Diversity of the Rap-Phr quorum-sensing systems in the Bacillus cereus group.

Bacteria of the Bacillus cereus group colonize several ecological niches and infect different hosts. Bacillus cereus, a ubiquitous species causing food poisoning, Bacillus thuringiensis, an entomopathogen, and Bacillus anthracis, which is highly pathogenic to mammals, are the most important species of this group. These species are closely related genetically, and their specific toxins are encoded by plasmids. The infectious cycle of B. thuringiensis in its insect host is regulated by quorum-sensing systems from the RNPP family. Among them, the Rap-Phr systems, which are well-described in Bacillus subtilis, regulate essential processes, such as sporulation. Given the importance of these systems, we performed a global in silico analysis to investigate their prevalence, distribution, diversity and their role in sporulation in B. cereus group species. The rap-phr genes were identified in all selected strains with 30% located on plasmids, predominantly in B. thuringiensis. Despite a high variability in their sequences, there is a remarkable association between closely related strains and their Rap-Phr profile. Based on the key residues involved in RapH phosphatase activity, we predicted that 32% of the Rap proteins could regulate sporulation by preventing the phosphorylation of Spo0F. These Rap are preferentially located on plasmids and mostly related to B. thuringiensis. The predictions were partially validated by in vivo sporulation experiments suggesting that the residues linked to the phosphatase function are necessary but not sufficient to predict this activity. The wide distribution and diversity of Rap-Phr systems could strictly control the commitment to sporulation and then improve the adaptation capacities of the bacteria to environmental changes.

Complete Genome Sequence of Streptococcus iniae UEL-Si1, Isolated in Diseased Nile Tilapia (Oreochromis niloticus) from Northern Paraná, Southern Brazil.

The Streptococcus iniae UEL-Si1 strain was isolated from diseased Nile tilapia within the Paranapanema River Basin, Northern Paraná, Brazil. This is an emerging infectious disease agent of fish from Brazil, and sequencing of the complete genome is fundamental to understanding aspects relative to pathogenesis, infection, epidemiology, and immunity.

Genome Sequence of the Mosquitocidal Bacillus thuringiensis Strain BR58, a Biopesticide Product Effective against the Coffee Berry Borer (Hypothenemus hampei).

Bacillus thuringiensis is an important microbial control agent against insect pests. The draft genome sequence of the Brazilian strain BR58 described here contains the insecticidal genes cry4A, cry4B, cry10A, cry11A, cry60A, cry60B, and cyt1A, which show toxicity to both Aedes aegypti and Hypothenemus hampei larvae.

Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches' broom pathogen Moniliophthora perniciosa.

Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches' broom disease (WBD) in cacao. Marked dimorphism characterizes this fungus, showing a monokaryotic or biotrophic phase that causes disease symptoms and a later dikaryotic or saprotrophic phase. A combined strategy of DNA microarray, expressed sequence tag, and real-time reverse-transcriptase polymerase chain reaction analyses was employed to analyze differences between these two fungal stages in vitro. In all, 1,131 putative genes were hybridized with cDNA from different phases, resulting in 189 differentially expressed genes, and 4,595 reads were clusterized, producing 1,534 unigenes. The analysis of these genes, which represent approximately 21% of the total genes, indicates that the biotrophic-like phase undergoes carbon and nitrogen catabolite repression that correlates to the expression of phytopathogenicity genes. Moreover, downregulation of mitochondrial oxidative phosphorylation and the presence of a putative ngr1 of Saccharomyces cerevisiae could help explain its lower growth rate. In contrast, the saprotrophic mycelium expresses genes related to the metabolism of hexoses, ammonia, and oxidative phosphorylation, which could explain its faster growth. Antifungal toxins were upregulated and could prevent the colonization by competing fungi. This work significantly contributes to our understanding of the molecular mechanisms of WBD and, to our knowledge, is the first to analyze differential gene expression of the different phases of a hemibiotrophic fungus.

Genetic diversity among Botryosphaeria isolates and their correlation with cell wall-lytic enzyme production

Nine isolates of Botryosphaeria spp. were evaluated for their growth and the production of cell wall-lytic enzymes (laccase, pectinase and beta-1,3-glucanase) when grown on basal medium in the absence and presence of the laccase inducer, veratryl alcohol (VA). The genetic relationship among the nine isolates collected from different host plants was determined by RAPD analyses. ITS sequence analysis showed eight closely related isolates classified as Botryosphaeria rhodina, and one isolate classified as Botryosphaeria ribis. RAPD analysis resolved the isolates into three main clusters based upon levels of laccase and beta-1,3-glucanase activity. There appears to be no correlation between pectinase production and genetic diversity among the nine isolates. However, the strain characterized as B. ribis, positioned out of the main cluster, was found to be the highest producer of pectinases in the presence of VA.

Nove isolados de Botryosphaeria spp foram avaliados quanto ao crescimento e produção de enzimas líticas da parede celular (lacase, pectinase e beta-1,3-glucanase) quando cultivados em meio basal na ausência e presença do indutor de lacase álcool veratrílico (VA). As relações genéticas entre os nove isolados coletados de diferentes plantas hospedeiras foram determinadas por RAPD. A análise das seqüências de nucleotídeos da região ITS mostrou oito isolados estreitamente relacionados, os quais foram classificados como Botryosphaeria rhodina e um isolado como Botryosphaeria ribis. A análise por RAPD agrupou os isolados em três grupos principais condizentes com os níveis de atividades de lacase e beta-1,3-glucanase. Nenhuma correlação foi detectada entre a produção de pectinase e a diversidade genética nos nove isolados. Entretanto, a linhagem caracterizada como B. ribis, posicionada fora dos grupos principais, se mostrou maior produtora de pectinase na presença de álcool veratrílico.

Distribution of a Ty3/gypsy-like retroelement on the A and B-chromosomes of Cestrum strigilatum Ruiz & Pav. and Cestrum intermedium Sendtn. (Solanaceae)

Retroelements are a diversified fraction of eukaryotic genomes, with the Ty1/copia and Ty3/gypsy groups being very common in a large number of plant genomes. We isolated an internal segment of the Ty3/gypsy retroelement of Cestrum strigilatum (Solanaceae) using PCR amplification with degenerate primers for a conserved region of reverse transcriptase. The isolated segment (pCs12) was sequenced and showed similarity with Ty3/gypsy retroelements of monocotyledons and dicotyledons. This segment was used as probe in chromosomes of C. strigilatum and Cestrum intermedium. Diffuse hybridization signals were observed along the chromosomes and more accentuated terminal signals in some chromosome pairs, always associated with nucleolus organizer regions (NORs). The physical relationship between the hybridization sites of pCs12 and pTa71 ribosomal probes was assessed after sequential fluorescence in situ hybridization (FISH). Hybridization signals were also detected in the B chromosomes of these species, indicating an entail among the chromosomes of A complement and B-chromosomes.

Structural characterization of the bglH gene encoding a beta-glucosidase-like enzyme in an endophytic Bacillus pumilus strain

A beta-glucosidase-like enzyme-encoding gene (bglH) of an endophytic Bacillus pumilus strain (CL16) was cloned using a shotgun genomic library constructed in Escherichia coli. The nucleotide sequence of the entire cloned fragment (2484 bp) was determined and characterized. An incomplete open reading frame (ORF) of 534 bp (ORF1) designated bglP and a complete ORF of 1419 bp (ORF2) designated bglH, located in the fragment, are organized in an operon. The protein deduced from 1419 bp (ORF2) had 472 amino acid residues without a characteristic signal peptide sequence, suggesting that the enzyme is localized in the cytoplasm. The amino acid sequence deduced from bglH gene had high similarity with beta-glucosidases from the glycosyl hydrolase family 1. Over-expression of the B. pumilus bglH gene in E. coli showed a 54 kDa protein whose identity was confirmed by mass spectrometry (MALDI-TOF).

Isolation and partial characterization of a mutant of Bacillus thuringiensis producing melanin

Um mutante (407-P) da linhagem Bacillus thuringiensis subsp. thuringiensis 407 produtor de melanina foi obtido após tratamento com o agente mutagênico etil-metano-sulfonato. Diversas propriedades microbiológicas e bioquímicas das duas linhagens foram analisadas e os resultados foram similares. O mutante 407-P foi incorporado em amostras de solo não esterilizado, recuperado, facilmente identificado e quantificado, possibilitando seu uso em estudos de ecologia de B. thuringiensis.