AbR gene E coli

3' copy of target gene

_ Integrant chromosome

Figure 2. Single-crossover recombination showing integration of an intact integration vector into the host chromosome at the site of the target gene. Part or all of the target gene is duplicated as a result of the integration event. The inclusion of a controllable promoter upstream of the target gene fragment on the vector allows for controllable gene expression of the 3' copy of the gene or any genes downstream in the same operon. AlP, selectable antibiotic resistance gene.

than 4,100 protein-coding genes (16). The genome shows evidence of 10 prophages or remnants of prophages, which may account for its susceptibility to lysis at the end of exponential growth in the absence of an energized membrane (33). Nearly half the protein-coding genes have paralogues in the genome, and several expanded gene families were detected, particularly for two-component signal transducers (34 genes) and ATP-binding transporters (77 genes).

Although analysis of a complete microbial genome sequence reveals many new ORFs, it is not possible to ascribe functions to all the detected ORFs on the basis of currently available sequence databases and bioinformatics tools. Even in well-characterized species such as E. coll and B. subtilis, URFs represent about 40% of the detected ORFs. Given the intensive in vitro studies on bacteria, it is likely that the function of a significant proportion of the URFs will relate to growth and survival in natural habitats.

The inability to ascribe function to more than a third of the ORFs in B. subtilis has required the development of new strategies for analyzing gene function. In the case of B. subtilis, a highly coordinated systematic functional analysis program involving groups with a wide range of technical expertise has been established. Approximately 2,000 unknown genes have been systematically disrupted with an integrational vector to generate a collection of isogenic mutants strains (34). The mutant construction strategy includes the introduction of a lacZ transcriptional reporter for monitoring the expression of the target gene and a controllable promoter that facilitates the expression of any genes downstream in the same operon (Fig. 2).

The availability of the B. subtilis genome sequence has revolutionized the methods of analysis and manipulation of this bacterium, and consequently traditional methods of genetic linkage mapping involving generalized transducing for long-range mapping and transformation for fine structure mapping have largely been rendered redundant (31). This is not the case for species such as B. megaterium for which generalized transducing phages such as MP13 and protoplast transformation remain important analytical tools (35).

Increasingly, integration systems are used for the analysis and manipulation of the B. subtilis genome. Transformation of cells with homologous DNA fragments is very efficient, and if heterologous sequences are flanked by sequences homologous to chromosomal DNA they can be inserted efficiently at the sites of homology via a double-crossover recombination event (Fig. 1). In the case of circular molecules, a single site of homology is all that is required and integration is via a single-crossover recombination event (Fig. 2).

Single or Campbell-type integration events generally use E. coli-based plasmid vectors that are not able to replicate in gram-positive bacteria but that include an antibiotic resistance gene that can be selected in B. subtilis. A fragment of B. subtilis DNA (>0.4 kb) is cloned via E. coli into the integrational vector, which is then transformed into B. subtilis. Selection for an antibiotic-resistant gene results in transformants in which the vector has integrated into the host chromosome via a single-crossover recombination between homologous regions on the vector and bacterial chromosomes. Integration results in a duplication of the cloned fragment at the flanking ends of the integrated vector. These tandem repeats represent an amplifiable unit; increasing the amounts of antibiotic used for selection can result in the amplification of the intervening sequences up to 70 copies (36).

The integrational vector system can be used to generate different mutational outcomes. If the cloned fragment carries an entire operon, including promoter and transcription terminator, two fully functional copies of the gene are present in the integrant. If 5'- or 3'-end fragments of an operon are used, only one functional copy of the target gene is generated. If the 5'-end fragment includes a ribosome-binding site and start codon, and is placed downstream of a controllable promoter, then the expression of the single functional copy of the target gene is under the control of this promoter (Fig. 2). Finally, if regions internal to the target gene are used, no functional copies are formed after integration.

With double-crossover integrations, chromosomal DNA is replaced by either mutationally altered homologous DNA (Fig. 1a) or heterologous DNA (Fig. 1b). Only a single copy of the target gene is introduced. The incoming fragment, which must encode a selectable phenotype, is flanked by DNA that is homologous to sequences at the chromosomal target site. The amyE gene, encoding a non-essential a-amylase, is often used as a target of double-crossover integrations because its disruption provides a positive selection phenotype on starch plates (Fig. 1b).

Cloning and Expression Systems

The development of recombinant DNA techniques for B. subtilis is now well advanced, and previously encountered problems of vector stability have largely been overcome.

Plasmid Vectors. B. subtilis 168, the strain that has been sequenced and that is used for most genetic studies, does not contain endogenous plasmids, whereas most plas-mids present in other Bacillus strains are cryptic. Consequently, plasmid vectors designed for use in B. subtilis have been taken from other gram-positive bacteria, notably Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus (18,37). Staphylococcal plasmids such as pUB110 and pE194, which formed the basis of the first generation of Bacillus vectors, are still in common use for B. subtilis (38). The kanamycin resistance plasmid pUB110 is the most widely used replicon but suffers from problems of structural and segregational instability that result from its single-stranded (rolling circle) mode of replication (39). The second generation of Bacillus vectors in volved the construction of chimeras between gram-positive and gram-negative replicons (40). These shuttle orbifunc-tional vectors enabled initial cloning experiments to be carried out in E. coli, exploiting this organism's highly efficient transformation system. In recent years, stable single-stranded replicating plasmids and theta-replicating plasmids have been identified and are being developed as a new generation of Bacillus vectors (18). The former includes derivatives of plasmids such as pWVO1 derived from Lactococcus lactis (41) and certain endogenous Bacillus plasmids, the latter plasmids based on pAM/1, originally isolated from Enterococcus faecalis (42,43).

An alternative to using autonomously replicating plas-mid vectors is to use plasmid integration vectors. Such integrants are usually stable; reversal of the process, by recombination in the duplicated sequences, occurs at a frequency of about 10β€”4 to 10 β€”5 per cell generation (44). Integration vectors have been reviewed by Albertini and Galizzi (45) and Perego (46).

Various special-purpose plasmid vectors have also been developed, including expression and secretion vectors. Although the expression of many genes has been studied in B. subtilis, relatively few promoter systems have been developed for controlled, high-level expression. The majority of extracellular enzymes synthesized for industrial purposes are expressed from native promoters induced at the end of exponential growth and for extended periods in the stationary phase (47). During this time they can be subjected to a variety of control pathways, including transition-state regulators and catabolite repression (48,49). Industry has developed promoter-expression systems (including growth regimes) that can direct the synthesis of extracellular proteins to concentrations of about 20 g/L, although for commercial reasons the details of these systems are not generally available. Three promoter systems that are widely used in research laboratories are discussed next.

Pspac Promoter. The Pspac promoter was constructed by fusing the 5' sequences of a promoter from SPO1, a lytic B. subtilis bacteriophage, and the 3' sequences of the lac promoter, including the entire lac operator. The point of fusion is within the β€”10 region of the hybrid promoter (50). The controllability of Pspac is dependent on the presence of the lac repressor, expressed in B. subtilis by placing it downstream of a promoter and ribosome-binding site (RBS) derived from a B. licheniformis penicillinase gene. The Pspac promoter can be induced 50 fold with the lactose analog IPTG.

XylR-Controlled Promoters. The genes for the utilization of xylose polymers are controlled in B. subtilis at the level of transcription (51) through the activity of a repressor, the product of the xylR gene. The xylose-inducible promoteroperator elements have been used without modification to control expression from chromosomal and plasmid locations. A copy of the xylR gene is usually included on high-copy-number expression vectors to maintain a balance between the number of repressor molecules and operator sites. Although genes in the xylose regulon are subject to catabolite repression, the catabolite-responsive element is located well downstream of the promoter and operator, and is therefore not included in the expression vectors. In con trast to IPTG, xylose is a cheap and readily available substrate and can be used for the induction of large-scale fermentations.

SacR-Controlled Promoters. The inducible expression of sacB, the gene encoding extracellular levansucrase by sucrose, involves a number of regulatory mechanisms, not all of which are fully understood. The sacB gene is controlled positively by sucrose, the sacY antiterminator, and the products of degQ and sacU and negatively by sacX, a putative PTS enzyme IIsucrose. The sacR regulatory region located upstream of the sacB promoter contains a target sequence for DegQ and SacU, which together enhance transcription. Downstream of this promoter, but before the sacB RBS, a termination-like stem-loop structure is present, which is acted on by the SacY antiterminator to mediate sucrose-induced expression (52). Expression vectors based on the sacB promoter have the advantage that this promoter can be activated during exponential growth when the expression of most Bacillus extracellular proteases is repressed and is not subject to catabolite repression. The level of induction is also geared to the level of sucrose in the medium; the promoter is only weakly induced in the presence of 1 mM sucrose and is fully induced at 30 mM.

Phage Vectors. The development of phage vectors for B. subtilis has lagged well behind that of E. coli, and no phage vectors with the range and versatility of those of lambda exist for this organism. Libraries have been generated in derivatives of Bacillus phages ^ 105 and SPfj, although the former has been used more extensively (53). Attempts to develop a cosmid system for B. subtilis have failed because of the lack of an efficient in vitro packaging system for any of the candidate phages.

Bacteriophages ^105 and PBSX (a defective phage of B. subtilis strain 168) have both been developed for the production of proteins in B. subtilis (53). In both cases the phages have been modified to make them temperature in-ducible by the introduction of mutations in the genes encoding the immunity repressor proteins. Target genes are introduced downstream of a strong prophage promoter. In the case of ^105MU333, temperature induction can lead to the production of the target gene product to 0.5 mg/mL culture supernatant (54). Bacteriophage-based expression systems have the advantage of being relatively stable because they are maintained as a single copy during growth. Copy number increases at the time of induction due to the replication of the phage. The promoter is tightly controlled but is induced cheaply and in a manner favored in industrial fermentations by increasing temperature. However, vector development is still required to maximize the full potential of this system for high-level protein production.

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