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Applied Microbiology and Biotechnology (v.83, #5)

Plasmid uptake by bacteria: a comparison of methods and efficiencies by Naoto Yoshida; Misa Sato (pp. 791-798).

The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation, (2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method, was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique. We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by which to transform bacteria.

Keywords: Biolistic transformation; Chemical transformation; Chrysotile; Electro-transformation; Plasmid; Sepiolite; Sonic transformation; Tribos transformation; Yoshida effect

Biosynthesis and biotechnological production of flavanones: current state and perspectives by Zachary L. Fowler; Mattheos A. G. Koffas (pp. 799-808).

Polyphenols produced in a wide variety of flowering and fruit-bearing plants have the potential to be valuable fine chemicals for the treatment of an assortment of human maladies. One of the major constituents within this chemical class are flavonoids, among which flavanones, as the precursor to all flavonoid structures, are the most prevalent. We review the current status of flavanone production technology using microorganisms, with focus on heterologous protein expression. Such processes appear as attractive production alternatives for commercial synthesis of these high-value chemicals as traditional chemical, and plant cell cultures have significant drawbacks. Other issues of importance, including fermentation configurations and economics, are also considered.

Keywords: Flavanones; Fermentation; Polyphenols; Cofactor engineering

Bioprocessing of plant cell cultures for mass production of targeted compounds by Milen I. Georgiev; Jost Weber; Alexandre Maciuk (pp. 809-823).

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered.

Keywords: Bioreactor(s); Flow cytometry; Operational mode; Optimization; Plant cell culture; Process monitoring; Secondary metabolite

Characteristics and biotechnological applications of microbial cholesterol oxidases by Noriyuki Doukyu (pp. 825-837).

Microbial cholesterol oxidase is an enzyme of great commercial value, widely employed by laboratories routinely devoted to the determination of cholesterol concentrations in serum, other clinical samples, and food. In addition, the enzyme has potential applications as a biocatalyst which can be used as an insecticide and for the bioconversion of a number of sterols and non-steroidal alcohols. The enzyme has several biological roles, which are implicated in the cholesterol metabolism, the bacterial pathogenesis, and the biosynthesis of macrolide antifungal antibiotics. Cholesterol oxidase has been reported from a variety of microorganisms, mostly from actinomycetes. We recently reported cholesterol oxidases from gram-negative bacteria such as Burkholderia and Chromobacterium. These enzymes possess thermal, detergent, and organic solvent tolerance. There are two forms of cholesterol oxidase, one containing a flavin adenine dinucleotide cofactor non-covalently bound to the enzyme (class I) and the other containing the cofactor covalently linked to the enzyme (class II). These two enzymes have no significant sequence homology. The phylogenetic tree analyses show that both class I and class II enzymes can be further divided into at least two groups.

Keywords: Cholesterol; Cholesterol oxidase; Chromobacterium ; Actinomycetes; Gram-negative bacteria; Diagnosis

A lytic enzyme cocktail from Streptomyces sp. B578 for the control of lactic and acetic acid bacteria in wine by V. Blättel; K. Wirth; H. Claus; B. Schlott; P. Pfeiffer; H. König (pp. 839-848).

Beside yeasts, lactic acid bacteria (LAB) are the most abundant microbes in must during vinification. Whereas Oenococcos oeni is commercially used as a starter culture for the biological acid reduction in wines, other species are responsible for different types of wine spoilage. Members of the genera Pediococcus, Weissella, Leuconostoc, and Lactobacillus are producers of exopolysaccharide slimes, biogenic amines, acetic acid, and other off-flavors. In order to control microbial growth, different procedures such as heating of must and addition of sulfite or lysozyme from egg white are generally applied. Yet, because of health risks, the application of sulfite should be reduced and lysozyme is not effective against all LAB. In this study, we describe exoenzymes from a Streptomyces sp. strain B578 lysing nearly all wine-relevant strains of LAB and Gram-negative acetic acid bacteria. The lytic enzymes were active under wine-making conditions, such as the presence of sulfite and ethanol, low temperatures, and low pH values. The analysis of the exoenzyme composition revealed a synergistic action of different cell wall hydrolases. In conclusion, the lytic cocktail of Streptomyces sp. B578 is a promising tool for the control of wine-spoiling bacteria.

Keywords: Lactic acid bacteria; Acetic acid bacteria; Wine spoilage; Cell wall hydrolases; Lysozyme; Inhibition; Streptomyces

P450_BM-3-catalyzed whole-cell biotransformation of α-pinene with recombinant Escherichia coli in an aqueous–organic two-phase system by Hendrik Schewe; Dirk Holtmann; Jens Schrader (pp. 849-857).

A recombinant Escherichia coli BL21 (DE3) strain overexpressing a variant of P450_BM-3 (V26T/R47F/A74G/F87V/L188K; abbreviated: BL21 (P450_BM-3 QM)) oxyfunctionalizes the bicyclic monoterpene α-pinene to α-pinene oxide, verbenol, and myrtenol. To address the low water solubility and the toxicity of terpenoids, an aqueous–organic two-phase bioprocess was developed. Diisononyl phthalate was selected as a biocompatible organic carrier solvent capable of masking the toxic effects mediated by α-pinene and of efficiently extracting the products enabling scale-up to the bioreactor. With an aqueous to organic phase ratio of 3:2 and 30% (v/v) of α-pinene in the organic phase, a biocatalytic product formation period of more than 4 h was achieved. A comparison of the biotransformation performance of BL21 (P450_BM-3 QM) and a strain with an additional heterologous NADPH regeneration system comprising glucose facilitator and dehydrogenase, but only expressing half the amount of P450_BM-3 QM, shows comparable product concentrations of 1,020 ± 144 and 800 ± 61 mg l _Aq ⁻¹ , respectively. The total product yields Y _P/P450 (µmol µmol _P450 ⁻¹ ) were 80% higher when the strain with the cofactor regeneration system was used. A total product concentration of over 1 g l _Aq ⁻¹ , corresponding to the highest value reported for microbial α-pinene oxyfunctionalization so far, marks a promising step forward toward a future application of recombinant microorganisms for the selective oxidation of terpenoids to value-added products.

Keywords: Aqueous–organic two-phase bioprocess; P450_BM-3 ; Whole-cell; Biotransformation; Cofactor regeneration; Pinene; Phthalate

Efficient synthesis of enantiomeric ethyl lactate by Candida antarctica lipase B (CALB)-displaying yeasts by Chiaki Inaba; Kenjiro Maekawa; Hironobu Morisaka; Kouichi Kuroda; Mitsuyoshi Ueda (pp. 859-864).

The whole-cell biocatalyst displaying Candida antarctica lipase B (CALB) on the yeast cell surface with α-agglutinin as the anchor protein was easy to handle and possessed high stability. The lyophilized CALB-displaying yeasts showed their original hydrolytic activity and were applied to an ester synthesis using ethanol and l-lactic acid as substrates. In water-saturated heptane, CALB-displaying yeasts catalyzed ethyl lactate synthesis. The synthesis efficiency increased depending on temperature and reached approximately 74% at 50°C. The amount of l-ethyl lactate increased gradually. l-Ethyl lactate synthesis stopped at 200 h and restarted after adding of l-lactic acid at 253 h. It indicated that CALB-displaying yeasts retained their synthetic activity under such reaction conditions. In addition, CALB-displaying yeasts were able to recognize l-lactic acid and d-lactic acid as substrates. l-Ethyl lactate was prepared from l-lactic acid and d-ethyl lactate was prepared from d-lactic acid using the same CALB-displaying whole-cell biocatalyst. These findings suggest that CALB-displaying yeasts can supply the enantiomeric lactic esters for preparation of useful and improved biopolymers of lactic acid.

Keywords: Candida antarctica lipase B (CALB); Whole-cell biocatalyst; Ethyl lactate; Biodegradable plastics; Enantiomer synthesis

Purification and functional characterization of endo-β-mannanase MAN5 and its application in oligosaccharide production from konjac flour by Min Zhang; Xiu-Lan Chen; Zhi-Hua Zhang; Cai-Yun Sun; Lei-Lei Chen; Hai-Lun He; Bai-Cheng Zhou; Yu-Zhong Zhang (pp. 865-873).

MAN5, the main extracellular saccharide hydrolase from Bacillus sp. MSJ-5, is an endo-β-mannanase with a demand of at least five sugar moieties for effective cleavage. It has a pH optimum of 5.5 and a temperature optimum of 50°C and is stable at pH 5–9 or below 65°C. MAN5 has a very high ability to hydrolyze konjac flour, 10 U/mg of which could completely liquefy konjac flour gum in 10 min at 50°C. HPLC analysis showed that most glucomannan in the konjac flour was hydrolyzed into a large amount of oligosaccharides with DP of 2–6 and a very small amount of monosaccharide. With the culture supernatant as enzyme source, the optimum condition to prepare oligosaccharides from konjac flour was obtained as 10 mg/ml konjac flour incubated with 10 U/mg enzyme at 50°C for 24 h. With this condition, more than 90% polysaccharides in the konjac flour solution were hydrolyzed into oligosaccharides and a little monosaccharide (2.98% of the oligosaccharides). Konjac flour is an underutilized agricultural material with low commercial value in China. With MAN5, konjac flour can be utilized to generate high value-added oligosaccharides. The high effectiveness and cheapness of this technique indicates its potential in industry.

Keywords: Endo-beta-mannanase; MAN5; Oligosaccharides preparation; Konjac flour; Bacillus sp. MSJ-5

A novel protease-resistant α-galactosidase with high hydrolytic activity from Gibberella sp. F75: gene cloning, expression, and enzymatic characterization by Yanan Cao; Yaru Wang; Kun Meng; Yingguo Bai; Pengjun Shi; Huiying Luo; Peilong Yang; Zhigang Zhou; Zhifang Zhang; Bin Yao (pp. 875-884).

A novel α-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino acid sequence showed highest identity (69%) with an α-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60°C and pH 4.0 and was stable at pH 3.0–12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline proteases. The enzyme K _m values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared with the commercial α-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant α-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity of Aga-F75 make it potentially useful in the food and feed industries.

Keywords: α-Galactosidase; Gibberella ; Protease-resistance; High hydrolytic activity

Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound by Dinesh Simkhada; Tae-Jin Oh; Binod Babu Pageni; Hei Chan Lee; Kwangkyoung Liou; Jae Kyung Sohng (pp. 885-895).

The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography–mass spectrometry.

Keywords: Biosynthesis; Calicheamicin; Dihydrochalcomycin; Streptomyces ; UDP-xylose; 5-O-xylosyl-chalconolide

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains by Daisy Rickli Binde; Pâmela Menna; Eliane Villamil Bangel; Fernando Gomes Barcellos; Mariangela Hungria (pp. 897-908).

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.

Keywords: Bacterial fingerprinting; Bacterial taxonomy; Biological nitrogen fixation; Culture collections; Inoculants; 16S rRNA

Proteomic insights into adaptive responses of Saccharomyces cerevisiae to the repeated vacuum fermentation by Jing-Sheng Cheng; Xiao Zhou; Ming-Zhu Ding; Ying-Jin Yuan (pp. 909-923).

The responses and adaptation mechanisms of the industrial Saccharomyces cerevisiae to vacuum fermentation were explored using proteomic approach. After qualitative and quantitative analyses, a total of 106 spots corresponding to 68 different proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differentially expressed proteins were involved in amino acid and carbohydrate metabolisms, various signal pathways (Ras/MAPK, Ras–cyclic adenosine monophosphate, and HOG pathway), and heat shock and oxidative responses. Among them, alternations in levels of 17 proteins associated with carbohydrate metabolisms, in particular, the upregulations of proteins involved in glycolysis, trehalose biosynthesis, and the pentose phosphate pathway, suggested vacuum-induced redistribution of the metabolic fluxes. The upregulation of 17 heat stress and oxidative response proteins indicated that multifactors contributed to oxidative stresses by affecting cell redox homeostasis. Taken together with upregulation in 14-3-3 proteins levels, 22 proteins were detected in multispots, respectively, indicating that vacuum might have promoted posttranslational modifications of some proteins in S. cerevisiae. Further investigation revealed that the elevations of the differentially expressed proteins were mainly derived from vacuum stress rather than the absence of oxygen. These findings provide new molecular mechanisms for understanding of adaptation and tolerance of yeast to vacuum fermentation.

Keywords: Proteome; Vacuum fermentation; Adaptation; Stress response; Saccharomyces cerevisiae

Effect of ultrafine-grained titanium surfaces on adhesion of bacteria by Vi Khanh Truong; Stuart Rundell; Rimma Lapovok; Yuri Estrin; James Y. Wang; Christopher C. Berndt; David G. Barnes; Christopher J. Fluke; Russell J. Crawford; Elena P. Ivanova (pp. 925-937).

The influence of the ultrafine crystallinity of commercial purity grade 2 (as-received) titanium and titanium modified by equal channel angular pressing (modified titanium) on bacterial attachment was studied. A topographic profile analysis of the surface of the modified titanium revealed a complex morphology of the surface. Its prominent micro- and nano-scale features were 100–200-nm-scale undulations with 10–15 μm spacing. The undulating surfaces were nano-smooth, with height variations not exceeding 5–10 nm. These surface topography characteristics were distinctly different from those of the as-received samples, where broad valleys (up to 40–60 μm) were detected, whose inner surfaces exhibited asperities approximately 100 nm in height spaced at 1–2 μm. It was found that each of the three bacteria strains used in this study as adsorbates, viz. Staphylococcus aureus CIP 68.5, Pseudomonas aeruginosa ATCC 9025 and Escherichia coli K12, responded differently to the two types of titanium surfaces. Extreme grain refinement by ECAP resulted in substantially increased numbers of cells attached to the surface compared to as-received titanium. This enhanced degree of attachment was accompanied with an increased level of extracellular polymeric substances (EPS) production by the bacteria.

Keywords: Titanium surfaces; Equal channel angular pressing (ECAP); Bacterial adhesion; Staphylococcus aureus ; Pseudomonas aeruginosa ; Escherichia coli

Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production by Zheng-Jun Li; Lei Cai; Qiong Wu; Guo-Qiang Chen (pp. 939-947).

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y _PHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y _PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.

Keywords: PHB; Polyhydroxyalkanoates; Cofactor engineering; NAD kinase; NADPH; E. coli

Feasibility of atmospheric methane removal using methanotrophic biotrickling filters by Sukhwan Yoon; Jeffrey N. Carey; Jeremy D. Semrau (pp. 949-956).

Methane is a potent greenhouse gas with a global warming potential ~23 times that of carbon dioxide. Here, we describe the modeling of a biotrickling filtration system composed of methane-consuming bacteria, i.e., methanotrophs, to assess the utility of these systems in removing methane from the atmosphere. Model results indicate that assuming the global average atmospheric concentration of methane, 1.7 ppmv, methane removal is ineffective using these methanotrophic biofilters as the methane concentration is too low to enable cell survival. If the concentration is increased to 500–6,000 ppmv, however, similar to that found above landfills and in concentrated animal feeding operations (factory farms), 4.98–35.7 tons of methane can be removed per biofilter per year assuming biotrickling filters of typical size (3.66 m in diameter and 11.5 m in height). Using reported ranges of capital, operational, and maintenance costs, the cost of the equivalent ton of CO₂ removal using these systems is $90–$910 ($2,070–$20,900 per ton of methane), depending on the influent concentration of methane and if heating is required. The use of methanotrophic biofilters for controlling methane emissions is technically feasible and, provided that either the costs of biofilter construction and operation are reduced or the value of CO₂ credits is increased, can also be economically attractive.

Keywords: Methane; Methanotroph; Biofilm; Biofiltration; Biotrickling filter

Citric acid wastewater as electron donor for biological sulfate reduction by Alfons J. M. Stams; Jacco Huisman; Pedro A. Garcia Encina; Gerard Muyzer (pp. 957-963).

Citrate-containing wastewater is used as electron donor for sulfate reduction in a biological treatment plant for the removal of sulfate. The pathway of citrate conversion coupled to sulfate reduction and the microorganisms involved were investigated. Citrate was not a direct electron donor for the sulfate-reducing bacteria. Instead, citrate was fermented to mainly acetate and formate. These fermentation products served as electron donors for the sulfate-reducing bacteria. Sulfate reduction activities of the reactor biomass with acetate and formate were sufficiently high to explain the sulfate reduction rates that are required for the process. Two citrate-fermenting bacteria were isolated. Strain R210 was closest related to Trichococcus pasteurii (99.5% ribosomal RNA (rRNA) gene sequence similarity). The closest relative of strain S101 was Veillonella montepellierensis with an rRNA gene sequence similarity of 96.7%. Both strains had a complementary substrate range.

Keywords: Desulfurization; Sulfate reduction; Citrate fermentation; Trichococcus ; Veillonella

Continuous power generation and microbial community structure of the anode biofilms in a three-stage microbial fuel cell system by Kyungmi Chung; Satoshi Okabe (pp. 965-977).

A mediator-less three-stage two-chamber microbial fuel cell (MFC) system was developed and operated continuously for more than 1.5 years to evaluate continuous power generation while treating artificial wastewater containing glucose (10 mM) concurrently. A stable power density of 28 W/m³ was attained with an anode hydraulic retention time of 4.5 h and phosphate buffer as the cathode electrolyte. An overall dissolved organic carbon removal ratio was about 85%, and coulombic efficiency was about 46% in this MFC system. We also analyzed the microbial community structure of anode biofilms in each MFC. Since the environment in each MFC was different due to passing on the products to the next MFC in series, the microbial community structure was different accordingly. The anode biofilm in the first MFC consisted mainly of bacteria belonging to the Gammaproteobacteria, identified as Aeromonas sp., while the Firmicutes dominated the anode biofilms in the second and third MFCs that were mainly fed with acetate. Cyclic voltammetric results supported the presence of a redox compound(s) associated with the anode biofilm matrix, rather than mobile (dissolved) forms, which could be responsible for the electron transfer to the anode. Scanning electron microscopy revealed that the anode biofilms were comprised of morphologically different cells that were firmly attached on the anode surface and interconnected each other with anchor-like filamentous appendages, which might support the results of cyclic voltammetry.

Keywords: A mediator-less three-stage two-chamber microbial fuel cell (MFC) system; Anode biofilm communities; Cyclic voltammetry; FISH

In vivo Tn5-based transposon mutagenesis of Streptomycetes by Lutz Petzke; Andriy Luzhetskyy (pp. 979-986).

This paper reports the in vivo expression of the synthetic transposase gene tnp(a) from a hyperactive Tn5 tnp gene mutant in Streptomyces coelicolor. Using the synthetic tnp(a) gene adapted for Streptomyces codon usage, we showed random insertion of the transposon into the Streptomycetes genome. The insertion frequency for the hyperactive Tn5 derivative is 98% of transformed S. coelicolor cells. The random transposition has been confirmed by the recovery of ~1.1% of auxotrophs. The Tn5 insertions are stably inherited in the absence of apramycin selection. The transposon contains an apramycin resistance selection marker and an R6Kγ origin of replication for transposon rescue. We identified the transposon insertion loci by random sequencing of 14 rescue plasmids. The majority of insertions (12 of 14) were mapped to putative open-reading frames on the S. coelicolor chromosome. These included two new regulatory genes affecting S. coelicolor growth and actinorhodin biosynthesis.

Keywords: Tn5 ; Actinomycetes; Mutagenesis; Rescue plasmid; Natural products

In vivo Tn5-based transposon mutagenesis of Streptomycetes by Lutz Petzke; Andriy Luzhetskyy (pp. 979-986).

Keywords: Tn5 ; Actinomycetes; Mutagenesis; Rescue plasmid; Natural products

Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR by David M. Stevenson; Paul J. Weimer (pp. 987-988).

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Applied Microbiology and Biotechnology (v.83, #5)

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Applied Microbiology and Biotechnology (v.83, #5)