Inhibitory effects of C2 to C10 1-alkynes on ammonia oxidation in two Nitrososphaera species.

A previous study showed that ammonia oxidation by the Thaumarchaeota Nitrosopumilus maritimus (group 1.1a) was resistant to concentrations of the C8 1-alkyne, octyne, which completely inhibits activity by ammonia-oxidizing bacteria. In this study, the inhibitory effects of octyne and other C2 to C10 1-alkynes were evaluated on the nitrite production activity of two pure culture isolates from Thaumarchaeota group 1.1b, Nitrososphaera viennensis strain EN76 and Nitrososphaera gargensis. Both N. viennensis and N. gargensis were insensitive to concentrations of octyne that cause complete and irreversible inactivation of nitrite production by ammonia-oxidizing bacteria. However, octyne concentrations (≥20 µM) that did not inhibit N. maritimus partially inhibited nitrite production in N. viennensis and N. gargensis in a manner that did not show the characteristics of irreversible inactivation. In contrast to previous studies with an ammonia-oxidizing bacterium, Nitrosomonas europaea, octyne inhibition of N. viennensis was: (i) fully and immediately reversible, (ii) not competitive with NH4 (+), and (iii) without effect on the competitive interaction between NH4 (+) and acetylene. Both N. viennensis and N. gargensis demonstrated the same overall trend in regard to 1-alkyne inhibition as previously observed for N. maritimus, being highly sensitive to ≤C5 alkynes and more resistant to longer-chain length alkynes. Reproducible differences were observed among N. maritimus, N. viennensis, and N. gargensis in regard to the extent of their resistance/sensitivity to C6 and C7 1-alkynes, which may indicate differences in the ammonia monooxygenase binding and catalytic site(s) among the Thaumarchaeota.

Altered precipitation regime affects the function and composition of soil microbial communities on multiple time scales.

Climate change models predict that future precipitation patterns will entail lower-frequency but larger rainfall events, increasing the duration of dry soil conditions. Resulting shifts in microbial C cycling activity could affect soil C storage. Further, microbial response to rainfall events may be constrained by the physiological or nutrient limitation stress of extended drought periods; thus seasonal or multiannual precipitation regimes may influence microbial activity following soil wet-up. We quantified rainfall-driven dynamics of microbial processes that affect soil C loss and retention, and microbial community composition, in soils from a long-term (14-year) field experiment contrasting "Ambient" and "Altered" (extended intervals between rainfalls) precipitation regimes. We collected soil before, the day following, and five days following 2.5-cm rainfall events during both moist and dry periods (June and September 2011; soil water potential = -0.01 and -0.83 MPa, respectively), and measured microbial respiration, microbial biomass, organic matter decomposition potential (extracellular enzyme activities), and microbial community composition (phospholipid fatty acids). The equivalent rainfall events caused equivalent microbial respiration responses in both treatments. In contrast, microbial biomass was higher and increased after rainfall in the Altered treatment soils only, thus microbial C use efficiency (CUE) was higher in Altered than Ambient treatments (0.70 +/- 0.03 > 0.46 +/- 0.10). CUE was also higher in dry (September) soils. C-acquiring enzyme activities (beta-glucosidase, cellobiohydrolase, and phenol oxidase) increased after rainfall in moist (June), but not dry (September) soils. Both microbial biomass C:N ratios and fungal:bacterial ratios were higher at lower soil water contents, suggesting a functional and/or population-level shift in the microbiota at low soil water contents, and microbial community composition also differed following wet-up and between seasons and treatments. Overall, microbial activity may directly (C respiration) and indirectly (enzyme potential) reduce soil organic matter pools less in drier soils, and soil C sequestration potential (CUE) may be higher in soils with a history of extended dry periods between rainfall events. The implications include that soil C loss may be reduced or compensated for via different mechanisms at varying time scales, and that microbial taxa with better stress tolerance or growth efficiency may be associated with these functional shifts.

Measuring T2 and T1, and imaging T2 without spin echoes.

During adiabatic excitation, the nuclear magnetization in the transverse plane is subject to T(2) (spin-spin) relaxation, depending on the pulse length τ. Here, this property is exploited in a method of measuring T(2) using the ratio of NMR signals acquired with short and long-duration self-refocusing adiabatic pulses, without spin-echoes. This Dual-τ method is implemented with B(1)-insensitive rotation (BIR-4) pulses. It is validated theoretically with Bloch equation simulations independent of flip-angle, and experimentally in phantoms. Dual-τT(2) measurements are most accurate at short T(2) where results agree with standard spin-echo measures to within 10% for T(2) ≤ 100 ms. Dual-τ MRI performed with a long 0° BIR-4 pre-pulse provides quantitative T(2) imaging of phantoms and the human foot while preserving desired contrast and functional properties of the rest of the MRI sequence. A single 0° BIR-4 pre-pulse can provide T(2) contrast-weighted MRI and serve as a "T(2)-prep" sequence with a lower B(1) requirement than prior approaches. Finally, a Tri-τ experiment is introduced in which both τ and flip-angle are varied, enabling measurement of T(2), T(1) and signal intensity in just three acquisitions if flip-angles are well-characterized. These new methods can potentially save time and simplify relaxation measurements and/or contrast-weighted NMR and MRI.

Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora.

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C(2)-C(9) alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of beta-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O(2) when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH(4)Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O(2) (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O(2), CuSO(4) (0.5 microM) repressed 1-butanol-dependent induction of beta-galactosidase activity. Under oxic conditions (20% O(2) [vol/vol]), significantly higher concentrations of CuSO(4) (2 microM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu(2+) reducing agent Na ascorbate (100 microM) and CuSO(4) (0.5 microM) repressed the induction of beta-galactosidase activity under oxic conditions to the same extent that 0.5 microM CuSO(4) alone repressed it under anoxic conditions. Under oxic conditions, 2 microM CuSO(4) repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO(4) repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

Propionate inactivation of butane monooxygenase activity in 'Pseudomonas butanovora': biochemical and physiological implications.

Butane monooxygenase (BMO) catalyses the oxidation of alkanes to alcohols in the alkane-utilizing bacterium 'Pseudomonas butanovora'. Incubation of alkane-grown 'P. butanovora' with butyrate or propionate led to irreversible time- and O2-dependent loss of BMO activity. In contrast, BMO activity was unaffected by incubation with lactate or acetate. Chloramphenicol inhibited the synthesis of new BMO, but did not change the kinetics of propionate-dependent BMO inactivation, suggesting that the propionate effect was not simply due to it acting as a repressor of BMO transcription. BMO was protected from propionate-dependent inactivation by the presence of its natural substrate, butane. Although both the time and O2 dependency of propionate inactivation of BMO imply that propionate might be a suicide substrate, no evidence was obtained for BMO-dependent propionate consumption, or 14C labelling of BMO polypeptides by [2-(14)C]propionate during inactivation. Propionate-dependent BMO inactivation was also explored in mutant strains of 'P. butanovora' containing single amino acid substitutions in the alpha-subunit of the BMO hydroxylase. Propionate-dependent BMO inactivation in two mutant strains with amino acid substitutions close to the catalytic site differed from wild-type (one was more sensitive and the other less), providing further evidence that propionate-dependent inactivation involves interaction with the BMO catalytic site. A putative model is presented that might explain propionate-dependent inactivation of BMO when framed within the context of the catalytic cycle of the closely related enzyme, soluble methane monooxygenase.

Product repression of alkane monooxygenase expression in Pseudomonas butanovora.

Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C2 to C8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even- versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following beta-oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 microM propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane- and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed.min(-1).mg protein(-1)) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls beta-galactosidase expression, only even-chain-length alcohols (C2 to C8) induced beta-galactosidase following growth on acetate or butyrate. In contrast, both even- and odd-chain-length alcohols (C3 to C7) were able to induce beta-galactosidase following the induction of propionate consumption by propionate or pentanoate.

Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium "Pseudomonas butanovora".

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of approximately 20 nmol mg protein(-1) of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein(-1). Oxidation of similar amounts of each DCE isomer ( approximately 20 nmol mg protein(-1)) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.

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 (approximately 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.

Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils.

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 alpha-Proteobacteria species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.

Ammonia-oxidizing bacteria along meadow-to-forest transects in the Oregon Cascade Mountains.

Although nitrification has been well studied in coniferous forests of Western North America, communities of NH(3)-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 NH(3) 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.

Biodegradation of monohalogenated alkanes by soil NH(3)-oxidizing bacteria.

Although cooxidative biodegradation of monohalogenated hydrocarbons has been well studied in the model NH(3)-oxidizing bacterium, Nitrosomonas europaea, virtually no information exists about cooxidation of these compounds by native populations of NH(3)-oxidizing bacteria. To address this subject, nitrifying activity was stimulated to 125-400 nmol NO(3)(-) produced g(-1) soil h(-1) by first incubating a Ca(OH)(2)-amended, silt loam soil (pH 7.0+/-0.2) at field capacity (270 g H(2)O kg(-1) soil) with 10 micro mol NH(4)(+) g(-1) soil for 14 days, followed by another 10 days of incubation in a shaken slurry (2:1 water:soil, v/w) with periodic pH adjustment and maintenance of 10 mM NH(4)(+). These slurries actively degraded both methyl bromide (MeBr) and ethyl chloride (EtCl) at maximum rates of 20-30 nmol ml(-1) h(-1) that could be sustained for approximately 12 h. Although the MeBr degradation rates were linear for the first 10-12 h of incubation, they could not be sustained regardless of NH(4)(+) level and declined to zero over 20 h of incubation. The transformation capacity of the slurry enrichments (~1 micro mol MeBr ml(-1) soil slurry) was similar to the value measured previously in cell suspensions of N. europaea with similar NH(3)-oxidizing activity. Several MeBr-degrading characteristics of the nitrifying enrichments were found to be similar to those documented in the literature for MeBr-degrading methanotrophs and facultatively methylotrophic bacteria.

Noninvasive quantitative measurement of bacterial growth in porous media under unsaturated-flow conditions.

Glucose-dependent growth of the luxCDABE reporter bacterium Pseudomonas fluorescens HK44 was monitored noninvasively in quartz sand under unsaturated-flow conditions within a 45- by 56- by 1-cm two-dimensional light transmission chamber. The spatial and temporal development of growth were mapped daily over 7 days by quantifying salicylate-induced bioluminescence. A nonlinear model relating the rate of increase in light emission after salicylate exposure to microbial density successfully predicted growth over 4 orders of magnitude (r(2) = 0.95). Total model-predicted growth agreed with growth calculated from the mass balance of the system by using previously established growth parameters of HK44 (predicted, 1.2 x 10(12) cells; calculated, 1.7 x 10(12) cells). Colonization expanded in all directions from the inoculation region, including upward migration against the liquid flow. Both the daily rate of expansion of the colonized zone and the population density of the first day's growth in each newly colonized region remained relatively constant throughout the experiment. Nonetheless, substantial growth continued to occur on subsequent days in the older regions of the colonized zone. The proportion of daily potential growth that remained within the chamber declined progressively between days 2 and 7 (from 97 to 13%). A densely populated, anoxic region developed in the interior of the colonized zone even though the sand was unsaturated and fresh growth medium continued to flow through the colonized zone. These data illustrate the potential of a light transmission chamber, bioluminescent bacteria, and sensitive digital camera technology to noninvasively study real-time hydrology-microbiology interactions associated with unsaturated flow in porous media.

Noninvasive quantification of total sodium concentrations in acute reperfused myocardial infarction using 23Na MRI.

The transport of sodium and potassium between the intra- and extracellular pools and the maintenance of the transmembrane concentration gradients are important to cell function and integrity. The early disruption of the sodium pump in myocardial infarction in response to the exhaustion of energy reserves following ischemia and reperfusion results in increased intracellular (and thus total) sodium levels. In this study a method for noninvasively quantifying myocardial sodium levels directly from sodium (23Na) MRI is presented. It was used to measure total myocardial sodium on a clinical 1.5T system in six normal dogs and five dogs with experimentally-induced myocardial infarction (MI). The technique was validated by comparing total sodium content measured by 23Na MRI with that measured by atomic absorption spectrophotometry (AAS) in biopsied tissue. Total sodium measured by 23Na MRI was significantly elevated in regions of infarction (81.3 +/- 14.3 mmol/kg wet wt, mean +/- SD) compared to noninfarcted myocardial tissue from both infarcted dogs (36.2 +/- 1.1, P < 0.001) and from normal controls (34.4 +/- 2.8, P < 0.0001). Myocardial tissue sodium content as measured by 23Na MRI did not vary regionally in the lateral, anterior, or inferior regions in normal hearts (ANOVA, P = NS). Sodium content measured by 23Na MRI agreed with the mean AAS estimates of 31.3 +/- 5.6 mmol/kg wet wt (P = NS) in normal hearts, and did not differ significantly from AAS measurements in MI (P = NS). Thus, local tissue sodium levels can be accurately quantified noninvasively using 23Na MRI in normal and acutely reperfused MI. The detection of regional myocardial sodium elevations may help differentiate viable from nonviable, infarcted tissue.

Superparamagnetic iron oxide MION as a contrast agent for sodium MRI in myocardial infarction.

An intravascular iron-based contrast agent was used as a sodium (23Na) MRI T2 relaxant in an effort to suppress the blood signal from the ventricular cavities in normal and infarcted canine myocardium in vivo. 23Na MRI signal decreases in blood were attributed to decreases in the fast (T2f) and slow (T2s) transverse relaxation components, which were quantified as a function of dose and MRI echo time (TE). In vivo 23Na MRI signal decreases up to 65% were noted in ventricular blood when imaging under dose and TE conditions of 10 mg/kg body weight and 5 ms, respectively. Contrast injection followed by subsequent 23Na MRI in canine myocardial infarction led to a clear delineation of the location of the injured tissue, as identified by postmortem triphenyltetrazolium chloride staining, and to an improvement in the contrast-to-noise ratio between the blood in the ventricular chamber and the infarcted tissue that was as high as 3.3-fold in the postcontrast images in comparison to the precontrast images.

Requirement of DNA repair mechanisms for survival of Burkholderia cepacia G4 upon degradation of trichloroethylene.

A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H(2)O(2). The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.