Genetic tools are required to take full advantage of the wealth of information generated by genome sequencing efforts and ensuing global gene and protein expression analyses. Bacterial genetics was originally developed and refined in Escherichia coli. As a consequence, elegant plasmid, cloning, expression, and mutagenesis systems were developed over the years and a good number of them are commercially available. This is not true for other bacteria. Although the development of genetic tools has generally not kept up with the sequencing pace, substantial progress has been made in this arena with many bacterial species. This short review highlights selected topics and achievements in the field over the past 25 years and presents some strategies that may help address future challenges. BioTechniques has played an integral part in the publication of important technological advances in the field over the first 25 years of its existence and it can be anticipated that it will continue to do so in the future.

Introduction

One may argue that among the technologies that had the greatest impact on the field of bacterial genetics in the last 25 years is the polymerase chain reaction (PCR) (1,2), for its effect on strategies for cloning and manipulation of genes (3,4,5). However, PCR is undoubtedly rivaled by the emergence of whole-genome shotgun sequencing technologies (6) that, to date, have allowed deciphering of several hundred bacterial genome sequences. Besides postgenomic methods such as transcriptome, proteome, and metabolome analyses, genetic tools are needed to take advantage of the wealth of information contained within these genomes. However, with the notable exception of PCR-based strategies, tools for genetic analysis have largely not kept up with the “sequencing pace.” Many bacteria are recalcitrant to genetic manipulation because of the lack of suitable selection markers, necessitating either identification of novel selection markers or use of markers that can be repeatedly used (“recycled”). Furthermore, tools for genetic manipulation are either scarce or nonexistent, or they exhibit a narrow host-range. This may be remedied by developing tools that are as broad-host-range as possible. The new technologies needed may not necessitate de novo identification of elements and tools that work in the bacterium of choice, but in most instances, can probably be derived by appropriate modification of what is already available for other bacteria. In this short paper, I review some of the technologies that have emerged over the last 25 years and how they may help meet the challenges the future of bacterial genetics holds. Because of space limitations, this review cannot be all-encompassing, but I hope that it will nonetheless be thought-provoking to the reader and encourage further searching and reading of the pertinent literature.

Cloning Vectors

Plasmid cloning vectors for Escherichia coli were developed many years ago. Most rely on the pMB1 (closely related to ColE1), p15A, or pSC101 replicons. Examples for pMB1-based vectors include the pBR series of vectors of intermediate copy number (15–20 per cell) that contain various antibiotic resistance markers for cloning via insertional inactivation (reviewed in Reference 7). Subsequent modifications by Messing and colleagues created the pUC series of vectors with an increased plasmid-copy number (>500 copies per cell) and facilitated cloning into a multiple cloning site (MCS) (8,9). This MCS is located within the lacZα gene segment of E. coli β-galactosidase (β-Gal) (8) and allows blue-white screening in appropriate host strains expressing the LacZΔM15 β-Gal protein (10). Other versions of similar high-copy number vectors are exemplified by the widely used pBluescript vectors (11). The pACYC vectors were the first examples of p15A-based vectors of lower copy number (∼10 copies per cell), which were also engineered for cloning by insertional inactivation of ampicillin, chloramphenicol, kanamycin, and tetracycline resistance markers (12). Later derivatives containing the p15A replicon included vectors that carry the pUC-derived MCSs and β-Gal-based blue-white screening elements (13,14). Lastly, pSC101 was one of the very first low-copy number (∼5 copies per cell) plasmids engineered for cloning of heterologous DNA fragments (15). The pSC101 replicon was later combined with the pBluescript selection markers and MCS to derive versatile low-copy number cloning vectors (16).

Some of these vectors were later modified to allow cloning of PCR fragments via TA (17) or Vaccinia DNA topoisomerase I based TOPO (18) cloning, which are commercially marketed technologies. Additionally, the commercially available Gateway system (Invitrogen, Carlsbad, CA, USA) allows rapid recombination-based cloning of DNA fragments into recombination-proficient, so-called destination vectors, by exploiting the efficient phage λ recombination machinery (19).

The ColE1, p15A, and pSC101 replicons only function in E. coli and some other closely related Enterobacteriaceae. There are basically two strategies for development of plasmid cloning vectors for bacteria other than E. coli: (i) one can search for cryptic plasmids present in these bacteria and exploit their replicons for development of cloning vectors (20,21) or (ii) one can exploit vectors that have been shown to be of broad-host-range such as pBBR1 (22), pRO1600 (20), RK2 (23), RSF1010 (24), or pVS1 (25). Because of the time-consuming nature of the former approach, the latter is more feasible and has been exploited by several groups for development of broad-host-range vectors using strategies similar to those illustrated in (Figure 1). These efforts resulted in small and well-characterized pBBR1-based vectors such as pBBR1MCS (26,27) or the pRO1600-based pUCP family of vectors (28,29,30,31). More comprehensive reviews of these and other replicon-based cloning vectors have been published (32,33). Because of the ease of manipulation of E. coli and the many reagents available for this bacterium, most cloning vectors available for other bacterial hosts are shuttle plasmids that replicate in both E. coli and other bacteria (e.g., contain a pMB1-derived replicon and the broad-host-range replicon). Besides a replicon for stable maintenance in the respective hosts, other basic requirements include a selection marker for identification of cells containing and maintaining the plasmid and restriction sites (e.g., a MCS) for cloning of DNA fragments. While not essential, other features are often included to increase the versatility of the vectors. These include genetic elements facilitating screening of recombinants (e.g., lacZα for blue-white screening); elements that facilitate interspecies transfer of recombinant plasmids (e.g., an origin of conjugal transfer, mob or oriT); promoters whose expression levels can be regulated (e.g., E. coli lac operon promoter P_lac or its derivatives P_tac and P_trc (34)); other E. coli promoters such as those derived from the arabinose (P_araBAD) (35,36) and rhamnose (P_rhaBAD) (37) operons; tags (e.g., oligohistidine (38,39,40)) that facilitate purification of recombinant proteins.