Quantitative RNA-seq Analysis Unveils Osmotic and Thermal Adaptation Mechanisms Relevant for Ectoine Production in Chromohalobacter salexigens

Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to “oxidative- and protein-folding- stress responses,” “modulation of respiratory chain and related components,” and “ion homeostasis.” A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in “protein-folding-stress response,” suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyectoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of ectoines, in C. salexigens.España Ministerio de Economía y Competitividad BIO2015-63949-RJunta de Andalucía P11-CVI-729

Draft Genome of Scalindua rubra, Obtained from the Interface Above the Discovery Deep Brine in the Red Sea, Sheds Light on Potential Salt Adaptation Strategies in Anammox Bacteria

Several recent studies have indicated that members of the phylum Planctomycetes are abundantly present at the brine-seawater interface (BSI) above multiple brine pools in the Red Sea. Planctomycetes include bacteria capable of anaerobic ammonium oxidation (anammox). Here, we investigated the possibility of anammox at BSI sites using metagenomic shotgun sequencing of DNA obtained from the BSI above the Discovery Deep brine pool. Analysis of sequencing reads matching the 16S rRNA and hzsA genes confirmed presence of anammox bacteria of the genus Scalindua. Phylogenetic analysis of the 16S rRNA gene indicated that this Scalindua sp. belongs to a distinct group, separate from the anammox bacteria in the seawater column, that contains mostly sequences retrieved from high-salt environments. Using coverage- and composition-based binning, we extracted and assembled the draft genome of the dominant anammox bacterium. Comparative genomic analysis indicated that this Scalindua species uses compatible solutes for osmoadaptation, in contrast to other marine anammox bacteria that likely use a salt-in strategy. We propose the name Candidatus Scalindua rubra for this novel species, alluding to its discovery in the Red Sea

Isolasi, Karakterisasi, dan Kloning Gen Penyandi α α α α α-Amilase Bakteri Halofil Moderat asal Bledug Kuwu

A moderately halophilic bacteria, BK-05, was isolated from Bledug Kuwu, a saline terrestrial area at Central Java. Based on partial sequence of 16S-rRNA gene, the isolate was closely related to Halobacillus litoralis. This isolate showed amylolytic activity when grown on saline media [15% (w/v) NaCl] suplemented with starch. A pair of primer was designed based on the sequence of amyH gene from Halomonas meridiana and Pseudoalteromonas haloplanktis. PCR amplification using these primers showed three DNA bands with each size approximately 1.50, 1.00, and 0.75 kb. Partial DNA sequencing analysis based on its deduced protein sequence revealed that the 1.50 kb band was closely related to the sequence of metalloprotease from Bacillus subtilis (approximately 54.3% identity in 184 amino acid overlap). Southern hybridization analysis showed that the 1.50 kb fragment was located within a 4.0 kb fragment of BamHI, 4.8 kb of EcoRI, 4.3 kb of HindIII, and 4.0 kb of XhoI digestion of BK-05 genomic DNA, respectively

Archaeal Communities in a Heterogeneous Hypersaline-Alkaline Soil

In this study the archaeal communities in extreme saline-alkaline soils of the former lake Texcoco, Mexico, with electrolytic conductivities (EC) ranging from 0.7 to 157.2 dS/m and pH from 8.5 to 10.5 were explored. Archaeal communities in the 0.7 dS/m pH 8.5 soil had the lowest alpha diversity values and were dominated by a limited number of phylotypes belonging to the mesophilic Candidatus Nitrososphaera. Diversity and species richness were higher in the soils with EC between 9.0 and 157.2 dS/m. The majority of OTUs detected in the hypersaline soil were members of the Halobacteriaceae family. Novel phylogenetic branches in the Halobacteriales class were detected in the soil, and more abundantly in soil with the higher pH (10.5), indicating that unknown and uncharacterized Archaea can be found in this soil. Thirteen different genera of the Halobacteriaceae family were identified and were distributed differently between the soils. Halobiforma, Halostagnicola, Haloterrigena, and Natronomonas were found in all soil samples. Methanogenic archaea were found only in soil with pH between 10.0 and 10.3. Retrieved methanogenic archaea belonged to the Methanosarcinales and Methanomicrobiales orders. The comparison of the archaeal community structures considering phylogenetic information (UniFrac distances) clearly clustered the communities by pH

Whole-genome sequencing and genome-scale metabolic modeling of Chromohalobacter canadensis 85B to explore its salt tolerance and biotechnological use

Salt tolerant organisms are increasingly being used for the industrial production of high‐value biomolecules due to their better adaptability compared to mesophiles. Chromohalobacter canadensis is one of the early halophiles to show promising biotechnology potential, which has not been explored to date. Advanced high throughput technologies such as whole‐genome sequencing allow in‐depth insight into the potential of organisms while at the frontiers of systems biology. At the same time, genome‐scale metabolic models (GEMs) enable phenotype predictions through a mechanistic representation of metabolism. Here, we sequence and analyze the genome of C. canadensis 85B, and we use it to reconstruct a GEM. We then analyze the GEM using flux balance analysis and validate it against literature data on C. canadensis. We show that C. canadensis 85B is a metabolically versatile organism with many features for stress and osmotic adaptation. Pathways to produce ectoine and polyhydroxybutyrates were also predicted. The GEM reveals the ability to grow on several carbon sources in a minimal medium and reproduce osmoadaptation phenotypes. Overall, this study reveals insights from the genome of C. canadensis 85B, providing genomic data and a draft GEM that will serve as the first steps towards a better understanding of its metabolism, for novel applications in industrial biotechnology

Haloferax volcanii for biotechnology applications: challenges, current state and perspectives

© 2019, The Author(s). Haloferax volcanii is an obligate halophilic archaeon with its origin in the Dead Sea. Simple laboratory culture conditions and a wide range of genetic tools have made it a model organism for studying haloarchaeal cell biology. Halophilic enzymes of potential interest to biotechnology have opened up the application of this organism in biocatalysis, bioremediation, nanobiotechnology, bioplastics and the biofuel industry. Functionally active halophilic proteins can be easily expressed in a halophilic environment, and an extensive genetic toolkit with options for regulated protein overexpression has allowed the purification of biotechnologically important enzymes from different halophiles in H. volcanii. However, corrosion mediated damage caused to stainless-steel bioreactors by high salt concentrations and a tendency to form biofilms when cultured in high volume are some of the challenges of applying H. volcanii in biotechnology. The ability to employ expressed active proteins in immobilized cells within a porous biocompatible matrix offers new avenues for exploiting H. volcanii in biotechnology. This review critically evaluates the various application potentials, challenges and toolkits available for using this extreme halophilic organism in biotechnology

Electricity generation and ethanol production using iron-reducing, haloalkaliphilic bacteria

Microbial life in extreme environments has been studied primarily for their metabolic activities. Very few commercial or industrial applications have been known from these systems. In this study, the metabolic pathways and properties of bacteria from a haloalkaliphilic environment of Soap Lake, Washington were employed in two research applications related to energy production. In the first study, the bacterial cultures that were known to reduce iron (III) were used in a Microbial Fuel Cell (MFC) reactor. Iron reducing bacteria have shown to shunt their electrons on to the surface of a carbon electrode of an MFC, and the electrons can be harvested to produce current...The second study focused on applying the fermentative mechanism of the bacterial cultures from Soap Lake. Bacteria were found to use glycerol as the substrate and ferment it to ethanol. Glycerol is a major by-product of biodiesel industry and therefore bacterial fermentation of this glycerol to ethanol would help to manage the waste as well as produce a value-added product --Abstract, page iv

Sequence, organization, and genes characteristics of ORFs identified in a metagenomic DNA fragment from microbial community of the deep brine environment of Atlantis II in the Red Sea.

Microbial communities that reside in different natural habitats, particularly those of extreme environments, constitute a rich source for novel industrial enzymes and bioactive compounds. Until the advent of metagenomics technique, extreme environments represented a locked area with huge genetic repertoire that remained unexplored. The Atlantis II brine pool of the Red Sea (ATII) is one of such unexplored extreme environment. The lower part of this pool, the lower convective layer (LCL), has a pH of 5.3, high temperature (68C), elevated concentration of toxic heavy metals, and extreme salinity (26% salt). To understand the metabolic and the physiological properties of proteins and enzymes that contribute to the survival of microorganisms in this extreme and hostile environment, the structure and characteristics of their genes should be determined. Metagenomics approach helped in this task through two different techniques: 1) mass sequencing of environmental DNA by high throughput sequencing technique such as pyrosequencing technique; 2) sequencing of environmental DNA fragments from metagenomic fosmid library. The advantage of the first approach is that it produces massive number of reads that can be assembled into long contigs. Its disadvantage is that the majority of the contigs are chimeric i.e. assembled from reads belong to genomes of different microbial species. The second technique has an advantage of establishing the sequence of a contiguous piece of genomic DNA of around 30 to 40 kb‚ that most probably is not a chimeric. The major disadvantages however are the high cost of the sequencing process, it involves elaborate steps, and it has a limited output of nucleotide sequences. In this work we sequenced a contiguous fragment of DNA from the microbial community of the ATII-LCL environment and presenting the structural and potential function of its annotated genes. Interestingly, out of the 39 identified ORFs, 10 ORFs (25%) have no matches in the database. The structure and the function of the potential annotated genes are presented and discussed. In addition, we were able to assembled 28.378 kb out of 33.819 kb of the insert in the recombinant fosmid. The unassembled 5.441 kb is most probably due to the detection of characteristic patterns of low complexity regions, simple repeats as well as gene duplication exists at the end of the assembled sequence

Surprising prokaryotic and eukaryotic diversity, community structure and biogeography of Ethiopian soda lakes

Soda lakes are intriguing ecosystems harboring extremely productive microbial communities in spite of their extreme environmental conditions. This makes them valuable model systems for studying the connection between community structure and abiotic parameters such as pH and salinity. For the first time, we apply high-throughput sequencing to accurately estimate phylogenetic richness and composition in five soda lakes, located in the Ethiopian Rift Valley. The lakes were selected for their contrasting pH, salinities and stratification and several depths or spatial positions were covered in each lake. DNA was extracted and analyzed from all lakes at various depths and RNA extracted from two of the lakes, analyzed using both amplicon- and shotgun sequencing. We reveal a surprisingly high biodiversity in all of the studied lakes, similar to that of freshwater lakes. Interestingly, diversity appeared uncorrelated or positively correlated to pH and salinity, with the most “extreme” lakes showing the highest richness. Together, pH, dissolved oxygen, sodium- and potassium concentration explained approximately 30% of the compositional variation between samples. A diversity of prokaryotic and eukaryotic taxa could be identified, including several putatively involved in carbon-, sulfur- or nitrogen cycling. Key processes like methane oxidation, ammonia oxidation and ‘nitrifier denitrification’ were also confirmed by mRNA transcript analyses