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Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata.

ABSTRACT: For osmoadaptation the halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. H. elongata does not rely entirely on synthesis but can accumulate ectoine by uptake from the surrounding environment with the help of the osmoregulated transporter TeaABC. Disruption of the TeaABC-mediated ectoine uptake creates a strain that is constantly losing ectoine to the medium. However, the efflux mechanism of ectoine in H. elongata is not yet understood. H. elongata possesses four genes encoding mechanosensitive channels all of which belong to the small conductance type (MscS). Analysis by qRT-PCR revealed a reduction in transcription of the mscS genes with increasing salinity. The response of H. elongata to hypo- and hyperosmotic shock never resulted in up-regulation but rather in down-regulation of mscS transcription. Deletion of all four mscS genes created a mutant that was unable to cope with hypoosmotic shock. However, the knockout mutant grew significantly faster than the wildtype at high salinity of 2 M NaCl, and most importantly, still exported 80% of the ectoine compared to the wildtype. We thus conclude that a yet unknown system, which is independent of mechanosensitive channels, is the major export route for ectoine in H. elongata.

SUBMITTER: Vandrich J

PROVIDER: S-EPMC7174268 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata.

Vandrich Jasmina J Pfeiffer Friedhelm F Alfaro-Espinoza Gabriela G Kunte Hans Jörg HJ

Extremophiles : life under extreme conditions 20200407 3

For osmoadaptation the halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. H. elongata does not rely entirely on synthesis but can accumulate ectoine by uptake from the surrounding environment with the help of the osmoregulated transporter TeaABC. Disruption of the TeaABC-mediated ectoine uptake creates a strain that is constantly losing ectoine to the medium. However, the efflux mechanism of ectoine in H. elongata is not yet unders ...[more]

PMID: 32266565

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Project description:The halophilic γ-proteobacterium Halomonas elongata DSM 2581 T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed.

Project description:The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13.

Project description:BackgroundEctoine and its derivative 5-hydroxyectoine are cytoprotectants widely synthesized by microorganisms as a defense against the detrimental effects of high osmolarity on cellular physiology and growth. Both ectoines possess the ability to preserve the functionality of proteins, macromolecular complexes, and even entire cells, attributes that led to their description as chemical chaperones. As a consequence, there is growing interest in using ectoines for biotechnological purposes, in skin care, and in medical applications. 5-Hydroxyectoine is synthesized from ectoine through a region- and stereo-specific hydroxylation reaction mediated by the EctD enzyme, a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenases. This chemical modification endows the newly formed 5-hydroxyectoine with either superior or different stress- protecting and stabilizing properties. Microorganisms producing 5-hydroxyectoine typically contain a mixture of both ectoines. We aimed to establish a recombinant microbial cell factory where 5-hydroxyectoine is (i) produced in highly purified form, and (ii) secreted into the growth medium.ResultsWe used an Escherichia coli strain (FF4169) defective in the synthesis of the osmostress protectant trehalose as the chassis for our recombinant cell factory. We expressed in this strain a plasmid-encoded ectD gene from Pseudomonas stutzeri A1501 under the control of the anhydrotetracycline-inducible tet promoter. We chose the ectoine hydroxylase from P. stutzeri A1501 for our cell factory after a careful comparison of the in vivo performance of seven different EctD proteins. In the final set-up of the cell factory, ectoine was provided to salt-stressed cultures of strain FF4169 (pMP41; ectD (+)). Ectoine was imported into the cells via the osmotically inducible ProP and ProU transport systems, intracellularly converted to 5-hydroxyectoine, which was then almost quantitatively secreted into the growth medium. Experiments with an E. coli mutant lacking all currently known mechanosensitive channels (MscL, MscS, MscK, MscM) revealed that the release of 5-hydroxyectoine under osmotic steady-state conditions occurred independently of these microbial safety valves. In shake-flask experiments, 2.13 g l(-1) ectoine (15 mM) was completely converted into 5-hydroxyectoine within 24 h.ConclusionsWe describe here a recombinant E. coli cell factory for the production and secretion of the chemical chaperone 5-hydroxyectoine free from contaminating ectoine.

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Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata.

Publications

Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata.

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