The analysis of protein sequences makes it possible to identify conserved domains in proteins, such as enzyme catalytic sites, cofactor binding sites, small molecule ligand domains, DNA binding domains and many others (Corpet et al., 1999; Bradley et al., 2008). Proteins or protein domains belonging to a particular family often share functional attributes, and therefore the grouping of proteins has been used in turn to characterize them at the functional level (Holm and Sander, 1996; Tatusov et al., 2001). Several approaches are available to define domains and protein families, and the availability of semi-automatic methods for profile construction, as well as their high sensitivity, has improved the efficiency and eased the process involved in the definition of protein families (Bucher and Bairoch, 1994; Bucher et al., 1996; Bateman et al., 2004; Ramos et al., 2005; Tobes and Ramos, 2005; Hulo et al., 2006). Profiles are not necessarily confined to small regions with high sequence similarity, but rather they attempt to characterize a protein family (or domain) based on full-length sequences (Rigali et al., 2002; Hulo et al., 2006; Molina-Henares et al., 2009).
Microorganisms in the environment are exposed to a large number of drugs of natural and xenobiotic origin and, as such, have developed strategies to cope with toxic compounds. The extensive use of some drugs in medicine, such as biocides and antibiotics, has lead to a major therapeutic problem as bacteria have developed resistance to multiple antibiotics (Zhang and Mah, 2008; Aminov, 2009; Baquero et al., 2009; Daniels and Ramos, 2009). Resistance-Nodulation-cell Division (RND) efflux pumps are common elements in multidrug resistance, and their wide substrate specificity explains cross-resistance between antibiotics, biocides, dyes and solvents in laboratory strains (Nikaido, 1996; 1998; 2000; Daniels and Ramos, 2009; Nikaido and Takatsuka, 2009). A number of RND efflux pumps have also been described that extrude heavy metals, and represent a major determinant in the proliferation of microorganisms at sites polluted with zinc, lead, mercury, cobalt and other metals (Checa et al., 2007). Although the entire suite of physiological functions of RND pumps has not yet been well established, a number of recent studies have shown that these pumps may be involved in the extrusion of intracellularly generated toxic compounds. These RND pump-excluded compounds may include, for example, formaldehyde produced from the metabolism of histidine and methoxylated chemicals (Roca et al., 2008); amino acids to maintain amino acid homeostasis (Herrera et al., 2010); and quorum sensing molecules (Pearson et al., 1999; Ueda et al., 2009). As well, RND pumps may also be important for protecting cells against toxic compounds in the cell's environment (Levy, 1998; Nikaido and Takatsuka, 2009).
A typical RND efflux pump consists of three components, with one of these components being the inner membrane protein, which acts as the extrusion element, and which is often more than 1000 amino acids long, consisting of 12 transmembrane helices (Murakami et al., 2002; Yu et al., 2005; Törnroth-Horsefield et al., 2007; Nikaido and Takatsuka, 2009). A second component of RND efflux pumps is the outer membrane protein that penetrates into the periplasmic space to form a channel (Koronakis et al., 2000; Nikaido, 2000; Wong et al., 2001). The third component is a lipoprotein that is linked to the inner membrane, and which plays a role in stabilizing the interactions between the two other elements (Zgurskaya and Nikaido, 1999; Mikolosko et al., 2006; Takatsuka and Nikaido, 2009). The best-studied antibiotic-extruding RND pump in structural and functional terms is the AcrAB-TolC system in Escherichia coli (Nikaido and Takatsuka, 2009). This pump was initially described as a transporter for the topical antiseptic acriflavin (hence the name Acr), but it was later shown to transport a large variety of other substrates (Nikaido, 1998; Fábrega et al., 2009). Another well-characterized RND pump is the CzcABC metal-extruding pump, which is involved in the extrusion of heavy metals such as zinc, cadmium and cobalt (Nies, 1999; 2003).
In this study we have constructed a stringent profile for RND efflux pumps and have used it to search for and identify members of this family within annotated genomes. With the protein sequences that we found we were able to group them, using Z-score values and phylogenetic analysis into four groups, which represents a novel method regarding the application of profiles. Based on gene annotation data, those within groups 1 and 3 appeared to be involved in extrusion of antibiotic and metals respectively. Using a series of P. putida KT24440 mutants, we were able to validate that the efflux pumps found in group 1 do in fact extrude a wide range of antibiotic compounds, and that those in group 3 extrude heavy metals. The paucity of information available for proteins in groups 2 and 4 made it necessary to carry out screens, using mutants, in order to assign functional activities to these groups. Our results show that the efflux pumps found in group 2 are involved in the extrusion of oxidative stress-causing agents, while the pumps in group 4 appear to be involved in the extrusion of organic and inorganic chemicals.