Growth conditions and identification
The growth of the strain-6 was measured at different temperatures of 4, 10, 15, 20, 25, 30, and 37°C. It can be seen that strain W-6 could growth at the range from 4 to 30°C, and the optimal growth temperature of was 15°C. So, the strain W-6 was psychrophilic rather than psychrotrophic bacterium. Based on 16S rRNA gene amplification, DNA sequencing and BLAST in GenBank, the isolate was identified as P. fluorescens.
Characteristic analysis of genome
After the sequence assembly and data quality assessment of sequencing samples, the circular map (Figure 1) of the W-6 genome is obtained by splicing, then subjected to basic analysis (Table S1). One scaffold is acquired through genomic sequencing and the assembled circular contig has a total length of 6,109,123 bp with GC content of 59.79%. The whole genome length and the GC content are more similar to other P. fluorescens strains (Table S2). The 19 rRNAs and 70 tRNAs are acquired from P. fluorescens W-6. Besides, 2 prophage sequences of 41,297 bp (phiW-6-1) and 38,126 bp (phiW-6-2) are acquired by Prophage Hunter prediction.
Function annotation of genes in genome
According to the KEGG annotation information, 3,063 genes were found to be annotated in W-6 with 115 pathways, which can be classified into five categories: metabolism, genetic information processing, environmental information processing, cellular processes and tissue systems (Figure 2a). The number of metabolic pathway genes was 1477, accounting for 48.23% of the annotated genes. A large number of secondary metabolic biosynthesis, microbial metabolism in different environments, antibiotic biosynthesis, ABC transporter system and two-component system were also annotated with 349, 289, 261, 241 and 199 genes, respectively. ABC transporter proteins are widely present in microorganisms. They play an important role in the nutrient uptake of ions, monosaccharides, amino acids, phospholipids, peptides, polysaccharides and proteins; while the two-component system regulates amino acid metabolism and mediates the stress response to the external environment. In addition, carbon metabolism and bacterial secretion system related pathways have a high number of genes.
Through the COG functional annotation of the W-6 genome (Figure 2b), it showed that the W-6 strain had the most proteins in the R class (general function prediction, 723), followed by the E class (amino acid transport and metabolism, 695), the T class (signal transduction mechanism, 646), the L class (replication, recombination and repair, 576), and the P class (inorganic ion transport and metabolism class, 444). These classes accounted for 49.48% of the total W-6 proteins, which is a significant proportion of all proteins.
The GO database has three major categories: molecular function, cellular component and biological process of genes, respectively (Figure 2c). The number of genes in the cellular component was 856, and among them 64 for both cellular and cytosolic functions. The number of genes in the molecular function category was the lowest, only 546, but among them 78 for the binding function. Overall, the genes involved in metabolic processes, cellular and cellular components, and binding functions were the most numerous, indicating that these functions play a key role in W-6.
In the functional annotation of W-6 encoded proteins, it was found that most of the proteins in W-6 strains are about amino acid transport and metabolism, signal transduction mechanism, replication, recombination and repair, and inorganic ion transport and metabolism, which accounts for 49.48% of the total W-6 proteins, which is a significant proportion of all proteins. Not only as the basic units of proteins, amino acids but also function in free form in cells. These free amino acids are either precursor or intermediate of metabolism or storage form of free ammonia to eliminate their toxic effects on the body, but the role of these proteins in bacteria is not clear and needs to be further explored.
Similar annotation results were obtained in the GO functional annotation of the W-6 genome. Up to 113 functional genes belonging to biological process genes involved in metabolic processes were found. Metabolic pathways accounted for 48.23% of the total number of genes annotated in the pathway annotation. Taken together, it can be seen that the largest proportion of protein functions in W-6 are metabolism-related proteins.
Comparative genomic analysis of P. fluorescens W-6
The information of 8 P. fluorescens strains is shown in Table S1. The collinearity analysis was performed by combining W-6 with the nucleic acid sequences of the other eight P. fluorescens strains in two combinations (Figure 3). It can be seen that W-6 has collinearity with all seven P. fluorescens strains, both forward and reverse collinearity, among which W-6 has stronger collinearity with SIK_W1 and SBW25; the weakest collinearity with NEP1; and no collinearity with Pf0-1. It shows that the differences between W-6, SIK_W1 and SBW25 genomes are small, while the differences with NEP1 and Pf0-1 are large, which may be more related to the climate and terrain of the bacterial survival habitat.
Mobile Genetic Elements
Mobile genetic elements played a crucial role in genome evolution, conferring bacterial adaptation to various environmental conditions. Mobile genetic elements also contributed greatly to horizontal gene transfer (HGT).
Two prophage loci was predicted in the chromosome, phiW-6-1 (41,297 bp; positions 1,571,724-1,613,020) and phiW-6-2 (38,126 bp; positions 1,603,523-1,641,648). Eleven and seventeen phage-related genes were identified in these regions, respectively (Table S3). The DNA synthesis genes were found in two prophage loci, indicating that these are not replication-defective (Table S4).
The transposon prediction of the W-6 genome identified only one transposon, located at 1,861,323-1,861,742 in the genome, indicating that the genetic plasticity of the strain might not be determined by intragenomic rearrangements.
CRISPRs were a component of many bacterial genomes, and CRISPRs functioned in the interference pathway to preserve genome integrity. In the W-6 chromosome, two CRISPRs were detected. CRISPR1 had one spacer and CRISPR2 had three spacers.
Drug resistance gene annotation of P. fluorescens W-6
Currently, due to antibiotic ubiquity and misuse, the problem of bacterial resistance has become a serious issue 19. It will be of great use for the analysis of bacterial resistance which isolated in the natural ecological environment. A total of 56 resistance genes were found in W-6 strain by predictive annotation in both ARDB and CARD databases (Table S5), which were classified into seven categories: Efflux pumps, Fluoroquinolones, Polypeptides, β-Lactams, Polyphosphate, Peptide antibiotics, and Elfamycin. Among them, there were 46 resistance genes in Efflux pumps, accounting for 82.14% of the total number of resistance genes (10 resistance genes). Efflux pumps genes have a considerable proportion in W-6 and may show a crucial role. It may be related to the environment W-6 located, and the exact relationship needs to be explored.
CAZyme-encoding genes in P. fluorescens W-6
Among the 5,474 identified protein-encoding genes in P. fluorescens W-6, 725 were significantly annotated and classified into CAZyme groups (GH, GT, CE, AA, CBM, and PL). It provided an insight into the carbohydrate utilization mechanisms of W-6. The 242 GHs, 261 GTs, 80 CEs, 16 AAs, 125 CBMs and 1 PL were distributed (Figure 4). These genes are related to amino acid transport, transcription, carbohydrate transport, and energy production/conversion, which suggests that the strain W-6 utilizes CAZymes for energy storage and carbohydrate metabolism. Most bacteria rely on cell respiration to catabolize carbohydrates, and then obtain the energy used during photosynthesis for converting carbon dioxide into carbohydrates. The energy is often stored in adenosine triphosphate (ATP) and used in several cell processes.
It was reported that Shigella sp. PAMC28760 could adapt and survive in cold environments through glycogen metabolism 20. Bacillus sp. TK-2 possessed cold evolution adaptability through CAZyme genes related to degradation of polysaccharides including cellulose and hemicellulose 10. The disruption of glycogen metabolism pathway compromised E. coli survival in cold environment 11. Also, Arthrobacter sp. PAMC26654 utilized polysaccharide or carbohydrate degradation as a source of energy to adapt and survive in in cold environments 21. Complete genomic analyses have revealed genomic information and evolutionary insights about different strains and species from cold environments. However, compared with eukaryotes, the characteristics of glycogen metabolism in prokaryotes are still not well studied, and the metabolism of low temperature microorganisms is not well understood 22. In this study, the role of CAZymes in cold adaptation was predicted, specifically those genes involved in glycogen and trehalose metabolism. It will provide valuable information on cold-active CAZymes, and they possess candidate biotechnological applications and the fundamental research values.
Glycogen metabolism and the trehalose pathway in P. fluorescens W-6
The pathways of glycogen metabolism and trehalose metabolism were studied in 14 Pseudomonas species. Similarities in different environments were analyzed based on the composition of GH, GT and other major enzymes in 14 selected genomes. The genome of W-6 contains genes that are different from those of other Pseudomonas species and therefore have slightly different pathways for energy acquisition or polysaccharide degradation than other′s Pseudomonas spp. (Table S6). Among the genes related to glycogen metabolism and trehalose metabolism, glgC, otsA, otsB and treT genes were missing in all 14 strains. Thus, most Pseudomonas spp. missing OtsA/B, but encoding TreS and TreY/Z 23. Among these 14 genomes, there was partial gene overlap between W-6 and the other 13 strains, but there were also differences, which could indicate that glycogen and trehalose metabolic pathways may exist in W-6 differently from the 13 strains, which may be one of the key factors for W-6 to adapt to low temperature environment.
The relationship between glycogen and trehalose metabolic pathways in bacteria is shown in Figure 5. There are three main pathways of glycogen metabolism. The most common glycogen metabolism pathway in bacteria involves in the glgC gene 11, while galU gene is generally more common in fungi 12. The metabolic pathways of trehalose are well known in bacteria, for example a defense strategy involving trehalose accumulation. It was reported that trehalose and glycogen were highly accumulated in Propionibacterium freudenreichii under cold condition 24. OtsBA, TreYZ and TreS existed in Arthrobacter sp. PAMC25564 23, Bacillus sp. TK2 10, P. freudenreichii 24 and Mycobacterium sp. 25, but OtsA/B missing in most Pseudomonas spp. 23. Five trehalose metabolic pathways have been reported in some bacteria 15, such as TPS/TPP, TreY-TreZ, TreP, TreS and TreT pathways, facilitating the survival in cold environments. Trehalose is essential for E. coli viability at low temperatures 26. Based on the CAZy gene annotation, P. fluorescens W-6 contains most of the genes involved in these two metabolic pathways, except for the glgC, otsA, otsB and treT genes. Gene glgC encodes a key enzyme in the glycogen metabolic pathway, so the glycogen metabolic pathway commonly involved in bacteria with glgC, but it is not present in W-6. The galU gene is generally involved in glycogen metabolism in fungi, but galU gene is present in W-6 genome, thus inferring that glycogen metabolism in W-6 may be different with those in general bacteria. There are four main trehalose metabolism pathways in S. acidocaldarius, including TPS/TPP, TreY-TreZ, TreH and TreT pathways 27. However, W-6 did not contain otsA, otsB and treT genes, so it can be inferred that three trehalose metabolism pathways involved in TreY-TreZ, TreS and TreP pathways in W-6. This may be a unique feature of W-6 in the adaptation to low temperature environments. At present, the roles of trehalose and glycogen are currently poorly understood in Pseudomonas spp.
Thus, the metabolic pathways of glucose, trehalose and maltose were interconnected. It provides an understanding of survival adaptation in cold environment by comparative analysis of glycogen metabolism and trehalose pathway from different Pseudomonas spp.
Other cold adaptation strategy
Membrane fluidity by changing unsaturated fatty acid profile is a universal strategy to adapt in cold environment 28. Eight genes (bdcA, bacC, fadB, fadJ, tesA, tesB, fabC and fabG) were identified which involved in the synthesis of unsaturated fatty acids, and these genes are most likely important for maintaining the membrane fluidity of W-6 under cold stress. Glutathione maintains cell redox homeostasis also protects membrane lipids from the oxidative stress induced at cold stress 29. Glutathione synthase (GshB, W-6-4258) was encoded in the W-6 genome, and two key genes, encoding glutathione peroxidase (Gpx2, W-6-2237) and glutathione reductase (Gor, W-6-1098), involved in the cycle of glutathione, were also identified, indicating that glutathione may facilitate psychrotolerance of the strain W-6.
Two-component regulatory systems are responsible for bacterial survival in cold environment 30. A two-component regulatory system is composed of a sensor kinase and a response regulator. In the W-6 genome, 199 pairs of sensor kinase and response regulator were found, which may act as a multifunctional sensory to control numerous cold-responsive genes as well as responses to osmotic, salt, and oxidative stress 31.
The sfsA (W-6-4762) was the cold-induced regulatory gene in W-6 strain, which was the DNA-binding transcriptional regulator, involved in regulation of sugar catabolism. The production of RNA helicases (deaD, hrpB, hrpA, pcrA, rapA, rhlE, rhlB, dbpA, uvrD, ywqA) also could be induced by low temperatures, then the formation of structured nucleic acids was prevented.
Anti-sense transcription may lead RNA secondary structure inaccuracy, and low efficiency, slow speed and false fidelity of transcription and translation under cold stress 31. In bacteria, nusG is a co-factor of Rho transcriptional terminator, and could diminish genome-wide anti-sense transcription combination with histone-like nucleoid-structuring protein (H-NS) and Rho-dependent transcriptional terminators 32. Therefore, the expression of nusA/nusB/nusG (W-6-4739, W-6-4562 and W-64489, respectively) may benefit W-6 to silence the anti-sense transcription for survival in a cold environment.
The rpsU gene (W-6-4435) in W-6 genome encoding the 30S ribosomal subunit protein, may play an important role in cold adaptation. As can be reported in Synechocystis, the rpsU gene was induced 10-fold under cold stress 33. The ribosome chaperone trigger factor (Tig, W-6-0528) in W-6 may help to early folding and prevents misfolding and aggregation of proteins. The SmpB protein (W-6-4717) in W-6 is needed to rescue ribosomes stalled on defective messages 33.
In sum, the analysis of W-6 genome suggested that cold-adapted bacterium W-6 had glycogen and trehalose metabolism pathways associated with CAZyme genes, and they were used as energy sources to adapt and survive in cold environments. The metabolic pathways of glucose, trehalose and maltose were interconnected. It provides an understanding of survival adaptation in cold environment by comparative analysis of glycogen metabolism and trehalose pathway from different Pseudomonas spp. Adaptations of the W-6 strain to low temperatures also are conferred by membrane fluidity by changing unsaturated fatty acid profile, the two-component regulatory systems, anti-sense transcription, the role played by rpsU genes in the translation process etc. These findings need to be verified by more detailed functional researches.