The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the ␣ subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoAbased T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA.The atmospheric trace gas methane (CH 4 ) is a prominent "greenhouse" gas. Its atmospheric concentration has been increasing until recently at a rate of about 1% a year (8). Up to 70 to 80% of atmospheric CH 4 is biogenic (55). Flooded rice fields are one of the major sources of biogenic CH 4 (34, 50). Estimations of the annual emission rate from flooded rice fields range between 60 and 110 Tg (8, 21, 45). The upper limit of this emission rate accounts for approximately 25% of the total annual CH 4 emission into the atmosphere (8,21).Approximately 90% of the CH 4 that is emitted from rice paddies escapes through the aerenchyma of the rice plants, whereas only 10% escapes through the floodwater (19, 52). However, the aerenchyma does not merely function as a gas transport system but rather constitutes a dynamic, oxygenated biofilter. The diffusive input of oxygen into the below-ground plant surface area enables aerobic methanotrophs to oxidize CH 4 . Gilbert and Frenzel (22) showed that the activities of methanotrophs were directly dependent on the oxygen availability in the rice root environment. It was shown that up to 30% of the CH 4 produced in rice paddy ...

In Salmonella enterica serovar Typhimurium, the Cra protein (catabolite repressor/activator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 ⌬cra mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (fbp, glpX, ppsA, and pckA), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an ⌬sdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 ⌬aspA mutant and an SR-11 ⌬frdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 ⌬fadD mutant and an SR-11 ⌬aceA mutant were both fully virulent.In sensitive mice, Salmonella enterica serovar Typhimurium causes a systemic, often fatal disease, similar to human typhoid fever (43). After ingestion, serovar Typhimurium survives passage through the acidic environment of the stomach and reaches the terminal ileum where, within 30 min, it invades M cells in the Peyer's patches (22). Within 60 min, the M cells are destroyed and serovar Typhimurium gains access to both adjacent enterocytes and to underlying lymphoid cells in the mesenteric lymph follicles of the Peyer's patches (22,24). Serovar Typhimurium grows logarithmically in Peyer's patches for 2 days (20) and simultaneously disseminates systemically to the liver and spleen, where it grows in macrophages (40, 43). Mice usually become ruffled and lethargic 3 to 5 days post-oral infection and usually die within 7 to 12 days. Despite much work relating to the relevant virulence mechanisms, little is known about the nutrition of serovar Typhimurium during infection.The Cra protein (catabolite repressor/activator) is a regulator of central carbon metabolism in salmonellae and Escherichia coli. Cra is a transcription factor that activates genes encoding key enzymes in the glyoxylate bypass, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and electron transport and represses genes encoding key enzymes in the EmbdenMeyerhof and Entner-Doudoroff pathways (42). It has recently been reported that a cra mutant of serovar Typhimurium SR-11 is totally avirulent and immunogenic in...

Experiments were conducted to determine if a novel pmoA-like gene (pmoA2) recently discovered in the methane-oxidizing bacterium Methylocystis strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of pmoA1 and pmoA2. By using this primer combination, we identified pmoA2 in a wide range of type II MOB of the Methylosinus-Methylocystis group. However, screening by PCR and by Southern hybridization using a newly developed pmoA2-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor pmoA2. No pmoA2 was detected in five type I MOB tested: Methylococcus capsulatus strain Bath, Methylocaldum strain E10A, Methylobacter luteus, Methylomicrobium album, and Methylomonas strain D1a. In comparative sequence analyses, all pmoA2-like sequences formed a coherent cluster clearly distinct from pmoA1 sequences of type I and type II MOB, and from amoA sequences of the Nitrosomonas-Nitrosospira group. Phylogenetic analysis using the paml model suggested that pmoA2 is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of Methylocystis strain SC2 for gene expression of pmoA. A strong signal was observed for pmoA1 in Northern hybridization, while the results obtained for pmoA2 were ambiguous. However, reverse transcription-PCR confirmed that pmoA2 was expressed, albeit at lower level than pmoA1. This provided experimental evidence that the gene product of pmoA2 may be a functionally active enzyme.Methane-oxidizing bacteria (MOB) are able to utilize methane (CH 4 ) as their sole source of carbon and energy for growth (13). Their ability to oxidize CH 4 released at the interface of methanogenic environments and to act as sink for atmospheric CH 4 makes these organisms key players in balancing the global CH 4 budget and mitigating global warming due to CH 4 (4, 28). Phylogenies based on 16S rRNA genes show that MOB form distinct lineages in the Gammaproteobacteria (type I MOB) and Alphaproteobacteria (type II MOB) (2,5,6,13,16).The first step in CH 4 oxidation, the conversion of methane to methanol, is carried out by a methane monooxygenase (MMO). This enzyme exists in two forms, a particulate, membrane-associated form (pMMO) and a soluble form (sMMO). The two forms of enzyme differ in their structures, kinetic properties, and ranges of substrates they utilize (26). Only a restricted number of MOB species harbor sMMO, while almost all MOB possess pMMO. The only MOB lacking pMMO is Methylocella palustris (5).Cloning and sequence analysis of genes encoding pMMO revealed three consecutive open reading frames (pmoC, pmoA, and pmoB) in both type I (30, 31) and type II MOB (11). The pmoA gene, which encodes the 27-kDa subunit (Pm...

A type II methanotrophic bacterium (Methylocystis strain SC2) was isolated from a polluted aquifer and identified based on morphology and on 16S rRNA gene phylogeny. Primers targeting the particulate methane monooxygenase subunit A gene (pmoA) were used to obtain a PCR product from DNA extract of strain SC2. Denaturing gradient gel electrophoresis of this PCR product demonstrated that strain SC2 contained two very different pmoA-like genes. One gene (pmoA1) had very high sequence homology to pmoA genes of other type II methanotrophic bacteria (identical amino acid sequence to pmoA of some other Methylocystis strains). The second gene (pmoA2) possessed only 73% identity with the first gene at the nucleotide level and 68.5% identity (83% similarity) at the amino acid level. The presence of both pmoA-like genes was verified by developing specific oligonucleotide probes for each and using these in Southern hybridisation of genomic DNA. Purity of the culture was exhaustively verified with a variety of methods to ensure that both genes were present in a single genospecies. These included microscopic examination, plating on various media, denaturing gradient gel electrophoresis of PCR products of the 16S rRNA gene (universal to bacteria) and of the methanol dehydrogenase alpha-subunit gene mxaF (universal to methylotrophic bacteria), and whole-cell hybridisation with fluorescently labelled 16S rRNA-targeted oligonucleotide probes specific for the genera Methylosinus and Methylocystis, or specific for strain SC2. Reverse transcription PCR of extracted RNA suggested that the novel pmoA2 gene was not expressed during growth under standard conditions used for the cultivation of these bacteria. The presence of multiple, diverse pmoA-like genes in a single genospecies of methanotrophic bacteria implies that pmoA must be cautiously applied as a phylogenetic marker in cultivation-independent molecular ecology studies.