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.