Methylphosphonate Degradation and Salt-Tolerance Genes of Two Novel Halophilic Marivita Metagenome-Assembled Genomes from Unrestored Solar Salterns

Aerobic bacteria that degrade methylphosphonates and produce methane as a byproduct have emerged as key players in marine carbon and phosphorus cycles. Here, we present two new draft genome sequences of the genus Marivita that were assembled from metagenomes from hypersaline former industrial salterns and compare them to five other Marivita reference genomes. Phylogenetic analyses suggest that both of these metagenome-assembled genomes (MAGs) represent new species in the genus. Average nucleotide identities to the closest taxon were <85%. The MAGs were assembled with SPAdes, binned with MetaBAT, and curated with scaffold extension and reassembly. Both genomes contained the phnCDEGHIJLMP suite of genes encoding the full C-P lyase pathway of methylphosphonate degradation and were significantly more abundant in two former industrial salterns than in nearby reference and restored wetlands, which have lower salinity levels and lower methane emissions than the salterns. These organisms contain a variety of compatible solute biosynthesis and transporter genes to cope with high salinity levels but harbor only slightly acidic proteomes (mean isoelectric point of 6.48).

Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes

Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol—∆sps, ∆ggpS, and ∆sps∆ggpS—were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis’ native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.

Halophiles

Halophiles are extremophilic salt-loving microorganisms that can survive in an extremely high level of salinity (10-30% NaCl). They belong to all three groups (i.e., bacteria, archaea, and eukaryotes). Halophiles tolerate high salt concentration due to unique cellular adaptations like salt-in strategy, compatible solute strategy, and enzyme adaptations. The chapter describes the classification, physiology, ecology, and mechanisms of adaptations and biotechnological applications of halophiles.

Integrative analysis of the salt stress response in cyanobacteria

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

Cysteinolic Acid Is a Widely Distributed Compatible Solute of Marine Microalgae

Phytoplankton rely on bioactive zwitterionic and highly polar small metabolites with osmoregulatory properties to compensate changes in the salinity of the surrounding seawater. Dimethylsulfoniopropionate (DMSP) is a main representative of this class of metabolites. Salinity-dependent DMSP biosynthesis and turnover contribute significantly to the global sulfur cycle. Using advanced chromatographic and mass spectrometric techniques that enable the detection of highly polar metabolites, we identified cysteinolic acid as an additional widely distributed polar metabolite in phytoplankton. Cysteinolic acid belongs to the class of marine sulfonates, metabolites that are commonly produced by algae and consumed by bacteria. It was detected in all dinoflagellates, haptophytes, diatoms and prymnesiophytes that were surveyed. We quantified the metabolite in different phytoplankton taxa and revealed that the cellular content can reach even higher concentrations than the ubiquitous DMSP. The cysteinolic acid concentration in the cells of the diatom Thalassiosira weissflogii increases significantly when grown in a medium with elevated salinity. In contrast to the compatible solute ectoine, cysteinolic acid is also found in high concentrations in axenic algae, indicating biosynthesis by the algae and not the associated bacteria. Therefore, we add this metabolite to the family of highly polar metabolites with osmoregulatory characteristics produced by phytoplankton.

CsrA Coordinates Compatible Solute Synthesis in Acinetobacter baumannii and Facilitates Growth in Human Urine

The opportunistic human pathogen Acinetobacter baumannii has become one of the leading causes of nosocomial infections around the world due to the increasing prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. Recently, it was found that CsrA, a global mRNA binding posttranscriptional regulator, plays a role in osmotic stress adaptation, virulence, and growth on amino acids of A. baumannii AB09-003 and 17961.

Exploring the Potential of Corynebacterium glutamicum to Produce the Compatible Solute Mannosylglycerate

The compatible solute mannosylglycerate (MG) has exceptional properties in terms of protein stabilization and protection under salt, heat, and freeze-drying stresses as well as against protein aggregation. Due to these characteristics, MG possesses large potential for clinical and biotechnological applications. To achieve efficient MG production, Corynebacterium glutamicum was equipped with a bifunctional MG synthase (encoded by mgsD and catalyzing the condensation of 3-phosphoglycerate and GDP-mannose to MG) from Dehalococcoides mccartyi. The resulting strain C. glutamicum (pEKEx3 mgsD) intracellularly accumulated about 111 mM MG (60 ± 9 mg gCDW−1) with 2% glucose as a carbon source. To enable efficient mannose metabolization, the native manA gene, encoding mannose 6-phosphate isomerase, was overexpressed. Combined overexpression of manA and mgsD from two plasmids in C. glutamicum resulted in intracellular MG accumulation of up to ca. 329 mM [corresponding to 177 mg g cell dry weight (CDW)−1] with glucose, 314 mM (168 mg gCDW−1) with glucose plus mannose, and 328 mM (176 mg gCDW−1) with mannose as carbon source(s), respectively. The product was successfully extracted from cells by using a cold water shock, resulting in up to 5.5 mM MG (1.48 g L−1) in supernatants. The two-plasmid system was improved by integrating the mgsD gene into the manA-bearing plasmid and the resulting strain showed comparable production but faster growth. Repeated cycles of growth/production and extraction of MG in a bacterial milking-like experiment showed that cells could be recycled, which led to a cumulative MG production of 19.9 mM (5.34 g L−1). The results show that the newly constructed C. glutamicum strain produces MG from glucose and mannose and that a cold water shock enables extraction of MG from the cytosol into the medium.

Influence of the compatible solute sucrose on thylakoid membrane organization and violaxanthin de-epoxidation

Main conclusion The compatible solute sucrose reduces the efficiency of the enzymatic de-epoxidation of violaxanthin, probably by a direct effect on the protein parts of violaxanthin de-epoxidase which protrude from the lipid phase of the thylakoid membrane. The present study investigates the influence of the compatible solute sucrose on the violaxanthin cycle of higher plants in intact thylakoids and in in vitro enzyme assays with the isolated enzyme violaxanthin de-epoxidase at temperatures of 30 and 10 °C, respectively. In addition, the influence of sucrose on the lipid organization of thylakoid membranes and the MGDG phase in the in vitro assays is determined. The results show that sucrose leads to a pronounced inhibition of violaxanthin de-epoxidation both in intact thylakoid membranes and the enzyme assays. In general, the inhibition is similar at 30 and 10 °C. With respect to the lipid organization only minor changes can be seen in thylakoid membranes at 30 °C in the presence of sucrose. However, sucrose seems to stabilize the thylakoid membranes at lower temperatures and at 10 °C a comparable membrane organization to that at 30 °C can be observed, whereas control thylakoids show a significantly different membrane organization at the lower temperature. The MGDG phase in the in vitro assays is not substantially affected by the presence of sucrose or by changes of the temperature. We conclude that the presence of sucrose and the increased viscosity of the reaction buffers stabilize the protein part of the enzyme violaxanthin de-epoxidase, thereby decreasing the dynamic interactions between the catalytic site and the substrate violaxanthin. This indicates that sucrose interacts with those parts of the enzyme which are accessible at the membrane surface of the lipid phase of the thylakoid membrane or the MGDG phase of the in vitro enzyme assays.