Genetic Manipulation

A wide range of genetic techniques has been developed for B. subtilis, making it among the most amenable bacteria to genetic manipulation. These techniques include classical in vitro and in vivo mutagenic methods and extremely efficient in vitro genome manipulation methods that are in advance of those available for other bacteria, including Escherichia coli (18). Bacillus vectors have been designed to allow recombinant or mutant genes to be maintained autonomously in the host or integrated into the chromo some by double-crossover (Fig. 1) or single-crossover (Fig. 2) recombinations. In the case of a single-crossover recombination, tandem copies of the gene, one wild type and one mutated, can be engineered into the chromosome, if required, each under the control of independently controlled promoters (Fig. 2). The techniques for the manipulation of other Bacillus species are limited by their transformabil-ity.

Mutagenesis

A number of DNA repair systems have been detected and studied in B. subtilis, including error-prone SOS-like translesion DNA synthesis but not photoreactivation (19). Consequently, classical in vivo mutagenic methods generate mutants efficiently. Limitations in whole-cell muta-genic techniques, including lack of specificity and the generation of multiple mutations that may influence the cell's phenotype in an unpredictable manner, can be overcome by use of an alternative approach in which cloned copies of the target DNA are subjected to in vitro mutagenesis and subsequently reintegrated into the chromosome (20). This approach is aided by the publication of the genome sequence of B. subtilis strain 168 (16).

Specific changes are currently introduced into DNA sequences by PCR techniques, with more extensive changes being engineered by splicing PCR methodologies (e.g., splicing by overlap extension [21]). The modified DNA can then be introduced into the host bacterium either on replicating or integration plasmids. The ability to generate insertion mutants is one of the great technical strengths of B. subtilis. The techniques rely on the ease with which Bacillus DNA can be cloned into plasmids that replicate in E. coli but not in B. subtilis and the high frequency with which homologous recombination occurs within B. subtilis.

The identification and analysis of B. subtilis genes involved in specific functions or responding to particular stimuli has been facilitated by the use of transposons and transpositional mutagenesis. Although the value of transposons as a tool for the analysis of B. subtilis has diminished with the publication of the genome sequence (16), nevertheless the technology remains important for the analysis of other Bacillus species. Because of the lack of suitable native Bacillus transposons, Tn917 from Entero-coccus faecalis and Tn 10 from E. coli have been used (2224). In addition to their value for mutagenesis, transposons have been used to clone DNA adjacent to the site of integration, to generate transcriptional fusions to reporter genes, to control the expression of adjacent genes, and as phenotypic tags for mapping and cloning studies.

Transposon Tn917 is a 5.3-kb Tn3-like transposon conferring inducible erythromycin resistance (MLS-type). This transposon has a number of properties that make it a valuable genetic tool, including (1) the ability to insert relatively randomly into bacterial chromosomes, (2) a relatively high transposition frequency, (3) the ability to accommodate at least 8 kb of DNA without markedly affecting transposition frequency or insertional stability, and (4) a host range that includes gram-positive and gramnegative bacteria. In addition to B. subtilis, Tn917 has been used in B. amyloliquefaciens, B. licheniformis, and B.

megaterium (23). Insertion of Tn917 is not entirely random, and a limited preference for specific loci has been observed in the case of B. subtilis and B. licheniformis (25,26).

An alternative to Tn917 is an engineered miniderivative of Tn10 (24). This transposon consists of a Staphylococcus aureus-derived cat (chloramphenicol resistance) gene flanked by 307-bp fragments derived from IS 10. The gene encoding the transposase is incorporated into a delivery vector rather than the minitransposon itself. Tn10-derived transposons have two potential advantages over Tn917-based transposons. They appear to transpose at a significantly higher frequency and insert randomly without bias to specific targets. A series of special-purpose delivery systems have been developed for gene inactivation, the recovery of adjacent chromosomal sequences, and transcription fusions.

Transformation

Bacillus subtilis was the first nonpathogenic bacterium to be successfully transformed with extracted DNA (27-29). The availability of methods for introducing DNA into cells is essential for recombinant DNA technology, and several transformation techniques have been developed for B. sub-tilis. The most frequently used method is transformation of naturally competent cells, although protoplasts of B. subtilis (30) and several other Bacillus species can be efficiently transformed. Current methods for electrotrans-formation of Bacillus species usually result in low yields of transformants.

Natural competence is one of several postexponential phase phenomena that are characteristic of B. subtilis strain 168. In glucose-minimal media, maximal competence develops shortly after the transition from exponential to stationary phase, and only about 10% of the cells are able to take up DNA. Natural competence has been observed with a limited number of B. subtilis strains, and few natural competence systems are known for Bacillus species. Unlike Haemophilus influenzae, competent B. sub-tilis cells do not discriminate between native and foreign DNA. In recombination-proficient strains, homologous chromosomal DNA is integrated in the host chromosome at the site of homology. If saturating amounts of DNA (> 1 1g/mL of culture) are used, the cotransfer of unlinked genetic markers occurs. This phenomenon, called congres-sion, can be used to introduce genes for which no selectable phenotype is known (31).

The frequency of plasmid-mediated transformation is usually between 0.001% and 0.1%, and this frequency is reduced still further with ligation mixtures. A major reason for these low efficiencies is that donor plasmid DNA becomes randomly fragmented and converted into single-stranded DNA before entry into the cell. Recirculation of the plasmid DNA requires a region of homology provided on fragments of opposite polarity, and consequently only plasmid multimers, present in most plasmid preparations, or monomers containing internal repeats are effective in transformation (32).

Bacillus protoplasts can incorporate DNA if treated with chaotropic agents such as polyethylene glycol. The

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