RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater. (33/69)

Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [(13)C(6)]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the (13)C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [(14)C]benzene showed that the labeled carbon was mostly incorporated into (14)CO(2) within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.  (+info)

Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp. (34/69)

The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifHi::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.  (+info)

Self-assembly of a group I intron from inactive oligonucleotide fragments. (35/69)

The Azoarcus group I ribozyme was broken into four fragments, 39-63 nucleotides long, that can self-assemble into covalently contiguous ribozymes via RNA-directed recombination events. The fragments have no activity individually yet can cooperate through base pairing and tertiary interactions to produce stable trans complexes at 48 degrees C. These complexes can then catalyze a sequence of energy-neutral recombination reactions utilizing other oligomers as substrates, assembling covalent versions of the ribozyme. Up to 17% of the original fragments are converted into approximately 200 nucleotide products in 8 hr. Assembly occurs primarily by only one of many possible pathways, and the reaction is driven in the correct and forward direction by the burial of key base-pairing regions in stems after recombination. Autocatalysis, and hence self-replication, is inferred by a reaction rate increase upon doping the reaction with full-length RNA.  (+info)

Heterologous expression and identification of the genes involved in anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) in Azoarcus anaerobius. (36/69)

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R(+), carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R(+) carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R(+), eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.  (+info)

Twitching motility is essential for endophytic rice colonization by the N2-fixing endophyte Azoarcus sp. strain BH72. (37/69)

Azoarcus sp. strain BH72, as an endophyte of grasses, depends on successful host colonization. Type IV pili are essential for mediating the initial interaction with rice roots. In the genome sequence analysis, the pilT gene was identified, which encodes for a putative type IV pilus retraction protein. PilT of Azoarcus sp. BH72 shares high similarity to PilT of the human pathogen Pseudomonas aeruginosa PAO1 (77% amino acid sequence identity) and contains a predicted nucleotide-binding motif. To gain more insights into the role of the type IV pili in the colonization process of Azoarcus spp., we constructed an insertional mutant of pilT and a deletion mutant of pilA, the major structural component of the pilus structure. The pilT mutant, as the pilin deletion mutant deltapilA, was abolished in twitching motility. Western blot analyses and electron microscopy studies demonstrated an enhanced piliation of the Azoarcus pilT mutant strain compared with the wild type, indicating that, indeed, PilT has a role in pilus retraction. Studies on rice root colonization in gnotobiotic cultures revealed that the establishment of microcolonies on the root surface was strongly reduced in the deltapilA mutant, whereas the surface colonization was reduced by only 50% in the nontwitching pilT mutant. However, endophytic colonization of rice roots was strongly reduced in both mutants. These results demonstrate that the retractile force mediated by PilT is not essential for the bacterial colonization of the plant surface, but that twitching motility is necessary for invasion of and establishment inside the plant. Thus, a novel determinant for endophytic interactions with grasses was identified.  (+info)

Structural metals in the group I intron: a ribozyme with a multiple metal ion core. (38/69)

Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg(2+) and K(+) ions. Five of the metals bind within 12 A of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function.  (+info)

Mechanisms of covalent self-assembly of the Azoarcus ribozyme from four fragment oligonucleotides. (39/69)

RNA oligomers of length 40-60 nt can self-assemble into covalent versions of the Azoarcus group I intron ribozyme. This process requires a series of recombination reactions in which the internal guide sequence of a nascent catalytic complex makes specific interactions with a complement triplet, CAU, in the oligomers. However, if the CAU were mutated, promiscuous self-assembly may be possible, lessening the dependence on a particular set of oligomer sequences. Here, we assayed whether oligomers containing mutations in the CAU triplet could still self-construct Azoarcus ribozymes. The mutations CAC, CAG, CUU and GAU all inhibited self-assembly to some degree, but did not block it completely in 100 mM MgCl(2). Oligomers containing the CAC mutation retained the most self-assembly activity, while those containing GAU retained the least, indicating that mutations more 5' in this triplet are the most deleterious. Self-assembly systems containing additional mutant locations were progressively less functional. Analyses of properly self-assembled ribozymes revealed that, of two recombination mechanisms possible for self-assembly, termed 'tF2' and 'R2F2', the simpler one-step 'tF2' mechanism is utilized when mutations exist. These data suggest that self-assembling systems are more facile than previously believed, and have relevance to the origin of complex ribozymes during the RNA World.  (+info)

New insights into the BzdR-mediated transcriptional regulation of the anaerobic catabolism of benzoate in Azoarcus sp. CIB. (40/69)

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