3.1.1. Differentially expressed protein analysis
A total of 2605 proteins were identified from three groups of A. johnsonii (A), S. putrefaciens (S) and co-culture A. johnsonii + S. putrefaciens (AS). As shown in Fig. 1A, B, there were 67 significant up-regulated proteins and 2295 down-regulated proteins in the A vs. AS group, while 36 up-regulated and 68 down-regulated proteins were in the S vs. AS group. This suggested those significantly regulated proteins are common response proteins of co-culture A. johnsonii and S. putrefaciens to cold adaptation levels. Based on the KEGG database classification of these differentially expressed proteins, showed that these proteins are mainly involved in ABC transporters, Cysteine and methionine metabolism, Nucleotide metabolism, Lipopolysaccharide biosynthesis, Histidine metabolism, DNA replication (Fig. 1(C)). The 60 differentially expressed proteins of co-culture A. johnsonii and S. putrefaciens were most significantly enriched in ABC transporters. It is interesting to note that the KEGG pathway analysis revealed an overlap in the ABC transporters pathway. In this pathway, there were 54 down-regulated proteins and 6 up-regulated proteins observed in the A vs. AS group, indicating a significant alteration in the expression levels of these proteins. Additionally, in the S vs. AS group, 2 proteins were found to be up-regulated in comparison to the AS group (Table 1). It suggested that ABC transporters was a valuable target for co-culturing and thus deserve further investigation.
Table 1
The differentially expression proteins related to ABC transporters pathway identified in A vs. AS and S vs. AS.
Accession | FC(A/AS) | P value(A vs AS) | Regulate |
A4YCA2 | 0.02784 | 0.002232 | down |
A0A8G0XB23 | 9.691 | 0.001542 | up |
A0A8G0XA39 | 7.076 | 0.001347 | up |
A0A8G0TVT9 | 7.281 | 1.00E-05 | up |
A4Y8D6 | 3.508 | 0.01615 | up |
A4Y9S0 | 7.98 | 0.04615 | up |
A0A8G0TYX7 | 0.4964 | 0.02763 | down |
E6XFU4 | 15.61 | 3.20E-05 | up |
A4Y3A6 | 0.00001 | 0 | down |
A4Y3C1 | 0.00001 | 0 | down |
A0A8G0TX59 | 0.00001 | 0 | down |
E6XK21 | 0.00001 | 0 | down |
A0A8G0TXJ8 | 0.00001 | 0 | down |
A0A8G0TXN0 | 0.00001 | 0 | down |
E6XKS3 | 0.00001 | 0 | down |
A0A8G0TY21 | 0.00001 | 0 | down |
A0A8G0TYB1 | 0.00001 | 0 | down |
A0A8G0TIS3 | 0.00001 | 0 | down |
A0A8G0TYT5 | 0.00001 | 0 | down |
A0A8G0TYV3 | 0.00001 | 0 | down |
A0A8G0TZD4 | 0.00001 | 0 | down |
A0A8G0TZN3 | 0.00001 | 0 | down |
A0A8G0U020 | 0.00001 | 0 | down |
E6XFU0 | 0.00001 | 0 | down |
E6XRP6 | 0.00001 | 0 | down |
A0A8G0U243 | 0.00001 | 0 | down |
A0A8G0U2D4 | 0.00001 | 0 | down |
A0A8G0X863 | 0.00001 | 0 | down |
A0A8G0X937 | 0.00001 | 0 | down |
A4Y5I9 | 0.00001 | 0 | down |
A0A8G0X9U4 | 0.00001 | 0 | down |
A4YA35 | 0.00001 | 0 | down |
A4Y383 | 0.00001 | 0 | down |
A0A8G0XAH0 | 0.00001 | 0 | down |
A4Y1F8 | 0.00001 | 0 | down |
A4YCA4 | 0.00001 | 0 | down |
A0A8G0XAV7 | 0.00001 | 0 | down |
A0A8G0XBB2 | 0.00001 | 0 | down |
E6XM61 | 0.00001 | 0 | down |
E6XLH4 | 0.00001 | 0 | down |
A0A8G0XCX6 | 0.00001 | 0 | down |
A0A8G0XDB8 | 0.00001 | 0 | down |
A0A8G0XFJ5 | 0.00001 | 0 | down |
E6XS07 | 0.00001 | 0 | down |
A4Y5Y4 | 0.00001 | 0 | down |
A4Y5E4 | 0.00001 | 0 | down |
A0A8G0TS61 | 0.00001 | 0 | down |
A0A8G0TST7 | 0.00001 | 0 | down |
A0A8G0TY32 | 0.00001 | 0 | down |
A0A8G0TTY6 | 0.00001 | 0 | down |
A0A8G0TUH0 | 0.00001 | 0 | down |
A0A8G0TVE5 | 0.00001 | 0 | down |
A0A8G0TVI6 | 0.00001 | 0 | down |
A0A8G0TW45 | 0.00001 | 0 | down |
A0A8G0TW66 | 0.00001 | 0 | down |
A0A8G0TWK8 | 0.00001 | 0 | down |
O52688 | 0.00001 | 0 | down |
A0A8G0TWS2 | 0.00001 | 0 | down |
Accession | FC(S/AS) | P value(S vs AS) | Regulate | |
A4Y5J0 | 32 | 0 | up | |
A0A8G0XC2 | 32 | 0 | up | |
3.1.2 Differentially expressed proteins related to ABC transporters by GO, Pfm, and KEGG enrichment analysis
Microbial behavior is a complex regulatory network in a co-culture caused by cold conditions [30–32]. The 4D quantitative proteomic analysis revealed that in response to cold stress, co-culture of A. johnsonii and S. putrefaciens exhibited varying degrees of up- or down-regulated expression in their ABC transporters pathway. This adaptive response allows them to better cope with the stress induced by cold conditions (Table 1).
GO enrichment-based cluster analysis of differentially expressed proteins involved in cell compartmentalization highlighted the involvement of ATP-binding cassette (ABC) transporter complex, ATPase-dependent transmembrane transport complex, plasma membrane protein complex, transporter complex, and transmembrane transporter complex (P < 0.001). Genes such as SmbFT, MlaFEDB, vltA, and vltB were identified within the ABC transporter complex, playing a role in organizing viologen transporter and providing protection against bacteria [33, 34]. The bacterial membrane has been noted for the metabolic status of the cell, as it provides selection in the presence of cellular homeostasis and metabolic energy transduction [35]. Besides, the membrane protein of co-culture in A. johnsonii and S. putrefaciens is the site for key processes, such as the active transport of nutrients, bacterial interaction, ATP generation, and biofilm formation. The differentially expressed molecular function proteins were involved in ABC-type transporter activity, active transmembrane transporter activity, carbohydrate derivative binding and primary active transmembrane transporter activity (P < 0.001). The study reported the key function of ABC-type transporter activity was also related to hydrophobic protein with structural similarities to the integral membrane components in Cyanobacterium Synechococcus elongatus [36].
Based on the Pfam domains database analysis of co-culture in A. johnsonii and S. putrefaciens, a total of 33 proteins (Pfam accession no. PF00005.30) were defined that included proven and potentially ABC transporter proteins (Fig. 2(B), Table S1). Pfam domains highlight the presence of the ABC transporter of co-culture in A. johnsonii and S. putrefaciens as supported by the higher number of dedicated molecular regions such as RecF/RecN/SMC N terminal domain (SMC_N), AAA domain, putative AbiEii toxin, Type IV TA system (AAA_21), membrane ABC transporter transmembrane region (ABC_membrane), commonly found in ABC transporter proteins associated with outer membrane structural components [37], and are most likely involved in regulating through the operon, which is activated upon may be associated with bacterial resistance [38].
Furthermore, the key pathway of ABC transporters associated with environmental information processing, which encompasses membrane transport and signal transduction, was identified in the co-culture of A. johnsonii and S. putrefaciens (Fig. 2C), This discovery suggests that this pathway plays a significant role in the response to cold stress by facilitating bacterial interaction, nutrient absorption, signal transduction, and the excretion of metabolic products.
3.1.3 The relationships between proteins and functional terms in co-culture A. johnsonii and S. putrefaciens
To assess the relationships between functional terms and differentially expressed proteins in the identified co-culture of A. johnsonii and S. putrefaciens, molecular function enrichment and protein-protein interaction (PPI) networks were analyzed. In order to visualize smaller subsets of high-dimensional data, KEGG enrichment was employed (Fig. 3(A)). The most representative functions in co-culture A. johnsonii and S. putrefaciens were involved in ABC transporters and sulfur metabolism. The protein expression showed that E6XFU4 had the highest log2FC (3.964). Co-culture A. johnsonii and S. putrefaciens responses to cold stress involve many ABC transporters proteins, in which of proteins with at log2FC in expression > 2 included A4Y9S0, A0A8G0XB23, A0A8G0XA39, E6XFU4.
A full list of the differentially expressed proteins in A. johnsonii group, S. putrefaciens group and co-culture in A. johnsonii and S. putrefaciens group evaluated are displayed in a heatmap (Fig. 3B). Within this subcluster, A. johnsonii exhibited higher expression levels of E6XKS3, A0A8G0TVT9, A4Y9S0, E6XFU4, A0A8G0XA39, A0A8G0XB23, A4Y8D6 proteins than the S, AS groups. Most proteins were involved in ABC transporters. The up-regulated proteins expression level of A0A8G0TTY6, A0A8G0TUH0, A0A8G0TYB1, A0A8G0TYT5, A0A8G0TYV3, A0A8G0TZD4 also occurred in the co-culture in A. johnsonii and S. putrefaciens group. Therefore, it could be concluded that the key proteins of ABC transporters exhibited a higher relative contribution from the ABC peptide transporter, lipoprotein-releasing ABC transporter permease subunit and UvrABC system protein A, which were key protein metabolic actors and functional interactions in co-culture in A. johnsonii and S. putrefaciens under cold stress.
An interactive network diagram of ABC protein is shown in Figure. 3(C). The network contains 85 DEPs were connected with no less than 2 proteins and 56 nodes. The co-culture in A. johnsonii and S. putrefaciens indicated that interaction proteins are involved in the ABC proteins including ABP74125.1, ABP74416.1, ABP74439.1, ABP74127.1, etc.. Protein ABP74454.1 was predicted to interact with five other proteins, namely ABP76703.1, ABP76704.1, ABP75875.1, lolD, and ABP74455.1. It is a component of the ABC transporter complex LolCDE, which is responsible for the translocation of mature outer membrane components. We found interaction networks for ABC transporter related (ABP75221.1, ABP76742.1, ABP77578.1) had the highest interaction degree with four neighbors in oligopeptide/dipeptide abc transporter (ABP75222.1), cationic peptide transport system substrate-binding protein (ABP75225.1), binding-protein-dependent transport systems inner membrane component (ABP75223.1) and lolD. Co-culture in A. johnsonii and S. putrefaciens respond to low temperatures by ABC transporter proteins, which result in phospholipid transport and inner membrane component.
3.2 Differential metabolites related to ABC transporters
A total of 259 metabolites in A vs AS and 186 metabolites in S vs AS were identified by metabolomics, respectively. The results of metabolomics analysis revealed an enrichment in the biosynthesis of ABC transporters, Aminoacyl-tRNA biosynthesis, Biosynthesis of various secondary metabolites - part 3, Cyanoamino acid metabolism, Purine metabolism pathways. ABC transporters has a significant effect on the accumulation and transmembrane transport of secondary metabolites of co-culture A. johnsonii and S. putrefaciens (Figure S1A, S2A). Fifteen differential metabolites related to ABC transporters in A vs AS were identified, and one (Glutathione) of these metabolites was significantly downregulated, while fourteen metabolites were significantly upregulated (FC > 2)(Fig. 6B-P). Such compounds as L-Arginine, Deoxyguanosine, L-Phenylalanine, L-Isoleucine, L-Glutamate, Choline, L-Threonine, L-Lysine, Xanthosine, L-Proline, Uridine, Indole-3-carboxaldehyde, L-Serine, and Guanosine showed an upregulation trend between S and AS (Figure S1 A-J). On the one hand, ABC transporters indeed play essential roles in bacteria growth and development, response to cold stressors, and interactions with their environment. On the other hand, some transporters are broadly conserved from the co-culture of bacteria, and most of them transport various metabolites and signalling molecules in an ATP-dependent (ABC transporters)[39], hence, ABC transporters contribute to the co-culture A. johnsonii and S. putrefaciens under cold stress.
3.3 Alkaline phosphatase (AKP) and ATPase activity analysis
The AKP enzyme is an intrinsic plasma membrane enzyme that plays a significant in hydrolyzing phosphate-containing compounds and oxygen-carrying systems [40]. Cold stress to the membrane properties of microorganisms could lead to a substantially changed in AKP activity [1, 9]. As shown in Fig. 3(D), the increased AKP activity observed in the mono-culture/co-culture of A. johnsonii and S. putrefaciens at 4°C can be attributed to cold adaptation and damage to the plasma membranes. The expression of AKP in bacterial cells is promoted, allowing AKP enzymes to breach the cell membrane and become released under cold conditions. This increased AKP activity serves as an indicator of cellular response to cold stress and potential membrane damage in these bacteria. The higher level of AKP activity observed in AS group than in other groups was stimulated under cold stress, indicating that the intrinsic plasma membrane integrity of co-culture in A. johnsonii and S. putrefaciens were cooperative interactions in response to metabolic cross-feeding to promote the cell morphology damage, and similar results of cell morphology damage were also obtained in Fig. 4(A-C2).
Changes in ATPase activity can be indicative of the integrity of the cell wall and cell membrane in the co-culture of A. johnsonii and S. putrefaciens in response to cold stress (Fig. 3(E)). ATPase is commonly utilized by bacteria to adapt to various stress conditions, including cold stress. It plays a crucial role in providing energy for cellular processes and maintaining microenvironmental homeostasis. The increased demand for energy during stress, such as cold stress, is essential for maintaining cell metabolisms and ensuring the overall functioning of the cells. Adequate energy supply through ATPase activity is vital for bacteria to cope with stress and sustain their cellular activities under challenging environmental conditions. As shown in Fig. 3(E), the ATPase activity of mono-culture/co-culture A. johnsonii and S. putrefaciens increased at 4°C, indicating that more ATP binding and hydrolysis was strongly affected by environmental stress which was utilized in the ABC transporter to drive the transport of ions to macromolecules across membranes [41]. Besides, the higher ATPase activity observed in the co-culture of A. johnsonii and S. putrefaciens, compared to the other groups, indicates that the external cold stimulus and the sustained high bacterial activity contribute to an increased ATPase yield through the cooperation of these two bacteria. However, the co-culture of A. johnsonii and S. putrefaciens exhibited a significant decreasing trend from 120 to 144 hours (p < 0.05). This finding suggests that a longer incubation time may lead to a reduced availability of energy supplementation.
3.2 Changes of ultramicrostructure in co-culture A. johnsonii and S. putrefaciens
The morphologies of mono-culture/co-culture A. johnsonii and S. putrefaciens under cold stress were depicted in Fig. 4(A-C2). The TEM images (Fig. 4A-C1) showed that mono-culture A. johnsonii and S. putrefaciens exhibited normal morphology with a regular and smooth surface. Bacteria are very sensitive to reactive cold stress, A. johnsonii and S. putrefaciens appeared to have slight damage in cell membrane thereby causing in cell fluid leakage phenomenon. Co-culture in A. johnsonii and S. putrefaciens existed in severe shrinkage and collapse, accompanied by cytoplasmic leakage. Especially, A. johnsonii seemed to be partially etched, deformed, and splintered cells. A large amount of cell membrane contents leaked out ultimately leading to bacterial death. Leakage of intracellular contents might be used to maintain its growth and metabolism to adapt at low temperature coupled coexistence, The microstructure of S. putrefaciens was a little damaged with some leakage of intracellular contents to maintain a relatively stable structure. Meanwhile, the leakage of intracellular contents also provides energy and nutrients for the co-culture in the two bacteria, which further indicated its synergistic effect.
SEM of mono-culture/co-culture A. johnsonii and S. putrefaciens under cold stress were shown in Fig. 4(A2-C2). The cell morphology of mono-culture A. johnsonii and S. putrefaciens was complete, and there was no obvious damage of the cell and no leakage of the contents (Fig. 4(A2, B2)). However, mono-culture A. johnsonii and S. putrefaciens might be present to a small part of unsmooth walls, and the cell body appeared to fold due to the low temperature stimulation. While, after incubating at 144 h under cold stress, many membrane defects on co-culture in A. johnsonii and S. putrefaciens surface combined with the presence of the leakage of intracellular contents, suggesting that the bacteria was severely damaged and wrinkled to absorb metabolic nutrients and adapt to cold temperatures. Such a co-culture process might induce bacterial death, more action than the mono-culture process effect played by ABC transporter proteins on the bacteria may be present to achieve strong spoilage ability.