Phylogenetic analysis of 16S rRNA genes in the genus Halomonas
Halomonas elongata 1H9 is a saline bacterium known for 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) producing abilities under extreme environmental condition.
RAST genome analysis of the Halomonas elongata 1H9 shows that various subsystem feature counts consists of different pathways (Figure 3). Similarly, other members of the genus Halomonas are known to code and produce molecular variants 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. Therefore, the diversity of ectoine coding Halomonas might form a cluster with a similar kind of Halomonas species. Hence, Phylogenetic analysis of 16S rRNA sequences of type strain amongst genus Halomonas revealed that type strains AJ261, 1H9, M8, 5-3, RS-16, AAD6, SS20, 11S, NTU-107, TBZ21, 5CR, F8-11, SL014B-69, TBZ202, KCTC 42685, Z-7009, SL014B-85, CIP 105456, 204, KMM 1376, 10-C-3, Hwa etc., (Figure 4) were clustered together. It suggests that those species have a similar gene pool regardless of their genome length forms cluster. Variation in some clusters may occur due to the use of single 16S rRNA genes for phylogenetic analysis. Hence, members of the genus Halomonas might possess similar single-copy ectoine coding genes reveals that apart from the 16S RNA gene.
Identification of protein families, single copy genes and pearson correlation
Whole-genome analyses and annotation have resolved the misery of uniqueness amongst Halomonas spp. The radar chart shows that existing genomic data of Halomonas spp., possesses complete genome sequences, reference genomes and some scaffolds (Figure 5). Available genomic sequence data shows a similar gene pool and all ectoine-coding sequences from 93 type strains not having sets of genes. To resolve this issue and find relevant species in the genus Halomonas, we, therefore, annotated all genomes and identified the single-copy gene that codes ectoine. It was noticed that few Halomonas species that more than 11 single copy ectoine-coding genes. Therefore, inferred ML tree (Figure 6) some type strains shows that ectoine biomarker (in 1H9, F9-6, AJ261, SP4, ACAM 71, 62, Hb3, DSM 15911, N12, NTU-107, G-16.1, ZJ2214, TBZ3, M29, 79, BJGMM-B45, LCB169, CFH 9008, AIR-2, DQD2-30, 4A, SL014B-69, TBZ202, DX6, 9-2 and MC28) possessed by species were more or less similar kind of representative species similar to concatenated sequence of 32 Halomonas species (Figure 1). It was observed that of the 93 annotated genome sequences, 31 + 1 (32) species have 11 ectoine coding genes (DoeA-DoeC-DoeX-EctC-EctD-EutB-EutC-TeaA-TeaB-TeaC-UspA) as single copy number genes (Figure 7; Table 1). Heatmap of 11 ectoine coding genes shows high degree of pearson correlation (Figure 2) value lies between 0.50 and ± 1 (0 = no correlation, 1 = high degree correlation).
Novel Universal stress protein in Ectoine TRAP cluster (UspA) and resistance mediated by UspA gene
Studies on genome sequence analyses and analysis of various ectoine coding in Halomonas spp., uncovered that type strains viz. H. aestuarii Hb2 (NZ_CP018139), H. anticariensis DSM 16096 (GCF_000409775), H. azerbaijanica TBZ202 (GCF_004551485), H. bachuensis DX6 (GCA_011742165), H. beimenensis NTU-111 (NZ_CP021435), H. campisalis SS10-MC5 (NZ_CP065435), H. caseinilytica DSM 18067 (GCF_001662285), H. cerina CECT 7282 (GCF_014192215), H. cupida (GCF_900142755), H. daqingensis CGMCC 1.6443 (GCF_900108215), H. denitrificans DSM 18045 (GCF_003056305), H. endophytica MC28 (GCF_002879615), H. eurihalina MS1 (GCF_008274785), H. gudaonensis (GCF_900100195), H. halmophila NBRC 15537 (GCF_006540005), H. heilongjiangensis 9-2 (GCF_003202165), H. huangheensis BJGMM-B45 (NZ_CP013106), H. kenyensis DSM 17331 (GCF_013697085), H. korlensis CGMCC 1.6981 (GCF_900116705), H. lactosivorans KCTC 52281 (GCF_003254665), H. litopenaei SYSU ZJ2214 (GCF_003045775), H. niordiana ATF 5.4 (GCF_004798965), H. organivorans CECT 5995 (GCF_014192055), H. pacifica (GCF_007989625), H. qijiaojingensis KCTC 22228 (GCF_014651875), H. saliphila LCB169 (GCF_002930105), H. stenophila CECT 7744 (GCF_014192275), H. taeanensis (GCF_900100755), H. urmiana TBZ3 (GCF_005780185), H. ventosae (GCF_004363555), H. xinjiangensis TRM 0175 (GCF_000759345) and H. zincidurans B6 (GCF_000731955) possess superfamily of conserved gene – UspA - suggests that the UspA gene/domain has been inherited from ancient protein family found in primitive bacteria. UspA protein helps Halomonas species provide support and assist Halomonas to function and produce ectoine in the saline environment under stressful conditions like high salt, low water activity and low temperature etc. Hence, UspA – stress protein – found in 32 species is a new report in the genus Halomonas.