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Bacillus megaterium was used in biotransformation studies to test the influence of several barbiturates towards the recently observed N-dealkylation and arylic hydroxylation of N,N-diethylaniline. Certain barbiturates (phenobarbital and amobarbital) induced increased enzyme activity in whole-cell incubations with B. megaterium resulting in an increasing amount of N-deethylated and hydroxylated arylic (ortho- and para-hydroxylation) products, whereas ortho-hydroxylation was slightly favored. Phenobarbital did not show any effects towards hydroxylation activity, but N-dealkylation activity was highly induced. Finally, metyrapone highly reduced hydroxylation and N-dealkylation activity.

Bacillus megaterium, isolated in a screening process from topsoil, was used for N-demethylation of natural N-methyl methyl anthranilate to produce natural methyl anthranilate. Maximal productivity of 70 mg/L/day was achieved under laboratory-scale conditions without further optimization. No byproducts were observed. Thus, production of “natural” methyl anthranilate using B. megaterium is a significant improvement over comparable already existing procedures. Keywords: Bacillus megaterium; bioflavor; biotransformation; methyl anthranilate; N-demethylation; N-methyl methyl anthranilate

Mutants ofListeria monocytogeneswith deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA.aroA,aroB, and in particulararoEmutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of thearomutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that thearomutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of thearomutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and allaromutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 104-fold for thearoA,aroB, andaroA/Bmutants and >105-fold for thearoEmutant compared to the parent strain. Nevertheless, mice preimmunized witharomutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 × 106aroA,aroB, andaroA/Bmutant bacteria were sufficient to obtain a protective T-cell response, while 5 × 108aroEoraroA/Emutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating thatListeriastrains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.

A neutrophilic, autotrophic bacterium that couples iron oxidation to nitrate reduction (iron-oxidizing bacteria [IOB]) under anoxic conditions was isolated from a working bioremediation site in Trail, British Columbia. The site was designed and developed primarily to treat high concentrations of Zn and As that originate from capped industrial landfill sites. The system consisted of two upflow biochemical reactor cells (BCR) followed by three vegetated wetland polishing cells with sub-surface flow and a holding pond. During a 5-year period (2003–2007), the system treated more than 19,100 m3 of contaminated water, removing and sequestering more than 10,700 kg of As, Zn and sulfate at average input water concentrations of: As, 58.6 mg l−1 (±39.9 mg l−1); Zn, 51.9 mg l−1 (±35.4 mg l−1) and SO4 2−, 781.5 mg l−1 (±287.8 mg l−1). The bacterium was isolated in order to better understand the mechanisms underlying the consistent As removal that took place in the system. Analysis using Basic Local Alignment Search Tool (BLAST) database showed that the closest homologies are to Candidatus accumulibacterphosphatis (95 % homology), Dechloromonas aromatica (94 %), and Sideroxydans lithotrophicus ES-1 (92 %) Within the BCR cells, the IOB oxidized Fe2+ generated by iron-reducing bacteria (IRB); the source of the iron was most likely biosolids and coatings of iron oxide on locally available sand used in the matrix. We have provisionally designated the novel bacterium as TR1.

This article summarizes general design principles for functional metagenomics. The focus is on Escherichia coli as an expression host, although alternative host–vector systems are discussed in relation to optimizing gene recovery in activity-based screens. Examples of DNA isolation and enrichment approaches, library construction and phenotypic read-out are described with special emphasis on the use of high throughput technologies for rapid isolation of environmental clones encoding phenotypic traits of interest.

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system. As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1]. As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3]. Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4]. The vector used here (pPICZαA) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the α-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Soil samples were screened to select microorganisms with the capability to oxidize organic sulfides into the corresponding sulfoxides with differential enantioselectivities. Several bacterial strains that preferentially produced the S- configured sulfoxide enantiomer were isolated. Surprisingly, one bacterial strain, genotypically and phenotypically characterized as Pseudomonas frederiksbergensis , selectively gave the R enantiomer. The finding that two apparently identical organisms displayed opposite enantioselectivities is novel for non-genetically modified organisms.