Biodegradation of MTBE by Achromobacter xylosoxidans MCM1/1 induces synthesis of proteins that may be related to cell survival

The gasoline additive methyl tert-butyl ether (MTBE) can contaminate groundwater and soil. In order to eliminate it, several methods are being developed, among which bioremediation – that is, the addition of microbial cultures that can degrade the compound – holds promise. Our laboratory has identified Achromobacter xylosoxidans MCM1/1 as an MTBE-degrading bacterial strain. It degrades 78% of this chemical in 5 days. In this study we also analyze the effects of MTBE on the biology of A. xylosoxidans MCM1/1 and compare its proteomic profile after incubation with MTBE with that of unchallenged bacteria. The 2D proteomic analysis shows that the following four proteins are induced by MTBE: 50S ribosomal protein L10, amino acid-binding periplasmic protein, ATP synthase and endoribonuclease L. Characterizing the bacterial response to MTBE at the biochemical level identifies proteins that can be used by biocatalysts for soil and water bioremediation.     

Biodegradation of fuel oxygenates and their effect on the expression of a newly identified cytochrome P450 gene in Achromobacter xylosoxidans MCM2/2/1

Achromobacter xylosoxidans MCM2/2/1 was enriched and isolated from gasoline-contaminated soil and was found to degrade ethyl tert-butyl ether (ETBE) and methyl tert-butyl ether (MTBE) by 41.48% and 34.15%, respectively, in 6 days. Furthermore, the effect of MTBE and TBA on the expression of cytochrome P450 (CYP) of A. xylosoxidans MCM2/2/1 was examined. The presence of the CYP gene in this organism was first confirmed by amplification of a putative 350 bp CYP gene fragment followed by identification of the entire gene by genome walking and DNA-sequencing. The identified CYP gene of A. xylosoxidans MCM2/2/1 shares a high similarity of about 88% with the thcB gene of A. xylosoxidans A8. Gene expression studies have shown that the CYP gene is expressed in A. xylosoxidans MCM2/2/1; however, the expression of this gene was altered at different concentrations of MTBE.     

Aerobic MTBE biodegradation in the presence of BTEX by two consortia under batch and semi-batch conditions

This study explores the effect of microbial consortium composition and reactor configuration on methyl tert-butyl ether (MTBE) biodegradation in the presence of benzene, toluene, ethylbenzene and p-xylenes(BTEX). MTBE biodegradation was monitored in the presence and absence of BTEX in duplicate batch reactors inoculated with distinct enrichment cultures: MTBE only (MO—originally enriched on MTBE) and/or MTBE BTEX (MB—originally enriched on MTBE and BTEX). The MO culture was also applied in a semi-batch reactor which received both MTBE and BTEX periodically in fresh medium after allowing cells to settle. The composition of the microbial consortia was explored using a combination of 16S rRNA gene cloning and quantitative polymerase chain reaction targeting the known MTBE-degrading strain PM1^T. MTBE biodegradation was completely inhibited by BTEX in the batch reactors inoculated with the MB culture, and severely retarded in those inoculated with the MO culture (0.18 ± 0.04 mg/L-day). In the semi-batch reactor, however, the MTBE biodegradation rate in the presence of BTEX was almost three times as high as in the batch reactors (0.48 ± 0.2 mg/L-day), but still slower than MTBE biodegradation in the absence of BTEX in the MO-inoculated batch reactors (1.47 ± 0.47 mg/L-day). A long lag phase in MTBE biodegradation was observed in batch reactors inoculated with the MB culture (20 days), but the ultimate rate was comparable to the MO culture (0.95 ± 0.44 mg/L-day). Analysis of the cultures revealed that strain PM1^T concentrations were lower in cultures that successfully biodegraded MTBE in the presence of BTEX. Also, other MTBE degraders, such as Leptothrix sp. and Hydrogenophaga sp. were found in these cultures. These results demonstrate that MTBE bioremediation in the presence of BTEX is feasible, and that culture composition and reactor configuration are key factors.     

<Emphasis Type="Italic">n</Emphasis>-Alkane assimilation and <Emphasis Type="Italic">tert</Emphasis>-butyl alcohol (TBA) oxidation capacity in <Emphasis Type="Italic">Mycobacterium austroafricanum</Emphasis> strains

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C₂ to C₁₆). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C₁ to C₁₆) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.     

Biodegradation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether using bacterial strains

Prospective methyl tert-butyl ether (MTBE) degrading bacterial strains and/or consortia were identified. The potential for aerobic degradation of MTBE was examined using bacterial isolates from contaminated soils and groundwater. Using the 16S rDNA protocol, two isolates capable of degrading MTBE (Rhodococcus pyridinivorans 4A and Achromobacter xylosoxidans 6A) were identified. The most efficient consortium of microorganisms was acquired from contaminated groundwater. The growth of both strains and the consortium on MTBE was supported by various organic substrates, and monitored using Bioscreen®. The biochemical oxygen demand of the cultures was measured using OxiTop®, and their MTBE concentrations were estimated by gas chromatography. After 3 weeks of aerobic cultivation using n-alkanes as cosubstrate, the concentration of MTBE in R. pyridinivorans 4A was reduced to 62.4 % of its initial amount (50 ppm).     

Microbial community analyses of three distinct, liquid cultures that degrade methyl tert-butyl ether using anaerobic metabolism

Methyl tert-butyl ether (MTBE) is a prevalent groundwater contaminant. In this study, three distinct MTBE-degrading, anaerobic cultures were derived from MTBE-contaminated aquifer material: cultures NW1, NW2 and NW3. The electron acceptors used are anthraquinone-2,6-disulfonate (AQDS; NW1), sulfate (NW2) and fumarate (NW3), respectively. About 1–2 mM MTBE is consistently degraded within 20–30 days in each culture. The 16S rDNA-based amplified ribosomal DNA restriction analysis (ARDRA) was used to analyze the microbial community in each culture. Results indicate novel microorganisms (i.e. no closely related known genera or species) catalyze anaerobic MTBE biodegradation, and microbial diversity varied with different electron acceptors. Tert-butyl alcohol (TBA) accumulated to nearly stoichiometric levels, and these cultures will be critical to understanding the factors that influence TBA accumulation versus degradation. The cultures presented here are the first stable anaerobic MTBE-degrading cultures that have been characterized with respect to taxonomy.     

Methyl <Emphasis Type="Italic">tert</Emphasis>-butyl Ether and <Emphasis Type="Italic">tert</Emphasis>-butyl Alcohol Degradation by <Emphasis Type="Italic">Fusarium solani</Emphasis>

Fusarium solani degraded methyl tert-butyl ether (MTBE) and other oxygenated compounds from gasoline including tert-butyl alcohol (TBA). The maximum degradation rate of MTBE was 16 mg protein h and 46 mg/g protein h for TBA. The culture transformed 77% of the total carbon to ¹⁴CO₂. The estimated yield for MTBE was 0.18 g dry wt/g MTBE.     

The mutagenicity testing of tertiary-butyl alcohol, tertiary-butyl acetate™ and methyl tertiary-butyl ether in Salmonella typhimurium

Tertiary-Butyl alcohol (TBA), tertiary-butyl acetate™ (TBAc™) and methyl tertiary-butyl ether (MTBE) are chemicals to which the general public may be exposed either directly or as a result of their metabolism. There is little evidence that they are genotoxic; however, an earlier publication reported that significant results were obtained in Salmonella typhimurium TA102 mutagenicity tests with both TBA and MTBE. We now present results of testing these chemicals and TBAc™ against S. typhimurium strains in two laboratories. The emphasis was placed on testing with S. typhimurium TA102 and the use of both dimethyl sulphoxide and water as vehicles. Dose levels up to 5000 μg/plate were used and incubations were conducted in both the presence and absence of liver S9 prepared from male rats treated with either Arochlor 1254 or phenobarbital-β-naphthoflavone. The experiments were replicated, but in none of them was a significant mutagenic response observed, thus the current evidence indicates the TBA, TBAc™ and MTBE are not mutagenic in bacteria.     

Biodegradation of methyl <Emphasis Type="Italic">t</Emphasis>-butyl ether by aerobic granules under a cosubstrate condition

Aerobic granules efficient at degrading methyl tert-butyl ether (MTBE) with ethanol as a cosubstrate were successfully developed in a well-mixed sequencing batch reactor (SBR). Aerobic granules were first observed about 100 days after reactor startup. Treatment efficiency of MTBE in the reactor during stable operation exceeded 99.9%, and effluent MTBE was in the range of 15–50 μg/L. The specific MTBE degradation rate was observed to increase with increasing MTBE initial concentration from 25 to 500 mg/L, which peaked at 22.7 mg MTBE/g (volatile suspended solids)·h and declined with further increases in MTBE concentration as substrate inhibition effects became significant. Microbial-community deoxyribonucleic acid profiling was carried out using denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S ribosomal ribonucleic acid. The reactor was found to be inhabited by several diverse bacterial species, most notably microorganisms related to the genera Sphingomonas, Methylobacterium, and Hyphomicrobium vulgare. These organisms were previously reported to be associated with MTBE biodegradation. A majority of the bands in the reactor represented a group of organisms belonging to the Flavobacteria–Proteobacteria–Actinobacteridae class of bacteria. This study demonstrates that MTBE can be effectively degraded by aerobic granules under a cosubstrate condition and gives insight into the microorganisms potentially involved in the process.     

Biodegradation of Methyl <Emphasis Type="Italic">tert</Emphasis>-Butyl Ether by Enriched Bacterial Culture

Degradation of methyl tert-butyl ether (MTBE) as a sole carbon and energy source was investigated utilizing an enriched bacterial consortium derived from an old environmental MTBE spill. This enriched culture grew on MTBE with concentration up to 500 mg/l, reducing the MTBE in medium to undetectable concentrations in 23 days. Traces of tert-butyl alcohol were detected during MTBE degradation. The degradation was not affected by additional cobalt ions, whereas low concentration of glucose enhanced the rate of degradation. The bacterial community consisted of numerous bacterial genera, the majority being members of the phylum Acidobacteria and genus Terrimonas. The alkane 1-monooxygenase (alk) gene was detected in this consortium. Our findings suggest that environmental degradation of MTBE proceeds along the previously proposed pathway.     

Enhanced Biodegradation of Methyl tert-butyl-ether by a Microbial Consortium

The widespread use of Methyl tert-butyl-ether (MTBE) as a gasoline additive has resulted in a higher detection rate of MTBE in groundwater systems. Therefore, the researchers show more concern about the bioremediation of MTBE-impacted aquifers. In this paper, a MTBE-direct-degrading bacterial consortium was enriched (named RS1) and further studied. In order to identify the microbial community of the consortium, 17 and 12 different single strains were isolated from nutrient medium and MSM media (with MTBE as the sole carbon source), respectively. 16S rDNA-based phylogenetic analysis revealed that these diverse bacteria belonged to 14 genera, in which Pseudomonas was dominant. Several strains which can grow with MTBE as the sole carbon and energy source were also identified, such as M1, related to MTBE-degrading Arthrobacter sp. ATCC27778. Furthermore, the appropriate addition of certain single strain in consortium RS1 (M1:RS1 = 1:2) facilitates MTBE degradation by increasing the quantity of efficient MTBE-degrading bacteria. This work will provide microbial source and theoretical fundament for further bioremediation of MTBE-contaminated aquifers, which has applied potential and environmental importance.     

饲用微生物的分离鉴定及其对杏鲍菇菌糠发酵的效果

【目的】菌糠的营养素含量齐全,但纤维素含量过高是阻碍其饲料化利用的主要因素。故本研究筛选适合于发酵杏鲍菇菌糠的微生物菌株,以改善其饲用品质。【方法】首先,本研究采用纤维素-刚果红、苯胺蓝和MRS-Ca (De Man, Rogosa, Sharpe-Ca)筛选培养基,结合纤维素、木质素酶活力及抑菌活性的测定,从EM (effective microorganisms)原液发酵的杏鲍菇菌糠中分离筛选具有较强纤维素、木质素降解能力及抑菌能力的细菌/真菌。通过细菌16S rRNA和真菌18S rDNA基因序列分析确定菌株所属种属。其次,将筛选出的菌株菌液等体积混合制成复合菌剂用于固态发酵杏鲍菇菌糠。测定不同发酵时长菌糠营养成分含量以确定最佳发酵时间,并与相同工艺条件下EM原液发酵的杏鲍菇菌糠进行饲用品质比较。【结果】筛选并鉴定得到纤维素酶活性较高的特基拉芽孢杆菌(Bacillus tequilensis)菌株P11、发酵毕赤酵母(Pichia fermentans)菌株R8和马克斯克鲁维应变酵母(Kluyveromyces marxianus)菌株MU5;木质素酶活性较高的解淀粉芽孢杆菌(Bacillus amyloliquefaciens subsp.plantarum)菌株MU7;抑菌活性较高的类肠膜魏斯氏菌(Weissella paramesenteroides)菌株R4和乳酸片球菌(Pediococcus acidilactici)菌株R9。使用以上菌株复合发酵杏鲍菇菌糠7 d后,各项指标达到稳定。与EM原液发酵的杏鲍菇菌糠相比,复合菌剂发酵杏鲍菇菌糠的NDF和ADF分别显著降低了19.6%和21.44%(P0.05);CP (crude protein)、CA (crude ash)和EE (ether extract)含量分别显著提高了10.44%、5.26%和123.53%(P0.05)。【结论】本研究筛选得到的芽孢杆菌、酵母菌和乳酸菌优势菌株复合后用于发酵杏鲍菇菌糠可以很好地改善其饲用品质,效果优于生产中常用市售EM原液。     

The Comparative and Combined Inhibitory Effects of MTBE and TBA on the Activities of Soil Exoenzymes

Methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) are major soil contaminants, and they have been actively investigated for their toxic effects on living organisms in soil ecosystems. Although previous studies have been used as tools to evaluate the health of soil, they have been limited in scope and ability to analyze the overall microbial activity. In the present study, the effects of MTBE and TBA on the activity of soil exoenzymes including urease, acid phosphatase, arylsulfatase, β-glucosidase, dehydrogenase, and fluorescein diacetate hydrolase, which are involved in nutrient cycles and overall microbial activities, were investigated. Soil samples were treated with 0–2% of MTBE and TBA solutions, and the comparative effects and combined effects on quantity of active soil exoenzymes were determined. The activity of six exoenzymes exposed solely to MTBE and TBA did not significantly change with dose concentration or exposure time, but did show significant changes when exposed to high concentrations of MTBE and TBA combined, with dehydrogenase being the most affected. Therefore, we proposed dehydrogenase as a potential biomarker to assess the risk of co-contamination of MTBE and TBA.     

POLYPHASIC STUDY OF ANTARCTIC CYANOBACTERIAL STRAINS1

We isolated 59 strains of cyanobacteria from the benthic microbial mats of 23 Antarctic lakes, from five locations in two regions, in order to characterize their morphological and genotypic diversity. On the basis of their morphology, the cyanobacteria were assigned to 12 species that included four Antarctic endemic taxa. Sequences of the ribosomal RNA gene were determined for 56 strains. In general, the strains closely related at the 16S rRNA gene level belonged to the same morphospecies. Nevertheless, divergences were observed concerning the diversity in terms of species richness, novelty, and geographical distribution. For the 56 strains, 21 operational taxonomic units (OTUs, defined as groups of partial 16S rRNA gene sequences with more than 97.5% similarity) were found, including nine novel and three exclusively Antarctic OTUs. Sequences of Petalonema cf. involvens and Chondrocystis sp. were determined for the first time. The internally transcribed spacer (ITS) between the 16S and the 23S rRNA genes was sequenced for 33 strains, and similar groupings were observed with the 16S rRNA gene and the ITS, even when the strains were derived from different lakes and regions. In addition, 48 strains were screened for antimicrobial and cytotoxic activities, and 17 strains were bioactive against the gram‐positive Staphylococcus aureus, or the fungi Aspergillus fumigatus and Cryptococcus neoformans. The bioactivities were not in coincidence with the phylogenetic relationships, but rather were specific to certain strains.     

新疆乌尔禾地区盐渍土壤耐盐细菌多样性与群落结构研究

研究新疆北部乌尔禾地区盐渍土壤中微生物群落结构及多样性,以期发现新的高盐环境耐盐性微生物资源菌株。采用传统分离培养法获得可培养耐盐菌株并对菌株形态学、16S rRNA基因测序、耐盐特性进行研究,同时结合高通量测序技术分析新疆乌尔禾地区盐渍土壤耐盐细菌的多样性与群落结构。共分离得到耐盐细菌11株,分属6个属,均为中度耐盐菌,以芽胞杆菌属(Bacillus)为优势菌。对盐渍土壤微生物16S rRNA(V3～V4)基因测序,共获得细菌序列290 952条,分属24个门410个属,变形菌门(Proteobacteria, 60.31%)、厚壁菌门(Firmicutes, 21.52%)、拟杆菌门(Bacteroidetes, 6.9%)和放线菌门(Actinobacteria, 6%)相对丰度较高。优势属为克吕沃尔菌属(Kluyvera,21%)、Hafnia-Obesumbacterium(19.6%)和假单胞菌属(Pseudomonas,7.5%)。结果表明,新疆乌尔禾地区盐渍土壤耐盐细菌优势菌群以芽胞杆菌属(Bacillus)居多,细菌群落结构较复杂,潜在可利用微生物资源较为丰富,对高盐极端环境耐盐微生物新资源有进一步研究的意义。     

Pathway,inhibition and regulation of methyl <Emphasis Type="Italic">tertiary</Emphasis> butyl ether oxidation in a filamentous fungus, <Emphasis Type="Italic">Graphium</Emphasis> sp.

The filamentous fungus Graphium sp. (ATCC 58400) co-metabolically oxidizes the gasoline oxygenate methyl tertiary butyl ether (MTBE) after growth on gaseous n-alkanes. In this study, the enzymology and regulation of MTBE oxidation by propane-grown mycelia of Graphium sp. were further investigated and defined. The trends observed during MTBE oxidation closely resembled those described for propane-grown cells of the bacterium Mycobacterium vaccae JOB5. Propane-grown mycelia initially oxidized the majority (∼95%) of MTBE to tertiary butyl formate (TBF), and this ester was biotically hydrolyzed to tertiary butyl alcohol (TBA). However, unlike M. vaccae JOB5, our results collectively suggest that propane-grown mycelia only have a limited capacity to degrade TBA. None of the products of MTBE exerted a physiologically relevant regulatory effect on the rate of MTBE or propane oxidation, and no significant effect of TBA was observed on the rate of TBF hydrolysis. Together, these results suggest that the regulatory effects of MTBE oxidation intermediates proposed for MTBE-degrading organisms such as Mycobacterium austroafricanum are not universally relevant mechanisms for MTBE-degrading organisms. The results of this study are discussed in terms of their impact on our understanding of the diversity of aerobic MTBE-degrading organisms and pathways and enzymes involved in these processes.     

Identification of <Emphasis Type="Italic">tertiary</Emphasis> butyl alcohol (TBA)-utilizing organisms in BioGAC reactors using <Superscript>13</Superscript>C-DNA stable isotope probing

Biodegradation of the gasoline oxygenates methyl tertiary-butyl ether (MTBE) and ethyl tertiary-butyl ether (ETBE) can cause tertiary butyl alcohol (TBA) to accumulate in gasoline-impacted environments. One remediation option for TBA-contaminated groundwater involves oxygenated granulated activated carbon (GAC) reactors that have been self-inoculated by indigenous TBA-degrading microorganisms in ground water extracted from contaminated aquifers. Identification of these organisms is important for understanding the range of TBA-metabolizing organisms in nature and for determining whether self-inoculation of similar reactors is likely to occur at other sites. In this study ¹³C-DNA-stable isotope probing (SIP) was used to identify TBA-utilizing organisms in samples of self-inoculated BioGAC reactors operated at sites in New York and California. Based on 16S rRNA nucleotide sequences, all TBA-utilizing organisms identified were members of the Burkholderiales order of the β-proteobacteria. Organisms similar to Cupriavidus and Methylibium were observed in both reactor samples while organisms similar to Polaromonas and Rhodoferax were unique to the reactor sample from New York. Organisms similar to Hydrogenophaga and Paucibacter strains were only detected in the reactor sample from California. We also analyzed our samples for the presence of several genes previously implicated in TBA oxidation by pure cultures of bacteria. Genes Mpe_B0532, B0541, B0555, and B0561 were all detected in ¹³C-metagenomic DNA from both reactors and deduced amino acid sequences suggested these genes all encode highly conserved enzymes. One gene (Mpe_B0555) encodes a putative phthalate dioxygenase-like enzyme that may be particularly appropriate for determining the potential for TBA oxidation in contaminated environmental samples.     

Rapid discrimination and classification of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> group based on a partial <Emphasis Type="Italic">dnaK</Emphasis> sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.     

An extractive membrane biofilm reactor as alternative technology for the treatment of methyl tert‐butyl ether contaminated water

Among the strategies developed for contaminated groundwater bioremediation, those based on the use of bacteria adhering to inert supports and establishing biofilms have gained great importance in this field. Extractive membrane biofilm reactor (EMBFR) technology offers productive solutions for the removal of volatile and semi‐volatile compounds. EMBFR technology is based on the use of extractive semipermeable membranes through which contaminants migrate to the biological compartment in which microorganisms with pollutant biotransformation and/or mineralization capacities can grow, forming an active biofilm on the membrane surface. The objective of this study was to assess the use of three bacterial strains (Paenibacillus sp. SH7 CECT 8558, Agrobacterium sp. MS2 CECT 8557, and Rhodococcus ruber EE6 CECT 8612), as inoculum in a lab‐scale EMBFR running for 28 days under aerobic conditions to eliminate methyl tert‐butyl ether (MTBE) from water samples. Three different hydraulic retention times (1, 6, and 12 h) were employed. MTBE degradation values were determined daily by a gas GC‐MS technique, as well as suspended bacterial growth. The biofilm established by the bacterial strains on the semipermeable membrane was detected by Field‐Emission Scanning Electron Microscopy (FESEM) at the end of each experiment. The acute toxicity of the treated effluents and biomedium was determined by Microtox^© assay (EC₅₀).The results achieved from the MTBE degradation, biofilm formation, and toxicity analysis indicated that bacterial strains MS2 and EE6 were the best options as selective inoculum, although further research is needed, particularly with regard to their possible use as a mixed culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1238–1245, 2016     

16S–23S rRNA Intergenic Spacer Region Variability in the Genus Frankia

16S–23S rRNA internally transcribed spacer (ITS) sequences from 53 Frankia strains were sequenced and sized from polymerase chain reaction amplification products and compiled with 14 selected 16S–23S ITS sequences from public database. Frankia genomes included two to three ITS copies lacking length polymorphism except for nine strains. No tRNA gene was encountered in this region. Frankia strains exhibited various lengths (369 to 452 nt) and a wide range of sequence similarity (35–100%) in the ITS region. The average pairwise distance varied from 0.368 (clusters 1 and 2) to 0.964 (clusters 3 and 4) and was 0.397, 0.138, 0.129, and 0.016, respectively, for cluster 4 (saprophytic non-infective/non-effective), clusters 1 and 3 (facultative symbiotic), and cluster 2 (obligate symbiotic). This suggests a gradual erosion of Frankia diversity concomitantly with a shift from saprophytic non-infective/non-effective to facultative and symbiotic lifestyle. Comparative sequence analyses of the 16S–23S rRNA intergenic spacer region of Frankia strains are not useful to assign them to their respective cluster or host infection group. Accurate assignment required the inclusion of the adjacent 16S and 23S rRNA gene fragments.