Peter J. Bottomley

Community Composition and Functioning of Denitrifying Bacteria from Adjacent Meadow and Forest Soils

ABSTRACT We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers. nosZ, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of nosZ terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225 nosZ clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of nosZ fragments sequenced from meadow or forest soils were most similar to nosZ from the Rhizobiaceae group in α-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.

Complete Genome Sequence of Nitrosospira multiformis, an Ammonia-Oxidizing Bacterium from the Soil Environment

ABSTRACT The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196T) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.

Community composition of ammonia‐oxidizing bacteria and archaea in soils under stands of red alder and Douglas fir in Oregon

This study determined nitrification activity and nitrifier community composition in soils under stands of red alder (Alnus rubra) and Douglas fir (Pseudotsuga menziesii) at two sites in Oregon. The H.J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon, has low net N mineralization and gross nitrification rates. Cascade Head Experimental Forest, in the Coast Range, has higher net N mineralization and nitrification rates and soil pH is lower. Communities of putative bacterial [ammonia-oxidizing bacteria (AOB)] and archaeal [ammonia-oxidizing archaea (AOA)] ammonia oxidizers were examined by targeting the gene amoA, which codes for subunit A of ammonia monooxygenase. Nitrification potential was significantly higher in red alder compared with Douglas-fir soil and greater at Cascade Head than H.J. Andrews. Ammonia-oxidizing bacteria amoA genes were amplified from all soils, but AOA amoA genes could only be amplified at Cascade Head. Gene copy numbers of AOB and AOA amoA were similar at Cascade Head regardless of tree type (2.3-6.0 x 10(6)amoA gene copies g(-1) of soil). DNA sequences of amoA revealed that AOB were members of Nitrosospira clusters 1, 2 and 4. Ammonia-oxidizing bacteria community composition, determined by terminal restriction fragment length polymorphism (T-RFLP) profiles, varied among sites and between tree types. Many of the AOA amoA sequences clustered with environmental clones previously obtained from soil; however, several sequences were more similar to clones previously recovered from marine and estuarine sediments. As with AOB, the AOA community composition differed between red alder and Douglas-fir soils.

Ammonia-Oxidizing Bacteria along Meadow-to-Forest Transects in the Oregon Cascade Mountains

ABSTRACT Although nitrification has been well studied in coniferous forests of Western North America, communities of NH3-oxidizing bacteria in these forests have not been characterized. Studies were conducted along meadow-to-forest transects at two sites (Lookout and Carpenter) in the H. J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon. Soil samples taken at 10- or 20-m intervals along the transects showed that several soil properties, including net nitrogen mineralization and nitrification potential rates changed significantly between vegetation zones. Nonetheless, terminal restriction fragment length polymorphism (T-RFLP) analysis of the PCR-amplified NH3 monooxygenase subunit A gene (amoA) showed the same DNA fragments (TaqI [283 bp], CfoI [66 bp], and AluI [392 bp]) to dominate ≥45 of 47 soil samples recovered from both sites. Two fragments (491-bp AluI [AluI491] and CfoI135) were found more frequently in meadow and transition zone soil samples than in forest samples at both sites. At the Lookout site the combination AluI491-CfoI135 was found primarily in meadow samples expressing the highest N mineralization rates. Four unique amoA sequences were identified among 15 isolates recovered into pure culture from various transect locations. Six isolates possessed the most common T-RFLP amoA fingerprint of the soil samples (TaqI283-AluI392-CfoI66), and their amoA sequences shared 99.8% similarity with a cultured species, Nitrosospira sp. strain Ka4 (cluster 4). The other three amoA sequences were most similar to sequences of Nitrosospira sp. strain Nsp1 and Nitrosospira briensis (cluster 3). 16S ribosomal DNA sequence analysis confirmed the affiliation of these isolates with Nitrosospira clusters 3 and 4. Two amoA clone sequences matched T-RFLP fingerprints found in soil, but they were not found among the isolates.

Complete genome sequence of Nitrobacter hamburgensis X14 and comparative genomic analysis of species within the genus Nitrobacter.

ABSTRACT The alphaproteobacterium Nitrobacter hamburgensis X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the Nitrobacter hamburgensis X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between N. hamburgensis and the finished and draft genome sequences of Nitrobacter winogradskyi and Nitrobacter sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to N. hamburgensis and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet ∼21 kb of a ∼28-kb “autotrophic” island on the largest plasmid was conserved in the chromosomes of Nitrobacter winogradskyi Nb-255 and Nitrobacter sp. strain Nb-311A. The N. hamburgensis chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome b561, and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A Nitrobacter “subcore” genome was also constructed by removing homologs found in strains of the closest evolutionary relatives, Bradyrhizobium japonicum and Rhodopseudomonas palustris. Among the Nitrobacter subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in Nitrobacter.

Responses of Nitrification and Ammonia-Oxidizing Bacteria to Reciprocal Transfers of Soil between Adjacent Coniferous Forest and Meadow Vegetation in the Cascade Mountains of Oregon

Despite the critical position of nitrification in N cycling in coniferous forest soils of western North America, little information exists on the composition of ammonia-oxidizing bacteria (AOB) in these soils, or their response to treatments that promote or reduce nitrification. To this end, an experiment was conducted in which a set of soil cores was reciprocally transplanted between adjacent forest (low nitrification potential) and meadow (high nitrification potential) environments, at two high-elevation (~1500 m) sites in the H.J. Andrews Experimental Forest located in the Cascade Mountains of Oregon. Half of the cores were placed in screened PVC pipe (closed) to prevent new root colonization, large litter debris inputs, and animal disturbance; the other cores were placed in open mesh bags. A duplicate set of open and closed soil cores was not transferred between sites and was incubated in place. Over the 2-year experiment, net nitrification increased in both open and closed cores transferred from forest to meadow, and to a lesser extent in cores remaining in the forest. In three of four forest soil treatments, net nitrification increases were accompanied by increases in nitrification potential rates (NPR) and 10- to 100-fold increases in AOB populations. In open cores remaining in the forests, however, increases in net nitrification were not accompanied by significant increases in either NPR or AOB populations. Although some meadow soil treatments reduced both net nitrification and nitrification potential rates, significant changes were not detected in most probable number (MPN)-based estimates of AOB population densities. Terminal restriction fragment profiles (T-RFs) of a PCR-amplified 491-bp fragment of the ammonia monooxygenase subunit A gene (amoA) changed significantly in response to some soil treatments, and treatment effects differed among locations and between years. A T-RF previously shown to be a specific biomarker of Nitrosospira cluster 4 (Alu390) was widespread and dominant in the majority of soil samples. Despite some treatments causing substantial increases in AOB population densities and nitrification potential rates, nitrosomonads remained undetectable, and the nitrosospirad AOB community composition did not change radically following treatment.

Soil microbial communities associated with Douglas-fir and red alder stands at high- and low-productivity forest sites in Oregon, USA.

Communities of archaea, bacteria, and fungi were examined in forest soils located in the Oregon Coast Range and the inland Cascade Mountains. Soils from replicated plots of Douglas-fir (Pseudotsuga menziesii) and red alder (Alnus rubra) were characterized using fungal ITS (internal transcribed spacer region), eubacterial 16S rRNA, and archaeal 16S rRNA primers. Population size was measured with quantitative (Q)-PCR and composition was examined using length heterogeneity (LH)-PCR for fungal composition, terminal restriction fragment length (T-RFLP) profiles for bacterial and archaeal composition, and sequencing to identify dominant community members. Whereas fungal and archaeal composition varied between sites and dominant tree species, bacterial communities only varied between sites. The abundance of archaeal gene copy numbers was found to be greater in coastal compared to montane soils accounting for 11% of the prokaryotic community. Crenarchaea groups 1.1a-associated, 1.1b, 1.1c, and 1.1c-associated were putatively identified. A greater abundance of Crenarchaea 1.1b indicator fragments was found in acidic (pH 4) soils with low C:N ratios under red alder. In coastal soils, 25% of fungal sequences were putatively identified as basidiomycetous yeasts belonging to the genus Cryptococcus. Although the function of these yeasts in soil is not known, they could significantly contribute to decomposition processes in coastal soils distinguished by rapid tree growth, high N content, low pH, and frequent water-saturation events.

Expression of a putative nitrite reductase and the reversible inhibition of nitrite-dependent respiration by nitric oxide in Nitrobacter winogradskyi Nb-255.

The nitrite oxidizing Alphaproteobacterium, Nitrobacter winogradskyi, primarily conserves energy from the oxidation of nitrite (NO(2)(-))to nitrate (NO(3)(-)) through aerobic respiration. Almost 20 years ago, NO-dependent NADH formation was reported to occur in both aerobic and anaerobic cell suspensions of N. winogradskyi strain agilis, suggesting that NO oxidation might contribute to energy conservation by Nitrobacter. Recently, the N. winogradskyi Nb-255 genome was found to contain a gene (Nwin_2648) that encodes a putative copper-containing nitrite reductase (NirK), which may reduce NO(2)(-) to NO. In this study, the putative nirK was found to be maximally transcribed under low O(2) (between zero and 4% O(2)) in the presence of NO(2)(-). Transcription of nirK was not detected under anaerobic conditions in the absence of NO(2)(-) or in the presence of NO(3)(-) and pyruvate. Although net production of NO could not be detected from either aerobically grown or anaerobically incubated cells, exogenous NO was consumed by viable cells and concomitantly inhibited NO(2)(-)-dependent O(2) uptake in a reversible, concentration dependent manner. Both NO(2(-)-dependent O(2) uptake and NO consumption were inhibited by 1 mM cyanide suggesting involvement of cytochrome oxidase with NO consumption. Abiotic consumption of NO was measured, yet, both the rates and kinetics of NO transformation in buffer alone, or by heat killed, or cyanide-treated cells differed from those of viable cells. In light of this new information, a modified model is proposed to explain how NirK and NO manage electron flux in Nitrobacter.

Involvement of BmoR and BmoG in n-alkane metabolism in 'Pseudomonas butanovora'

Pseudomonas butanovora uses an alcohol-inducible alkane monooxygenase (BMO) to grow on C(2)-C(9) n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative sigma(54)-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C(2)-C(5) n-alkanes and showed little to no growth on C(6)-C(8) n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the P. butanovora bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C(2)-C(8) carbon sources. In acetate- or lactate-grown cells, C(2)-C(8) n-alcohols failed to induce beta-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced approximately 45 % of the beta-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C(2)-C(5) n-alcohols induced beta-galactosidase activity, whereas C(7) and C(8) n-alcohols did not. BmoR may act as a sigma(54)-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e. </=C(6)). The bmoG-deficient mutant did not grow on C(2)-C(8) n-alkanes; however, it was capable of transcribing bmoX and bmoC of the BMO operon. BmoG may act as a chaperonin to assemble competent BMO.

Responses of soil bacterial and fungal communities to reciprocal transfers of soil between adjacent coniferous forest and meadow vegetation in the Cascade Mountains of Oregon

Little information exists on the responses of soil fungal and bacterial communities in high elevation coniferous forest/open meadow ecosystems of the northwest United States of America to treatments that impact vegetation and soil conditions. An experiment was conducted in which soil cores were reciprocally transplanted between immediately adjacent forests and meadows at two high elevation (∼1,600xa0m) sites (Carpenter and Lookout) in the H.J. Andrews Experimental Forest located in the Cascade Mountains of Oregon. Half of the cores were placed in PVC pipe (closed) to prevent new root colonization, whereas the other cores were placed in mesh bags (open) to allow recolonization by fine roots. A duplicate set of open and closed soil cores was not transferred between sites and was incubated in place. After 2xa0year, soil cores were removed and changes in fungal and bacterial biomasses determined using light microscopy, and changes in microbial community composition determined by PLFA analysis, and by length heterogeneity PCR of the internal transcribed spacer region of fungal ribosomal DNA. At both sites soil microbial community structures had responded to treatments after 2xa0year of incubation. At Carpenter, both fungal and bacterial community structures of forest soil changed significantly in response to transfer from forest to meadow, with the shift in fungal community structure being accompanied by a significant decrease in the PLFA biomarker of fungal biomass,18:2ω6,9. At Lookout, both fungal and bacterial community structures of forest soil changed significantly in response to open versus closed core treatments, with the shift in the fungal community being accompanied by a significant decrease in the 18:2ω6,9 content of closed cores, and the shift in the bacterial community structure being accompanied by a significant increase in bacterial biomass of closed cores. At both sites, fungal community structures of meadow soils changed differently between open and closed cores in response to transfer to forest, and were accompanied by increases in the18:2ω6,9 content of open cores. Although there were no significant treatment effects on the bacterial community structure of meadow soil at either site, bacterial biomass was significantly higher in closed versus open cores regardless of transfer.