3.3 Stress-driven resilience and competition in a nascent marine
plastisphere
Proteins associated with the regulation of oxidative stress, nutrient
limitation, and interspecies competition were abundantly expressed,
reflecting the plastisphere’s resilience to environmental stress and the
intense competitive dynamics within these biofilms (Figure 4). Evidence
of nutrient starvation was observed through the expression of cobalamin
biosynthesis protein, CobW (Marinomonas ), and stringent
starvation protein (Marinomonas, Psychromonas ) on D7.
Additionally, poly(3-hydroxyalkanoate) granule-associated proteins PhaF,
Phal and poly(R)-hydroxyalkanoic acid (Pha) synthase expressed byPseudomonas on D3 (Figure 4), provided evidence for starvation
responses due to nutrient limitation. Moreover, evidence for alternative
carbon source utilisation was observed, including three enzymes
associated with the alkane degradation pathway. Including,
catalase-peroxidase (Kat) expressed by Marinomonas (>D7; FC 2.96), Psychromonas (D7), andPseudomonas (D3), aldehyde dehydrogenase (ALDH) expressed byPseudomonas (D3; D7), Thioclava (D7), Rhodobacter (D7), and Marinomonas (D7), and alcohol dehydrogenase (AD)
expressed by Marinomonas (D7; Figure 4). Protein identification
using PlasticDB - a database containing proteins specifically mediating
plastic biodegradation – identified the polyethylene-degrading enzyme
laccase expressed by Psychromonas , on D7 (Figure 2; Supplementary
file S2).
Proteins associated with carbon metabolism, under the functional
classification ‘carbohydrate transport and metabolism’ (Figure 3), were
consistently upregulated by Pseudomonas on D3, and byMarinomonas on D7. CreA family proteins and response regulatory
domain-containing proteins were also expressed by Pseudomonas on
D3. Tricarboxylic transporters were expressed by Pseudomonas on
D3, and by Pseudomonas, Pseudorhodobacter, Rhodobacter andMarinomonas on D7 (Supplementary file S3). Interestingly, enzymes
involved in aromatic hydrocarbon biodegradation were characterised,
including dienelactone hydrolase, mostly expressed by Marinomonas on D7 (FC 2.03; Figure 3), and by Pseudomonas on D3. Nine other
enzymes associated with this process were discovered on both days,
including 3-oxoadipate-CoA transferase, 3-hydroxyacyl-CoA dehydrogenase,
acetyl-CoA carboxylase, acetyl-CoA acetyltransferase, acetyl-CoA
dehydrogenase, pyruvate aldolase, p-cresol methylhydroxylase (PCMH)-type
protein, thiolase I, and thiopurine s-methyltransferase. Of these,
acetyl-CoA acetyltransferase and acetyl-CoA dehydrogenase were most
expressed on D7 (FC 4.51 and 2.48, respectively). Evidence for nitrogen
cycling was found through the constitutive expression of nitrate
reductase (Pseudomonas , D3; Pseudomonas, Marinomonas, D7),
nitrous-oxide reductase (Pseudomonas, D7), and nitrogen
regulatory protein PII (Pseudomonas , D3; Pseudomonas,
Acinetobacter , D7) on D3 and D7. Nitrogen and urea transporters were
also expressed by Pseudomonas, Marinomonas, and Paracoccus on both days.
In response to intracellular ROS, the plastisphere communities expressed
a variety of stress-response proteins, including alkyl hydroperoxide
reductase (Ahp), catalase (CAT), catalase peroxidase (Kat), glutathione
peroxidase (Gpx), thiol peroxidase (TP), and thioredoxin peroxidase
(TPx) in response to hydrogen peroxide
(H2O2), flavohemoprotein (Fhp) to
respond to nitric oxide (NO), and superoxide dismutase (SOD) to
neutralise superoxide radicals. The most frequently expressed
stress-response proteins were Ahp (Pseudomonas , D3;Pseudomonas , Paracoccus , D7; >D7Acinetobacter , FC 5.83; >D7 Marinomonas , FC
4.32), Fhp (Pseudomonas , D3; Acinetobacter , D7;
>D7 Marinomonas , FC 3.5), Kat (Pseudomonas, D3; Psychromonas , D7; >D7 Marinomonas , FC
2.96), and SOD (Paracoccus , D3; Acinetobacter ,Psychromonas , Rhodobacter , D7; >D3Pseudomonas , FC 3.92; >D7 Marinomonas , FC
2.74) due to their consistent expression across multiple genera (Figure
4; Supplementary file S3). In response to metals and anions,Marinomonas (D7), and Paracoccus (D7) expressed copper
resistance protein A (CopA), and toxic anion resistance protein (TelA),
respectively, and Pseudomonas (D3) expressed tellurium resistance
protein (TerD) (Figure 4). Osmotic stress proteins OsmC
(Pseudomonas, Marinomonas, D7), and glucans biosynthesis protein
C (Pseudomonas , D3) were also found. The downstream effects of
stress were apparent in the expression of membrane repair proteins
ATP-dependent zinc metalloprotease (FtsH), α-2 macroglobulin (α2m), and
phage shock protein (Psp). Additionally, transcriptional response
proteins such as HU, RecA, RdgC, cold shock-domain (CSD) proteins, and
DNA repair proteins such as GrpE, Lon protease, and methionine sulfoxide
reductase were also adundant. Further stress mitigation was supported by
thioredoxin/thioredoxin reductase systems, along with a suite of
chaperones and chaperonins (e.g., ClpA, ClpB, DnaJ, DnaK, GroEL, GroES,
HtpG, SurA), and the redox enzyme glucose-6-phosphate dehydrogenase
(Figure 4). Among these, the cold shock-domain protein was the most
abundantly expressed protein, found in the proteomes ofPseudomonas (>D3 FC 3.8), Marinomonas (D7),Acinetobacter (D7), Paracoccus (D7), and Shewanella (D7). Further protective mechanisms were also identified, such as
dipicolinate synthase expression by Pseudomonas on D3 and
capsular biosynthesis protein on D7. Interestingly, proteins linked to
reactive oxygen species (ROS) generation, including sarcosine oxidase
(Pseudomonas , D3) and Na(+)-translocating NADH-quinone reductase
(Marinomonas ; > D7 FC 7.01), were also expressed.
Competition for resources was evidenced by the expression of proteins
associated with competitive stress. This included the type VI secretion
system (TVISS) proteins, such as TssM, contractile sheath large subunit,
and secretion proteins Evp, IcmF, and VasK, expressed byPseudomonas (D3; FC 1.74 ± 0.12), Pseudoalteromonas (D7;
FC 3.54), and Acinetobacter (D7) (Figure 4; Supplementary file S3).
Competitive advantage was also indicated by the expression of
hemolysin-related proteins and the actin cross-linking toxin VgrG2 byPseudomonas on D3. In potential response to antibiotic
production, resistance mechanisms were expressed, such as polymyxin
resistance protein ArnA and OprM by Pseudomonas on D3, and
β-lactamase by Pseudomonas and colicin-I receptor proteins byShewanella on D7.
Interestingly, the potential pathogens Pseudomonas syringae and Pseudomonas aeruginosa , expressed two
TVISS proteins on D3, while P. syringae also expressed
tol-pal system proteins and a FeADH domain-containing protein. These
results indicate a complex competition network within the biofilm, where
both resource limitation and interspecies antagonism shape the microbial
community structure and resilience.