Plasmid curing and the loss of grip – The 65-kb replicon of Phaeobacter inhibens DSM 17395 is required for biofilm formation, motility and the colonization of marine algae
Introduction
Biofilms are assemblages of microorganisms encased in a slimy matrix of exopolysaccharides, proteins and extracellular DNA that function as a cooperative consortium [22]. This surprisingly complex microenvironment contains “social life” at a microscopic level and was hence designated as the “city of microbes” [46]. Biofilms are found in oligotrophic habitats, such as water pipes, and on nutrient-rich organic surfaces where they act as ultimate recycling yards. Their formation depends on extracellular polymeric substances (EPS) with various functions [9], but the natural variety of matrix biopolymers is so vast and difficult to analyze that the slime has been termed as “dark matter” [8]. Biofilms of hydrothermal hot springs, freshwater rivers and laboratory flow cells have been shown to exhibit a similar structure and their formation is a key factor for survival in diverse habitats [16]. Moreover, they represent hot spots for horizontal gene transfer (HGT) and may thus promote rapid genetic adaptations in natural environments [40]. Biofilm formation is an integral trait of the contemporary prokaryotic life style and fossil records indicate that it already existed more than 3 billion years ago [16]. The establishment of a functional biofilm and its dispersal is a dynamic process that depends on the successful interplay between many extra- and intracellular factors [22]. One key element for the switch from the sessile to planktonic life style is the ubiquitous bacterial second messenger cyclic di-GMP [18], which is also responsible for the regulation of flagellar motility during biofilm formation [36], [15].
The marine Roseobacter group exhibits a conspicuous repertoire of physiological capacities that facilitate the adaptation to a broad spectrum of ecological niches [44], [25]. Many roseobacters have been isolated from dinoflagellates and the genus Phaeobacter produces troponoid compounds (“roseobacticides”) as modulators of algal-bacterial symbiosis [38], [39]. A recently discovered roseobacticide of Silicibacter sp. TM1040 acts as an inducer of Roseobacter motility while disrupting symbiosis with its algal host [42]. Representatives of this alphaproteobacterial group harbor up to a dozen stable coexisting plasmids that can constitute a third of the bacterial genome [33]. The importance of extrachromosomal elements is shown, for example, by their essential role for anaerobic growth in Dinoroseobacter shibae DSM 16493T [7], and the presence of complete photosynthesis gene clusters in Roseobacter litoralis DSM 6996T and Sulfitobacter guttiformis DSM 14458T [32], [20], [29]. Horizontal exchange of plasmids via conjugation could be documented for D. shibae DSM 16493T as a donor and the distantly related recipient strain Phaeobacter inhibens DSM 17395 (unpublished data). The GC content and codon usage of the three extrachromosomal replicons of P. inhibens are comparable to those of the chromosome and they can hence be classified as “chromids” [17], [30]. All three replicons are also present in the sister species Phaeobacter gallaeciensis [10], thus documenting their stable maintenance over a long evolutionary period. The presence of important genes – the largest 262-kb replicon contains the complete pathway for the biosynthesis of the antibiotic tropodithietic acid (TDA) and siderophores are encoded on the 78-kb plasmid [43] – allow us to conclude that these extrachromosomal elements are indispensable for bacterial survival in natural habitats [30].
Genome-guided physiological analyses of the manually re-annotated species R. litoralis DSM 6996T and P. inhibens DSM 17395 have revealed two plasmids with a size of approximately 65-kb that exhibited a conspicuous accumulation of genes for polysaccharide synthesis and transport, including a complete rhamnose operon [20], [43]. This operon contains four genes for the conversion of glucose-1-phosphate into dTDP-l-rhamnose (rmlA to rmlD [13]) and has previously been identified in the rfb (O-antigen) gene cluster of E. coli [41]. Rml mutants documented that l-rhamnose was essential for biofilm formation in different gammaproteobacterial pathogens [34], [19] and it was also required for maize colonization of the betaproteobacterium Herbaspirillum seropedicae [2]. With respect to Alphaproteobacteria, several knock-out mutants of rhamnose biosynthesis genes in Rhizobium documented the requirement of rhamnan O-antigens for effective symbiosis (nodulation) with legumes [3]. Within the Roseobacter group, more than 20 completely sequenced strains contain plasmids with a rhamnose operon.
The current study was initiated based on working hypotheses that the 65-kb plasmid of P. inhibens DSM 17395 was required for biofilm formation and docking on algal surfaces. In order to test these predictions a holistic approach was chosen, with the RepA-I type replicon being eliminated with an established curing strategy and the consequences for surface attachment were studied. A second aim was the comparison of flagellar swimming motility between the wild type and a curing mutant due to the known tight coupling of both capacities (“swim or stick” lifestyle [42]). Finally, the effect of plasmid curing on the host-interaction was investigated with macro- and microalgae. The thalloid green alga Ulva lactuca was chosen as a natural host for colonization experiments with GFP-labeled bacteria. Moreover, P. inhibens DSM 17395 was co-cultivated with the axenic dinoflagellate Prorocentrum minimum and their interaction was visualized via scanning electron microscopy.
Section snippets
Genetic techniques
The complete RepA-I replication module of the 154-kb plasmid of Dinoroseobacter shibae DSM 16493T was amplified with two specific primers (P039: 5′-TAACCGCGAAGCTCACCACCC-3′, P040: 5′-TCGCCTTCTGTCCCAATGTCA-3′) and it was cloned into the SmaI site of a modified pBluescript II SK(+) vector, which contained an additional gentamicin resistance gene (Gm; [28]). Control sequencing of the 4637-bp amplificate revealed the absence of PCR errors.
Plasmid curing
Preparation of electrocompetent cells from Phaeobacter
Curing of the 65-kb replicon
Plasmid curing of P. inhibens DSM 17395 was performed in order to test the working hypothesis that the 65-kb RepA-I type replicon (NC_018288) played an essential role in biofilm formation. This hypothesis was essentially based on the prevalence of genes involved in polysaccharide biosynthesis, transfer or export (23 of 47 genes) and the structural similarity with the rfb gene cluster of Escherichia coli (nine conserved orthologs; Fig. 1, Supplementary Table S1) required for the formation of
Conclusion
In the current study, the 65-kb replicon of P. inhibens DSM 17395 was shown to be a biofilm-plasmid by a curing approach. Furthermore, its functional role was investigated using attachment assays on artificial and natural surfaces. Future work should be focused on the characterization of specific genes of this plasmid and their impact for the “all or nothing” phenotypes, which was beyond the scope of the current study. Loss of attachment in combination with the observed immotility is an
Acknowledgements
We would like to thank Thorsten Brinkhoff for providing us with the GFP-vector pBBR1MCS-1gfp, as well as Irene Wagner-Döbler, Henner Brinkmann and two anonymous reviewers for very helpful comments on the manuscript, and the Biologische Anstalt Helgoland (Alfred-Wegener-Institut) for the provision of a guest laboratory. This work included a PhD stipend for O.F. and the position of V.M. was supported by the Transregional Collaborative Research Center “Roseobacter” (Transregio TRR 51) of the
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