Dendroctonus rhizophagus Thomas and Bright (Curculionidae: Scolytinae) is an endemic economically important insect of the Sierra Madre Occidental in Mexico. This bark beetle has an atypical behavior within the genus because just one beetle couple colonizes and kills seedlings and young trees of 11 pine species. In this work, the bacteria associated with the Dendroctonus rhizophagus gut were analyzed by culture-dependent and culture-independent methods. Analysis of 16S rRNA sequences amplified directly from isolates of gut bacteria suggests that the bacterial community associated with Dendroctonus rhizophagus, like that of other Dendroctonus spp. and Ips pini, is limited in number. Nine bacterial genera of γ-Proteobacteria and Actinobacteria classes were detected in the gut of Dendroctonus rhizophagus. Stenotrophomonas and Rahnella genera were the most frequently found bacteria from Dendroctonus rhizophagus gut throughout their life cycle. Stenotrophomonas maltophilia, Ponticoccus gilvus, and Kocuria marina showed cellulolytic activity in vitro. Stenotrophomonas maltophilia, Rahnella aquatilis, Raoultella terrigena, Ponticoccus gilvus, and Kocuria marina associated with larvae or adults of Dendroctonus rhizophagus could be implicated in nitrogen fixation and cellulose breakdown, important roles associated to insect development and fitness, especially under the particularly difficult life conditions of this beetle.

The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (γ- and α-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (K(m) of 8.9 µM for NADPH and 3.7 µM for dihydrofolate and IC(50) of 20 µM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences.

An obligate methanotrophic bacterium, strain MTS, was isolated from a methane-fed microaerobic denitrifying bioreactor. 16S rRNA and DNA-DNA hybridization analysis revealed that this organism was most closely related to Methylocystis parvus, a Type II methanotroph, belonging to the α-subclass of the Proteobacteria. The metabolism of the bacterium under microaerobic and anaerobic conditions was studied by (13) C-NMR. (13) C-labelled poly-β-hydroxybutyrate (PHB) formation occurred in cell suspensions incubated with (13) C-labelled methane at low (5-10%) oxygen concentration. Under these conditions low levels of succinate, acetate and 2,3-butanediol were formed and excreted into the culture medium. Intracellular PHB degradation was observed in intact cells under anaerobic conditions in the absence of an exogenous carbon source during a long-term incubation of 90 days. Multiple (13) C-labelled β-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were identified as products in in vivo(13) C-NMR spectra and in the spectra of culture medium during the dynamic PHB degradation. The isolated obligate methanotroph clearly shows a fermentative metabolism of PHB under anaerobic conditions. The excreted products may serve as substrates for denitrifying bacteria.

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The nature reserve of Tehuacan-Cuicatlan in central Mexico is known for its diversity and endemism mainly in cactus plants. Although the xerophytic flora is reasonably documented, the bacterial communities associated with these species have been largely neglected. We assessed the diversity and composition of bacterial communities in bulk (non-rhizospheric) soil and the rhizosphere of three cactus plant species: Mammillaria carnea, Opuntia pilifera and Stenocereus stellatus, approached using cultivation and molecular techniques, considering the possible effect of dry and rainy seasons. Cultivation-dependent methods were focused on putative N(2)-fixers and heterotrophic aerobic bacteria, in the two media tested the values obtained for dry season samples grouped together regardless of the sample type (rhizospheric or non-rhizospheric), these groups also included the non-rhizospheric sample for rainy season, on each medium. These CFU values were smaller and significantly different from those obtained on rhizospheric samples from rainy season. Genera composition among isolates of the rhizospheric samples was very similar for each season, the most abundant taxa being α-Proteobacteria, Actinobacteria and Firmicutes. Interestingly, the genus Ochrobactrum was highly represented among rhizospheric samples, when cultured in N-free medium. The structure of the bacterial communities was approached with molecular techniques targeting partial 16S rRNA sequences such as denaturing gradient gel electrophoresis and serial analysis of ribosomal sequence tags. Under these approaches, the most represented bacterial phyla were Actinobacteria, Proteobacteria and Acidobacteria. The first two were also highly represented when using isolation techniques.

The phylogenetic positions of four rhizobial strains obtained from nodules of common bean plants (PhaseoZus vulgaris L.) grown in an Austrian soil and of the Mexican bean isolate FL27 are described. Analysis of the 16s rRNA genes revealed sequences almost identical to that of the Rhizobium gallicum type strain, R602sp, with a maximum of two nucleotide substitutions. Comparison of the 16s rRNA gene sequences with those from other bacteria indicated highest similarity to Rhizobium sp. strain OK-50, Rhizobium Zeguminosarum IAM 12609, and Rhizobium etli. DNA homology determined by DNA-DNA hybridization was high among the Austrian isolates and R602spT (45 to 90%) and ranged from 21 to 65% with FL27, but hybridization analysis revealed very low homology to the recognized common bean-nodulating species, R. Zeguminosarum bv. phaseoli, R. etli, and Rhizobium tropici. Ribosomal gene organization was studied by Southern hybridization with the 16s rRNA gene and temperature gradient gel electrophoresis, indicating identical organizations and the presence of three identical 16s rRNA copies in the genome of this species. The six strains investigated showed different plasmid profiles based on their geographical origins. We propose that the Austrian isolates and the Mexican strain FL27 are members of the species R. gallicum.Bacteria of the genus Rhizobium that are able to nodulate common bean plants (Phaseolus vulgaris L.) have been traditionally classified as Rhizobium leguminosarum bv. phaseoli (13) on the basis of the host plant they infect. Strains belonging to the other subdivisions of this species, R. leguminosarum bv. viciae and bv. trifolii, nodulate peas and clovers, respectively, and their symbiotic plasmids carry genes with different host specificities. Nevertheless, rhizobia from common bean plants have been found to be phylogenetically diverse based on different criteria, such as protein profiles (32), multilocus enzyme electrophoresis patterns (6, 28), results of DNA relatedness analysis (16,37,44), and differences in their 16s rRNA gene (rDNA) sequences (9,16,44). In addition to R. leguminosarum bv. phaseoli, two new species, Rhizobium etli (38) and Rhizobium tropici (20), have been described. Both R. leguminosarum bv. phaseoli and R. etli carry multiple copies of the nitrogenase reductase gene ( n i m on their symbiotic plasmids, but they have different 16s rRNA sequences (17,30,38). In contrast, R. tropici maintains only a single nifH gene copy on its symbiotic plasmid (20). R. etli and R. tropici show a broad host range, but they nodulate different hosts (9, 20). Several new species among bean-nodulating strains, including Rhizobium gallicum and Rhizobium giardinii, which comprise the French isolates (1, 16), as well as Rhizobium sp. (Phaseolus) RCR 3618D of unknown geographical origin, have been proposed (44). The partial 16s rDNA sequence of the R. gallicum type strain, R602sp, was found to be identical to that of strain FL27 (16), a Mexican isolate from the common bean which does not fixate N2 well (28). In ...

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Ten bacterial strains were isolated by enrichment culture, using as carbon sources either aliphatics or an aromatic-polar mixture. Oxygen uptake rate was used as a criterion to determine culture transfer timing at each enrichment stage. Biodegradation of aliphatics (10,000 mg L(-1)) and an aromatic-polar mixture (5000 mg L(-1), 2:1) was evaluated for each of the bacterial strains and for a defined culture made up with a standardized mixture of the isolated strains. Degradation of total hydrocarbons (10,000 mg L(-1)) was also determined for the defined mixed culture. Five bacterial strains were able to degrade more than 50% of the aliphatic fraction. The most extensive biodegradation (74%) was obtained with strain Bs 9A, while strains Ps 2AP and UAM 10AP were able to degrade up to 15% of the aromatic-polar mixture. The defined mixed culture degraded 47% of the aliphatics and 6% of the aromatic-polar mixture. The defined mixed culture was able to degrade about 40% of the aliphatic fraction and 26% of the aromatic fraction when grown in the presence of total hydrocarbons, while these microorganisms did not consume the polar hydrocarbons fraction. The proposed strategy that combines enrichment culture together with oxygen uptake rate allowed the isolation of bacterial strains that are able to degrade specific hydrocarbons fractions at high consumption rates.