Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.

Genomic islands: tools of bacterial horizontal gene transfer and evolution.

Abstract Bacterial genomes evolve through mutations, rearrangements or horizontal gene transfer. Besides the core genes encoding essential metabolic functions, bacterial genomes also harbour a number of accessory genes acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. The horizontal gene transfer contributes to the diversification and adaptation of microorganisms, thus having an impact on the genome plasticity. A significant part of the horizontal gene transfer is or has been facilitated by genomic islands (GEIs). GEIs are discrete DNA segments, some of which are mobile and others which are not, or are no longer mobile, which differ among closely related strains. A number of GEIs are capable of integration into the chromosome of the host, excision, and transfer to a new host by transformation, conjugation or transduction. GEIs play a crucial role in the evolution of a broad spectrum of bacteria as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes leading to generation of hospital 'superbugs', as well as catabolic genes leading to formation of new metabolic pathways. Depending on the composition of gene modules, the same type of GEIs can promote survival of pathogenic as well as environmental bacteria.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.

Time-related action of Lactobacillus plantarum in the bacterial microbiota of shrimp digestive tract and its action as immunostimulant

The objective of this work was to assess the time-related action of probiotic Lactobacillus plantarum in the bacterial microbiota of the digestive tract of Litopenaeus vannamei, and the relation of total haemocyte count and serum phenol oxidase activity of shrimp challenged with Vibrio harveyi. Shrimps were fed with a probiotic-supplemented diet, for eight days, then shifted to a commercial diet. Shrimps fed only with the commercial diet served as control. Evaluations were made on the 8th day of experiment and repeated two, four, six and eight days later. Total lactic bacteria in the digestive tract was higher until the 4th day of evaluation in the probiotic-supplemented group. Vibrio spp. counts were higher in the control at days zero and two. Until the 4th day of evaluation, the total haemocyte counts in shrimps after challenge with V. harveyi were higher in probiotic-supplemented group than in control group. Significant difference was not observed in phenol oxidase activity. On the 6th day after shifting from supplemented to control diet, all parameters were equal in both groups, suggesting that the time-related action of L. plantarum in shrimp is short.

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

Molecular characterization of grapevine from Santa Catarina, Brazil, using microsatellite markers

The objective of this work was to characterize the grape germplasm in Santa Catarina, Brazil, using microsatellite DNA markers (simple sequence repeats - SSR). The DNA samples were collected from leaves and shoots of accessions of public and private collections from the counties Urussanga, Nova Trento, Rodeio, São Joaquim, Campos Novos, Videira, and Água Doce. Ten SSR loci (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79, VVMD25, VVMD28, VVMD31, and VVMD32) were analysed by capillary electrophoresis. Molecular profiling was conducted for 190 grapevines (European, American, and hybrids), and 67 genotypes were obtained. The data were compared with each other and with those from the literature and from online databases, in order to identify varieties and discover cases of synonymy and homonymy. Forty molecular profiles corresponded to known varieties, while 27 genotypes were described for the first time. The existence of typical germplasm composed mainly of American and hybrid varieties is an important finding for local viticulture. Applications of the results rely on quality control and certification at the nursery level. Increasing precision in the characterization of grapevine genotypes may help breeding programs.

Bacterial Monitoring of Iowa's Beaches, 2000

Summary of bacterial monitoring of Iowa's beaches during 2000.

Virulent bacterial infection improves aversive learning performance in Drosophila melanogaster.

Virulent infections are expected to impair learning ability, either as a direct consequence of stressed physiological state or as an adaptive response that minimizes diversion of energy from immune defense. This prediction has been well supported for mammals and bees. Here, we report an opposite result in Drosophila melanogaster. Using an odor-mechanical shock conditioning paradigm, we found that intestinal infection with bacterial pathogens Pseudomonas entomophila or Erwinia c. carotovora improved flies' learning performance after a 1h retention interval. Infection with P. entomophila (but not E. c. carotovora) also improved learning performance after 5 min retention. No effect on learning performance was detected for intestinal infections with an avirulent GacA mutant of P. entomophila or for virulent systemic (hemocoel) infection with E. c. carotovora. Assays of unconditioned responses to odorants and shock do not support a major role for changes in general responsiveness to stimuli in explaining the changes in learning performance, although differences in their specific salience for learning cannot be excluded. Our results demonstrate that the effects of pathogens on learning performance in insects are less predictable than suggested by previous studies, and support the notion that immune stress can sometimes boost cognitive abilities.

Exposure to bioaerosols in poultry houses at different stages of fattening: use of real-time PCR for airborne bacterial quantification

Previous studies have demonstrated that poultry-house workers are exposed to very high levels of organic dust and consequently have an increased prevalence of adverse respiratory symptoms. However, the influence of the age of broilers, on bioaerosol concentrations has not been investigated. To evaluate the evolution of bioaerosol concentration during the fattening period, bioaerosol parameters (inhalable dust, endotoxin and bacteria) were measured in 12 poultry confinement buildings in Switzerland, at 3 different stages of the birds' growth; Samples of air taken from within the breathing zones of individual poultry-house employees as they caught the chickens ready to be transported for slaughter, were also analysed. Quantitative PCR (Q-PCR) was used to assess the quantity of total airborne bacteria and total airborne Staphylococcus species. Bioaerosol levels increased significantly during the fattening period of the chickens. During the task of catching mature birds, the mean inhalable dust concentration for a worker was 31 ± 4.7 mg/m3, and endotoxin concentration was 11'080 ± 3436 UE/m3 air, more than ten-fold higher than the Swiss occupational recommended value (1000 UE/m3). The mean exposure level of bird catchers to total bacteria and Staphylococcus species measured by Q-PCR is also very high, respectively reaching values of 72 (± 11) x107 cells/m3 air and 70 (± 16) x106/m3 air. It was concluded that in the absence of wearing protective breathing apparatus, chicken catchers in Switzerland risk exposure beyond recommended limits for all measured bioaerosol parameters. Moreover, the use of Q-PCR to estimate total and specific numbers of airborne bacteria is a promising tool for evaluating any modifications intended to improve the safety of current working practices.

Architectural features of bacterial genomes and their applications

Résumé -Caractéristiques architecturales des génomes bactériens et leurs applications Les bactéries possèdent généralement un seul chromosome circulaire. A chaque génération, ce chromosome est répliqué bidirectionnellement, par deux complexes enzymatiques de réplication se déplaçant en sens opposé depuis l'origine de réplication jusqu'au terminus, situé à l'opposé. Ce mode de réplication régit l'architecture du chromosome -l'orientation des gènes par rapport à la réplication, notamment - et est en grande partie à l'origine des pressions qui provoquent la variation de la composition en nucléotides du génome, hors des contraintes liées à la structure et à la fonction des protéines codées sur le chromosome. Le but de cette thèse est de contribuer à quantifier les effets de la réplication sur l'architecture chromosomique, en s'intéressant notamment aux gènes des ARN ribosomiques, cruciaux pour la bactérie. D'un autre côté, cette architecture est spécifique à l'espèce et donne ainsi une «identité génomique » aux gènes. Il est démontré ici qu'il est possible d'utiliser des marqueurs «naïfs » de cette identité pour détecter, notamment dans le génome du staphylocoque doré, des îlots de pathogénicité, qui concentrent un grand nombre de facteurs de virulence de la bactérie. Ces îlots de pathogénicité sont mobiles, et peuvent passer d'une bactérie à une autre, mais conservent durant un certain temps l'identité génomique de leur hôte précédent, ce qui permet de les reconnaître dans leur nouvel hôte. Ces méthodes simples, rapides et fiables seront de la plus haute importance lorsque le séquençage des génomes entiers sera rapide et disponible à très faible coût. Il sera alors possible d'analyser instantanément les déterminants pathogéniques et de résistance aux antibiotiques des agents pathogènes. Summary The bacterial genome is a highly organized structure, which may be referred to as the genome architecture, and is mainly directed by DNA replication. This thesis provides significant insights in the comprehension of the forces that shape bacterial chromosomes, different in each genome and contributing to confer them an identity. First, it shows the importance of the replication in directing the orientation of prokaryotic ribosomal RNAs, and how it shapes their nucleotide composition in a tax on-specific manner. Second, it highlights the pressure acting on the orientation of the genes in general, a majority of which are transcribed in the same direction as replication. Consequently, apparent infra-arm genome rearrangements, involving an exchange of the leading/lagging strands and shown to reduce growth rate, are very likely artifacts due to an incorrect contig assembly. Third, it shows that this genomic identity can be used to detect foreign parts in genomes, by establishing this identity for a given host and identifying the regions that deviate from it. This property is notably illustrated with Staphylococcus aureus: known pathogenicity islands and phages, and putative ancient pathogenicity islands concentrating many known pathogenicity-related genes are highlighted; the analysis also detects, incidentally, proteins responsible for the adhesion of S. aureus to the hosts' cells. In conclusion, the study of nucleotide composition of bacterial genomes provides the opportunity to better understand the genome-level pressures that shape DNA sequences, and to identify genes and regions potentially related to pathogenicity with fast, simple and reliable methods. This will be of crucial importance when whole-genome sequencing will be a rapid, inexpensive and routine tool.

Bacterial spot and early blight biocontrol by epiphytic bacteria in tomato plants

The objective of this work was to evaluate in vitro and in vivo biocontrol of bacterial spot (Xanthomonas vesicatoria) and early blight (Alternaria solani) by the epiphytic bacteria Paenibacillus macerans and Bacillus pumilus. Tomato plants were previously sprayed with epiphytic bacteria, benzalkonium chloride and PBS buffer and, after four days, they were inoculated with A. solani and X. vesicatoria. To determine the phytopathogenic bacteria population, leaflet samples were collected from each treatment every 24 hours, for seven days, and plated on semi-selective medium. The effect of epiphytic bacteria over phytopathogens was performed by the antibiosis test and antagonistic activity measured by inhibition zone diameter. The epiphytic and benzalkonium chloride drastically reduced the severity of early blight and bacterial spot in comparison to the control (PBS). In detached leaflets, the epiphytic bacteria reduced in 70% the number of phytopathogenic bacteria cells in the phylloplane. The antibiosis test showed that the epiphytic bacteria efficiently inhibit the phytopathogens growth. In all the bioassays, the epiphytic bacteria protect tomato plants against the phytopathogens

Obtaining interspecific hybrids, and molecular analysis by microsatellite markers in grapevine

The objective of this work was to assess the potential of interspecific hybridization of Vitis labruscana and Muscadinia rotundifolia by using artificial cross-pollinations. Microsatellite markers were used to confirm interspecific hybridizations and the identity of the parental genotypes. In crosses in which M. rotundifolia was used as the female parent, no true hybrids were obtained. In the reciprocal crosses, 114 seedlings were identified as true V. labruscana x M. rotundifolia hybrids. Self pollination occurred in direct and in reciprocal crosses. The crossings between 'Bordo' x 'Carlos', 'Magnolia', 'Regale' and' Roanoke', and between' Isabel' x 'Bountiful', 'Carlos', 'Magnolia', 'Regale' and 'Roanoke' were confirmed. The 15 markers evaluated showed that two M. rotundifolia parental genotypes had the same fingerprint profile, indicating a like lyplanting error. The success of hybridization depends mainly on the species and on the cultivar used as the female parent. Microsatellite markers are efficient to confirm the paternity of interspecific F1 hybrids and to determine the correct identity of M. rotundifolia cultivars.