Bacillus subtilis cloning

We routinely use a mix of five mRNAs that are derived from the lys (ATCC no. 87482), trp (ATCC no. 87485), dap (ATCC no. 87486), thr (ATCC no. 87484), andphe (ATCC no. 87483) clones from the bacterium Bacillus subtilis cloned into a vector that contains a stretch of As. These RNAs are generated by in vitro transcription using a T3 in vitro transcription kit (e.g., MEGAscript from Ambion) of the linearized DNA template with the appropriate restriction enzyme. 

Cloned esterase (isolated from Bacillus subtilis and cloned in E. coli), cheap and easy to produce... 

Effective cloning systems are available for a variety of bacterial hosts, including Bacillus subtilis, Streptomyces spp., and Agrobacter tumefaciens. Cloning systems have also been developed for eukaryotic hosts such as the yeast Saccharomyces cerevisiae, mammalian cells in tissue culture, and plant cells. 

Ebbole, D. J., and Zalkin, H. (1987). Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis. J. Biol. Chem., 262, 8274-8287. 

We chose subtilisin E to test our prediction that directed evolution makes mutations at uncoupled positions (Voigt et al., 2000b). Directed evolution increased the temperature optimum for activity, T pt, of Bacillus subtilis subtilisin E from 59° to 76°C, with eight mutations (Zhao and Arnold, 1999). In an independent study, thirteen mutations improved the activity toward the hydrolysis of su c c i iivI-A 1 a-A1 a-Pro-Phe- >-nitroanilide (s-AAPF-j Na) in the organic solvent dimethylformamide (DMF). The mutants were found by screening 2000 to 5000 clones from... 

La Vallie ER, Stahl ML, Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vhro-derived deletion mutation, J. Bacteriol., 171 3085-3094, 1989. 

Archaea contain a stable 7S RNA species of unknown function, which is not associated with the ribosome. The 7S RNA gene from Halobacterium halobium NRC817 has been cloned and sequenced [120]. The sequence resembles the 7SL RNA of eucarya [85] and the 4.5S Escherichia coli) or sc Bacillus subtilis) RNAs of bacteria [121] in potential secondary structure. The 7SL RNA forms part of the signal-recognition particle, involved in protein translocation. The function of the 4.5S RNA is not known exactly, but it is known to be essential [122]. 

RNase P, which removes the 5 leader from tRNA transcripts, contains an RNA moiety in bacteria, eucarya, and archaea. The enzyme from Haloferax volcanii has been purified and the 345-nucleotide RNA portion used to generate a probe for cloning of the corresponding gene [123]. The sequence can be folded into a structure similar to that of bacterial RNase P RNAs. SI nuclease and primer extension localize the 5 end of the transcript adjacent to four potential archaeal promotor sequences. An in vitro transcript corresponding to the native RNA plus twenty 5 and nine 3 flanking nucleotides did not by itself exhibit RNase P activity (as bacterial RNase P RNAs do), under a variety of conditions. It was, however, able to reconstitute an active enzyme in combination with the protein moiety from Bacillus subtilis. This indicates that in Haloferax volcanii the RNase P RNA is the catalytic part of the enzyme but it may require some structural help [123]. 

More recently acids have been resolved with enzymes cloned and over-expressed in their own organisms, such as an esterase from Bacillus subtilis that resolves ibuprofen methyl ester 138 to give ibuprofen 139 of 99% ee. A range of anti-inflammatory arylpropionic esters, including 138, could also be resolved with a cell-free extract from Pseudomonas fluorescens showing that purified enzymes are not essential.34... 

Kreiswirth B, Handley J, Schlievert P, Novick R Cloning and expression of streptococcal pyrogenic exotoxin A and staphylococcal toxic shock syndrome toxin-1 in Bacillus subtilis. Mol Gen Genet 1987 208 84-87. 

LC—MS) analysis of culture broth extracts, and reporter gene activation have all been successfully used as readouts in high-throughput assays designed to find small-molecule-producing clones (see Section 2.13.4). The most frequently used functional assays have been color production and antibacterial activity. While any assay strain can he selected for an overlay assay, Bacillus subtilis is commonly used due to its sensitivity to most known classes of antibiotics. Using simple functional assays, clones that produce new natural products have been recovered from both Escherichia coli- and Streptomyces lividans-h seA eDNA libraries (see Section 2.13.4). 

Figure 8 Compound 24 is a C3-isocyanide functional indole antibiotic that was isolated from an antibacterially active eDNA clone containing soil DNA. The active clone was identified in a top-agar overlay screen using Bacillus subtilis as the test organism. The isonitrile in this metabolite is biosynthesized in a single enzymatic step by IsnA using tryptophan and ribulose-...

Vrljic et al. cloned a new gene lysE from Corynebacterium glutamicum and showed that it encodes the translocator which specifically exports L-lysin out of the cell [10]. Recently they analyzed the membrane topology of the gene product and showed that it is a member of a family of proteins found in some bacteria -Escherichia coli, Bacillus subtilis, Mycobacterium tuberculosis, and Helicobacter pylori. The authors suggested that LtsE superfamily members will prove to catalyze the export of a variety of biologically important solutes including amino acids [11-13]. 

Wanker E, Schwab H (1993) The cloned Bacillus subtilis levanase gene as a potent system for the exploitation of inulin in biotechnological processes. In Fuchs A (ed) Inulin and inulin-containing crops, studies in plant ccience, vol 3. Elsevier, Amsterdam, pp 289-296... 

The Bacillus subtilis rib genes were identified and cloned by plasmid library screening with a guess-a-mer probe synthesized according to peptide sequences from purified B. subtilis riboflavin synthase. Independently, the... 

To ascertain the mechanism of heme A biosynthesis, we cloned HOS and HAS from Bacillus subtilis and heterologously expressed them in Escherichia coli. In addition to observing the production of both heme O and heme A, we also observed two additional previously unidentified heme products. Utilizing a number of techniques including optical spectroscopy, NMR spectroscopy, tandem mass spectroscopy, and chemical synthesis, we identified these two hemes as an alcohol intermediate and an overoxidized carboxylate product 19, 20). The carboxylate derivative, however, has only been identified when HAS is heterologously expressed in E. coli no carboxylate derivative has been observed when HAS is expressed under native conditions. 

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