106) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.

Predominant African-derived mtDNA in Caribbean and Brazilian Creole cattle is also found in Spanish cattle (Bos taurus).

African-derived mitochondrial DNA (mtDNA) have been described in South American and Caribbean native cattle populations, which could have been introduced into America from Iberia or by direct importation from Africa. However, the similarity among described haplotypes is not known. We examined mtDNA variation in Guadeloupe Creole and Spanish cattle in an attempt to identify African-derived mtDNA haplotypes and compare them with those previously described. Eleven haplotypes clustered into the European taurine haplogroup (T3), two haplotypes into the African taurine (T1) haplogroup, and three haplotypes into the African-derived American haplogroup (AA). The AA1 and Eucons haplotypes were the most frequently observed. The presence of the AA haplogroup in Spanish cattle confirms historical records and genetic evidence of Iberian cattle as the main source of American native cattle origin. The possible origin of African-derived mitochondrial haplotypes in Iberian and Creole cattle is discussed, and the accumulated evidence does not support a founder effect from African ancestral cattle by direct importations. The presence of taurine AA and T3 haplotypes in Brazilian Nellore may indicate introgression by local European-derived cattle. Data presented in this work will contribute to the understanding of the origin of Guadeloupe Creole cattle.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Coordinated, network-based research as a strategic component of science in Brazil

Scientific research plays a fundamental role in the health and development of any society, since all technological advances depend ultimately on scientific discovery and the generation of wealth is intricately dependent on technological advance. Due to their importance, science and technology generally occupy important places in the hierarchical structure of developed societies, and they receive considerable public and private investment. Publicly funded science is almost entirely devoted to discovery, and it is administered and structured in a very similar way throughout the world. Particularly in the biological sciences, this structure, which is very much centered on the individual scientist and his own hypothesis-based investigations, may not be the best suited for either discovery in the context of complex biological systems, or for the efficient advancement of fundamental knowledge into practical utility. The adoption of other organizational paradigms, which permit a more coordinated and interactive research structure, may provide important opportunities to accelerate the scientific process and further enhance its relevance and contribution to society. The key alternative is a structure that incorporates larger organizational units to tackle larger and more complex problems. One example of such a unit is the research network. Brazil has utilized such networks to great effect in genome sequencing projects, demonstrating their relevance to the Brazilian research community and opening the possibility of their wider utility in the future

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa.

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Growth hormone mRNA expression in the pituitary of Bos indicus and Bos taurus x Bos indicus crossbred young bulls treated with recombinant bovine somatotropin

The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 x 2]-factorial arrangement, using two levels of rbST (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A cDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus cDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbST treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone mRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone mRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9 higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds

Comparative genomic analysis of plant-associated bacteria.

This review deals with a comparative analysis of seven genome sequences from plant-associated bacteria. These are the genomes of Agrobacterium tumefaciens, Mesorhizobium loti, Sinorhizobium meliloti, Xanthomonas campestris pv campestris, Xanthomonas axonopodis pv citri, Xylella fastidiosa, and Ralstonia solanacearum. Genome structure and the metabolism pathways available highlight the compromise between the genome size and lifestyle. Despite the recognized importance of the type III secretion system in controlling host compatibility, its presence is not universal in all necrogenic pathogens. Hemolysins, hemagglutinins, and some adhesins, previously reported only for mammalian pathogens, are present in most organisms discussed. Different numbers and combinations of cell wall degrading enzymes and genes to overcome the oxidative burst generally induced by the plant host are characterized in these genomes. A total of 19 genes not involved in housekeeping functions were found common to all these bacteria.

Comparison of the genomes of two Xanthomonas pathogens with differing host specificities.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Effect of heat exposure on the thermoregulatory responses of selected naked neck chickens

The variation in cloacal temperature, body weight loss and expression of the 70 kDa heat shock protein (Hsp70) in three naked neck broiler genotypes during heat stress were studied. Twelve birds of each genotype (Na/Na, Na/na and na/na) were reared to market weight (approximately 2.1kg) at thermoneutral temperature. Six birds from each group served as controls and the remaining six underwent gradual heat stress (from 28oC to 36oC). Cloacal temperature and body weight were measured before and after exposure to heat. Liver samples were collected and Hsp70 levels were quantified using western blotting with monoclonal anti-chicken Hsp70 antibody. Heterozygous (Na/na) birds had a significantly lower cloacal temperature variation and less body weight loss during heat stress than the other genotypes. There was no significant difference in the Hsp70 levels among the genotypes. Heterozygous birds (Na/na) appeared to have a slight advantage over the other genotypes during gradual heat stress, perhaps because of a heterotic effect

Growth hormone mRNA expression in the pituitary of Bos indicus and Bos taurus x Bos indicus crossbred young bulls treated with recombinant bovine somatotropin.

The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 x 2]-factorial arrangement, using two levels of rbST (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A cDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus cDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbST treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone mRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone mRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9% higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds.

African-derived mitochondria in South American native cattle breeds (Bos taurus): evidence of a new taurine mitochondrial lineage.

This article reports the nucleotide diversity within the control region of 42 mitochondrial chromosomes belonging to five South American native cattle breeds (Bos taurus). Analysis of these data in conjunction with B. taurus and B. indicus sequences from Africa, Europe, the Near East, India, and Japan allowed the recognition of eight new mitochondrial haplotypes and their relative positions in a phylogenetic network. The structure of genetic variation among different hypothetical groupings was tested through the molecular variance decomposition, which was best explained by haplotype group components. Haplotypes surveyed were classified as European-related and African-related. Unexpectedly, two haplotypes within the African cluster were more divergent from the African consensus than the latter from the European consensus. A neighbor-joining tree shows the position of two haplotypes compared to European/African mitochondrial lineage splitting. This different and putatively ancestral mitochondrial lineage (AA) is supported by the calibration of sequence divergence based on the Bos-Bison separation. The European/African mitochondria divergence might be subsequent (67,100 years before present) to that between AA and Africans (84,700 years before present), also preceding domestication times. These genetic data could reflect the haplotype distribution of Iberian cattle five centuries ago.

High-level production of functional muscle alpha-tropomyosin in Pichia pastoris.

Although numerous studies have reported the production of skeletal muscle alpha-tropomyosin in E. coli, the protein needs to be modified at the amino terminus in order to be active. Without these modifications the protein does not bind to actin, does not exhibit head-to-tail polymerization, and does not inhibit the actomyosin Mg(2+)-ATPase in the absence of troponin. On the other hand, the protein produced in insect cells using baculovirus as an expression vector (Urbancikova, M., and Hitchcock-DeGregori, S. E., J. Biol. Chem., 269, 24310-24315, 1994) is only partially acetylated at its amino terminal and therefore is not totally functional. In an attempt to produce an unmodified functional recombinant muscle alpha-tropomyosin for structure-function correlation studies we have expressed the chicken skeletal alpha-tropomyosin cDNA in the yeast Pichia pastoris. Recombinant protein was produced at a high level (20 mg/L) and was similar to the wild type muscle protein in its ability to polymerize, to bind to actin and to regulate the actomyosin S1 Mg(2+)-ATPase.

Restriction fragment length polymorphism (RFLP) in exon 2 of the BoLA-DRB3 gene in South American cattle.

The Bola-DRB3 gene participates in the development of the immune response and is highly polymorphic. For these reasons, it has been a candidate gene in studies of the genetic basis of disease resistance and in population genetic analysis. South American native cattle breeds have been widely replaced by improved exotic breeds leading to a loss of genetic resources. In particular, South American native breeds have high levels of fertility and disease resistance. This work describes genetic variability in the BoLA-DRB3 gene in native (Caracu, Pantaneiro, Argentinean Creole) and exotic (Holstein, Jersey, Nelore, Gir) cattle breeds in Brazil and Argentina. PCR-RFLP alleles were identified by combining the restriction patterns for the BoLA-DRB3.2 locus obtained with RsaI, BstY and HaeIII restriction enzymes. Allelic frequencies and deviations from the Hardy-Weinberg equilibrium were also calculated. Analysis of the 24 BoLA-DRB3 PCR-RFLP alleles identified showed differences in the allele distributions among breeds.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Structure and genetic relationship among Brazilian naturalized and imported goat breeds.

Fourteen goat populations were studied regarding their genetic relationship and structure. Parameters of genetic diversity (HT, HS and GST) and F statistic (FIS, FIT and FST) were estimated. Undefined breed populations presented high homogeneity, as did imported breed populations. Naturalized breed populations showed high differentiation. The genetic distances separating these 14 goat populations were calculated from gene frequency data for eight blood genetic markers (esterase D, phosphoglucomutase 1, carbonic anhydrase II, peptidase B, amylase, haemoglobin, transferrin, and protein X). Working with the genetic distance matrix of Nei corrected for small samples (DA), we constructed a dendrogram using the unweighted pair group method with arithmetic mean. DA values ranged from 0.0027 to 0.1518. The dendrogram divided the populations into two groups, one consisting of three populations of naturalized breeds, and another including the other populations (imported breeds, undefined breeds and some other naturalized breeds).

Hepatic concentration of heat shock protein 70 kD (Hsp70) in broilers subjected to different thermal treatments.

1. The relationship between repeated thermal treatments and hepatic synthesis of Hsp 70 was studied in broiler chickens. 2. Sixty broilers were submitted to 5 different treatments (12 birds each) from day 1 to day 42. Four groups were kept in a thermoneutral environment and subjected to 0, 1, 2 and 3 heat stress episodes at 35 degrees C for 4 h per week (TN-0, TN-1, TN-2 and TN-3, respectively). The last group (HT-35) was reared at a room temperature of 35 degrees C. 3. From 39 to 42 old, the birds experienced acute heat stress at 41 degrees C. Resistance to heat stress was evaluated by the time taken for rectal temperature to increase by 3 degrees C above the pre-treatment value. Livers were collected (before and after heat stress) and Hsp70 was determined using Western Blot analysis with monoclonal anti-Hsp70 antibody. 4. Resistance to heat stress and concentration of Hsp70 were higher in those birds subjected to more heat stress episodes during the experimental period (TN-3) and HT-35. A positive correlation was observed between Hsp70 concentration and the time taken for a 3 degrees C increase in rectal temperature (r = 0.42; P<0.01). 5. Exposing birds to episodes of heat stress (35 degrees C) during rearing may improve their resistance to acute heat stress, but the previous thermal history did not seem to influence the hepatocyte Hsp70 content after exposure to more severe heat stress (41 degrees C).