Bacteriain nature are most often found associated with surfaces in communities known asbiofilms 1. Biofilm has been defined as an aggregate of microorganisms inwhich cells are surrounded by a self-produced matrix of extracellular polymericsubstances (EPS) and adhere to each other and/or to a surface. Theself-organization of EPS molecules in the matrix is based on intermolecularinteractions between the EPS components, which also determine the mechanicalproperties of the matrix, and the physiological activity of the organisms inthe biofilm 2. Althoughthe precise chemical and physical composition of the EPS varies according tothe species and the growth conditions, the main biofilm matrix building blocksare bacterial proteins, extracellular DNA (eDNA) 3, lipids andpolysaccharides 2.
eDNA is a critical component of the biofilm matrix of severalbacteria 3, it is apparently derived from stochastic lysis of a subpopulationof the bacteria within the biofilm. The amount of eDNA accumulating in theextracellular matrix of aggregates and biofilms varies highly from strain tostrain. Although the eDNA is a major structural component of the biofilm matrixformed by Staphilococcus aureus (Mannet al., 2009)4, whereas it is only a minor component of biofilmsformed by the closely related S.epidermidis (Izano etal., 2008)5, the eDNA is neverthelessimportant for biofilm formation in both species. The eDNA has multiplefunctions in biofilm formation and stability: in most situations, it is acontributing component of biofilm matrix able to interact with proteins orpolysaccharides, it promotes biofilm formation by aiding in initialbacterium-surface adhesion by mediating cell-substrate interactions 6, it influences Three-dimensional biofilm architecture andstability by acting as a cell-cell interaction polymer 7. Theimportance of proteins in biofilm structure and function is increasinglyrecognized.
Indeed, for many organisms, the structure and the mechanicalresistance properties of biofilms can be reduced by protease treatments 8.Matrix proteins include not only the outer membrane proteins and the secretedproteins, but also peculiar classes of proteins such as adhesins or motilityorganelles like type IV pili proteins or curli pili 2.Extracellularpolysaccharides are considered as the major structural components of the biofilmmatrix. Most of the matrix exopolysaccharides are very long, with a molecularweight of 500-2000 kDa; they can be homo-polymers such as cellulose, curdlan anddextran, or hetero-polymers like alginate, emulsan, gellan and xanthan 9.Exopolysaccharide chains can be linear or branched and they are generallyconstituted by monosaccharides and some non-carbohydrate substituents such asacetate, pyruvate, succinate, and phosphate. Biofilmarchitectures are highly variable, ranging from open structures containingchannels and columns of bacteria, to structures with no obvious pores anddensely packed regions of cells 10. To date mostattention has focused on biofilms arising from the colonization ofsolid-liquid (S–L) interfaces (i.e.
submerged biofilms), butseveral other kinds of surface and interface also provide ecologicalopportunities for the bacterial colonization. The most common of these is theinterface between air and liquid (A-L); the biofilms formed on thesurface of static liquids are usually referred to as pellicles or floatingbiofilms 10. Regardlessof the interface where bacteria aggregate, the biofilm formation consists ofseveral stages and involve numerous conserved and/or species-specific factors 1.
In any case, biofilm formation can be described as a developmental process withdistinct stages: an ‘initial adhesion’, in which microorganisms adhere tobiotic or abiotic surfaces 11; an ‘early biofilm formation’, during which themicroorganisms begin to produce extracellular polymeric substances (EPS),; a’biofilm maturation’, involving the development of three-dimensional structureswhere the EPS component provides a multifunctional and protective scaffold; andfinally a ‘dispersal’, whereby cells leave the biofilm to re?enter theplanktonic phase 12. This is a dynamic and complex process and requires aconsiderable energetic cost 13; however, this cost may be evolutionarilyacceptable due to the structural and physicochemical advantages deriving from thebiofilm formation. First, the capacity to colonize a surface provides a highlevel of stability in the growth environment, moreover the matrix enables thebiofilm to capture resources such as the nutrients that are present in the eniviromentor that are associated with the substratum on which the biofilm is growing 2.
Second, biofilm formation affords protection from a wide range of environmentalchallenges 14, such as UV exposure, metaltoxicity, acid exposure, dehydration and salinity, phagocytosis and severalantimicrobial agents. Third, spatial organization of cells in biofilms allowshigh biodiversity and complex, dynamic and synergistic interactions, includingcell-to-cell communication and enhanced horizontal gene transfer 14. Therefore,the ability to form biofilm is a selective advantage for bacteria, correspondinglybacteria living in extreme environments, like Antarctica, can be found asbiofilms and this ability is believed to aid their adaptation and survival inthe environment 15. Cold-adapted bacteria capability to live and proliferateat low temperature is the results of a wide range of adaptive features since lowtemperatures place severe physicochemical constraints on several cellularfunctions 16. Several adaptation strategies including the higher flexibilityand lower thermal stability of their enzymes, the up-regulation of genesencoding for cold-shock proteins, the modification of cellular membranecomposition, and the production of anti-freeze proteins and glyco-proteins andpolysaccharides, are reported 17-19.Inthis paper, the attention was focused on the biofilm structure of Pseudoalteromonas haloplanktis TAC125 (PhTAC125) 20, one of the modelorganisms of cold-adaptation, in order to assess if environmental conditions, suchas temperature and nutrient abundance, influence a complex and multicellularstructure like the biofilm. PhTAC125, a bacterium isolated from Antarctic sea water, is oneof the most intensively investigated cold-adapted bacteria. The increasinginterest in PhTAC125 has led to the accumulation ofdifferent data types for this bacterium in the last few years, including itscomplete genome sequence 20, its proteome 21, detailed growth phenotypes 22-24and the construction of a genome scale metabolic model 25.
Inthis paper, a characterization of PhTAC125biofilm in different environmental conditions wasperformed. In detail, the response to different temperatures (15°C vs 0°C) and nutrient abundance (richmedium vs synthetic medium) was analysed. ThePhTAC125 biofilm wascharacterized in terms of biofilm typology and matrix composition by several classical experimental approaches, like Confocallaser scanning microscopy, and by Raman microspectroscopy, a techniquewhich has been recently used to provide molecular details of the chemicalcomposition of bacterial biofilms 26-28. Moreover, the carbohydratescomposition of matrix in the different studied condition was assessed,revealing the presence of cellulose as main polysaccharidic component.
The results reported demonstrate the adaptation of PhTAC125 biofilmstructure in response to different environmental conditions, introducing thebiofilm as a topic (one of the principal target) within the broader context ofcold/environmental adaptation.