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Applied Microbiology and Biotechnology (v.88, #2)

Antimicrobial properties of nitric oxide and its application in antimicrobial formulations and medical devices by Mitchell Lawrence Jones; Jorge Gabriel Ganopolsky; Alain Labbé; Christopher Wahl; Satya Prakash (pp. 401-407).

This review describes the antimicrobial properties of nitric oxide (NO) and its application as an antimicrobial agent in different formulations and medical devices. We depict the eukaryotic biosynthesis of NO and its physiologic functions as a cell messenger and as an antimicrobial agent of the cell-mediated immune response. We analyze the antimicrobial activity of NO and the eukaryotic protective mechanisms against NO for the purpose of delineating the therapeutic NO dosage range required for an efficacious and safe antimicrobial activity. We also examine the role of NO produced by virulent bacteria in lessening the efficacy of traditional antimicrobials. In addition, we discuss the efficacy of NO in the healing of infected wounds, describing different NO-producing devices by category, analyzing therapeutic levels, duration of NO production, as well as commercial considerations. Finally, we provide current and future prospects for the design and use of NO-producing devices.

Keywords: Nitric oxide; Antimicrobial; Bactericidal; Fungicidal; Bacteria; Infection; Wound healing; Patch; Dressing; Device

Threonine aldolases—screening, properties and applications in the synthesis of non-proteinogenic β-hydroxy-α-amino acids by Nina Dückers; Katrin Baer; Sabine Simon; Harald Gröger; Werner Hummel (pp. 409-424).

Threonine aldolases (TAs) constitute a powerful tool for catalyzing carbon–carbon bond formations in synthetic organic chemistry, thus enabling an enantio- and diastereoselective synthesis of β-hydroxy-α-amino acids. Starting from the achiral precursors glycine and an aldehyde, two new stereogenic centres are formed in this catalytic step. The resulting chiral β-hydroxy-α-amino acid products are important precursors for pharmaceuticals such as thiamphenicol, a l-threo-phenylserine derivative or l-threo-3,4-dihydroxyphenylserine. TAs are pyridoxal-5-phosphate-dependent enzymes, which, in nature, catalyze the cleavage of l-threonine or l-allo-threonine to glycine and acetaldehyde in a glycine biosynthetic pathway. TAs from a broad number of species of bacteria and fungi have been isolated and characterised as biocatalysts for the synthesis of β-hydroxy-α-amino acids. In this review, screening methods to obtain novel TAs, their biological function, biochemical characterisation and preparative biotransformations with TAs are described.

Keywords: Threonine aldolases; Carbon–carbon bond formation; Racemic resolution; Asymmetric synthesis; Phenylserine; Review

Methods in mammalian cell line engineering: from random mutagenesis to sequence-specific approaches by Oliver Krämer; Sandra Klausing; Thomas Noll (pp. 425-436).

Due to the increasing demand for recombinant proteins, the interest in mammalian cell culture, especially of Chinese hamster ovary cells, grows rapidly. This is accompanied by the desire to improve cell lines in order to achieve higher titers and a better product quality. Until recently, most cell line development procedures were based on random integration and gene amplification, but several methods for targeted genetic modification of cells have been developed. Some of those are homologous recombination, RNA interference and zinc-finger nucleases. Especially the latter two have evolved considerably and will soon become a standard for cell line engineering in research and industrial application. This review presents an overview of established as well as new and promising techniques for targeted genetic modification of mammalian cells.

Keywords: Cell line engineering; siRNA; miRNA; Zinc-finger nuclease (ZFN); Chinese hamster ovary (CHO) cells

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures by Jian Wen Wang; Jian Yong Wu (pp. 437-449).

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.

Keywords: Salvia miltiorrhiza ; Tanshinones; Production processes; Biosynthesis; Tissue cultures; Elicitation

Prevention of bacterial adhesion by Per Klemm; Rebecca Munk Vejborg; Viktoria Hancock (pp. 451-459).

Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach that imposes selection pressure for resistant bacteria. New approaches are urgently needed. Targeting bacterial virulence functions directly is an attractive alternative. An obvious target is bacterial adhesion. Bacterial adhesion to surfaces is the first step in colonization, invasion, and biofilm formation. As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will become valuable weapons for preventing pathogen contamination and fighting infectious diseases in the future.

Keywords: Adhesins; Bacterial attachment; Biofilms; Fimbriae

MDCK and Vero cells for influenza virus vaccine production: a one-to-one comparison up to lab-scale bioreactor cultivation by Yvonne Genzel; Christian Dietzsch; Erdmann Rapp; Jana Schwarzer; Udo Reichl (pp. 461-475).

Over the last decade, adherent MDCK (Madin Darby canine kidney) and Vero cells have attracted considerable attention for production of cell culture-derived influenza vaccines. While numerous publications deal with the design and the optimization of corresponding upstream processes, one-to-one comparisons of these cell lines under comparable cultivation conditions have largely been neglected. Therefore, a direct comparison of influenza virus production with adherent MDCK and Vero cells in T-flasks, roller bottles, and lab-scale bioreactors was performed in this study. First, virus seeds had to be adapted to Vero cells by multiple passages. Glycan analysis of the hemagglutinin (HA) protein showed that for influenza A/PR/8/34 H1N1, three passages were sufficient to achieve a stable new N-glycan fingerprint, higher yields, and a faster increase to maximum HA titers. Compared to MDCK cells, virus production in serum-free medium with Vero cells was highly sensitive to trypsin concentration. Virus stability at 37 °C for different virus strains showed differences depending on medium, virus strain, and cell line. After careful adjustment of corresponding parameters, comparable productivity was obtained with both host cell lines in small-scale cultivation systems. However, using these cultivation conditions in lab-scale bioreactors (stirred tank, wave bioreactor) resulted in lower productivities for Vero cells.

Keywords: Influenza virus; Vaccine production; TCID₅₀ stability; Trypsin; Glycosylation; Bioreactor

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16 by Torben Hoefel; Eva Wittmann; Liv Reinecke; Dirk Weuster-Botz (pp. 477-484).

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L⁻¹ on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L⁻¹ on a liter scale after 2 days of cultivation.

Keywords: Microbioreactor; 2-Hydroxyisobutyric acid; Cupriavidus necator H 16; Industrial biotechnology; 2-Hydroxyisobutyryl-CoA mutase; PHB

Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56 by Yogan Khatri; Marco Girhard; Anna Romankiewicz; Michael Ringle; Frank Hannemann; Vlada B. Urlacher; Michael C. Hutter; Rita Bernhardt (pp. 485-495).

Sesquiterpenes are particularly interesting as flavorings and fragrances or as pharmaceuticals. Regio- or stereoselective functionalizations of terpenes are one of the main goals of synthetic organic chemistry, which are possible through radical reactions but are not selective enough to introduce the desired chiral alcohol function into those compounds. Cytochrome P450 monooxygenases are versatile biocatalysts and are capable of performing selective oxidations of organic molecules. We were able to demonstrate that CYP109D1 from Sorangium cellulosum So ce56 functions as a biocatalyst for the highly regioselective hydroxylation of norisoprenoids, α- and β-ionone, which are important aroma compounds of floral scents. The substrates α- and β-ionone were regioselectively hydroxylated to 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively, which was confirmed by ¹H NMR and ¹³C NMR. The results of docking α- and β-ionone into a homology model of CYP109D1 gave a rational explanation for the regio-selectivity of the hydroxylation. Kinetic studies revealed that α- and β-ionone can be hydroxylated with nearly identical V _max and K _m values. This is the first comprehensive investigation of the regioselective hydroxylation of norisoprenoids by CYP109D1.

Keywords: Terpenoids; Norisoprenoids; Cytochromes P450; CYP109D1; Sorangium cellulosum So ce56

Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha by Surisa Suwannarangsee; Doo-Byoung Oh; Jeong-Woo Seo; Chul Ho Kim; Sang Ki Rhee; Hyun Ah Kang; Warawut Chulalaksananukul; Ohsuk Kwon (pp. 497-507).

The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 °C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l⁻¹. Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.

Keywords: Alcohol dehydrogenase·ADH1; Hansenula polymorpha ; Ethanol production; Glycerol fermentation

High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris by Chengwei Hua; Qiaojuan Yan; Zhengqiang Jiang; Yinan Li; Priti Katrolia (pp. 509-518).

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l⁻¹ with an activity of 55,300 U ml⁻¹ in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp.

Keywords: Cloning; Efficient expression; β-1,3-1,4-Glucanase; Lichenase; Paecilomyces thermophila ; Pichia pastoris

Characterization of the novel antifungal chitosanase PgChP and the encoding gene from Penicillium chrysogenum by Andrea Rodríguez-Martín; Raquel Acosta; Susan Liddell; Félix Núñez; María José Benito; Miguel A. Asensio (pp. 519-528).

The protein PgChP is a new chitosanase produced by Penicillium chrysogenum AS51D that showed antifungal activity against toxigenic molds. Two isoforms were found by SDS-PAGE in the purified extract of PgChP. After enzymatic deglycosylation, only the smaller isoform was observed by SDS-PAGE. Identical amino acid sequences were obtained from the two isoforms. Analysis of the molecular mass by electrospray ionization-mass spectrometry revealed six major peaks from 30 to 31 kDa that are related to different levels of glycosylation. The pgchp gene has 1,146 bp including four introns and an open reading frame encoding a protein of 304 amino acids. The translated open reading frame has a predicted mass of 32 kDa, with the first 21 amino acids comprising a signal peptide. Two N glycosylation consensus sequences are present in the protein sequence. The deduced sequence showed high identity with fungal chitosanases. A high level of catalytic activity on chitosan was observed. PgChP is the first chitosanase described from P. chrysogenum. Given that enzymes produced by this mold species are granted generally recognized as safe status, PgChP could be used as a food preservative against toxigenic molds and to obtain chitosan oligomers for food additives and nutraceuticals.

Keywords: Antifungal protein; Food preservative; Glycosylation; Protective culture; Toxigenic mold

Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium by Simon Stammen; Franziska Schuller; Sylvia Dietrich; Martin Gamer; Rebekka Biedendieck; Dieter Jahn (pp. 529-539).

Gene “7” of Escherichia coli phage K1E was proposed to encode a novel DNA-dependent RNA polymerase (RNAP). The corresponding protein was produced recombinantly, purified to apparent homogeneity via affinity chromatography, and successfully employed for in vitro RNA synthesis. Optimal assay conditions (pH 8, 37°C, 10 mM magnesium chloride and 1.3 mM spermidine) were established. The corresponding promoter regions were identified on the phage genome and summarized in a sequence logo. Surprisingly, next to K1E promoters, the SP6 promoter was also recognized efficiently in vitro by K1E RNAP, while the T7 RNAP promoter was not recognized at all. Based on these results, a system for high-yield in vitro RNA synthesis using K1E RNAP was established. The template plasmid is a pUC18 derivative, which enables blue/white screening for positive cloning of the target DNA. Production of more than 5 μg of purified RNA per microgram plasmid DNA was achieved. Finally, in vivo protein production systems for Bacillus megaterium were established based on K1E and SP6 phage RNAP transcription. Up to 61.4 mg g _CDW ⁻¹ (K1E RNAP) of the reporter protein Gfp was produced in shaking flask cultures of B. megaterium.

Keywords: Bacillus megaterium ; In vitro transcription; Protein production; DNA-dependant RNA polymerase; Escherichia coli phage K1E

Differential gene expression of ligninolytic enzymes in Pleurotus ostreatus grown on olive oil mill wastewater by A. Goudopoulou; A. Krimitzas; Milton A. Typas (pp. 541-551).

The expression of laccase and manganese peroxidase genes of a selected strain of Pleurotus ostreatus were studied in olive oil mill wastewater (OMW). The fungal strain decolourized 50% OMW in a linear way for 21 days and, at the same time, degraded the phenol compounds by 85%. Transcripts of laccase genes poxa1b, pox2, poxa3, and sspoxa3a, sspoxa3b coding for the small subunits of POXA3, were estimated by qRT-PCR, at different time intervals, together with β-tubulin gene used as internal control, from fungal cultures grown in a chemically-defined complete medium (CM), a supplemented CM with the addition of Cu⁺² and Mn⁺² (CM-plus) and 50% OMW in distilled water. The most abundant transcripts in both OMW and CM-plus were those of the poxa3, whereas pox2 transcripts were induced only in OMW and those of poxa1b at a strict time-window (14 days) in both OMW and CM-plus. Interestingly enough, the transcripts of genes sspoxa3a and sspoxa3b were up-regulated between 14–21 days, at a time at which the large subunit of the enzyme coded by poxa3 was down-regulated. The manganese peroxidase gene mnp2 exhibited a strong and specific transcriptional induction in OMW after 12 and 14 days, followed by a drastic drop after 18 days and a complete cease of expression at day 21, whereas mnp3 transcripts were at maximum level in OMW at day 10 but where thereafter reduced.

Keywords: Ligninolytic enzymes; Laccases; Manganese peroxidases; Gene expression; Quantitative PCR; Olive-oil mill wastewater; Pleurotus ostreatus

Bacterial diversities on unaged and aging flue-cured tobacco leaves estimated by 16S rRNA sequence analysis by Jingwen Huang; Jinkui Yang; Yanqing Duan; Wen Gu; Xiaowei Gong; Wei Zhe; Can Su; Ke-Qin Zhang (pp. 553-562).

Flue-cured tobacco leaves (FCTL) contain abundant bacteria, and these bacteria play very important roles in the tobacco aging process. However, bacterial communities on aging FCTL are not fully understood. In this study, the total microbial genome DNA of unaged and aging flue-cured tobacco K326 were isolated using a culture-independent method, and the bacterial communities were investigated by restriction fragment length polymorphism analysis. Comparison of the number of operational taxonomic units (OTUs) between the cloned libraries from the unaged and aging FCTL showed that the microbial communities between the two groups were different. Fifty and 42 OTUs were obtained from 300 positive clones in unaged and aging FCTL, respectively. Twenty-seven species of bacteria exist in both the unaged and aging FCTL, Bacillus spp. and Pseudomonas spp. were two dominant genera in FCTL. However, 23 bacterial species were only identified from the unaged FCTL, while 15 species were only identified from the aging FCTL. Interestingly, more uncultured bacteria species were found in aging FCTL than in unaged FCTL.

Keywords: Flue-cured tobacco leaves (FCTL); Bacterial diversity; 16S rRNA gene library; RFLP analysis; Phylogenetic analysis

Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants by Pablo I. Nikel; M. Julia Pettinari; Miguel A. Galvagno; Beatriz S. Méndez (pp. 563-573).

Several biotechnological processes rely on the utilization of high-copy-number plasmids for heterologous gene expression, and understanding the interactions between plasmid DNA and bacterial hosts is highly relevant for bioprocess optimization. We assessed metabolic modifications and physiological changes exerted by expression of a plasmid-encoded alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides (adhE Lm) in Escherichia coli redox mutants. Plasmid pET Lm, a pBluescript II KS(–)-derivative carrying adhE Lm, was introduced in E. coli CT1061 [arcA creC(Con)]. This recombinant was able to attain a higher ethanol concentration in glycerol cultures compared to the parental strain. pBluescript II KS(–) was rapidly lost in 72-h bioreactor cultures (7.8 ± 1.2% of plasmid-bearing cells), while pET Lm was present in 92.4 ± 7.2% of the cells. In E. coli CT1061 carrying pBluescript II KS(–) the plasmid copy number steadily diminished in bioreactor cultures to reach 334 ± 45 copies per chromosome at 72 h, while pET Lm was stably maintained, reaching 498 ± 18 copies per chromosome at the end of the cultivation. Plasmid pETΩ Lm, bearing a defective copy of adhE Lm interrupted by cat, reached 293 ± 62 copies per chromosome, implying a functional role of adhE Lm on plasmid maintenance. The intracellular NADH/NAD⁺ content suggest that regeneration of oxidized co-factors by the heterologous bioreaction might play a relevant role in plasmid maintenance.

Keywords: Escherichia coli ; Plasmid copy number; Redox mutants; ArcAB; CreBC; Microaerobiosis

Advanced tool for characterization of microbial cultures by combining cytomics and proteomics by Nico Jehmlich; Thomas Hübschmann; Manuela Gesell Salazar; Uwe Völker; Dirk Benndorf; Susann Müller; Martin von Bergen; Frank Schmidt (pp. 575-584).

Flow cytometry approaches are applicable to recover sub-populations of microbial cultures in a purified form. To examine the characteristics of each sorted cell population, Omics technologies can be used for comprehensively monitoring cellular physiology, adaptation reactions, and regulated processes. In this study, we combined flow cytometry and gel-free proteomic analysis to investigate an artificial mixed bacterial culture consisting of Escherichia coli K-12 and Pseudomonas putida KT2440. Therefore, a filter-based device technique and an on-membrane digestion protocol were combined in conjunction with liquid chromatography and mass spectrometry. This combination enabled us to identify 903 proteins from sorted E. coli K-12 and 867 proteins from sorted P. putida KT2440 bacteria from only 5 × 10⁶ cells of each. Comparative proteomic analysis of sorted and non-sorted samples was done to prove that sorting did not significantly influence the bacterial proteome profile. We further investigated the physicochemical properties, namely M _r, pI, hydropathicity, and transmembrane helices of the proteins covered. The on-membrane digestion protocol applied did not require conventional detergents or urea, but exhibited similar recovery of all protein classes as established protocols with non-sorted bacterial samples.

Keywords: Filter-based concentration; Flow cytometry; Microbial cultures; Mass spectrometry

Bioremediation of glyphosate-contaminated soils by Inna T. Ermakova; Nina I. Kiseleva; Tatyana Shushkova; Mikhail Zharikov; Gennady A. Zharikov; Alexey A. Leontievsky (pp. 585-594).

Based on the results of laboratory and field experiments, we performed a comprehensive assessment of the bioremediation efficiency of glyphosate-contaminated soddy-podzol soil. The selected bacterial strains Achromobacter sp. Kg 16 (VKM B-2534D) and Ochrobactrum anthropi GPK 3 (VKM B-2554D) were used for the aerobic degradation of glyphosate. They demonstrated high viability in soil with the tenfold higher content of glyphosate than the recommended dose for the single in situ treatment of weeds. The strains provided a two- to threefold higher rate of glyphosate degradation as compared to indigenous soil microbial community. Within 1–2 weeks after the strain introduction, the glyphosate content of the treated soil decreased and integral toxicity and phytotoxicity diminished to values of non-contaminated soil. The decrease in the glyphosate content restored soil biological activity, as is evident from a more than twofold increase in the dehydrogenase activity of indigenous soil microorganisms and their biomass (1.2-fold and 1.6-fold for saprotrophic bacteria and fungi, respectively). The glyphosate-degrading strains used in this study are not pathogenic for mammals and do not exhibit integral toxicity and phytotoxicity. Therefore, these strains are suitable for the efficient, ecologically safe, and rapid bioremediation of glyphosate-contaminated soils.

Keywords: Glyphosate; Bioremediation; Herbicide; Biodegradation; Microorganism degraders; Toxicology

(Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., isolated from an underground gas storage by Melike Balk; Farrakh Mehboob; Antonie H. van Gelder; W. Irene C. Rijpstra; Jaap S. Sinninghe Damsté; Alfons J. M. Stams (pp. 595-603).

A mesophilic bacterium, strain An4, was isolated from an underground gas storage reservoir with methanol as substrate and perchlorate as electron acceptor. Cells were Gram-negative, spore-forming, straight to curved rods, 0.5–0.8 μm in diameter, and 2–8 μm in length, growing as single cells or in pairs. The cells grew optimally at 37°C, and the pH optimum was around 7. Strain An4 converted various alcohols, organic acids, fructose, acetoin, and H₂/CO₂ to acetate, usually as the only product. Succinate was decarboxylated to propionate. The isolate was able to respire with (per)chlorate, nitrate, and CO₂. The G+C content of the DNA was 42.6 mol%. Based on the 16S rRNA gene sequence analysis, strain An4 was most closely related to Sporomusa ovata (98% similarity). The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell-free extracts.

Keywords: Sporomusa sp.; Perchlorate; Underground gas storage

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Applied Microbiology and Biotechnology (v.88, #2)