Current development in genetic engineering strategies of Bacillus species

Abstract

The complete sequencing and annotation of the genomes of industrially-important Bacillus species has enhanced our understanding of their properties, and allowed advances in genetic manipulations in other Bacillus species. Post-genomic studies require simple and highly efficient tools to enable genetic manipulation. Here, we summarize the recent progress in genetic engineering strategies for Bacillus species. We review the available genetic tools that have been developed in Bacillus species, as well as methods developed in other species that may also be applicable in Bacillus. Furthermore, we address the limitations and challenges of the existing methods, and discuss the future research prospects in developing novel and useful tools for genetic modification of Bacillus species.

Figure 1

Operator-repressor system-based genetic engineering strategy. (A) In the presence of the R gene, the constitutively expressed R protein specifically binds to the P_r promoter and represses promoter activity. (B) When the R gene is excised from the genome, the P_r promoter is activated and mutants can be selected by antibiotic resistance. (C) Diagrams of selection and counter-selection marker cassettes. R: Repressor, AR: Antibiotic resistance, P_r: Promoter. [AR1^R], [AR1^S], [AR2^R], and [AR2^S] are AR1-resistant, AR1-sensitive, AR2-resistant, and AR2-sensitive bacterial phenotypes, respectively.

Figure 2

Summary of pyrimidine metabolism pathway and mechanisms of action of pyrimidine analogs. Orotate is metabolized to UMP by OPRTase and OMPdecase, encoded by pyrE and pyrF, respectively. These enzymes also convert 5-fluoroorotate to 5-fluoro-UMP. pyrR encodes an mRNA-binding attenuator (PyrR) that negatively regulates pyr expression by sensing UMP or UTP. UMP is also produced from uracil by UPRTase, encoded by upp. UPRTase converts 5-FU to 5-fluoro-UMP, which is further metabolized to the toxic metabolite 5-fluoro-dUMP. 5-fluoro-dUMP is a strong inhibitor of thymidylate synthetase, which provides the sole de novo source of dTMP for DNA biosynthesis.

Figure 3

Site-specific recombination-based genetic engineering strategy. Selectable genes are often antibiotic resistance genes. White regions represent recognition sites of site-specific recombinases, and blue regions represent sequences homologous between the genome and the integration cassette.

Figure 4

Toxin gene-based genetic engineering strategies. (A) Schematic representation of the toxin gene cassette. P: Commonly-used constitutive promoter. Pr: Promoter of operator-repressor system, which is repressed by the repressor and activated by an inducer e.g. P_spac, P_xyl. The repressor gene in the cassette can also be deleted in some methods. (B) Two different strategies based on toxin gene cassettes. A: upstream sequence; B: downstream sequence; C: sequence for integration of the toxin gene cassette, in combination with A in (2); DR: direct repeat sequence. After integration of the toxin gene cassette into a target chromosome locus via double-crossover recombination [A and B in (1) or A and C in (2)] and positive selection for antibiotic resistance, the cassette is removed by a single crossover event between two DR sequences in (1) or B sequences in (2).

Figure 5

Classification of genetic modification strategies according to different procedures.