Structural development of Haloferax volcanii biofilms and biopolymers of the extracellular matrix

The present study and that of Fröls and coworkers [38] demonstrate a propensity of haloarchaeal species to form biofilms and indicate this may be a predominant natural mode of growth for this group of organisms. While haloarchaea are considered extremophiles, the ability of any microbial group to form biofilms is consistent with the emerging view that biofilm formation is an adaptation common to most if not all species [16]–[19]. In this particular case, the benefits associated with a community lifestyle match the environmental conditions that challenge microbial life in hypersaline environments. For example, encapsulation within a hydrated nutrient-dense ECM and a biofilm structure with areas that surround developed microcolonies (Figure 1E-H), which likely act as channels to facilitate waste and nutrient exchange, may help explain the persistence of haloarchaeal populations through prolonged periods of starvation [73]. Overall, the delicate and motile nature of multicellular structures imaged in static liquid (Figure 1C-I and Figure 4) may reflect low levels of natural circulation and water activity within hypersaline environments and a selective advantage associated with movement towards favorable conditions.

Our staining experiments with H. volcanii H1206(pJAM1020) allowed for accurate discrimination between cellular structures emitting endogenous GFP signal and ECM and support previous evidence showing that polysaccharide and eDNA are major biofilm components (Figure 2B-E) [38]. Species of the genus Haloferax have long been known to produce exopolysaccharide [74] and recent studies of H. mediterranei (average nucleotide identity of 86.6% with H. volcanii[57]) have identified genes essential for exopolysaccharide synthesis and export [75]. Homologs of genes within an H. mediterranei operon shown to be required for exopolysaccharide production [75] are present in the H. volcanii genome. Several putative EPS operons have been identified: HVO_2056-HVO_2057 on the main chromosome, and HVO_A0216-A0221 and HVO_A0594-0595 on the mini-chromosome pHV4. While the function(s) of extracellular DNA within H. volcanii ECM is not yet clear, in bacterial biofilms, eDNA is known to be involved in attachment to surfaces, microcolony formation, overall structural integrity and even spatial self-organization [13],[76]–[78]. In several crenarchaeal species, eDNA is present within biofilms but is not a structural component [32]. Similar attempts at DNase treatment of eDNA in haloarchaeal biofilms were unsuccessful and may have been complicated by high salt concentrations within the growth medium [38]. Interestingly, ECM is also known to be a mechanism for nutrient storage [5], and we have previously demonstrated that H. volcanii is capable of utilizing eDNA as a nutrient [79],[80], suggesting that eDNA may contribute towards the growth of cells within the biofilm.

Bacterial appendages involved in attachment and biofilm formation are often composed of amyloid proteins [27],[64],[81],[82]. Previous studies have surveyed the presence of amyloid in natural biofilms using the dyes CR and ThT and found that it is an abundant and widely distributed ECM component [66],[67]. Those same studies reported that amyloid production by archaea could not be determined due to low recovery of archaeal cells from sample sites [66]. On the basis of visible staining with CR, CRF and ThT fluorescence, we have identified putative amyloid protein that was colocalized with H. volcanii microcolonies (Figure 2F-M). Although it is most commonly used to stain amyloid fibrils, it is important to note that CR may stain other biopolymers such as polysaccharide [83]. However, because it was colocalized with microcolonies and larger biofilm structures (i.e., not individual cells), CR likely stained a major ECM component and not an internally stored polymer. The gene HVO_1403 is homologous to a known amyloidogenic factor in Sulfolobus solfataricus[84] and is a potential genetic determinant of amyloid protein within H. volcanii biofilms.

Social motility of static liquid biofilms formed by Haloferax volcanii as a multicellular or collective behavior Macroscopic time-lapse images collected following disruption of SL-biofilms (Figure 4A) demonstrate a propensity for H. volcanii cells to re-aggregate within hours, rather than remaining suspended in liquid culture or evenly precipitating. During and after reformation, multicellular filaments or extensions formed, appearing to self-organize and explore the surface area along the bottom of the culture plate (see Additional files 3, 4, 5, 6 and 7: Movies 2–6). This activity was not seen in identically prepared SL-biofilms that had been heat-treated or treated with formaldehyde (see Additional files 6 and 7: Movies 5 and 6, respectively), indicating it is an active biological process, and suggesting the existence of an archaeal system evocative of the elaborate swarming behavior characterized in the soil bacterium Myxococcus xanthus[11],[85],[86] and in Pseudomonas aeruginosa[13]. Although swarming is strictly defined as migration across a solid surface [87] and is typically imaged in these species at a solid–air interface, early studies of rippling and wave-formation in M. xanthus swarms were also conducted in submerged liquid culture [88],[89]. The existence of a system for social motility in H. volcanii is supported by the known production of type IV pili-like structures responsible for surface adhesion [90], the prior observation of a hypermotile phenotype [91] and the presence of a complete set of chemotaxis genes (including CheA, CheB, CheC, CheD, CheR and CheW) positioned adjacent and interspersed with archaeal flagella gene clusters (i.e., flgA1 and flgA2) [43],[90],[91]. A predicted operon including four genes on the main chromosome (HVO_1221 to 1224) is of particular interest as HVO_1222, HVO_1223 and HVO_1224 proteins are homologs of bacterial frizzy aggregation (Frz)/defective in fruiting (Dif) proteins FrzF, FrzE/DifE and FrzG, respectively, factors that are essential determinants of social motility and ECM production in M. xanthus[92]. The ecological role of social motility in H. volcanii is undetermined at this point. It is plausible that multicellular groups might migrate towards a nutrient influx during known bloom events [93], or that social motility is associated with an additional activity, such as cooperative feeding.

Haloferax volcanii biofilms are hotspots for gene transfer While H. volcanii is often noted for its unique gene transfer system [56], the mechanism and genes underpinning this phenomenon remain largely uncharacterized [57],[94]. Here we report that mating occurs within biofilms at the same frequency as in earlier reports (Figure 5), consistent with the known cell-contact-dependent nature of the mechanism. Mating was discovered and has been routinely observed in the laboratory by several groups by co-precipitating cells or co-filtering them on nitrocellulose filters [56]–[58],[94],[95]. These experimental methods for bringing cells together in close proximity can be thought of as proxies for naturally formed biofilms. We show that H. volcanii biofilms contain a biologically formed layer of pleomorphic adherent cells at high density and in close association (Figure 3B,C,D and Additional file 1: Figure S2). The observed spatial arrangement and diversity of cellular structure is consistent with the model of cytoplasmic bridge formation and membrane fusion during mating [95], previous electron micrographs showing elongated cells with intercellular structures [39], and/or the involvement of additional contact-dependent cargo-transfer mechanisms not described in H. volcanii, such as lipoprotein transfer [96], vesicular trafficking [97] and cell-to-cell secretion systems [98]. Based on these observations, we suggest biofilms are the microenvironments where mating likely occurs in nature, and propose this mode of growth as an excellent way for studying and visualizing HGT mechanisms in live cells and in real time.