[Figure 2]
Key functional categories driving early plastisphere development
The proteins expressed on D3 and D7 were classified into functional categories, with ’translation, ribosomal structure and biogenesis,’ ’energy production and conversion,’ and ’amino acid transport and metabolism’ being the most abundant across most genera (Figure 3a). These categories were then followed by functions associated with ‘cell wall/membrane/envelope biogenesis’, ‘cell motility’, and biofilm formation.
Within ’energy production and conversion,’ proteins linked to the TCA cycle were prominent and differentially expressed between time points. These include succinate dehydrogenase, expressed by Pseudomonas (>D3; FC 2.83), Marinomonas (>D7; FC 4.53), Paracoccus (>D7; FC 2.72),Acinetobacter (>D7; FC 4.27), and Vibrio on both days, as well as by Rhodobacter and Shewanella on D7. Additionally, citrate synthase was expressed by Pseudomonas (D3; D7), Marinomonas (D7), Acinetobacter (D7), andPsychromonas (D7), while malate dehydrogenase was expressed byPseudomonas , Marinomonas (>D7; FC 3.6),Paracoccus (>D7; FC 2.92), andPseudoalteromonas (>D7; FC 4.13) on both days. Isocitrate dehydrogenase was expressed by Pseudomonas (>D7; FC 3.01 ± 0.79), Marinomonas (>D7; FC 3.21 ±0.52), Psychromonas (>D7; FC 3.85), andFlavobacterium (>D7; FC 4.07) (Supplementary File S3).
Interestingly, proteins associated with ’amino acid transport and metabolism’ were differentially regulated across nine genera (Figure 3a), accounting for 11.5% (± 1.5) of the total differentially regulated proteins. These include enzymes involved in glutamate cycling (e.g. glutamate methylesterase, >D7 Marinomonas , FC 3.03; glutamine synthetase, >D7 Marinomonas , FC 2.7; >D7 Paracoccus , FC 2.1), arginine cycling (e.g. acetylornithine aminotransferase, >D7 Marinomonas , FC 3.79), and the regulation and transport of branched-chain amino acids (e.g. acetohydroxy-acid isomeroreductase, >D7Flavobacterium , FC 4.95).
Within the categories of ‘cell wall/membrane/envelope biogenesis’, and ‘cell motility’,
several membrane proteins facilitating substrate binding and biofilm formation were expressed. Key adhesive structures include lipoprotein (expressed by Pseudomonas on D3 and Marinomonas on D7), large adhesive protein (LAD; expressed by Pseudomonas on D3), and outer membrane protein A (OmpA; expressed by Thioclava ,Shewanella , Pseudomonas , Rheinheimera on D7), withAcinetobacter showing the highest expression on D7 (FC 4.4 ± 0.14; Figure 3). Additionally, Type V secretory adhesin AidA (FC 2.92 ± 0.76) and elements of the Type II secretion system (TIISS) were expressed by Pseudomonas on D3 (Figure 4). Pili were also expressed by Pseudomonas on both D3 and D7, with pili assembly proteins PilZ (Pseudomonas , D3) and FimV (Marinomonas , D7) similarly expressed. In parallel, the curli assembly/transport component CsgG was identified in Marinomonas on D7 (Figure 4), indicating potential involvement in biofilm formation. (Figure 4) Additionally, flagellin, which may facilitate substrate adhesion, exhibited higher expression on D7 compared to D3. This increase was particularly noted inMarinomonas (FC 4.86 ± 0.79), Pseudoalteromonas (FC 4.5 ± 0.13), Rheinheimera (FC 4.02), and Pseudomonas (FC 2.96 ± 0.32). However, certain Pseudomonas species demonstrated elevated flagellin expression on D3 (FC 3.35 ± 0.31), suggesting variation in flagellar activity between strains across different time points.
Proteins associated with biofilm arrangement including components of calcium-gated (EF-hand domain-containing protein, dCache_2 domain-containing protein, Pseudomonas ), and potassium channels (potassium uptake protein TrkA, Marinomonas ) were expressed on D3 and D7, respectively. One instance of quorum sensing, via the expression of the autoinducer 2-binding periplasmic protein LuxP, was found on D7, expressed by an uncharacterised species of Vibrio . Further examples of cell-cell interactions were identified through the expression of competence protein ComEA (Pseudomonas , D3), involved in horizontal gene transfer (HGT), the OAA-family lectin-sugar binding domain-containing protein, (>D3 Pseudomonas , FC 4.72), and TIVSS protein (Marinomonas , D7). Proteins involved in purine nucleotide biosynthesis (e.g. bifunctional purine biosynthesis protein PurH), which are also thought to play a role in early biofilm formation, were expressed on both days (Figure 3; Supplementary file S3).