Laurival A. Vilas-Boas

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

A Molecular Method for Detection of Aspergillus carbonarius in Coffee Beans

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7809, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F809 and OPX7R809) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.

Molecular identification of Aspergillus spp. isolated from coffee beans

Some species belonging to the genus Aspergillus are potential producers of ochratoxin A (OA), a mycotoxin with nephrotoxic, immunosuppressive, teratogenic and carcinogenic effects. The aim of the present study was to identify the species of Aspergillus that contaminate the inside of coffee beans collected in the stage of maturation and drying, from 16 producing areas located in the northern region of the State of Parana, in the South of Brazil. A total of 108 isolates of Aspergillus spp. was identified at the species level, by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The results revealed the presence of potentially ochratoxigenic species in 82% of the geographic regions studied, among which Aspergillus niger was the species most frequently detected, followed by A. ochraceus and A. carbonarius. The presence of A. carbonarius in immature coffee fruits harvested from trees is reported for the first time.

Detection of Salmonella spp, Salmonella Enteritidis and Typhimurium in naturally infected broiler chickens by a multiplex PCR-based assay

The presence of Salmonella in the intestinal tract, on the chickens skin and among their feathers, may cause carcasses contamination during slaughtering and processing and possibly it is responsible by the introduction of this microorganism in the slaughterhouses. A rapid method to identify and monitor Salmonella and their sorovars in farm is becoming necessary. A pre-enriched multiplex polymerase chain reaction (m-PCR) assay employing specific primers was developed and used to detect Salmonella at the genus level and to identify the Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) in broiler chicken swab samples. The method was validated by testing DNA extract from 90 fresh culture cloacal swab samples from poultry chicken cultured in phosphate buffer peptone water at 37 °C for 18 h. The final results showed the presence of Salmonella spp. in 25% of samples, S. Enteritidis was present in 12% of the Salmonella-positive samples and S. Typhimurium in 3% of the samples. The m-PCR assay developed in this study is a specific and rapid alternative method for the identification of Salmonella spp. and allowed the observation of specific serovar contamination in the field conditions within the locations where these chickens are typically raised.

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.

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

A mutant (407-P) of Bacillus thuringiensis subsp. thuringiensis strain 407 producing a melanin was obtained after treatment with the mutagenic agent ethyl-methane-sulfonate. Several microbiological and biochemical properties of the two strains were analyzed and the results were similar. The mutant 407-P was also incorporated into non-sterilized soil samples, recovered, easily identified, and quantified, what enables its use in ecology of B. thuringiensis.

A New Approach to Chromosomal Evolution in the Giant Water Bug (Heteroptera: Belostomatidae)

The genus Belostoma, known colloquially as “giant water bugs,” presents striking cytogenetic diversity and extensive chromosome variability. Notwithstanding, its karyotype evolution is not well understood. We analyzed 8 species of Belostoma (77 samples). The meiotic analysis revealed 2n = 14 + XY for Belostoma horvathi and Belostoma candidulum; 2n = 22 + XY for Belostoma cummings; 2n = 26 + X1X2Y for Belostoma dentatum, Belostoma elongatum, and Belostoma discretum; and 2n = 26 + X1X2X3Y for Belostoma testacopallidum and Belostoma dilatatum. All species showed holokinetic chromosomes. Based on heterochromatin distribution patterns and 18S rDNA, the species of the genus Belostoma were separated into four groups. The analysis of C0t-1 DNA showed that the repetitive DNA, partly composed of microsatellite DNA, was absent on the Y chromosome. Fluorescent in situ hybridization (FISH) using a microdissected X chromosome in species with simple sex system presents uniform hybridization in the nuclear region corresponding to the X chromosome. Species with multiple systems revealed discrete markings. The present data in conjunction with the existing literature led us to propose a new evolutionary hypothesis for the group, with an ancestral karyotype with a low diploid number, simple sex determination system, and nucleolus organizer regions (NORs) on the sex chromosomes. That karyotype would have originated other karyotypes through agmatoploidy, simploidy, heterochromatinization, and movement of the 18S rDNA.

Genetic diversity of bovine papillomavirus types, including two putative new types, in teat warts from dairy cattle herds

Teat papillomatosis affects dairy cows worldwide. Milking can become difficult due to teat warts, and maintaining affected cows in the herds may diminish economic profit in the dairy industry. Currently, 13 bovine papillomavirus (BPV) types have been fully characterized, and numerous putative BPV types have been identified through partial L1 gene PCR. In order to identify the viral types present in warts on the udders of dairy cows, 40 teat lesions from 24 cows from 13 cattle farms in three States of Brazil were evaluated by PV L1 gene PCR. The warts that were evaluated contained sequences from BPVs 6-10, the putative BPV types BAPV9 and BAPV4, and two unreported putative papillomavirus (PV) types, named BPV/BR-UEL6 and BPV/BR-UEL7. In addition, mixed infections and coinfections were identified, since more than one lesion was observed on the udders of 13 cows. Phylogenetic analysis showed that BPV/BR-UEL6 is closely related to BPVs belonging to the genus Xipapillomavirus, while BPV/BR-UEL7 clustered with the previously reported strains Cervus timorensis and Pudu puda PVs, which represent a putative new PV type, and it was only distantly related to xi-, epsilon-, delta- and dyoxi-PVs. These results provide information that will assist in the understanding of the association of BPVs 6, 7, 8, 9, and 10, as well as putative BPV types BAPV4 and BAPV9, with mammary papillomatosis. This is the first characterization of putative novel PV types BPV/BR-UEL6 and BPV/BR-UEL7 in teat warts of dairy cows, highlighting the high genetic diversity of BPVs associated with teat papillomatosis.

Streptococcus iniae: an unusual important pathogen fish in Brazil.

The current paper contains a report on the isolation of Streptococcus iniae in Nile tilapia (Oreochromis niloticus) in Brazil and South America. S. iniae is an important pathogen often associated with significant losses in fish production. It is also known for causing invasive infections in humans. An outbreak of infections characterized by exophthalmos, erratic swimming, ascites and melanosis occurred in intensive fish farming. Samples of kidney, brain and liver of the fish seeded into the culture medium yielded beta-hemolytic, gram-positive cocci isolate from the colonies. The partial sequencing of the 16S ribosomal gene was performed to identify the isolate. The sequence obtained showed 99% identity to 16S sequences of S. iniae present in the Genbank. A phylogenetic analysis was accomplished to confirm the species. Although S. iniae is frequently present in almost all continents, this work disclosed the second case of isolation of this pathogen in farmed fish both in Brazil and South America.

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 b-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).