Binbin Liu

Denitrification gene pools, transcription and kinetics of NO, N2O and N2 production as affected by soil pH

The N(2)O : N(2) product ratio of denitrification is negatively correlated with soil pH, but the mechanisms involved are not clear. We compared soils from field experiments where the pH had been maintained at different levels (pH 4.0-8.0) by liming (> or = 20 years), and quantified functional gene pools (nirS, nirK and nosZ), their transcription and gas kinetics (NO, N(2)O and N(2)) of denitrification as induced by anoxic incubation with and without a carbon substrate (glutamate). Denitrification in unamended soil appeared to be based largely on the activation of a pre-existing denitrification proteome, because constant rates of N(2) and N(2)O production were observed, and the transcription of functional genes was below the detection level. In contrast, glutamate-amended soils showed sharp peaks in the transcripts of nirS and nosZ, increasing the rates of denitrification and pH-dependent transient accumulation of N(2)O. The results indicate that the high N(2)O : N(2) product ratio at low pH is a post-transcriptional phenomenon, because the transcription rate of nosZ relative to that of nirS was higher at pH 6.1 than at pH 8.0. The most plausible explanation is that the translation/assembly of N(2)O reductase is more sensitive to low pH than that of the other reductases involved in denitrification.

Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils

Denitrifying prokaryotes use NOx as terminal electron acceptors in response to oxygen depletion. The process emits a mixture of NO, N2O and N2, depending on the relative activity of the enzymes catalysing the stepwise reduction of NO3− to N2O and finally to N2. Cultured denitrifying prokaryotes show characteristic transient accumulation of NO2−, NO and N2O during transition from oxic to anoxic respiration, when tested under standardized conditions, but this character appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in their propensity to emit N2O, it may be difficult to predict such characteristics by analysis of the community composition. A common feature of strains tested in our laboratory is that the relative amounts of N2O produced (N2O/(N2+N2O) product ratio) is correlated with acidity, apparently owing to interference with the assembly of the enzyme N2O reductase. The same phenomenon was demonstrated for soils and microbial communities extracted from soils. Liming could be a way to reduce N2O emissions, but needs verification by field experiments. More sophisticated ways to reduce emissions may emerge in the future as we learn more about the regulation of denitrification at the cellular level.

Strains in the genus Thauera exhibit remarkably different denitrification regulatory phenotypes.

Denitrifiers differ in how they handle the transition from oxic to anoxic respiration, with consequences for NO and N2O emissions. To enable stringent comparisons we defined parameters to describe denitrification regulatory phenotype (DRP) based on accumulation of NO2(-) , NO and N2O, oxic/anoxic growth and transcription of functional genes. Eight Thauera strains were divided into two distinct DRP types. Four strains were characterized by a rapid, complete onset (RCO) of all denitrification genes and no detectable nitrite accumulation. The others showed progressive onset (PO) of the different denitrification genes. The PO group accumulated nitrite, and no transcription of nirS (encoding nitrite reductase) was detected until all available nitrate (2 mM) was consumed. Addition of a new portion of nitrate to an actively denitrifying culture of a PO strain (T. terpenica) resulted in a transient halt in nitrite reduction, indicating that the electron flow was redirected to nitrate reductase. All eight strains controlled NO at nano-molar concentrations, possibly reflecting the importance of strict control for survival. Transient N2O accumulation differed by two orders of magnitude between strains, indicating that control of N2O is less essential. No correlation was seen between phylogeny (based on 16S rRNA and functional genes) and DRP.

Impaired Reduction of N2O to N2 in Acid Soils Is Due to a Posttranscriptional Interference with the Expression of nosZ

ABSTRACT Accumulating empirical evidence over the last 60 years has shown that the reduction of N2O to N2 is impaired by low soil pH, suggesting that liming of acid soils may reduce N2O emissions. This option has not gained much momentum in global change research, however, possibly due to limited understanding of why low pH interferes with N2O reductase. We hypothesized that the reason is that denitrifying organisms in soils are unable to assemble functional N2O reductase (N2OR) at low pH, as shown to be the case for the model strain Paracoccus denitrificans. We tested this by experiments with bacteria extracted from soils by density gradient centrifugation. The soils were sampled from a long-term liming experiment (soil pH 4.0, 6.1, and 8.0). The cells were incubated (stirred batches, He atmosphere) at pH levels ranging from 5.7 to 7.6, while gas kinetics (NO, N2O, and N2) and abundances of relevant denitrification genes (nirS, nirK, and nosZ) and their transcripts were monitored. Cells from the most acidic soil (pH 4.0) were unable to reduce N2O at any pH. These results warrant a closer inspection of denitrification communities of very acidic soils. Cells from the neutral soils were unable to produce functional N2OR at pH values of ≤6.1, despite significant transcription of the nosZ gene. The N2OR expressed successfully at pH 7.0, however, was functional over the entire pH range tested (5.7 to 7.6). These observations lend strong support to our hypothesis: low soil pH diminishes/prevents reduction of N2O, primarily by precluding a successful assembly of functional N2O reductase. IMPORTANCE Impaired N2O reduction in acid soils was first observed ~60 years ago, and the phenomenon has been rediscovered several times since then. The practical implication would be that the emissions of N2O from cropped soils could be controlled by soil pH management, but this option has largely been ignored till now. One reason for this could be that the mechanisms involved have remained obscure. Here, we provide compelling evidence that the primary reason is that low pH interferes with the making of the enzyme N2O reductase rather than the function of the enzyme if properly assembled. The implications are important for understanding how pH controls the kinetics of N2O and N2 production by denitrification. The improved understanding provides credibility for soil pH management as a way to mitigate N2O emissions. Impaired N2O reduction in acid soils was first observed ~60 years ago, and the phenomenon has been rediscovered several times since then. The practical implication would be that the emissions of N2O from cropped soils could be controlled by soil pH management, but this option has largely been ignored till now. One reason for this could be that the mechanisms involved have remained obscure. Here, we provide compelling evidence that the primary reason is that low pH interferes with the making of the enzyme N2O reductase rather than the function of the enzyme if properly assembled. The implications are important for understanding how pH controls the kinetics of N2O and N2 production by denitrification. The improved understanding provides credibility for soil pH management as a way to mitigate N2O emissions.

Isolation, genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO(3)(-) and NO(2)(-)) to N(2) via the intermediates NO and N(2)O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.

Structural differentiation of bacterial communities in indole-degrading bioreactors under denitrifying and sulfate-reducing conditions.

The acclimated, anaerobic microbial community is an efficient method for indole-containing wastewater treatment. However, our understanding of the diversity of indole-degrading communities is still limited. We investigated two anaerobic, indole-decomposing microbial communities under both denitrifying and sulfate-reducing conditions. Utilizing a near full-length 16S rRNA gene clone library, the most dominant bacteria in the denitrifying bioreactor identified was β-proteobacteria. Among these, bacteria from genera Alicycliphilus, Acaligenes and Thauera were abundant and thought responsible for indole degradation. However, in the sulfate-reducing bioreactor, Clostridia and Actinobacteria were the dominant bacterial class found and likely the main degrading species. Microbial communities in these bioreactors shared only two operational taxonomic units (OTUs). Differences in the electron acceptors of denitrification or sulfate reduction may be responsible for the higher indole removal capacity in the denitrifying bioreactor (80%) than the capacity in the sulfate-reducing bioreactor (52%). This study is the first detailed analysis of an anaerobic indole-degrading community.

Pattern extraction of structural responses of gut microbiota to rotavirus infection via multivariate statistical analysis of clone library data

This study developed a new statistical strategy for analyzing clone library data to observe whether there is a defined pattern in structural responses of gut microbiota to environmental perturbations. A large clone library of genus Bacteroides was constructed with fecal samples for each subject in rotavirus-infected (Group R) and healthy children (Group H). In all, 665 clones of the 12 Group H subjects and 284 clones of the nine Group R subjects were sequenced and classified into 34 operational taxonomic units (OTUs) with a similarity cutoff at 98%. Partial least squares-discriminant analysis was used to observe the change of the Bacteroides spp. composition caused by rotavirus infection and to identify the most relevant species contributing to this shift. It was revealed that H subjects and R subjects were well separated. Bacteroides vulgatus, Bacteroides stercoris and Bacteroides fragilis were identified as the most important discriminating OTUs between two groups. The increased abundance of B. fragilis and the decreased populations of B. vulgatus and B. stercoris in infected guts observed in this study were in agreement with previous culture-based studies. The strategy developed in this work can be used to reveal patterns in structural responses of gut microbiota to environmental perturbations from large-scale 16S rRNA gene-based sequencing data.

Development of group-specific PCR-DGGE fingerprinting for monitoring structural changes of Thauera spp. in an industrial wastewater treatment plant responding to operational perturbations

A Thauera-specific nested-PCR denaturing gradient gel electrophoresis (DGGE) method was developed, and its usefulness was demonstrated by monitoring the structural shifts of Thauera spp. in an anaerobic-anoxic-oxic fixed-biofilm coking wastewater treatment plant (WWTP) responding to operational perturbations. The specificity of the PCR method was confirmed by the fact that all 16 S rRNA gene sequences, cloned from the amplicons of a biofilm sample, belonged to Thauera genus. 16 S rRNA gene V3 region was then amplified from the first round Thauera-specific PCR product and applied for DGGE analysis. All Thauera clones, with 13 different V3 regions, migrated into 10 positions on DGGE gel, which demonstrated the high resolution of this DGGE method. When the WWTP experienced a gradual deterioration in chemical oxygen demand (COD) removal function due to a mechanical failure of the recirculation pump, biofilm samples were collected from the reactor and analyzed by this method. Principal component analysis (PCA) of the DGGE fingerprinting data showed that the composition of Thauera group exhibited a time related trajectory when the plants COD removal rate decreased from 84.1+/-2.7% in the first 4 weeks to less than 75% at week 5 and 6, suggesting a concomitant shift of Thauera composition and the systems COD removal function. This group-specific PCR DGGE fingerprinting technology has the potential to be a profiling tool for monitoring structural shifts of Thauera spp. in industrial WWTPs.

Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov., isolated from root nodules of different agroforestry legume trees

A total of 18 strains, representing members of the genus Mesorhizobium, obtained from root nodules of woody legumes growing in Ethiopia, have been previously shown, by multilocus sequence analysis (MLSA) of five housekeeping genes, to form three novel genospecies. In the present study, the phylogenetic relationship between representative strains of these three genospecies and the type strains of their closest phylogenetic neighbours Mesorhizobium plurifarium, Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium huakuii was further evaluated using a polyphasic taxonomic approach. In line with our earlier MLSA of other housekeeping genes, the phylogenetic trees derived from the atpD and glnII genes grouped the test strains into three well-supported, distinct lineages that exclude all defined species of the genus Mesorhizobium. The DNA-DNA relatedness between the representative strains of genospecies I-III and the type strains of their closest phylogenetic neighbours was low (≤59 %). They differed from each other and from their closest phylogenetic neighbours by the presence/absence of several fatty acids, or by large differences in the relative amounts of particular fatty acids. While showing distinctive features, they were generally able to utilize a wide range of substrates as sole carbon and nitrogen sources. The strains belonging to genospecies I, II and III therefore represent novel species for which we propose the names Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov. The isolates AC39a(T) ( = LMG 26966(T) = HAMBI 3295(T)), AC99b(T) ( = LMG 26968(T) = HAMBI 3301(T)) and AC98c(T) ( = LMG 26967(T) = HAMBI 3306(T)) are proposed as type strains for the respective novel species.

A novel protein protects bacterial iron-dependent metabolism from nitric oxide.

Reactive nitrogen species (RNS), in particular nitric oxide (NO), are toxic to bacteria, and bacteria have mechanisms to allow growth despite this stress. Understanding how bacteria interact with NO is essential to understanding bacterial physiology in many habitats, including pathogenesis; however, many targets of NO and enzymes involved in NO resistance remain uncharacterized. We performed for the first time a metabolomic screen on NO-treated and -untreated bacteria to define broadly the effects of NO on bacterial physiology, as well as to identify the function of NnrS, a previously uncharacterized enzyme involved in defense against NO. We found many known and novel targets of NO. We also found that iron-sulfur cluster enzymes were preferentially inhibited in a strain lacking NnrS due to the formation of iron-NO complexes. We then demonstrated that NnrS is particularly important for resistance to nitrosative stress under anaerobic conditions. Our data thus reveal the breadth of the toxic effects of NO on metabolism and identify the function of an important enzyme in alleviating this stress.