Marine Biofilm - BEMA 513 - 2020-2021 PDF

Quorum sensing and Biofilm formation in marine microbial environments BEMA 513- Marine Microbial Ecology Faculty of Science - LU Dr Claude Daou  Limitation of growth by environmental factors  Complex environment, exposure of microorganisms to...

Quorum sensing and Biofilm formation in marine microbial environments BEMA 513- Marine Microbial Ecology Faculty of Science - LU Dr Claude Daou  Limitation of growth by environmental factors  Complex environment, exposure of microorganisms to numerous overlapping gradients of nutrients and other environmental factors.  Growth in biofilms: microenvironments, limiting factor  Liebig's Law of the Minimum  The total biomass of an organism will be determined by the nutrient present in a smaller amount relative to the requirements of the organism. 2 BEMA 513_Dr DAOU_LU  Limitation of growth by environmental factors  Shelford Tolerance Law  There are limits in the environmental factors below and above which an organism can not survive and grow regardless of nutrient intake.  Ex: temperature, pH, etc.  The development of a microorganism depends on the supply of nutrients and its tolerance to environmental conditions. 3 BEMA 513_Dr DAOU_LU  Limitation of growth by environmental factors  To respond to low levels of nutrients (oligotrophic medium):  Improved nutrient uptake and exploitation of available resources.  The morphology of the microorganism will change to increase the surface area and nutrient absorption capacity  Changing from the rod shape to the mini and ultramicron form  Stops step by step metabolism with the exception of maintenance genes.  Sequestration of a limiting factor for other microorganisms such as iron (siderophores). 4 BEMA 513_Dr DAOU_LU  Limitation of growth by environmental factors  Vegetative prokaryotes viable but not culturable  A viable microorganism is defined by its ability to develop rapidly, producing a colony or visible turbidity in a liquid medium.  Mr Postgate (England) was the first to observe the presence of viable but non-culturable bacteria.  Development of microscopic techniques, molecular techniques, such as PCR. 5 BEMA 513_Dr DAOU_LU The biofilm  The search for relations between biodiversity and the functioning of communities within ecosystems is one of the major issues of contemporary ecology of the last ten years.  In this context, biofilms constitute a relevant model of study because of the complexity of microbial community structures and their spatial and temporal dynamics, and the variety of hosted functions.  These microbial aggregates consist of heterotrophic microorganisms (bacteria, protozoa) and phototrophic microorganisms (diatoms, cyanobacteria). 6 BEMA 513_Dr DAOU_LU The biofilm  The term microbial mat, which is used in the field of microbial ecology, refers to assemblies where the coherence between the microorganisms is very strong and is maintained even when separating the mat from its hooking support.  The photosynthetic biofilm includes the idea of the presence of algal communities, an autotrophic fraction capable of carrying out photosynthesis reactions.  The term periphyton was defined by R.G. Wetzel (1983) as "a complex community of microorganisms (algae, bacteria, fungi, inorganic and organic debris) attached to a substrate. The substrates can be inorganic or organic, living or dead.  Periphyton ≈ Biofilm. 7 BEMA 513_Dr DAOU_LU Biofilm The formation of biofilms concerns:  all natural aquatic environments (aquifers, lakes, rivers, seas)  living tissues (epithelial tissues, teeth, roots...),  medical biomaterials (medical tools, protheses...),  industrial and sanitary devices (air conditioners, drinking water distribution networks, etc.). 8 BEMA 513_Dr DAOU_LU Biofilm VBNC  From a medical point of view,  biofilms can be a major source of nuisance.  Indeed, bacteria organized in biofilms are often more resistant to antibiotics or immune defenses than bacteria in free form.  This difference in sensitivity towards antibacterial agents, between free bacteria and fixed bacteria into biofilms, suggests that the establishment of this structure is accompanied by physiological changes. 9 BEMA 513_Dr DAOU_LU Biofilm A biofilm can be formed by: a single bacterial species (eg Pseudomonas aeruginosa model) Or a diverse assemblage of heterotrophic or photosynthetic microorganisms. 10 BEMA 513_Dr DAOU_LU Biofilm  They constitute an essential functional compartment by their contribution to the realization of the biogeochemical cycles.  The structure of the communities within these aggregates is conditioned by abiotic parameters (physico-chemistry of the water, pollutants,...) and biotic parameters (competition, predation,...).  In return, the main functions expressed by these communities derive from their structure (taxonomic composition) and their dynamics (ecological succession). 11 BEMA 513_Dr DAOU_LU  Photos of marine biofilm 12 BEMA 513_Dr DAOU_LU Photos of marine biofilm Biofilm on carbon steel after immersion in seawater for 14 days. 13 BEMA 513_Dr DAOU_LU Biofilms Conditions for biofilm development: The presence of an available substrate that is stable enough to withstand the ocean current Resources, abiotic and biotic factors : In contrast to abiotic and biotic factors, resources are directly consumed for metabolism and reproduction. Abiotic and biotic factors directly influence the ability of the microbial community to consume these resources (enzymatic activity or the physical integrity of cells). This set of factors is controlled by other indirect factors such as climate, geology, land use, hydrology, nutrient loading, sedimentation, and biotic interactions. 14 BEMA 513_Dr DAOU_LU  Biofilms Growth cycle: 15 BEMA 513_Dr DAOU_LU The biofilms Growth Cycle :  The theoretical evolution of the total biomass of the biofilm with time follows a general growth model and describes a sigmoidal curve corresponding to the following successive phases:  colonization and adaptation (latency phase); -  growth;  balance;  decrease in biomass. 16 BEMA 513_Dr DAOU_LU The biofilms a) Attachment The bacteria first colonize the surfaces. Microbial adhesion is a physico-chemical process that depends on: 1) surface properties (surface energy, roughness), 2) microorganisms themselves 3) surrounding media (pH, ionic strength); Adhesion is possible when electrostatic repulsions are reduced. 17 BEMA 513_Dr DAOU_LU The biofilms a) Attachment  A cell does not colonize a "clean" surface but a so-called "conditioned" surface  Layer of adsorbed organic or mineral material (ions, small molecules of proteins, polysaccharides,...).  Coming from the cell itself (biosurfactant, protons, exopolymer substances) or from the environment (cells of animal or plant tissues).  The adhesion on the surface begins with a single layer of microbial cells (assembly of micro-colonies between which there are empty zones). 18 BEMA 513_Dr DAOU_LU The biofilms 19 BEMA 513_Dr DAOU_LU The biofilms  Attachment rate correlated to:  the concentration of suspended bacteria  the substrate structure  the water velocity.  Adhesion to surfaces through a network of extracellular matrix called "glycocalyx" or "EPS" for "Extracellular Polymeric Substances".  This matrix, composed of polysaccharides, glycoproteins and lipids, is a mucosal complex that will act as a trap for microorganisms (and detritic residues) circulating in the surrounding environment of the biofilm. 20 BEMA 513_Dr DAOU_LU The biofilms b) Growth phase Two successive stages: Exponential growth then linear growth (role played by the existing diffusion boundary layer at the limit between the biofilm surface and the running water). This boundary layer, or "Diffusive Boundary Layer" (DBL), strongly influences the internal and external exchanges of nutrients, and the chemical environment in the vicinity of periphytic communities may be very different from the surrounding conditions. 21 BEMA 513_Dr DAOU_LU Biofilms - Diffusive Boundary Layer b) Growth phase 22 BEMA 513_Dr DAOU_LU Biofilms b) Growth Phase  Within this boundary layer, molecular diffusion becomes the predominant mode of transfer of dissolved compounds to the biofilm  Existence of a concentration gradient between the medium and the biofilm is required and the rate of molecular diffusion is proportional to the slope of this gradient.  The consumption of nutrients by the biofilm for its development ensures the maintenance of this gradient. 23 BEMA 513_Dr DAOU_LU The biofilms b) Growth Phase  When the biofilm is thin, the concentration of nutrients above remains high and the active transport, dominating with respect to diffusion, implies an exponential growth.  At the end of exponential growth and before the equilibrium phase, growth becomes progressively linear because the newly synthesized biomass is smaller compared to the biomass already accumulated 24 BEMA 513_Dr DAOU_LU The biofilms c) Senescence and detachment of the biofilm The cause of the stalling of all or part of the biofilm may be due to: The death of organisms within the biofilm, stronger hydrodynamic conditions or the bioturbation activity of aquatic invertebrates The particles thus detached circulate within the water column by participating in the pool of SS (Suspended Solids). The decomposition of the deepest cells and the probable existence of anaerobic conditions in the base layers of the periphyton promote its unhooking of the support. 25 BEMA 513_Dr DAOU_LU  The biofilms Within the system, microorganisms interact with each other on the basis of mutualism or competitive relationships. Between algal and bacterial communities, several types of relationships have been described:  competitive relationship to nutrient resources (mineral nitrogen and orthophosphates)  mutualism: the algal exsudates (source of C) ensure the bacterial nutrition, in return the algae use the mineral compounds resulting from the bacterial degradation;  inhibition phenomena: inhibitory effects of algae on bacterial development or the opposite have been also described. 26 BEMA 513_Dr DAOU_LU  The biofilms  The biofilm compartment has been widely recognized as being useful in water quality assessment approaches.  Many characteristics of biofilms make it possible to emphasize their effectiveness in terms of bioindication:  their ubiquity;  their sensitivity to environmental changes, including small variations;  their low generation time;  the ability to easily quantify them. 27 BEMA 513_Dr DAOU_LU  The biofilms  To follow the development of biofilms, two approaches coexist:  an analysis of the structural parameters (organic matter, chlorophyll a, taxonomic composition,...)  and an analysis of the functional parameters (respiration, activity of the alkaline phosphatase,...) 28 BEMA 513_Dr DAOU_LU  The biofilms Factors controlling the development of biofilms The geological and topographical nature of the environment, the climate, three other types: physical, chemical and biological. Physical parameters: hydrodynamics, light penetration, water temperature and the nature of colonization substrates. Chemical factors: the water quality and more particularly the availability of nutrients. Biological parameters: action of grazers (or grazing) 29 BEMA 513_Dr DAOU_LU Biofilms Schéma de l’architecture d’un biofilm (d’après Costerton et al., 1995) 30 BEMA 513_Dr DAOU_LU  Ecologie Microbienne - C. Daou Les biofilms 31 BEMA 513_Dr DAOU_LU Biofilms 1) Physical factors a) Hydrological parameters  Ocean Current flow  Benthic area stability 32 BEMA 513_Dr DAOU_LU  Biofilms 1) Physical factors b) Temperature  The water temperature presents daily and seasonal variations.  Increase the production rates of the biofilm in case of high temperatures.  The environmental temperature has a strong impact on the selection and development of the different species present in the water.  An increase in periphyton biomass is observed over the range of 0 to 30°C and a decrease of 30°C to 40°C, although cyanobacteria mats can survive at 75 ° C. 33 BEMA 513_Dr DAOU_LU Les biofilms 1) Physical factors c) Lighting  Light is a structuring parameter for the autotrophic fraction of the biofilm.  This parameter, defined by its intensity and spectral composition, may become limiting in some ecosystems. 34 BEMA 513_Dr DAOU_LU  Biofilms 1) Physical facteurs physiques d) Nature of colonization support materials The characteristics of the substrates that can influence the development of biofilms are: - the structure of the substrate: an open structure, that is to say allowing water and organisms to circulate easily, promotes the attachment and development of organisms; - stability of the substrate: a stable substrate is more hospitable for the development of organisms; - the roughness of the support material: a smooth support disadvantages the attachment of the constituent organisms of the biofilm. 35 BEMA 513_Dr DAOU_LU Les biofilms 2) Biological Factors  The periphyton is at the base of the food chain and serves mainly as food for the macroinvertebrates which will then be consumed by fish and birds.  The action taken by the animals for their food by plant and microbial biomass is called "grazing" 36 BEMA 513_Dr DAOU_LU Biofilms 37 BEMA 513_Dr DAOU_LU  Biofilms 3) Chemical Factors : Interactions Biofilm – Nutrients a) Effects of nutrients on biofilm development The chemical determinants of periphyton growth may be: phosphorus, nitrogen, carbon, trace elements, combination of factors when light is not limiting, nutrients are the most important resource for the biofilm development Nutrient enrichments stimulate periphyton growth. 38 BEMA 513_Dr DAOU_LU Biofilms 3) Chemical Factors: Interactions Biofilm – Nutrients a) Effects of nutrients on biofilm development  At the level of the internal composition of the periphyton, in oligotrophic situations, a nutrient supply results in an increase in biodiversity.  In eutrophic to hypereutrophic situations, there is a specialization and reduction of species richness 39 BEMA 513_Dr DAOU_LU Biofilms 3) Chemical Factors : Interactions Biofilm – Nutrients b) Biofilm action on nutrient levels Biofilms are considered key elements in the process of improving water quality. They ensure the recycling of dissolved nutrients and have a role in the retention of pollutants, such as heavy metals or pesticides. The properties that are attributed to the periphyton are therefore mainly those of a purifying role of the aquatic environment. Profit in an industrial environment (decrease in nitrogen and phosphorus levels in wastewater treatment by fixed biomass). 40 BEMA 513_Dr DAOU_LU  Biofilms 41 BEMA 513_Dr DAOU_LU  Biofilms 3) Chemical Factors : Interactions Biofilm – Nutrients b) Biofilm action on nutrient levels  It is the heterotrophic microorganisms that are the main agents of biological purification and thus their abundance indicates a strong organic pollution.  Microbial activity ensures the essential biogeochemical processes of elements recycling and enables the release of mineral elements to primary producers. 42 BEMA 513_Dr DAOU_LU  Marine biofilm  Marine biofilms easily colonize man-made surfaces, accelerating corrosion, biofouling, and may even influence the buoyance (flottabilité) of polyethylene plastic.  Together with diatoms and other microorganisms, bacteria are responsible for microfouling, allowing the adhesion of larger organisms such as algae, mussels and barnacles which cause macrofouling.  The costs to several industries are substantial. The environmental impact of biofouling is also significant. Biofouling organisms reduce water flow and increase biodeposition beneath aquaculture farms, and may be fish pathogens.  The strategies to prevent cell adhesion and biofilm formation usually involve the use of an antimicrobial/antiadhesion coating, release of a toxic agent such as metal ions at the surface or smart surfaces. The environmental fate and effects of these biocides should also be considered during biofouling management and control (Anti-fouling). 43 BEMA 513_Dr DAOU_LU  Marine biofouling, the settlement of organisms on marine man-made structures, generate various and extensive challenges for industries developing technologies and working in the marine environment worldwide. The intrusion of invasive aquatic species to new environments by ships is now identified as a major threat to the world’s oceans. 44 BEMA 513_Dr DAOU_LU Quorum sensing and biofilm  Secretion of acyl-homoserine lactones (AHL) = «signals » molecules → control structured biofilm formation  Davies et al., 1998 45 BEMA 513_Dr DAOU_LU  The perception of quorum or self-induction or "quorum sensing” (QS)  QS is a mechanism by which bacteria can coordinate gene expression and cooperate with one another.  QS bacteria use small, diffusible chemical signals to gauge the density of their clonal population and initiate group-beneficial behaviors at high population densities.  QS-regulated phenotypes include the production of exopolysaccharides required for adhesion and biofilm formation, the production of extracellular hydrolytic enzymes, and the production of antibiotic compounds.  These phenotypes aid in colonization, nutrient acquisition, and collective defense 46 BEMA 513_Dr DAOU_LU  The perception of quorum or self-induction or "quorum sensing” (QS)  The term quorum sensing was originally coined to describe the mechanism underlying the onset of luminescence production in cultures of the marine bacterium Vibrio fischeri.  Luminescence and, more generally, quorum sensing are important for V. fischeri to form a mutualistic symbiosis with the Hawaiian bobtail squid, Euprymna scolopes.  The symbiosis is established when V. fischeri cells migrate via flagella-based motility from the surrounding seawater into a specialized structure injuvenile squid called the light organ.  The cells grow to high cell densities within the light organ where the infection persists over the lifetime of the animal.  A hallmark of a successful symbiosis is the luminescence produced by V. fischeri that camouflages the squid at night by eliminating its shadow within the water column. 47 BEMA 513_Dr DAOU_LU  The perception of quorum or self-induction or "quorum sensing” (QS) 48  The perception of quorum or self-induction or "quorum sensing” (QS) Slide 47: The QS network of V. fischeri. V. fischeri has three QS systems: LuxI-LuxR,AinS-AinR, and LuxS-LuxP/Q. In the absence of C8-HSL and AI-2 autoinducers, LuxO is phosphorylated by the kinase activities of the histidine kinases AinR and LuxQ. Phosphorylated LuxO activates expression of the sRNA Qrr1, which degrades via Hfq the mRNA of litR, thereby reducing the level of the transcription factor LitR. Accumulation of C8-HSL and AI-2 at high cell density results in decreased phosphorylation of LuxO, which enhances the level of LitR. LitR activates transcription of luxR, which encodes the transcription factor that, when bound by the autoinducer 3-oxo-C6-HSL, directly regulates expression of the luminescence (lux) genes. C8-HSL can also affect luminescence by directly binding to LuxR. The LuxR/C8-HSL complex can activate transcription of the lux genes, although less effectively than the LuxR/3-oxo-C6-HSL complex. In addition to encoding the light-producing enzyme luciferase, the lux operon contains luxI, which encodes the synthase LuxI that synthesizes 3-oxo-C6-HSL. Synthesis of both C8-HSL and 3-oxo-C6- HSL is autoregulated by separate positive feedback loops. OM = outer membrane, IM = inner membrane. 49 BEMA 513_Dr DAOU_LU  QS in biofilm 50 BEMA 513_Dr DAOU_LU BEMA 513_Dr DAOU_LU Schematic of a generic LuxI-LuxR quorum sensing (QS) circuit. The signal synthase gene luxI encodes a signal synthase protein, LuxI, and the luxR gene encodes a response regulator protein, LuxR. LuxI catalyzes the synthesis of acylated homoserine lactones (AHLs) (small red circles). Generally, AHLs freely diffuse through the cell membrane into the extracellular environment and back into the cells. The concentration of intracellular AHLs serves as a proxy for cell density. At a threshold concentration, LuxR binds its specific cognate AHL, and the LuxR-AHL complex transcriptionally activates the genes 51 under QS control, which in turn leads to expression of QS-regulated traits. Biofilms 52 BEMA 513_Dr DAOU_LU  QS signals  A variety of classes of QS signals have been identified in different bacteria.  Those most relevant to marine bacterial systems are acylated homoserine lactones (AHLs), furanosyl-borate diesters [autoinducer-2 (AI-2) molecules], and α- hydroxyketones.  AHLs are the most common QS signal produced by Proteobacteria, the most abundant bacteria in the oceans, and therefore they dominate most discussions of QS in marine microbial systems. 53 BEMA 513_Dr DAOU_LU BEMA 513_Dr DAOU_LU Examples of quorum sensing (QS) signals. Examples of quorum sensing (QS) signals. The acylated homoserine lactone (AHL) family of QS signals includes the canonical forms (designated AHL, 3-oxo-AHL, and 3-OH-AHL), which vary according to the substitution on the 3- carbon of the acyl side chain and by the length of the acyl side chain (designated by R). p-Coumaroyl-homoserine lactone (p-coumaroyl-HSL) is a representative aryl-HSL, produced by several marine bacteria, in which the acyl side chain is replaced with a p-coumaric acid moiety. The cholera autoinducer-1 (CAI-1) family, which is produced only in Vibrio species, is composed of several molecules, including the CAI-1 molecule and structural variations such as Ea-8-CAI-1. The autoinducer-2 (AI-2) family is composed of two molecules, (2S,4S)-2-methyl-2,3,3,4- tetrahydroxytetrahydrofuran 54 borate (S-THMF) and (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate (R-THMF). S-THMF is the form produced by marine bacteria.  Biofilms Different N-acyl-L-homosérine lactones : 55 BEMA 513_Dr DAOU_LU  QS in the ocean  AHL-producing bacteria, most often Alpha- and Gammaproteobacteria, have been isolated from a range of densely colonized marine environments, including marine snow, corals, sponges, dinoflagellates, and Trichodesmium colonies.  In addition, luxI homologs are readily identified in the Global Ocean Sampling metagenome (Doberva et al. 2015). Although both the isolation of AHL-producing bacteria from marine environments and the presence of AHL biosynthesis genes in marine metagenomes BEMA 513_Dr DAOU_LU indicate that a genetic capability for AHL-QS exists in this environment, these observations do not provide insight into in situ QS activity. Doberva M, Sanchez-Ferandin S, Toulza E, Lebaron P, Lami R. 2015. Diversity of quorum sensing autoinducer synthases in the Global Ocean 56 Sampling metagenomic database. Aquat. Microb. Ecol. 74:107–19  Marine snow  Marine snow originates in the surface waters of the ocean, primarily composed of phytoplankton produced through photosynthesis and microbes.  As the material sinks, it collects other floating debris, including fecal material (poop), dead and decaying animals, suspended sediments, and other organic material that may have been transported from the land to the sea.  We call this material "marine snow », because it looks a bit like the white fluffy stuff that falls on land. When diving in the deep, sometimes we see a lot of marine snow and sometimes we see very little. Differences in the amount of marine snow falling through the water column, or density of this snowfall, is influenced by many factors, including production of phytoplankton in surface waters, consumption and decomposition rates of the organic matter en route to the seafloor, and the movement of this material via currents (horizontal and vertical). 57 BEMA 513_Dr DAOU_LU  QS in the ocean  AHL-QS is often regarded as the most common type of QS in the oceans.  Another signal produced by marine bacteria is AI-2, which plays an accessory role in certain Vibrio species as a cell-cell communication signal.  AI-2 signaling is mediated by two genes: luxS (encoding the signal synthase) and luxP (encoding the signal receptor).  AI-2-QS frequently occurs in parallel with other QS systems, such as AHL-QS. BEMA 513_Dr DAOU_LU  In marine bacteria, the active AI-2 molecule is the furanosyl- borate diester (2S,4S)-2-methyl-2,3,3,4- tetrahydroxytetrahydrofuran borate (S-THMF) 58  Biofilms  Example of Pseudomonas aeruginosa  In P. aeruginosa, two main quorum sensing systems have been described:  the las system and the rhl system, controlling together the transcription of several genes involved in the formation of biofilm and more broadly in virulence. 59 BEMA 513_Dr DAOU_LU  Biofilms  Example of Pseudomonas aeruginosa  Las system :  the lasR gene encoding the LasR regulatory protein and the lasI gene coding for an enzyme, autoinducer synthase, LasI, necessary for the synthesis of a type of HSL: N- (3- oxododecanoyl) -L-homoserine lactone (3-oxo-C12-HSL).  3-oxo-C12-HSL has the property of easily crossing bacterial membranes and thus constitutes a real tool of communication between bacteria.  When the concentration of 3-oxo-C12-HSL reaches a critical threshold, indicating a high bacterial concentration, it causes the activation of an "R" type transcriptional regulator which will then trigger the expression of several target genes (Figure).  Many of these target genes are involved in the virulence of P. aeruginosa. lasI is also part of these targets allowing amplification of the signal by auto-induction. 60 BEMA 513_Dr DAOU_LU  Biofilms  Example of Pseudomonas aeruginosa  rhl system:  The second system rhl operates according to the same scheme includes:  the rhlR gene, coding for the regulatory protein RhlR and  the rhlI gene, coding for a self-inducing enzyme synthase, rhlI necessary for the synthesis of a second type of HSL: N-butyryl-L homoserine lactone, C4-HSL  RhlR-C4-HSL complex controls the expression of several genes including rhlI (self-induction). 61 BEMA 513_Dr DAOU_LU  Biofilms Mécanisme moléculaire BEMA 513_Dr DAOU_LUdu quorum sensing chez P. aeruginosa 62 (Ruimy et Andremont, 2004). Biofilms Facteurs génétiques et phénotypiques intervenant lors de la formation du biofilm à P. aeruginosa (Davey et O'Toole, 2000). 63 BEMA 513_Dr DAOU_LU Biofilms P. aeruginosa is not the only bacterium capable of forming biofilms. Most opportunistic pathogens and bacteria in general are able to develop in this form and resist a large number of external aggression. 64 BEMA 513_Dr DAOU_LU Biofilms  It is now widely accepted that biofilm is the most common form of development in the environment for all microorganisms.  The planktonic form is only a transitional stage before the colonization of new surfaces.  Here are some examples of pathogenic bacteria known and studied for their biofilm formation: Staphylococcus spp., Escherichia coli, Mycobacterium spp., Vibrio cholerae, Bacillus spp. , Legionella pneumophila. 65 BEMA 513_Dr DAOU_LU