Genetics for all bacteria.

KEY WORDS: conjugation, transduction, transformation, electroporation, mapping, whole genome analysis, physical and genetic maps

CONTENTS

 
INTRODUCTION AND HISTORICAL PERSPECTIVE                        660 
THE AIMS OF GENETIC ANALYSIS                                   661 
  The Historical Approach to Bacterial Genetic Analysis and 
          Its Problems                                         662 
  In Vivo Systems of Genetic Exchange                          662 
OVERCOMING THE PROBLEMS OF GENETIC ANALYSIS                    665 
  The Role of New Technology                                   666 
  Complementation Mapping                                      667 
  Physical Analysis of Genomes                                 669 
  Physical and Genetic Maps                                    670 
  Whole Genome Structure                                       673 
USES OF GENOME ANALYSIS OF BACTERIA                            675 
SUMMARY                                                        676 

The availability of genetic analysis has now been extended to a wide variety of bacteria. While the traditional methods of conjugation, transduction, and transformation have made major contributions to microbiology and genetics, new recombinant DNA techniques and the development of new equipment for characterization and isolation of DNA fragments have enabled genome analysis of many bacteria for which no genetic information was previously available. These new procedures have enabled the construction of detailed physical/genetic maps as well as precise measurements of genome size, and provided new data on functional arrangements of genes in the bacterial genome. Such information is proving increasingly valuable for many aspects of microbiology as well as for the genetic manipulation of bacteria important in human disease, agriculture, and biotechnology.

INTRODUCTION AND HISTORICAL PERSPECTIVE

For many years the literature on bacterial genetics was dominated by work on Escherichia coli K12, Salmonella typhimurium LT2, Bacillus subtilis 168, and Streptomyces coelicolor A3(2). The accumulation of an impressive body of information has influenced aspects of biological research not restricted to microbiology. The most notable example is the development of recombinant DNA techniques that have, and will continue to have, a pervasive influence on the understanding of all living organisms. Microbiologists soon recognized the value of genetic analysis for understanding and investigating microbial phenomena. Workers studying organisms other than those listed above sought to use these newly developed techniques to investigate other genetic characteristics of their bacteria of interest. The predominance of conjugation in genetic analysis during those early days meant that the F factor was almost the only agent known to promote conjugal exchange in bacteria. As it turned out, the F factor of E. coli is very organism specific in its action. In addition, the host range of bacteriophages is very strain specific and limits the range of organisms for which transduction is available. Finally, the techniques for obtaining transformation competent cells were found to be highly fastidious and needed to be precisely defined in each organism examined. Development of systems of genetic analysis for individual strains of other bacteria was very labor intensive, and investigators devoted considerable effort to searching for new conjugative plasmids with chromosome mobilizing ability (cma), and bacteriophages with transducing activity, and to devising more general techniques for establishing competence to enable transformation analysis in a wider range of bacteria.

These efforts are described below. The overall conclusion is that genetic analysis using one or another of these three systems is now possible in a wide range of different bacteria. Two factors have encouraged the extension of genetic analysis to a wider range of bacteria. The first is the use of newly isolated organisms in biotechnology applications and the need to analyze and manipulate their genome. To this activity has been added a greater interest in genetics by those bacteriologists studying plant bacterial interactions, notably nitrogen fixation and plant disease caused by bacteria. The second factor is the development and use of recombinant DNA techniques that have enabled genomic analysis of bacteria without the classical mechanisms of gene exchange, the need to have selective markers to obtain recombinants, or even the need to isolate mutants, although, in the immortal words of Salvadore Luria, "When you have a mutant you are better off than when you don't."

This review does not aim to provide an encyclopedic coverage of all the bacteria for which genetic analysis is now possible, nor does it attempt to describe genetic analysis of the four organisms identified above, except where such work is important for specific aspects of genetic analysis for other bacteria. Numerous reviews cover the genetic analysis of these organisms including those by Neidhardt et al (124), Hopwood & Chater (83), and Lovett (108). The focus here is to understand genomic analysis, mapping, and those general approaches that are or will be important for an even wider understanding of bacterial genome structure in a greater number of organisms than presently exists. This review does not include any of the numerous papers describing the cloning and characterization of individual bacterial genes or chromosomal segments, the procedures by which this has been achieved, or the significance of this large body of data. Previous reviews on genetic exchange processes in bacteria include those by Low & Porter (109), Ball (6), Scaife et al (142), Birge (11), and Holloway (74). A recent compendium of papers (120) provides an excellent outline of methods for use in both in vivo and molecular genetic systems of bacterial genetics for a variety of bacteria.

THE AIMS OF GENETIC ANALYSIS

Any traditional system of genetic analysis for bacteria must include the ability to (a) produce and select desired mutants, (b) introduce DNA into bacterial cells, and (c) provide for the selection and characterization of recombinants or mutants that have undergone complementation. Given that these requirements have been achieved, a number of common aims for the genetic analysis of bacteria can be identified:

1. The understanding of genome structure, the measurement of genome size, the number and size of individual replicons, and the distinction between chromosomally located genes and plasmid-borne genes.

2. The identification of particular gene arrangements on both chromosomes and plasmids that have significance for biological properties.

3. The use of genetic data to explain biological characteristics and phenomena, particularly in regard to disease, natural habitat, regulation, evolution, and taxonomy.

4. The manipulation of bacterial strains to produce novel recombinant forms with special properties, which can be used to solve problems in biotechnology, medical research, or agriculture.

The extent and success of these aims will depend upon the focus of the investigator, the nature of the organism, and the genetic data sought. Clearly it has been much easier to obtain more sophisticated and extensive data with E. coli than with some newly isolated autotroph. Such limitations have encouraged the development of methods of genetic analysis that are applicable to a wider range of organisms. These methods have exploited particular recombinant DNA techniques and new equipment that together enable one to obtain data on genome structures and linkage relationships for all bacteria, and these data are not restricted by biological factors dependent on in vivo genetic transfer techniques.

The Historical Approach to Bacterial Genetic Analysis and Its Problems

Traditionally, mutants have been isolated by the use of chemical mutagens, but as shown by the example of N-methyl-N-nitrosoguanidine, inherent sources of error can result in multiple genetic changes. Nevertheless, numerous excellent mutants have been produced by one or another chemical mutagen. The availability of transposon mutagenesis has made possible the production of mutants that have inbuilt selectivity and are likely to be at a single site; and should physical genetic analysis be available, the mutation can be located with the utmost precision. Problems that arise include the type of selection inherent in the transposon. This is commonly antibiotic resistance, but other types, e.g. substrate utilization, are available, and in some situations the transposon insertion may be unstable (59). Techniques of transposon mutagenesis and related topics have been comprehensively reviewed by Berg et al (9).

In Vivo Systems of Genetic Exchange

In what must now be called classical mechanisms, the three principal ways by which bacteria can exchange genetic information are conjugation, transduction, and transformation. The critical feature of these mechanisms for any individual bacteria is the frequency of exchange, which must be at least [10.sup.-6] recombinants per donor cell. The second critical feature is a selection mechanism to identify these rare recombinants in the presence of an excess of parental bacterial cells. The elegance of the use of auxotrophs in E. coli by Lederberg & Tatum (100) created high expectations for other bacteria, but this system is not universally available because not all bacteria grow on a defined medium. The innovative skills of bacterial geneticists are best displayed in seeking new ways of selecting recombinants. Antibiotic resistance, use of carbon and nitrogen sources for growth, and conditional lethal mutants have all been used, but the lack of an effective recombinant selection procedure for individual species has often limited the effectiveness of a gene exchange system.

Conjugation systems of genetic exchange have in practice been the most likely to produce extensive knowledge of chromosomal gene arrangement in bacteria. The prime requirement of a such system is usually a plasmid that will promote chromosome transfer. The first such plasmid was F, a native plasmid found in E. coli K12, but conjugative plasmids capable of promoting chromosome transfer have been difficult to find in most organisms. One exception is P. aeruginosa, whose clinical strains frequently carry plasmids capable of acting as sex factors (37). Fortunately, the ubiquitous drug resistance plasmids have proved to be the principle means by which conjugation can be used as a genetic tool in various bacteria, and as early as 1962, investigators showed that antibiotic resistance plasmids could promote chromosome transfer in E. coli (167). The next key discovery was that of Lowbury et al (110), who demonstrated the first plasmids that could replicate in a range of unrelated gram-negative bacteria and be transferred freely among different genera. These IncP1 plasmids, as they are now described (174), have a host range that extends to most gram-negative organisms including Acinetobacter, Agrobacterium, Azospirillum, Azobacteria, Erwinia, Escherichia, Pseudomonas, Rhizobium, Rhodobacter, Vibrio, and Zymomonas (73)(174). IncP1 plasmids can promote chromosome transfer (161), but their effectiveness to do so can be increased by the construction of variants with enhanced frequencies of gene transfer, such as R68.45 (64)(65) or pMO514 (129). Wide host-range plasmids do not necessarily display conjugal competence in a variety of species. The IncP10 plasmid R91-5 has a wide host range of transfer among many gram-negative genera but has only a narrow host range of replication, namely P. aeruginosa (22). R91-5 is excellent for mapping in P. putida PPN (36)(166), but less effective in other strains of P. putida (M. I. Sinclair, unpublished data) and in P. syringae (126).

Transposable elements that were mainly derived from plasmids carrying antibiotic resistance markers have also been used to construct chromosome mobilizing systems for a range of bacteria. The concept has been to mimic the native E. coli system in which chromosome transfer takes place because of limited homology between the conjugative F plasmid and regions of the chromosome where common insertion sequences (IS) are located. Using transposable elements, an artificial homology can be constructed by the presence of a transposable element on both a plasmid and the chromosome. This has been achieved using well-known transposons such as Tn1, Tn3, Tn5, Tn10, Tn501, and Mu to create chromosome mobilizing systems…