Genome Analysis Provides Insights into the Osmoadaptation Mechanisms of Halomonas titanicae

Abstract

Here, we report the osmoadaptation strategies adopted by the halotolerant species Halomonas titanicae BH1(T) inferred from genome sequence analysis. BH strain was isolated in 2010 from a rusticated sample collected in 1991 from the wreck of the Titanic, genome deposited in the database under the accession number (CP059082.1). It showed a high salt tolerance ranging from 0.5 to 25% NaCl (w/v) (optimal growth at 10% NaCl) with no growth in the absence of NaCl. The phylogenomic analysis showed that the BH1 strain is more closely related to the Halomonas sedementi QX-2, a strain isolated from deep-sea sediments. The RAST (Rapid Annotation using Subsystem Technology) annotation revealed divergent mechanisms involved in the primary and secondary response to osmotic stress citing protein implicated in potassium transport, periplasmic glucan synthesis, choline and betaine upake system, biosynthesis of glycine-betaine, ectoine, and proline. These findings provide an overview of the osmoadaptive mechanisms of H. titanicae BH1, and could offer helpful information to future biotechnological applications like osmolyte synthesis and related applications.

1. Introduction

Halomonas titanicae BH1 type strain was isolated from rusticlesis collected from the RMS Titanic wreck site [1]. It is a Gram-negative, heterotrophic, aerobic rod, and motile bacterium. It belongs to Halomonadaceae family [2]. Currently, there are 119 Halomonas species (https://lpsn.dsmz.de/) and interestingly, most of them are isolated from marine or hypersaline habitats [3]. They are slightly to moderately halophilic and oligotrophic organisms [4]. H. titanicae BH1 strain showed an ability to grow in media with 0.5–25% NaCl with no growth in the absence of NaCl [1], which reflects an ability to tolerate osmotic stress fluctuations. Generally, to cope with osmotic stress halophilic microorganisms deploy multiple strategies to regulate their internal osmotic pressure through the accumulation of organic or inorganic compatible solutes [5, 6, 7] citing (i) The primary response to osmotic stress: the early response consists of water influx either in or out of the cells through dedicated membrane channels called aquaporin. This mechanism is coupled with a salt-in strategy [5]. These changes are detected by osmosensors. It was suggested that these osmosensors are the aquaporins themselves and the stretch-activated/mechanosensitive channels (sensitive to cell turgor and intracellular tension) in eukaryotic cells [8]; (ii) the salt-in strategy: this strategy involves the accumulation of KCl. The exclusion of Na + from the cytoplasm is achieved through a Na+/H+ antiport (NhaC) located at the cytoplasmic membrane. Generally, K+ ions move in passively via a trkH system under the impulse of the membrane pressure. The main source of energy for the expulsion of Na+ and the accumulation of K+ in cells is the electrochemical potential difference of protons. This potential difference is due to both the transport of electrons in the respiratory chain, as well as the proton gradient formed during ATP synthesis by membrane ATPases and K+ antiporters (KdpABC). The influx of cations must be balanced by an equivalent number of anions. The movement of anions such as chloride is coupled to the energy of the membrane potential. It penetrates through a Na+/Cl− symport [5, 7]; (iii) The compatible osmolytes accumulation strategy: it employs the exclusion of intracellular salt ions simultaneously synthesizing or accumulating high concentrations of compatible solutes [5]. Compatible solutes can be sugars (sucrose, trehalose) and their derivatives (sulfotrehalose, glucosylglycerol), some amino acids and derivatives (proline, glutamic acid, glutamine, and glycine betaine), ectoine (and derivatives), or polyalcohols (glycerol, arabitol, and mannitol) [5]. These osmoprotectants can be synthesized or taken from the external environment without interfering with cellular metabolism. These solutes help to maintain turgor, pressure, cell volume, and electrolyte concentration. They can be released into the external environment either by other producing microorganisms following a drop in osmolarity or by decaying plant, animal, or bacterial cells [5, 7, 9, 10].

Here, we attempt to unravel the mechanisms of osmoadaption of adopted by H. titanicae BH1(T) strain to cope with the osmotic stress, based on in silico whole genome sequence analyses.

2. Material and method

2.1 H. titanicae BH1 genome sequence analysis

H. titanicae BH1 bacterium was isolated from a sample of rusticles collected from the RMS Titanic wreck site [1]. BH1 is Gram-negative cell, heterotrophic, aerobic rod, and motile by peritrichous flagella. Phylogenetically this organism belongs to the Gammaproteobacteria class within Halomonadaceae family [1]. The genome sequence of BH1 strain was retrieved from GenBank database under the accession number NZ_AOPO00000000 [11]. Cluster of Orthologous Groups (COG) functional categories was performed based on database of clusters of ortholgous genes.

2.2 Phylogenomic analysis of H. titanicae BH1

The whole-genome-based taxonomic analysis was performed by type strain genome server (TYGS) (at https://tygs.dsmz.de) [12]. The phylogenomic tree assessment was carried out using FastME algorithm [13] from the Genome BLAST Distance Phylogeny (GBDP). All pairwise-genome comparisons were conducted using Genome BLAST Distance Phylogeny approach (GBDP) under the algorithm “coverage” and distance formula d5 [14]. The trees were rooted at the midpoint [15]. Branch supports were inferred from 100 pseudo-bootstrap replicates.

2.3 Identification of genes involved in halotolerance in H. titanicae BH1

Genome sequence annotation was uploaded in Rapid Annotations using Subsystems Technology server (RAST, http://rast.nmpdr.org/) to identify potential genes and subsystems involved in the osomoadaptation pathways.

3. Results and discussions

3.1 Genome features and phylogenomic analysis

H. titanicae BH1 has been deposited in several culture collections as ATCC BAA-1257, CECT 7585, JCM 16411, and LMG 25388. It was able to grow in media with 0.5 to 25% NaCl and no growth occurs in the absence of NaCl [1]. The draft genome of H. titanicae BH1 (NZ_AOPO00000000.1) [11] includes 5,28 Mb with a G + C content of 54.6% and is composed of 4,759 putative protein-coding genes (Figure 1) [11].

Figure 1.

The COG functional categories repartition of H. titanicaea BH1.

Whole genome-based phylogenetic tree conducted by TYGS illustrated that strain BH1 forms a distinct clade with and forms a distinct cluster with Halomonas sedimenti QX-2’(NZ_JACCGK000000000.1) (Figure 2). H. sedimenti QX-2 is a halophilic gram-negative bacterium that was isolated from deep-water sediments in the southwest Indian Ocean at a depth of 2699 m [16].

Figure 2.

Whole-genome-based phylogenetic tree highlighting the position of H. titanicaea strain BH1 to other closely related bacterial taxa. Trees are generated with fastme 2.1.6.1. The numbers above 60% from 100 replications. The tree was rooted at the midpoint.

The distribution of predicted genes based on RAST annotation among the subsystem database (Figure 3) revealed that subsystems with “Amino Acids and Derivatives (414)” and “Carbohydrates (281)” were the most represented subsystem features. In addition, the annotated subsystem features denoted 107 genes associated with “Stress response.”

Figure 3.

Subsystem distribution of H. titanicaea strain BH1 genome based on RAST annotation server.

3.2 Genes related to osmoadaptation strategies in H. titanicae strain BH1

3.2.1 Primary responses to osmotic stress

3.2.1.1 Salt-in strategy: potassium transport

The Trk-type transporter system: Mining of the BH1 genome sequence showed three Trk-type transporters (Table 1) (i) TrkA system potassium uptake protein, serves to control the activity of the potassium translocating subunit (ii) TrkH system potassium uptake protein, exhibits only a low affinity for K+ (iii) TrkI system potassium uptake protein, the main K+ transporter in osmotically adapted cells, exhibits medium affinity for K+. Several studies reported the role of K⁺ uptake via Trk-like transporter in the primary response to osmotic stress in bacteria [17, 18].

Subsystem	Gene	Predicted protein
Primary response to osmotic stress
Trk-like transporter	trkA	Potassium transporter TrkA
	TrkI	Trk system potassium uptake protein
	TrkH	Trk system potassium uptake protein
kdpABC operon	KdpE	transcriptional regulatory protein
	KdpD	Histidine kinase
Synthesis of osmoregulated periplasmic glucans	mdoH	Glucan biosynthesis glucosyltransferase H
	mdoG	Glucan biosynthesis protein G precursor
Secondary response to osmotic stress—use of osmolytes
Ectoine biosynthesis and uptake	Asd	L-aspartate-β-semialdehyde dehydrogenase
	Ask	L-aspartate kinase
	ectA	L-2,4-diaminobutyric acid acetyltransferase
	ectB	Diaminobutyrate-pyruvate aminotransferase
	ectC	L-ectoine synthase
	ectD	Ectoine hydroxylase
	TeaA	Ectoine-binding periplasmic protein
	TeaC	Ectoine TRAP transporter large permease protein
	TeaD	TRAP-T-associated universal stress protein
	TeaB	Ectoine TRAP transporter small permease protein
	eutD/doeB	Xaa-Pro dipeptidase
Choline and Betaine Uptake and Betaine Biosynthesis	BetT2	Choline transporter
	betI
	BetL	Glycine betaine transporter
	betA	Choline dehydrogenase (EC 1.1.99.1)
	betB	betaine aldehyde dehydrogenase
	betC	Choline-sulfatase (EC 3.1.6.6)
	betI	HTH-type transcriptional regulator BetI
	DgcA	dimethylglycine demethylation protein
	Sarcosine oxydase	SoxBDAG
	OpuD	Glycine betaine transporter
	opuA	Glycine betaine ABC transport system, glycine betaine-binding protein
	proU	L-proline glycine betaine binding ABC transporter protein
	GbuC	Glycine betaine/carnitine transport binding protein
	ATPase	Quaternary-amine-transporting ATPase
	YehY	Glycine betaine uptake system permease protein
	YehW	Glycine betaine uptake system permease protein
	BCCT transporter	L-carnitine/gamma-butyrobetaine antiporter
Proline synthesis and uptake	proZ	proline/glycine betaine ABC transporter permease
	proA	glutamate semialdehyde dehydrogenase
	proJ	the glutamate 5-kinase
	proH	Glutamate-5-semialdehyde dehydrogenase
	proV	proline/glycine betaine ABC transporter permease
	Proline racemase	Proline, 4-hydroxyproline uptake and utilization
	PutR for proline utilization,	Proline, 4-hydroxyproline uptake and utilization

Table 1.

Genes and encoded proteins implicated in halotolerance in Halomonas titanicae BH1.

Trk-type transporter systems have an important role in controlling the flux of potassium (K+) ions into cells. Generally, K+ ions enter passively via a uniport system (trkH) under the impulse of the membrane potential. The difference in potential is mainly due to the transport of electrons in the respiratory chain, as well as to the proton gradient generated during ATP synthesis via membrane ATPases and K+ antiport transporters (KdpABC). Here, BH1 genome sequence contains three genes (i) KdpE: transcriptional regulatory protein involved in the regulation of the kdp operon; (ii) Histidine kinase KdpD, involved in the regulation of the kdp operon; and (iii) KdpD may function as a membrane-associated protein kinase that phosphorylates KdpE in response to environmental signals [19].

3.2.1.2 Periplasmic glucans synthesis

Periplasmic glucans synthesis (OPG) in H. titanicae strain BH1 is carried out by the products of two genes, mdoG and mdoH (Table 1). MdoH catalyzes the production of linear β-1,2 polyglucose chains from the precursor UDP-glucose. MdoG function is unclear. Earlier studies showed that in some gram-negative bacteria, OPGs are synthesized at low osmolarity, which suggests that they play a part in the initial response to osmotic stress [20].

3.2.2 Secondary response to osmotic stress: biosynthesis and uptake of osmolytes

Osmolytes are organic molecules with low molecular weight, which can accumulate in cells at elevated concentrations without interfering with cell function, thanks to their high solubility and non-interaction with proteins [5, 7]. Analysis of the BH1 genome sequence revealed several osmolytes implicated in osmodapatation.

3.2.2.1 Ectoine biosynthesis and uptake

The complete biosynthetic pathway of ectoine/hydroxyectoine was identified in BH1 genome sequence (Table 1, Figure 4). In fact, the biosynthesis of ectoine from aspartate is catalyzed successively by five enzymes: L-aspartate kinase (Ask), L-aspartate-β-semialdehyde dehydrogenase (Asd), L-2,4-diaminobutyrate aminotransferase (EctB), L-2,4-diaminobutyrate acetyltransferase (EctA), and ectoine synthase (EctC). In addition, ectoine hydroxylase (EctD), which catalyzes the conversion of ectoine to hydroxyectoine, has been identified. The genes encoding EctB, EctA, and EctC are typically present as a gene cluster (ectABC) in some cases with ectD [21, 22].

Figure 4.

Biosynthetic pathway for ectoines from aspartate in H. titanicaea strain BH1. The enzymes involved are Aspartate kinase (Ask), Aspartate semialdehyde dehydrogenase (Asd), L-diaminobutyric acid transaminase (EctB), L-diaminobutyric acid acetyl transferase (EctA), ectoine synthase (EctC), and ectoine hydroxylase (EctD).

In addition, the TeaABCD gene cluster, involved in the uptake of ectoine as a response to an osmotic shock, has also been identified (Figure 4, Table 1). TeaABC, is an osmoregulated transporter that catalyzes the uptake of ectoine and hydroxyectoine as a response to osmotic shock. TeaD (ATP-binding protein) negatively regulates the activity of the tripartite ATP-independent periplasmic ectoine transport system (TeaABC) [23].

3.2.2.2 Choline and glycine betaine uptake and biosynthesis

Osmotic adaptation can also be achieved by the uptake of betaine/choline osmolytes available in the environment. It should be noted that the uptake strategy is favored as it is significantly less energetically expensive than the de novo synthesis [7].

3.2.2.3 Uptake of choline and glycine betaine

Choline uptake is achieved by the BetT2 osmo-dependent choline transporter, transcribed via the betIBA operon (Table 1). For glycine uptake, several membrane transporters have been identified, notably (i) the glycine betaine transporter (BetL), a member of the BCCT family of transporters, displays a high affinity for glycine betaine uptake, (ii) OpuAB, a permease protein for the glycine betaine transport system. It is part of the binding protein-dependent transport family, (iii) glycine betaine transporter OpuD, is a single-component BCCT-type transporter, (iv) glycine betaine/carnitine transport binding protein GbuC, part of the ABC transporter complex GbuABC involved in glycine betaine and carnitine uptake, (v) quaternary-amine-transporting ATPase: involved in a multicomponent binding-protein-dependent transport system for glycine betaine, (vi) glycine betaine uptake system permease protein (YehY), part of ABC transporter complex involved in low-affinity glycine betaine uptake, (vii) glycine betaine uptake system permease protein (YehW), part of an ABC transporter complex involved in low-affinity glycine betaine uptake, and (viii) L-carnitine/gamma-butyrobetaine antiporter, which catalyzes the exchange of L-carnitine for gamma-butyrobetaine and related betaines, it belongs to the BCCT transporter family [24].

3.2.2.4 Glycine betaine biosynthesis

In silico whole genome analysis reveals that the biosynthesis of glycine betaine (GB) can be achieved via choline oxydation and then glycine demethylation (Table 1, Figure 5) [24]. The reaction is as follows: choline is first oxidized into GB by a pair of enzymes from the same operon, choline oxidase (BetA) and betaine aldehyde dehydrogenase (BetB). Then, the GB is demethylated via the oxygenase activity of GbcAB enzyme (Glycine betaine demethylase subunit A and B) to dimethylglycine (DMG). Next, the heterodimeric flavin-linked oxidoreductase DgcA (dimethylglycine demethylation protein) catalyzes the conversion of DMG to sarcosine. Finally, oxidative demethylation of sarcosine is conducted by a heterotetrameric enzyme (SoxBDAG) that generates glycine. Genes of this catabolic pathway have been recognized based on comparative genomic analysis. In fact, the same pathway was identified in the Pseudomonas aeruginosa species [25].

Figure 5.

Schematic representation of choline catabolic pathway of H. titanicae BH1.

3.2.2.5 Proline biosynthesis and uptake

Glutamate is considered as the primary precursor amino acid for proline synthesis. The biosynthesis is catalyzed by three enzymes (Table 1, Figure 6) [26, 27]: First, glutamate 5-kinase (proJ), catalyzes the transfer of a phosphate group to glutamate to form L-glutamate 5-phosphate. Then, glutamate semialdehyde dehydrogenase (proA), catalyzes the NADPH-dependent reduction of L-glutamate 5-phosphate to L-glutamate 5-semialdehyde. Next, Pyrroline-5-carboxylate reductase (proH) catalyzes the reduction of 1-pyrroline-5-carboxylate (PCA) to L-proline. The uptake of proline is under osmotic control and is mediated by the osmoregulated glycine betaine transport systems and 4-hydroxyproline uptake and utilization (Table 1).

Figure 6.

Proline biosynthesis from glutamate in H. titanicae BH1.

4. Conclusion

In conclusion, the analysis of the whole genome sequence gave us an insight into the osmotic adaptation mechanisms adopted by the H. titanicae BH1 strain face to osmotic stress. In fact, two main strategies were identified, the salt-in-cytoplasm and the solute accumulation or biosynthesis strategies. Genes implicated in several pathways were identified as well. This funding may facilitate an in-depth understanding of the transcription or the regulation of the metabolic pathways under stressful conditions.