Social Features of an Archaeon: Biofilm Formation, Social Motility, Extracellular DNA Metabolism and Gene Transfer in Haloferax volcanii

This thesis seeks to advance the field of archaeal biofilms and related social behaviors through the study of the haloarchaeon Haloferax volcanii. Biofilms are multicellular microbial communities enmeshed within an extracellular matrix. Close proximity between biofilm cells facilitates many emergent behaviors and phenotypes, from communication mechanisms, to cellular differentiation, collective activities, and gene transfer. For this reason, microbial biofilms offer a simplified system for studying development, the evolution of multicellularity, and social behaviors. However, almost all investigations of biofilms to date have been conducted in a handful of model bacterial systems. My work has reported on the structural development and extracellular matrix composition of Hfx. volcanii biofilms and establishes this species as an excellent model for studying archaeal biofilm biology. Furthermore, several related biological phenomena were discovered in Hfx. volcanii, including an ability to metabolize extracellular DNA (an abundant community environmental resource and biofilm component), cellular differentiation, social motility, and genetic exchange in biofilms through a cell-contact-dependent mechanism known as mating. These studies lay a foundation for characterizing genetic determinants of archaeal biofilms, as well as novel archaeal collective-behaviors, cell-to-cell interactions and functional cell types, and the molecular machinery for extracellular DNA processing

DNA as a phosphate storage polymer and the alternative advantages of polyploidy for growth or survival

Haloferax volcanii uses extracellular DNA as a source for carbon, nitrogen, and phosphorous. However, it can also grow to a limited extend in the absence of added phosphorous, indicating that it contains an intracellular phosphate storage molecule. As Hfx. volcanii is polyploid, it was investigated whether DNA might be used as storage polymer, in addition to its role as genetic material. It could be verified that during phosphate starvation cells multiply by distributing as well as by degrading their chromosomes. In contrast, the number of ribosomes stayed constant, revealing that ribosomes are distributed to descendant cells, but not degraded. These results suggest that the phosphate of phosphate-containing biomolecules (other than DNA and RNA) originates from that stored in DNA, not in rRNA. Adding phosphate to chromosome depleted cells rapidly restores polyploidy. Quantification of desiccation survival of cells with different ploidy levels showed that under phosphate starvation Hfx. volcanii diminishes genetic advantages of polyploidy in favor of cell multiplication. The consequences of the usage of genomic DNA as phosphate storage polymer are discussed as well as the hypothesis that DNA might have initially evolved in evolution as a storage polymer, and the various genetic benefits evolved later

<i>Hfx. volcanii</i> consumes high molecular weight chromosomal DNA.

<p>Three <i>Hfx. volcanii</i> cultures were grown in synthetic medium with chromosomal DNA as sole source of phosphorous and a growth curve was recorded (solid line, squares). As negative controls three non-inoculated cultures were incubated under identical conditions (solid line, circles). At the indicated times the optical densities were recorded and aliquots were removed for the quantification of the DNA content. Average optical densities and their standard deviations are shown (solid lines). The cells were pelleted by centrifugation and the DNA content of the supernatants was analyzed by analytical agarose gel electrophoresis (compare B and C). The DNA concentration was quantified using ImageJ, and average values and their standard deviations are shown (dotted lines, circles for the mock-treated non-inoculated control, squares for the inoculated culture). <b>B</b>. The supernatants of the aliquots of non-inoculated negative control cultures were dialyzed to remove salts and analyzed by analytical agarose gel electrophoresis. One representative gel is shown. For comparison the input DNA (gDNA) and a size marker (1 kb plus) were included. <b>C</b>. The supernatants of the aliquots of cultures grown with genomic DNA as phosphate source were dialyzed to remove salts and analyzed by analytical agarose gel electrophoresis. One representative gel is shown. For comparison the input DNA (gDNA) and a size marker (1 kb plus) were included.</p

Phosphate balance in cells prior to and after growth in the absence of external phosphate.

<p>A preculture in complex medium was grown to mid-exponential phase. Aliquots were harvested, washed, and used to inoculate synthetic medium lacking any added phosphate source. Aliquots were removed at the beginning of the experiment and after growth in the absence of phosphate ceased. The cell densities were quantified using a counting chamber, the genome copy numbers were quantified by Real Time PCR, and the numbers of ribosomes were quantified after RNA isolation and two DNase treatments as described in the text. The figure gives a schematic overview of the phosphate balance prior to and after growth during phosphate starvation.</p

<i>Hfx. volcanii</i> uses external DNA as a nutrient source and contains internal P and N storages.

<p><i>Hfx. volcanii</i> was grown in microtiter plates in synthetic medium with added carbon (C), nitrogen (N), and phosphate (P) as positive control (diaments). In additional cultures each one of the three nutrients was replaced with genomic DNA (dotted lines), i.e. C was replaced (squares), N was replaced (circles), and P was replaced (triangles). In further cultures each one of the respective nutrients was omitted without replacement (solid lines), i.e. C was omitted (squares), N was omitted (circle), and P was omitted (triangles). To verify that spill over did not occur, for each medium also non-inoculated controls (sterile controls) were performed (open symbols). In each case average values of three independent cultures and their standard deviations are shown.</p

Desiccation resistances of cells of with different ploidy levels.

<p>Cultures were grown to mid-exponential phase in synthetic medium with casamino acids and 1 mM K₂HPO₄ to generate cells with 20 copies of the chromosome. For comparison, cultures were grown in synthetic medium in the absence of phosphate to generate cells with 2 copies of the chromosome. Both types of cells were exposed to a desiccation period of 12 days. Colony forming units (CFU) were quantified before and after desiccation, and the survival rates were calculated. Average results of three independent experiments and their standard deviations are shown. Left columns, prior to desiccation, right columns, after desiccation.</p

Chromosome copy numbers after re-addition of phosphate to starved cells.

<p>Stationary phase, phosphate-starved, chromosome-depleted cells were resuspended in medium containing 1 mM phosphate. At various times, as indicated, aliquots were removed and the chromosome copy number was determined using Real Time PCR. Three biological replicates were performed and average values and standard deviations are shown.</p

Phosphate content of the four <i>Hfx. volcanii</i> chromosomes before and after growth in the absence of phosphate.

<p>Phosphate content of the four <i>Hfx. volcanii</i> chromosomes before and after growth in the absence of phosphate.</p

Chromosomal copy numbers during and after growth with and without added phosphate.

<p><i>Hfx. volcanii</i> was grown in synthetic medium in the presence of 10 mM and 1 mM phosphate and in the absence of phosphate, respectively. Aliquots were removed during mid-exponential growth phase and at stationary phase (compare text). An aliquot from the pre-culture used for inoculation was also included. Cells were harvested by centrifugation and the chromosome copy number was quantified using Real Time PCR. Three biological replicates were performed and average values and standard deviations are shown, from left to right 10 mM phosphate, 1 mM phosphate, and no externally added phosphate.</p