Molecular and Culture-Based Analyses of Aerobic Carbon Monoxide Oxidizer Diversity†

Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants. DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase (coxL). CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum), from novel CO-oxidizing isolates (e.g., Aminobacter sp. strain COX, Burkholderia sp. strain LUP, Mesorhizobium sp. strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp. strain LUP, and Xanthobacter sp. strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti, Stappia stellulata, and Stappia aggregata). PCR products from several taxa, e.g., O. carboxidovorans, B. japonicum, and B. fungorum, yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases. Aligned sequences formed two phylogenetically distinct groups. Group OMP contained sequences from previously known CO oxidizers, including O. carboxidovorans and Pseudomonas thermocarboxydovorans, plus a number of closely related sequences. Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and other α-Proteobacteria. PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group. CO oxidation by M. loti, for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.     

Physiological, ecological, and phylogenetic characterization of Stappia, a marine CO-oxidizing bacterial genus

Bacteria play a major role in marine CO cycling, yet very little is known about the microbes involved. Thirteen CO-oxidizing Stappia isolates obtained from existing cultures, macroalgae, or surf samples representing geographically and ecologically diverse habitats were characterized using biochemical, physiological, and phylogenetic approaches. All isolates were aerobic chemoorganotrophs that oxidized CO at elevated (1,000 ppm) and ambient-to-subambient concentrations (<0.3 ppm). All contained the form I (OMP) coxL gene for aerobic CO dehydrogenase and also the form II (BMS) putative coxL gene. In addition, some strains possessed cbbL, the large subunit gene for ribulose-1,5-bisphosphate carboxylase/oxygenase, suggesting the possibility of lithotrophic or mixotrophic metabolism. All isolates used a wide range of sugars, organic acids, amino acids, and aromatics for growth and grew at salinities from 5 to 45 ppt. All but one isolate denitrified or respired nitrate. Phylogenetic analyses based on 16S rRNA gene sequences indicated that several isolates could not be distinguished from Stappia aggregata and contributed to a widely distributed species complex. Four isolates (of strains GA15, HI, MIO, and M4) were phylogenetically distinct from validly described Stappia species and closely related genera (e.g., Ahrensia, Pannonibacter, Pseudovibrio, and Roseibium). Substrate utilization profiles, enzymatic activity, and membrane lipid composition further distinguished these isolates and supported their designations as new Stappia species. The observed metabolic versatility of Stappia likely accounts for its cosmopolitan distribution and its ability to contribute to CO cycling as well as other important biogeochemical cycles.     

Molecular analysis of carbon monoxide-oxidizing bacteria associated with recent Hawaiian volcanic deposits

Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.     

Analysis of the distribution and diversity in recent Hawaiian volcanic deposits of a putative carbon monoxide dehydrogenase large subunit gene

A putative carbon monoxide dehydrogenase large subunit gene (BMS putative coxL) was amplified from genomic DNA extracts of four recent (42-300 year old) Hawaiian volcanic deposits by polymerase chain reaction (PCR). Sequence databases derived from clone libraries constructed using PCR products were analysed phylogenetically and statistically. These analyses indicated that each of the deposits supported distinct BMS putative coxL gene assemblages. Statistical analyses also showed that the youngest deposit (42 years old) contained the least diverse sequences (P<0.05), but that diversity did not vary significantly among three older deposits with ages from about 108-300 years. Although diversity indices did not vary among the older deposits, mismatch analyses suggested population structures increased in complexity with increasing deposit age. At each of the sites, most of the clone sequences appeared to originate from Proteobacteria not currently represented in culture or recognized as CO oxidizers.     

The phylogenetic distribution and ecological role of carbon monoxide oxidation in the genus Burkholderia

Burkholderia is a physiologically and ecologically diverse genus that occurs commonly in assemblages of soil and rhizosphere bacteria. Although Burkholderia is known for its heterotrophic versatility, we demonstrate that 14 distinct environmental isolates oxidized carbon monoxide (CO) and possessed the gene encoding the catalytic subunit of form I CO dehydrogenase (coxL). DNA from a Burkholderia isolate obtained from a passalid beetle also contained coxL as do the genomic sequences of species H160 and Ch1-1. Isolates were able to consume CO at concentrations ranging from 100 ppm (vol/vol) to sub-ambient (< 60 ppb (vol/vol)). High concentrations of pyruvate inhibited CO uptake (> 2.5 mM), but mixotrophic consumption of CO and pyruvate occurred when initial pyruvate concentrations were lower (c. 400 lM). With the exception of an isolate most closely related to Burkholderia cepacia, all CO-oxidizing isolates examined were members of a nonpathogenic clade and were most closely related to Burkholderia species, B. caledonica, B. fungorum, B. oxiphila, B. mimosarum, B. nodosa, B. sacchari, B. bryophila, B. ferrariae, B. ginsengesoli, and B. unamae. However, none of these type strains oxidized CO or contained coxL based on results from PCR analyses. Collectively, these results demonstrate that the presence of CO oxidation within members of the Burkholderia genus is variable but it is most commonly found among rhizosphere inhabitants that are not closely related to B. cepacia.     

Nitrate-dependent anaerobic carbon monoxide oxidation by aerobic CO-oxidizing bacteria

Two dissimilatory nitrate-reducing (Burkholderia xenovorans LB400 and Xanthobacter sp. str. COX) and two denitrifying isolates (Stappia aggregata IAM 12614 and Bradyrhizobium sp. str. CPP), previously characterized as aerobic CO oxidizers, consumed CO at ecologically relevant levels (<100 ppm) under anaerobic conditions in the presence, but not absence, of nitrate. None of the isolates were able to grow anaerobically with CO as a carbon or energy source, however, and nitrate-dependent anaerobic CO oxidation was inhibited by headspace concentrations >100-1000 ppm. Surface soils collected from temperate, subtropical and tropical forests also oxidized CO under anaerobic conditions with no lag. The observed activity was 25-60% less than aerobic CO oxidation rates, and did not appear to depend on nitrate. Chloroform inhibited anaerobic but not aerobic activity, which suggested that acetogenic bacteria may have played a significant role in forest soil anaerobic CO uptake.     

Burkholderia fungorum DBT1: a promising bacterial strain for bioremediation of PAHs-contaminated soils

An extensive taxonomic analysis of the bacterial strain Burkholderia sp. DBT1, previously isolated from an oil refinery wastewater drainage, is discussed here. This strain is capable of transforming dibenzothiophene through the 'destructive' oxidative pathway referred to as the Kodama pathway. Burkholderia DBT1 has also been proved to use fluorene, naphthalene and phenanthrene as carbon and energy sources, although growth on the first two compounds requires a preinduction step. This evidence suggests that the strain DBT1 exerts a versatile metabolism towards polycyclic aromatic hydrocarbons other than condensed thiophenes. Phylogenetic characterization using a polyphasic approach was carried out to clarify the actual taxonomic position of this strain, potentially exploitable in bioremediation. In particular, investigations were focused on the possible exclusion of Burkholderia sp. DBT1 from the Burkholderia cepacia complex. Analysis of the sequences of 16S, recA and gyrB genes along with the DNA-DNA hybridization procedure indicated that the strain DBT1 belongs to the species Burkholderia fungorum, suggesting the proposal of the taxonomic denomination B. fungorum DBT1.     

Development of a species-specific recA-based PCR test for Burkholderia fungorum

The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B. fungorum. Previous analysis of several novel rhizospheric, environmental isolates belonging to the B. cepacia complex suggested they may be closely related to B. fungorum. To discover any relationship between these isolates and B. fungorum we set out to clone and sequence a portion of the B. fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay. Using a similar procedure we extended the recA-based PCR assay to identify B. sacchari and B. caledonica, two additional soil-inhabiting Burkholderia spp.     

Uptake of carbon monoxide and hydrogen at environmentally relevant concentrations by mycobacteria

Liquid culture assays revealed a previously unreported capacity for Mycobacterium bovis BCG, M. gordonae, and M. marinum to oxidize CO and for M. smegmatis to consume molecular hydrogen. M. bovis BCG, M. gordonae, M. smegmatis, and M. tuberculosis H37Ra oxidized CO at environmentally relevant concentrations (<50 ppm); H2 oxidation by M. gordonae and M. smegmatis also occurred at environmentally relevant concentrations (<10 ppm). CO was not consumed by M. avium or M. microti, although the latter appeared to possess CO dehydrogenase (CODH) genes based on PCR results with primers designed for the CODH large subunit, coxL. M. smegmatis and M. gordonae oxidized CO under suboxic (10 and 1% atmospheric oxygen) and anoxic conditions in the presence of nitrate; no oxidation occurred under anoxic conditions without nitrate. Similar results were obtained for H2 oxidation by M. smegmatis. Phylogenetic analyses of coxL PCR products indicated that mycobacterial sequences form a subclade distinct from that of other bacterial coxL, with limited differentiation among fast- and slow-growing strains.     

Diazotrophic Burkholderia species isolated from the Amazon region exhibit phenotypical, functional and genetic diversity

Forty-eight Burkholderia isolates from different land use systems in the Amazon region were compared to type strains of Burkholderia species for phenotypic and functional characteristics that can be used to promote plant growth. Most of these isolates (n=46) were obtained by using siratro (Macroptilium atropurpureum - 44) and common bean (Phaseolus vulgaris - 2) as the trap plant species; two isolates were obtained from nodules collected in the field from Indigofera suffruticosa and Pithecellobium sp. The evaluated characteristics were the following: colony characterisation on "79" medium, assimilation of different carbon sources, enzymatic activities, solubilisation of phosphates, nitrogenase activity and antifungal activity against Fusarium oxysporium f. sp. phaseoli. Whole cell protein profiles, 16S rRNA, gyrB, and recA gene sequencing and multilocus sequence typing were used to identify the isolates. The isolates showed different cultural and biochemical characteristics depending on the legume species from which they were obtained. Except for one isolate from I. suffruticosa, all isolates were able to solubilise calcium phosphate and present nitrogenase activity under free-living conditions. Only one isolate from common beans, showed antifungal activity. The forty four isolates from siratro nodules were identified as B. fungorum; isolates UFLA02-27 and UFLA02-28, obtained from common bean plants, were identified as B. contaminans; isolate INPA89A, isolated from Indigofera suffruticosa, was a close relative of B. caribensis but could not be assigned to an established species; isolate INPA42B, isolated from Pithecellobium sp., was identified as B. lata. This is the first report of nitrogenase activity in B. fungorum, B. lata and B. contaminans.     

Physiological, Ecological, and Phylogenetic Characterization of Stappia, a Marine CO-Oxidizing Bacterial Genus

Bacteria play a major role in marine CO cycling, yet very little is known about the microbes involved. Thirteen CO-oxidizing Stappia isolates obtained from existing cultures, macroalgae, or surf samples representing geographically and ecologically diverse habitats were characterized using biochemical, physiological, and phylogenetic approaches. All isolates were aerobic chemoorganotrophs that oxidized CO at elevated (1,000 ppm) and ambient-to-subambient concentrations (<0.3 ppm). All contained the form I (OMP) coxL gene for aerobic CO dehydrogenase and also the form II (BMS) putative coxL gene. In addition, some strains possessed cbbL, the large subunit gene for ribulose-1,5-bisphosphate carboxylase/oxygenase, suggesting the possibility of lithotrophic or mixotrophic metabolism. All isolates used a wide range of sugars, organic acids, amino acids, and aromatics for growth and grew at salinities from 5 to 45 ppt. All but one isolate denitrified or respired nitrate. Phylogenetic analyses based on 16S rRNA gene sequences indicated that several isolates could not be distinguished from Stappia aggregata and contributed to a widely distributed species complex. Four isolates (of strains GA15, HI, MIO, and M4) were phylogenetically distinct from validly described Stappia species and closely related genera (e.g., Ahrensia, Pannonibacter, Pseudovibrio, and Roseibium). Substrate utilization profiles, enzymatic activity, and membrane lipid composition further distinguished these isolates and supported their designations as new Stappia species. The observed metabolic versatility of Stappia likely accounts for its cosmopolitan distribution and its ability to contribute to CO cycling as well as other important biogeochemical cycles.     

Stappia alba sp. nov., isolated from Mediterranean oysters

Three bacterial strains isolated from oysters recovered at the Spanish Mediterranean coast have been phenotypically and genetically characterized. The results of the phylogenetic analysis based on almost complete 16S rDNA sequences clustered all three strains together with 99.9% average sequence similarity and situated them in the neighbourhood of the genera Stappia, Roseibium and Pannonibacter, Stappia aggregata being their closest neighbour with sequence similarities between 98.8% and 98.9%. DNA-DNA hybridization experiments using DNA of strains 5OM6T and S. aggregata CECT 4269T as reference DNAs confirmed the independent status at species level of the oyster isolates. Phenotypically, they can be distinguished from the closest relatives by the ionic requirements, growth temperatures and use of carbon compounds. We propose these oyster strains constitute a new species of Stappia, for which the name Stappia alba sp. nov. has been chosen, and strain 5OM6T (= CECT 5095T = CIP 108402T) as its type strain.     

Genetic diversity of endophytic diazotrophs of the wild rice, Oryza alta and identification of the new diazotroph, Acinetobacter oryzae sp. nov

Thirty-three endophytic diazotrophs were isolated from surface-sterilized leaves, stem, and roots of wild rice Oryza alta. The SDS-PAGE profile of total protein and insertion sequence-based polymerase chain reaction (IS-PCR) fingerprinting grouped the isolates into four clusters (I-IV). The 16S rRNA gene sequence homology of the representative strains B21, B31, B1, and B23 of clusters I, II, III, and IV were assigned to Pseudomonas oleovorans (99.2% similarity), Burkholderia fungorum (99.4% similarity), Enterobacter cloacae (98.9% similarity), and Acinetobacter johnsonii (98.4% similarity), respectively. The results showed wide genetic diversity of the putative diazotrophic strains of the wild rice, O. alta, and the strains of cluster IV are the first report of nitrogen-fixing Acinetobacter species. The cell size, phenotypic characters, total protein profile, genomic DNA fingerprinting, DNA-DNA hybridization, and antibiotic resistance differentiated strain B23(T) from its closest relatives A. johnsonii LMG999(T) and Acinetobacter haemolyticus LMG996(T). The DNA-DNA hybridization also distinguished the strain B23(T) from the closely related Acinetobacter species. Based on these data, a novel species, Acinetobacter oryzae sp. nov., and strain B23(T) (=LMG25575(T)?=?CGMCC1.10689(T)) as the type strain were proposed.     

Diversity of transconjugants that acquired plasmid pJP4 or pEMT1 after inoculation of a donor strain in the A- and B-horizon of an agricultural soil and description of Burkholderia hospita sp. nov. and Burkholderia terricola sp. nov

We examined the diversity of transconjugants that acquired the catabolic plasmids pJP4 or pEMT1, which encode degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), in microcosms with agricultural soil inoculated with a donor strain (Dejonghe, W., Goris, J., El Fantroussi, S., H?fte, M., De Vos, P., Verstraete, W., and Top, E. M. Appl. Environ. Microbiol. 2000, p. 3297-3304). Using repetitive element PCR fingerprinting, eight different rep-clusters and six separate isolates could be discriminated among 95 transconjugants tested. Representative isolates were identified using 16S rDNA sequencing, cellular fatty acid analysis, whole-cell protein analysis and/or DNA-DNA hybridisations. Plasmids pJP4 and pEMT1 appeared to have a similar transfer and expression range, and were preferably acquired and expressed in soil by indigenous representatives of Ralstonia and Burkholderia. Two rep-clusters were shown to represent novel Burkholderia species, for which the names Burkholderia hospita sp. nov. and Burkholderia terricola sp. nov. are proposed. When easily degradable carbon sources were added together with the plasmid-bearing donor strain, also a significant proportion of Stenotrophomonas maltophilia isolates were found. The transconjugant collections isolated from A- (0-30 cm depth) and B-horizon (30-60 cm depth) soil were similar, except for B. terricola transconjugants, which were only isolated from the B-horizon.     

Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov

The nodulation of legumes has for more than a century been considered an exclusive capacity of a group of microorganisms commonly known as rhizobia and belonging to the alpha-Proteobacteria. However, in the last 3 years four nonrhizobial species, belonging to alpha and beta subclasses of the Proteobacteria, have been described as legume-nodulating bacteria. In the present study, two fast-growing strains, LUP21 and LUP23, were isolated from nodules of Lupinus honoratus. The phylogenetic analysis based on the 16S and 23S rRNA gene sequences showed that the isolates belong to the genus Ochrobactrum. The strains were able to reinfect Lupinus plants. A plasmid profile analysis showed the presence of three plasmids. The nodD and nifH genes were located on these plasmids, and their sequences were obtained. These sequences showed a close resemblance to the nodD and nifH genes of rhizobial species, suggesting that the nodD and nifH genes carried by strain LUP21T were acquired by horizontal gene transfer. A polyphasic study including phenotypic, chemotaxonomic, and molecular features of the strains isolated in this study showed that they belong to a new species of the genus Ochrobactrum for which we propose the name Ochrobactrum lupini sp. nov. Strain LUP21T (LMG 20667T) is the type strain.     

Molecular Analysis of Carbon Monoxide-Oxidizing Bacteria Associated with Recent Hawaiian Volcanic Deposits

Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.     

Physiological and phylogenetic diversity of bacteria growing on resin acids

Resin acids are tricyclic diterpenes which are synthesized by trees and are a major cause of toxicity of pulp mill effluents. Bacterial strains isolated from three different sources and which grow on resin acids were physiologically characterized. Eleven strains, representating distinct groups, were further characterized physiologically and phylogenetically. The isolates had distinct specificities for use, as growth substrates, of the different resin acids tested. The isolates also used fatty acids but were generally limited in use of other diverse substrates tested. According to their 16S rDNA sequences, the representative isolates are related to members of the genera, Sphingomonas, Zoogloea, Ralstonia, Burkholderia, Pseudomonas and Mycobacterium. Analysis of whole-cell fatty acid profiles generally supported those phylogenetic relationships. However, most of the isolated did not have high similarities to reference strains in the Microbial Identification System database of fatty acid profiles or in the Biolog database of substrate oxidation patterns. Described species of Sphingomonas, Zoolgoea, Burkholderia Pseudomonas, most closely related to the isolates we characterized, failed to grow on, or degrade, resin acids. We propose recognition of Zoogloea resiniphila sp. nov., Pseudomonas vancouverensis sp. nov., P. abietaniphila sp. nov. and P. multiresinivorans sp. nov.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

Intergeneric Coaggregation among Drinking Water Bacteria: Evidence of a Role for Acinetobacter calcoaceticus as a Bridging Bacterium

Intergeneric coaggregation of drinking water bacteria was tested. Acinetobacter calcoaceticus was found not only to autoaggregate but also to coaggregate with four of the five other isolates (Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata, and Staphylococcus sp.). In its absence, no coaggregation was found. Interactions were lectin-saccharide mediated. The putative bridging function of A. calcoaceticus was evidenced by multispecies biofilm studies, through a strain exclusion process.