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Applied Microbiology and Biotechnology (v.94, #1)

On the evolutionary ecology of symbioses between chemosynthetic bacteria and bivalves by Guus Roeselers; Irene L. G. Newton (pp. 1-10).

Mutualistic associations between bacteria and eukaryotes occur ubiquitously in nature, forming the basis for key ecological and evolutionary innovations. Some of the most prominent examples of these symbioses are chemosynthetic bacteria and marine invertebrates living in the absence of sunlight at deep-sea hydrothermal vents and in sediments rich in reduced sulfur compounds. Here, chemosynthetic bacteria living in close association with their hosts convert CO₂ or CH₄ into organic compounds and provide the host with necessary nutrients. The dominant macrofauna of hydrothermal vent and cold seep ecosystems all depend on the metabolic activity of chemosynthetic bacteria, which accounts for almost all primary production in these complex ecosystems. Many of these enigmatic mutualistic associations are found within the molluscan class Bivalvia. Currently, chemosynthetic symbioses have been reported from five distinct bivalve families (Lucinidae, Mytilidae, Solemyidae, Thyasiridae, and Vesicomyidae). This brief review aims to provide an overview of the diverse physiological and genetic adaptations of symbiotic chemosynthetic bacteria and their bivalve hosts.

Keywords: Clams; Hydrothermal vents; Methane; Sulfur; Oxidizing bacterial symbionts; Chemoautotrophy

New developments in the trace analysis of organic water pollutants by Klaus Fischer; Elke Fries; Wolfgang Körner; Christina Schmalz; Christian Zwiener (pp. 11-28).

Challenging tasks, increasing demands, and new generations of powerful analytical instruments initiated considerable progress in aquatic environmental analysis and led to a considerable improvement of analytical performance during the last few years. The ever growing number of emerging pollutants is tackled by specific and highly sensitive analytical methods with detection limits of a few nanogram per liter and even lower. Wide-scope monitoring techniques and multiclass and multiresidue analysis allow for the simultaneous determination of hundreds of compounds. The high mass resolution capability and mass accuracy of advanced mass spectrometric instruments, i.e., time-of-flight (TOF) MS or Fourier transform (FT)–Orbitrap MS, enable combined target and non-target analysis, including the identification of metabolites and abiotic degradation products. This minireview highlights some of the most recent developments in the trace analysis of important organic water pollutants and focuses on some specific groups of emerging contaminants, i.e., pharmaceuticals, flame retardants, disinfection by-products, surfactants, per- and polyfluorinated compounds, benzotriazoles, and benzothiazoles, as well as on the identification of transformation products and on non-target analysis. References were selected according to their exemplary and innovative character and to their practical relevance.

Keywords: Water analysis; Emerging pollutants; Pharmaceuticals; Flame retardants; Disinfection by-products; Perfluorinated compounds; Benzotriazoles; Transformation products; Multiresidue analysis; Non-target analysis

Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates by Nedeljka N. Rosic (pp. 29-37).

Mycosporine-like amino acids (MAAs) are multifunctional secondary metabolites involved in photoprotection in many marine organisms. As well as having broad ultraviolet (UV) absorption spectra (310–362 nm), these biological sunscreens are also involved in the prevention of oxidative stress. More than 20 different MAAs have been discovered so far, characterized by distinctive chemical structures and a broad ecological distribution. Additionally, UV-screening MAA metabolites have been investigated and used in biotechnology and cosmetics. The biosynthesis of MAAs has been suggested to occur via either the shikimate or pentose phosphate pathways. Despite their wide distribution in marine and freshwater species and also the commercial application in cosmetic products, there are still a number of uncertainties regarding the genetic, biochemical, and evolutionary origin of MAAs. Here, using a transcriptome-mining approach, we identify the gene counterparts from the shikimate or pentose phosphate pathway involved in MAA biosynthesis within the sequences of the reef-building coral symbiotic dinoflagellates (genus Symbiodinium). We also report the highly similar sequences of genes from the proposed MAA biosynthetic pathway involved in the metabolism of 4-deoxygadusol (direct MAA precursor) in various Symbiodinium strains confirming their algal origin and conserved nature. Finally, we reveal the separate identity of two O-methyltransferase genes, possibly involved in MAA biosynthesis, as well as nonribosomal peptide synthetase and adenosine triphosphate grasp homologs in symbiotic dinoflagellates. This study provides a biochemical and phylogenetic overview of the genes from the proposed MAA biosynthetic pathway with a focus on coral endosymbionts.

Keywords: UV; MAAs; Symbiodinium ; Zooxanthellae; Coral–algal symbiosis; Coral bleaching

Dengue virus-like particles: construction and application by Weilong Shang; Jie Liu; Jie Yang; Zhen Hu; Xiancai Rao (pp. 39-46).

Virus-like particles (VLPs) are shell-like viruses that lack virus-specific genetic materials. Many viral-structured proteins can assemble into VLPs, which mimic the overall structure of virus particles and can elicit strong immune responses in a host. Dengue viruses (DENVs), from the genus Flavivirus, are transmitted to humans through the bites of an infected Aedes mosquito. DENVs cause several diseases that prevailed mainly in tropical and subtropical areas. However, effective treatment measures and preventive strategies for dengue diseases are still lacking. The present minireview summarized the assembly and maturation of DENVs, the strategies and effective factors for dengue VLP construction, and the application of DENV VLPs.

Keywords: Dengue virus; Virus-like particles; Vaccine; Adjutant; Antigen

Red mold, diabetes, and oxidative stress: a review by Yeu-Ching Shi; Tzu-Ming Pan (pp. 47-55).

Type 2 diabetes is a major health concern and a rapidly growing disease with a modern etiology, which produces significant morbidity and mortality. The optimal management of type 2 diabetes aims to control hyperglycemia, hypertension, and dyslipidemia to reduce overall risks. Diabetes and its complications usually develop as oxidative stress increases. Monascus-fermented rice, also called red mold rice or red mold dioscorea are used in China to enhance food color and flavor. Red mold-fermented products are popular health foods that are considered to have antiobesity, antifatigue, antioxidation, and cancer prevention effects. This review article describes the antidiabetic and antioxidative stress effects on humans and animals of red mold-fermented products or their secondary metabolites.

Keywords: Monascus ; Red mold; Secondary metabolite; Diabetes; Oxidative stress

Quercetin treatment changes fluxes in the primary metabolism and increases culture longevity and recombinant α₁-antitrypsin production in human AGE1.HN cells by Jens Niklas; Yannic Nonnenmacher; Thomas Rose; Volker Sandig; Elmar Heinzle (pp. 57-67).

Addition of the flavonoid quercetin to cultivations of the α₁-antitrypsin (A1AT) producing human AGE1.HN.AAT cell line resulted in alterations of the cellular physiology and a remarkable improvement of the overall performance of these cells. In a first screening in 96-well plate format, toxicity and the effect of quercetin on the lactate/glucose ratio was analyzed. It was found that quercetin treatment reduced the lactate/glucose ratio dose dependently. An increase in culture longevity, viable cell density (160% of control), and A1AT concentration (from 0.39 g/L in the control to 0.76 g/L with quercetin, i.e., 195% of the control) was observed in batch cultivation with 10 μM quercetin compared to the control. A detailed analysis of quercetin effects on primary metabolism revealed dose-dependent alterations in metabolic fluxes. Quercetin addition resulted in an improved channeling of pyruvate into the mitochondria accompanied by reduced waste product formation and stimulation of TCA cycle activity. The observed changes in cellular physiology can be explained by different properties of quercetin and its metabolites, e.g., inhibition of specific enzymes, stimulation of oxidation of cytoplasmic, and mitochondrial NADH resulting in reduced NADH/NAD⁺ ratio, and cytoprotective activity. The present study shows that the addition of specific effectors to the culture medium represents a promising strategy to improve the cellular metabolic phenotype and the production of biopharmaceuticals. The provided results contribute, additionally, to an improved understanding of quercetin action on the metabolism of human cells in a general physiological context.

Keywords: Flavonoid; Biopharmaceutical; Therapeutic protein; Metabolic flux; Neuronal cell; Mammalian cell

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells by Masayoshi Onitsuka; Wook-Dong Kim; Hiroyuki Ozaki; Akira Kawaguchi; Kohsuke Honda; Hiroyuki Kajiura; Kazuhito Fujiyama; Ryutaro Asano; Izumi Kumagai; Hisao Ohtake; Takeshi Omasa (pp. 69-80).

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.

Keywords: Chinese hamster ovary (CHO) cell; Antibody production; Glycosylation control; Sialylation; Beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I); Bispecific diabody

Perstraction of intracellular pigments by submerged cultivation of Monascus in nonionic surfactant micelle aqueous solution by Zhiqiang Hu; Xuehong Zhang; Zhenqiang Wu; Hanshi Qi; Zhilong Wang (pp. 81-89).

“Milking processing” describes the cultivation of microalgae in a water-organic solvent two-phase system that consists of simultaneous fermentation and secretion of intracellular product. It is usually limited by the conflict between the biocompatibility of the organic solvent to the microorganisms and the ability of the organic solvent to secret intracellular product into its extracellular broth. In the present work, submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle aqueous solution for pigment production is exploited, in which the fungus Monascus remains actively growing. Permeabilization of intracellular pigments across the cell membrane and extraction of the pigments to the nonionic surfactant micelles of its fermentation broth occur simultaneously. “Milking” the intracellular pigments in the submerged cultivation of Monascus is a perstraction process. The perstractive fermentation of intracellular pigments has the advantage of submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizing the pigments into nonionic surfactant micelles. It is shown as the marked increase of the extracellular pigment concentration by the submerged cultivation of Monascus in the nonionic surfactant Triton X-100 micelle solution.

Keywords: Perstraction; Extractive fermentation; Nonionic surfactant; Intracellular product; Monascus pigments

Modelling growth of, and removal of Zn and Hg by a wild microalgal consortium by Cristina M. Monteiro; Teresa R. S. Brandão; Paula M. L. Castro; F. Xavier Malcata (pp. 91-100).

Microorganisms isolated from sites contaminated with heavy metals usually possess a higher removal capacity than strains from regular cultures. Heavy metal-containing soil samples from an industrial dumpsite in Northern Portugal were accordingly collected; following enrichment under metal stress, a consortium of wild microalgae was obtained. Their ability to grow in the presence of, and their capacity to recover heavy metals was comprehensively studied; the datasets thus generated were fitted to by a combined model of biomass growth and metal uptake, derived from first principles. After exposure to 15 and 25 mg/L Zn²⁺ for 6 days, the microalgal consortium reached similar, or higher cell density than the control; however, under 50 and 65 mg/L Zn²⁺, 71% to 84% inhibition was observed. Growth in the presence of Hg²⁺ was significantly inhibited, even at a concentration as low as 25 μg/L, and 90% inhibition was observed above 100 μg/L. The maximum amount of Zn²⁺ removed was 21.3 mg/L, upon exposure to 25 mg/L for 6 day, whereas the maximum removal of Hg²⁺ was 335 μg/L, upon 6 day in the presence of 350 μg/L. The aforementioned mechanistic model was built upon Monod assumptions (including heavy metal inhibition), coupled with Leudeking–Piret relationships between the rates of biomass growth and metal removal. The overall fits were good under all experimental conditions tested, thus conveying a useful tool for rational optimisation of microalga-mediated bioremediation.

Keywords: Heavy metals; Metal uptake; Toxicity; Bioremediation; Monod model; Leudeking–Piret model

LysA2, the Lactobacillus casei bacteriophage A2 lysin is an endopeptidase active on a wide spectrum of lactic acid bacteria by Pedro Ribelles; Isabel Rodríguez; Juan Evaristo Suárez (pp. 101-110).

The lysin gene (lysA2) of the Lactobacillus casei bacteriophage A2 was cloned and expressed in Escherichia coli. LysA2 is an endopeptidase that hydrolyzes the bond between the terminal d-alanine of the peptidoglycan tetrapeptide and the aspartic acid residue that forms the bridge with the l-lysine of a neighboring peptidoglycan chain, characteristic of Gram-positive bacteria included into the A4 peptidoglycan subgroup. This includes most lactobacilli, Lactococcus lactis, Pediococcus acidilactici, and Pediococcus pentosaceus, the walls of all of which were substrates for the enzyme. Specific binding of LysA2 to the wall of these bacteria is mediated by its C-terminal moiety, does not need the N-terminal catalytic domain for recognition, and is stable: at least 88% of the molecules were still bound to L. casei after 3 days in phosphate buffer at 4°C. The enzyme acts as a monomer, is active at pH values between 4 and 6, and at temperatures ranging between 18°C and 50°C while being independent of divalent cation addition. The enzyme showed strong resistance to incubation at high and low pH values but became progressively inactivated at 50°C and above. LysA2 is bactericidal, the viability of L. casei cultures dropping to 1% in 10 min, under the standard conditions used for the enzymatic assay.

Keywords: Bacteriophage; Lactic acid bacteria; Lactobacillus ; Lysin; Peptidoglycan

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-d-glucanase by Jian-Wen Huang; Ya-Shan Cheng; Tzu-Ping Ko; Cheng-Yen Lin; Hui-Lin Lai; Chun-Chi Chen; Yanhe Ma; Yingying Zheng; Chun-Hsiang Huang; Peijian Zou; Je-Ruei Liu; Rey-Ting Guo (pp. 111-121).

1,3-1,4-β-d-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-d-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

Keywords: Cellobiose; Cellotetraose; β-Glucanase; Crystal structure; Synchrotron radiation

The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase by Hernán Costa; Ana Julia Distéfano; Cristina Marino-Buslje; Aurelio Hidalgo; José Berenguer; Mirtha Biscoglio de Jiménez Bonino; Susana Alicia Ferrarotti (pp. 123-130).

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in biotechnology because of their ability to produce cyclodextrin (CD) mixtures from starch whose relative composition depends on enzyme source. A multiple alignment of 46 CGTases and Shannon entropy analysis allowed us to find differences and similarities that could be related to product specificity. Interestingly, position 179 has Gly in all the CGTases except in that from Bacillus circulans DF 9R which possesses Gln. The absence of a side chain at that position has been considered as a strong requirement for substrate binding and cyclization process. Therefore, we constructed two mutants of this enzyme, Q179L and Q179G. The activity and kinetic parameters of Q179G remained unchanged while the Q179L mutant showed a different CDs ratio, a lower catalytic efficiency, and a decreased ability to convert starch into CDs. We show that position 179 is involved in CGTase product specificity and must be occupied by Gly—without a side chain—or by amino acid residues able to interact with the substrate through hydrogen bonds in a way that the cyclization process occurs efficiently. These findings are also explained on the basis of a structural model.

Keywords: CGTase; Product specificity; Structural aspects; Bacillus circulans ; Directed mutagenesis; Cyclodextrins

Engineering pH-tolerant mutants of a cyanide dihydratase by Lan Wang; Jean M. Watermeyer; Andani E. Mulelu; B. Trevor Sewell; Michael J. Benedik (pp. 131-140).

Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynD_pum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynD_pum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.

Keywords: Nitrilase; Cyanide; Bioremediation; Cyanide dihydratase; pH tolerance; Protein stability

The short form of the recombinant CAL-A-type lipase UM03410 from the smut fungus Ustilago maydis exhibits an inherent trans-fatty acid selectivity by Henrike Brundiek; Stefan Saß; Andrew Evitt; Robert Kourist; Uwe T. Bornscheuer (pp. 141-150).

The Ustilago maydis lipase UM03410 belongs to the mostly unexplored Candida antarctica lipase (CAL-A) subfamily. The two lipases with […] the highest identity are a lipase from Sporisorium reilianum and the prototypic CAL-A. In contrast to the other CAL-A-type lipases, this hypothetical U. maydis lipase is annotated to possess a prolonged N-terminus of unknown function. Here, we show for the first time the recombinant expression of two versions of lipase UM03410: the full-length form (lipUMf) and an N-terminally truncated form (lipUMs). For comparison to the prototype, the expression of recombinant CAL-A in E. coli was investigated. Although both forms of lipase UM03410 could be expressed functionally in E. coli, the N-terminally truncated form (lipUMs) demonstrated significantly higher activities towards p-nitrophenyl esters. The functional expression of the N-terminally truncated lipase was further optimized by the appropriate choice of the E. coli strain, lowering the cultivation temperature to 20 °C and enrichment of the cultivation medium with glucose. Primary characteristics of the recombinant lipase are its pH optimum in the range of 6.5–7.0 and its temperature optimum at 55 °C. As is typical for lipases, lipUM03410 shows preference for long chain fatty acid esters with myristic acid ester (C14:0 ester) being the most preferred one. More importantly, lipUMs exhibits an inherent preference for C18:1Δ9 trans and C18:1Δ11 trans-fatty acid esters similar to CAL-A. Therefore, the short form of this U. maydis lipase is the only other currently known lipase with a distinct trans-fatty acid selectivity.

Keywords: Ustilago maydis ; Secreted fungal lipase; Transmembrane domain; CAL-A type; Trans-fatty acid selectivity

Production, characterisation and immunogenicity of a plant-made Plasmodium antigen—the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 by Charles Ma; Lina Wang; Diane E. Webster; Alison E. Campbell; Ross L. Coppel (pp. 151-161).

Development of a safe, effective and affordable malaria vaccine is central to global disease control efforts. One of the most highly regarded proteins for inclusion in an asexual blood stage subunit vaccine is the 19-kDa C-terminal fragment of merozoite surface protein 1 (MSP1₁₉). As production of vaccine antigens in plants can potentially overcome cost and delivery hurdles, we set out to produce MSP1₁₉ in plants, characterise the protein and test its immunogenicity using a mouse model. Plasmodium yoelii MSP1₁₉ (PyMSP1₁₉) was produced in Nicotiana benthamiana using the MagnICON® deconstructed TMV-based viral vector. PyMSP1₁₉ yield of at least 23% total soluble protein (TSP;3–4 mg/g Fwt) were achieved using a codon-optimised construct that was targeted to the apoplast. Freeze-dried leaf powder contained at least 20 mg PyMSP1₁₉ per gram dry weight and the protein retained immunogenicity in this form for more than 2 years. Characterisation studies, including SDS-PAGE, mass spectrometry and circular dichroism, indicated that the plant-expressed PyMSP1₁₉ was similar to its Escherichia coli- and Saccharomyces cerevisiae-expressed counterparts. Purified plant-made PyMSP1₁₉ induced strong immune responses following intraperitoneal immunisation, although titres were lower than those induced by an equivalent dose of purified E. coli-expressed PyMSP1₁₉. The reason for this is uncertain but may be due to differences in the oligomerisation profile of the vaccines. The plant-made PyMSP1₁₉ vaccine was also found to be orally immunogenic when delivered alone or following immunisation with a PyMSP1₁₉ DNA vaccine. This study adds to an increasing body of research supporting the feasibility of plants as both a factory for the production of malaria antigens, and as a safe and affordable platform for oral delivery of a temperature-stable malaria vaccine.

Keywords: Malaria; Plasmodium yoelii ; Plant-made vaccines; PyMSP1₁₉

Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties by Antonio Valle; Sylvie Le Borgne; Jorge Bolívar; Gema Cabrera; Domingo Cantero (pp. 163-171).

Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2′-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% respectively).

Keywords: Biotransformation; Escherichia coli ; Benzohydroxamic acids; Nitroreductases NfsA and NfsB; Flavin reductase NemA; D-DIBOA

Ixr1p and the control of the Saccharomyces cerevisiae hypoxic response by Ángel Vizoso-Vázquez; Mónica Lamas-Maceiras; Manuel Becerra; M. Isabel González-Siso; Esther Rodríguez-Belmonte; M. Esperanza Cerdán (pp. 173-184).

In Saccharomyces cerevisiae, adaptation to hypoxia/anaerobiosis requires the transcriptional induction or derepression of multiple genes organized in regulons controlled by specific transcriptional regulators. Ixr1p is a transcriptional regulatory factor that causes aerobic repression of several hypoxic genes (COX5B, TIR1, and HEM13) and also the activation of HEM13 during hypoxic growth. Analysis of the transcriptome of the wild-type strain BY4741 and its isogenic derivative Δixr1, grown in aerobic and hypoxic conditions, reveals differential regulation of genes related not only to the hypoxic and oxidative stress responses but also to the re-adaptation of catabolic and anabolic fluxes in response to oxygen limitation. The function of Ixr1p in the transcriptional regulation of genes from the sulfate assimilation pathway and other pathways producing α-keto acids is of biotechnological importance for industries based on yeast-derived fermentation products.

Keywords: Transcriptional regulation; Oxidative stress; Sulfate assimilation pathway

Conditional confined oscillatory dynamics of Escherichia coli strain K12-MG1655 in chemostat systems by Irina Dana Ofiţeru; Mariana Ferdeş; Charles W. Knapp; David W. Graham; Vasile Lavric (pp. 185-192).

A series of continuous- and sequencing-batch reactor experiments were performed to assess the growth dynamics of Escherichia coli strain K12-MG1655 in chemostat systems. Previous mathematical predictions and early experimental results had shown that confined oscillatory dynamics ensue in bioreactor populations, which relates to “group birth and death” events within the population. New results are reported here that generally verify the predictions of the model and show that confined oscillations occur under different initial conditions, but the characteristics of the oscillatory dynamics vary as a function of the hydraulic retention time (HRT). Bioreactors were operated at HRTs ranging from 2.7 to 35 h and, regardless of initial conditions or the imposition of transient operational instabilities, highly patterned oscillations developed when HRT was between ∼3 and 8 h. However, outside of this range, bioreactor populations tended to form biofilms on the reactor walls (although the majority of the cells remained suspended in the bulk solution) and stable oscillations were not seen in the bulk phase. This suggests that alternate operating “states” might exist in chemostat populations with biofilm formation and non-homogenous spatial growth influencing “system” dynamics at very low and high HRTs. Although the model accurately predicts a confined dynamic equilibrium for mid-range HRT operations, experimental data show that model predictions do not extend outside of this range, when an alternate stable-state seems to be attained.

Keywords: Confined dynamics; Escherichia coli ; Synchronicity; Optical density; Dissolved oxygen

Alteration in cell surface properties of Burkholderia spp. during surfactant-aided biodegradation of petroleum hydrocarbons by Sagarika Mohanty; Suparna Mukherji (pp. 193-204).

Chemical surfactants may impact microbial cell surface properties, i.e., cell surface hydrophobicity (CSH) and cell surface charge, and may thus affect the uptake of components from non-aqueous phase liquids (NAPLs). This work explored the impact of Triton X-100, Igepal CA 630, and Tween 80 (at twice the critical micelle concentration, CMC) on the cell surface characteristics of Burkholderia cultures, Burkholderia cepacia (ES1, aliphatic degrader) and Burkholderia multivorans (NG1, aromatic degrader), when grown on a six-component model NAPL. In the presence of Triton X-100, NAPL biodegradation was enhanced from 21% to 60% in B. cepacia and from 18% to 53% in B. multivorans. CSH based on water contact angle (50–52°) was in the same range for both strains while zeta potential at neutral pH was −38 and −31 mV for B. cepacia and B. multivorans, respectively. In the presence of Triton X-100, their CSH increased to greater than 75° and the zeta potential decreased. This induced a change in the mode of uptake and initiated aliphatic hydrocarbon degradation by B. multivorans and increased the rate of aliphatic hydrocarbon degradation in B. cepacia. Igepal CA 630 and Tween 80 also altered the cell surface properties. For B. cepacia grown in the presence of Triton X-100 at two and five times its CMC, CSH increased significantly in the log growth phase. Growth in the presence of the chemical surfactants also affected the abundance of chemical functional groups on the cell surface. Cell surface changes had maximum impact on NAPL degradation in the presence of emulsifying surfactants, Triton X-100 and Igepal CA630.

Keywords: Direct interfacial uptake; Contact angle; Cell surface charge; Zeta potential; Surface functional groups

Anaerobic xylose fermentation by Spathaspora passalidarum by X. Hou (pp. 205-214).

A cost-effective conversion of lignocellulosic biomass into bioethanol requires that the xylose released from the hemicellulose fraction (20–40% of biomass) can be fermented. Baker’s yeast, Saccharomyces cerevisiae, efficiently ferments glucose but it lacks the ability to ferment xylose. Xylose-fermenting yeast such as Pichia stipitis requires accurately controlled microaerophilic conditions during the xylose fermentation, rendering the process technically difficult and expensive. In this study, it is demonstrated that under anaerobic conditions Spathaspora passalidarum showed high ethanol production yield, fast cell growth, and rapid sugar consumption with xylose being consumed after glucose depletion, while P. stipitis was almost unable to utilize xylose under these conditions. It is further demonstrated that for S. passalidarum, the xylose conversion takes place by means of NADH-preferred xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Thus, the capacity of S. passalidarum to utilize xylose under anaerobic conditions is possibly due to the balance between the cofactor’s supply and demand through this XR–XDH pathway. Only few XRs with NADH preference have been reported so far. 2-Deoxy glucose completely inhibited the conversion of xylose by S. passalidarum under anaerobic conditions, but only partially did that under aerobic conditions. Thus, xylose uptake by S. passalidarum may be carried out by different xylose transport systems under anaerobic and aerobic conditions. The presence of glucose also repressed the enzymatic activity of XR and XDH from S. passalidarum as well as the activities of those enzymes from P. stipitis.

Keywords: Xylose fermentation; Cofactor balance; Xylose reductase; Xylitol dehydrogenase; Glucose repression

Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants by Yasuhiro Inoue; Seiji Tsuge (pp. 215-222).

We have designed a new vector- and marker-free site-directed deletion system for gram-negative bacteria. In this system, a specific DNA fragment is amplified from a parental strain by using polymerase chain reaction (PCR), then circularized and introduced back into the parental strain for homologous recombination. The recombinant mutant is then detected and isolated by PCR-based sib selection. Unlike conventional methods, our Simple Deletion method requires no cloning procedures, and no foreign genes such as antibiotic-resistance genes are introduced as selection markers. The resulting mutant is, therefore, the same as the parental strain except for the lack of the target region. This method is categorized as a type of “self-cloning,” and the resulting mutant can be used for laboratory research without restrictions. Using this method, we generated a mutant of a plant pathogenic bacterium, Xanthomonas campestris pv. campestris, in which the 20.4-kb hrp gene cluster involved in the type III secretion system and in pathogenicity was deleted. In addition, we proved that this method can also be used to delete smaller DNA regions of X. campestris pv. campestris and to generate deletion mutants of the bacterium Ralstonia solanacearum.

Keywords: Site-directed deletion method; Gram-negative bacteria; Self-cloning; Genetically modified organisms

Quantitative colorimetric measurement of cellulose degradation under microbial culture conditions by Rembrandt J. F. Haft; Jeffrey G. Gardner; David H. Keating (pp. 223-229).

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay “Congo Red Analysis of Cellulose Concentration,” or “CRACC.” CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications.

Keywords: Cellulase; Cellulose degradation; Enzyme secretion; Bioprocessing; Cellvibrio japonicus

Phosphorus removal characteristics of granular and flocculent sludge in SBR by Xing Li; Dawen Gao; Hong Liang; Lin Liu; Yuan Fu (pp. 231-236).

Aerobic granulation technology has become a novel biotechnology for wastewater treatment. However, the distinct properties and characteristics of phosphorus removal between granules and flocculent sludge are still sparse in enhanced biological phosphorus removal process. Two identical sequencing batch reactors (SBRs) were operated to compare phosphorus removal performance with granular sludge (R1) and flocculate activated sludge (R2). Results indicated that the start-up period was shorter in R2 than R1 for phosphorus removal, which made R2 reach the steady-state condition on day 21, while R1 was on day 25, and R2 released and took up more phosphorus than R1. As a result, the phosphorus removal was around 90% in R2 while 80% in R1 at the steady-state system. The special phosphorus release rate and special phosphorus uptake rate were 8.818 mg P/g volatile suspended solids (VSS)/h and 9.921 mg P/g VSS/h in R2, which were consistently greater than those (0.999 and 3.016 mg P/g VSS/h) in R1. The chemical oxygen demand removal in two reactors was similar. The granular SBR had better solid-separation performance and higher removal efficiency of NH ₄ ⁺ –N than flocculent SBR. Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragment analysis revealed that the diversity and the level of phosphorus-accumulating bacteria in flocculent sludge were much more than those in the granular sludge.

Keywords: Granular sludge; Flocculent sludge; Phosphorus removal; Sequencing batch reactors; Wastewater treatment

Intermediate accumulation of metabolites results in a bottleneck for mineralisation of the herbicide metabolite 2,6-dichlorobenzamide (BAM) by Aminobacter spp. by Allan Simonsen; Nora Badawi; Gitte G. Anskjær; Christian N. Albers; Sebastian R. Sørensen; Jan Sørensen; Jens Aamand (pp. 237-245).

Degradation and mineralisation of the groundwater contaminant 2,6-dichloro-benzamide (BAM) was investigated in two Aminobacter strains focussing on the induction of BAM degradation and mineralisation and occurrence of intermediate metabolites. The BAM degradation rate was independent of whether the cells were pre-grown in the absence or presence of BAM, thus indicating that the first step in the degradation pathway was constitutively expressed. In contrast, ¹⁴CO₂ production was stimulated when cells were pre-grown in the presence of BAM, suggesting that one or more of the subsequent steps in the degradation pathway were inducible. Accumulation of 2,6-dichlorobenzoate (DCBA) during degradation of BAM demonstrated that the first step involved amidase activity. Mass balance calculations and thin-layer chromatography coupled with autoradiographic detection indicated that degradation of DCBA and at least one unknown metabolite may comprise a bottleneck for BAM mineralisation by Aminobacter spp. The study thus provides novel information about the BAM degradation pathway and points to the involvement of unknown intermediate metabolites in degradation of this important groundwater contaminant.

Keywords: Aminobacter spp.; Metabolites; 2,6-Dichlorobenzoate; 2,6-Dichlorobenzamide

Community shift of ammonia-oxidizing bacteria along an anthropogenic pollution gradient from the Pearl River Delta to the South China Sea by Huiluo Cao; Yiguo Hong; Meng Li; Ji-Dong Gu (pp. 247-259).

The phylogenetic diversity and abundance of ammonia-oxidizing beta-proteobacteria (beta-AOB) was analyzed along an anthropogenic pollution gradient from the coastal Pearl River Delta to the South China Sea using the ammonia monooxygenase subunit A (amoA) gene. Along the gradient from coastal to the open ocean, the phylogenetic diversity of the dominant genus changed from Nitrosomonas to Nitrosospira, indicating the niche specificity by these two genera as both salinity and anthropogenic influence were major factors involved. The diversity of bacterial amoA gene was also variable along the gradient, with the highest in the deep-sea sediments, followed by the marshes sediments and the lowest in the coastal areas. Within the Nitrosomonas-related clade, four distinct lineages were identified including a putative new one (A5-16) from the different sites over the large geographical area. In the Nitrosospira-related clade, the habitat-specific lineages to the deep-sea and coastal sediments were identified. This study also provides strong support that Nitrosomonas genus, especially Nitrosomonas oligotropha lineage (6a) could be a potential bio-indicator species for pollution or freshwater/wastewater input into coastal environments. A suite of statistical analyses used showed that water depth and temperature were major factors shaping the community structure of beta-AOB in this study area.

Keywords: Ammonia monooxygenase α-subunit (amoA) gene; Ammonia-oxidizing bacteria (AOB); South China Sea; Anthropogenic pollution gradient

Identification of the flavin monooxygenase responsible for ipso substitution of alkyl and alkoxyphenols in Sphingomonas sp. TTNP3 and Sphingobium xenophagum Bayram by A. W. Porter; B. R. Campbell; B. A. Kolvenbach; P. F.-X. Corvini; D. Benndorf; G. Rivera-Cancel; A. G. Hay (pp. 261-272).

We previously showed that opdA from Sphingomonas sp. PWE1 encodes a putative flavin monooxygenase capable of transforming octylphenol (OP) via type II ipso substitution. Here, we demonstrate that an opdA homolog is responsible for OP and related alkyl/alkoxyphenol degradation in the nonylphenol degrader Sphingomonas sp. TTNP3. PCR and Southern blot analyses revealed that TTNP3 contained an opdA homolog, while a TTNP3 derivative unable to grow on nonylphenol (TTNP3d) did not. OpdA expression was confirmed in wild-type TTNP3 via two dimensional gel electrophoresis. Activity was restored to TTNP3d following complementation with opdA. Sequence analysis of an opdA homolog from another nonylphenol degrader, Sphingobium xenophagum Bayram, revealed that the predicted protein sequences from PWE1 and Bayram were identical, but differed from TTNP3 by four amino acids. In order to assess differences, we heterologously expressed the two unique opdA homologs and compared their effect on the disappearance of five alkyl/alkoxyphenol substrates and subsequent appearance of hydroquinone. For all substrates, except OP, the levels of substrate disappearance and hydroquinone appearance were significantly lower in cultures expressing odpA _TTNP3 than those expressing opdA _PWE1/Bayram. These differences in substrate specificity were consistent with an in silico model which predicted that two of the amino acid differences between odpA _TTNP3 and opdA _PWE1/Bayram lay in a putative substrate binding pocket. While these strains are known to use the same type II ipso substitution mechanism for alkylphenol degradation, this work provides the first preliminary evidence that opdA homologs also encode the type I ipso substitution activity responsible for the degradation of alkoxyphenols.

Keywords: Octylphenol monooxygenase; Ipso substitution; Alkylphenol biodegradation; Octylphenol

Improvement of tolerance of Saccharomyces cerevisiae to hot-compressed water-treated cellulose by expression of ADH1 by Lahiru N. Jayakody; Kenta Horie; Nobuyuki Hayashi; Hiroshi Kitagaki (pp. 273-283).

Hot-compressed water treatment of cellulose and hemicellulose for subsequent bioethanol production is a novel, economically feasible, and nonhazardous method for recovering sugars. However, the hot-compressed water-treated cellulose and hemicellulose inhibit subsequent ethanol fermentation by the yeast Saccharomyces cerevisiae. To overcome this problem, we engineered a yeast strain with improved tolerance to hot-compressed water-treated cellulose. We first determined that glycolaldehyde has a greater inhibitory effect than 5-HMF and furfural and a combinational effect with them. On the basis of the hypothesis that the reduction of glycolaldehyde to ethylene glycol should detoxify glycolaldehyde, we developed a strain overexpressing the alcohol dehydrogenase gene ADH1. The ADH1-overexpressing strain exhibits an improved fermentation profile in a glycolaldehyde-containing medium. The conversion ratio of glycolaldehyde to ethylene glycol is 30 ± 1.9% when the control strain is used; this ratio increases to 77 ± 3.6% in the case of the ADH1-overexpressing strain. A glycolaldehyde treatment and the overexpression of ADH1 cause changes in the fermentation products so as to balance the metabolic carbon flux and the redox status. Finally, the ADH1-overexpressing strain shows a statistically significantly improved fermentation profile in a hot-compressed water-treated cellulose-containing medium. The conversion ratio of glycolaldehyde to ethylene glycol is 33 ± 0.85% when the control strain is used but increases to 72 ± 1.7% in the case of the ADH1-overexpressing strain. These results show that the reduction of glycolaldehyde to ethylene glycol is a promising strategy to decrease the toxicity of hot-compressed water-treated cellulose. This is the first report on the improvement of yeast tolerance to hot-compressed water-treated cellulose and glycolaldehyde.

Keywords: Saccharomyces cerevisiae ; Furfural; 5-hydroxymethylfurfural; Glycolaldehyde; ADH1 ; Bioethanol

Erratum to: The short form of the recombinant CAL-A-type lipase UM03410 from the smut fungus Ustilago maydis exhibits an inherent trans-fatty acid selectivity by Henrike Brundiek; Stefan Saß; Andrew Evitt; Robert Kourist; Uwe T. Bornscheuer (pp. 285-285).

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Applied Microbiology and Biotechnology (v.94, #1)

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Applied Microbiology and Biotechnology (v.94, #1)