Research Article
Austin J Proteomics Bioinform & Genomics. 2014;1(1): 11.
Comparative Genomic Analysis of Halophiles Reveals New Clues to Their Adaptation Strategies in Hypersaline Environments
Shaoxing Chen1,2*, Jian Yang1, Yanhong Liu1, Chuangming Wang1 and Zhu L Yang2
1University Key Laboratory of Crop High Quality and High Effective Cultivation and Safety Control in Yunnan Province, Honghe University, Mengzi 661100, China
2Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
*Corresponding author: Shaoxing Chen, Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, 132# Lanhei Road, Heilongtan, Kunming 650201, Yunnan, China
Received: July 30, 2014; Accepted: September 27, 2014; Published: September 29, 2014
Abstract
Background: Halophiles, which have many potential applications in the biomaterial, bioremediation, and nanotechnology arenas, are microorganisms that live in environments with high salt concentrations. To elucidate the adaptive strategies that allow them to live in such hypersaline environments, the genome sequences of 66 strains of halophiles and non-halophiles (including 27 strains of halophilic bacteria, 24 strains of haloarchaea, and 15 strains of non-halophilic bacteria) were subjected to comparative genomic analysis.
Results: The G+C content of the genomic DNA sequence and acidic amino acid composition of the gene product of the haloarchaea were higher than those of both the halophilic and non-halophilic bacteria. In addition, the probability of occurrence and proportion of extra chromosomal genetic elements in the haloarchaea outweighed those of the halophilic and non-halophilic bacteria. Further, proteasome, the mRNA surveillance pathway, and basal transcription factors were present in the haloarchaea but absent in the other two groups of microorganisms. Carotenoid, sesqui-terpenoid, and tri-terpenoid were common in the haloarchaea, but occurred with a relatively low degree of frequency in the halophilic and non-halophilic bacteria. In contrast, some D-amino acids (i.e., D-glutamine, D-glutamate, D-arginine, D-ornithine, and D-alanine) and lipopolysaccharide, fluorobenzoate, limonene, and pinene were widely distributed in both types of bacteria, but absent in the family Halobacteriaceae.
Conclusion: Large-scale comparative genomic analysis of the genomes of haloarchaea, halophilic bacteria, and non-halophilic bacteria provided a novel perspective on the strategies that microorganisms adopt to adapt to hypersaline environments. Although both haloarchaea and halophilic bacteria require a high concentration of sodium chloride for growth, they employ different mechanisms of adaptation. Haloarchaea, which contain a significantly high G+C content and proportion of acidic amino acids to with stand their harsh environment, use sun light as an energy resource to balance intracellular and extracellular osmotic pressure, thus allowing them to live in hypersaline environments the same way that non-halophilic bacteria live in more common environments.
Keywords: Haloarchaea; Halobacteriaceae; Halophilic bacteria; Hypersaline environment; Carotenoids
Introduction
Halophiles, including haloarchaea and halophilic bacteria, are commonly found in salt lakes, salt mines, saline soils, artificial salterns, heavily salted hides, meats, fishes, and sauces with a high concentration of sodium chloride (NaCl) [1-3]. They have a number of useful applications in biotechnological and biomedical research [4]. Most halophiles use organic solutes to provide an osmotic balance between their cytoplasm and the surrounding medium [5].
The first genome-sequenced organism of haloarchaea is Halobacterium sp. NRC-1, which gave researchers an opportunity to probe the mechanisms of adaptation to hypersaline brine [6,7]. A surprising finding was that the overwhelming majority of predicted proteins were highly acidic, with a pI mode of 4.2, and very few neutral or basic proteins [8,9]. In contrast, the predicted proteins of most other non-haloarchaeal and bacterial organisms had equal fractions of acidic and basic components. The implication is that an increase in protein acidity and GC-bias in the genome is an important factor in tolerance to extreme salinity. The negatively charged residues in the haloarchaeal proteins were predominantly found at the protein surface and predicted to function as enhancers of solubility and stability in environments with high salt concentrations [10-12].
An additional characteristic observed in most haloarchaeal genomes is the presence of large megaplasmids or minichromosomes that often harbor important or even essential genes [13]. Analyses of the gene content of these large extra chromosomal elements have resulted in the discovery of expanded gene families for replication and transcription initiation [14], a variety of genes involved in cell survival, e.g., an aminoacyl transfer RNA (tRNA) synthetase [7], arsenic resistance [15], and the production of buoyant gas vesicles [7].
However, no study to date has investigated the metabolic pathways of and other differences between the halophiles (haloarchaea and halophilic bacteria) and non-halophilic bacteria. In this paper, we present the results of comprehensive analysis of the genomes of these three groups of organisms, which was carried out to obtain an in-depth understanding of the genomic characteristics that allow for survival in harsh natural environments.
Methods
Group information
The complete genomes of the haloarchaea, halophilic bacteria, and non-halophilic bacteria used for statistical analysis in this study were downloaded from the public database of the Kyoto Encyclopedia of Genes and Genomes (KEGG) (www.genome.jp/kegg/) [16] and GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The genomes of the sequenced halophilic bacteria were grouped as Group I, which contained 27 strains; those of the haloarchaea as Group II, comprising 24 strains; and those of the non-halophilic bacteria or normal bacteria, in which NaCl is not required for regular growth, as Group III, which included 15 strains (see Supplementary Data Table S1).
No
Full name
T number
Original DB
Accession numbers
Number of nucleotides (bp)
Number of coding genes
Number of amino acids (aa)
1
Nitrosococcus halophilus
4,145,260
3,817
1,163,230
2
Halorhodospira halophila
2,678,452
2,407
815,991
3
Halothiobacillus neapolitanus
2,582,886
2,357
761,013
4
Chromohalobacter salexigens
3,696,649
3,298
1,099,708
5
Max-Planck
4,061,296
3,474
1,171,559
6
2,909,567
2,526
811,068
7
Max-Planck
4,170,008
4,126
1,135,727
8
Zhejiang U
3,948,887
3,881
1,167,725
9
4,202,352
4,065
1,189,286
10
NITE
2,562,720
2,555
734,179
11
JGI
4,321,753
4,011
1,211,838
12
U Toronto
3,069,953
2,978
871,612
13
U Toronto
3,092,048
2,980
878,404
14
2,578,146
2,342
742,032
15
2,469,596
2,282
704,792
16
2,649,255
2,468
768,933
17
Bielefeld U
3,222,008
2,865
905,617
18
JGI
4,179,170
3,708
1,181,058
19
1,469,720
1,580
438,843
20
1,395,502
1,458
417,476
21
1,341,892
1,371
400,087
22
1,413,462
1,439
424,784
23
UFZ
1,452,335
1,529
436,722
24
UFZ
1,431,902
1,477
427,122
25
1,686,510
1,659
489,618
26
2,012,424
1,987
589,941
27
2,406,232
2,254
626,180
28
2,571,010
2,622
594,376
29
2,668,776
2,749
603,761
30
4,274,642
4,243
899,535
31
CAS
3,890,005
3,859
872,570
32
3,179,361
2,646
777,797
33
3,260,476
2,743
798,092
34
2,749,696
2,820
781,219
35
Max-Planck
2,912,573
2,749
816,750
36
3,692,576
3,560
787,569
37
3,116,795
2,998
911,740
38
3,332,349
3,349
898,173
39
5,440,782
5,113
1,087,273
40
4,443,643
4,212
1,046,649
41
JCVI
4,012,900
4,015
817,712
42
CAS
3,904,707
3,863
837,224
43
Kyung Hee U
3,698,650
3,873
827,912
44
3,944,467
3,898
820,148
45
4,355,268
4,221
1,048,160
46
Nankai China
3,793,615
4,302
1,053,462
47
4,354,100
4,199
1,055,529
48
3,788,356
3,656
1,037,158
49
3,223,876
3,099
914,088
50
4,314,118
4,154
1,121,232
51
3,643,158
3,476
809,071
52
4,639,675
4,145
1,319,938
53
Salmonella enterica subsp. enterica serovar Typhimurium U288
U Nottingham
5,017,059
4,798
1,405,169
54
China CDC
4,812,922
3,781
1,241,676
55
6,264,404
5,571
1,860,283
56
5,199,401
4,344
1,406,846
57
Virginia Tech
1,968,651
1,624
502,086
58
Bielefeld U
2,227,255
1,941
572,960
59
Washington U
7,231,415
6,070
1,134,954
60
U Tokyo
161,66,17
1,520
485,985
61
Goettingen
4,215,610
4,188
1,226,616
62
Hvidovre Hospital
2,891,564
2,727
794,042
63
DTU
2,776,847
3,150
756,718
64
2,024,476
1,799
538,540
65
KAGOME
2,587,877
2,582
692,723
66
JGI
4,990,707
4,469
1,288,879
Table S1: General genomic characteristics of strains.
Statistical items
General genomic information, including species name, genomic accession number, T number in KEGG [16], total number of nucleotides and amino acids, original database, and number of coding genes, is provided in table S1 of the Supplementary Data. The optimum NaCl concentration for growth, G+C content (G: Guanine; C: Cytosine), acidic amino acids, tRNA, gene density, and average gene length can be found in table S2 of the Supplementary Data, and plasmid information (i.e., name, accession number, nucleotides, percentage of plasmids in total genetic elements, and megaplasmid or minichromosome) in Table S3. Finally, comparisons of the KEGG pathways [16] of Groups I, II, and III were performed (data not shown). The pathways shared by all strains were omitted from the study.
No
Full name
G+C%
Optimum NaCl for growth (%)
Proportion of acidic amino acids (%)
Number of RNAs
Gene coding density (%)
Average gene length (bp/gene)
1
Nitrosococcus halophilus
51.58
4
12
55
85.5
1086
2
Halorhodospira halophila
67.98
9
14
55
91.4
1113
3
Halothiobacillus neapolitanus
54.71
4
11
52
88.4
1096
4
Chromohalobacter salexigens
63.91
3
12
90
89.2
1121
5
63.61
9
13
81
86.5
1169
6
57.54
8
12
60
84.6
1152
7
41.82
3
13
91
81.9
1011
8
61.37
3
12
53
88.8
1017
9
43.69
6
13
105
84.9
1034
10
36.04
5
13
79
85.9
1003
11
44.97
5.8
12
99
84.1
1077
12
44.61
14
12
60
85.2
1031
13
44.31
0.1
12
60
85.2
1038
14
37.88
10
13
70
86.3
1101
15
36.63
15
15
85
85.6
1082
16
32.46
8.4
13
90
87.1
1073
17
68.44
10 (KCl)
13
65
86.4
1125
18
42.92
6
12
58
84.8
1127
19
48.85
3
12
51
89.6
930
20
47.03
2
11
52
89.7
957
21
47.17
2
12
51
89.4
979
22
47.27
2
11
51
90.2
982
23
47.28
2.5
12
49
90.2
950
24
47.07
2.55
12
49
89.5
969
25
55.04
2
12
52
87.1
1017
26
42.62
2.3
14
64
87.9
1013
27
36.63
10
14
59
83.2
1068
28
67.91
25.2
16
52
86.0
981
29
68.01
22.8
16
52
87.1
971
30
62.36
21
17
61
84.5
1007
31
63.69
23.4
17
60
86.0
1008
32
47.86
18
15
52
74.3
1202
33
47.78
18
15
56
75.8
1189
34
63.44
20.5
18
51
86.8
975
35
64.53
18
17
50
84.1
1060
36
66.72
20
17
61
83.8
1037
37
62.90
27
17
51
87.8
1040
38
65.63
20.6
17
60
86.3
995
39
65.83
20
18
64
81.3
1064
40
61.42
20
18
60
83.1
1055
41
66.64
15
17
58
85.1
999
42
61.12
20
17
64
84.7
1011
43
64.96
20
17
52
83.8
955
44
61.06
20
17
57
86.0
1012
45
65.98
25
18
60
84.9
1032
46
64.25
17.6
17
58
85.3
882
47
64.93
15.2
18
58
82.8
1037
48
62.24
20.5
18
59
82.1
1036
49
64.34
20
17
52
85.1
1040
50
64.94
23
18
68
83.2
1039
51
63.90
20
17
51
82.6
1048
52
50.79
NR
11
176
85.3
1119
53
Salmonella enterica subsp. enterica serovar Typhimurium U288
52.18
NR
11
107
86.7
1046
54
47.63
NR
11
89
80.1
1273
55
66.56
NR
11
106
89.1
1124
56
46.19
NR
11
141
83.2
1197
57
32.30
NR
11
49
76.5
1212
58
51.84
NR
12
67
77.2
1147
59
67.85
NR
11
71
82.8
1191
60
38.73
NR
12
42
90.2
1064
61
43.51
NR
12
123
87.3
1007
62
32.42
NR
12
61
83.0
1060
63
38.10
NR
13
85
81.8
882
64
39.85
NR
13
70
79.8
1125
65
46.12
NR
10
ND
86.0
1002
66
30.58
NR
12
109
77.5
1117
Table S2: Statistical items of comparative genomic analysis.
No
Full name
Plasmid number
Plasmid name (Accession number)
Nucleotides
(bp)
Percentage of each plasmid (%)
Percentage of plasmid out of its total genetic elements (%)
1
Nitrosococcus halophilus
1
pNHAL01 (NC_013958)
65,833
1.59
1.59
2
Halorhodospira halophila
0
—
0
0
0
3
Halothiobacillus neapolitanus
0
—
0
0
0
4
Chromohalobacter salexigens
0
—
0
0
0
5
0
—
0
0
0
6
1
pDRET01 (NC_013224)
45,263
1.56
1.56
7
2
PL16 (NC_017669)
16,047
0.38
0.46
PL3 (NC_017670)
3,329
0.08
8
1
pPHB2 (NC_016079)
4,050
0.10
0.10
9
0
—
0
0
0
10
0
—
0
0
0
11
0
—
0
0
0
12
0
—
0
0
0
13
0
—
0
0
0
14
0
—
0
0
0
15
0
—
0
0
0
16
0
—
0
0
0
17
1
pCha1 (NC_020303)
86,256
2.68
2.6
18
0
—
0
0
0
19
0
—
0
0
0
20
0
—
0
0
0
21
0
—
0
0
0
22
0
—
0
0
0
23
0
—
0
0
0
24
0
—
0
0
0
25
0
—
0
0
0
26
0
—
0
0
0
27
1
pMETEV01 (NC_014254)
16,3915
6.81
6.81
28
2
pNRC200 (NC_002608)
36,5425
14.21
21.65
pNRC100 (NC_001869)
19,1346
7.44
29
4
PHS1 (NC_010366)
14,7625
5.53
25.02
PHS2 (NC_010369)
19,4963
7.31
PHS3 (NC_010368)
28,4332
10.65
PHS4 (NC_010367)
40,894
1.53
30
8
Chromosome II (NC_006397)
28,8050
6.74
17.12
pNG700 (NC_006395)
410,554
9.60
pNG600 (NC_006394)
155,300
3.63
pNG500 (NC_006393)
132,678
3.10
pNG400 (NC_006392)
50,060
1.17
pNG300 (NC_006391)
39,521
0.92
pNG200 (NC_006390)
33,452
0.78
pNG100 (NC_006389)
33,303
0.78
31
2
Chromosome II (NC_015943)
488,918
12.57
23
pHH400 (NC_015944)
405,816
10.43
32
1
PL47 (NC_008213)
46,867
1.47
1.47
33
3
PL100 (NC_017457)
100,258
3.07
3.45
PL6A (NC_017460)
6,129
0.19
PL6B (NC_017458)
6,056
0.19
34
2
PL131 (NC_007427)
130,989
4.76
5.52
PL23 (NC_007428)
23,486
0.85
35
0
—
0
0
0
36
2
Chromosome II (NC_012028)
52,5943
14.24
25.92
pHLAC01 (NC_012030)
431,338
11.68
37
0
—
0
0
0
38
1
pHmuk01 (NC_013201)
221,862
6.66
6.66
39
6
pHTUR01 (NC_013744)
698,495
12.84
28.52
pHTUR02 (NC_013745)
413,648
7.60
pHTUR03 (NC_013746)
180,781
3.32
pHTUR04 (NC_013747)
171,943
3.16
pHTUR05 (NC_013748)
71,062
1.31
pHTUR06 (NC_013749)
15,815
0.29
40
3
pNMAG01 (NC_013923)
378,348
8.51
15.57
pNMAG02 (NC_013924)
254,950
5.74
pNMAG03 (NC_013925)
58,487
1.32
41
4
pHV1 (NC_013968)
85,092
2.12
29.03
pHV2 (NC_013965)
6,359
0.16
pHV3 (NC_013964)
437,906
10.91
pHV4 (NC_013966)
635,786
15.84
42
3
pHM100 (NC_017942)
129,210
3.31
24.48
pHM300 (NC_017943)
321,908
8.24
pHM500 (NC_017944)
504,705
12.93
43
6
p1 (NC_014298)
406,285
10.98
24.05
p2 (NC_014299)
363,534
9.83
p3 (NC_014300)
44,576
1.21
p4 (NC_014301)
44,459
1.20
p5 (NC_014302)
23,727
0.64
p6 (NC_014303)
6,951
0.19
44
5
pHBOR01 (NC_014735)
362,194
9.18
28.49
pHBOR02 (NC_014731)
339,010
8.59
pHBOR03 (NC_014736)
210,350
5.33
pHBOR04 (NC_014732)
194,834
4.94
pHBOR05 (NC_014737)
17,535
0.44
45
3
pHALXA01 (NC_015658)
436,718
10.03
15.78
pHALXA02 (NC_015667)
181,778
4.17
pHALXA03 (NC_015659)
68,763
1.58
46
1
pJ7-1 (NC_018225)
95,989
2.53
2.53
47
2
pNATPE01 (NC_019967)
28,7800
6.61
12.94
pNATPE02 (NC_019963)
27,5821
6.33
48
0
—
0
0
0
49
0
—
0
0
0
50
2
p1 (NC_019975)
12,939
0.30
6.97
p2 (NC_019976)
287,963
6.67
51
2
phalar01 (NC_015955)
705,810
19.37
20.02
phalar02 (NC_015959)
23,659
0.65
52
0
—
0
0
0
53
Salmonella enterica subsp. enterica serovar Typhimurium U288
3
pSTU288-1 (NC_021155)
148,711
2.96
3.18
pSTU288-2 (NC_021156)
11,067
0.22
pSTU288-3 (NC_021157)
4,675
0.09
0.09
54
3
pPCY1 (NC_017156)
9,611
0.20
3.58
pCD1 (NC_017153)
68,342
1.42
pMT1 (NC_017155)
94,249
1.96
55
0
—
0
0
0
56
2
pSBAL17501 (NC_017570)
72,392
1.39
2.56
pSBAL17502 (NC_017572)
60,958
1.17
57
0
—
0
0
0
58
0
—
0
0
0
59
1
Chromosome II (NC_017832)
3,138,747
43.40
43.40
60
0
—
0
0
0
61
0
—
0
0
0
62
1
pSK67-M1 (NC_021060)
27,439
0.95
0.95
63
0
—
0
0
0
64
0
—
0
0
0
65
9
pKB290-1 (NC_020820)
42,449
1.64
6.09
pKB290-2 (NC_020821)
35,388
1.37
pKB290-3 (NC_020826)
35,340
1.37
pKB290-4 (NC_020822)
25,335
0.98
pKB290-5 (NC_020823)
17,882
0.69
pKB290-6 (NC_020827)
11,627
0.45
pKB290-7 (NC_020824)
10,300
0.40
pKB290-8 (NC_020828)
8,556
0.33
pKB290-9 (NC_020825)
5,866
0.23
66
0
—
0
0
0
Table S3: Ratio of plasmid containing strains in each groups and plasmid properties.
Statistical methods
The main statistical analyses were carried out using Sigma Plot 12.2 (scatter plot or box plot) (https://www.sigmaplot.com/products/ sigmaplot/sigmaplot-details.php) or Origin 7.5, whereas the proportion of acidic amino acids was calculated online (https://www. bio-soft.net/sms/index.html). The probability values (p-values) were obtained via a t-test performed using Statistical Product and Service Solutions (SPSS) statistical software.
Results
NaCl requirements
The prokaryotes, including Archaea and Bacteria, can be divided into two main groups, a NaCl-dependent group and a NaCl-independent group, based on their requirements for NaCl for growth. Most of the strains in Group I needed a relatively low NaCl concentration (~5% in W/V) for optimum growth (Figure 1), whereas the 27 strains in Group III were NaCl-independent (Supplementary Data Table S1). However, the NaCl concentration needed for the optimum growth of the Group II strains was 20%, significantly higher than that for the other two groups (Figure 1).
Figure 1 : NaCl concentration for optimum growth. Some strains of conventional bacteria (Group III) have an NaCl tolerance, but NaCl is not needed for their optimum growth. These strains are not presented here.
G+C content
It is well known that haloarchaea (the Group II strains) possess a high G+C content (> 60%), except for the strains from genus Haloquadratum (see Figure 2 and Supplementary Data Table S2). In this study, the G+C content of the haloarchaea was markedly higher than that of the halophilic bacteria (~45%) and non-halophilic bacteria (~45%) (p < 0.0001), although there was no significant difference in average G+C content between Groups I and III (p = 0.3762) (Figure 2). Moreover, several strains in Group I (Nos. 2, 4, 5, 8, and 17) and Group III (Nos. 55 and 59) had a similar G+C content to the Group II strains (Figure 2 and Table S2).
Figure 2 : Comparison of G+C content. The genomic sequences were downloaded from KEGG, and calculated by DNASTAR.
Proportion of acidic amino acids
The proportion of the 20 natural amino acids was analyzed (data not shown). Acidic amino acids (or negatively charged amino acids), which mainly include asparagine (Asp, D) and glutamate (Glu, E), play a crucial role in osmotic regulation, particularly in haloarchaea and halophilic bacteria. The proportion of acidic amino acids in Group II (average = 17.0%) was found to be significantly higher than that in Group I (average = 12.5%) (p < 0.0001) or Group III (average = 11.5%) (p = 0.0029) (see Table S2). The proportion of NaCl concentration for growth (Figure 3), whereas the proportion of positive amino acids decreased with such an increase (Supplementary Data Figure S1).
Figure 3 : Proportion of acidic amino acids. The gene products were downloaded from KEGG, and the proportion of amino acids was analyzed online (https://www.bio-soft.net/sms/index.html).
Figure S1 : Proportion of positive amino acids.Genome size, gene number, and tRNA number
The average genome size in Group II was larger than that in Group I (Figure 4A), as was the number of genes (Figure 4B). See Table S1 in addition to the two figures. The average genome size and number of genes in Group III varied more widely (Figure 4), which rendered these strains unsuitable for direct comparison. Interestingly, the range of variation in tRNA number in the Group II was much narrower than that in either Group I or Group III (Figure 4C and Table S2). There are generally 61 tRNAs in all microorganisms (the exception being the several tRNAs in rare codons), but the tRNA numbers presented here are those given by KEGG (www.genome.jp/ kegg/), which are predicted by the software based on analysis of the genomic sequence. Hence, the tRNA number does not depend on the tRNA type.
Figure 4 :Genome size, gene number, and tRNA number. A. Genome size; B. Gene number; C. tRNA number.Figure 4 : Genome size, gene number, and tRNA number. A. Genome size; B. Gene number; C. tRNA number.Gene length and density
The gene coding density in Group II was very similar to that in Group III (p > 0.5), and much lower than that in Group I (p < 0.05) (Figure 5A). The halophilic bacteria (Group I) featured the highest gene coding density of the three groups. However, the average gene length in Group I was quite similar to that in Group II (p = 0.2624 > 0.05), albeit much lower than that in Group III (p < 0.05) (Figure 5B). Hence, we concluded haloarchaea and halophilic bacteria share a similar average gene length, which differs considerably from that of non-halophilic bacteria.
Figure 5 :Gene length and density. Genes from the extra-chromosome and the main genome were combined for gene length and density analysis. A. Gene coding density; B. Average gene length.Figure 5 : Gene length and density. Genes from the extra-chromosome and the main genome were combined for gene length and density analysis. A. Gene coding density; B. Average gene length.Proportion of plasmid-containing strains and plasmid properties
The plasmids used for statistical analysis in this study included conventional plasmids, megaplasmids, and minichromosomes. The percentage (ratio) of plasmid-presenting strains among the haloarchaea, halophilic bacteria, and non-halophilic bacteria was 22.2% (6/27) for Group I, 40% (6/15) for Group III, and 83.3% (20/24) for Group II (Supplementary Data Table S3). Hence, the proportion of plasmid-containing strains in Group II was markedly higher than that in the other two groups. It seems that plasmids are widely distributed in haloarchaea, but limited in both halophilic and non-halophilic bacteria.
In addition, the average number of plasmids (ratio of plasmid-containing strains) in Groups I, II, and III were 0.26 (7/27), 2.54 (61/24), and 1.27 (19/15), respectively (Supplementary Data Figure S2). If the Lactobacillus brevis KB290 strain in Group III, which harbored the richest plasmids (9/1) of the 66 genome-sequenced strains, was excluded from plasmid calculation, then the average number (ratio) of plasmids in Group III would immediately decrease to 0.71 (10/14). The proportion of plasmids in the total genetic elements in the three groups differed significantly. Those in each of the Group I and III strains accounted for less than 10% of the total number of genetic elements, with the exception of the Burkholderia pseudomallei 1026b strain (Figure 6B and Supplementary Data Table S3). In contrast, most of the plasmids in Group II accounted for more than 20% of the total number of such elements (Figure 6).
Figure 6 :Proportion of plasmid-containing strains and general properties of plasmids. A. Ratio of plasmid-containing strains; B. Number of plasmids in plasmid-containing strains.Figure 6 : Proportion of plasmid-containing strains and general properties of plasmids. A. Ratio of plasmid-containing strains; B. Number of plasmids in plasmid-containing strains.
Figure S2 :The average number of plasmid.Figure S2 : The average number of plasmid.Comparative analysis of metabolic pathways
The metabolic pathways that were widely distributed among the Group I, II, and III strains are not presented in this paper, leaving 61 distinct pathways for comparison (Figure 7). The pathways of fluorobenzoate degradation (row 12), D-glutamine and D-glutamate metabolism (row 20), D-arginine and D-ornithine metabolism (row 21), D-alanine metabolism (row 22), lipopolysaccharide biosynthesis (row 26), peptidoglycan biosynthesis (row 27), limonene and pinene degradation (row 46), flagellar assembly (row 55), homologous recombination (row 60), and non-homologous end-joining (row 61) were shared by the Group I and III strains, whereas that of polycyclic aromatic hydrocarbon degradation (row 35) was shared primarily by those in Groups I and II. The pathway of carbon fixation in photosynthetic organisms (row 42) was found in Group I alone, whereas those of carotenoid biosynthesis (row 47), sesquiterpenoid and triterpenoid biosynthesis (row 48), mRNA surveillance (row 57), basal transcription factors (row 58), and proteasome (row 59) were found primarily in Group II (Figure 7).
Figure 7 :Comparative analysis of metabolic pathways. The pathways shared by all halophilic bacteria (Group I), haloarchaea (Group II), and non-halophilic bacteria (Group III) were omitted from the study. The pathways boxed in red (the pathways of 12, 20-22, 26-27, 46, 55, and 60-61) were common to both the halophilic (Group I) and non-halophilic bacteria (Group III). That boxed in yellow (the pathway of 35) was mainly shared by halophilic bacteria (Group I) and haloarchaea (Group II). The pathway boxed in blue (the pathway of 42) was found only in the halophilic bacteria (Group I), and those boxed in green (the pathways of 47-48 and 57-59) were found primarily in the haloarchaea (Group II).
A1. Nitrosococcus halophilus; A2. Halorhodospira halophila; A3. Halothiobacillus neapolitanus; A4. Chromohalobacter salexigens; A5. Halomonas elongata; A6. Desulfohalobium retbaense; A7. Halobacillus halophilus; A8. Pelagibacterium halotolerans; A9. Bacillus halodurans; A10. Tetragenococcus halophilus; A11. Desulfitobacterium dehalogenans; A12. Dehalobacter sp. DCA; A13. Dehalobacter sp. CF; A14. Halothermothrix orenii; A15. Acetohalobium arabaticum; A16. Halobacteroides halobius; A17. Corynebacterium halotolerans; A18. Halothece sp. PCC 7418; A19. Methanohalophilus mahii; A20. Methanohalobium evestigatum; B1. Halobacterium sp. NRC-1; B2. Halobacterium salinarum R1; B3. Haloarcula marismortui; B4. Haloarcula hispanica; B5. Haloquadratum walsbyi DSM 16790; B6. Haloquadratum walsbyi C23; B7. Natronomonas pharaonis; B8. Natronomonas moolapensis; B9. Halorubrum lacusprofundi; B10. Halorhabdus utahensis; B11. Halomicrobium mukohataei; B12. Haloterrigena turkmenica; B13. Natrialba magadii; B14. Haloferax volcanii; B15. Haloferax mediterranei; B16. Halalkalicoccus jeotgali; B17. Halogeometricum borinquense; B18. Halopiger xanaduensis; B19. Natrinema sp. J7-2; B20. Natrinema pellirubrum; B21. Natronobacterium gregoryi; B22. Halovivax ruber; B23. Natronococcus occultus; B24. Halophilic archaeon; C1. Escherichia coli K-12 MG1655; C2. Yersinia pestis D106004; C3. Pseudomonas aeruginosa PAO1; C4. Shewanella baltica BA175; C5. Francisella tularensis TIGB03; C6. Neisseria meningitidis WUE 2594 (serogroup A); C7. Burkholderia pseudomallei 1026b; C8. Helicobacter pylori OK113; C9. Bacillus subtilis subsp. subtilis 6051-HGW; C10. Staphylococcus aureus M1; C11. Listeria monocytogenes N53-1; C12. Lactobacillus brevis KB290.
1. Ascorbate and aldarate metabolism; 2. Fatty acid biosynthesis; 3. Fatty acid metabolism; 4. Synthesis and degradation of ketone bodies; 5. Geraniol degradation; 6. Lysine degradation; 7. Histidine metabolism; 8. Tyrosine metabolism; 9. Phenylalanine metabolism; 10. Chlorocyclohexane and chlorobenzene degradation; 11. Benzoate degradation; 12. Fluorobenzoate degradation; 13. Tryptophan metabolism; 14. Phenylalanine, tyrosine and tryptophan biosynthesis; 15. Novobiocin biosynthesis; 16. beta-Alanine metabolism; 17. Taurine and hypotaurine metabolism; 18. Phosphonate and phosphinate metabolism; 19. Cyanoamino acid metabolism; 20. D-Glutamine and D-glutamate metabolism; 21. D-Arginine and D-ornithine metabolism; 22. D-Alanine metabolism; 23. Glutathione metabolism; 24. Other glycan degradation; 25. Polyketide sugar unit biosynthesis; 26. Lipopolysaccharide biosynthesis; 27. Peptidoglycan biosynthesis; 28. Glycerolipid metabolism; 29. Inositol phosphate metabolism; 30. Arachidonic acid metabolism; 31. alpha-Linolenic acid metabolism; 32. Dioxin degradation; 33. Xylene degradation; 34. Toluene degradation; 35. Polycyclic aromatic hydrocarbon degradation; 36. Chloroalkane and chloroalkene degradation; 37. Naphthalene degradation; 38. Aminobenzoate degradation; 39. Nitrotoluene degradation; 40. Styrene degradation; 41. C5-Branched dibasic acid metabolism; 42. Carbon fixation in photosynthetic organisms; 43. Lipoic acid metabolism; 44. Atrazine degradation; 45. Porphyrin and chlorophyll metabolism; 46. Limonene and pinene degradation; 47. Carotenoid biosynthesis; 48. Sesquiterpenoid and triterpenoid biosynthesis; 49. Sulfur metabolism; 50. Caprolactam degradation; 51. Biosynthesis of unsaturated fatty acids; 52. Nonribosomal peptide structures; 53. Degradation of aromatic compounds; 54. Bacterial chemotaxis; 55. Flagellar assembly; 56. Phosphotransferase system (PTS); 57. mRNA surveillance pathway; 58. Basal transcription factors; 59. Proteasome; 60. Homologous recombination; 61. Non-homologous end-joining.
Figure 7 :Comparative analysis of metabolic pathways. The pathways shared by all halophilic bacteria (Group I), haloarchaea (Group II), and non-halophilic bacteria (Group III) were omitted from the study. The pathways boxed in red (the pathways of 12, 20-22, 26-27, 46, 55, and 60-61) were common to both the halophilic (Group I) and non-halophilic bacteria (Group III). That boxed in yellow (the pathway of 35) was mainly shared by halophilic bacteria (Group I) and haloarchaea (Group II). The pathway boxed in blue (the pathway of 42) was found only in the halophilic bacteria (Group I), and those boxed in green (the pathways of 47-48 and 57-59) were found primarily in the haloarchaea (Group II).
A1. Nitrosococcus halophilus; A2. Halorhodospira halophila; A3. Halothiobacillus neapolitanus; A4. Chromohalobacter salexigens; A5. Halomonas elongata; A6. Desulfohalobium retbaense; A7. Halobacillus halophilus; A8. Pelagibacterium halotolerans; A9. Bacillus halodurans; A10. Tetragenococcus halophilus; A11. Desulfitobacterium dehalogenans; A12. Dehalobacter sp. DCA; A13. Dehalobacter sp. CF; A14. Halothermothrix orenii; A15. Acetohalobium arabaticum; A16. Halobacteroides halobius; A17. Corynebacterium halotolerans; A18. Halothece sp. PCC 7418; A19. Methanohalophilus mahii; A20. Methanohalobium evestigatum; B1. Halobacterium sp. NRC-1; B2. Halobacterium salinarum R1; B3. Haloarcula marismortui; B4. Haloarcula hispanica; B5. Haloquadratum walsbyi DSM 16790; B6. Haloquadratum walsbyi C23; B7. Natronomonas pharaonis; B8. Natronomonas moolapensis; B9. Halorubrum lacusprofundi; B10. Halorhabdus utahensis; B11. Halomicrobium mukohataei; B12. Haloterrigena turkmenica; B13. Natrialba magadii; B14. Haloferax volcanii; B15. Haloferax mediterranei; B16. Halalkalicoccus jeotgali; B17. Halogeometricum borinquense; B18. Halopiger xanaduensis; B19. Natrinema sp. J7-2; B20. Natrinema pellirubrum; B21. Natronobacterium gregoryi; B22. Halovivax ruber; B23. Natronococcus occultus; B24. Halophilic archaeon; C1. Escherichia coli K-12 MG1655; C2. Yersinia pestis D106004; C3. Pseudomonas aeruginosa PAO1; C4. Shewanella baltica BA175; C5. Francisella tularensis TIGB03; C6. Neisseria meningitidis WUE 2594 (serogroup A); C7. Burkholderia pseudomallei 1026b; C8. Helicobacter pylori OK113; C9. Bacillus subtilis subsp. subtilis 6051-HGW; C10. Staphylococcus aureus M1; C11. Listeria monocytogenes N53-1; C12. Lactobacillus brevis KB290.
1. Ascorbate and aldarate metabolism; 2. Fatty acid biosynthesis; 3. Fatty acid metabolism; 4. Synthesis and degradation of ketone bodies; 5. Geraniol degradation; 6. Lysine degradation; 7. Histidine metabolism; 8. Tyrosine metabolism; 9. Phenylalanine metabolism; 10. Chlorocyclohexane and chlorobenzene degradation; 11. Benzoate degradation; 12. Fluorobenzoate degradation; 13. Tryptophan metabolism; 14. Phenylalanine, tyrosine and tryptophan biosynthesis; 15. Novobiocin biosynthesis; 16. beta-Alanine metabolism; 17. Taurine and hypotaurine metabolism; 18. Phosphonate and phosphinate metabolism; 19. Cyanoamino acid metabolism; 20. D-Glutamine and D-glutamate metabolism; 21. D-Arginine and D-ornithine metabolism; 22. D-Alanine metabolism; 23. Glutathione metabolism; 24. Other glycan degradation; 25. Polyketide sugar unit biosynthesis; 26. Lipopolysaccharide biosynthesis; 27. Peptidoglycan biosynthesis; 28. Glycerolipid metabolism; 29. Inositol phosphate metabolism; 30. Arachidonic acid metabolism; 31. alpha-Linolenic acid metabolism; 32. Dioxin degradation; 33. Xylene degradation; 34. Toluene degradation; 35. Polycyclic aromatic hydrocarbon degradation; 36. Chloroalkane and chloroalkene degradation; 37. Naphthalene degradation; 38. Aminobenzoate degradation; 39. Nitrotoluene degradation; 40. Styrene degradation; 41. C5-Branched dibasic acid metabolism; 42. Carbon fixation in photosynthetic organisms; 43. Lipoic acid metabolism; 44. Atrazine degradation; 45. Porphyrin and chlorophyll metabolism; 46. Limonene and pinene degradation; 47. Carotenoid biosynthesis; 48. Sesquiterpenoid and triterpenoid biosynthesis; 49. Sulfur metabolism; 50. Caprolactam degradation; 51. Biosynthesis of unsaturated fatty acids; 52. Nonribosomal peptide structures; 53. Degradation of aromatic compounds; 54. Bacterial chemotaxis; 55. Flagellar assembly; 56. Phosphotransferase system (PTS); 57. mRNA surveillance pathway; 58. Basal transcription factors; 59. Proteasome; 60. Homologous recombination; 61. Non-homologous end-joining.
Discussion
Microorganisms that inhabit hypersaline environments are designated halophiles. Depending upon the salt concentration they require for optimum growth, they are classified as haloarchaea (Group II), which grow optimally in media with 15%-30% (2.5 M--5.2 M) NaCl, or halophilic bacteria (Group I), which grow optimally in media with 3%-15% (0.5 M-2.5 M) NaCl (Figure 1). Non-halophilic bacteria, in contrast, are microorganisms that achieve optimal growth in media with less than 1% (0.2 M) NaCl.
Halophiles have evolved two major strategies to cope with the high osmotic pressure in their hypersaline environments. Most aerobic halophilic bacteria produce compatible solutes, such as betaine, ectoine, β-carotene, or trehalose, whereas haloarchaea take advantage of the accumulation of intracellular potassium to balance that pressure. However, several species of halophilic bacteria, namely, Salinibacter and Salisaeta, have a similar mechanism to haloarchaea, coping with their hypersaline environments via a low degree of water activity [17,18]. A number of the current study's findings are worthy of particular note.
First, the G+C content of the haloarchaea far outweighed that of either the halophilic or non-halophilic bacteria, although a few exceptions were found in all three groups of microorganisms (Figure 2). Examination of the protein coding genes showed that the use of codons with G or C in the third codon position in the haloarchaea was over 90%, which may be attributable to the high G+C content of haloarchaea [19]. That content may contribute to the stability of genetic materials through replication, transcription, and gene expression. However, the reason that G or C always appears in the third codon position in haloarchaea requires elucidation.
Second, as is well known, high concentrations of salt lead to protein aggregation. Cations can capture the combined H2O from the protein molecule, and denature it. However, the proteins in haloarchaea are unlikely to be denatured by the universal denaturing NaCl concentration, as on the contrary, they need a high NaCl concentration to perform their biological activity. The proportion of acidic amino acids in the haloarchaeal proteins in this study reached 17% or even higher, which was markedly higher than those in the proteins from the halophilic or non-halophilic bacteria (Figure 3). Acidic amino acids (glutamate or aspartate), combined with the intracellular or extracellular cations needed to avoid a configuration change in proteins, may play a critical role in the mechanism of adaptive evolution.
Third, the distribution range of tRNA number (Figure 4A), genome size (Figure 4B), gene number (Figure 4C), gene coding density (Figure 5A), and gene length (Figure 5B) among the various strains of haloarchaea was much narrower than that among the halophilic and non-halophilic bacteria strains. Unlike haloarchaea, halophilic and non-halophilic bacteria are represented by a wide variety of species in different phylogenetic lineages (orders), thus reflecting a broad range of major genetic information [20].
Fourth, like most bacterial genomes, the haloarchaeal genomes in this study ranged from 2.5-5.4 Mbp (Supplementary Data Table S1), with a single main circular chromosome and, on occasion, accessory plasmids or extra chromosomal elements (Supplementary Data Table S3). Some large plasmids or megaplasmids containing several important or essential genes are classified as minichromosome or chromosome II (Supplementary Data Table S3; see also [7]). Our results show that the probability of extra chromosomal elements occurring in haloarchaea is greater than 83% and that the percentage of extra chromosomal elements in their total genetic elements is between 12% and 30%. Both figures far outweigh those for halophilic or non-halophilic bacteria (Figure 6). Studies of the megaplasmids in Halobacterium sp. NRC-1 and similar plasmids in other Halobacterium strains suggest that they are highly dynamic and rapidly evolving [21]. The widely distributed and highly dynamic extra chromosomal elements in haloarchaea can be attributed to the high frequency of homologous recombination [22] and imprecise excision.
Fifth, the plasma membranes of Archaea differ from those of Bacteria, and exhibit remarkable structural and chemical diversity. The realization in the early 1970s that these cell walls do not contain peptidoglycan, a major component of bacterial cell walls, was an initial pillar on which the establishment of Archaea as a distinct phylogenetic kingdom rested [23]. The lipopolysaccharide-containing outer membranes that are a typical characteristic of gram-negative bacteria are absent in Archaea [24]. In Archaea, including haloarchaea, flagellar biosynthesis is reminiscent of bacterial type IV pilus biosynthesis [25]. Apart from the flagella, the functional roles played by the putative archaeal pili and pilus-like structures are unknown. However, in the current study, few flagellins, which are related to the flagellar assembly, were found in the haloarchaea in comparative analysis of the metabolic pathways (Figure 7), suggesting that the flagellins of haloarchaea are quite different from those of halophilic or non-halophilic bacteria.
Sixth, Soloshonok and Izawa [26] indicated that many D-amino acids or secondary metabolites can be found in the cell walls of microorganisms. In this study, we did not find the metabolic pathways of D-glutamine, D-glutamate, D-arginine, D-ornithine, or D-alanine (Figure 7). Hence, we hypothesize that these D-amino acid pathways are most likely absent in haloarchaea. In addition to D-amino acids, we found fluorobenzoate, limonene, and pinene to be widely distributed in the two types of bacteria (Figure 7), yet absent in the family Halobacteriaceae. As there are few reports on other groups of Archaea, this absence may constitute a selective hallmark of the distinction between Archaea and Bacteria.
Seventh, homologous recombination or non-homologous end-joining frequently occurs in Archaea, including haloarchaea [27]. However, our survey of the comparative metabolic pathways of haloarchaea, halophilic bacteria, and non-halophilic bacteria found no such occurrence in haloarchaea. This finding suggests that the DNA or protein sequences of the homologous recombination-or non-homologous end-joining-related enzymes or proteins of haloarchaea are distinct from those of others.
Eighth, the proteasome that occurred in the haloarchaea was absent in the halophilic and non-halophilic bacteria (Figure 7). An ATP-dependent protease in Bacteria appears to be homologous to Archaea and the eukaryotic proteasome, and that in Archaea shares the simple architecture of bacterial proteases. However, the subunits are homologous to the eukaryotic proteasome, thus suggesting the existence of a bridge between bacteria and eukaryotic organisms [28]. Moreover, the mRNA surveillance pathway and basal transcription factors in haloarchaea are similar to proteasome, but neither was found in the halophilic or non-halophilic bacteria in this study.
Finally, carotenoid is a major component of halorhodopsin (light-driven chloride pump) [29] and bacteriorhodopsin (light-driven proton pump) [30]. In addition to carotenoid, sesquiterpenoid and triterpenoid were widely distributed in the haloarchaea, but barely present in either the halophilic or non-halophilic bacteria (Figure 7). Because haloarchaea can utilize sunlight to perform photosynthesis, whereas halophilic bacteria cannot, these bacteria need to take advantage of the synthesis of a compatible solute to cope with the hypersaline environment and achieve growth. From this perspective, it is obvious that the strategy evolved by haloarchaea is considerably more beneficial than that evolved by halophilic bacteria. In the condition of ever-present solar irradiance, the haloarchaea living in hypersaline environments thrive equally well to non-halophilic bacteria living in less hostile environments.
In sum, the genome composition of haloarchaea, including their high G+C and acidic amino acid content, reveal apparent traits of adaptive evolution when these species live in a hypersaline environment for long periods. The higher G+C content in haloarchaea leads to greater sequence similarity, which means that haloarchaea have a higher probability of homologous recombination than do halophilic or non-halophilic bacteria. As a result, the plasmid-containing ratio and plasmid proportion in haloarchaea are higher than those in the other two groups. In a harsh environment lacking in nutrition and full of salt, haloarchaea use their purple membrane structure to cope. Haloarchaea are one of the very few microorganisms that lack chloroplast but are able to draw upon the sun for energy synthesis.
Acknowledgements
We would like to thank Dr. Yan-Jia Hao from the Kunming Institute of Botany at the Chinese Academy of Sciences for assistance with data analysis. We are also grateful for the support we received for the study from the CAS/SAFEA International Partnership Program for Creative Research Teams and the Hundred Talents Program of the Chinese Academy of Sciences, and the National Natural Science Foundation of China (31460003).
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