Rep-PCR of tropical rhizobia for strain fingerprinting, biodiversity appraisal and as a taxonomic and phylogenetic tool

With more than 30 million doses of rhizobial inoculants marketed per year, it is probable that Brazilian agriculture benefits more than any other country from symbiotic N₂ fixation. As a result of strain-selection programs, 142 strains of rhizobia are officially recommended for use in commercial inoculants for ninety-six leguminous crops. In this study, sixty-eight of these elite strains were characterized by rep-PCR with the BOX-primer. Reproducibility of the DNA profiles was confirmed, suggesting efficacy of BOX-PCR both for control of quality of inoculants and for preliminary characterization of rhizobial culture collections. Strains of different species never showed similarity higher than 70% in the BOX-PCR analysis, however, some strains of the same species fit into more than one cluster, and correlation between BOX-PCR products and l6S rRNA sequences was low (7.6%). On the other hand, a polyphasic approach — 20%∶80% of BOX-PCR:16S rRNA which correlated well with the l6S rRNA analysis (95%), and provided higher definition of the genotypes, resulting in clearer indications of the taxonomic groups — might expedite rhizobial diversity studies.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

The composition of the gut microbiota shapes the colon mucus barrier

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free-living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.     

A genomic walking method for screening sequence length polymorphism

We adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.