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Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism with implications for function in soil environments.

A disturbance gradient was created to evaluate the relationship between changes in soil physical and chemical properties with microbial community composition. Plots of a 12-yr fallow Ultisol in Northeastern Brazil were arranged in a Latin Square design and disturbed by tillage events applied zero, one, two, three, or four times over a 3-mo period. Sixty days after the last event, plots were sampled (0-20 cm) and soil analyses performed. Tillage resulted in a linear disturbance gradient characterized by significant declines in soil available water, saturated hydraulic conductivity, soil aggregation, organic C, and microbial biomass. The disturbance gradient also affected soil microbial communities, but the changes observed were dependent on the profiling technique used. The application of one tillage event significantly altered terminal restriction fragment length polymorphism (T-RFLP) profiles, which remained unchanged on further disturbances. In contrast, community level physiological profiles (CLPPs) continued to change as disturbance levels increased and were significantly correlated with most soil properties associated with soil degradation. Phospholipid fatty acid (PLFA)-based assessment of microbial community structure was the least sensitive to disturbance, showing significant changes only between the control plots and those subjected to four tillage events. Nevertheless, the linear shift observed in a PLFA biomarker of bacterial stress (ratio of 19:0cy to 18:lw7c) in response to the disturbance gradient was consistent with physiological changes in the CLPPs. The results clearly illustrate that microbial community responses to increasing levels of soil physical disturbance can be detected at physiological (CLPPs), biochemical (PLFA), and genotypic (T-RFLP) levels; however, they are not synchronously coupled. Further studies are needed to determine if there is functional significance to the physiological responses to disturbance that are, or are not, accompanied by community composition changes. Abbreviations: CEC, cation exchange capacity; CLPP, community level physiological profile; FAME, fatty acid methyl ester; H' Shannon index; NMS, non-metric multidimensional scaling; PLFA, phospholipid fatty acid; R, richness index; SOC, soil organic carbon; SOM, soil organic matter; T-RFLP, terminal restriction fragment length polymorphism; T-RFs, terminal restriction fragments; WSA; water stable aggregates.

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the [C.sub.2]-[C.sub.9] alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for [C.sub.1]-[C.sub.5] alkanes and product inhibition profiles, sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by [[kappa].sub.cat]: [[kappa].sub.m] ratios). Relative rates of oxidation for [C.sub.1]-[C.sub.5] alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate [[kappa].sub.m] values. The low micromolar [[kappa].sub.m] for linear [C.sub.2]-[C.sub.5] alkanes and the millimolar [[kappa].sub.m] for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar [[kappa].sub.m] and [[kappa].sub.cat] values to those of methane. This inability to kinetically discriminate between the [C.sub.1] alkane and [C.sub.1] alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length [C.sub.2]-[C.sub.5] do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of [C.sub.3]-[C.sub.5] alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes [greater than or equal to] [C.sub.2], despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO.

Nitrobacter hamburgensis X14 is a facultative lithoautotroph that conserves energy from the oxidation of nitrite (N[O.sup.-.sub.2]) and fixes carbon dioxide (C[O.sub.2]) as its sole source of carbon. The availability of the N. hamburgensis X14 genorne sequence initiated a re-examination of its mixotrophic and organotrophic potential, as genes encoding three flavin-dependent oxidases were identified that may function to oxidize lactate, providing energy and carbon for growth. The response of N. harnburgensis to D- and L-lactate in the presence (mixotrophy) and absence (organotrophy) of N[O.sup.-.sub.2] was examined. L-Lactate did not support organotrophic growth or stimulate mixotrophic growth. In contrast, D-lactate enhanced the growth rate and yield of N. hamburgensis in the presence of N[O.sup.-.sub.2], and served as the sole carbon and energy source for growth in the absence of N[O.sup.-.sub.2] with ammonium as the sole nitrogen source. Lithoautotrophically grown cells immediately consumed D-lactate, suggesting that a lactate metabolic pathway is constitutively expressed. Nevertheless, a physiological adaptation to lactate occurred, as D-lactate-grown cells consumed and assimilated lactate at a faster rate than N[O.sup.-.sub.2]-grown cells, and the D-lactate-dependent [O.sub.2] uptake rate was significantly greater in cells grown either organotrophically or mixotrophically compared with cells grown lithoautotrophically. Although D-lactate was assimilated and metabolized to C[O.sub.2] in the presence or absence of N[O.sup.-.sub.2], exposure to atmospheric C[O.sub.2] or the addition of 0.75 mM sodium carbonate was required for mixotrophic growth and for optimum organotrophic growth on D-lactate.

Abstract: Meadows are natural dynamic features of forested mountain landscapes of the Pacific Northwest. Proportions of meadows and forests change with environmental conditions and disturbance history. We investigated the belowground [...]

'Pseudomonas butanovora' uses an alcohol-inducible alkane monooxygenase (BMO) to grow on [C.sub.2]--[C.sub.9] n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative [[sigma].sup.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.sub.2]-[C.sub.5] n-alkanes and showed little to no growth on [C.sub.6]-[C.sub.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.sub.2]-[C.sub.8] carbon sources. In acetate- or lactate-grown cells, [C.sub.2]-[C.sub.8] n-alcohols failed to induce [beta]-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced ~45 % of the [beta]-galactosidase activity observed in the control bmoX::lacZ strain. In propionate-grown cells, [C.sub.2]-[C.sub.5] n-alcohols induced [beta]-galactosidase activity, whereas [C.sub.7] and [C.sub.8] n-alcohols did not. BmoR may act as a [[sigma].sup.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. [less than or equal to][C.sub.6]). The bmoG-deficient mutant did not grow on [C.sub.2]-[C.sub.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.

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the [alpha]-subunit of butane monooxygenase hydroxylase (BMOH-[alpha]) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes ICEs) were reduced in mutant strain Gll3N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain Gll3N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G 113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain GI13N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent [O.sub.2] uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-[alpha] mutants might allow insights into the catalytic mechanism of BMO to be obtained.

Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase [alpha] subunit of butane monooxygenase (BMOH-[alpha]) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-[alpha] were altered to the corresponding residues from the o~ subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains GI13N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G 113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to l-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 [micro]M methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 [micro]M. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-[alpha]

The genome sequencing and analysis of the alphaproteobacterium Nitrobacter winogradskyi revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. Approximately 10 percent of the Nitrobacter winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules.

The soluble fraction of ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot.] straw comprises a major component of residue C and its presence or absence should influence the succession of decomposer communities. Changes in phospholipid fatty acid (PLFA) profiles are indicative of shifts in microbial community structure. We used [sup.13]C-labeled ryegrass to track substrate-derived C into microbial lipids during decomposition in a microcosm-based study. Treatments were unleached straw, leached straw, and leachate, plus an unamended control. Destructive sampling took place after 0.6, 1.6, 15, 18, 50, and 80 d of incubation. Phospholipid fatty acids were extracted from bulk soil and isolated straw (detritusphere), and analyzed by gas chromatography combustion isotope ratio mass spectroscopy (GC-C-IRMS). Distinct temporal shifts occurred in PLFAs in bulk soil samples; cy 19:0 was an indicator for late succession communities in unleached straw and leachate treatments, and 18:2[omega]6,9 characterized late samples in the leached straw treatment. The temporal shill was affected by the presence of the soluble fraction of strays. Microscale spatial effects were clear. Bulk soil and detritusphere communities were different and more [sup.13]C was detected in the 16:0 and 18:2[omega]6,9 PLFAs of the detritusphere than bulk soil. Carbon-13 was slowest to appear in PLFAs of leached straw samples, illustrating the importance of the soluble fraction in promoting growth of the biomass in bulk soil and in detritusphere. The 18:2[omega]6,9 PLFA, a fungal biomarker, was the most highly labeled in all treatments. Carbon-13 PLFA analysis provides greater insight into microbial community structure and functioning, facilitating more concrete conclusions regarding the role of functional groups of organisms.

An assumption inherent in isotope dilution methodologies is that of homogeneous distribution of label. This assumption may not hold, however because of mass transfer limitations in most soil systems. The effects of mass transfer limitations on isotope dilution in soil aggregates with radii up to 0.36 cm were examined using spherical diffusion-reaction models designed to simulate [sup.15]N[H.sub.4.sup.+] isotope dilution experiments measuring gross production and consumption of N[H.sub.4.sup.+] across a 24-h period. Equations that described transport and reaction of N[H.sub.4.sup.+] assumed Fickian diffusion, linear, equilibrium adsorption, zero-order production of natural abundance [sup.15]N, and either pseudo-first-order or zero-order consumption of N[H.sub.4.sup.+]. In the case of pseudo-first-order consumption, rate calculations were sensitive to the adsorption coefficient (emphasizing the need to interpret results as apparent rates), but not to other transport parameters. In the case of zero-order consumption, both production and consumption rates were always underestimated. Errors increased as aggregate size increased and as effective diffusivity decreased. Increasing the consumption to production rate ratio increased the error in production rate estimates. Allowing the applied label to diffuse into soil aggregates for 24 h prior to initial time sampling decreased errors by a factor of about three (to

Research has been conducted on the stable N and C isotopes used for soil C and N cycling process examination. The application of the time-of-flight secondary ion mass spectrometry in detecting N and C assimilation by different bacterial cells and in quantification of N isotope ratios in bacterial samples is described.

Trichloroethylene (TCE) oxidation by Burkholderia cepacia G4 is toxic to the bacterium. This occurs because reactive intermediates generated during TCE oxidation can inactivate not only the oxygenase, but also other cellular constituents as well.