A Broad Brush, Global Overview of Bacterial Sexuality

Bacterial sexuality is confusing, even for experts! I used to be such an expert on one mechanism of sexuality, conjugation, but that was over 30 years ago. At that time, extra-chromosomal elements, so-called plasmids, were known to encode multiple proteins that together enabled cell-to-cell contacts, which were then used to transfer single-stranded DNA from donor to recipient, thus providing the plasmid with a new host. Transfer of the plasmid resulted in concomitant transfer of any genes that it happened to include, such as genes encoding resistance to antibiotics or virulence factors. On unusual and rare occasions, the plasmid integrated into the chromosome, resulting in the conjugative transfer of chromosomal DNA. What was particularly confusing was the plethora of plasmids that encoded genes for conjugation, each apparently different from the other, and the corresponding large variety of differences between mechanisms of conjugation associated with different plasmids. Some plasmids didn’t even encode genes for conjugation, but simply hitchhiked with the help of conjugation proteins expressed by other plasmids, a phenomenon called mobilization. Fast forward 30 years, and things became even more confusing. We have learned that conjugation doesn’t even need plasmids. So-called integrative conjugative elements (ICEs) are capable of conjugation, but unlike plasmids, which are predominantly free in the cytoplasm, ICEs are integrated into the bacterial chromosome(s). The ends of ICEs contain short stretches that can recombine via sitespecific recombination, similar to the excision of bacteriophages or transposons. Like plasmids, conjugation transmits the ICE itself, which first excises from the chromosome within the donor and finally integrates into the recipient genome [1]. But occasionally the ICE also transfers chromosomal DNA, which can correspond to a significant proportion of the entire bacterial genome [2]. And bacterial chromosomes can contain still other transmissible elements, including some that can be mobilized by ICEs, such as integrative and mobilizable elements (IMEs) [1]. Conjugation (and other forms of sexuality such as transduction and transformation) can have dramatic evolutionary consequences. The use of methicillin for medical treatment of staphylococcal disease is now endangered due to the repeated selection [3] of independent staphylococcal variants that contain a methicillin resistance gene that probably evolved in non-pathogenic staphylococci [4]. The repeated acquisition of genomic islands (and the parallel loss of others) has resulted in ‘‘open’’ pan-genomes in some bacterial species [5], such as Escherichia coli, in which the variable (dispensable) portion of its genome is more than ten times as large as the conserved core genome [6]. Homologous recombination is as frequent as mutation in many microbial taxa [7], potentially facilitating selective sweeps of novel genes or particularly fit combinations of nucleotides throughout a species. Horizontal gene transfer between taxa is thought to be especially frequent between the inhabitants of a common environmental niche, and can blur or even eliminate patterns of phylogenetic descent [8]. But which particular genetic elements are responsible for these inundations with foreign genes? Plasmid-encoded conjugation can be subdivided into three genetic modules. The first, increasingly referred to as MOB, consists of a relaxase that nicks double-stranded, super-coiled DNA at a specific oriT site. The relaxase forms a socalled relaxosome complex with the terminal nucleotide of the nicked DNA, a single strand of which is then transferred by conjugation. The biochemical details of this nicking and coupling reaction are becoming clearer [9], more so than for the two other modules. The second module consists of a Type IV secretion system, often abbreviated as T4SS. The T4SS is a protein pore through the cell surface, whose magnificently beautiful, basic structure has recently been elucidated in Gramnegative bacteria, in which it connects the inner and outer membrane through the periplasm [10]. T4SS genes are essential for conjugation, and are often genetically linked to genes encoding a pilus, a protein grappling hook that can bind to other cells, or to surface adhesins [11]. T4SS are also sometimes misused by malicious pathogens to inject proteins and DNA into eukaryotic cells and to secrete them into the environment [11,12]. Finally, the complexed relaxase plus the single-stranded DNA end are transferred to the T4SS secretion system by the third module, consisting of a coupling protein, the T4CP, which links the relaxase-DNA complex to the T4SS and translocates the entire DNA single strand to the recipient. The transferred molecule is then re-ligated by the relaxase. These three modules are associated with a bewildering variety of different gene and protein families in plasmids, whose gene designations are arcane leftovers from the time when I was still an expert in this area. The basis of conjugation by ICEs is more poorly understood, except that the conjugation proteins encoded by some ICEs are quite distinct from those encoded by plasmids [1,12]. Due to two recent publications from groups led by Eduardo Rocha and Fernando de la Cruz, order is beginning to emerge from chaos, allowing a broad brush overview of the genes that are responsible for conjugation, and of the organisms in which they can be found. In their earlier publication [13], de la Cruz and Rocha examined 1,730 plasmid genomes, half of which were from proteobacteria, and the other half of which were primarily from firmicutes, spirochetes, and actinobacteria. A bioinformatic pipeline