Analyses of genomic sequences of bacterial pathogens have given an unprecedented view into their biology and evolutionary processes [1, 2]. A conclusion from these studies is that highly similar genes, many of which are associated with bacterial virulence, are found across great phylogenetic distances and in different genetic elements, which is indicative of horizontal gene transfer. These families of virulence factors - including toxins, transport systems, adhesins, and antibiotic resistance factors - have evolved by adaptive radiation of a functional progenitor molecule to and within other strains and species to support survival in differing ecological niches . The adaptation, or "evolutionary fine-tuning" of virulence factors that results in increased fitness, can involve modification of catalytic efficiency or substrate specificity of an enzyme, or alteration of bacterial interactions with target cells . The mechanisms of horizontal gene transfer and the functional diversity of bacterial toxin families and protein transport systems have been documented [2–4] but adaptive molecular evolution of bacterial virulence factors remains less understood in terms of altered structure/function relationships.
Yersinia pestis is the causative agent of plague, a zoonotic disease transmitted to humans usually by the bite of an infected flea . The bacterium spreads from the intradermal infection site into lymph nodes, causing bubonic plague, and subsequently to blood and to lungs, leading to pneumonic plague. The abilities to disseminate in the host and to cause high bacteremia are central for the transmission of the bacterium by the flea vector which feeds on contaminated blood. Y. pestis has been responsible for three human pandemics, which are estimated to have resulted in deaths of ca. 200 million humans . As a bacterial species Y. pestis is young, and recent population genetic studies have shown that the bacterium diverged from its ancestral species, the gastrointestinal pathogen Yersinia pseudotuberculosis, only ca. 13 000 years ago [6, 7]. The genome of Y. pestis has evolved through gene decay, recombination, single nucleotide changes, genome rearrangements, and horizontal gene transfer by acquisition of two Y. pestis-specific plasmids, of which the plasmid pPCP1 (pPst/pPla) potentiates bacterial dissemination from the primary intradermal infection site into lymph nodes [8, 9]. The decisive virulence factor encoded by pPCP1 is the surface protease plasminogen activator Pla. Deletion of pla attenuates Y. pestis millionfold in subcutaneously infected mice, whereas no difference is seen in intravenously infected mice . Pla is specifically needed for establishment of bubonic plague [10, 11], and a critical role of Pla has been described in pneumonic plague where it enables localized growth of Y. pestis in the lungs .
Pla belongs to an outer membrane protease family of omptins that have been detected in several Gram-negative bacteria of different phylogenetic groups; these bacteria commonly infect animals or plants . The omptin genes have spread through horizontal gene transfer by different mechanisms, and at least 16 members are known to date [13–16]. As omptin sequences are over 50% identical, they very likely fold similarly to the two structurally resolved members of the family, OmpT of E. coli  and Pla of Y. pestis . Both OmpT and Pla form a 70-Å long β-barrel of elliptical cross-section with ten antiparallel transmembrane β-strands, five surface-exposed loops (L1-L5) and four short periplasmic turns (T1-T4). The catalytic residues and the acidic, substrate-binding pocket, as well as the lipopolysaccharide-binding site are conserved in omptins [13, 17, 19]. This is in accordance with the finding that the cleavage site preferences of omptin proteolysis, analyzed mostly with short peptide substrates, are very similar: omptins cleave after basic residues, preferably between two basic amino acids [20–23]. With larger substrates, however, omptins are functionally diverse, and their genes have undergone adaptive evolution to support the life style of the bacterial host [14, 15, 24]. Insertions and deletions that give slight variation in the molecular sizes of omptins are mostly located in the solvent-accessible surface structures L1-L5, and the differential substrate selectivities of individual omptin proteases appear to be dictated by these surface loops, as observed by loop swapping and omptin chimeras [16, 19, 25, 26].
During infection, Pla seems to increase virulence mainly by interfering with the human plasminogen/fibrinolytic system that is critical in cell migration across tissue barriers. Pla cleaves the circulating, abundant zymogen plasminogen by a single cut to active plasmin  and also degrades two natural inhibitors of the plasminogen system, the serpins α2-antiplasmin (α2AP) and plasminogen activator inhibitor 1 (PAI-1) [16, 19]. Altogether, these activities result in uncontrolled plasmin activity. Plasmin is a broad-substrate protease involved in a number of (patho)physiological processes, of which fibrinolysis and damage of extracellular matrices contribute to bacterial dissemination within the host [12, 27, 28]. Pla has also non-proteolytic functions: it mediates invasion of Y. pestis into human epithelial and endothelial cells by an unknown mechanism as well as into mouse monocytes by binding to CD205 [29–31]. The Epo omptin is a close homolog of Pla, exhibiting 77% sequence identity, and it is encoded on a plasmid of the plant pathogen Erwinia pyrifoliae that causes tissue-destructive infection in pear trees . The functions of Epo in plant pathogenesis have not been systematically studied, but serpinolytic activity in vitro was recently shown .
The claim of directed evolution and adaptive molecular evolution is that natural selection generates particularly evolvable enzymes in response to rapidly fluctuating selective conditions and that proteins that require fewest mutations to adapt to novel conditions are the most likely to survive environmental changes . Evolvability has been argued to be a function of robustness, i.e., the capacity of a protein to withstand sequence variations without disruption of its binding or catalytic properties [33, 34]. Mutational robustness is dependent on a protein's intramolecular interactions, and the omptin molecule fulfils the criteria of an evolvable protein as defined by O'Loughlin et al. ; the β-barrel is a sturdy membrane-embedded molecule with flexible surface loops that can tolerate deletions and insertions without compromising molecular stability [35, 36]. Manifestations of the robustness of omptins include their unhurt catalytic activity after autocatalytic cleavage at L4 and the proteolytic specificity of omptin loop chimeras, which together have indicated that the five loops offer a flexible scaffold for recognition of different substrates [16, 19, 25, 26].
Evolutional studies on bacterial virulence factors highlight the importance of environment as an immense reservoir of evolvable potential progenitors of virulence factors . Pla of Y. pestis and Epo of E. pyrifoliae belong to the same subfamily of omptins which also contains the omptins PgtE of Salmonella enterica, Kop of Klebsiella pneumoniae, and the omptin of Enterobacter [15, 16]. These bacterial species infect mammals or plants or both, and have encountered mutual horizontal gene transfer. In this study we used the existing knowledge of omptin structure and function as a starting point for substitutional analysis, and addressed two specific questions on molecular adaptation: how many substitutions are needed and at which locations in Epo to gain a proteolytic function (plasminogen activation) and a non-proteolytic function (invasiveness) expressed by Pla.