Haloferax volcanii cells develop into structured colony biofilms and static liquid biofilms

Planktonic H. volcanii DS2 cells grown in shaking culture (Figure 1A) readily formed biofilms in typical rich media types Hv-YPC and Hv-Ca within several experimental systems that provided a solid plastic or glass substratum. Colony biofilms [7] developed on the surface of polycarbonate filters placed on solid media (Figure 1B) and were cryo-processed and cross-sectioned, exposing a surface structure containing crevices bounded by globular structures (Figure 1B). The greatest level of structural complexity was observed when biofilms were grown in static liquid (SL-biofilms; Figure 1C). Cultivating SL-biofilms within chamber slides and on the surface of borosilicate glass coupons placed in six-well plates permitted direct staining and optimized imaging of delicate biofilm structure that was visible macroscopically (Figure 1C).

Figure 1 Growth and development of Haloferax volcanii static liquid biofilms. (A) Cells within typical shaken culture of H. volcanii DS2 under transmitted light. Scale bar equals 10 μm. (B) Cross section of cryo-processed H. volcanii colony biofilm grown on CA medium for 5days and stained with CellMask Orange (CMO). Scale bar equals 30 μm. (C) Photographs of SL-biofilms routinely grown and analyzed within chamber slides (top left; scale bar equals 2 mm) and on glass coupons within six-well plates (bottom left; scale bar equals 1 cm). A macroscopic photograph of biofilm growth on a 12.7-mm glass coupon is shown in the center (scale bar equals 2 mm) with an area magnified on the right (shown in white box). (D-H) CLSM of biofilms grown on glass coupons (within a six-well plate in Hv-Ca medium; scale bars equal 30 μm). Biofilms stained with FM 1-43 were imaged directly in bulk Hv-starve medium using a 63× water-immersion objective after 2 days (D), 5 days (E) and 7 days (F-H). (I) Biofilm development shown through orthogonal views of SL-biofilms on glass coupons stained with CMO. CLSM, confocal laser scanning microscopy; CMO, CellMask Orange; SL-biofilm, static liquid biofilm. Full size image

We began by examining the development of wild-type H. volcanii DS2 biofilms in static liquid using CLSM and the cellular membrane dyes FM 1-43 and CellMask Orange (CMO; Figure 1D–I, Additional file 1: Table S1). Circular microcolonies formed within 48 h (Figure 1D), which led to well-defined clusters/aggregates after 5 days (Figure 1E). SL-biofilms reached maturation after 7 days of incubation at 42°C having developed into multi-layer towers with flake-like morphology (Figure 1F,G,H). Large towers were surrounded by a dense layer of smaller clusters or microcolonies and were separated by areas with little or no cell density (Figure 1E–H). Overall structural integrity was maintained as large clusters surrounded by smaller microcolonies for several weeks, although a comparison of orthogonal views of CMO-stained SL-biofilms showed that after 2 weeks the height of most structures was diminished and less of the total surface area was covered with microcolonies (Figure 1I, 2 weeks). Large clusters/towers, like the example shown in Figure 1F,G,H, varied in height and width, with a maximum measured height of 148 μm.

Microcolonies within biofilms formed by a GFP-expressing Haloferax volcanii strain bind stains targeting polysaccharide, DNA and amyloid protein

Several H. volcanii strains were engineered to express GFP to study ECM composition with the goal of reinforcing and expanding staining experiments conducted by Fröls and coworkers [38]. Confirmation of GFP expression was first accessed within colonies formed by two GFP-expression strains produced in this study. Colonies formed by the parental H. volcanii H1206 strain (Figure 2A; left panel) did not autofluoresce with blue excitation (Figure 2A, center panel), while those formed by the H. volcanii H1206(pJAM1020) strain based on the previously developed plasmid pJAM1020 (see Additional file 1: Table S2) [59] showed uniform and high levels of GFP fluorescence (Figure 2A; right panel). An additional GFP-expressing strain H. volcanii H1206(pSC409GFP), containing the same red-shifted gfp gene as in pJAM1020 but cloned into the plasmid pTA409, produced colonies with GFP signals that differed in both intensity and spatial distribution, resulting in an assortment of patterns of GFP signal within developed colonies (Additional file 1: Figure S1). The H1206(pJAM1020) strain was therefore selected for biofilm compositional studies to ensure stable GFP expression throughout the cellular population.

Figure 2 Visualizing the extracellular matrix of Haloferax volcanii biofilms. (A) Development of a GFP-expressing strain for use in biofilm visualization: colonies formed by the parental strain H. volcanii H1206 under transmitted light (left), under blue excitation (center), and by the strain H. volcanii H1206(pJAM1020) under blue excitation (right). Scale bar equals 250 μm. (B) Seven-day H. volcanii H1206(pJAM1020) SL-biofilm stained with concanavalin A-Texas Red collected with blue excitation (left), green excitation (center), and shown as an overlay (right). Scale bar equals 20 μm. (C,D,E) Top-down projections of a Z-stack for a H. volcanii H1206(pJAM1020) SL-biofilm stained with DAPI. (C) GFP signal under blue excitation. (D) DAPI-stained material with violet excitation. (E) Overlay of (C) and (D). Orthogonal views from CLSM analysis are shown below each panel and the plane of the orthoslice is shown in (E). Scale bars equal 20 μm. (F,G,H) Seven-day H. volcanii H1206(pJAM1020) SL-biofilm stained with Congo red with blue excitation (F), and green excitation for CR fluorescence (G), with an overlay of (F) and (G) shown in (H). Scale bar equals 20 μm. (I) Bright-field view of 7-day CR-stained SL-biofilm within a chamber slide. Scale bar equals 1 mm. Area outlined by black box is shown in (J). (K,L) Ten-day SL-biofilm within Petri dish grown at 25°C in medium containing CR with CR-stained string or web-like structures magnified in (L). Scale bar equals 1 cm. (M) Seven-day H1206 SL-biofilms grown in Hv-YPC medium stained with thioflavin T under blue excitation (top down 3D projection of a Z-stack; scale bar equals 20 μm). CLSM, confocal laser scanning microscopy; CR, Congo red; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; SL-biofilm, static liquid biofilm. Full size image

Cellular clusters visible in GFP-expressing biofilms were colocalized with the signal from a Texas Red conjugate of the lectin concanavalin A (ConA; Figure 2B) and with the DNA binding dye 4',6-diamidino-2-phenylindole (DAPI; Figure 2C,D,E). ConA is known to bind a-manopyranosyl and a-glucopyranosyl residues within glyconjugates of haloarchaeal biofilms [38]. DAPI was selected as an extracellular DNA stain for our CLSM study because it is known to stain eDNA preferentially in haloarchaeal biofilms [38]. Fröls and coworkers [38] used three nucleic acid dyes to distinguish between extracellular and intracellular DNA in biofilms formed by H. volcanii and additional haloarchaeal strains, and showed that: (a) only acridine orange stained individual live cells, appearing similar our use of the cell permeable nucleic acid dye SYTO 9 (Additional file 1: Figure S2), (b) the signal from DAPI appeared nebulous and granular and was colocalized with microcolonies, (c) simultaneous staining with 7-hydroxy-9H-1-3-dichloro-9,9-dimenthylacridin-2-one (DDAO), a nucleic acid dye considered completely impermeable to cells, led to an essentially identical pattern of fluorescence signals, and (d) very few non-viable cells are present within H. volcanii biofilms (even after 30 days of incubation), suggesting that the observed DAPI signal was not from dead cells. Our CLSM analysis also showed colocalization of DAPI-stained material with microcolonies and a lack of signal from individual cells. Further three-dimensional reconstruction of DAPI-stained GFP-expressing biofilms revealed that DNA was concentrated in the basal layer of the biofilm and dispersed as plume-like structures at the top of larger towers (Figure 2C,D,E; lower panels).

Larger structures observed in mature biofilms with transmitted light and through GFP fluorescence were also colocalized with the signal from Congo red (CR) and thioflavin T (ThT). These stains are routinely used as characteristic tests for the presence of a wide variety of amyloid proteins, including for diagnosis and study of disease-causing plaques formed by amyloidosis [60]–[63], and in many investigations of microbial adhesion and biofilm formation [27],[64]–[71].

Congo red fluorescence (CRF) was used as it has been proposed as the most sensitive and reliable method for amyloid detection when staining with CR [60] and has been applied to biofilm compositional studies [72]. CRF was sharply defined and granular in appearance and was colocalized with the large biofilm cellular clusters and towers shown in Figure 2F,G,H. Overlays where single GFP-producing cells are visible and images of CRF at 600× magnification (Additional file 1: Figure S3) indicated that CR did not stain individual cells. CR-stained biofilm aggregates were also orange-red under transmitted light and were visible macroscopically (Figure 2I–L). Further, green fluorescence signal was detected within ThT-stained mature biofilms formed by a non-GFP strain with blue excitation (Figure 2M; no signal detected in control without ThT staining).

Haloferax volcanii undergoes morphological differentiation during biofilm growth

The implementation of GFP for biofilm visualization led to the unexpected observation of increased variability in length of cellular structures within biofilms compared to planktonic cells (Figure 3). H. volcanii DS2 cells are known to be pleomorphic, appearing spherical, disk-like or as short rods 1 to 3 × 2 to 3 μm in size in liquid culture [39] (Figures 1A and 3A). However, during examination of GFP-expressing H. volcanii H1206(pJAM1020) biofilm cells, we observed large cellular structures composed of long rods in chains sometimes approaching 30 μm in length (Figure 3B,C,D). These structures were attached to the surface and were found within developing H1206(pJAM1020) biofilms at all observed time points (Figure 3B,C,D; Additional file 2: Movie 1), in several independently transformed H1206(pJAM1020) strains, as well as in biofilms formed by H. volcanii strains that do not have the GFP expression plasmid (H53 and H98; see Additional file 1: Figure S2).

Figure 3 Cellular morphology within developing Haloferax volcanii H1206(pJAM1020) static liquid biofilms. (A) Planktonic H. volcanii H1206(pJAM1020) cells from exponential phase shaking Hv-YPC culture. (B,C,D) Cells within a developing H1206(pJAM1020) SL-biofilm grown in a chamber slide (in Hv-YPC medium) after 12 h (B), 2 days (C) and 5 days (D). (E) Table listing the number of cells binning into 2.5-μm categories and summarized statistics for 2,000 cells measured for planktonic and biofilm cell populations (left) and histogram showing distribution of length at 0.2-μm binning intervals for planktonic (black) and biofilm (green) cells (right). Cell length was measured with Fiji particle analysis using images collected from three independent exponential phase shaking cultures and three 12 h biofilms grown in chamber slides. Mean length of populations was statistically different with P < 0.0001. Scale bars equal 20 μm. SD, standard deviation; SL-biofilm, static liquid biofilm. Full size image

Further experimentation verified the relationship between the chained long rod morphotype and biofilm formation in the H1206(pJAM1020) strain. A culture of planktonic cells with an average length of 1.6 μm underwent a morphological shift during the first 12 h of biofilm formation in replicate chamber slide wells (Figure 3E). The difference in length between populations of 2,000 planktonic and 2,000 biofilm cells was statistically significant (P < 0.0001 in unpaired t test). Cellular structures within biofilms were on average greater than twice as long, were more variable in size (with a standard deviation in length of 3.1 μm) and reached a maximum length ten times greater than that measured within the planktonic culture from which the biofilm was derived (Figure 3E).

Haloferax volcanii exhibits social motility following disruption of static liquid biofilms

An investigation of biofilm dynamics in static liquid began by disrupting mature 7-day biofilms through mechanical homogenization followed by time-lapse photography over a reformation period. To our surprise, this led to the discovery of rapid cellular re-aggregation and sustained coordinated social motility (Figure 4; see Additional files 3, 4, 5, 6 and 7: Movies 2–6). Structured cellular aggregates were visible 3 h following homogenization, after which cell density was incrementally concentrated within a central developing SL-biofilm, with the surrounding medium becoming visibly transparent after 48 h (Figure 4A). Samples were collected from the homogenized biofilm at time zero, and from the central biofilm and surrounding medium at 48 h. The homogenized biofilm was composed of single cells and cellular aggregates; after 48 h, the surrounding medium contained few planktonic cells and the SL-biofilm contained a high density of cells in large aggregates. Images collected during reformation at intervals of 3 h (Figure 4A), 10 min (Figure 4B) and 1 min (see Additional files 3 and 4: Movies 2 and 3) captured the coordination of large filaments of cells into dynamic web-like branches. Rippling or wave-like streaming was observed during extension and retraction of these structures, particularly evident at low frame rates (e.g., see Additional file 4: Movie 3).

Figure 4 Time-lapse macroscopic photography of static liquid biofilm reformation. An established 7-day SL-biofilm grown in a plastic Petri dish in rich medium (Hv-YPC) was mechanically homogenized and left to incubate at 42°C while photographs were taken at regular time intervals. (A) Biofilm reformation over a 2-day period, with images shown at 3 h intervals. Inlay: Cellular vitality controls shown as still images from time-lapse series (see Additional files 6 and 7: Movies 5 and 6) of a SL-biofilm treated with heat (left) and with 4% formaldehyde (right). (B) SL-biofilm imaged at 10-min intervals over a 50-min period (a series from between 45 h and 48 h above). Scale bar equals 1 cm. SL-biofilm, static liquid biofilm. Full size image

Social motility was also triggered by physical agitation or partial disruption of previously undisturbed developing SL-biofilms (see Additional files 5, 6 and 7: Movies 4, 5 and 6). Ring-shaped SL-biofilms, which developed 3 days after an exponential phase culture was left to incubate in replicate Petri dishes under static conditions, formed extensions and migrated towards the perimeter of the dish through a network of apparent cellular streams for over 1.5 h after the culture vessel was gently moved (see Additional file 5: Movie 4). Additional SL-biofilms from the same replicate set retained this sensitivity through day 5 of incubation. As SL-biofilms became denser and reached a stationary phase of growth after 10 days of incubation, identical applications of physical agitation or partial disruption no longer induced social motility. Coordinated motility as shown in Figure 4 and in Movies 2 to 6 (Additional files 3, 4, 5, 6 and 7) has been observed in many independent experiments and is absent in identically prepared heat-treated SL-biofilms (Figure 4A; Additional file 6: Movie 5) and SL-biofilms chemically fixed with formaldehyde (Figure 4A; Additional file 7: Movie 6).

Genetic transfer occurs at high frequency within Haloferax volcanii biofilms

A screen for gene transfer within H. volcanii biofilms through the known mating mechanism was conducted by cultivating biofilms composed of two strains, each with a unique auxotrophic marker. Gene transfer frequency of the auxotrophic markers was calculated as the number of colonies forming on selective medium (auxotrophs that had regained prototrophy), divided by the number of total viable cells recovered for each culture condition, as described previously [56],[57]. A comparison of transfer frequency was conducted for mixed shaking cultures, colony biofilms and SL-biofilms in chamber slides.

Transfer frequencies indicated that the mating mechanism was active within colony biofilms and SL-biofilms (Figure 5). For chamber slide SL-biofilms, an average transfer frequency of 2.90 × 10-6 was measured and is within the range reported in Mevarech and Werczberger's 1985 study in which mating was first discovered through co-filtering cells on nitrocellulose filters [56]. The transfer frequency for a shaking culture control was low as expected, and mating was not quantifiable in uni-culture biofilms of either H53 or H98 cells (as there is no available source for transfer of the gene required for growth on the selective medium).

Figure 5 Recombination within Haloferax volcanii biofilms. Two double auxotrophic strains (H53, ΔpyrE2/ΔtrpA; H98, ΔpyrE2/ΔhdrB) were mixed at equal cell densities and grown together in Hv-YPC + thymidine medium for 7 days under shaking conditions (black triangles), or as colony biofilms (green squares) and chamber slide SL-biofilms (green circles). Cultures and biofilms were then harvested, washed and plated on defined medium with uracil alone, to select for recombinants, which are either H53 cells that have regained tryptophan prototrophy through a transfer event with a H98 cell(s) or H98 cells that have gained thymidine prototrophy from a H53 cell(s). Average frequency of transfer is shown for three replicates per condition. The transfer frequency range reported in the study in which this HGT mechanism was discovered, traditionally conducted using nitrocellulose filters and known as mating, is shown as a grey box [56]. Vertical bars equal one standard deviation. HGT, horizontal gene transfer. Full size image

Planktonic (shaking) and SL-biofilm cells were visualized through microscopy in parallel prior to plating to determine whether the cellular morphology was consistent with known models for mating, i.e., that it requires cell-to-cell contact and involves the formation of cytosolic bridges and/or cellular fusion events [58]. Cells from a shaken culture were small and disk-shaped while those in chamber slide biofilms had formed a densely packed basal layer of morphologically diverse cellular structures in close physical association (Additional file 1: Figure S2). Natural transformation has not been reported in H. volcanii. In fact, an increase in transfer frequency was reported after adding DNase during mating experiments on filters, indicating that gene transfer in H. volcanii biofilms does not occur through transformation [56].