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-grapegrowing 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.Different microorganisms are able to survive in and to colonize plant water-conductive vessels (xylem). The result of this association is either beneficial or detrimental to the plant host.Of the latter, an example is the association of Xylella fastidiosa (38) with diverse plant hosts. X. fastidiosa is a fastidious, insecttransmitted, xylem-inhabiting bacterium known to cause several economically important diseases of both monocotyledonous and dicotyledonous plants (14,17,29). These diseases include Pierce's disease (PD) of grapevine and citrus variegated chlorosis (CVC), which have rather distinct symptoms and geographical distributions.PD, caused by certain strains of X. fastidiosa, is characterized by wilted, shriveled, raisin-like fruit and scorched leaves that detach, leaving bare petioles attached to the canes (37). The bark of affected canes may lignify or mature irregularly, leaving

PCR has been used to analyze the distribution of REP (Repetitive Extragenic Palindromic) and ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences (rep-PCR) found within the genome of the bacterium Bacillus thuringiensis, with the purpose to analyze the genetic similarities among 56 subspecies samples and 95 field isolates. The PCR products were analyzed by EB-AGE (ethidium bromide-agarose electrophoresis) and then submitted to banding comparisons, based on the Phyllip software algorithm. When the banding similarities were considered for comparison purposes among all the strains, the phylogenic tree patterns varied according to the rep-PCR primers considered, but, from a broader point of view, the ERIC sequences produced better results, which, together with electron microscopy analysis of the released parasporal bodies and colony morphology characteristics, allowed to detect two possible new subspecies of B. thuringiensis.

Detecção de

The entomopathogenic bacterium Bacillus thuringiensis is a control agent with toxic and environmental characteristics that allows the control of pest insects according to the Integrate Pest Management (IPM) precepts. In order to find new strains, potentially toxic to Sitophilus oryzae L. 1763 (Coleoptera: Curculinidae), 1.073 strains of B. thuringiensis from parts of Brazil were used. Genetic material was extracted with InstaGene Matrix kit, used for the amplification of sequences in Polymerase chain reaction (PCR), and viewed in 1.5% agarose gel. The gene cry35Ba class was represented by 60 B. thuringiensis isolates (5.6%), which were then subjected to bioassays with S. oryzae larvae. Among the isolates studied, four caused more than 50% mortality in pathogenicity tests, and the isolates 544 and 622 were the most virulent, as determined by CL 50 estimates. The four toxic isolates had spherical, bi-pyramidal and cuboid crystals, and a 44-kDa protein was found in sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE), which coded for the product of cry35Ba genes. These data demonstrate the potential of B. thuringiensis for the management of S. oryzae larvae.

There are several genes involved in Bacillus thuringiensis sporulation. The regulation and expression of these genes results in an upregulation in Cry protein production, and this is responsible for the death of insect larvae infected by Bacillus thuringiensis. Gene expression was monitored in Bacillus thuringiensis during three developmental phases. DNA macroarrays were constructed for selected genes whose sequences are available in the GenBank database. These genes were hybridized to cDNA sequences from B. thuringiensis var. kurstaki HD-1. cDNA probes were synthesized by reverse transcription from B. thuringiensis RNA templates extracted during the exponential (log) growth, stationary and sporulation phases, and labeled with 33 PadCTP. Two genes were differentially expressed levels during the different developmental phases. One of these genes is related to sigma factor (sigma 35), and the other is a cry gene (cry2Ab). There were differences between the differential levels of expression of various genes and among the expression detected for different combinations of the sigma factor and cry2Ab genes. The maximum difference in expression was observed for the gene encoding sigma 35 factor in the log phase, which was also expressed at a high level during the sporulation phase. The cry2Ab gene was only expressed at a high level in the log phase, but at very low levels in the other phases when compared to the sigma 35. EXPRESSÃO DOS GENES SIGMA 35 E CRY2AB ENVOLVIDOS NA VIRULÊNCIA DE Bacillus thuringiensis RESUMO: Muitos genes estão envolvidos nos mecanismos de esporulação da bactéria Bacillus thuringiensis. A regulação e expressão desses genes resultam em uma produção massiva da proteína Cry, responsável pela morte das larvas de muitos insetos. Neste trabalho monitorou-se a expressão de genes de Bacillus thuringiensis, ao longo de três fases de seu desenvolvimento. Foram construídos macroarrays de DNA dos genes selecionados, cujas seqüências estão disponibilizadas no GenBank. Estes genes foram hibridizados com cDNAs obtidos de B. thuringiensis kurstaki HD-1. As sondas de cDNA foram sintetizadas a partir da transcrição reversa do RNA da bactéria, extraído durante as fases de crescimento logarítmico, estacionária e esporulativa, marcadas com 33 PadCTP. A expressão diferencial encontrada foi significativa para dois genes de B.thuringiensis, um relacionado aos fatores sigma (sigma 35) e outro ao gene cry (cry2Ab). Detectaram-se diferenças entre as médias de expressão do fator sigma e do gene cry2Ab. Os valores máximos de expressão diferencial foram obtidos para o gene codificador do fator sigma 35 na fase log e na fase esporulativa. Na análise de médias observou-se expressão do gene cry2Ab apenas na fase log; no entanto, de forma bem mais baixa quando comparado com a expressão de sigma 35 , nas três fases.

Some pest management programs employ PCR to identify cry1 genes from Bacillus thuringiensis to predict bacterial toxicity towards different insect pests. However, due to changes on the mode of action of the Cry proteins, new primers had to be designed to detect the new genes. Therefore, an 'in-silico' study of genetic sequences from five cry1 subclasses was carried out and characterized by molecular tools. The design of new primers allows for more precise selection of B. thuringiensis isolates, helping to better direct the programs employing biological control.

RESUMO O presente trabalho teve como objetivo caracterizar geneticamente 1.073 isolados de Bacillus thuringiensis, de três coleções brasileiras, provenientes da UNESP, Jaboticabal, da ESALQ/ Piracicaba e da EMBRAPA. Sete Lagoas, analisando os tipos de genes cry1 apresentados pelos isolados. Para isso, foram elaborados oligonucleotídeos iniciadores a partir de 16 regiões conservadas e 4 regiões não conservadas das seqüências de cada uma das 16 subclasses do gene cry1. Essas seqüências foram amplificadas por PCR e a presença de amplicons para cada subclasse foi calculada em porcentagem por gene e por coleção. Nessa análise, 55,7% dos isolados apresentaram amplificação para o gene cry1, e as subclassescry1Aa, cry1Ab, cry1Ac, cry1Ad, cry1Ae, cry1Af, cry1Ag, e cry1Bf, cry1Ca e cry1Fa estão presentes em alta proporção de isolados, variando de 43,4% a 54,9%. Verificou-se que existe uma distribuição das subclasses dentro do banco de isolados de B. thuringiensis em estudo, com maior porcentagem de isolados portadores dos genes cry1Ab (42,12%) e com menor porcentagem de representantes da subclasse cry1Db (0,6%). A variabilidade gênica, nas coleções analisadas, destaca as coleções de Jaboticabal e Piracicaba como fontes de isolados promissores para uso em programas de Controle Biológico de pragas da ordem Lepidoptera. A coleção de Sete Lagoas, na qual as freqüências das subclasses estudadas foram relativamente baixas (abaixo de 20%), destaca somente o gene cry1Ab, presente em 38,5% dos isolados desta coleção.

RESUMO O objetivo deste trabalho foi analisar a sensibilidade antimicrobiana in vitro de cepas de Staphylococcus aureusisoladas de tetos de vacas e mãos de retireiros, além de verificar o polimorfismo entre elas pela técnica de PCR-RAPD. Os testes foram realizados pela técnica de difusão em discos e, após a extração do material genético foram desenvolvidas as técnicas de PCR e RAPD, usando para isso 40 iniciadores diferentes. A análise do polimorfismo foi realizada empregando-se o programa de taxonomia numérica NTSYS. As sensibilidades dos antimicrobianos nas cepas obtidas de tetos de vacas foram 4% para a penicilina, 88% para a tetraciclina, 92% para a gentamicina, 96% para a vancomicina e 100% ao cloranfenicol. Para as cepas provenientes das mãos de retireiros, os resultados de sensibilidade foram zero para a penicilina, 70% para a tetraciclina e 90% para a vancomicina e 100% para os antimicrobianos gentamicina e cloranfenicol. A realização do E-teste indicou uma concentração inibitória mínima (CIM) maior que 256 mg/mL para as cepas resistentes ao antimicrobiano vancomicina. Os estudos permitiram detectar a resistência dos S. aureus mediante o uso dos antimicrobianos testados e determinar a diversidade genética entre as cepas de estafilococos devido à presença de muitas bandas polimórficas encontradas em todos os iniciadores.