Hidden diversity and potential ecological function of phosphorus acquisition genes in widespread terrestrial bacteriophages.

Phosphorus (P) limitation of ecosystem processes is widespread in terrestrial habitats. While a few auxiliary metabolic genes (AMGs) in bacteriophages from aquatic habitats are reported to have the potential to enhance P-acquisition ability of their hosts, little is known about the diversity and potential ecological function of P-acquisition genes encoded by terrestrial bacteriophages. Here, we analyze 333 soil metagenomes from five terrestrial habitat types across China and identify 75 viral operational taxonomic units (vOTUs) that encode 105 P-acquisition AMGs. These AMGs span 17 distinct functional genes involved in four primary processes of microbial P-acquisition. Among them, over 60% (11/17) have not been reported previously. We experimentally verify in-vitro enzymatic activities of two pyrophosphatases and one alkaline phosphatase encoded by P-acquisition vOTUs. Thirty-six percent of the 75 P-acquisition vOTUs are detectable in a published global topsoil metagenome dataset. Further analyses reveal that, under certain circumstances, the identified P-acquisition AMGs have a greater influence on soil P availability and are more dominant in soil metatranscriptomes than their corresponding bacterial genes. Overall, our results reinforce the necessity of incorporating viral contributions into biogeochemical P cycling.

In situ enrichment of sulphate-reducing microbial communities with different carbon sources stimulating the acid mine drainage sediments.

The applications of sulphate-reducing microorganisms (SRMs) in acid mine drainage (AMD) treatment systems have received extensive attention due to their ability to reduce sulphate and stabilize metal(loid)s. Despite great phylogenetic diversity of SRMs, only a few have been used in AMD treatment bioreactors. In situ enrichment could be an efficient approach to select new effective SRMs for AMD treatment. Here, we performed in situ enrichment of SRMs in highly stratified AMD sediment cores using different kinds of carbon source mixture. The dsrAB (dissimilatory sulfite reductase) genes affiliated with nine phyla (two archaeal and seven bacterial phyla) and 26 genera were enriched. Remarkably, those genes affiliated with Aciduliprofundum and Vulcanisaeta were enriched in situ in AMD-related environments for the first time, and their relative abundances were negatively correlated with pH. Furthermore, 107 dsrAB-containing metagenome-assembled genomes (MAGs) were recovered from metagenomic datasets, with 14 phyla (two archaeal and 12 bacterial phyla) and 15 genera. The relative abundances of MAGs were positively correlated with total carbon and sulphate contents. Our findings expanded the diversity of SRMs that can be enriched in AMD sediment, and revealed the physiochemical properties that might affect the growth of SRMs, which provided guidance for AMD treatment bioreators.

Diverse Methylmercury (MeHg) Producers and Degraders Inhabit Acid Mine Drainage Sediments, but Few Taxa Correlate with MeHg Accumulation.

Methylmercury (MeHg) is a notorious neurotoxin, and its production and degradation in the environment are mainly driven by microorganisms. A variety of microbial MeHg producers carrying the gene pair hgcAB and degraders carrying the merB gene have been separately reported in recent studies. However, surprisingly little attention has been paid to the simultaneous investigation of the diversities of microbial MeHg producers and degraders in a given habitat, and no studies have been performed to explore to what extent these two contrasting microbial groups correlate with MeHg accumulation in the habitat of interest. Here, we collected 86 acid mine drainage (AMD) sediments from an area spanning approximately 500,000 km2 in southern China and profiled the sediment-borne putative MeHg producers and degraders using genome-resolved metagenomics. 46 metagenome-assembled genomes (MAGs) containing hgcAB and 93 MAGs containing merB were obtained, including those from various taxa without previously known MeHg-metabolizing microorganisms. These diverse MeHg-metabolizing MAGs were formed largely via multiple independent horizontal gene transfer (HGT) events. The putative MeHg producers from Deltaproteobacteria and Firmicutes as well as MeHg degraders from Acidithiobacillia were closely correlated with MeHg accumulation in the sediments. Furthermore, these three taxa, in combination with two abiotic factors, explained over 60% of the variance in MeHg accumulation. Most of the members of these taxa were characterized by their metabolic potential for nitrogen fixation and copper tolerance. Overall, these findings improve our understanding of the ecology of MeHg-metabolizing microorganisms and likely have implications for the development of management strategies for the reduction of MeHg accumulation in the AMD sediments. IMPORTANCE Microorganisms are the main drivers of MeHg production and degradation in the environment. However, little attention has been paid to the simultaneous investigation of the diversities of microbial MeHg producers and degraders in a given habitat. We used genome-resolved metagenomics to reveal the vast phylogenetic and metabolic diversities of putative MeHg producers and degraders in AMD sediments. Our results show that the diversity of MeHg-metabolizing microorganisms (particularly MeHg degraders) in AMD sediments is much higher than was previously recognized. Via multiple linear regression analysis, we identified both microbial and abiotic factors affecting MeHg accumulation in AMD sediments. Despite their great diversity, only a few taxa of MeHg-metabolizing microorganisms were closely correlated with MeHg accumulation. This work underscores the importance of using genome-resolved metagenomics to survey MeHg-metabolizing microorganisms and provides a framework for the illumination of the microbial basis of MeHg accumulation via the characterization of physicochemical properties, MeHg-metabolizing microorganisms, and the correlations between them.

Phytostabilization mitigates antibiotic resistance gene enrichment in a copper mine tailings pond.

Mining-impacted environments are distributed globally and have become increasingly recognized as hotspots of antibiotic resistance genes (ARGs). However, there are currently no reports on treatment technologies to deal with such an important environmental problem. To narrow this knowledge gap, we implemented a phytostabilization project in an acidic copper mine tailings pond and employed metagenomics to explore ARG characteristics in the soil samples. Our results showed that phytostabilization decreased the total ARG abundance in 0-10 cm soil layer by 75 %, which was companied by a significant decrease in ARG mobility, and a significant increase in ARG diversity and microbial diversity. Phytostabilization was also found to drastically alter the ARG host composition and to significantly reduce the total abundance of virulence factor genes of ARG hosts. Soil nutrient status, heavy metal toxicity and SO42- concentration were important physicochemical factors to affect the total ARG abundance, while causal mediation analysis showed that their effects were largely mediated by the changes in ARG mobility and microbial diversity. The increase in ARG diversity associated with phytostabilization was mainly mediated by a small subgroup of ARG hosts, most of which could not be classified at the genus level and deserve further research in the future.

Functional Guilds, Community Assembly, and Co-occurrence Patterns of Fungi in Metalliferous Mine Tailings Ponds in Mainland China.

Metalliferous mine tailings ponds are generally characterized by low levels of nutrient elements, sustained acidic conditions, and high contents of toxic metals. They represent one kind of extreme environments that are believed to resemble the Earth's early environmental conditions. There is increasing evidence that the diversity of fungi inhabiting mine tailings ponds is much higher than previously thought. However, little is known about functional guilds, community assembly, and co-occurrence patterns of fungi in such habitats. As a first attempt to address this critical knowledge gap, we employed high-throughput sequencing to characterize fungal communities in 33 mine tailings ponds distributed across 18 provinces of mainland China. A total of 5842 fungal phylotypes were identified, with saprotrophic fungi being the major functional guild. The predictors of fungal diversity in whole community and sub-communities differed considerably. Community assembly of the whole fungal community and individual functional guilds were primarily governed by stochastic processes. Total soil nitrogen and total phosphorus mediated the balance between stochastic and deterministic processes of the fungal community assembly. Co-occurrence network analysis uncovered a high modularity of the whole fungal community. The observed main modules largely consisted of saprotrophic fungi as well as various phylotypes that could not be assigned to known functional guilds. The richness of core fungal phylotypes, occupying vital positions in co-occurrence network, was positively correlated with edaphic properties such as soil enzyme activity. This indicates the important roles of core fungal phylotypes in soil organic matter decomposition and nutrient cycling. These findings improve our understanding of fungal ecology of extreme environments.

Metagenomic and metatranscriptomic insights into sulfate-reducing bacteria in a revegetated acidic mine wasteland.

The widespread occurrence of sulfate-reducing microorganisms (SRMs) in temporarily oxic/hypoxic aquatic environments indicates an intriguing possibility that SRMs can prevail in constantly oxic/hypoxic terrestrial sulfate-rich environments. However, little attention has been given to this possibility, leading to an incomplete understanding of microorganisms driving the terrestrial part of the global sulfur (S) cycle. In this study, genome-centric metagenomics and metatranscriptomics were employed to explore the diversity, metabolic potential, and gene expression profile of SRMs in a revegetated acidic mine wasteland under constantly oxic/hypoxic conditions. We recovered 16 medium- to high-quality metagenome-assembled genomes (MAGs) containing reductive dsrAB. Among them, 12 and four MAGs belonged to Acidobacteria and Deltaproteobacteria, respectively, harboring three new SRM genera. Comparative genomic analysis based on seven high-quality MAGs (completeness >90% and contamination <10%; including six acidobacterial and one deltaproteobacterial) and genomes of three additional cultured model species showed that Acidobacteria-related SRMs had more genes encoding glycoside hydrolases, oxygen-tolerant hydrogenases, and cytochrome c oxidases than Deltaproteobacteria-related SRMs. The opposite pattern was observed for genes encoding superoxide reductases and thioredoxin peroxidases. Using VirSorter, viral genome sequences were found in five of the 16 MAGs and in all three cultured model species. These prophages encoded enzymes involved in glycoside hydrolysis and antioxidation in their hosts. Moreover, metatranscriptomic analysis revealed that 15 of the 16 SRMs reported here were active in situ. An acidobacterial MAG containing a prophage dominated the SRM transcripts, expressing a large number of genes involved in its response to oxidative stress and competition for organic matter.

Globally distributed mining-impacted environments are underexplored hotspots of multidrug resistance genes.

Mining is among the human activities with widest environmental impacts, and mining-impacted environments are characterized by high levels of metals that can co-select for antibiotic resistance genes (ARGs) in microorganisms. However, ARGs in mining-impacted environments are still poorly understood. Here, we conducted a comprehensive study of ARGs in such environments worldwide, taking advantage of 272 metagenomes generated from a global-scale data collection and two national sampling efforts in China. The average total abundance of the ARGs in globally distributed studied mine sites was 1572 times per gigabase, being rivaling that of urban sewage but much higher than that of freshwater sediments. Multidrug resistance genes accounted for 40% of the total ARG abundance, tended to co-occur with multimetal resistance genes, and were highly mobile (e.g. on average 16% occurring on plasmids). Among the 1848 high-quality metagenome-assembled genomes (MAGs), 85% carried at least one multidrug resistance gene plus one multimetal resistance gene. These high-quality ARG-carrying MAGs considerably expanded the phylogenetic diversity of ARG hosts, providing the first representatives of ARG-carrying MAGs for the Archaea domain and three bacterial phyla. Moreover, 54 high-quality ARG-carrying MAGs were identified as potential pathogens. Our findings suggest that mining-impacted environments worldwide are underexplored hotspots of multidrug resistance genes.

Remarkable effects of microbial factors on soil phosphorus bioavailability: A country-scale study.

Low soil phosphorus (P) bioavailability causes the widespread occurrence of P-limited terrestrial ecosystems around the globe. Exploring the factors influencing soil P bioavailability at large spatial scales is critical for managing these ecosystems. However, previous studies have mostly focused on abiotic factors. In this study, we explored the effects of microbial factors on soil P bioavailability of terrestrial ecosystems using a country-scale sampling effort. Our results showed that soil microbial biomass carbon (MBC) and acid phosphatase were important predictors of soil P bioavailability of agro- and natural ecosystems across China although they appeared less important than total soil P. The two microbial factors had a positive effect on soil P bioavailability of both ecosystem types and were able to mediate the effects of several abiotic factors (e.g., mean annual temperature). Meanwhile, we revealed that soil phytase could affect soil P bioavailability at the country scale via ways similar to those of soil MBC and acid phosphatase, a pattern being more pronounced in agroecosystems than in natural ecosystems. Moreover, we obtained evidence for the positive effects of microbial genes encoding these enzymes on soil P bioavailability at the country scale although their effect sizes varied between the two ecosystem types. Taken together, this study demonstrated the remarkable effects of microbial factors on soil P bioavailability at a large spatial scale, highlighting the importance to consider microbial factors in managing the widespread P-limited terrestrial ecosystems.

Physiological, Genomic and Transcriptomic Analyses Reveal the Adaptation Mechanisms of Acidiella bohemica to Extreme Acid Mine Drainage Environments.

Fungi in acid mine drainage (AMD) environments are of great concern due to their potentials of decomposing organic carbon, absorbing heavy metals and reducing AMD acidity. Based on morphological analysis and ITS/18S high-throughput sequencing technology, previous studies have provided deep insights into the diversity and community composition of fungi in AMD environments. However, knowledge about physiology, metabolic potential and transcriptome profiles of fungi inhabiting AMD environments is still scarce. Here, we reported the physiological, genomic, and transcriptomic characterization of Acidiella bohemica SYSU C17045 to improve our understanding of the physiological, genomic, and transcriptomic mechanisms underlying fungal adaptation to AMD environments. A. bohemica was isolated from an AMD environment, which has been proved to be an acidophilic fungus in this study. The surface of A. bohemica cultured in AMD solutions was covered with a large number of minerals such as jarosite. We thus inferred that the A. bohemica might have the potential of biologically induced mineralization. Taking advantage of PacBio single-molecule real-time sequencing, we obtained the high-quality genome sequences of A. bohemica (50 Mbp). To our knowledge, this was the first attempt to employ a third-generation sequencing technology to explore the genomic traits of fungi isolated from AMD environments. Moreover, our transcriptomic analysis revealed that a series of genes in the A. bohemica genome were related to its metabolic pathways of C, N, S, and Fe as well as its adaptation mechanisms, including the response to acid stress and the resistance to heavy metals. Overall, our physiological, genomic, and transcriptomic data provide a foundation for understanding the metabolic potential and adaptation mechanisms of fungi in AMD environments.

A comprehensive synthesis unveils the mysteries of phosphate-solubilizing microbes.

Phosphate-solubilizing microbes (PSMs) drive the biogeochemical cycling of phosphorus (P) and hold promise for sustainable agriculture. However, their global distribution, overall diversity and application potential remain unknown. Here, we present the first synthesis of their biogeography, diversity and utility, employing data from 399 papers published between 1981 and 2017, the results of a nationwide field survey in China consisting of 367 soil samples, and a genetic analysis of 12986 genome-sequenced prokaryotic strains. We show that at continental to global scales, the population density of PSMs in environmental samples is correlated with total P rather than pH. Remarkably, positive relationships exist between the population density of soil PSMs and available P, nitrate-nitrogen and dissolved organic carbon in soil, reflecting functional couplings between PSMs and microbes driving biogeochemical cycles of nitrogen and carbon. More than 2704 strains affiliated with at least nine archaeal, 88 fungal and 336 bacterial species were reported as PSMs. Only 2.59% of these strains have been tested for their efficiencies in improving crop growth or yield under field conditions, providing evidence that PSMs are more likely to exert positive effects on wheat growing in alkaline P-deficient soils. Our systematic genetic analysis reveals five promising PSM genera deserving much more attention.

Metagenomic Assembly: Reconstructing Genomes from Metagenomes.

Assembly of metagenomic sequence data into microbial genomes is of critical importance for disentangling community complexity and unraveling the functional capacity of microorganisms. The rapid development of sequencing technology and novel assembly algorithms have made it possible to reliably reconstruct hundreds to thousands of microbial genomes from raw sequencing reads through metagenomic assembly. In this chapter, we introduce a routinely used metagenomic assembly workflow including read quality filtering, assembly, contig/scaffold binning, and postassembly check for genome completeness and contamination. We also describe a case study to reconstruct near-complete microbial genomes from metagenomes using our workflow.

Novel phosphate-solubilizing bacteria enhance soil phosphorus cycling following ecological restoration of land degraded by mining.

Little is known about the changes in soil microbial phosphorus (P) cycling potential during terrestrial ecosystem management and restoration, although much research aims to enhance soil P cycling. Here, we used metagenomic sequencing to analyse 18 soil microbial communities at a P-deficient degraded mine site in southern China where ecological restoration was implemented using two soil ameliorants and eight plant species. Our results show that the relative abundances of key genes governing soil microbial P-cycling potential were higher at the restored site than at the unrestored site, indicating enhancement of soil P cycling following restoration. The gcd gene, encoding an enzyme that mediates inorganic P solubilization, was predominant across soil samples and was a major determinant of bioavailable soil P. We reconstructed 39 near-complete bacterial genomes harboring gcd, which represented diverse novel phosphate-solubilizing microbial taxa. Strong correlations were found between the relative abundance of these genomes and bioavailable soil P, suggesting their contributions to the enhancement of soil P cycling. Moreover, 84 mobile genetic elements were detected in the scaffolds containing gcd in the 39 genomes, providing evidence for the role of phage-related horizontal gene transfer in assisting soil microbes to acquire new metabolic potential related to P cycling.

A simple slash-and-char system to mitigate climate change and environmental pollution.

Agriculture-based climate change mitigation may occur through enhancing the carbon sink or through reducing greenhouse gases (GHGs) emissions from agricultural residue treatment, as open burning of agricultural residues produces millions of tons of GHGs and air pollutants annually worldwide. Charring slashed biomass, termed as slash-and-char, has been considered as a promising alternative to open burning in dealing with agricultural residues such as rice straw. Previous studies, however, focused on relatively sophisticated slash-and-char systems, which could not be practiced easily by smallholder farmers in developing countries. Here we introduce a simple slash-and-char system to mitigate the environmental problems associated with open burning of rice straw. This system could convert 30.7% of the initial carbon in rice straw into biochar, much higher than that retained in the ash generated by open burning (3.95%). It could also cut GHGs, particulate matters and polycyclic aromatic hydrocarbons (PAHs) emissions by 26.9%, 99.0% and 99.4%, respectively. If open burning of rice straw was replaced by the slash-and-char, the annual emissions of GHGs, particulate matters and PAHs in China would decrease by at least 15.4 Tg, 1.51 Tg and 1.27 Gg, correspondingly. This decrease is nearly twice the size of China's estimated forest C sink (8.81 Tg).

Single-Homology-Arm Linear DNA Recombination by the Nonhomologous End Joining Pathway as a Novel and Simple Gene Inactivation Method: a Proof-of-Concept Study in Dietzia sp. Strain DQ12-45-1b.

Nonhomologous end joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNA in vivo Here, we established a proof-of-concept single-homology-arm linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNA in vivo by NHEJ could be used to generate nonreplicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach in Dietzia sp. strain DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA single homolog to the sequence in the chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% of prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, an Escherichia coli strain also showed NHEJ activity when the Ku and LigD of Dietzia sp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for nonmodel microorganisms.IMPORTANCE Many nonmodel Gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway in Dietzia sp. DQ12-45-1b was evaluated and was used to successfully knock out 11 genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the single-homology-arm linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse nonmodel prokaryotic organisms.

Fine-scale spatial patterns in microbial community composition in an acid mine drainage.

Microbial community composition is essential for aquatic ecosystem functions and has been explored across diverse environments and various spatial scales. However, documented patterns are often based on samples from spatially/geographically separated locations or sites. Here, we define sampling volume as spatial scale and examine (by Illumina 16S rRNA sequencing) microbial community composition over a scale of 1 mL to 10 L in an acid mine drainage. ß-Diversity analysis revealed that all samples grouped very tightly according to spatial scales and variations between every two scales were significant. Notably, mean ß-diversity within each group was negatively correlated with spatial scales, indicating patchy microbial distribution. Partition of ß-diversity further revealed that it was the relative abundances of some microbial taxa that largely changed among spatial scales. Phylogenetic analysis showed that microbial lineages were not randomly distributed, but displayed a tendency of more phylogenetically clustering at smaller spatial scales. Thus, we documented fine-scale spatial patterns in microbial community composition within a continuous aquatic environment, which may have practical implications for adequate sampling of aquatic systems in future studies.

Crystal Structure of TetR Family Repressor AlkX from Dietzia sp. Strain DQ12-45-1b Implicated in Biodegradation of n-Alkanes.

n-Alkanes are ubiquitous in nature and are widely used by microorganisms as carbon sources. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of n-alkanes, which plays important roles in natural alkane attenuation and is used in industrial and environmental applications. The alkane oxidation operon, alkW1-alkX, in the alkane-degrading strain Dietzia sp. strain DQ12-45-1b is negatively autoregulated by the TetR family repressor AlkX via a product positive feedback mechanism. To predict the gene regulation mechanism, we determined the 3.1-Å crystal structure of an AlkX homodimer in a non-DNA-bound state. The structure showed traceable long electron density deep inside a hydrophobic cavity of each monomer along the long axis of the helix bundle, and further gas chromatography-mass spectrometry analysis of AlkX revealed that it contained the Escherichia coli-derived long-chain fatty acid molecules as a ligand. Moreover, an unusual structural feature of AlkX is an extra helix, α6', forming a lid-like structure with α6 covering the inducer-binding pocket and occupying the space between the two symmetrical DNA-binding motifs in one dimer, indicating a distinct conformational transition mode in modulating DNA binding. Sequence alignment of AlkX homologs from Dietzia strains showed that the residues involved in DNA and inducer binding are highly conserved, suggesting that the regulation mechanisms of n-alkane hydroxylation are possibly a common characteristic of Dietzia strains.IMPORTANCE With n-alkanes being ubiquitous in nature, many bacteria from terrestrial and aquatic environments have evolved n-alkane oxidation functions. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of n-alkanes, which plays important roles in natural alkane attenuation and petroleum-contaminating environment bioremediation. The gene regulation of the most common alkane hydroxylase, AlkB, has been studied widely in Gram-negative bacteria but has been less explored in Gram-positive bacteria. Our previous study showed that the TetR family regulator (TFR) AlkX negatively autoregulated the alkane oxidation operon, alkW1-alkX, in the Gram-positive strain Dietzia sp. strain DQ12-45-1b. Although TFRs are one of the most common transcriptional regulator families in bacteria, the TFR involved in n-alkane metabolism has been reported only recently. In this study, we determined the crystal structure of AlkX, which implies a distinct DNA/ligand binding mode. Our results shed light upon the regulation mechanism of the common alkane degradation process in nature.

Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b.

CYP153, one of the most common medium-chain n-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed in n-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading various n-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrum n-alkane-degrading Gram-positive bacterium, Dietzia sp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the -10 and -35 regions in Actinobacteria. Further analysis of the ß-galactosidase activity in the CYP153 gene promoter-lacZ fusion cell indicated that the CYP153 gene promoter was induced by n-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed a cypR mutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence of n-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements in Actinobacteria and Proteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by many Actinobacteria.

Regulation of alkane degradation pathway by a TetR family repressor via an autoregulation positive feedback mechanism in a Gram-positive Dietzia bacterium.

n-Alkanes are ubiquitous in nature and serve as important carbon sources for both Gram-positive and Gram-negative bacteria. Hydroxylation of n-alkanes by alkane monooxygenases is the first and most critical step in n-alkane metabolism. However, regulation of alkane degradation genes in Gram-positive bacteria remains poorly characterized. We therefore explored the transcriptional regulation of an alkB-type alkane hydroxylase-rubredoxin fusion gene, alkW1, from Dietzia sp. DQ12-45-1b. The alkW1 promoter was characterized and so was the putative TetR family regulator, AlkX, located downstream of alkW1 gene. We further identified an unusually long 48 bp inverted repeat upstream of alkW1 and demonstrated the binding of AlkX to this operator. Analytical ultracentrifugation and microcalorimetric results indicated that AlkX formed stable dimers in solution and two dimers bound to one operator in a positive cooperative fashion characterized by a Hill coefficient of 1.64 (± 0.03) [k(D) = 1.06 (± 0.16) µM, k(D) ' = 0.05 (± 0.01) µM]. However, the DNA-binding affinity was disrupted in the presence of long-chain fatty acids (C10-C24), suggesting that AlkX can sense the concentrations of n-alkane degradation metabolites. A model was therefore proposed where AlkX controls alkW1 expression in a metabolite-dependent manner. Bioinformatic analysis revealed that the alkane hydroxylase gene regulation mechanism may be common among Actinobacteria.

Diverse alkane hydroxylase genes in microorganisms and environments.

AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature.

Characterization of a CYP153 alkane hydroxylase gene in a Gram-positive Dietzia sp. DQ12-45-1b and its "team role" with alkW1 in alkane degradation.

CYP153 and AlkB-like hydroxylases were recently discovered in Gram-positive alkane-degrading bacteria. However, it is unclear whether they cooperate with each other in alkane degradation as they do in Gram-negative bacteria. In this paper, we cloned the CYP153 gene from a representative Gram-positive alkane-degrading bacterium, Dietzia sp. DQ12-45-1b. The CYP153 gene transcription in Dietzia sp. DQ12-45-1b and heterologous expression in alkB gene knockout mutant strain Pseudomonas fluorescens KOB2∆1 both confirmed the functions of CYP153 on C6-C10 n-alkanes degradation, but not on longer chain-length n-alkanes. In addition, substrate-binding analysis of the purified CYP153 protein revealed different substrate affinities to C6-C16 n-alkanes, confirming n-alkanes binding to CYP153 protein. Along with AlkW1, an AlkB-like alkane hydroxylase in Dietzia sp. DQ12-45-1b, a teamwork pattern was found in n-alkane degradation, i.e. CYP153 was responsible for hydroxylating n-alkanes shorter than C10 while AlkW1 was responsible for those longer than C14. Further sequence analysis suggested that the high horizontal gene transfer (HGT) potential of CYP153 genes may be universal in Gram-positive alkane-degrading actinomycetes that contain both alkB and CYP153 genes.