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Applied Microbiology and Biotechnology (v.76, #6)

Bacterial metabolism of long-chain n-alkanes by Alexander Wentzel; Trond E. Ellingsen; Hans-Kristian Kotlar; Sergey B. Zotchev; Mimmi Throne-Holst (pp. 1209-1221).

Degradation of alkanes is a widespread phenomenon in nature, and numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing these substrates as a carbon and energy source have been isolated and characterized. In this review, we summarize recent advances in the understanding of bacterial metabolism of long-chain n-alkanes. Bacterial strategies for accessing these highly hydrophobic substrates are presented, along with systems for their enzymatic degradation and conversion into products of potential industrial value. We further summarize the current knowledge on the regulation of bacterial long-chain n-alkane metabolism and survey progress in understanding bacterial pathways for utilization of n-alkanes under anaerobic conditions.

Keywords: Bacterial alkane metabolism; Alkane degradation; Long-chain n-alkanes; Anaerobic; Aerobic; Wax ester

The state of the art in the analysis of two-dimensional gel electrophoresis images by Matthias Berth; Frank Michael Moser; Markus Kolbe; Jörg Bernhardt (pp. 1223-1243).

Software-based image analysis is a crucial step in the biological interpretation of two-dimensional gel electrophoresis experiments. Recent significant advances in image processing methods combined with powerful computing hardware have enabled the routine analysis of large experiments. We cover the process starting with the imaging of 2-D gels, quantitation of spots, creation of expression profiles to statistical expression analysis followed by the presentation of results. Challenges for analysis software as well as good practices are highlighted. We emphasize image warping and related methods that are able to overcome the difficulties that are due to varying migration positions of spots between gels. Spot detection, quantitation, normalization, and the creation of expression profiles are described in detail. The recent development of consensus spot patterns and complete expression profiles enables one to take full advantage of statistical methods for expression analysis that are well established for the analysis of DNA microarray experiments. We close with an overview of visualization and presentation methods (proteome maps) and current challenges in the field.

Keywords: 2-D gel electrophoresis; Image analysis; Proteome maps; Warping; Statistics

The state of the art in the analysis of two-dimensional gel electrophoresis images by Matthias Berth; Frank Michael Moser; Markus Kolbe; Jörg Bernhardt (pp. 1223-1243).

Keywords: 2-D gel electrophoresis; Image analysis; Proteome maps; Warping; Statistics

Biofilms: strategies for metal corrosion inhibition employing microorganisms by Rongjun Zuo (pp. 1245-1253).

Corrosion causes dramatic economic loss. Currently widely used corrosion control strategies have disadvantages of being expensive, subject to environmental restrictions, and sometimes inefficient. Studies show that microbial corrosion inhibition is actually a common phenomenon. The present review summarizes recent progress in this novel strategy: corrosion control using beneficial bacteria biofilms. The possible mechanisms may involve: (1) removal of corrosive agents (such as oxygen) by bacterial physiological activities (e.g., aerobic respiration), (2) growth inhibition of corrosion-causing bacteria by antimicrobials generated within biofilms [e.g., sulfate-reducing bacteria (SRB) corrosion inhibition by gramicidin S-producing Bacillus brevis biofilm], (3) generation of protective layer by biofilms (e.g., Bacillus licheniformis biofilm produces on aluminum surface a sticky protective layer of γ-polyglutamate). Successful utilization of this novel strategy relies on advances in study at the interface of corrosion engineering and biofilm biology.

Keywords: Bacterial biofilm; Corrosion control

Single-species microbial biofilm screening for industrial applications by Xuan Zhong Li; Bernhard Hauer; Bettina Rosche (pp. 1255-1262).

While natural microbial biofilms often consist of multiple species, single-species biofilms are of great interest to biotechnology. The current study evaluates biofilm formation for common industrial and laboratory microorganisms. A total of 68 species of biosafety level one bacteria and yeasts from over 40 different genera and five phyla were screened by growing them in microtiter plates and estimating attached biomass by crystal violet staining. Most organisms showed biofilm formation on surfaces of polystyrene within 24 h. By changing a few simple conditions such as substratum characteristics, inoculum and nutrient availability, 66 strains (97%) demonstrated biofilm formation under at least one of the experimental conditions and over half of these strains were classified as strong biofilm formers, potentially suitable as catalysts in biofilm applications. Many non-motile bacteria were also strong biofilm formers. Biofilm morphologies were visualized for selected strains. A model organism, Zymomonas mobilis, easily established itself as a biofilm on various reactor packing materials, including stainless steel.

Keywords: Biofilm; Screening; Bacteria; Yeast; Biotransformation; Industrial application

Succinic acid production from wheat using a biorefining strategy by Chenyu Du; Sze Ki Carol Lin; Apostolis Koutinas; Ruohang Wang; Colin Webb (pp. 1263-1270).

The biosynthesis of succinic acid from wheat flour was investigated in a two-stage bio-process. In the first stage, wheat flour was converted into a generic microbial feedstock either by fungal fermentation alone or by combining fungal fermentation for enzyme and fungal bio-mass production with subsequent flour hydrolysis and fungal autolysis. In the second stage, the generic feedstock was converted into succinic acid by bacterial fermentation by Actinobacillus succinogenes. Direct fermentation of the generic feedstock produced by fungal fermentation alone resulted in a lower succinic acid production, probably due to the low glucose and nitrogen concentrations in the fungal broth filtrate. In the second feedstock production strategy, flour hydrolysis conducted by mixing fungal broth filtrate with wheat flour generated a glucose-rich stream, while the fungal bio-mass was subjected to autolysis for the production of a nutrient-rich stream. The possibility of replacing a commercial semi-defined medium by these two streams was investigated sequentially. A. succinogenes fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of almost 16 g l^–1 with an overall yield of 0.19 g succinic acid per g wheat flour. These results show that a wheat-based bio-refinery employing coupled fungal fermentation and subsequent flour hydrolysis and fungal autolysis can lead to a bacterial feedstock for the efficient production of succinic acid.

Keywords: Bio-refinery; Succinic acid fermentation; Wheat; Actinobacillus succinogenes ; Aspergillus awamori ; Flour hydrolysis; Fungal autolysis

Azoreductase from Rhodobacter sphaeroides AS1.1737 is a flavodoxin that also functions as nitroreductase and flavin mononucleotide reductase by Guangfei Liu; Jiti Zhou; Hong Lv; Xuemin Xiang; Jing Wang; Mi Zhou; Yuanyuan Qv (pp. 1271-1279).

Previously reported azoreductase (AZR) from Rhodobacter sphaeroides AS1.1737 was shown to be a flavodoxin possessing nitroreductase and flavin mononucleotide (FMN) reductase activities. The structure model of AZR constructed with SWISS-MODEL displayed a flavodoxin-like fold with a three-layer α/β/α structure. With nitrofurazone as substrate, the optimal pH value and temperature were 7.0 and 50°C, respectively. AZR could reduce a number of nitroaromatic compounds including 2,4-dinitrotoluene, 2,6-dinitrotoluene, 3,5-dinitroaniline, and 2,4,6-trinitrotoluene (TNT). TNT resulted to be the most efficient nitro substrate and was reduced to hydroxylamino-dinitrotoluene. Both NADH and NADPH could serve as electron donors of AZR, where the latter was preferred. Externally added FMN was also reduced by AZR via ping-pong mechanism and was a competitive inhibitor of NADPH, methyl red, and nitrofurazone. AZR with broad substrate specificity is a member of a new nitro/FMN reductase family demonstrating potential application in bioremediation.

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol by Shijin Wu; Jiajia Shen; Xiaoyun Zhou; Jianmeng Chen (pp. 1281-1287).

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.

Keywords: Bacillus sp. Z018; Epoxide hydrolase; (R)-3-phenoxy-1,2-propanediol; Transformation

Bacillus circulans WZ-12—a newly discovered aerobic dichloromethane-degrading methylotrophic bacterium by S. J. Wu; L. L. Zhang; J. D. Wang; J. M. Chen (pp. 1289-1296).

A novel dichloromethane (DCM)-degrading bacterial strain named WZ-12 (GenBank accession no. EF100968) was isolated and identified as Bacillus circulans based on standard morphological and physiological properties and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. DCM dehalogenase from B. circulans WZ-12 was purified to 8.27-fold with a yield of 34.83%. The electrophoretically homogeneous-purified enzyme exhibited a specific activity of 118.82 U/mg. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of purified DCM dehalogenase gave a distinct band with an estimated molecular mass of 20,000 ± 1,000.

Keywords: Dichloromethane; Bacillus circulans ; Dehalogenase; Biodegradation

Cloning, sequencing, overexpression and characterization of l-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production by Wayoon Poonperm; Goro Takata; Hiromi Okada; Kenji Morimoto; Tom Birger Granström; Ken Izumori (pp. 1297-1307).

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.

Keywords: l-Rhamnose isomerase; Bacillus pallidus ; Rare sugar

Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita by Zhongwei Gan; Jinkui Yang; Nan Tao; Lianming Liang; Qili Mi; Juan Li; Ke-Qin Zhang (pp. 1309-1317).

The nematophagous fungus Lecanicillium psalliotae (syn. Verticillium psalliotae) is a well-known biocontrol agent. In this study, a chitinase gene Lpchi1 was isolated for the first time from L. psalliotae using degenerate primers and DNA-walking technique. The cloned gene Lpchi1 encoding 423 amino acid residues shares a high degree of homology with other pathogenicity-related chitinases from entomopathogenic and mycoparasitic fungi. The complementary DNA sequence of the mature chitinase was amplified via reverse transcription polymerase chain reaction and expressed well in Pichia pastoris GS115. Through gel filtration, the recombinant chitinase was purified as a protein of ca. 45 kDa with an optimal activity at pH 7.0 and 37.6°C. The purified chitinase LPCHI1 was found degrading chitinous components of eggs of the root-knot nematode Meloidogyne incognita and significantly influence its development. Moreover, our results also demonstrate that the protease Ver112 and the chitinase LPCHI1 from the same fungus interacted on the egg infection.

Keywords: Chitinase; Cloning; Expression; Lecanicillium psalliotae ; Meloidogyne incognita ; Pichia pastoris

Biochemical characterization and phylogenetic analysis of UDP-glucose dehydrogenase from the gellan gum producer Sphingomonas elodea ATCC 31461 by Ana Teresa Granja; Alma Popescu; Ana Rita Marques; Isabel Sá-Correia; Arsenio M. Fialho (pp. 1319-1327).

Sphingomonas elodea ATCC 31461 synthesizes in high yield the exopolysaccharide gellan, which is a water-soluble gelling agent with many applications. In this study, we describe the cloning and sequence analysis of the ugdG gene, encoding a UDP-glucose dehydrogenase (47.2 kDa; UDPG-DH; EC 1.1.1.22), required for the synthesis of the gellan gum precursor UDP-glucuronic acid. UgdG protein shows homology to members of the UDP-glucose/GDP-mannose dehydrogenase superfamily. The Neighbor-Joining method was used to determine phylogenetic relationships among prokaryotic and eukaryotic UDPG-DHs. UgdG from S. elodea and UDPG-DHs from Novosphingobium, Zymomonas, Agrobacterium, and Caulobacter species form a divergent phylogenetic group with a close evolutionary relationship with eukaryotic UDPG-DHs. The ugdG gene was recombinantly expressed in Escherichia coli with and N-terminal 6-His tag and purified for biochemical characterization. The enzyme has an optimum temperature and pH of 37°C and 8.7, respectively. The estimated apparent K _m values for UDP-glucose and NAD⁺ were 0.87 and 0.4 mM, respectively. DNA sequencing of chromosomal regions adjacent to ugdG gene and sequence similarity studies suggests that this gene maps together with others presumably involved in the biosynthesis of S. elodea cell wall polysaccharides.

Keywords: Sphingomonas elodea ; Gelling agents; Gellun gum; UDP-glucosedehydrogenase; ugdG gene

Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata by Bing Feng; Wei Hu; Bai-ping Ma; Yong-ze Wang; Hong-ze Huang; Sheng-qi Wang; Xiao-hong Qian (pp. 1329-1338).

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of 12.34 U mg of total protein⁻¹ under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin.

Keywords: Glucoamylase; Curvularia lunata ; Steroidal saponin-rhamnosidase activity; Steroidal saponins

Effective production of retinal from β-carotene using recombinant mouse β-carotene 15,15′-monooxygenase by Yeong-Su Kim; Nam-Hee Kim; Hye-Jung Kim; Jung-Kul Lee; Seon-Won Kim; Deok-Kun Oh (pp. 1339-1345).

The gene encoding β-carotene 15,15′-monooxygenase from Mus musculus (house mouse), which cleaves β-carotene into two molecules of retinal, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by His-tag affinity and resource Q ion exchange chromatography columns to a final specific activity of 0.51 U mg⁻¹. The optimum pH, temperature, substrate and detergent concentrations, and enzyme amount for effective retinal production were determined to be 9.0, 37°C, 200 mg l⁻¹ β-carotene, 5% (w/v) Tween 40, and 0.2 U ml⁻¹ enzyme, respectively. Under optimum conditions, the recombinant enzyme produced 72 mg l⁻¹ retinal in a 15-h reaction time, with a conversion yield of 36% (w/w). The specific activity of the purified enzyme and retinal production obtained in the present study were the highest results ever reported.

Keywords: β-Carotene; β-Carotene 15,15′-monooxygenase; Optimization of reaction conditions; Retinal production

Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R by Anna Kotrbova-Kozak; Pavel Kotrba; Masayuki Inui; Jiri Sajdok; Hideaki Yukawa (pp. 1347-1356).

Corynebacterium glutamicum R adhA gene encodes a homodimeric, NAD-dependent, 345 amino acid residue alcohol dehydrogenase with two zinc ions per subunit. Chromosomal inactivation of the adhA gene rendered the strain incapable of growth on either ethanol or n-propanol as the sole carbon source. RNA hybridization analysis revealed that adhA transcription was not only induced by these two substrates, but it was also subject to glucose catabolite repression. Accordingly, both induction of AdhA activity and ethanol utilization were detected only after depletion of glucose. Deletion of either or both of potential cyclic adenosine monophosphate (cAMP) receptor binding site and an inverted repeat of sequence 5′-GCAATTGATG-N ₈-CACAATTGC-3′ in the promoter region of adhA strongly suggested that IR, which does not share significant similarity with other regulatory DNA elements of C. glutamicum, represents a transcriptional repressor binding site. Purified recombinant AdhA displayed the highest substrate specificities towards ethanol and n-propanol and their corresponding aldehydes.

Keywords: Alcohol dehydrogenase; Ethanol; Corynebacterium glutamicum ; Catabolite repression

Heterologous expression of the kanamycin biosynthetic gene cluster (pSKC2) in Streptomyces venezuelae YJ003 by Laxmi Prasad Thapa; Tae-Jin Oh; Hei Chan Lee; Kwangkyoung Liou; Je Won Park; Yeo Joon Yoon; Jae Kyung Sohng (pp. 1357-1364).

The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides antibiotic. The isolated compound was analyzed by electrospray ionization–mass spectrometry, liquid chromatography–mass spectrometry, high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A.

Keywords: Heterologous expression; Kanamycin; Streptomyces kanamyceticus ATCC 12853; Streptomyces venezuelae YJ003

Molecular cloning and comparative analysis of four β-galactosidase genes from Bifidobacterium bifidum NCIMB41171 by Theodoros K. Goulas; Athanasios K. Goulas; George Tzortzis; Glenn R. Gibson (pp. 1365-1372).

Bifidobacterium bifidum NCIMB41171 carries four genes encoding different β-galactosidases. One of them, named bbgIII, consisted of an open reading frame of 1,935 amino acid (a.a.) residues encoding a protein with a multidomain structure, commonly identified on cell wall bound enzymes, having a signal peptide, a membrane anchor, FIVAR domains, immunoglobulin Ig-like and discoidin-like domains. The other three genes, termed bbgI, bbgII and bbgIV, encoded proteins of 1,291, 689 and 1,052 a.a. residues, respectively, which were most probably intracellularly located. Two cases of protein evolution between strains of the same species were identified when the a.a. sequences of the BbgI and BbgIII were compared with homologous proteins from B. bifidum DSM20215. The homologous proteins were found to be differentiated at the C-terminal a.a. part either due to a single nucleotide insertion or to a whole DNA sequence insertion, respectively. The bbgIV gene was located in a gene organisation surrounded by divergently transcribed genes putatively for sugar transport (galactoside-symporter) and gene regulation (LacI-transcriptional regulator), a structure that was found to be highly conserved in B. longum, B. adolescentis and B. infantis, suggesting optimal organisation shared amongst those species.

Keywords: B. bifidum ; Bifidobacteria; Galactosidase; Beta-galactosidase; Protein evolution

Bioconversion of 12-, 14-, and 16-membered ring aglycones to glycosylated macrolides in an engineered strain of Streptomyces venezuelae by Won Seok Jung; Ah Reum Han; Jay Sung Joong Hong; Sung Ryeol Park; Cha Yong Choi; Je Won Park; Yeo Joon Yoon (pp. 1373-1381).

To develop a system for combinatorial biosynthesis of glycosylated macrolides, Streptomyces venezuelae was genetically manipulated to be deficient in the production of its macrolide antibiotics by deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of thymidine diphosphate (TDP)-d-quinovose or TDP-d-olivose in conjunction with the glycosyltransferase–auxiliary protein pair DesVII/DesVIII derived from S. venezuelae were expressed in the mutant strain. Feeding the representative 12-, 14-, and 16-membered ring macrolactones including 10-deoxymethynolide, narbonolide, and tylactone, respectively, to each mutant strain capable of producing TDP-d-quinovose or TDP-d-olivose resulted in the successful production of the corresponding quinovose- and olivose-glycosylated macrolides. In mutant strains where the DesVII/DesVIII glycosyltransferase–auxiliary protein pair was replaced by TylMII/TylMIII derived from Streptomyces fradiae, quinovosyl and olivosyl tylactone were produced; however, neither glycosylated 10-deoxymethynolide nor narbonolide were generated, suggesting that the glycosyltransferase TylMII has more stringent substrate specificity toward its aglycones than DesVII. These results demonstrate successful generation of structurally diverse hybrid macrolides using a S. venezuelae in vivo system and provide further insight into the substrate flexibility of glycosyltransferases.

Keywords: Combinatorial biosynthesis; Streptomyces; Macrolide; Glycosyltransferase; Auxiliary protein

Development of a yeast biosensor–biocatalyst for the detection and biodegradation of the organophosphate paraoxon by David A. Schofield; Caroline Westwater; Jeremy L. Barth; Augustine A. DiNovo (pp. 1383-1394).

Organophosphate (OP) poisoning can occur through unintentional exposure to OP pesticides, or by the deliberate release of OP nerve agents. Consequently, there is considerable interest in the development of systems that can detect and/or biodegrade these agents. The aim of this study was to generate a prototype fluorescent reporter yeast biosensor that could detect and biodegrade the model OP pesticide, paraoxon, and subsequently detect paraoxon hydrolysis. Saccharomyces cerevisiae was engineered to hydrolyze paraoxon through the heterologous expression of the Flavobacterium species opd (organophosphate degrading) gene. Global transcription profiling was subsequently used to identify yeast genes, which were induced in the presence of paraoxon, and genes, which were associated with paraoxon hydrolysis. Paraoxon-inducible genes and genes associated with paraoxon hydrolysis were identified. Candidate paraoxon-inducible promoters were cloned and fused to the yeast-enhanced green fluorescent protein (yEGFP), and candidate promoters associated with paraoxon hydrolysis were fused to the red fluorescent protein (yDsRed). The ability of the yeast biosensor to detect paraoxon and paraoxon hydrolysis was demonstrated by the specific induction of the fluorescent reporter (yEGFP and yDsRed, respectively). Biosensors responded to paraoxon in a dose- and time-dependent manner, and detection was rapid (15 to 30 min). yDsRed induction occurred only in the recombinant opd ⁺ strains suggesting that yDsRed induction was strictly associated with paraoxon hydrolysis. Together, these results indicate that the yeast biocatalyst–biosensor can detect and degrade paraoxon and potentially also monitor the decontamination process.

Functional analysis of the pBC1 replicon from Bifidobacterium catenulatum L48 by Pablo Álvarez-Martín; Mary O’Connell-Motherway; Douwe van Sinderen; Baltasar Mayo (pp. 1395-1402).

To determine the minimal replicon of pBC1 (a 2.5-kb cryptic plasmid of Bifidobacterium catenulatum L48) and to check the functionality of its identified open reading frames (ORFs) and surrounding sequences, different segments of pBC1 were amplified by polymerase chain reaction (PCR) and cloned into pBif, a replication probe vector for bifidobacteria. The largest fragment tested in this manner encompassed most of the pBC1 sequence, while the shortest just included the repB gene and its immediate upstream sequences. Derivatives were all shown to allow replication in bifidobacteria. Surprisingly, both the transformation frequency and segregational stability in the absence of antibiotic selection decreased with reducing plasmid length. The relative copy number of the constructs (ranging from around 3 to 23 copies per chromosome equivalent, as compared to 30 copies for the original pBC1) was shown to be strain dependent and to decrease with reducing plasmid length. These results suggest that, although not essential, the copG-like and orfX-like genes of pBC1 play important roles in pBC1 replication. Interruption of repB produced a construct incapable of replicating in bifidobacteria. The analysis of pBC1 will allow its use in the construction of general and specific cloning vectors.

Keywords: Plasmid; Bifidobacteria; Probiotics; Bifidobacterium catenulatum ; Cloning vectors

Sequencing and expression analysis of sakacin genes in Lactobacillus curvatus strains by Luca Cocolin; Kalliopi Rantsiou (pp. 1403-1411).

In this study, we focused our investigation on two strains of Lactobacillus curvatus, L442 and LTH1174, which are able to produce bacteriocins. L. curvatus LTH1174 is widely studied for its capability to produce curvacin A, while L. curvatus L442 was isolated from traditional Greek fermented sausages and was shown to possess a strong inhibitory activity toward Listeria monocytogenes. By polymerase chain reaction, we were able to target in both strains the genes for the production of sakacin P and sakacin Q, sppA and sppQ, respectively, both encoded chromosomally. While sppA was found to be conserved when compared with other sakacin P genes, sppQ showed a deletion of about 15 nucleotides when aligned with sequences obtained from Lactobacillus sakei. This difference did not affect the activity of sakacin Q as determined by testing sensitive strains. Expression analysis highlighted that sakacin P was expressed in L. curvatus L442 but not in L. curvatus LTH1174. Curing experiments were performed on L. curvatus LTH1174 to study the effect of the megaplasmid, present in this strain. In the plasmid-cured strain, expression of the sppA gene was detected. sppQ was expressed in both plasmid-cured and wild-type L. curvatus LTH1174, although expression was higher in the plasmid-cured strain.

Keywords: Bacteriocins; Expression analysis; Gene sequencing; Bioprotection

A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of Escherichia coli by Mario Kraft; Uwe Knüpfer; Rolf Wenderoth; André Kacholdt; Patricia Pietschmann; Björn Hock; Uwe Horn (pp. 1413-1422).

The functional analysis of individual proteins or of multiprotein complexes—since the completion of several genome sequencing projects—is in focus of current scientific work. Many heterologous proteins contain disulfide-bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degP-promoter-based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies.

Adaptation of Rhodococcus erythropolis cells to high concentrations of toluene by Carla C. C. R. de Carvalho; Vanessa Fatal; Sebastião S. Alves; M. Manuela R. da Fonseca (pp. 1423-1430).

Cells of Rhodococcus erythropolis DCL14 were adapted to increasing toluene concentrations in a mechanically stirred reactor. When the initial non-adapted cells were placed in contact with toluene, only 10.5% of cells remained viable after 1 h in the presence of 20% (v/v) toluene, while 8.6% of cells were viable after 28 h in the presence of an organic phase containing 80% (v/v) toluene in n-dodecane. Cell adaptation was studied by following the toluene consumption rate, the viability of the cell population, and the composition of the bacteria cellular membrane in the presence of increasing concentrations of toluene in the reactor. A maximum toluene concentration of 4.9 M, which corresponds to 52.4% (v/v) toluene in the organic phase, was achieved, toluene being consumed at 10.7 mg/(h mg protein). The adapted cells showed a substantially increased resistance to 50% ethanol and to concentrations of Betadine® and Micropur® tablets currently used in water purification, when compared to non-adapted cells.

Keywords: Xenobiotic; Bioremediation; Degradation; Xylene; Toluene; Adaptation; Tolerance

A new kinetic approach to microbial storage process by Bing-Jie Ni; Han-Qing Yu (pp. 1431-1438).

In this work, a new kinetic approach was proposed to describe the microbial growth, substrate consumption, and formation and utilization of the intracellular storage products (X _STO) in activated sludge. It was found that the formation of X _STO was coupled with energy generation and respiration and that the X _STO formation rate was proportional to the substrate utilization rate. A high amount of external substrate resulted in a relatively rapid storage process with a large fraction of substrate electrons for X _STO formation. The maximum growth rate of active biomass on X _STO and the yield coefficient for growth on the storage polymers were estimated as 0.12 h⁻¹ and 0.60 g chemical oxygen demand (COD) X g⁻¹ COD_STO, respectively. This established model was verified with the experimental results from two different case studies with pure and mixed cultures. Results showed that this kinetic model was able to accurately and mechanistically describe the microbial storage processes.

Self-excreted mediator from Escherichia coli K-12 for electron transfer to carbon electrodes by Yung-Fu Wang; Seiya Tsujimura; Sheng-Shung Cheng; Kenji Kano (pp. 1439-1446).

Escherichia coli K-12 was cultured under anaerobic conditions to form biofilm on carbon fiber electrodes in glucose-containing medium. The anodic current increased with the development of the biofilm and depended on the glucose concentration. Cyclic voltammetric results support the presence of a redox compound(s) excreted from E. coli cells in the biofilm. The compound remained in the film under conditions of continuous flow and gave a couple of oxidation and reduction waves, which may be assigned to a menaquinone-like compound based on the mid-point potential (−0.22 V vs Ag|AgCl at pH 7.1) and its pH dependence. The catalytic current started to increase around the anodic peak potential of the redox compound and also increased by the permeabilization of the E. coli cell membranes with ethylenediamine tetraacetic acid-treatment. The results indicate that the E. coli-excreted redox compound works as a mediator for the electron transfer from the E. coli cells to the electrode as the final electron acceptor. The activity of the redox compound in the E. coli-biofilm as a mediator with some mobility was also verified for diaphorase-catalyzed electrochemical oxidation of NADH.

Keywords: Electron transfer mediator; Escherichia coli K-12; Biofilm; Endogenous redox compound; Bioanode; Glucose oxidation

Benzoate-driven dehalogenation of chlorinated ethenes in microbial cultures from a contaminated aquifer by Michael Bunge; Jutta Kleikemper; Ciro Miniaci; Laurence Duc; Margje G. Muusse; Gerd Hause; Josef Zeyer (pp. 1447-1456).

Microbial dehalogenation of tetrachloroethene (PCE) and cis-dichloroethene (cis-DCE) was studied in cultures from a continuous stirred tank reactor initially inoculated with aquifer material from a PCE-contaminated site. Cultures amended with hydrogen and acetate readily dechlorinated PCE and cis-DCE; however, this transformation was incomplete and resulted in the accumulation of chlorinated intermediates and only small amounts of ethene within 60 days of incubation. Conversely, microbial PCE and cis-DCE dechlorination in cultures with benzoate and acetate resulted in the complete transformation to ethene within 30 days. Community fingerprinting by denaturing gradient gel electrophoresis (DGGE) revealed the predominance of phylotypes closely affiliated with Desulfitobacterium, Dehalococcoides, and Syntrophus species. The Dehalococcoides culture VZ, obtained from small whitish colonies in cis-DCE dechlorinating agarose cultures, revealed an irregular cell diameter between 200 and 500 nm, and a spherical or biconcave disk-shaped morphology. These organisms were identified as responsible for the dechlorination of cis-DCE to ethene in the PCE-dechlorinating consortia, operating together with the Desulfitobacterium as PCE-to-cis-DCE dehalogenating bacterium and with a Syntrophus species as potential hydrogen-producing partner in cultures with benzoate.

Keywords: Reductive dechlorination; PCE; Benzoate; Dehalococcoides ; Desulfitobacterium ; Syntrophus

Ammonium removal performance of anaerobic ammonium-oxidizing bacteria immobilized in polyethylene glycol gel carrier by Kazuichi Isaka; Yasuhiro Date; Tatsuo Sumino; Satoshi Tsuneda (pp. 1457-1465).

Anaerobic ammonium-oxidizing (anammox) bacteria were immobilized in polyethylene glycol gel carriers. A small amount of seed sludge [0.24% (w/v)] was entrapped in the carriers, and continuous feeding tests were performed. Nitrogen removal activity increased gradually, reaching 3.7 kg N/m³ reactor per day on day 67. The average of nitrogen conversion rate was calculated as 3.4 kg N/m³ reactor per day. Microscopic examination clearly showed that small red clusters formed in the gel carrier. Moreover, fluorescence in situ hybridization analysis revealed that these clusters consisted of anammox bacteria. From real-time polymerase chain reaction analysis, the growth of anammox bacteria in the gel carriers was clearly shown by increased concentration of 16S rRNA gene of planctomycete from 4.3 × 10⁸ to 4.2 × 10⁹ copies/ml between days 41 and 55. To determine the effects of inoculation on the start-up of the reactor, the amount of seed sludge in the gel carrier was varied and it was found that the start-up period could be reduced to as little as 25 days when a sludge concentration of 1.4% (w/v) was used. This is the first report of successful immobilization and cultivation of anammox bacteria in a gel carrier.

Keywords: Immobilization; Anammox; Ammonium; Nitrite; Nitrogen removal; Gel entrapment

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Applied Microbiology and Biotechnology (v.76, #6)

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Applied Microbiology and Biotechnology (v.76, #6)