Plasmid-encoded biofilm factors
The typical plasmid genome can be divided into backbone and accessory regions. The traits encoded in the backbone include replication, partition, stability and mobilization functions and can be thought of as the essentials of a functionally stable minimal plasmid entity. Genes that encode functions, which enhance the fitness of the plasmid's host under a given selective pressure, are typically described as accessory genes (Smillie et al., 2010). Examples of functions of accessory genes include resistance towards antibiotics, metals, bacteriocines, metabolic functions and attachment to specific surfaces, to name a few (Norman et al., 2008).
Biofilm-associated factors (BAFs) can be encoded both by genes in the backbone and the accessory regions of plasmids. This indicates that biofilm formation may be of importance for some plasmids. This is especially true for the BAFs encoded by the backbone genes (e.g. conjugal pili), as such genes are well-integrated parts of the plasmids biology.
Although only a few BAFs of plasmids have been studied in details, we will give a few examples to illustrate the variety of factors that can be involved. As the understanding of the interconnectedness between biofilms and plasmids is further explored, many more examples will, no doubt, be uncovered.
The plasmid backbone and biofilm formation – the conjugative pili
Conjugation is, in itself, aggregative in nature, promoting cell–cell contact between donors and recipients, thus demonstrating that the backbone of conjugative plasmids by default promotes interactions, which in time may lead to biofilm formation. Surprisingly, Ghigo (2001) found that all investigated conjugative plasmids could also prime surface-associated biofilms by providing cell-surface adhesive properties. These results were supported by Reisner et al. (2006) who found that biofilm formation was most common for natural E. coli isolates that harboured conjugative plasmids. It was also shown that biofilm formation was most pronounced during derepression of plasmids. This phenomenon has mostly been studied using incF-type plasmids. Naturally repressed incF plasmids also promote biofilm formation, but to a lesser degree. Research on biofilm formation primed by conjugative incF plasmids indicates that the expression of the conjugative pili is implicated in biofilm priming, but it seems that the pili is not the main factor directly facilitating the adherence. Cell-surface adherence may, however, be initiated mainly by activating the host biofilm system. This was shown to be the case by May & Okabe (2008), who discovered that expression of colonic acid and curli in E. coli was induced by a natural incF plasmid. They therefore proposed that the conjugative pili promotes cell–cell contact whilst the induction of colonic acid and curli production enables cell-surface adherence in addition to overall stability and structure of the biofilm.
The genetics and mechanics behind biofilm priming by conjugative incF plasmids are not well understood as the interactions between host chromosome and plasmid have proven to be very complex (González Barrios et al., 2005; Yang et al., 2008; May et al., 2010; Nuk et al., 2011). The connection is, nevertheless, well documented, and unveiling such interactions is important to understand the interconnectedness between biofilm formation and plasmid biology. The example of conjugative incF biofilm priming shows us that what can be considered the plasmid backbone may have evolved in such a manner that biofilm priming is intrinsic to many plasmids.
Biofilm factors encoded in accessory regions of plasmids
In the accessory regions of plasmids, different examples of biofilm priming factors have been found. The best understood examples of these are fimbriae and nonconjugative pili that are known to mediate cell–cell (bacteria and/or eukaryotic) contact but also cell-surface adherence. Fimbriae and some types of pili are structures that are dedicated to mediating adherence and thus biofilm priming. Looking at members of Enterobacteriaceae, three assembly pathways of surface-associated fimbriae have been identified: the type IV pili pathway, the nucleation pathway (curli assembly) and the chaperone/usher pathway (Clegg et al., 2011). These three pathways have given rise to a wealth of fimbriae structures that show enormous diversity in genetic structure and regulation, but also in specificity and function. It is typical to identify multiple different types of chaperone/usher fimbriae amongst different genera and also in a single bacterial genome (Clegg et al., 2011). Within E. coli genomes, there are fimbriae-encoding genes both on mobile elements such as plasmids and amongst core genes of the chromosome. Plasmid-encoded fimbriae are identified both on conjugative and nonmobilizable plasmids. We have, in our group, characterized different biofilm-enabling plasmids that encode type 3 fimbriae. Some of these plasmids were isolated from environmental samples based on a PCR screening for IncX plasmid replicons, whereas others were isolated because of the enhanced ability of their host to attach to abiotic surfaces (Norman et al., 2008). Based on complete nucleotide sequencing of a number of these plasmids, the origin of the type 3 fimbriae encoding mrkABCDF cassette was identified. Investigations of the composite transposons, which mobilize the mrkABCDF cassette, revealed the likely modular mobilization of this gene cassette from a Klebsiella pneumoniae chromosome to incX1 type plasmids (Norman et al., 2008; unpublished data). Besides enhancing attachment and biofilm formation, the mrkABCDF cassette of the incX1 plasmid, pOLA52, also resulted in elevated transfer frequencies (when compared to a mrk-knock-out mutant of pOLA52; Burmølle et al., 2006). Finding biofilm primers such as fimbriae in the accessory regions of plasmids from many independent origins indicates that the adhesive properties must be advantageous to the host bacterium.
Another adhesion structure protruding from bacterial surfaces are the type 4 pili. Type 4 pili are interesting biofilm primers because these pili are found widely distributed amongst Bacteria. Research even suggests that type 4 pili-like organelles are found in Archaeae (Pohischroder et al., 2011). Type 4 pili are remarkable in that they enable not only attachment but also a variety of other functions including gliding motility, twitching motility, DNA uptake and signal transduction (Craig & Li, 2008). In E. coli, we see examples of type 4 pili encoded in the chromosome but also on plasmids and other MGEs (Pelicic, 2008). Intriguingly, the type 4 pili often found on conjugative plasmids of E. coli (the incI-cluster), a type associated with a so-called shufflon (Gyohda et al., 2004), seem to have been partly incorporated into the conjugative apparatus and are required for conjugation between cells in liquid culture (Sampi et al., 2010). Regulation of the type 4 pili of the incI1 plasmid R64 shares regulatory genes with the conjugative pili (Kim et al., 1993). The related IncI2 plasmid R721 has fragmented type 4 pili where two of its genes are located away from the type 4 pili operon and situated amongst genes of the conjugative pili (Kim & Komano, 1992; accession number: AP002527). These type 4 pili thus represent a biofilm priming module that is somewhere between an accessory and a backbone-related element. This implies that biofilm priming associated with the accessory region might be incorporated to become part of the backbone. It is most likely that accessory genes are gained and lost at relatively higher rates than backbone genes, and for genes to be incorporated into the backbone of a plasmid implies that the function of the genes is of general importance for the success of the plasmid.
It is noteworthy that plasmids that have been associated with biofilm formation belong to the incF (incFI – incFV), incX and the incI cluster and have also been shown to be amongst the most relatively abundant plasmids harboured by E. coli (Reisner et al., 2006, unpublished data), a bacterium that, in the absence of these plasmids, shows weak biofilm forming capabilities on abiotic surfaces.
Many of the aforementioned examples of biofilm-enabling plasmids originate from E. coli, but biofilm priming encoded on plasmids has been identified in both Gram-negative and Gram-positive bacteria such as the Pseudomonas putida TOL plasmid (D'Alvise et al., 2010), Lactococcus lactis pAMβ1 (Lou et al., 2005), Azospirillum brasilense plasmids (Pentrova et al., 2010) and the Enterococcus faecialis pBEE99. Similar to E. coli, E. faecialis commonly occupy a commensal niche in the gastrointestinal tract but also appear as opportunistic pathogens. Random mutagenesis revealed that knocking out the bee cassette of conjugative plasmid pBEE99 lowered biofilm formation of the parent strain by 70%. The bee cassette seems to be located on an element resembling a transposon. It is likely that bee encodes a pilus-like structure that showed distant relatedness to genes found in the chromosome of Leuconostoc mesenteroides (Tendolkar et al., 2006; Coburn et al., 2010).
Above we discuss the role of pili and fimbriae in biofilm priming. These are the examples that are best studied in connection with MGEs. Reports of plasmids that enable biofilm formation through increased EPS production were recently made; pO157 is such an example and enables a hyper-adherent E. coli variant (Lim et al., 2010). If biofilm priming/formation is a lucrative strategy for a plasmid and its host, then we can expect alternative BAFs to exist and function in various ways as a result of convergent adaptation. Hence, new plasmid-encoded BAFs will be identified as more focus is directed towards the interconnectedness between plasmid and biofilm biology.
Box 3. Social bacterial behaviours
Social behaviours are classified into four categories according to their effect on the direct fitness (fitness gained through reproduction) of the actor and the recipient. If both the actor and the recipient increase their direct fitness as a consequence of the social behaviour, this is defined as mutualism. If the behaviour increases the direct fitness of the actor but decreases that of the recipient, this would be selfishness. Spite occurs when the behaviour of an actor reduces both its own and the recipient's direct fitness. The last category, altruism, is seen when the actor's direct fitness decreases, whilst the recipient's increases (Hamilton, 1964).
The social behaviours classification scheme is applicable in social interactions between bacteria, but can also be applied when considering the relationship between plasmid and bacterium. The lifestyle strategy outcome of the specific plasmid can be defined according to the classification of social behaviours where the plasmid serves as the actor and the host as the recipient. Illustration of the four categories of social behaviours
From chromosome to plasmid and back – regulation of gene expression
One of the better-studied examples of proteins that are believed to have gained new functions through HGT by MGEs is that of some antibiotic resistance functions. We refer to Martínez (2008) for a fuller perspective on this subject. The basic idea is that proteins that confer antibiotic resistance have developed from household genes of various functions, mainly though HGT and selection. One such example is that of the OqxAB pump. The OqxAB pump is encoded in the chromosome of K. pneumonia where its function is unknown but it does not confer resistance to antibiotics (Hansen et al., 2007). The OqxAB pump does, however, provide multidrug resistance when overexpressed on plasmids in a variety of enterobacteria (Hansen et al., 2007). Such natural plasmids have been isolated from pigs that were treated with olaquindox, which is one of the drugs to which the OqxAB renders its host resistant (Hansen et al., 2007). Plasmids encoding OqxAB have, in addition to farm animals, also been isolated from humans (Kim et al., 2009; Zhao et al., 2010). Although this example is not biofilm related per se, it helps to illustrate an event where few proteins can change the host cell phenotype if they are taken out of their normal chromosomal context and expressed on a plasmid.
Another example is the transcriptional factor SoxR. Recently, Dietrich & Kiley (2011) argued that SoxR has different functions in enteric and nonentric bacteria, although the genes are clearly related. In enteric bacteria (E. coli), SoxR regulates only one gene, soxS, that subsequently regulates many (> 100) targets. In nonenteric bacteria, SoxR, which has been identified in Alpha-, Beta-, Delta- and Gammaproteobacteria and Actinobacteria (Dietrich et al., 2008), regulates multiple targets directly, including aspects of biofilm development (Pseudomonas aeruginosa and Streptomyces coelicolor). The soxR gene is believed to have been transferred horizontally from a nonenteric to an enteric bacterium. This represents an example of a single gene that has been transferred horizontally and, because of this and recombination events, subsequently functions differently in the new host bacterium (Dietrich et al., 2008).
Looking at the biofilm priming mrkABCDF system previously mentioned has proven to be very convenient because the nucleotide sequence of chromosomally encoded mrkABCDF in K. pneumoniae and plasmid-encoded mrkABCDF are almost identical, indicating a relatively recent mobilization of the mrkABCDF cassette to the incX1 plasmids. Lately, important regulatory elements of type 3 fimbriae encoded in the chromosome of K. pneumoniae have been revealed (Johnson & Clegg, 2010; Johnson et al., 2011; Wilksch et al., 2011). These regulatory key elements are not present on the mrkABCDF encoding plasmids (Norman et al., 2008; Ong et al., 2009, unpublished data), indicating that either new regulatory functions have progressed or constitutive expression occurs. This example illustrates how plasmids can function as evolutionary and adaptive templates for biofilm-related mechanisms. The regulatory networks directly connected to the BAFs found on plasmids are generally less complex than the ones found in the chromosome.
More complex examples of BAFs encoded on plasmids include type 4 pili as mentioned earlier. Tracking the evolutionary events of type 4 pili is complicated because these pili are very diverse and widespread. The ubiquity and the diversity do, however, indicate that the horizontal spread and evolution (modular included) of such BAFs have shaped the type 4 pili to have many properties related to biofilm formation and microbial socialness (Craig & Li, 2008; Pelicic, 2008).
The presence of biofilm-enabling incF plasmids in E. coli has been shown to affect the expression of numerous genes of the chromosome (González Barrios et al., 2005; Yang et al., 2008; May et al., 2010; Nuk et al., 2011). Further expanding the knowledge about how the innate biofilm system works in bacteria will help in the assessment of the importance and implications that genes encoded on MGEs have in cross-regulating and interacting with biofilm-related genes of the rest of the genome.
The pAA plasmids enable aggregative adherence amongst enteroaggregative E. coli (EAEC). The aggregative pAA behaviour is controlled through an AggR regulon. Intriguingly, there are indications that the plasmid-encoded AggR regulon can regulate a chromosomal operon of a pathogenic island of its host (Harrington et al., 2005). In turn, this indicates interactions across the genome. When BAFs of the communal gene pool are integrated into the chromosome, such as in the case of genomic islands, these MGEs are expected to become less independent and more integrated with the innate biofilm system of the bacterium.