[Figure 4]
Discussion
In this study, we analysed 2,948 non-redundant proteins from the LDPE plastisphere, providing near-complete proteomes for the two dominant genera Pseudomonas and Marinomonas within the first days of colonisation. This extensive dataset offered valuable insights into the metabolic activity of these key taxa, as well as the broader functioning of the young plastisphere community. In particular, we revealed the intense competition between taxa within the rapidly developing community and identified the molecular mechanisms underpinning plastisphere resilience to nutrient limitation and oxidative stress. Our results also revealed molecular interactions between the early plastic colonisers and the plastic itself, with important implications for the modulation of biofilm formation and the exploration of plastic biodegradation. These results provide new insights into the dynamics of the nascent plastisphere and their biotechnological potential.
Dominance of key taxa in the young marine plastisphere
The activity of the young marine plastisphere was dominated by heterotrophic bacteria, particularly Gammaproteobacteria, consistent with our previous studies examining plastisphere formation and function [6, 9, 16], and with studies investigating the taxonomic diversity of the plastisphere [7, 28, 29]. Herein and in our previous work, plastisphere samples were collected from the Scottish coast, and biogeographic location appears to play a significant role in shaping plastisphere communities, as noted in several studies including our own [6, 9, 16]. Community development in the present study mirrored previous observations demonstrating that plastisphere diversity increases over time [10-13]. Indeed, we observed a plastisphere community whose activity was dominated by Pseudomonas after three days of colonisation, shifting to an active community comprised of Bacteroidetes after seven days. Pseudomonas , the pioneering genus in this study and one of two high-quality MAGs recovered from the marine plastisphere, is known for its metabolic versatility and biotechnological potential. Its species span biogeochemical cyclers, symbionts, decomposers, denitrifying bacteria, and pathogens [30]. This genus is particularly known for its biofilm-forming capabilities, facilitated by the secretion of specialised membrane proteins and secretion systems which likely facilitate interactions with plastic (Section 4.3) [31]. We have previously observed Pseudomonas within the marine plastisphere, but at < 25% of the active community [9, 16], indicating that the functional role ofPseudomonas is most significant within the initial stages of plastisphere development. As such, the expressed proteome of this organism provides important insights into microbe-plastic interactions at the time of most significance, where colonisers specifically attracted to the plastic surface may utilise it as a substrate [32]. Nevertheless, the most abundant and active genera found on D7,Marinomonas , was the second high-quality MAG recovered and is also a marine-adapted genus typically associated with pollutant bioremediation [33, 34]. Its enrichment on the LDPE substrate suggests that beached plastic provided an ideal niche for colonisation, but likely as a secondary coloniser. Consistent with this, in our previous work, Marinomonas was also the dominant and active taxa following 1-2 weeks LDPE colonisation, but it was less important within the mature marine plastisphere. Additionally, further bioremediative genera such as Thioclava [28] were active on D7 (relative abundance 1.5%), alongside the presence of potential pathogens P. aeruginosa, and P. syringae which represented 4.6% and 17.5% of all annotated species, equalling 285 non-redundant proteins [35, 36]. Indeed, P. syringae was the most annotated species in the D7 plastisphere, indicating a concerning enrichment of potential pathogens within the portion of the plastisphere (19%) that could be annotated to this level. The observed diversity within this early-stage plastisphere highlights the biofilm as a dynamic environment, initially dominated by one or two taxa, likely shaped by significant competitive interactions.
Biofilm formation
Biofilm formation is a key survival strategy for microbial proliferation in challenging, nutrient-poor marine environments [37, 38]. Several proteins involved in surface adhesion were found to be repetitively identified across different taxa known for their propensity to form biofilms. In this way, biofilm formation might have been promoted inPseudomonas , Marinomonas , Acinetobacter , andVibrio through the expression of secretion systems I, II, and IV, as well as flagellin, adhesin, curli, Omps, lipoprotein, and LAD [39-41]. These surface adhesion mechanisms were predominantly expressed by Pseudomonas , potentially aiding their dominance and rapid colonisation of plastic at D3. The succession ofMarinomonas after the initial colonisers further supports their biofilm-forming ability, which is well-documented in marine environments [34]. The detection of quorum sensing molecules expressed byVibrio on D7 could indicate that this genus was involved in processes potentially related to surface adhesion, such as the regulation of membrane sorting proteins, which are often associated with coordinated biofilm development [42].
Biofilm formation may have also been supported by the upregulation of purine biosynthesis, the TCA cycle, arginine, and glutamate cycling proteins, which are crucial for energy and resource allocation within the biofilm community [37, 38, 43]. Moreover, our previous findings highlighted the critical role of glutamine in biofilm proliferation [16]. Its regulation was found to be closely linked with ammonium availability and oxidative stress within low-nutrient environments, withMarinomonas showing distinct glutamine metabolism within the plastisphere [9]. This suggested a differential nutrient strategy between biofilm-associated and planktonic communities, further supporting the notion of a resilient and metabolically adaptive plastisphere biofilm [16]. The observed increase in microbial diversity over time implies that the resources produced by pioneering species (e.g., carbon, amino acids) may have facilitated the growth of subsequent colonisers, creating a more complex and resilient biofilm [15, 44].
Importantly, plastisphere biofilms offer distinct advantages to microorganisms in these environments. For instance, the expression of TIVSS and a competence protein suggests the potential for HGT within the plastisphere [39], allowing microbes to share adaptive traits. Moreover, the expression of calcium-gated channels (e.g., EF-hand and dCache_2 domain-containing proteins) and potassium uptake proteins (e.g., TrkA) points to a potential cooperative exchange of nutrients between neighbouring cells, further enhancing the biofilm’s capacity to thrive under nutrient-limited conditions [45]. Moreover, nitrogen sufficiency is suggested by the expression of nitrogen cycling proteins like nitrate reductase, nitrogen regulatory protein PII, and glutamine. Carbohydrates and amino acids were metabolised and transported within the community, indicating possible resource sharing through symbiosis or cross-feeding, contributing to the overall resilience and stability of the biofilm [15, 44].
However, it is important to note that biofilm communities are not only cooperative but also competitive, as microorganisms compete for dominance and resources within these complex ecosystems.
Resilience and competition
Proteins associated with the regulation of metabolic stressors were among the most abundant expressed non-housekeeping proteins found in our datasets. Enzymes responsible for mitigating oxidative stress were frequently expressed by multiple genera (e.g., Pseudomonas ,Marinomonas , Acinetobacter , Paracoccus ,Psychromonas , and Rhodobacter ), with nearly the entire process of reactive ROS suppression (antioxidant production → DNA regulation → DNA repair) being characterised [46]. Oxidative stress in multi-species biofilms may be triggered by spatial constraints, forced oxygen gradients, competition, nutrient limitation, and the accumulation of abiotic stressors such as metals (e.g., CopA, TelA, TerD) [6, 47]. The plastic itself could also contribute to oxidative stress through the accumulation of ROS under UV-light exposure [48, 49]. The multifunctionality of proteins identified here, such as the frequently expressed CSD protein (Pseudomonas ,Marinomonas , Acinetobacter , Paracoccus ,Shewanella ), suggests that they may have been induced by a combination of these stressors. Indeed, in addition to their role in cold adaptation, cold shock proteins are crucial for maintaining protein stability and proper folding under stress conditions, extending their protective function against oxidative and metabolic stress [50].
Granule storage, competition, and the expression of nutrient-limitation-related proteins highlight nutrient stress during both sampling days. Interestingly, granule formation is often triggered by the scarcity of other essential nutrients outside carbon sources [51]. Additionally, we found evidence for the utilisation of diverse potential carbon sources through the expression of enzymes associated with alkene and consequentially polyethylene degradation (AD, ALDH, Kat, laccase), also as noted by Delacuvellerie et al. (2022).Pseudomonas enriched at D3 are also known to be associated with the biodegradation of polyethylene [52-54]. Enzymes related to aromatic hydrocarbon metabolism, such as dienelactone hydrolase [55, 56], were also detected, though no definitive evidence of plastic or aromatic hydrocarbon biodegradation was found. The synthesis of granules and the presence of carbohydrate metabolism proteins suggest an available carbon source, though potentially through cross-feeding.
Nutrient limitation is known to cause dysregulation of core metabolic processes, leading to the accumulation of ROS, as well as an increased competition for resources [57, 58]. In support of this, the expression of hemolysin and VgrG2 toxins by Pseudomonas may suggest competitive interactions within the plastisphere biofilm. The production of these toxins, alongside other virulence factors, could potentially induce membrane damage in neighbouring microorganisms through the action of the TVISS [59, 60]. Interestingly, we identified a near-complete annotation of the TVISS machinery (i.e. TVISS proteins IcmF, VasK, VipB, membrane subunit TssM, contractile sheath small subunit, and large subunit, Hcp1 family TVISS effector, EvpB family TVISS protein) expressed by active members of the marine plastisphere [8, 16, 17]. These findings suggest that interspecies competition within the biofilm could be a significant factor in shaping plastisphere community structure and dynamics.
Using the TVISS as an indication of virulence [61],Pseudomonas were most competitive on D3 within a predominantly single genera biofilm. Previously, Pseudomonas , andPseudoalteromonas have also been found to outcompete other microbial species through the secretion of toxins [62-64], perhaps actively antagonising other members of the biofilm. The expression of proteins used to convey antibiotic resistance (i.e. b-lactamase, efflux RND transporters, capsule protein) [39, 64, 65] byPseudomonas and Marinomonas on D7, highlight the intense competition within the diversified plastisphere community. These mechanisms are important for maintaining cell integrity in a stressful environment, but their expression puts great demand on the cell’s intracellular resources [39, 57, 61, 62], including those which may be used in key metabolic processes. The deregulation of these processes can further precipitate oxidative stress – similar to nutrient deprivation [57, 58] – and limit the growth of impacted bacteria [46]. Interspecies competition may have therefore contributed to this plastisphere’s function and composition, confirming the observations made by Herschend et al. (2017) and Guillonneauet al. (2018) in model biofilm communities [14, 15].
Metabolic versatility for plastic degradation
The proteins expressed here which relate to the utilisation of alternative carbon sources are of interest within the context of plastic biodegradation. As stated in Section 4.3 , we do not find this to be conclusive evidence of pollutant biodegradation within these plastispheres. However, it is interesting to note that the two enzymes expressed here which align with Delacuvellerie’s (2022) depiction of the alkene degradation pathway (AD, ALDH) are expected to be involved in the initial depolymerisation process of polyethylene degradation by Tao (2023) [8, 66]. Catalase peroxidase, also found here, but not in the alkane degradation pathway, is expected to play a role in the first steps of polyethylene biodegradation. Our recent paper on the late-stage plastisphere also revealed the expression of acyl-CoA dehydrogenase [16], another element of the alkene degradation pathway, which was also expressed in Delacuvellerie’s (2022) study alongside ferredoxin and ferredoxin reductase [8]. In combination, five enzymes associated with the alkene degradation pathway have now been annotated from within the marine plastisphere, plus an additional enzyme which may directly depolymerise polyethylene.
In alignment with our previous investigation, 10 proteins involved in fatty acid beta-oxidation (i.e. acetyl-CoA carboxylase, acetyltransferase, and dehydrogenase) were found in these plastispheres [16]. This process, like the alkene degradation pathway, has been linked to both plastic biodegradation and the biodegradation of other xenobiotic hydrocarbons due to the need to dismantle their similarly structured hydrocarbon base [67]. Metabolites from xenobiotic degradation are also likely funnelled directly into the fatty acid beta-oxidation pathway once initially depolymerised [66]. Dienelactone hydrolase is not linked to this pathway but is associated with the final stages of aromatic hydrocarbon (chlorophenol) degradation [55, 56]. The discovery of such enzymes is interesting because no such xenobiotics were present in these samples, and the plastics used were purportedly free of additives. Another trend of note is the upregulation of these proteins by Marinomonas . Even if these organisms did not actively biodegrade aromatic hydrocarbons in these samples, this may reveal a little of their adaptability to other xenobiotics such as crude oil in the marine environment [33].
The combined expression of enzymes which may be used by bacteria to biodegrade plastics, and other noted xenobiotics here may not indicate that plastic biodegradation is occurring in this scenario. However, it may suggest that organisms found here and in other marine plastispheres have the capacity to perform this biodegradation. Interestingly, the incomplete annotation of these processes can rarely be attributed to a singular genus. Instead, several genera express these enzymes in tandem. This may have significant implications for the field of plastic bioremediation, encouraging the exploration of complex microbial communities rather than relying on single species for more effective plastic degradation.
Conclusion
To date, the molecular mechanisms underpinning the initial days of microbial colonisation of marine plastic debris have remained uncharacterised. To resolve the interactions between the earliest colonisers and plastic surfaces, we utilised multi-omics to investigate the activity of the marine plastisphere community grown on LDPE for 3 and 7 days. Our findings reveal a complex relationship between pioneering and secondary colonisers, shaped by stressors such as reactive oxygen species (ROS) and nutrient limitation, while also highlighting the interplay of cooperation and competition among microorganisms. Consistent with previous research, the majority of these nascent plastisphere microorganisms were primarily composed of heterotrophic bacteria, indicative of marine plastisphere communities found in colder environments. The near-complete assembly of metagenome-assembled genomes (MAGs) for Pseudomonas andMarinomonas significantly enhanced our ability to characterise the associated proteomes and elucidate their precise functions within the dynamic young plastisphere. In this study, Pseudomonas, the pioneering genera, was likely aided through its known ability to form biofilms, while the genera detected in the later plastisphere diversified over time, potentially fuelled by metabolites produced within these biofilms. Proteins related to the biodegradation of plastic and other xenobiotics were expressed by Pseudomonas ,Marinomonas , and three additional genera, highlighting the potential for these microorganisms to contribute to plastic biodegradation processes. Further studies exploring the functioning of the nascent plastisphere are required, to determine their role in those processes. Overall, this study significantly enhances our understanding of the early formation of the plastisphere and its interactions with plastic, providing valuable insights essential for addressing the growing environmental challenge of plastic pollution.
Associated data
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org/) via the MassIVE partner repository [68, 69] with the dataset identifier PXD056358 (for reviewers only: ftp://MSV000095986@massive.ucsd.edu).
Acknowledgements
The authors would like to thank Dr. Johannes Werner for the updated version of mPies.
Funding
This research was funded by the joint UKRI Natural Environment Research Council (NERC) and the National Research Foundation Singapore (NRF), project, “Sources impacts and solutions to plastics in South-East Asia coastal environments”. L.F.M and S.M.-S were supported by the UKRI NERC/NRF project (NERC Award No. NE/V009621/1, NRF Award No. NRF-SEAP-2020-0001). The Bioprofiling platform used for proteomic analysis was supported by the European Regional Development Fund and the Walloon Region, Belgium.
CL is the recipient of a studentship funded by the NERC Scottish Universities Partnership for Environmental Research (SUPER) Doctoral Training Partnership (DTP) (Grant reference number NE/S007342/1). Additionally, CL received a travel mobility fund through the MASTS-SFC Saltire Emerging Researcher Scheme (MASTS-SERS) to support her travel expenses to Belgium for her research visit at UMons.