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.     

Molecular cloning and sequencing of the Na+/K+-ATPase α-subunit of the medical leech Hirudo medicinalis (Annelida) – implications for modelling protostomian evolution

Within the past few years, the phylogenetic tree is discussed controversially regarding the position of the different bilaterian groups. There are two varying views of evolution: the classical one based on morphological structures where the annelids and arthropods are combined in the group of Articulata, and a new hypothesis based on molecular data sets, which divides the protostomian groups in Ecdysozoa (e.g. arthropods, and nematodes) and Lophotrochozoa (e.g. annelids, molluscs, and platyhelminthes). The Na⁺/K⁺-ATPase is a highly conserved protein and fulfils a very important role in physiology and maintaining the homeostasis of cells and can be found in almost all eukaryotic animals. Due to its similar molecular structure throughout the animal kingdom the Na⁺/K⁺-ATPase is an excellent marker for phylogenetic studies. Here we report the molecular cloning, sequencing and phylogenetic analysis of Na⁺/K⁺-ATPase complementary deoxyribonucleic acid (cDNA) of the medical leech Hirudo medicinalis . The cloned cDNA codes for a polypeptide of 1022 amino acids and possesses a predicted molecular mass of 113.33 kDa. Phylogenetic analysis of the complete Na⁺/K⁺-ATPase α -subunit of H. medicinalis and sequence data from other Na⁺/K⁺-ATPases supports the previously developed 'Ecdysozoa concept' with high posterior probabilities. A common clade comprising annelids and platyhelminthes can be defined, whereas nematodes are in a basal position at the arthropod stem line.     

Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants

Nitrogen is often a limiting nutrient, therefore the sustainability of food crops, forages and green manure legumes is mainly associated with their ability to establish symbiotic associations with stem and root-nodulating N2-fixing rhizobia. The selection, identification and maintenance of elite strains for each host are critical. Decades of research in Brazil resulted in a list of strains officially recommended for several legumes, but their genetic diversity is poorly known. This study aimed at gaining a better understanding of phylogenetic relationships of 68 rhizobial strains recommended for 64 legumes, based on the sequencing of the 16S rRNA genes. The strains were isolated from a wide range of legumes, including all three subfamilies and 17 tribes. Nine main phylogenetic branches were defined, seven of them related to the rhizobial species: Bradyrhizobium japonicum, B. elkanii, Rhizobium tropici, R. leguminosarum, Sinorhizobium meliloti/S. fredii, Mesorhizobium ciceri/M. loti, and Azorhizobium caulinodans. However, some strains differed by up to 35 nucleotides from the type strains, which suggests that they may represent new species. Two other clusters included bacteria showing similarity with the genera Methylobacterium and Burkholderia, and amplification with primers for nifH and/or nodC regions was achieved with these strains. Host specificity of several strains was very low, as they were capable of nodulating legumes of different tribes and subfamilies. Furthermore, host specificity was not related to 16S rRNA, therefore evolution of ribosomal and symbiotic genes may have been diverse. Finally, the great diversity observed in this study emphasizes that tropics are an important reservoir of N2-fixation genes.     

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.     

Molecular phylogeny of fungus gnats (Diptera: Mycetophilidae) revisited: position of Manotinae,Metanepsiini, and other enigmatic taxa as inferred from multigene analysis

The phylogeny of selected genera from four subfamilies of fungus gnats (Diptera: Mycetophilidae) – Manotinae, Leiinae, Sciophilinae and Gnoristinae (including Metanepsiini) – is reconstructed based on the combined analysis of five mitochondrial (12S, 16S, COI, COII, cytB) and two nuclear (28S, ITS2) gene markers. Results of the different analyses all support Manotinae as a monophyletic group, with Leiinae as the sister group. Allactoneura DeMeijere is nested in the monophyletic and strongly supported clade of Leiinae. The tribe Metanepsiini is revealed as paraphyletic and the genera Metanepsia Edwards and Chalastonepsia Søli do not appear to be closely related. The genera Docosia Winnertz, Ectrepesthoneura Enderlein, Novakia Strobl and Syntemna Winnertz were placed with a group of genera included traditionally in the Gnoristinae. The monophyly of Dziedzickia Johannsen and Phthinia Winnertz is not supported. The genera of Sciophilinae (excluding Paratinia Mik but including Eudicrana Loew) form a monophyletic group in the Bayesian model.     

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.     

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.