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Applied Microbiology and Biotechnology (v.72, #2)
Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems by Kay Terpe (pp. 211-222).
During the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. Bacterial systems remain most attractive due to low cost, high productivity, and rapid use. However, the rational choice of the adequate promoter system and host for a specific protein of interest remains difficult. This review gives an overview of the most commonly used systems: As hosts, Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Caulobacter crescentus, other strains, and, most importantly, Escherichia coli BL21 and E. coli K12 and their derivatives are presented. On the promoter side, the main features of the l-arabinose inducible araBAD promoter (PBAD), the lac promoter, the l-rhamnose inducible rhaP BAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter p L , and the anhydrotetracycline-inducible tetA promoter/operator are summarized.
Microbial degradation of nonylphenol and other alkylphenols—our evolving view by P. F. X. Corvini; A. Schäffer; D. Schlosser (pp. 223-243).
Because the endocrine disrupting effects of nonylphenol (NP) and octylphenol became evident, the degradation of long-chain alkylphenols (AP) by microorganisms was intensively studied. Most NP-degrading bacteria belong to the sphingomonads and closely related genera, while NP metabolism is not restricted to defined fungal taxa. Growth on NP and its mineralization was demonstrated for bacterial isolates, whereas ultimate degradation by fungi still remains unclear. While both bacterial and fungal degradation of short-chain AP, such as cresols, and the bacterial degradation of long-chain branched AP involves aromatic ring hydroxylation, alkyl chain oxidation and the formation of phenolic polymers seem to be preferential elimination pathways of long-chain branched AP in fungi, whereby both intracellular and extracellular oxidative enzymes may be involved. The degradation of NP by sphingomonads does not proceed via the common degradation mechanisms reported for short-chain AP, rather, via an unusual ipso-substitution mechanism. This fact underlies the peculiarity of long-chain AP such as NP isomers, which possess highly branched alkyl groups mostly containing a quaternary α-carbon. In addition to physicochemical parameters influencing degradation rates, this structural characteristic confers to branched isomers of NP a biodegradability different to that of the widely used linear isomer of NP. Potential biotechnological applications for the removal of AP from contaminated media and the difficulties of analysis and application inherent to the hydrophobic NP, in particular, are also discussed. The combination of bacteria and fungi, attacking NP at both the phenolic and alkylic moiety, represents a promising perspective.
Keywords: Bacteria; Degradation; Fungi; Laccase; Nonylphenol isomers; Sphingomonas
Biodegradability and biodegradation of poly(lactide) by Yutaka Tokiwa; Buenaventurada P. Calabia (pp. 244-251).
Poly(lactide) (PLA) has been developed and made commercially available in recent years. One of the major tasks to be taken before the widespread application of PLA is the fundamental understanding of its biodegradation mechanisms. This paper provides a short overview on the biodegradability and biodegradation of PLA. Emphasis is focused mainly on microbial and enzymatic degradation. Most of the PLA-degrading microorganisms phylogenetically belong to the family of Pseudonocardiaceae and related genera such as Amycolatopsis, Lentzea, Kibdelosporangium, Streptoalloteichus, and Saccharothrix. Several proteinous materials such as silk fibroin, elastin, gelatin, and some peptides and amino acids were found to stimulate the production of enzymes from PLA-degrading microorganisms. In addition to proteinase K from Tritirachium album, subtilisin, a microbial serine protease and some mammalian serine proteases such as α-chymotrypsin, trypsin, and elastase could also degrade PLA.
Keywords: Poly(lactide); Biodegradability; Biodegradation; Amycolatopsis; Protease; Lipase
Enzymes and genes involved in the aerobic biodegradation of methyl tert-butyl ether (MTBE) by Nicolas Lopes Ferreira; Cédric Malandain; Françoise Fayolle-Guichard (pp. 252-262).
Fuel oxygenates, mainly methyl tert-butyl ether (MTBE) but also ethyl tert-butyl ether (ETBE), are added to gasoline in replacement of lead tetraethyl to enhance its octane index. Their addition also improves the combustion efficiency and therefore decreases the emission of pollutants (CO and hydrocarbons). On the other hand, MTBE, being highly soluble in water and recalcitrant to biodegradation, is a major pollutant of water in aquifers contaminated by MTBE-supplemented gasoline during accidental release. MTBE was shown to be degraded through cometabolic oxidation or to be used as a carbon and energy source by a few microorganisms. We have summarized the present state of knowledge about the microorganisms involved in MTBE degradation and the MTBE catabolic pathways. The role of the different enzymes is discussed as well as the rare and recent data concerning the genes encoding the enzymes involved in the MTBE pathway. The phylogeny of the microorganisms isolated for their capacity to grow on MTBE is also described.
A multicomponent reaction–diffusion model of a heterogeneously distributed immobilized enzyme by J. L. van Roon; M. M. H. D. Arntz; A. I. Kallenberg; M. A. Paasman; J. Tramper; C. G. P. H. Schroën; H. H. Beeftink (pp. 263-278).
A physical model was derived for the synthesis of the antibiotic cephalexin with an industrial immobilized penicillin G acylase, called Assemblase. In reactions catalyzed by Assemblase, less product and more by-product are formed in comparison with a free-enzyme catalyzed reaction. The model incorporates reaction with a heterogeneous enzyme distribution, electrostatically coupled transport, and pH-dependent dissociation behavior of reactants and is used to obtain insight in the complex interplay between these individual processes leading to the suboptimal conversion. The model was successfully validated with synthesis experiments for conditions ranging from heavily diffusion limited to hardly diffusion limited, including substrate concentrations from 50 to 600 mM, temperatures between 273 and 303 K, and pH values between 6 and 9. During the conversion of the substrates into cephalexin, severe pH gradients inside the biocatalytic particle, which were previously measured by others, were predicted. Physical insight in such intraparticle process dynamics may give important clues for future biocatalyst design. The modular construction of the model may also facilitate its use for other bioconversions with other biocatalysts.
Comparative analysis of HIV-1 recombinant envelope glycoproteins from different culture systems by S A. Jeffs; S. Goriup; G. Stacey; C-T. Yuen; H. Holmes (pp. 279-290).
The productivity of stable Chinese hamster ovary cell lines secreting HIV-1 monomeric (IIIB gp120) and oligomeric (UG21 gp140) recombinant envelope glycoproteins was compared in serum-containing (S+), serum-free (S−) and protein-free (P−) culture media. UG21 gp140 expression was greatest in S+ medium, while IIIBgp120 production was lower than gp140 in all three media but highest in S−. UG21 gp140 production was highest in standard 850-cm2 roller bottle cultures in S+ media, peaking after 14 days of incubation, while expression levels in the three media were 0.5 (S+), 0.4 (S−) and 0.2 (P−) mg/l, from which 90, 80 and 12% of gp140, respectively, could be purified by immunoaffinity chromatography. Purified UG21 gp140 from S+ and S− media possessed biological functionality as evidenced by CD4 and monoclonal antibody (Mab) binding. In contrast, UG21 gp140 from P− medium appears to be misfolded and non-functional. Despite the possession of a different N-linked glycan profile, UG21 gp140 from S− media shows very similar CD4 and Mab binding characteristics to S+ UG21 gp140. The relevance of these findings to HIV vaccine development is discussed.
The utilization of sugar cane molasses with/without the presence of lignosulfonate for the production of bacterial cellulose by Sherif Keshk; Kazuhiko Sameshima (pp. 291-296).
Production of bacterial cellulose (BC) using sugar cane molasses (MO) with/without the presence of lignosulfonate (MOL) as a sole carbon source in a Hestrin–Schramm medium (HS) was investigated. Six strains of Acetobacter xylinum [American Type Culture Collection 10245 and Institute of Fermentation in Osaka (IFO) 13693, 13772, 13773, 14815, and 15237] were screened for their BC production. The yield of the BC among all the strains from both the MO and MOL media was much higher than that from the HS medium. Acetobacter xylinum IFO 13772 was the best BC producer for all media. Furthermore, physical properties of these BC from the HS, MO, and MOL media were studied using Fourier-transform infrared spectroscopy, X-ray diffractometer, and cross polarization/magic angle spinning 13C nuclear magnetic resonance. There are no significant differences in the crystallinity and the recorded I α fraction among the BC produced from the different media. A remarkable difference was only recorded in terms of viscosity. These results indicate that MO is a better carbon source than glucose for most of the strains investigated.
Pichia pastoris fermentation optimization: energy state and testing a growth-associated model by Bradley A. Plantz; Jayanta Sinha; Lorelie Villarete; Kenneth W. Nickerson; Vicki L. Schlegel (pp. 297-305).
A growth-associated model was applied to the production of recombinant ovine interferon-τ (rOvIFN-τ) with Pichia pastoris for the purpose of manufacturing preclinical and clinical active material. This model predicts that product yields will be the greatest when the specific growth of the culture is maintained at a steady and optimal rate. However, rOvIFN-τ yields did not meet the expected linear model but most closely corresponded to a polynomial relationship. After transitioning from glycerol to methanol, product accumulated for 31–45 h, and then the yield decreased. This production shift, which has been termed decoupling, was clearly related to time on methanol and not culture density. It was determined that a correlation exists between the decoupling point and a drop in energy state of the cell when expressing β-galactosidase. By assigning decoupling as a constraint that limits productivity and by reformulating the growth medium, the time prior to decoupling increased to 46.8±2.4 h, product yield improved for rOvIFN-τ from 203 to 337 mg l−1, and the coefficient of variation for yield decreased from 67.9 to 23.3%. A robust and stable fermentation process was realized, resulting in a 210% improvement in total yield from 557±357 to 1,172±388 mg.
Phenylacetylene reversibly inhibits the phenol hydroxylase of Pseudomonas sp. CF600 at high concentrations but is oxidized at lower concentrations by Jeanne Kagle; Anthony G. Hay (pp. 306-315).
Alkynes are mechanism-based inhibitors of several bacterial monooxygenases, including the soluble methane monooxygenase (sMMO) of Methylococcus capsulatus and the toluene o-monooxygenase (TOM) of Burkholderia cepacia G4. In this paper, we investigated the inhibition of the phenol hydroxylase of Pseudomonas sp. CF600 by the alkyne phenylacetylene. Growth of CF600 on phenol and phenol hydroxylase activity were inhibited by phenylacetylene concentrations greater than 1.0 mM. Unlike other alkynes, which irreversibly inhibit a number of monooxygenases, inhibition of phenol hydroxylase by phenylacetylene was reversible, as demonstrated by the ability of washed cells to regain phenol hydroxylase activity. Additionally, phenylacetylene was metabolized by phenol-grown cells, yielding a yellow meta-ring fission product which absorbed light maximally at 412 nm. Phenol-grown CF600 transformed phenylacetylene to hydroxyphenylacetylene and 2-hydroxy-6-oxo-octa-2,4-dien-7-ynoic acid as detected by gas chromatography–mass spectroscopy and high-performance liquid chromatography (HPLC), respectively, while neither a derivative of CF600 with a non-functional phenol hydroxylase nor wild-type CF600 grown on acetate transformed phenylacetylene. These results demonstrate that the phenol hydroxylase of CF600 has broader substrate specificity than previously reported. They also suggest that phenylacetylene acts as a competitive inhibitor rather than as a mechanism-based inhibitor of this phenol hydroxylase.
High-level secretion and purification of recombinant acetylcholinesterase from human cerebral tissue in P. pastoris and identification by chromogenic reaction by Xingyuan Ma; Jianhua Tan; Dongzhi Wei; Pin Zhu; Manji Sun (pp. 316-322).
The gene encoding human cerebral tissue acetylcholinesterase (AChE) was cloned from an 18-week fetal cerebral tissue and expressed in Pichia pastoris. Twenty-two positive transformants were obtained by Mut+/Muts phenotypes screening in MD/MM medium and polymerase chain reaction amplification, and four recombinant P. pastoris strains that could secrete active AChE at high level were identified by simple and specific development reaction with indoxyl acetate as the chromogenic substrate. In shake-flask culture induced with methanol, the recombinant human AChE (rhAChE) content was about 76% of the total secreted proteins, and rhAChE activity in supernatant was 40 U/ml. The enzyme was purified through anion-exchange and affinity chromatography. Purity of the rhAChE was up to 96% after the simple purification procedure. The enzymatic activity reached 200 U/mg.
Heterologous expression of the benzoate para-hydroxylase encoding gene (CYP53B1) from Rhodotorula minuta by Yarrowia lipolytica by Andreas Shiningavamwe; George Obiero; Jacobus Albertyn; Jean-Marc Nicaud; Martie Smit (pp. 323-329).
There is currently an increasing number of cytochrome P450 (CYP450) monooxygenase encoding genes becoming available from various genome-sequencing projects. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. In this study, the CYP53B1 gene, which encodes a benzoate para-hydroxylase, was successfully cloned from Rhodotorula minuta and overexpressed in Yarrowia lipolytica E150. Multiple copies of the CYP53B1 cDNA were cloned under the POX2 promoter, while the Y. lipolytica CPR was cloned under the isocitrate lyase promoter. Whole cell biotransformation of benzoic acid to para-hydroxybenzoic acid (pHBA) was used to analyse the hydroxylase activity of the recombinant Y. lipolytica UOFS Y-2366. Different induction conditions were tested in shake flask cultures. The highest concentration of pHBA produced by UOFS Y-2366 was 1.6 g l−1 after 200 h when stearic acid was repeatedly added to the media. R. minuta accumulated up to 1.8 g l−1 of pHBA within only 24 h. Thus, the specific hydroxylase activity of Y. lipolytica UOFS Y-2366 [approximately 0.07 U (g dry wt.)−1] was about 30 times lower than the specific hydroxylase activity of R. minuta [2.62 U (g dry wt.)−1]. However, the hydroxylation activity obtained with Y. lipolytica was one of the highest hydroxylation activities thus reported for whole cell biotransformation studies carried out with yeasts expressing foreign CYP450s.
Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli by Ha-Kun Kim; Dae-Sik Chun; Joon-Sik Kim; Cheol-Ho Yun; Ju-Hoon Lee; Soon-Kwang Hong; Dae-Kyung Kang (pp. 330-338).
Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide–lactoferricin fusion gene. The monomeric acidic peptide–lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-β-d-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.
Aspergillus oryzae strains with a large deletion of the aflatoxin biosynthetic homologous gene cluster differentiated by chromosomal breakage by Yun-Hae Lee; Mihoko Tominaga; Risa Hayashi; Kazutoshi Sakamoto; Osamu Yamada; Osamu Akita (pp. 339-345).
Recently we divided Aspergillus oryzae RIB strains into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-1, aflR, norA, avnA, verB, and vbs), and group 2, having three homologues (avnA, verB, and vbs). Here, partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8 kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups' strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage.
Biofilm development of the polyethylene-degrading bacterium Rhodococcus ruber by A. Sivan; M. Szanto; V. Pavlov (pp. 346-352).
We have recently isolated a biofilm-producing strain (C208) of Rhodococcus ruber that degraded polyethylene at a rate of 0.86% per week (r 2=0.98). Strain C208 adheres to polyethylene immediately upon exposure to the polyolefin. This initial biofilm differentiates (in a stepwise process that lasts about 20 h) into cell-aggregation-forming microcolonies. Further organization yields “mushroom-like” three-dimensional structures on the mature biofilm. The ratio between the population densities of the biofilm and the planktonic C208 cells after 10 days of incubation was about 60:1, indicating a high preference for the biofilm mode of growth. Analysis of extracellular polymeric substances (EPS) in the biofilm of C208 revealed that the polysaccharides level was up to 2.5 folds higher than that of the protein. The biofilm showed a high viability even after 60 days of incubation, apparently due to polyethylene biodegradation.
Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases by Mimmi Throne-Holst; Sidsel Markussen; Asgeir Winnberg; Trond E. Ellingsen; Hans-Kristian Kotlar; Sergey B. Zotchev (pp. 353-360).
A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C10H22) to tetracontane (C40H82) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.
Motility influences biofilm architecture in Escherichia coli by Thomas K. Wood; Andrés F. González Barrios; Moshe Herzberg; Jintae Lee (pp. 361-367).
Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms (∼43 μm thick, 21 to 34% surface coverage). JM109, DH5α, and MG1655 motA formed intermediate biofilms (∼13 μm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5α) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).
Measurement of chlorine dioxide penetration in dairy process pipe biofilms during disinfection by Am Jang; Jeffrey Szabo; Ahmed A. Hosni; Michael Coughlin; Paul L. Bishop (pp. 368-376).
Biofilms are considered a significant health risk in the food and dairy industries because they can harbor pathogens, and direct contact with them can lead to food contamination. Biofilm control is often performed using strong oxidizing agents like chlorine and peracetic acid. Although chlorine dioxide (ClO2) is being used increasingly to control microbiological growth in a number of different industries, not much is known about disinfection in biofilms using chlorine dioxide. In this study, a microelectrode originally made for chlorine detection was modified to measure the profiles of chlorine dioxide in biofilm as a function of depth into the biofilm. In addition, discarded microelectrodes proved useful for in situ direct measurement of biofilm thicknesses. The chlorine dioxide microelectrode had a linear response when calibrated up to a ClO2 concentration of 0.4 mM. ClO2 profiles showed depletion of disinfectant at 100 μm in the biofilm depth, indicating that ClO2 may not reach bacteria in a biofilm thicker than this using a 25 mg/l solution.
Redirection of the NADH oxidation pathway in Torulopsis glabrata leads to an enhanced pyruvate production by Liming Liu; Yin Li; Guocheng Du; Jian Chen (pp. 377-385).
This study aimed at increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting NADH oxidation from adenosine triphosphate (ATP)-production pathway (oxidative phosphorylation pathway) to non-ATP production pathway (fermentative pathway). Two respiratory-deficient mutants, RD-17 and RD-18, were screened and selected after ethidium bromide (EtBr) mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, cytochrome aa 3 and b in electron transfer chain (ETC) of RD-18 and cytochrome b in RD-17 were disrupted. As a consequence, the activities of key ETC enzymes of the mutant RD-18, including F0F1-ATP synthase, complex I, complex I + III, complex II + III, and complex IV, decreased by 22.2, 41.6, 53.1, 23.6, and 84.7%, respectively. With the deficiency of cytochromes in ETC, a large amount of excessive cytosolic NADH was accumulated, which hampered the further increase of the glycolytic flux. An exogenous electron acceptor, acetaldehyde, was added to the strain RD-18 culture to oxidize the excessive NADH. Compared with the parent strain, the concentration of pyruvate and the glucose consumption rate of strain RD-18 were increased by 26.5 and 17.6%, respectively, upon addition of 2.1 mM of acetaldehyde. The strategy for increasing the glycolytic flux in T. glabrata by redirecting the NADH oxidation pathway may provide an alternative approach to enhance the glycolytic flux in yeast.
The survival response of Escherichia coli K12 in a natural environment by S. H. Na; K. Miyanaga; H. Unno; Y. Tanji (pp. 386-392).
To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4°C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4°C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28°C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28°C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28°C.
TGGE analysis of the diversity of ammonia-oxidizing and denitrifying bacteria in submerged filter biofilms for the treatment of urban wastewater by B. Gómez-Villalba; C. Calvo; R. Vilchez; J. González-López; B. Rodelas (pp. 393-400).
The spatial and temporal diversity of the bacterial community-forming biofilms in a pilot-scale submerged biofilter used for the treatment of urban wastewater was analyzed by a temperature-gradient gel electrophoresis (TGGE) approach. TGGE profiles based on partial sequence of the 16S rRNA gene showed that the community composition of the biofilms remained fairly stable along the column system and during the whole time of operation of the biofilter (more than 1 year). Community-profiling based on the amplification and separation of partial ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes demonstrated that ammonia-oxidizing and denitrifying bacteria coexisted in both the anoxic and the aerated parts of the system. Several amoA and nosZ bands separated by TGGE were reamplified and sequenced, in order to further analyze the composition of these microbial communities in the biofilm. Phylogeny inferred from amoA/AmoA revealed the prevalence of Nitrosomonas species with five sequences affiliated to Nitrosomonas oligotropha, six sequences affiliated to Nitrosomonas europaea, and three sequences that showed only 75.7–76.1% identity of the DNA sequence with the closest described species (Nitrosomonas nitrosa). According to literature, this low identity value is indicative of previously undiscovered species. Eighteen new partial nosZ sequences were obtained which were mostly related to nosZ of gamma-proteobacteria (Pseudomonas) or clustered in the periphery of previously known denitrifying alpha-proteobacteria (Bradyrhizobium and Azospirillum).
Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes by Toru Shigematsu; Shinobu Era; Yuko Mizuno; Kana Ninomiya; Yukiko Kamegawa; Shigeru Morimura; Kenji Kida (pp. 401-415).
We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d −1 with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d −1 were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d −1 , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d −1 . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d −1 . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.
Novel lysine biosynthetic gene sequences (LYS1 and LYS5) used as PCR targets for the detection of the pathogenic Candida yeast by Shujuan Guo; J. K. Bhattacharjee (pp. 416-420).
We report here a sensitive and specific polymerase chain reaction (PCR) detection assay for the pathogenic Candida yeast based on the novel LYS1 [encoding saccharopine dehydrogenase (SDH)] and LYS5 [encoding phosphopantetheinyl transferase (PPTase)] gene sequences of the fungal unique lysine biosynthetic pathway. Both LYS1 and LYS5 DNA-specific PCR primers SG1, SG2 and SG3, SG4, respectively, amplified predicted 483 and 648-bp fragments from Candida albicans genomic DNA but not from other selected fungal, bacterial, or human DNA. The 18S rDNA control primers exhibited positive amplifications in all PCR assays. The LYS1-and LYS5-specific primers strongly amplified C. albicans and Candida tropicalis target sequences; however, the LYS1 primers also weakly amplified fragments from Candida kefyr and Candida lusitaniae DNA. Both sets of primers amplified target sequences from less than 10 pg of serially diluted C. albicans DNA, and the LYS1 specific primers also detected DNA isolated from serially diluted 50 C. albicans cells. The PCR primers reported here are sufficiently sensitive and specific for the potential early detection of Candida infections with no possibility of false positive results from cross-contamination with bacterial or human DNA.