Molecular Biology Reports (2021) 48:1045–1053

https://doi.org/10.1007/s11033-020-06131-w

ORIGINAL ARTICLE

Genomic analysis of a novel species Halomonas shambharensis

isolated from hypersaline lake in Northwest India
Kapilesh Jadhav1 · Bijayendra Kushwaha2 · Indrani Jadhav2 · Prem Shankar3 · Anjali Geethadevi4 · Gaurav Kumar5 ·
Sonam Mittal6 · Guru Prasad Sharma7 · Madhuri Parashar2,8 · Deepak Parashar2,4 

Received: 13 May 2020 / Accepted: 24 December 2020 / Published online: 21 January 2021
© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021

Abstract
Genome analysis of Halomonas shambharensis, a novel species, was performed to understand the osmoprotectant strategies
used by the strain to overcome the salinity stress and to explore the prospective industrial uses. It will also help to better
understand the ecological roles of Halomonas species in hypersaline habitats. Ultrastructure of the cell was determined by
using transmission electron microscopy. Standard microbiological methods were used to find out growth parameters and het-
erotrophic mode of nutrition. For Genome analysis, complete bacterial genome sequencing was performed using the Oxford
Nanopore MinION DNA Sequencer. Assembly, annotation and finishing of the obtained sequence were done by using a
Prokaryotic Genome Annotation Pipeline (PGAP) (SPAdes v. 3.10.1). Predicted Coading sequences (CDSs) obtained through
the PGAP were used for functional annotation using Clusters of Orthologous Groups and Kyoto Encyclopedia of Genes and
Genomes (KEGG) platforms. The H. shambharensis was found to be a Gram-stain-negative, rod-shaped bacterium, motile
with a peritrichous flagella. The H. shambharensis bacterium can grow in a wide range of temperature (from 25 to 65 °C),
pH (pH 4 to pH 12.0) and salt concentration (5.0% NaCl to 30.0% NaCl). After annotation and assembly, the total genome
size obtained was 1,533,947 bp, which revealed 146 subsystems, 3847 coding sequences, and 19RNAs with G+C content of
63.6%. Gene annotation identified the genes related to various metabolic pathways, including carbohydrate metabolism, fatty
acid metabolism and stress tolerance. The genomic dataset of H. shambharensis will be useful for analysis of protein-coding
gene families and how these coding genes are significant for the survival and metabolism among the different species of
Halomonas. The complete genome sequence presented here will help to unravel the biotechnological potential of H. shamb-
harensis for production of the high-value products such as betaine, or as a source of gene-mining for individual enzymes.

Keywords  Genome analysis · Genome annotation · Halomonas shambharensis · Oxford Nanopore Technology · Sambhar
Salt Lake

Supplementary Information  The online version contains

supplementary material available at https​://doi.org/10.1007/s1103​
3-020-06131​-w.

4
* Kapilesh Jadhav Department of Obstetrics and Gynecology, Medical College
jadhavkapilesh@gmail.com of Wisconsin, Milwaukee, WI 53226, USA
5
* Deepak Parashar Department of Biochemistry, University of Delhi, South
dparashar@mcw.edu Campus, New Delhi 110021, India
6
1 School of Biotechnology, Jawaharlal Nehru University,
School of Engineering and Technology, Jaipur National
New Delhi 110067, India
University, Jaipur, India
7
2 Blood Center of Wisconsin, Milwaukee, WI 53233, USA
School of Life Sciences, Jaipur National University, Jaipur,
8
India Department of Internal Medicine, UT Southwestern Medical
3 Center, Dallas, TX 75390, USA
Department of Laboratory Medicine, All India Institute
of Medical Sciences, New Delhi, New Delhi, India

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Introduction
Members of the genus Halomonas are moderately halo-
philic to halotolerant, Gram-negative rod like bacteria,
which are widely distributed in the saline environment [1,
2]. During the course of evolution, Halomonas species
have developed some important characteristics which help
them to survive in extreme saline conditions [3]. Many of
these characteristics features may exploit industrially [4].
Compatible solutes, halo- and thermotolerant hydrolys-
ing enzymes, extracellular polymers and polyhydroxyal-
kanoates, are now commercially used in the pharmaceuti-
cal, food-processing and biotechnological industries [3,
5, 6].
As reported in our previous findings, the bacterium
Halomonas shambharensis (strain: SBS 10) was isolated
from the Sambhar Salt Lake [7, 8]. The Sambhar Salt Lake
was selected as a sampling site because of its athalassic
hypersaline environment and rich microbial diversity. The
H. shambharensis can grow well across a wide range of
the salinity and temperatures and showed physiological
adaptation in accordance to the environmental conditions
which prevail in the Sambhar Salt Lake [8]. We performed
whole genome sequencing to understand the osmoprotect-
ant strategies being used by the strain in order to overcome
the salinity stress and to explore its prospective industrial
applications. It will also help in the discovery of new pro-
tein families, expand the diversity of known functions, and
improve the recruitment and phylogenetic assignment of
existing metagenomic sequences [9–12].
Here, we summarized the classification and features
of the H. shambharensis using genome sequencing and
annotation. Genome sequencing of H. shambharensis
was undertaken with the aim to identify the enzymes and
their adaptability in the hypersaline conditions to better
understand the ecological roles of Halomonas species in
hypersaline habitats.
Materials and methods
Growth, morphology and phylogenetic position
Halomonas shambharensis was isolated from Sambhar
Salt Lake, which is located 80 km southwest of the city
of Jaipur and 64 km northeast of Ajmer, Rajasthan with
Latitude and Longitude (WGS84) of 26° 56′ 38″ North,
75° 5′ 30″ East respectively. The enrichment, isolation,
and growth conditions of H. shambharensis are reported
in our previous findings [7, 8]. The utilization of vari-
ous carbon sources was checked using the minimal salt
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Molecular Biology Reports (2021) 48:1045–1053
medium as proposed by Ventosa et al. [13] containing
­(l−1): 80.0 g NaCl, 2.0 g KCl, 0.2 g M ­ gSO4·7H2O, 1.0 g
­KNO3, 1.0 g (­ NH4)2HPO4, 0.5 g K ­ H2PO4. Substrates were
added (0.2 μm filter sterilized solutions) to achieve the
final concentration of 1.0 g ­l−1, except for carbohydrates,
which were used at 2.0 g ­l−1.
The ultrastructure of the H. shambharensis (Strain SBS
10) was determined by transmission electron microscopy
(TEM). The TEM was used as described by Bouchotroch
et al. [14]. To determine the cell size, cell morphology and
the presence of flagella, samples were examined under the
TEM [Tecnai G2 20 (FEI) S-Twin 200 kV]. For phylogenetic
analysis, genomic DNA (gDNA) was extracted using a pro-
cedure as described previously [15]. Amplification of the
16S Ribosomal RNA (rRNA) genes was carried out using
universal bacterial primers 27F (5′-AGA GTT TGA TCC
TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG
ACT T-3′) using a PCR thermal cycler (Bio-Rad). Ampli-
fied 16S rRNA gene sequences were submitted to the NCBI
database (Accession No.: KT796562). The taxonomic posi-
tion of the H. shambharensis and the identification of near-
est neighbors were performed using the MEGA software
version 7 [16].
Genome sequencing, assembly and annotation
Genome project history
Bacterium H. shambharensis was selected on the basis of
its ability to survive in polyextreme conditions. The genome
project is deposited in the Genome Online Database (GOLD)
with GOLD project ID Gp0400756, and completed draft of
the genome sequence has been deposited in the Gene Bank
(Accession number RXHI00000000) [17]. A summary of
the genome project information is shown in Table 1. The
project information is according to the compliance of MIGS
version 7 [18].
Genome sequencing
The  gDNA was extracted using a GenElute Bacterial
Genomic DNA Kit (Sigma-Aldrich, UK) from 50  ml
broth culture grown in Tryptic Soy Broth, followed by the
protocol for Gram-negative bacteria. Using the extracted
gDNA, sequencing library was prepared by RAD004
Rapid Sequencing Kit, which contains transposase for
simultaneous cleavage of template DNA and attaches tags
to the cleaved ends. Rapid Sequencing Adapters were then
added to the tagged ends (Supplementary File-1). Resulted
ultra-long reads were sequenced. The Oxford Nanopore
MinION was used for the whole genome sequence of H.
shambharensis. The Oxford Nanopore MinION is a nano-
pore DNA sequencer, which is a real time, long-read, low
Molecular Biology Reports (2021) 48:1045–1053 1047

Table 1  Genome project MIGS ID Property Term

information
MIGS-31 Finishing quality Improved high-quality draft
MIGS-28 Libraries used RAD004 rapid
MIGS-29 Sequencing platform Oxford NanoporeMinION
MIGS-31.2 Fold coverage 316×
MIGS-30 Assembles SPAdes v. 3.10.1
MIGS-32 Gene calling method GeneMarkS-2+
Locus tag RXHI01000000
GenBank ID RXHI00000000.1
GenBank release date 27th December, 2018
GOLD Study ID Gs0141947
Bio-project PRJNA479678
Project relevance Distinguishing features from the closet species
and its ability to survive in polyextreme condi-
tions
MIGS-13 Source material identifier Halomonas sp. (Strain SBS 10)

cost device developed by Oxford Nanopore Technologies

Ltd., Oxford, U.K. The advantage of Oxford Nanopore
MinION sequencing is its ability to read ultra-long reads
up to 2 Mb. The device is scalable and results in highly-
contiguous de-novo assembly, which can close the gaps
in assembling genome sequence and allow read through
of repetitive regions. The quality of reads generated was
assessed using Fast QC v0.11.5 (https​://www.bioin​forma​
tics.babra​ham.ac.uk/proje​cts/fastq​c/).

Genome assembly and annotation

Assembly, annotation and finishing were performed using

a Prokaryotic Genome Annotation Pipeline (PGAP)
(SPAdes v. 3.10.1), which was designed to annotate the
bacterial genome. The PGAP is a multi-level process
that includes prediction of protein-coding genes, as
well as other functional genome units such as structural
RNAs (sRNAs), Transfer RNAs (tRNAs), and small RNAs
[19, 20]. To assign functions to the genes, predict subsys-
tem in the Genome and to predict metabolic functions of
the assigned genes, we have used Rapid Annotations using
Subsystems Technology (RAST) [21–23]. The RAST uses
FIG fams protein family subset to predict gene functions.
MicroScope Microbial Genome Annotation and Analysis
Platform (MaGe, Genoscope, Évry, France) were used for
the construction of a circular genomic map using Circular
Genome Viewer (CGView) [24]. Predicted CDSs obtained
through the PGAP were used for functional annotation
using Clusters of Orthologous Groups (COG) and KEGG
platforms. All these data sets were combined to assert
Fig. 1  TEM image of H. shambharensis. A cell is rod-shaped with
product description for each set of genes. an outer capsulated layer and peritrichous flagella. Scale bar: 5.0 μm
(×1,00,000 magnifications)

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Results (98.20% similarity) as weighted by Basic Local Alignment

Cell morphology and phylogenetic position
Cells of H. shambharensis are capsulated, rod shaped,
0.3–0.4 × 0.75–1.65 µm, motile by peritrichous flagella and
do not form endospores (Fig. 1). The H. shambharensis is
aerobic, heterotrophic, and utilizes a wide range of carbon
sources including ethanol, fructose, galactose, glucose, glyc-
erol, lactose, and sucrose. A summary of the features of H.
shambharensis is given in Table 2.
For taxonomic position, 16S rRNA gene sequence of H.
shambharensis (1452 bp, gene accession no. KT796562) was
compared with other sequences in NCBI databases. Phylo-
genetic tree was plotted by MEGA software version 7 using
Maximum Likelihood (ML) and Maximum Parsimony (MP)
methods (Fig. 2) [16]. Among the different related species,
the closest species was found to be Halomonas gudaonensis
Search Tool (BLAST) scores.
Genomic features
Whole genome sequencing of the H. shambharensis
yielded a genome of 1,533,947 bp in length after assem-
bly of 5,30,458 bp of raw sequence producing 31 scaffolds
[7]. All the scaffolds were larger than 1219 bp with the
largest at 1,10,994 bp. Annotation of the assembled con-
tigs using PGAP and GeneMarkS+ annotation method and
RAST revealed 146 subsystems, 3847 coding sequences,
and 19 RNAs. Sequencing and assembly metrics for the
H. shambharensis is represented in Table 3. The resulting
genome consists of only one ~1.5 Mb circular chromo-
some, with a high G+C content of 63.60% (Fig. 3). For
functional annotation, a number of functional and uni-
genes were categorized as COG [30] and KEGG functional
categories [31, 32] (Fig. 4).

Table 2  General features MIGS ID Property Term Evidence ­codea

and classification of H.
shambharensis  Classification Domain Bacteria TAS [25]
Phylum Proteobacteria TAS [26]
Class Gamma proteo bacteria TAS [26]
Order Oceanospirillales TAS [27]
Family Halomonadaceae TAS [27]
Genus Halomonas TAS [27]
Strain SBS 10
Gram stain Negative TAS [28]
Cell shape Rods IDA
Motility Motile TAS [29]
Sporulation Non-sporulating NAS
Temperature Range: 25–65 °C IDA
Optimum: 37 °C
pH range Optimum: 5.0–9.0 pH IDA
Carbon source Monosaccharides and polysaccharides IDA
MIGS-6 Habitat Hypersaline lake IDA
MIGS-6.3 Salinity Range: 5.0–30.0% NaCl IDA
Optimum: 8.0% NaCl
MIGS-22 Oxygen requirement Aerobic TAS [29]
MIGS-15 Biotic relationship Free living NAS/IDA
MIGS-14 Pathogenicity Not reported IDA
MIGS-4 Geographic location Sambhar Salt Lake, Jaipur, India IDA
MIGS-5 Sample collection January, 2013 IDA
MIGS-4.1 Latitude 26° 56′ 38″ North IDA
MIGS-4.2 Longitude 75° 5′ 30″ East IDA
MIGS-4.4 Altitude 360 m IDA
a
 Evidence code–IDA: inferred from direct assay; TAS: traceable author statement (i.e., a direct report
exists in the literature); NAS: non-traceable author statement (i.e., not directly observed for the living, iso-
lated sample, but based on a generally accepted property for the species, or anecdotal evidence). These
evidence codes are from the Gene Ontology

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Fig. 2  Phylogenetic tree analysis of 16S rRNA of the H. shambharensis (Strain SBS 10). ML and MP approaches were used for constructing the
tree (MEGA Version 7.0 [16])

Table 3  Genome features of H. shambharensis  portion of stress tolerance genes including, 26 genes for oxi-
Attributes Values
dative stress. The majority of these are related to protection
from reactive oxygen species, including manganese super-
Genome size (bp) 1,533,947 oxide dismutase Sod A (EC 1.15.1.1), superoxide dismutase
DNAs (G+C) concentration (%) 63.60 [Fe] Sod B (EC 1.15.1.1), catalase HPII (EC 1.11.1.6), per-
Plasmid 0 oxidase HPI (EC 1.11.1.7) and cytochrome c551 peroxidase
DNA scaffolds 31 CCP (EC 1.11.1.5). Role and description of genes related to
rRNA 03 osmotic and oxidative stress are illustrated in Table 4.
tRNA 18
Other non-coding RNA 02 Genes for metabolism of carbohydrate and aromatic
Protein coding genes 829 intermediates
Pseudogenes 957
Total genes 1809 The genome of H. shambharensis revealed 118 genes related
to carbohydrate metabolism and are responsible for utiliza-
tion of different carbon sources found in various environ-
Genes for stress responses ments, which suggest variability of product formation and
hence may be of great biotechnological importance. These
Upon analysis, 56 stress tolerant genes were identified as in macromolecules are hydrolyzed into small molecules that
H. shambharensis. Out of these, 17 genes were responsible can be absorbed and metabolized by H. shambharensis
for osmotic stress, 29 genes were identified for oxidative and other microorganisms in the hypersaline environment.
stress and 2 genes were found to belong to the universal Majority of carbohydrate metabolism genes found on func-
stress protein family. As reported in our previous findings, tional annotation are related to central carbohydrate metab-
betaine is the major compatible solute used by H. shamb- olism, including pyruvate dehydrogenase E1 component
harensis to overcome the salinity stress and its endogenous Pyr DH (EC 1.2.4.1), pyruvate oxidase POX (EC 1.2.3.3),
accumulation increases with an increase in the salinity in acetyl-coenzyme A synthetase ACS-AMP (EC 6.2.1.1),
the external environment [15]. Among the 17 genes which aldehyde dehydrogenase ADH (EC 1.2.1.3), and acetalde-
were responsible for osmotic stress tolerance, 16 were identi- hyde dehydrogenase AADH (EC 1.2.1.10).
fied as genes responsible for choline and betaine uptake and Apart from carbohydrate metabolism, the genome of H.
betaine biosynthesis. shambharensis also contains genes for the beta-ketoadipate
Halophilic environments generally have diminished pathway which is a chromosomally encoded convergent
oxygen tension; nevertheless they are sufficient to gener- pathway for aromatic compound degradation and is widely
ate active oxygen intermediate. As with other members of distributed in soil bacteria and fungi. One of its branches
halophiles, H. shambharensis uses a different set of genes converts catechol, generated from various aromatic hydro-
that helps in neutralizing potentially lethal active oxygen carbons, aromatic amino acids, and lignin monomers into
intermediates. These unigenes comprise the second major beta-ketoadipate. Two additional steps accomplish the

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Fig. 3  An illustration of circular map of the H. shambharensis.
Genome map was constructed by using CGView software. The
skew is represented on the distance scale (in kbp) on the inner map.
conversion of beta-ketoadipate to tricarboxylic acid cycle
intermediates making the catechol degradation pathway an
effective route for utilization of carbon and energy. The pres-
ence of this pathway shows that the species H. shambharen-
sis can be used for bioremediation of aromatic compounds,
especially in marine environments where other bacterial
species cannot survive.
Discussion
Halomonas shambharensis was isolated from Sambhar
Salt Lake, a hypersaline lake situated in the north-western
region of India [15]. Recently, we reported this species as
a novel species on the basis of its phenotypic features, fatty
acid profile, G+C content and having distinct characteris-
tics with related species [8]. The species are polyextrem-
ophile with the capability to survive at high temperature,
pH and saline conditions. On the basis of these observa-
tions, our primary objective was to understand the survival
strategies being used by H. shambharensis through genome
annotation. Hence, we performed the whole genome analy-
sis using the Oxford Nanopore MinION DNA Sequencer.
Functional annotation using different assembly and anno-
tation platforms demonstrated that, the H. sambharensis
genome contains two sets of stress genes (osmotic stress
and oxidative stress) which help to survive in the hypersaline
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Molecular Biology Reports (2021) 48:1045–1053
Deduced ORFs and their orientations are represented by blue and red
arrows around the map
conditions. Inconsistent with our previous study, we found
that H. sambharensis contains complete Operon for bio-
synthesis of glycine betaine, which acts as an osmoprotect-
ant to combat high osmotic pressure [15]. Betaine is either
transported from the external environment or synthesized
endogenously via oxidation of choline in H. shambharensis
through a de-novo pathway. Choline dehydrogenase Bet A
(EC 1.1.99.1), betaine aldehyde dehydrogenase Bet B (EC
1.2.2.8) and high affinity choline uptake protein Bet T are
part of betaine biosynthesis operon and are preceded by
choline ABC transporter periplasmic binding proteins in H.
shambharensis [37].
Intense solar radiations and high alkalinity in addition
to the hyper osmotic condition creates unique environment
for Sambhar Lake. Species surviving in these conditions
must have adopted to overcome the oxidative stress gen-
erated by such extreme conditions. We found that, the H.
sambharensis genome bear set of enzymes; manganese
superoxide dismutase, superoxide dismutase, catalase, per-
oxidase and Cytochrome c551 peroxidase, which help in
neutralizing potentially lethal effect of these scavenging
radicals generated by oxidative stress [38]. Species of the
Halomonas are capable of utilizing a wide variety of car-
bon sources [39]. Similarly, we found that, H. sambharen-
sis contains 118 genes related to carbohydrate transport,
metabolism, and synthesis of pyruvate dehydrogenase,
pyruvate oxidase, acetyl-coenzyme A synthetase, aldehyde
Molecular Biology Reports (2021) 48:1045–1053 1051

Fig. 4  Functional annotation summary of the H. shambharensis genome. The functional classification of annotated genes was done by (a) COG
and (b) KEGG

Table 4  Genes from H. shambharensis predicted to be involved in stress tolerance and detoxification

Metabolic pathways Genes Proteins Functions

Betaine biosynthesis Bet A Choline dehydrogenase An osmolyte responsible for osmotic balance [33]
Bet B Betaine aldehyde dehydrogenase
Bet C Choline-sulfatase
Glucan biosynthesis Mdo H Glucans biosynthesis glucosyl transferase Osmoregulation and feedback control [34]
Mdo G Glucans biosynthesis protein G precursor
Mdo C Glucans biosynthesis protein C
Mdo B Phosphoglycerol transferase I
Superoxide dismutase Sod B Superoxide dismutase (Fe) Protects the organism against the toxic effects of
Sod C Superoxide dismutase (Cu–Zn) precursor the superoxide anion [35]
Radical scavangers HP I Catalase Helps in scavenging of toxic radical molecules
HP II Peroxidase produce by cells [36]
CCP Cytochrome c551 peroxidase

dehydrogenase and acetaldehyde dehydrogenase. Variabil- by-products generated are utilized by other species in the
ity of substrate utilization and product formation may be same environment and might be essential for maintaining
of great biotechnological importance. Additionally, the the ecological balance and homeostasis [15, 40, 41].

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Data availability  The genome sequence and associated data for Halo-
monas sp. SBS 10 was deposited under GenBank accession number
RXHI00000000, BioProject accession number PRJNA479678, SRA
accession number SRP224914, and BioSample accession number
SAMN0​96016​49.
References
1. Arahal DR, Ventosa A (2006) The family Halomonadaceae. In:
Dworkin M, Falkow S, Rosenberg E, Schleifer K.-H., Stacke-
brandt E (eds) The prokaryotes, Springer, New York, pp 811–835.
2. de la Haba RR, Sánchez-Porro C, Ventosa A (2011) Taxonomy,
phylogeny, and biotechnological interest of the family Halomona-
daceae. In: Ventosa A, Oren A, Ma Y (eds) Halophiles and hyper-
saline environments. Springer, Heidelberg, pp 27–64
3. Lentzen G, Schwarz T (2006) Extremolytes: natural compounds
from extremophiles for versatile applications. Appl Microbiol
Biotechnol 72:623–634
4. Jadhav K, Kushwah B, Jadhav I (2018) Insight into compat-
ible solutes from halophiles: exploring significant applications
in biotechnology. In: Singh J, Sharma D, Kumar G, Sharma
N (eds) Microbial bioprospecting for sustainable develop-
ment. Springer-Nature, Singapore, pp 291–307. https​: //doi.
org/10.1007/978-981-13-0053-0_16
5. Satpute SK, Banat IM, Dhakephalkar PK, Banpurkar AG, Cho-
pade BA (2010) Biosurfactants, bioemulsifiers and exopolysaccha-
rides from marine microorganisms. Biotechnol Adv 28:436–450
6. Sharma A, Paul D, Dhotre D, Jani K, Pandey A, Shouche Y (2017)
Deep sequencing analysis of bacterial community structure of Sol-
dhar hot spring, India. Microbiology 86:126–132
7. Kushwaha B, Sharma GP, Sharma A, Shankar P, Geethadevi A,
Sharma N, Sharma MK, Jadhav I, Parashar D, Jadhav K (2020a)
Whole-genome shotgun sequence of Halomonas sp. strain SBS
10, isolated from a hypersaline lake in India. Microbiol Resour
Announce 9:e01270. https​://doi.org/10.1128/MRA.01270​-19
8. Kushwaha B, Jadhav I, Jadhav K (2020b) Halomonas sambharen-
sis sp. nov., a moderately halophilic bacterium isolated from the
saltern crystallizer ponds of the Sambhar Salt Lake in India. Curr
Microbiol 77:1125. https​://doi.org/10.1007/s0028​4-020-01892​-w
9. Gao XY, Zhi XY, Li HW, Zhou Y, Lapidus A, Han J, Haynes
M, Lobos E, Huntemann M, Pati A, Ivanova NN, Mavromatis
K, Tindall BJ, Markowitz V, Woyke T, Klenk HP, Kyrpides NC,
Li WJ (2015) Draft genome sequence of Halomonas lutea strain
YIM 91125(T) (DSM 23508(T)) isolated from the alkaline Lake
Ebinur in Northwest China. Stand Genomic Sci 10:1. https​://doi.
org/10.1186/1944-3277-10-1
10. Jadhav K, Jadhav I (2017) Sulfur oxidation by Achromobacter
xylosoxidans strain wsp05 reveals ecological widening over which
thiotrophs are distributed. World J Microbiol Biotechnol 30:192.
https​://doi.org/10.1007/s1127​4-017-2359-6
11. Tatiana T, Anne E, Alexis B, Stéphane B (2019) Complete
genome sequence of the halophilic PHA-producing bacterium
Halomonas sp. SF2003: insights into its biotechnological poten-
tial. World J Microbiol Biotechnol 35:50. https​://doi.org/10.1007/
s1127​4-019-2627-8
12. Williamson A, De Santi C, Altermark B, Karlsen C, Hjerde E
(2016) Complete genome sequence of Halomonas sp. R5-57.
Stand Genomic Sci 11(1):62. https​: //doi.org/10.1186/s4079​
3-016-0192-4
13. Ventosa A, Quesada E, Rodrı´guez-Valera F, Ruiz-Berraquero F,
Ramos-Cormenzana A (1982) Numerical taxonomy of moderately
halophilic Gram-negative rods. J Gen Microbiol 128:959–1968
14. Bouchotroch S, Quesada E, Ad M, Llamas I, Bejar V (2001)
Halomonasmaura sp. nov., a novel moderately halophilic,
13
Molecular Biology Reports (2021) 48:1045–1053
exopolysaccharide-producing bacterium. Int J Syst Evol Microbiol
51:1625–1632
15. Kushwah B, Jadhav I, Verma HN, Geethadevi A, Parashar D,
Jadhav K (2019) Betaine accumulation suppresses the de-novo
synthesis of ectoine at a low osmotic concentration in Halomonas
sp SBS 10, a bacterium with broad salinity tolerance. Mol Biol
Rep 46(5):4779–4786. https:​ //doi.org/10.1007/s11033​ -019-04924​
-2
16. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolu-
tionary genetics analysis version 7.0 for bigger datasets. Mol Biol
Evol 33:1870–1874
17. Mukherjee S, Stamatis S, Bertsch J, Ovchinnikova G, Katta HY,
Mojica A, Chen AMI, Kyrpides CN, Reddy TBK (2018) Genomes
OnLine database (GOLD) v.7: updates and new features. Nucleic
Acids Res 47:D649. https​://doi.org/10.1093/nar/gky97​7
18. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P,
Tatusova T, Thomson N, Allen MJ, Angiuoli SV, Ashburner M,
Axelrod N, Baldauf S, Ballard S, Boore J, Cochrane G, Cole J,
Dawyndt P, De Vos P, DePamphilis C, Edwards R, Faruque N,
Feldman R, Gilbert J, Gilna P, Glöckner FO, Goldstein P, Gural-
nick R, Haft D, Hancock D et al (2008) The minimum information
about a genome sequence (MIGS) specification. Nat Biotechnol
26:541–547
19. Haft DH, DiCuccio M, Badretdin A, Brover V, Chetvernin V,
O’Neill K, Li W, Chitsaz F, Derbyshire MK, Gonzales NR,
Gwadz M, Lu F, Marchler GH, Song JS, Thanki N, Yamashita
RA, Zheng C, Thibaud-Nissen F, Geer LY, Marchler-Bauer A,
Pruitt KD (2018) RefSeq: an update on prokaryotic genome anno-
tation and curation. Nucleic Acids Res 46(1):851–860. https:​ //doi.
org/10.1093/nar/gkx10​68
20. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki
EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell
J (2016) NCBI prokaryotic genome annotation pipeline. Nucleic
Acids Res 44(14):6614–6624. https​://doi.org/10.1093/nar/gkw56​
9
21. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA,
Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ,
Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D,
Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O,
Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid
annotations using subsystems technology. BMC Genomics 9:75.
https​://doi.org/10.1186/1471-2164-9-75
22. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ,
Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thoma-
son JA, Stevens R, Vonstein V, Wattam AR, Xia F (2015) RASTtk:
a modular and extensible implementation of the RAST algorithm
for building custom annotation pipelines and annotating batches
of genomes. Sci Rep 5:8365. https​://doi.org/10.1038/srep0​8365
23. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz
T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V,
Wattam AR, Xia F, Stevens R (2014) The SEED and the Rapid
Annotation of microbial genomes using Subsystems Technology
(RAST). Nucleic Acids Res 42:D206. https:​ //doi.org/10.1093/nar/
gkt12​26
24. Vallenet D, Calteau A, Cruveiller S, Gachet M, Lajus A, Josso A,
Mercier J, Renaux A, Rollin A, Rouy Z, Roche D, Scarpelli C,
Médigue C (2017) MicroScope: an expanding and evolving inte-
grated resource for community expertise of microbial genomes.
Nucleic Acids Res 45(1):517–528. https​://doi.org/10.1093/nar/
gkw11​01
25. Woese CR, Kandler O, Wheelis ML (1990) Towards a natural
system of organisms: proposal for the domains archaea, bacteria,
and eucarya. Proc Natl Acad Sci 87:4576–4579
26. Garrity GM, Bell JA, Lilburn T (2005) Oceanospirillales ord. nov.
In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, Boone DR,
De Vos P (eds) Bergey’s Manual® of systematic bacteriology:
Molecular Biology Reports (2021) 48:1045–1053 1053
volume two the Proteobacteria part B the Gammaproteobacteria.
Springer, Boston, pp 270–323
27. Franzmann PD, Wehmeyer U, Stackebrandt E (1988) Halo-
monadaceae fam. nov., a new family of the class Proteobacteria
to accommodate the genera Halomonas and Deleya. Syst Appl
Microbiol 11:16–19
28. Vreeland RH, Litchfield CD, Martin EL, Elliot E (1980) Halo-
monas elongata, a new genus and species of extremely salt-toler-
ant bacteria. Int J Syst Evol Microbiol 30:485–495
29. Mata JA, Martinez-Canovas J, Quesada E, Bejar V (2002) A
detailed phenotypic characterisation of the type strains of Halo-
monas species. Syst Appl Microbiol 25:360–375
30. Tatusov RL, Galperin MY, Natale DA, Koonin EV (2000) The
COG database: a tool for genome-scale analysis of protein func-
tions and evolution. Nucleic Acids Res 28(1):33–36. https​://doi.
org/10.1093/nar/28.1.33
31. Kanehisa M, Goto S (2000) KEGG: Kyoto encyclopedia of genes
and genomes. Nucleic Acids Res 28:27–30
32. Kanehisa M, Sato Y, Furumichi M, Morishima K, Tanabe M
(2019) New approach for understanding genome variations in
KEGG. Nucleic Acids Res 47:D590–D595
33. Galinski EA (1995) Osmoadaptation in bacteria. Adv Microb
Physiol 37:272–328
34. Bohin JP (2000) Osmoregulated periplasmic glucans in Proteo-
bacteria. FEMS Lett 186:11–19
35. Daily OP, Debell RM, Joseph SW (1978) Superoxide dis-
mutase and catalase levels in halophilic vibrios. J Bacteriol
134(2):375–380
36. Mishra S, Imlay J (2012) Why do bacteria use so many enzymes to
scavenge hydrogen peroxide? Arch Biochem Biophys 525(2):145–
160. https​://doi.org/10.1016/j.abb.2012.04.014
37. Meyer JL, Dillard BA, Rodgers JM, Ritchie KB, Paul VJ, Teplitski
M (2015) Draft genome sequence of Halomonas meridiana R1t3
isolated from the surface microbiota of the Caribbean Elkhorn
coral Acropora palmata. Stand Genomic Sci 10:75
38. Kottemann M, Kish A, Iloanusi C, Bjork S, DiRuggiero J (2005)
Physiological responses of the halophilic archaeon Halobacterium
sp. strain NRC1 to desiccation and gamma irradiation. Extremo-
philes 9:219–227. https​://doi.org/10.1007/s0079​2-005-0437-4
39. Oren A (2015) Pyruvate: a key nutrient in hypersaline environ-
ments? Microorganisms 3(3):407–416
40. Garrity GM, Bell JA, Lilburn T. Class I (2005) Alphaproteobacte-
ria class. Nov. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s
manual of systematic bacteriology: volume two, the Proteobac-
teria part C the alpha-, beta-, delta-, and epsilonproteobacteria,
Springer , Boston , US, pp 1–574.
41. León MJ, Hoffmann T, Sánchez-Porro C, Heider J, Ventosa A,
Bremer E (2018) Compatible solute synthesis and import by the
moderate halophile Spiribacter salinus: physiology and genomics.
Front Microbiol 9:108. https:​ //doi.org/10.3389/fmicb.​ 2018.00108​
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