Molecular analysis of a consortium of ruminal microbes that detoxify pyrrolizidine alkaloids

Members of a consortium of bacteria, isolated from the rumen of sheep, that degrades pyrrolizidine alkaloids (PAs) found in tansy ragwort (Senecio jacobaea) were characterized. An enrichment of ruminal bacteria was isolated from a sample of ruminal fluid using standard anaerobic techniques. The PA degradative capacity of the enrichment was tested by spiking purified PA extract from tansy ragwort. Length heterogeneity analysis by PCR (LH-PCR) and restriction fragment length polymorphism (RFLP) analysis was used to identify members of the consortium. Phylogenetic analysis of the 16S rDNA gene revealed differing results based on the molecular method used. LH-PCR identified 7 different organisms in 3 groups while RFLP identified 6 organisms with differing banding patterns in 5 groups. After the phylogenetic analyses of both methods were combined, the combined isolates represented 6 groups. The majority of the members of this consortium are <97.0% homologous with known bacteria, indicating this consortium may contain novel organisms able to detoxify PAs found in tansy ragwort. Further understanding of the metabolic pathways used by this consortium to degrade PAs could lead to the use of the consortium as a probiotic therapy for livestock and horses afflicted with tansy ragwort toxicosis.     

DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.     

Continuous Enrichment Culture and Molecular Monitoring to Investigate the Microbial Diversity of Thermophiles Inhabiting Deep-Sea Hydrothermal Ecosystems

The microflora developing during a continuous enrichment culture from a hydrothermal chimney sample was investigated by molecular methods. The culture was performed in a gas-lift bioreactor under anaerobic conditions, at 90°C and pH 6.5, on a complex medium containing sulfur as the terminal electron acceptor. Archaeal and bacterial diversity was studied. Microorganisms affiliated with the genera Pyrococcus, Marinitoga, and Bacillus were detected through DGGE analysis of 16S rDNA. Additional sequences phylogenetically related to Thermococcus and -Proteobacteria were detected by cloning and sequencing of 16S rDNA from two samples of the enrichment culture. In comparison, the sequences retrieved from cloning analysis from an enrichment culture performed in a flask (batch condition) using the same culture medium showed that only members of the genus Thermococcus were cultivated. Therefore, continuous enrichment culture using the gas-lift bioreactor can be considered as an efficient and improved method for investigating microbial communities originating from deep-sea hydrothermal vents.     

Management practices for control of ragwort species

The ragwort species common or tansy ragwort (Jacobaea vulgaris, formerly Senecio jacobaea), marsh ragwort (S. aquaticus), Oxford ragwort (S. squalidus) and hoary ragwort (S. erucifolius) are native in Europe, but invaded North America, Australia and New Zealand as weeds. The abundance of ragwort species is increasing in west-and central Europe. Ragwort species contain different groups of secondary plant compounds defending them against generalist herbivores, contributing to their success as weeds. They are mainly known for containing pyrrolizidine alkaloids, which are toxic to grazing cattle and other livestock causing considerable losses to agricultural revenue. Consequently, control of ragwort is obligatory by law in the UK, Ireland and Australia. Commonly used management practices to control ragwort include mechanical removal, grazing, pasture management, biological control and chemical control. In this review the biology of ragwort species is shortly described and the different management practices are discussed.     

Anaerobic degradation of p-xylene in sediment-free sulfate-reducing enrichment culture

Anaerobic degradation of p-xylene was studied with sulfate-reducing enrichment culture. The enrichment culture was established with sediment-free sulfate-reducing consortium on crude oil. The crude oil-degrading consortium prepared with marine sediment revealed that toluene, and xylenes among the fraction of alkylbenzene in the crude oil were consumed during the incubation. The PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene for the p-xylene degrading sulfate-reducing enrichment culture showed the presence of the single dominant DGGE band pXy-K-13 coupled with p-xylene consumption and sulfide production. Sequence analysis of the DGGE band revealed a close relationship between DGGE band pXy-K-13 and the previously described marine sulfate-reducing strain oXyS1 (similarity value, 99%), which grow anaerobically with o-xylene. These results suggest that microorganism corresponding to pXy-K-13 is an important sulfate-reducing bacterium to degrade p-xylene in the enrichment culture.     

Electricity generation coupled to oxidation of propionate in a microbial fuel cell

Propionate was used as fuel to enrich an electrochemically-active microbial consortium in a microbial fuel cell, and the bacterial consortium was analyzed by culture-independent methods including denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA, and by fluorescent in situ hybridization (FISH). MFCs fed with propionate produced a current of 4.88 ± 0.1 mA stably on 100 mg propionate/l as COD within 3 weeks of the enrichment. When the MFCs were fed with H₂-saturated fuel containing propionate, the current dropped to 3.82 ± 0.07 mA. The maximum current generated was up to 8.8 mA when MFCs were fed with 200 mg propionate/l as COD. The DGGE of 16S rDNA showed that propionate-enriched MFCs have a different bacterial population from that enriched with acetate and from the inoculum used for enrichment. The major member (42%) of the consortium was an unidentified bacterium followed by γ, β, and δ-proteobacteria.     

Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype

Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, β- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

     

Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.     

The differential impact of a native and a non‐native ragwort species (Senecioneae) on the first and second trophic level of the rhizosphere food web

Whereas the impact of exotic plant species on above‐ground biota is relatively well‐documented, far less is known about the effects of non‐indigenous plants on the first and second trophic level of the rhizosphere food web. Here, rhizosphere communities of the invasive narrow‐leaved ragwort Senecio inaequidens and the native tansy ragwort Jacobaea vulgaris, co‐occurring in three semi‐natural habitats are compared. For both species, two life stages were taken into consideration. Quantitative PCR assays for the analyses of bacterial and fungal communities at a high taxonomic level were optimized, and it was investigated whether changes in the primary decomposer community were translated in alterations in bacterivorous and fungivorous nematode communities. In contrast to J. vulgaris, small but significant reductions were observed for Actinobacteria and Bacteroidetes (both p < 0.05) in case of the invasive S. inaequidens. More pronounced changes were detected for the overall nematode community density, and, more specifically, for the bacterivorous genus Anaplectus and the family Monhysteridae (both p < 0.05), as well as the necromenic Pristionchus (p < 0.001). At high taxonomic level, no differences were observed in fungal rhizosphere communities between native and non‐native ragwort species. The impact of plant developmental stages on rhizosphere biota was prominent. The overall bacterial and fungal biomasses, as well as a remarkably consistent set of constituents (Actinobacteria, α‐ and β‐Proteobacteria and Bacteroidetes) were negatively affected by plant stage for both ragwort species. Although later developmental stages of plants generally coincided with lower levels for individual nematode taxa, densities of the fungivorous genera Diphtherophora and Tylolaimophorus remain unaltered. Hence, even at a high taxonomic level, differential effects of native and non‐native ragwort could be pinpointed. However, plant developmental stage has a more prominent impact and this impact was similar in nature for both native and non‐native ragwort species.     

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines, and feces of earthworms

The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing Gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other straisn, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.     

Coamplification of Eukaryotic DNA with 16S rRNA Gene-Based PCR Primers: Possible Consequences for Population Fingerprinting of Complex Microbial Communities

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V₃, V₃-V₅, and V₆-V₈ hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V₃ and V₃-V₅ primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.     

Bacterial community structure and bamA gene diversity in anaerobic degradation of toluene and benzoate under denitrifying conditions

Aim: To characterize the microbial community structure and bamA gene diversity involved in anaerobic degradation of toluene and benzoate under denitrifying conditions. Methods and Results: Nitrate‐reducing enrichment cultures were established on either toluene, benzoate or without additional substrate. Bacterial community structures were characterized by 16S rRNA gene–based PCR‐DGGE analysis. bamA gene diversity was analysed using DGGE and cloning methods. The results showed that bamA gene related to bamA of Thauera chlorobenzoica was dominant in toluene and benzoate cultures. However, a greater diversity of sequences was obtained in benzoate cultures. Low diversity of bamA gene was observed, and some similarities of bamA were also found between active cultures and backgrounds, suggesting that potential natural attenuation of aromatic compounds might occur. Conclusions: The combined analysis of 16S rRNA and bamA genes suggests that the species related to genera Thauera dominated toluene‐ and benzoate‐degrading cultures. The combination of multiple methods (DGGE and cloning) provides a more complete picture of bamA gene diversity. Significance and Impact of the Study: To our knowledge, this is the first report of bamA gene in denitrifying enrichments using DGGE and cloning analysis.     

A novel n-alkane-degrading bacterium as a minor member of p-xylene-degrading sulfate-reducing consortium

A p-xylene-degrading, sulfate-reducing enrichment culture was characterized by analyzing the response of its members to changes in the available substrate. The culture was inoculated into media containing other substrates, resulting in the establishment of benzoate-, acetate-, and lactate-utilizing enrichment cultures. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the enriched cultures targeting 16S rRNA genes showed quite simple band patterns. The predominant band from the benzoate-utilizing enrichment culture was identical to that from the original enrichment culture utilizing p-xylene. A single, dominant DGGE band was observed in common from the acetate- and lactate-utilizing enrichment cultures. A novel sulfate-reducing bacterium, strain PL12, was isolated from the lactate-utilizing enrichment culture. The 16S rRNA gene sequence of strain PL12 was identical to that of the dominant DGGE band in the acetate- and lactate-utilizing enrichment cultures and distinct from the dominant sequences in the original p-xylene-degrading and benzoate-utilizing enrichment cultures. Phylogenetic analysis of the 16S rRNA gene sequences showed that the isolate belonged to the family Desulfobacteraceae in the class Deltaproteobacteria. The isolated strain PL12 could utilize n-hexane and n-decane as substrates, but could not utilize benzoate, p-xylene and other aromatic hydrocarbons. These results suggest that the p-xylene degradation observed in the original enrichment culture was performed by the dominant bacterium corresponding to DGGE band pXy-K-13 (Nakagawa et al. 2008). The novel strain PL12 might have been utilizing metabolites of p-xylene.     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

Bacterial community diversity associated with four marine sponges from the South China Sea based on 16S rDNA-DGGE fingerprinting

Culture-independent 16S rDNA-DGGE fingerprinting and phylogenetic analysis were used to reveal the community structure and diversity of the predominant bacteria associated with the four sponges Stelletta tenui, Halichrondria, Dysidea avara, and Craniella australiensis from the South China Sea for the first time. Sponge total community DNA extracted with a direct grinding disruption based method was used successfully after series dilution for 16S rDNA PCR amplification, which simplifies the current procedure and results in good DGGE banding profiles. 16S rDNA-V3 fragments from 42 individual DGGE bands were sequenced and the detailed corresponding bacteria were found in sponges for the fist time based on BLAST results. The sponge-associated bacteria are sponge host-specific because each of the tested four sponges from the same geographical location has different predominant bacterial diversity. Proteobacteria, e.g. α, β and γ subdivisions, make up the majority of the predominant bacteria in sponges and are perhaps in close symbiotic relationship with sponges. Though similar bacteria with close phylogenetic relationships were found among different sponges, the sponge-associated predominant bacterial community structures differ. Sponge C. australiensis has the greatest bacterial diversity, with the four bacteria phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, followed by the sponge D. avara with the two phyla Proteobacteria and Bacteroidetes, and the sponges S. tenui and Halichrondria with the phylum Proteobacteria. DGGE fingerprint-based analysis should ideally be integrated with band cloning and sequencing, phylogenetic analysis and molecular techniques to obtain precise results in terms of the microbial community and diversity.     

Microbial Diversity in High Arsenic Groundwater in Hetao Basin of Inner Mongolia,China

The microbial diversity and community structure in twenty-one groundwater samples from high arsenic shallow aquifers of Hetao Basin, Inner Mongolia, China was investigated with an integrated approach including polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene phylogenetic analyses. A total of 25 bacterial and 32 archaeal DGGE bands were exercised for sequencing. Phylogenetic analyses showed that the bacterial DGGE bands were dominated by Proteobacteria, and the archaeal bands were dominated by Thaumarchaeota and Euryarchaeota. Based on arsenic concentrations, three samples (corresponding to low, medium, and high level of arsenic, respectively) were selected for construction of 16S rRNA gene clone libraries. A total of 912 (468 and 444 for bacteria and archaea, respectively) 16S rRNA gene clone sequences were obtained and subjected to phylogenetic analyses. The results showed that bacterial communities of these samples were dominated by Acinetobacter, Pseudomonas, Massilia, Dietzia, Planococcus, Brevundimonas, Aquabacterium and Geobacter, and archaeal communities by Nitrosophaera, Thermoprotei and Methanosaeta. The relative abundance of major groups varied as a function of changes in groundwater geochemistry. Acinetobacter, Brevundimonas, Geobacter, Thermoprotei and Methanosaeta dominated in high arsenic samples with high concentrations of methane and Fe(II), and low concentrations of SO^2? ₄ and NO^? ₃, while Pseudomonas and Nitrosophaera were abundant in low arsenic groundwater. These results imply that microbes play an important role in arsenic mobilization in the shallow aquifers of Hetao Basin, Inner Mongolia.     

Community Structure of Actively Growing Bacterial Populations in Plant Pathogen Suppressive Soil

The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil.     

Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat.

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.     

Biodegradation of 4-chlorobenzoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PA01 NC

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.     

Biodegradation of 2-Nitrotoluene by <Emphasis Type="Italic">Micrococcus</Emphasis> sp. strain SMN-1

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.