Uncultured Soil Bacteria Research Articles

An antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Discovery, synthesis, and optimization of teixobactin, a novel antibiotic without detectable bacterial resistance.

Discovering new antibiotics with novel chemical scaffolds and antibacterial mechanisms presents a challenge for medicinal scientists worldwide as the ever-increasing bacterial resistance poses a serious threat to human health. A new cyclic peptide-based antibiotic termed teixobactin was discovered from a screen of uncultured soil bacteria through iChip technology in 2015. Teixobactin exhibits excellent antibacterial activity against all the tested gram-positive pathogens and Mycobacterium tuberculosis, including drug-resistant strains. Given that teixobactin targets the highly conserved lipid II and lipid III, which induces the simultaneous inhibition of both peptidoglycan and teichoic acid synthesis, the emergence of resistance is considered to be rather difficult. The novel structure, potent antibacterial activity, and highly conservative targets make teixobactin a promising lead compound for further antibiotic development. This review provides a comprehensive treatise on the advances of teixobactin in the areas of discovery processes, antibacterial activity, mechanisms of action, chemical synthesis, and structural optimizations. The synthetic methods for the key building block l-allo-End, natural teixobactin, representative teixobactin analogs, as well as the structure-activity relationship studies will be highlighted and discussed in details. Finally, some insights into new trends for the generation of novel teixobactin analogs and tips for future work and directions will be commented.

Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

Biodiversity and Dynamics (Rate of Change) of Bacterial Communities Involved in the Biodegradation of Petroleum Refinery Sludge in Contaminated Soils

Aim: The study assessed the bio treatability of the petroleum refinery sludge in contaminated soils by indigenous bacterial communities and the effects of the sludge contamination and bio stimulants on the biodiversity and dynamics (rate of change) of the bacterial communities involved in the biodegradation of the sludge, using the molecular biology technique, Denaturing Gradient Gel Electrophoresis (DGGE).
 Study Design: The randomnized block design was used for the study.
 Place and duration of the Study: The research was conducted in the biology laboratory of Flinders University, Adelaide, South Australia.
 Methodology: The percentage of total petroleum hydrocarbons (TPH) degraded and the bacterial load in the test microcosms was assessed tri-weekly for 12 weeks. The percentage TPH was assessed using Gas chromatography, while the bacterial count was determined as gene copies using the culture independent molecular tool, quantitative real-time PCR (qRt-PCR) analysis. The effects of the experimental treatments on the biodiversity and dynamics (rate of change) of the bacterial communities involved in the biodegradation of the sludge in the soils was determined by the culture-independent molecular biology technique, DGGE. Moving Windows Analysis (MWA) and Shannon Weaver diversity index were used to determine the dynamics (rate of change) and biodiversity of the bacterial communities respectively.
 Results: Results obtained for the Moving Window Analysis (MWA) which is used to determine the dynamics (Dy), or rate of change of the bacterial communities, showed that, the 1% and 5% sludge contaminated soils biostimulated with compost, recorded the highest Dy of 86.0 ± 1.90% and 87.0 ± 2.20% respectively.NPK biostimilated soil microcosms however recorded a lower Dy of 33.75± 3.20 and 32.50 ± 4.68% for 1% and 5% sludge contamination respectively. The biodiversity of the bacterial communities expressed as Shannon -Weaver index (H1), recorded the highest value of 2.76 ±0.02 for the compost biostimulated microcosm in the 1% sludge treatment, while for the 5% sludge contamination, the treatment with NPK and surfactant enhanced the bacterial biodiversity most with a value of 2.76 ±0.07%. In the test soils with 1% sludge contamination, bio stimulation with NPK gave the highest % TPH degradation (78.25%) while the treatment with NPK and Triton-X 100 had the highest TPH degradation (46.55%) for the 5% sludge contaminated soils. There was insignificant difference in the % sludge degradation between the control and other treatments at P > 0.05 and F = 4.07 for the 1% sludge treated soils, while for the soils treated with 5% sludge there was significant difference between the control and other treatments at P < 0.05 and F= 4.07.
 Conclusion: Bacteria species identified in the sludge by molecular biology techniques included; Pseudomonas sp. ITRI77, Uncultured Thauera sp., Uncultured Pseudomonas sp., Flavobacterium sp., Bacillaceae bacterium, Uncultured soil bacterium, Clostridium sp., most of which are Gram negative. Biostimulation with compost enhanced a higher biodiversity (H i) and dynamics (Dy) of the bacterial communities involved in the biodegradation of the sludge. Though the NPK treated soils enhanced the biodegradation of the sludge most, degradation started declining by the 9th week while that of compost continued to rise steadily till the 12th week. Results obtained indicate that compost is as good as NPK in the biodegradation of petroleum sludge especially at 1% sludge contamination, since there was no statistical difference between the % TPH degraded and the use of compost is environmentally friendly and economically sustainable.

Phylogenetic analysis of hydrocarbon degrading bacteria associated with crude oil polluted soil from Mesogar community, Delta State, Nigeria

This study was carried out to isolate hydrocarbon-degrading bacteria associated with oil polluted soil samples collected from Mesogar community of Delta State, Nigeria. The samples were aseptically collected and the bacteria isolated according to standard microbiological techniques. The isolates with hydrocarbon biodegradative ability were screened on MSM supplemented with 2% crude oil using spectrophotometric method. The amount of crude oil degraded by the highest hydrocarbon degrader was determined using gas chromatographic (GC) assay. A total of seven bacterial isolates were molecularly identified using the 16S rRNA gene sequencing method. The sequences were compared to those deposited in NCBI using the basic local alignment tool (BLAST) algorithm. Phylogenetic analysis of 16S rRNA gene sequences was carried out to determine the evolutionary relationships of the isolated bacterial species. The isolates were identified to have remote similarities with Alcaligenes faecalis SH179a, Alcaligenes faecalis subsp. Phenolicus, Bacillus thuringiensis serovar konkukian, Ochrobactrum anthropi, Alcaligenes faecalis SH179b, Uncultured soil bacterium clone and Alcaligenes faecalis IVN45. Strain OFBR 4 had the highest degrading ability. The use of molecular methods for rapid and accurate detection of diverse strains of hydrocarbon-degraders is of utmost necessity in bioremediation.
 Keywords: Phylogenetic analysis, oil polluted soil, biodegradative, hydrocarbon degrader, 16S rRNA gene and crude oil.

Structures of teixobactin-producing nonribosomal peptide synthetase condensation and adenylation domains.

The recently discovered antibiotic teixobactin is produced by uncultured soil bacteria. The antibiotic inhibits cell wall synthesis of Gram-positive bacteria by binding to precursors of cell wall building blocks, and therefore it is thought to be less vulnerable to development of resistance. Teixobactin is synthesized by two nonribosomal peptide synthetases (NRPSs), encoded by txo1 and txo2 genes. Like other NRPSs, the Txo1 and Txo2 synthetases are large, multifunctional, and comprised of several modules. Each module is responsible for catalysis of a distinct step of teixobactin synthesis and contains specific functional units, commonly including a condensation (C) domain, an adenylation (A) domain, and a peptidyl carrier protein (PCP) domain. Here we report the structures of the C-A bidomains of the two L-Ser condensing modules, from Txo1 and Txo2, respectively. In the structure of the C domain of the L-Ser subunit of Txo1, a large conformational change is observed, featuring an outward swing of its N-terminal α-helix. This repositioning, if functionally validated, provides the necessary conformational change for the condensation reaction in C domain, and likely represents a regulatory mechanism. In an Acore subdomain, a well-coordinated Mg2+ cation is observed, which is required in the adenylation reaction. The Mg2+-binding site is defined by a largely conserved amino acid sequence motif and is coordinated by the α-phosphate group of AMP (or ATP) when present, providing some structural evidence for the role of the metal cation in the catalysis of A domain.

Development of a novel cultivation technique for uncultured soil bacteria

In this study, a new diffusion bioreactor was developed to cultivate hidden bacterial communities in their natural environment. The newly developed method was investigated to cultivate microbial communities from the forest soil, and the results were evaluated against traditional culture methods and compared to the results of a pyrosequencing-based molecular survey. The molecular analysis revealed that a diverse bacterial population was present in the soil sample. However, both the newly developed method and the traditional method recovered more than 400 isolates, which belonged to only four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Although these isolates were distributed over only four major phyla, the use of the newly developed technique resulted in the successful cultivation of 35 previously uncultured strains, whereas no such strains were successfully cultivated by the traditional method. Furthermore, the study also found that the recovery of uncultured bacteria and novel isolates was related to sampling season, incubation period, and cultivation media. The use of soil collected in summer, a prolonged incubation period, and low-substrate modified media increased the recovery of uncultured and novel isolates. Overall, the results indicate that the newly designed diffusion bioreactor can mimic the natural environment, which permits the cultivation of previously uncultured bacteria.

Experimental Setup for a Diffusion Bioreactor to Isolate Unculturable Soil Bacteria.

Unculturable bacteria are those bacteria which proliferate in their native habitat but unable to grow or thrive in the normal laboratory media and conditions. The molecular techniques have revealed the significance of these uncultured bacteria in terms of their functional diversity and potential to produce secondary metabolites. To achieve these benefits, scientists have attempted to isolate and cultivate unculturable bacteria in the laboratory using transwell plates, optical tweezers, laser microdissection, microbioreactors, and diffusions bioreactors. However, these techniques are still inadequate to resolve the difficulties of cultivating unculturable bacteria. Therefore, it is essential to develop new cultivation method that enables growth of diverse range of bacteria in the laboratory conditions. Diffusion bioreactor is a membrane bound chamber which allows microbes to proliferate in their native environment by providing the excess to naturally occurring nutrients and signaling compounds. This paper presents efficient and reliable protocol to construct a diffusion bioreactor and its utilization to isolate and cultivate unculturable soil bacteria in laboratory.

Effective Soil Extraction Method for Cultivating Previously Uncultured Soil Bacteria.

Here, a new medium, named intensive soil extract medium (ISEM), based on new soil extract (NSE) using 80% methanol, was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n = 258), respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts made using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological, and functional properties and even roles of unknown genes.IMPORTANCE Both metagenomics and single-cell sequencing can detect unknown genes from uncultured microbial strains in environments, and either method may find the significant potential metabolites and roles of these strains. However, such gene/genome-based techniques do not allow detailed investigations that are possible with cultures. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture.

Profiling of heavy metal(loid)-resistant bacterial community structure by metagenomic-DNA fingerprinting using PCR–DGGE for monitoring and bioremediation of contaminated environment

Frequent exposure of microbes to hazardous metalloids/heavy metals in contaminated environment results in the development of heavy metal(loid)-resistance properties. The study attempted to assess the profile of elevated arsenic (As), cadmium (Cd) and mercury (Hg)—resistant bacterial community structures of sludge (S1, India), sludge and sediment (S2 and S3, Japan) and sediment (S4, Vietnam) samples by metagenomic-DNA fingerprinting using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) for monitoring and bioremediation of hazardous metal(loid) contamination in environment. The results revealed that As-resistant bacteria were dominant compared to Cd- and Hg-resistant bacteria with higher species diversity (Lysinibacillus sp., Uncultured soil bacterium clone, Staphylococcus sciuri, Bacillus fastidiosus, Bacillus niacini, Clostridium sp. and Bacillus sp.) in S1 and S4 than that of S2 and S3 samples. The occurrence of dominant As-resistant bacteria may indicate arsenic contamination in the investigated coastal habitats of India, Japan and Vietnam. The As-, Cd- and Hg-resistant bacteria/bacterial consortiums showed appreciable uptake ability of respective metal(loid) (0.042–0.125 mg As/l, 0.696–0.726 mg Cd/l and 0.34–0.412 mg Hg/l). Therefore, it might be concluded that the profiling of metalloids/heavy metal-resistant bacterial community structure by metagenomic-DNA fingerprinting using PCR–DGGE could be used to explore high metal(loid)-resistant bacteria for applying in metal(loid) bioremediation and as an indicator for monitoring hazardous metal(loid) contamination in environment.

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Effect of chromium (VI) on the multiple nitrogen removal pathways and microbial community of aerobic granular sludge

ABSTRACTThe frequent appearance of Cr(VI) significantly impacts the microbial metabolism in wastewater. In this study, long-term effects of Cr(VI) on microbial community, nitrogen removal pathways and mechanism of aerobic granular sludge (AGS) were investigated. AGS had strong resistance ability to 1.0 mg/L Cr(VI). 3.0 mg/L Cr(VI) increased the heterotrophic-specific ammonia uptake rate (HSAUR) and heterotrophic-specific nitrate uptake rate (HSNUR) transiently, whereas 5.0 mg/L Cr(VI) sharply decreased the specific ammonia uptake rate (SAUR), specific nitrate uptake rate (SNUR) and simultaneous nitrification denitrification rate (SNDR). It was found that Cr (VI) has a greater inhibitory effect on autotrophic nitrification (ASAUR), and the maximal inhibition rate (IR) was 139.19%. Besides, the inhibition of Cr (VI) on nitrogen removal process belongs to non-competitive inhibition. Cr(VI) had a weaker negative impact on heterotrophic bacteria compared with that on autotrophic bacteria. Denaturing gradient gel electrophoresis analyses suggest that Acidovorax sp., flavobacterium sp., uncultured soil bacterium, uncultured nitrosospira sp., uncultured prokaryote, uncultured β-proteobacterium and uncultured pseudomonas sp. were the dominant species. The inhibition of Cr(VI) on nitrite-oxidizing bacteria was the strongest, followed by ammonia-oxidizing bacteria and denitrifying bacteria. Linear correlations between bacterial count and biomass-specific uptake rate were observed when the Cr(VI) concentration exceeded 3 mg/L. This study revealed the effect of Cr(VI) on nitrification is more serious than that on denitrification. Autotrophic and heterotrophic nitrification, heterotrophic denitrification and simultaneous nitrification denitrification played a significant role on nitrogen removal under Cr(VI) stress.

Characterization of Uncultured Genome Fragment from Soil Metagenomic Library Exposed Rare Mismatch of Internal Tetranucleotide Frequency.

Exploring the genomic information of a specific uncultured soil bacterium is vital to understand its function in the ecosystem but is still a challenge due to the lack of culture techniques. To examine the genomes of uncultured bacteria, a metagenomic bacterial artificial chromosome library derived from a soil sample was screened for 16S rDNA-containing clones. Five clones (4C6, 5E7, 5G4, 5G12, and 5H7) containing uncultured soil bacteria genome fragment (with low 16S rDNA similarity to isolated bacteria) were selected for sequencing. Clone 5E7 and 5G4 showed only 82 and 83% of 16S rDNA identity to known sequences. Phylogenetic analysis of 16S rDNA indicated that 5E7 and 5G4 were potentially from new class of Chloroflexi. Only one-third of the 5G4 open reading frames have significant hits against HMMER. Internal tetranucleotide frequency analysis indicated that the unknown region of 5G4 was poorly correlated with other parts of the clone, indicating that this section might be obtained through lateral transfer. It was suggested that this region rich for unknown genes is under fast evolution.

Looking at the 99% in nature to stop drug-resistant bacteria

New antibiotics without detectable resistance were discovered in unculturable soil bacteria.

Antibacterial drugs: a new drug for resistant bugs.

ba st ia n K au lit zk i/ A la m y The paucity of novel antimicrobial compounds in the drug discovery pipeline is one of the main hurdles to combatting the current antibiotic resistance crisis. Now, Lewis and colleagues report the discovery of the new antibiotic teixobactin, which has a completely novel mechanism of action and does not seem to select for resistance. Most antibiotics in clinical use were discovered by screening cultivable soil microorganisms, but this resource is limited and alternative synthetic approaches have had little success. As up to 99% of environmental bacteria are uncultured and are a potentially untapped reservoir of novel compounds, the authors developed a high-throughput, multichannel device called the iChip to screen previously uncultured soil bacteria. This approach involves isolating an individual bacterial cell in one of 96 chambers that is surrounded by a semi-permeable membrane; bacteria can then be grown in situ as they have access to the nurients and growth factors present in their natural environment. The authors examined the antimicrobial activity of extracts obtained from 10,000 soil bacteria and found that an extract from a new species of the Gram-negative β-proteobacteria, provisionally named Eleftheria terrae, showed promising inhibitory activity against the Gram-positive pathogen Staphylococcus aureus. Structural analysis showed that the antimicrobial compound, termed teixobactin, has a unique chemical scaffold and thus represents a completely new antibiotic class. In addition to S. aureus, teixobactin is a potent inhibitor of other drug-resistant Gram-positive pathogens, including Mycobacterium tuberculosis, Clostridium difficile and Bacillus anthracis, but is ineffective against most Gram-negative bacteria. Moreover, teixobactin showed promising in vivo activity, resulting in a substantial reduction in bacterial burden in mice infected with methicillin-resistant S. aureus or Streptococcus pneumoniae. Further experiments revealed that the antibiotic inhibits peptidoglycan synthesis by targeting the peptidogycan precursor lipid II and the cell wall teichoic acid precursor lipid III. This explains why teixobactin is ineffective against Gram-negative bacteria, as they lack teichoic acids and have an outer membrane that shields peptidoglycan. Importantly, the authors were unable to isolate teixobactin-resistant mutants, probably due to the fact that the drug has two targets, neither of which is a protein. The targeting of lipids rather than proteins is less likely to select for resistance as protein-encoding genes can evolve mutations that block the binding of antibiotics more readily than the emergence of lipid variants that preclude antibiotic binding. Whether resistance mechanisms already exist in nature is unclear, but as the producer of teixobactin is a Gramnegative species and is therefore intrinsically resistant, it is unlikely to encode a transferrable resistance mechanism for Gram-positive species. This study is a proof-of-principle of the value of natural product discovery for identifying promising drug candidates, and the authors conclude that similar compounds with a low propensity to select for resistance await to be discovered.

Isolation of Paenibacillus pinesoli sp. nov. from forest soil in Gyeonggi-Do, Korea

Using a new culture method for unculturable soil bacteria, strain NB5(T) was isolated from forest soil at Kyonggi University, and characterized taxonomically on the basis of 16S rRNA gene sequence as well as phenotypic and chemotaxonomic characteristics. The novel strain was a Gram- and catalase-positive, rod-shaped bacterium, which grew in the pH range 6.0-9.5 (optimum, 6.5-9.5) and at temperatures between 15°C and 45°C (optimum, 25-40°C). Growth was possible at 0-5% NaCl (optimum, 0% to 3%) in nutrient, Luria-Bertani, and trypticase soy broths (TSB), as well as R2A medium (with optimal growth in TSB). A phylogenetic analysis of the 16S rRNA gene sequence showed that the novel strain was affiliated with the genus Paenibacillus and had 96.8% and 96.5% similarity to P. nanensis MX2-3(T) and P. agaridevorans DSM 1355(T), respectively. The predominant menaquinone in NB5(T) was MK-7; the major fatty acids were anteiso-C15:0 and iso-C16:0; and the DNA G+C content was 54.5 mol%. We propose this strain as a novel species of the genus Paenibacillus, and suggest the name Paenibacillus pinesoli sp. nov. (type strain, KACC 17472(T)=KEMB 9005-025(T)=JCM 19203(T)).

A Novel Mercuric Reductase from the Unique Deep Brine Environment of Atlantis II in the Red Sea

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg(2+), and efficiently detoxifies Hg(2+) in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

Bacillus thaonhiensis sp. nov., a New Species, was Isolated from the Forest Soil of Kyonggi University by Using a Modified Culture Method

Using a new culture method for unculturable soil bacteria, we discovered a novel species, NHI-38(T), from the forest soil of Kyonggi University campus, South Korea. It was a Gram-positive, rod-shaped, and endospore-forming bacterial strain. It grew over a wide pH range (6.5-9.5), with an optimum range of pH 7-9, and in a wide range of temperatures (15-60°C), with an optimum range of 35-45°C. Growth was possible at 0-2% NaCl concentration, and the optimal range was between 0.5 and 1.5% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that this new species clustered within the genus Bacillus; it was closely related to "Bacillus abyssalis" SCSIO 15042(T) (98.86%), B. methanolicus NCIMB 13113(T) (95.97%), B. vietnamensis 15-1(T) (95.8%), B. seohaeanensis BH724(T) (95.5%), B. timonensis MM10403188(T) (95.33%), and B. subtilis subsp. subtilis NCIB 3610(T) (94.87%). The main fatty acid components of this bacterium were iso-C15:0 (35.92%), summed feature 3 (C16:1ω7c/C16:1ω6c; 16.92%), and anteiso-C15:0 (14.19%). The predominant quinone in this bacterial strain was MK-7. The polar lipid profile primarily comprised phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The genomic DNA G+C composition of the isolate was 40.7mol%. The DNA-DNA hybridization results indicated that this strain was distinct from other Bacillus species, the degree of similarity being 50% with "B. abyssalis", 56% with B. methanolicus, 47% with B. vietnamensis, 43% with B. seohaeanensis, 46% with B. timonensis, and 32% with B. subtilis. Based on our results, we regard strain NHI-38(T) as a novel member of the Bacillus genus, and we propose the name Bacillus thaonhiensis (=KACC 17216(T)=KEMB 9005-019(T)=JCM 18863(T)).

Biotreatment of heavy oil wastewater by combined upflow anaerobic sludge blanket and immobilized biological aerated filter in a pilot-scale test

A field pilot study has been constructed in the Liaohe oilfield, China to treat heavy oil wastewater with large amounts of dissolved recalcitrant organic compounds and low nutrient of nitrogen and phosphorus by an upflow anaerobic sludge blanket (UASB) coupled with immobilized biological aerated filters (I-BAFs). By operating the system for 252 days (including the start-up of 128 days), the chemical oxygen demand (COD), ammonia nitrogen (NH3-N) and suspended solid (SS) in the wastewater were removed by 74%, 94% and 98%, respectively. GC–MS analysis indicated that most of alkanes were degraded by the UASB process, while the I-BAF played important roles both in degrading organic compounds and in removing the NH3-N and SS. The bacterial community structural analysis based on the PCR-DGGE technology reveals that the predominant bacteria in the UASB reactor belong to the Bacillales and Rhodobacterales, and that in the I-BAF was identified as uncultured soil bacterium. Our results suggest that the combined biotreatment system has immense potential in large-scale treatment of heavy oil wastewater.

Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil

Ring-hydroxylating dioxygenases (RHDs) catalyze the initial oxidation step of a range of aromatic hydrocarbons including polycyclic aromatic hydrocarbons (PAHs). As such, they play a key role in the bacterial degradation of these pollutants in soil. Several polymerase chain reaction (PCR)-based methods have been implemented to assess the diversity of RHDs in soil, allowing limited sequence-based predictions on RHD function. In the present study, we developed a method for the isolation of PAH-specific RHD gene sequences of Gram-negative bacteria, and for analysis of their catalytic function. The genomic DNA of soil PAH degraders was labeled in situ by stable isotope probing, then used to PCR amplify sequences specifying the catalytic domain of RHDs. Sequences obtained fell into five clusters phylogenetically linked to RHDs from either Sphingomonadales or Burkholderiales. However, two clusters comprised sequences distantly related to known RHDs. Some of these sequences were cloned in-frame in place of the corresponding region of the phnAIa gene from Sphingomonas CHY-1 to generate hybrid genes, which were expressed in Escherichia. coli as chimerical enzyme complexes. Some of the RHD chimeras were found to be competent in the oxidation of two- and three-ring PAHs, but other appeared unstable. Our data are interpreted in structural terms based on 3D modeling of the catalytic subunit of hybrid RHDs. The strategy described herein might be useful for exploring the catalytic potential of the soil metagenome and recruit RHDs with new activities from uncultured soil bacteria.