3.1. Identification of the DEGs That Are Responsible for the Various Environmental Conditions

We first used GEO2R to identify the DEGs under the various environments, and overall 763, 1635, 1039, and 1130 unique DEGs were involved in cold shock, heat shock, high pressure, and salt shock conditions, respectively. Because proteins need interactions to perform their functions, we constructed the corresponding PPI networks for the proteins encoded by these DEGs. Subsequently, considering that enrichment analysis can be used to extract biological insight from a given gene/protein set, we first performed KEGG pathway enrichment analysis to reveal the biological functions and differences of the largest connected components in these PPI networks.

Overall, 44, 54, 36, and 16 KEGG pathways were enriched under the abovementioned conditions, respectively. As illustrated in Figure 2, the most common enriched pathways across these environments included metabolism-related (e.g., metabolic pathways, biosynthesis of secondary metabolites, and microbial metabolism in diverse environments) and protein translation-related (e.g., biosynthesis of amino acids, ribosomes, aminoacyl-tRNA biosynthesis, and protein export) pathways, indicating that metabolism largely acclimates to changing environmental conditions (cold shock, heat shock, high pressure, and salt shock here), and, accordingly, S. piezotolerans WP3 needs to produce new proteins that acclimate to these environments. These results are in good agreement with previous studies in other Shewanella species (see, e.g., [Reference Barchinger, Pirbadian and Sambles24, Reference Ding and Sun25]).

Figure 2 KEGG pathway enrichment analysis for the DEGs (differentially expressed genes) under the various environmental conditions. False discovery rate <0.05.

On the other hand, apart from these commonly enriched pathways, some pathways presented large differences across three of these conditions (there were no unique enriched pathways under salt shock condition; see Figure 2). First, the largest difference was derived from the heat shock condition, and there were 12 enriched pathways exclusively presented under this condition, indicating that S. piezotolerans WP3 is more sensitive to heat shock than the other shocks examined in this study. This point is in good agreement with the fact that S. piezotolerans WP3 was isolated from West Pacific sediment at a depth of ∼1914 m [Reference Xiao, Wang, Xiao, Bartlett and Wang15], where the most remarkable environmental features are low temperature, high pressure, and high salt, but not high temperature. Second, there were 6 pathways exclusively presented under the cold shock condition, including glutathione metabolism (FDR: 1.35E − 05), inositol phosphate metabolism (FDR: 8.54E − 03), beta-alanine metabolism (FDR: 1.62E − 02), pentose and glucuronate interconversions (FDR: 2.29E − 02), lysine biosynthesis (FDR: 2.78E − 02), and fructose and mannose metabolism (FDR: 4.58E − 02). Third, only one pathway (degradation of aromatic compounds, FDR: 2.86E − 02) was exclusively presented under the high-pressure condition. Due to microorganisms need to produce different proteins to cope with the changing environmental conditions, these exclusively enriched pathways and the involved proteins will be helpful to understand the different metabolic processes under various environmental conditions in S. piezotolerans WP3.

3.2. Construction of the PPI Networks That Are Involved in the Various Environmental Conditions

It is generally believed that the global protein network defines the collective function of cells. In contrast, a condition-specific PPI network contains a lot of interactions that are enriched for processes relevant to the specific condition and thereby may be more relevant for characterizing specific biological processes, such as identifying protein complexes or stimulus-response processes [Reference Ideker and Krogan26, Reference Wang, Peng, Li and Pan27].

To construct the PPI networks that are involved in the various environments, we first identified condition-specific protein-coding genes under these conditions by using a Venn diagram. As shown in Figure 3, 167, 647, 330, and 428 unique DEGs were involved in the abovementioned environments, respectively. These gene sets were then used to construct condition-specific PPI networks from the background PPI network for S. piezotolerans WP3.

Figure 3 Venn diagram of the differentially expressed genes involved in the various environmental conditions. The figure is produced by using an online Venn diagrams tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).

As a result, although there were many isolated proteins in the network, each network contains many interactions that form several interconnected parts. Numerically speaking, all of these condition-specific networks have significantly more interactions than were expected (the PPI enrichment P values were 1.66E − 2, 7.21E − 4, 2.74E – 2, and 9.96E − 3, respectively). Since proteins mainly carry out biological functions through interactions, it is generally considered that the hubs (i.e., proteins with a high degree value) in the PPI network are more important. To this end, we used the degree value to rank the importance of the proteins in the networks and thereby identified the top 10 key proteins in each network (Table 1). As shown in Table 1, these key proteins are mostly linked to the electron transport processes of S. piezotolerans WP3 under various environmental conditions (except for the heat shock condition; see below).

Table 1 The top 10 key proteins in the condition-specific PPI networks of S. piezotolerans WP3 under various environmental conditions.

Note: as many proteins have the same degree value, we used the clustering coefficient as the second measurement to eliminate the proteins that are not ranked in the top 10.

First, the top 10 key proteins under cold shock were mostly relevant to flagellar proteins, including flagellar biosynthesis sigma factor (swp_5124), flagellar basal body rod protein (swp_5105, swp_5110), flagellar basal body L-ring protein (swp_1504, swp_5111), flagellar hook-associated protein FlgL (swp_1508), and flagellar ATPase FliI/YscN (swp_5095). A transcriptional study suggested that the flagellar operon of Escherichia coli is inducible in response to cold shock [Reference Phadtare and Inouye28]; in addition, a proteomic study showed that flagellar biosynthesis and motility proteins presented significant changes under cold shock [Reference Kim, Han, Lee and Lee29]. Therefore, the key proteins of S. piezotolerans WP3 under cold shock are mostly flagellar proteins and are expectable. Furthermore, the correlation between oxidative electron transport and anaerobic flagellum assembly of Escherichia coli was reported long ago [Reference Tana, Howlett and Koshland30], and, more importantly, several recent experimental studies from different groups showed that flagellar proteins were involved in the electron transfer activity in Geobacter sulfurreducens and Shewanella oneidensis (e.g., [Reference Liu, Liu, Zhou and Jin31–Reference Wang, Wu and An33], raising a speculation that flagellar proteins may be also involved in the electron transfer activity in S. piezotolerans WP3.

Second, the top 10 key proteins under high pressure also included several flagellar proteins, such as flagellar biosynthesis protein (swp_1531) and two flagellar motor switch proteins (swp_1521, swp_1527). Motor switch proteins have been shown to be directly associated with some energy-linked enzymes and thereby lead to higher rates of ATP synthesis, ATP hydrolysis, and electron transport [Reference Zarbiv, Li and Wolf34]. Other important proteins in this environment are also electron transfer-related, including a flavocytochrome c (swp_4352) and a flavoprotein (swp_0430), which are homologous proteins for periplasmic fumarate reductase FccA (63% identity) and quinol:fumarate reductase FAD-binding subunit FrdA (88% identity) from Shewanella oneidensis MR-1, respectively. Formate dehydrogenase subunits swp_4312 and swp_2139 are homologous proteins for molybdopterin-binding oxidoreductase (SO_0988, 67% identity) and formate dehydrogenase cytochrome b subunit FdhC (47% identity) from Shewanella oneidensis MR-1. These proteins play an important role in electron transfer processes of Shewanella oneidensis MR-1 [Reference Hau and Gralnick35]. For example, periplasmic fumarate reductase FccA can promote the periplasmic electron transfer process by rapid transient interactions [Reference Sturm, Richter, Doetsch, Heide, Louro and Gescher36].

Third, the most important proteins that are listed in the top 10 key proteins under salt shock are several mannose-sensitive haemagglutinin- (MSHA-) related proteins, including MSHA pilin protein (swp_0495) and MSHA biogenesis proteins (swp_0501, swp_0486), which have been shown to be involved in electron transfer processes in Shewanella oneidensis MR-1 [Reference Fitzgerald, Petersen and Ray37]. Other notable proteins are Cbb ₃ -type cytochrome oxidase (the oxidase subunit swp_4145 and the cytochrome c subunit swp_4144), which are homologous proteins for Cbb ₃ -type cytochrome c oxidase subunit CcoN and Cbb ₃ -type cytochrome c oxidase subunit CcoO from Shewanella oneidensis MR-1. These proteins have previously been shown to be involved in electron transfer in various organisms [Reference Gao, Barua and Liang38], which made swp_4145 and swp_4144 probable candidates for electron transfer studies in S. piezotolerans WP3.

Finally, the top 10 key proteins under heat shock conditions were mainly metabolism-related, including glutamate synthase subunit (swp_3803), pyruvate kinase II (swp_2388), oxaloacetate decarboxylase (swp_1379), multifunctional fatty acid oxidation complex subunit (swp_3139), 2-isopropylmalate synthase (swp_2213), isopropylmalate isomerase small subunit (swp_2216), chorismate mutase (swp_3758), Na⁺-transporting methylmalonyl-CoA/oxaloacetate decarboxylase subunit (swp_1380), sodium pump decarboxylase subunit (swp_1378), and malate dehydrogenase (swp_0481). These results are consistent with previous KEGG enrichment results, indicating that cell metabolism undergoes tremendous changes (from their native living environment) under the heat shock condition. These metabolic proteins may be helpful for identifying which forms of metabolism are more important in response to heat shock.

3.3. Important Functional Modules Underlying the Various Environmental Conditions

To identify the functional modules underlying these condition-specific networks and thereby reveal the mechanisms by which S. piezotolerans WP3 uses to respond to environmental changes, we used the ClusterONE algorithm to analyze these networks and obtained 6, 13, 10, and 5 functional modules from the abovementioned networks, respectively. We then performed enrichment analysis for these functional modules to reveal their potential functions (see Tables 2–5).

Table 2 The most representative KEGG pathway that was enriched in the modules identified from the cold shock condition.

Note: the bold enrichment term indicates that there was no KEGG enrichment and InterPro enrichment was used alternatively.

Table 3 The most representative KEGG pathway that was enriched in the modules identified from the heat shock condition.

Note: the bold enrichment terms indicate that there was no KEGG enrichment and InterPro enrichment was used alternatively; — indicate that the enrichment analysis returned with no results.

Table 4 The most representative KEGG pathway that was enriched in the modules identified from the high-pressure condition.

Note: the bold enrichment term indicates that there was no KEGG enrichment, and InterPro enrichment was used alternatively; — indicate that the enrichment analysis returned no results.

Table 5 The most representative KEGG pathway that was enriched in the modules identified from the salt shock condition.

Note: the bold enrichment terms indicate that there was no KEGG enrichment and InterPro enrichment was used alternatively; — indicate that the enrichment analysis returned no results.

First, what interested us was that the different flagellar assembly modules appeared in three of these conditions (Tables 2–5; Figure 4). As we discussed in Section 3.2, flagellar assembly-related proteins are of great concern under cold shock and high-pressure conditions. Although flagellar assembly-related proteins are not key proteins under the heat shock condition, the flagellar assembly module identified here suggests that these proteins should also be important under this condition. As mentioned earlier, flagella are important for biofilm formation, energy production, and electron transfer. The results reported here further illustrate that proteins of flagellar assembly systems will, in fact, form interacting flagellar assembly modules to achieve their purpose. Although they play an important role in a variety of environments, the bacteria can use different flagellar assembly modules under diverse environments, suggesting that diversified flagellar systems are required under diverse environments. In addition, the flagellar assembly module under cold shock contains an important polar flagellum gene (swpt_1508), and the flagellar assembly module under high pressure contains a lateral flagella gene (swp_3616). These results coincide with the fact that S. piezotolerans WP3 can express a polar flagellum and multiple lateral flagella systems with distinguishing characteristics [Reference Jian, Wang, Zeng, Xiong, Wang and Xiao39], which can partly explain how the expression of these systems is differentially regulated under low temperature and high pressure conditions [Reference Wang, Wang and Jian40].

Figure 4 The flagellar assembly module underlying (a) cold shock, (b) heat shock, and (c) high pressure conditions.

Furthermore, since the use of a variety of electron acceptors is the most important feature of the Shewanella species, we then focus on electron transfer processes, and we found several electron transfer-relevant modules in these various environmental conditions (Figure 5).

Figure 5 Electron transfer-relevant modules underlying the various environmental conditions. (a) Molybdenum cofactor biosynthesis module under the cold shock condition, (b) multihaem cytochrome module under the heat shock condition, (c) NrfD family module under the high pressure condition, and (d) cytochrome c oxidase module under the salt shock condition.

First, we identified a molybdenum cofactor biosynthesis module under the cold shock condition (Figure 5(a)), which could be linked to the fact that some molybdenum cofactor-included enzymes (such as sulfurase) can be used to modulate cold stress-responsive gene expression [Reference Xiong, Ishitani, Lee and Zhu41]. Furthermore, the molybdenum cofactor has also been shown to be a redox active prosthetic group, which is an essential component of numerous enzymes for facilitating electron transfer, such as dithiolene complexes [Reference Armstrong, Austerberry and Birks42], aldehyde oxidoreductase [Reference Krippahl, Palma, Moura and Moura43], and dimethyl sulfide dehydrogenase [Reference Creevey, McEwan and Bernhardt44].

Second, a multihaem cytochrome module was identified under the heat shock condition (Figure 5(b)). Multihaem cytochromes are major electron transfer proteins in Shewanella species. These proteins often contain several closely arranged haem cofactors, which can mediate rapid and long-distance electron transfer by reversible changes between the reduced and oxidized states of iron atoms in haem cofactors [Reference Breuer, Rosso, Blumberger and Butt10, Reference Pirbadian, Barchinger and Leung12]. Typically, from the inner membrane through the periplasm and outer membrane to the extracellular space, they can form electron transfer pathways [Reference Ding, Xu, Li, Xie and Sun11]. Recently, we also identified a multihaem cytochrome module in Shewanella oneidensis MR-1 under altered O₂ conditions [Reference Ding and Sun25]. Therefore, the multihaem cytochrome module of S. piezotolerans WP3 identified here should also be responsible for electron transfer processes under this condition. In addition, the nitrite reductase (cytochrome c ₅₅₂ ) swp_0613 and swp_3403 can catalyze the reduction of nitrite to ammonia. The reduction of nitrates and nitrites by the shock heating supplies reduced nitrogen under nonreducing conditions, which are necessary for amino acids and nucleic acids, and thus the origin of life under such conditions [Reference Summers45].

Third, an NrfD family module was identified under the high-pressure condition (Figure 5(c)), including the formate-dependent nitrite reductase NrfGCD (swp_4654∼swp_4652) and the polysulfide reductase NrfD (swp_1240). Periplasmic nitrite reductase Nrf can obtain electrons from other cytochrome c molecules located in the inner membrane and then use them to reduce soluble substances such as nitrites, a process that has been widely studied in Shewanella oneidensis MR-1 [Reference Gao, Yang and Barua46]. Polysulfide reductase NrfD could also be used to transfer electrons from the quinone pool.

Finally, we identified a cytochrome c oxidase module under the salt shock condition (Figure 5(d)). The module mainly contains two cbb ₃ -type cytochrome c oxidase subunits (swp_4145∼swp_4144) and one class I cytochrome c subunit (swp_4143), which are homologous to CcoN, CcoO, and CcoP from Shewanella oneidensis MR-1, respectively. Considering that cco family of proteins plays a key role in both aerobic and anaerobic respiration in Shewanella oneidensis MR-1, and they have also been shown to be involved in the generation of electric current [Reference Gao, Barua and Liang38], the cytochrome c oxidase module is thereby also considered to be related to these processes in S. piezotolerans WP3.