[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.