Profile of the complete genome of Arthrobacter sp. PAMC25564
As shown in Table 1, the complete genome of A. sp. PAMC25564 is composed of a circular chromosome of 4,170,970 bp with a 66.74% GC content. The chromosome is predicted to include 3,829 genes, from which 3,613 protein-encoding genes were functionally assigned, whereas the remaining genes were predicted as hypothetical proteins. We annotated 147 pseudogenes, 15 rRNA genes, and 51 tRNA genes distributed through the genome. From the predicted genes, 3,449 (90.08%) were classified into 20 functional COG categories, whereas the remaining 380 (9.92%) remained un-classified. The most numerous COG categories were S genes with unknown function (705 genes), K (298 genes), E (280 genes), G (276 genes), and C (259 genes) (Fig. 1). Many of these genes are related to amino acid transport, transcription, carbohydrate transport, and energy production/conversion, which suggests that this strain utilizes CAZymes for energy storage and carbohydrate metabolism. Most bacteria rely on cell respiration to catabolize carbohydrates to obtain the energy used during photosynthesis for converting carbon dioxide into carbohydrates. The energy is stored temporarily in the form of high-energy molecules such as ATP and used in several cell processes [33, 34]. Therefore, we predicted that the PAMC25564 strain could utilize carbohydrate degradation to obtain energy.
Table 1
Genome features of Arthrobacter sp. PAMC25564.
Feature
|
Value
|
A; Genome Statistics
|
|
Contigs
|
1
|
Total length bp;
|
4,170,970
|
N50
|
4,170,970
|
L50
|
1
|
GC %;
|
66.74
|
B; Genome features
|
|
Assembly level
|
Complete genome
|
Chromosome genes
|
3,829
|
Protein coding genes
|
3,613
|
Pseudogenes
|
147
|
rRNA genes
|
15
|
tRNA genes
|
51
|
16S rRNA phylogenetic analysis and ANI values
The identification of A. sp. PAMC25564 was verified using 16S rRNA sequence analysis (Fig. 2). This strain is phylogenetically placed among Arthrobacter and Pseudarthrobacter species. The results from phylogenetic analysis, BLAST analysis, and EzBio Cloud analysis revealed closely related strains such as P. sulfonivirans ALL (T) (99.09%), P. siccitolerans 4J27 (T) (98.48%), A. ginsengisoli DCY81 (T) (98.23%), and P. phenanthrenivorans Sphe3 (T) (98.13%). These results confirmed that isolate PAMC25564 belongs to the family Micrococcaceae, phylum Actinobacteria. Recently, several Arthrobacter species have been reclassified into new genera, based on 16S rRNA sequence similarities and chemotaxonomic traits such as peptidoglycan types, quinone systems, and/or polar lipid profiles [35]. Therefore, it has been proposed to reclassify within the genus Arthrobacter members of these five genera: Paenarthrobacter gen. nov., Pseudarthrobacter gen. nov., Glutamicibacter gen. nov., Paeniglutamicibacter gen. nov., and Pseudoglutamicibacter gen. nov. Among them, Pseudarthrobacter group would be reclassified into the genus Arthrobacter as: A. chlorophenolicus, A. defluvii, A. equi, A. niigatensis, A. oxydans, A. phenanthrenivorans, A. polychromogenes, A. scleromae, A. siccitolerans, and A. sulfonivorans [36]. Therefore, the PAMC25564 strain will probably be reclassified into the genus Pseudarthrobacter. As shown in Fig. 3, each ANI values ranged from 70.67 to 98.46%. So, we were confirmed that comparative genome results much lower than the common ANI values of 92–94%. In general, bacterial comparative genome analysis uses this method. The ANI analysis shows the average nucleotide identity of all bacterial orthologous genes that are shared between any two genomes and offers a robust resolution between bacterial strains of the same or closely related species (i.e., species showing 80–100% ANI) [37]. However, ANI values do not represent genome evolution, because orthologous genes can widely vary between the genomes being compared. Nevertheless, ANI closely reflects the traditional microbiological concept of DNA-DNA hybridization relatedness for defining species, so many researchers used this method, since it takes into account the fluid nature of bacterial gene pool and hence implicitly considers shared functions [38]. So, this mean the PAMC25564 strain could either belong to the species from which Arthrobacter diverged, or this could be a Pseudarthrobacter closely related new species.
CAZyme-encoding genes in Arthrobacter sp. PAMC25564
Among the 3,613 identified protein-encoding genes in PAMC25564, 108 were significantly annotated and classified into CAZyme groups: GH, GT, CE, AA, CBM, and PL using dbCAN2. The results provided an insight into the carbohydrate utilization mechanisms of PAMC25564. The signal P analysis predicted that 11 genes contained signal peptides. We found that proteins were distributed as follows: 33 GHs, 45 GTs, 23 CEs, 5 AAs, and 2 CBMs. However, no protein was assigned to the PL group. Most annotation results of GH genes revealed that the PAMC25564 genome has genes involved in glycogen and trehalose metabolism pathways such as β-glucosidase (GH1), glycogen debranching proteins (CBM48 and GH13_11), (1→4)-α-D-glucan 1-α-D-glucosylmutase (GH13_26), α-glucosyltransferase (GH13), α-trehalose phosphorylase (GH65), and 4-α-glucanotransferase (GH77) (Table 2). Previous studies showed the complex interplay of glycogen metabolism in colony development of Streptomycetes (in Actinomycetes species was only reported), showing that spore germination is followed by an increase in glycogen metabolism [39]. The underlying genetic and physiological mechanisms of spore germination remain unknown, but some mechanisms associated with the accumulation of nutrients as biomass and storage materials in the substrate mycelium during morphological phases of development have been reported [40]. Recently, Shigella sp. PAMC 28760, of pathogens isolated from Antarctica, was also reported to be able to adapt and survive in cold environments through glycogen metabolism [64]. Nonetheless, glycogen metabolism in bacteria remains unknown, even though it has been well-studied in eukaryotes [41]. However, we could predict the specificity of PAMC25564 strain genes involved in glycogen and trehalose metabolism.
Table 2
List of CAZyme GH enzymes from Arthrobacter sp. PAMC25564.
CAZyme
group
|
Enzyme activity
|
Gene position
|
EC
number
|
Number
|
GH1
|
β-Glucosidase
|
1206507_1208054
|
EC 3.2.1.21
|
2
|
1548357_1546930
|
GH2
|
β-Glucuronidase
|
230633_228825
|
EC 3.2.1.31
|
1
|
GH3
|
β-Glycosyl hydrolase
|
226049_223737
|
-
|
2
|
1615559_1617091
|
GH4
|
6-Phospho-β-glucosidase
|
1608453_1606978
|
EC 3.2.1.86
|
1
|
GH13
|
Malto-oligosyltrehalose/
trehalohydrolaseGH13_10;
|
1599543_1601333
|
EC 3.2.1.141
|
8
|
Limit dextrin α-1,6-maltotetraose-hydrolase GH13_11;
|
4158486_4156372
|
EC 3.2.1.196
|
Trehalose synthase
α-Amylase GH13_16;
|
4150667_4148871
|
EC 5.4.99.16
EC 3.2.1.1
|
Malto-oligosyltrehalose synthase GH13_26;
|
1597186_1599498
|
EC 5.4.99.15
|
Glucanase glge
GH13_3;
|
4152718_4150673
|
EC 3.2.1.-
|
α-Glucosidase
GH13_30;
|
1490553_1492259
|
EC 3.2.1.20
|
Limit dextrin α-1,6-maltotetraose-hydrolase
CBM48 + GH13_11;
|
1594748_1597189
|
EC 3.2.1.196
|
1,4-α-Glucan glycogen; branching enzyme CBM48 + GH13_9;
|
4148869_4145174
|
EC 2.4.1.18
|
GH15
|
Glucoamylase
|
2725735_2726796
|
EC 3.2.1.3
|
4
|
1210063_1211832
|
2262201_2264033
|
Trehalose-6-phosphate phosphatase
|
943180_945807
|
EC 3.1.3.12
|
GH23
|
Peptidoglycan-binding lysm
|
3355397_3356797
|
-
|
2
|
Membrane-bound lytic murein transglycosylase
|
3043759_3043151
|
EC 4.2.2.-
|
GH25
|
1,4-β-N-Acetylmuramidase
|
1863081_1865612
|
EC 3.2.1.92 -
|
1
|
GH30
|
Endo-1,6-β-galactosidase
|
731722_733167
|
EC 3.2.1.164
|
1
|
GH32
|
Sucrose-6-phosphate hydrolase
|
3442058_3440550
|
EC 3.2.1.26
|
2
|
β-Fructosidase
|
1383596_1384843
|
EC 3.2.1.26
|
GH33
|
Sialidase
|
613011_614603
|
EC 3.2.1.18
|
1
|
GH38
|
α-Mannosidase
|
4082745_4079713
|
EC 3.2.1.24
|
1
|
GH53
|
Galactosidase
|
2955779_2956930
|
-
|
1
|
GH65
|
Maltose phosphorylase/
Trehalose phosphorylase
|
392560_390227
|
EC 2.4.1.8
EC 2.4.1.64
|
2
|
Trehalose-6-phosphate phosphatase
|
342376_339176
|
EC 3.1.3.12
|
GH76
|
Fructose-bisphosphate aldolase
|
137645_138862
|
EC 4.1.2.13
|
1
|
GH77
|
4-α-Glucanotransferase amylomaltase;
|
2730522_2728363
|
EC 2.4.1.25
|
1
|
GH109
|
Gluconokinase
|
787401_788513
|
EC 2.7.1.12
|
2
|
236269_235103
|
Comparison of Arthrobacter sp. PAMC25564 genome characteristics with those from closely related species
We compared CAZyme genes from Arthrobacter species to speculate about their bacterial lifestyles and identified relevant CAZymes for potential applications in biotechnology. Considering the accessibility of available genome data, the complete genomes of 26 strains were chosen for the comparative analysis of CAZymes: 19 genomes of Arthrobacter spp., 1 genome of A. crystallopoietes, 3 genomes of A. alpinus, and 3 genomes of Pseudarthrobacter spp. (Table 3). Our results showed that the number of genes encoding glycogen and trehalose metabolism-associated CAZymes ranged from a minimum of 56 (A. sp. YC-RL1) to a maximum of 177 (A. sp. YN). We predicted that genes such as CE14, CE9, GH23, GH65, GT2, GT20, GT28, GT39, GT4, and GT51 were in each of the 26 genomes. In addition, GH13, GH65, GH77, GT5, and GT20 (glycogen and trehalose-related genes) are involved in energy storage. These genes are involved in glycogen degradation and trehalose pathways and were found in strains PAMC25564, 24S4-2, FB24, Hiyo8, KBS0702, MN05-02, PGP41, QXT-31, U41, UKPF54-2, A6, Ar51, and sphe3. These genes code for proteins with a strong ability to store and release energy. We found that strain PAMC25564 had the largest number of CAZyme genes. In general, CAZymes are large group of protein and this is mainly responsible for the degradation and biosynthesis/modification of polysaccharide but not all the members of this group are secreted proteins. This confirmed the little difference through results (Fig. 4).
Table 3
Genome information and comparative data of CAZymes from 26 strains including Arthrobacter sp. PAMC25564.
Species
|
Strain
|
Size
Mb;
|
GC %;
|
Replicons
|
Plasmid
|
Gene
|
Protein
|
tRNAs
|
rRNAs
|
References
|
Arthrobacter sp.
|
PAMC25564
|
4.17097
|
66.70
|
NZ_CP039290.1
|
0
|
3,829
|
3,613
|
51
|
15
|
This study
|
24S4-2
|
5.56375
|
65.10
|
NZ_CP040018.1
|
0
|
5,152
|
4,522
|
50
|
15
|
Unpublished
|
YN
|
5.06355
|
62.70
|
NZ_CP022436.1
|
0
|
4,673
|
4,387
|
55
|
18
|
Unpublished
|
QXT-31
|
5.04157
|
66.00
|
NZ_CP019304.1
|
0
|
4,593
|
4,379
|
54
|
18
|
Unpublished
|
Rue61a
|
5.08104
|
62.23
|
NC_018531.1/CP003203.1
|
2
|
4,693
|
4,568
|
53
|
18
|
[55]
|
FB24
|
5.07048
|
65.42
|
NC_008541.1/CP000454.1
|
3
|
4,623
|
4,486
|
51
|
15
|
[56]
|
PAMC25486
|
4.59358
|
62.80
|
NZ_CP007595.1
|
0
|
4,154
|
3,995
|
53
|
18
|
Unpublished
|
ZXY-2
|
5.05871
|
63.35
|
NZ_CP017421.1
|
5
|
4,700
|
4,505
|
54
|
18
|
Unpublished
|
U41
|
4.79263
|
66.38
|
NZ_CP015732.1
|
3
|
4,407
|
4,134
|
51
|
15
|
Unpublished
|
DCT-5
|
4.53075
|
66.22
|
NZ_CP029642.1
|
1
|
4,040
|
3,816
|
50
|
15
|
Unpublished
|
PGP41
|
4.27024
|
65.40
|
NZ_CP026514.1
|
0
|
3,917
|
3,760
|
49
|
12
|
Unpublished
|
ERGS1:01
|
4.93669
|
65.41
|
NZ_CP012479.1
|
2
|
4,481
|
4,232
|
41
|
6
|
[57]
|
YC-RL1
|
4.01864
|
64.04
|
NZ_CP013297.1
|
2
|
3,754
|
3,606
|
66
|
19
|
[58]
|
Hiyo4
|
3.77925
|
65.00
|
AP014718.1
|
0
|
5,182
|
5,120
|
50
|
12
|
[59]
|
KBS0702
|
3.64955
|
67.90
|
NZ_CP042172.1
|
0
|
3,373
|
3,243
|
51
|
15
|
Unpublished
|
UKPF54-2
|
3.51782
|
68.50
|
NZ_CP040174.1
|
0
|
3,238
|
3,110
|
50
|
15
|
Unpublished
|
MN05-02
|
3.64342
|
68.81
|
AP018697.1
|
1
|
3,608
|
3,543
|
52
|
12
|
Unpublished
|
Hiyo8
|
5.02672
|
63.76
|
AP014719.1
|
2
|
7,108
|
7,038
|
53
|
15
|
[59]
|
ATCC21022
|
4.43490
|
63.40
|
NZ_CP014196.1
|
0
|
4,078
|
3,910
|
53
|
12
|
[60]
|
Arthrobacter crystallopoietes
|
DSM 20117
|
5.03270
|
64.36
|
NZ_CP018863.1
|
2
|
4,634
|
4,425
|
48
|
15
|
Unpublished
|
Arthrobacter alpinus
|
R3.8
|
4.04645
|
62.20
|
NZ_CP012677.1
|
0
|
3,732
|
3,523
|
51
|
18
|
Unpublished
|
ERGS4:06
|
4.33365
|
60.59
|
NZ_CP013200.1
|
1
|
3,850
|
3,581
|
53
|
25
|
[61]
|
A3
|
4.45829
|
60.64
|
NZ_CP013745.1
|
1
|
4,033
|
3,902
|
52
|
19
|
Unpublished
|
Pseudarthrobacter phenanthrenivorans
|
Sphe3
|
4.53532
|
65.38
|
NC_015145.1/CP002379.1
|
2
|
4,278
|
4,052
|
50
|
12
|
[62]
|
Pseudarthrobacter chlorophenolicus
|
A6
|
4.98087
|
65.98
|
NC_011886.1/CP001341.1
|
2
|
4,685
|
4,505
|
49
|
15
|
[63]
|
Pseudarthrobacter sulfonivorans
|
Ar51
|
5.04376
|
64.70
|
NZ_CP013747.1
|
1
|
4,640
|
4,408
|
50
|
12
|
Unpublished
|
Bacterial glycogen metabolism in a cold environment
Glycogen is an energy source for plants, animals, and bacteria and is one of the most common carbohydrates. Glycogen consists of D-glucose residues joined by α (1→4) links; and it is a structural part of cellulose and dextran [42]. Glycogen is a polymer with approximately 95% of α-1, 4 linkages, and 5% of α-1, 6 branching linkages. In bacteria, glycogen metabolism includes five essential enzymes: ADP-glucose pyrophosphorylase (GlgC), glycogen synthase (GlgA), glycogen branching enzyme (GlgB), glycogen phosphorylase (GlgP), and glycogen debranching enzyme (GlgX) [43]. To adapt and survive in a cold environment, organisms need well-developed functional energy storage systems, one of which is glycogen synthesis. Bacteria have a passive energy saving strategy to adapt to cold environmental conditions such as nutrient deprivation, by using a slow glycogen degradation. Glycogen has the hypothesis of durability energy reserves, which have been reported as a Durable Energy Storage Mechanism (DESM) to account for the long-term survival of some bacteria in cold environments [44]. Metabolism of maltodextrin has been linked with osmoregulation and sensitivity of bacterial endogenous induction to hyperosmolarity, which is related to glycogen metabolism. Glycogen-generated maltotetraose is dynamically metabolized by maltodextrin phosphorylase (MalP) and maltodextrin glucosidase (MalZ), while 4-α-glucanotransferase (MalQ) is responsible for maltose recycling to maltodextrins [45]. Maltotetraose is produced using GlgB, MalZ, MalQ, and glucokinase (Glk), which act on maltodextrin and glucose. On the other hand, glucose-1-phosphate can be formed by MalP for glycogen synthesis or glycolysis [46]. Thus, glycogen degradation can play an essential role in bacterial adaptation to the environment. Additionally, maltose may form capsular α-glucan, which plays a role in environmental adaptation through the (TreS)-Pep2-GlgE-GlgB pathway [47, 48]. Previous studies indicate that trehalose is involved in bacterial adaptation to temperature fluctuation, hyperosmolarity, and desiccation resistance. Recently, the accumulation of trehalose and glycogen under cold conditions in Propionibacterium freudenreichii has been reported [49, 50]. Therefore, the role of glycogen in bacterial energy metabolism is closely linked to several metabolic pathways associated to bacterial persistence under environmental stresses such as starvation, drying, temperature fluctuations, and hyperosmolarity. Maltodextrin and trehalose pathways are examples of the relationship between glycogen and other metabolic pathways, as shown in Fig. 5. However, further exploration is needed to elucidate the relationship of glycogen with other compounds, and the mechanisms involved in bacterial persistence strategy [45]. The comparative analysis of predicted pathways for glycogen metabolism in Arthrobacter isolates (Additional file 1: Table S1), showed that in PAMC25564 the trehalose biosynthesis follows three metabolic pathways (OtsAB, TreYZ, and TreS) as in Mycobacterium [51]. The trehalose biosynthesis is well-known in numerous bacteria, for example, a defense strategy involving the accumulation of trehalose and three metabolic pathways to regulate osmotic stress is been reported in Corynebacterium glutamicum [52]. These three metabolic pathways are used for producing trehalose in C. glutamicum, where the gene galU/otsAB allows the increase of trehalose levels up to six times [53, 54]. This pathway was found in A. sp. PAMC25564, and it was predicted that such isolate could produce energy in cold environments.
Glycogen metabolism and trehalose pathway in Arthrobacter species
We investigated the glycogen metabolic pathways in each Arthrobacter strains (Fig. 6). To determine the 3 pathways of glycogen metabolism and trehalose pathway in Arthrobacter species, the level of dissimilarity was analyzed based on the composition of GH, GT, and other major enzymes from the 16 genomes. The analysis showed that only QXT-31, U41, and PGP41 shared with our strain the same genes and pathway, but other strains have a little different pattern. Therefore, we assumed that the PAMC25564 strain uses different pathways to obtain energy or degrade polysaccharides. Based on the above mentioned pathway-related genes, we confirmed that strains YN, Rue61, PAMC25486, ZXY-2, ERGS1:01, YC-RL1, ATCC21022, R.3.8, and A3 lack the malQ gene, which is responsible for maltose recycling to maltodextrins. Therefore, the energy supply may be compromised in such isolates. These Arthrobacter species showed a low number of genes for the three main pathways of trehalose. Therefore, we assumed that the PAMC25564 strain uses different pathways to obtain energy or degrade polysaccharides. Although most strains showed GalU/OtsAB genes, strains Hyo8 and ERGS1:01 lack the otsB gene (Fig. 6; Additional file 1: Table S1). So, we predicted that these strains would produce a significantly lower amount of trehalose, compared with the isolates having the OtsB gene. Additionally, we investigated the phosphotransferase system-related genes in strains R.3.8 and A3. These enzymes constitute another method used by bacteria for sugar uptake when the source of energy is phosphoenolpyruvate. As a result, these two strains were expected to produce polysaccharides by themselves or from an external source using phosphoenolpyruvate rather than consuming energy. Most of the compared strains were isolated from low temperature environments (-18 to 15 ℃), and most of them are known to adapt quickly to environmental changes. We expect that our results on glycogen metabolism, trehalose, and maltodextrin pathways will have a significant impact in industrial applications. The predicted trehalose metabolism in bacteria is important since they could be used for bioremediation. Additionally, these isolates would be an alternative for a cost-effective production of trehalose. We also predicted that trehalose metabolism varies among bacteria, depending on their metabolism and environmental conditions.