During the past 25 years, a nearly exponential increase has occurred in nucleotide sequences available from databases. The first microbial genomes were published in 1995 (Fleischmann et al., 1995; Fraser et al., 1995; Himmelreich et al., 1996); the NCBI database currently contains 534 complete bacterial chromosome sequences. The genomes have been determined not only from different species, but also from different strains of the same species. This has paved the way for comparative genomics and has allowed detailed analysis of genetic differences between strains (Wren, 2000; Dobrindt and Hacker, 2001; Edwards, Olsen, and Maloy, 2002; Raskin, Seshadri, Pukatzki, and Mekalanos, 2006). Since infectious diseases are a major health threat worldwide and range among the most frequent causes of death worldwide (WHO Health Statistics 2006), it is not surprising that the first two organisms to be sequenced were pathogenic bacteria.

During the past decade many attempts have been made to understand the molecular basis of microbial pathogenicity, and our knowledge has advanced quickly, in particular through the use of methods of cellular biology, genomics, and proteomics. Various events in the pathogenesis of microbial infections, such as adherence to and entry into human and animal host, invasion of host cells, toxin production, establishment and dissemination of bacterial populations in the host, and the role of the host immune system, have been studied in detail for many host-pathogen interactions.

INTRODUCTION

Despite our interest and motivation, bacteria are not particularly easy organisms to study; their niches are complex and poorly understood and the vast majority of these species are difficult to culture or to manipulate in the laboratory. Of all bacteria, it is pathogens whose physical, social, and economic impact on our day-to-day lives garners the most attention, from both scientists and non-scientists alike. As a result, pathogens are among the best-studied bacteria, and lessons we learn from them are often generalized to other, non-pathogenic bacteria. Not surprisingly, the first lessons learned in the so-called genomic era came from pathogens, which were the first organisms with fully sequenced genomes. The promise of genomics was that the limitations of conventional microbiology could be overcome by studies of genome sequences and careful analysis of the genes contained therein. Here we examine how genomics has shaped our understanding of microbial genome evolution and ask how extensible these lessons may be. Among the notions that attracted widespread attention was the finding that certain clusters of genes are specifically responsible for virulence and that these loci are often obviously of foreign origin, having been introduced by the then under-appreciated process of lateral gene transfer or LGT. Coming more than a decade after these findings, this volume is focused on the hugely influential role of LGT in the evolution of genomes, particularly those of pathogenic bacteria.

INTRODUCTION

The bacterial genome, that is, the entirety of all genes of a bacterium, was once viewed as a rather stable entity. However, the observation of spreading resistance to antibiotics led to the discovery of extra-chromosomal elements encoding this property. Obviously, plasmids are able to transfer genes from one bacterium to another not only among one species but also from one species to another. Such transfer of genes is not restricted to antibiotic resistance genes. Examples of further traits often encoded by plasmids include resistance to heavy metals and production of toxins.

Another example of mobile genetic elements is phages, the viruses of bacteria. Phages are not just able to infect and finally lyse the bacterial host cell. Certain phages infect and then integrate their whole genome into the bacterial chromosome and thereby become a prophage. This may add another important factor to the property of the infected bacteria. In the case of pathogenic bacteria the production of toxins is frequently encoded by a prophage. A few medically important examples are bacteriophage β of Corynebacterium diphtheriae encoding diphtheria toxin, phage C1 of Clostridium botulinum coding for the C1 neurotoxin, and phage H-19B of Escherichia coli, which harbors the gene for Shiga toxin Stx1 (for a recent review, see Brüssow et al., 2004).

Smaller but still important mobile genetic units are insertion sequence (IS) elements. IS elements mediate DNA rearrangements by transposition, resulting in off/on switching of gene expression by insertion into, and excision from, open reading frames (ORFs), respectively.

INTRODUCTION

In eukaryotes, the great majority of genetic recombination takes place during the complex and highly organized process of meiotic division, a part of sexual reproduction. As a consequence there are a number of constraints on patterns of variability in the recombination process. Recombination takes place only between organisms that are similar enough for their offspring to be viable, and therefore it is generally limited in the novelty it can introduce. Within species, the most common cause of reproductive isolation is geographical separation. In most higher animals and plants, the number of crossovers per chromosome is predictably a number between 1 and 5 in both sexes. Even where individuals differ in the amount of recombination that they initiate, for example because of the absence of crossing over in male Drosophila, the existence of a common mating pool will tend to homogenize the population with respect to the amount of genetic exchange that has occurred in the ancestry of each individual. In summary, while the mating process is elegant, eukaryotic recombination is typically quite predictable with minimal differences in genetic patterns between individuals in the same species.

In bacteria, there are no such rules. Recombination is never obligate and occurs by three distinct mechanisms; transformation, transduction, and conjugation, each of which in their nature can vary enormously between lineages.

INTRODUCTION

During the past 20,000 years the most striking change in the lifestyle of humans was the transition from the hunter-gatherer culture to the introduction of agriculture and animal husbandry associated with stable settlement in the Neolithic. With sufficient food resources, the human population, which had been scattered, started to grow. Larger urban communities were formed, which was the starting point of culture and technology.

Humans have evolved with bacterial communities (microecological systems) as colonizers (skin, mucosa, gastrointestinal tract) and as conditional pathogens. For true pathogens the more dense human communities became of particular interest, especially for specialization in the human hosts (McKeown, 1988).

The so-called technical revolution that began at the end of the 19th century created a need for energy as a permanent concern. Because of continuing urbanization and the growing human population in the Third World, sustainability of food supply remains an important issue.

When, 200 years ago, a more productive agriculture began that was based on the then-young agricultural sciences, the principle of enduring means of production long endured, especially with regard to recycling of energy. These principles began to fade with the invention of mineral fertilizers, followed by mechanization and energy-consuming (wasting) means of production that were more independent of seasons. By the middle of the last century this had led to a high degree of mechanization of agriculture, where animals and plants were regarded more as “work pieces” than as living beings.

INTRODUCTION

Like most eubacteria, S. aureus possesses a variety of mobile genetic elements (MGEs) that contribute in major ways to pathogenesis and its evolution. In addition to the typical MGEs carried by most bacteria, that is, prophages, transposons, and plasmids, S. aureus possesses two types of novel elements that have not been described for other bacteria, namely the superantigen-encoding pathogenicity islands and the resistance-encoding SCCmec elements. In this chapter, the general properties of these various MGEs are summarized, with special emphasis on the two novel types and on their contributions to pathogenesis and its evolution.

MOLECULAR GENETICS OF THE STAPHYLOCOCCAL MGEs

Plasmids

For a comprehensive review of plasmid origins and interactions, see Firth and Skurray (2006). Staphylococcal plasmids range in size from 1.2 to more than 100 kb; all known staphylococcal plasmids are circular duplex DNA, using either of the two standard modes of replication, theta and rolling circle (RC), with the latter being used principally by those of less than 10 kb, and the former by those larger, though this is only an approximate dividing line. As with all other plasmids, replication of staphylococcal plasmids is negatively autoregulated. For the known small RC plasmids, this is accomplished by cis-encoded antisense RNAs, sometimes with the assistance of small proteins. Theta plasmids of the pSK41/pGO1 family also appear to use an antisense mechanism.

INTRODUCTION

Intracellular bacteria (symbionts and parasites) are characterized by a genome reduction syndrome that, when compared to their free-living relatives, leads us to the conclusion that they are evolving anomalously. Is this right? The notion of anomaly has an anthropocentric connotation, and from such a viewpoint we cannot state that genome reduction is an evolutionary anomaly; likewise we cannot state that parasitic organisms represent a degenerate stage of evolution as compared to their non-parasitic ancestors. By contrast, we can affirm that they represent an anomaly if we are unable to explain their origin and evolution, given there is no suitable theory to explain both the increase in genome size and evolutionary complexity as well as the genome reduction process in endosymbionts. The question is: Do we have such a theory? In the past few years Michel Lynch and collaborators have published a series of papers on this particular issue, trying to integrate the evolution of genome size and concomitantly genome complexity of prokaryotes and eukaryotes into a single theoretical framework following the basic principles of population genetics.

In this chapter, we would like to deal with how basic principles, such as mutation, selection, and effective population size, can give us an acceptable explanation, empirically founded, of the genome reduction process in endosymbiotic bacteria. We propose a model that both explains the huge transformation of endosymbiotic genomes at nucleotide level and accounts for genome reduction.

INTRODUCTION

It appears that one of the first things that occurred to Felix d'Herelle when he discovered bacteriophages in 1917 was that these mysterious objects might provide a means of killing bacteria that are pathogenic to humans (Summers, 1999). The still ongoing story of phage therapy, as this approach was called, has been told elsewhere and will not be retold here, but it serves to point out that scientists have been interested in the effects of phages on their hosts since their discovery. d'Herelle believed, and eventually established, that phages are viruses that infect bacteria. However, it was not until the experimental investigations of phages at the dawn of molecular biology in the 1940s and 1950s that it became clear that phages - and for that matter their bacterial hosts - are genetic organisms (Luria and Delbrück, 1943; Hershey and Rotman, 1949; Hershey and Chase, 1952; Stent, 1963), just like fruit flies, corn, and humans, and so could be expected to mutate and evolve.

Although some work was done on the evolution of phages in the 1960s, 1970s, and 1980s, a more detailed understanding of the genetic mechanisms of phage evolution had to wait until the advent of high-throughput DNA sequencing in the 1990s. This is because the genetic history of a phage, while it is to a significant extent encoded in the phage's genome sequence, is largely invisible to our analysis until we can compare that sequence to the genome sequences of other phages.

INTRODUCTION

The evolution of bacterial pathogens from non-pathogens or from avirulent strains is a major cause for concern in agriculture. As exemplified by the explosion of antibiotic resistance in human pathogens, bacteria can rapidly overcome control strategies and host resistance. As we are now discovering, the intrinsic plasticity of the bacterial genome combined with horizontal gene transfer is the major determinant influencing the expression of pathogenicity. Many of the disease symptoms caused by pathogens on plants, including blights, galls, chlorosis, scabs, leaf spots, and wilting, are attributable to genes that are often clustered together and, in some cases, acquired from distantly related bacteria. One mechanism that affects the virulence of plant pathogens is the loss or gain of DNA regions called genomic islands (GEI).

GEI were first described as pathogenicity islands (PAI) in human pathogenic Escherichia coli by Hacker et al. (1990), who discovered that a region of chromosomally located, virulence-associated genes of uropathogenic E. coli was absent from some E. coli isolates (Blum et al., 1994). The term GEI is now more appropriate given that the features of PAI are displayed by a number of regions of DNA with functions other than pathogenicity, for example, symbiosis, metabolic, or resistance islands (Hacker and Kaper, 2000; Hentschel and Hacker, 2001).

INTRODUCTION

Bacterial genomes are no longer considered a stable and rigid DNA structure carrying the essential genetic information for survival, fitness, and transmission of the species. With the development of genomic high-throughput techniques such as sequencing of whole bacterial genomes (with a total in November 2007 of 597 complete microbial genomes that are sequenced and 879 genomes in progress; http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) and the bioinformatics tools, our view of bacterial genome plasticity has changed. Bacterial genomes are now regarded as highly flexible and dynamic structures that change in size, genetic content, and organization over time (Hanage et al., 2006). This flexibility is also known as genome evolution (Groisman and Casadesus, 2005). It is thought that this evolution is driven by the adaptation of microorganisms to environmental niches, changes, or stresses and occurs via several mechanisms including point mutations, deletions, and gene acquisition/loss via horizontal gene transfer (Albiger et al., 1999; Chen et al., 2005). This latter process is associated with mobile genetic elements such as conjugative plasmids, bacteriophages, transposons, insertion sequence (IS) elements, and genomic islands (Albiger et al., 1999; Frost et al., 2005).

In Gram-negative bacteria, pathogenicity islands (PAI) are genomic regions that harbor one or more gene clusters encoding virulence-associated properties (reviewed in Gal-Mor and Finlay, 2006; Schmidt and Hensel, 2004). They are present in the genomes of pathogenic bacteria and usually absent from the same or closely related non-pathogenic species.

INTRODUCTION

Acquisition of genetic elements by horizontal transfer has played a major role in the evolution, virulence, and transmission of many bacteria. The genus Yersinia represents a very good example of a bacterial group whose pathogenicity and transmission progressively evolved with the gradual acquisition of foreign genetic elements.

Yersinia are Gram-negative bacteria that belong to the family Enterobacteriaceae. The genus is composed of 12 species that can be differentiated into pathogenic (Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis) and non-pathogenic (Y. intermedia, Y. kristensenii, Y. fredericksenii, Y. aldovae, Y. rohdei, Y. bercovieri, Y. mollaretii, and Y. aleksiciae) species (Figure 8.1; see also color plate after p. 174). Y. ruckeri is not discussed here because the inclusion of this fish pathogen in the genus Yersinia is still controversial. Like several other species of Enterobacteriaceae, Y. enterocolitica and Y. pseudotuberculosis are true enteropathogens, while Y. pestis is the causative agent of plague. This chapter focuses on the evolution and lateral gene transfer in these three pathogenic species.

Y. enterocolitica and Y. pseudotuberculosis are widely spread in countries with temperate climates. They are transmitted by the fecal-oral route and cause intestinal symptoms such as abdominal pain (especially Y. pseudotuberculosis), diarrhea (especially Y. enterocolitica), and fever, usually of moderate intensity.

The species Y. enterocolitica is subdivided into six biotypes (1A, 1B, and 2 to 5). All strains are pathogenic except those of biotype 1A. Pathogenic Y. enterocolitica can be further subdivided into low- and high-pathogenicity strains.

INTRODUCTION

Salmonellae are enteric pathogenic bacteria that infect a vast spectrum of animal species from reptiles to mammals. The genus is highly diversified, comprising more than 2,500 serovars. An even greater diversity exists at the strain level as a result of countless combinations of genomic differences in individual isolates. Strain-specific variations are likely to influence aspects of the organism biology, such as the adaptation to specific hosts or environments, the tropism for certain organs or tissues, and the degree of pathogenicity. The emergence in recent years of epidemic clones that have become predominant, displacing pre-existing strains, confirms that strain diversification is an ongoing process. A leading mechanism promoting diversity in Salmonella genomes is lysogenization by temperate phages. Most strains harbor multiple prophages in variable numbers and combinations. Prophages modify the properties of the host bacterium in various ways, from expressing functions that directly influence pathogenicity, to improving the bacterium's ability to outgrow competitors or to resist killing by superinfecting phages. Unlike toxin-producing phages of other bacterial species, most Salmonella prophages contribute to virulence through the synergistic action of multiple factors playing subtle, often redundant roles. The scope of this chapter is to review the various facets of phage-mediated modification of Salmonella, as well as recent advances in the characterization of phage-borne virulence determinants.

SALMONELLA DIVERSITY

Current classification divides the genus Salmonella into two species, Salmonella bongori and Salmonella enterica.