Revision and reannotation of the Halomonas elongata DSM 2581T genome.

The genome of the Halomonas elongata type strain DSM 2581, an industrial producer, was reevaluated using the Illumina HiSeq2500 technology. To resolve duplication-associated ambiguities, PCR products were generated and sequenced. Outside of duplications, 72 sequence corrections were required, of which 24 were point mutations and 48 were indels of one or few bases. Most of these were associated with polynucleotide stretches (poly-T stretch overestimated in 19 cases, poly-C underestimated in 15 cases). These problems may be attributed to using 454 technology for original genome sequencing. On average, the original genome sequence had only one error in 56 kb. There were 23 frameshift error corrections in the 29 protein-coding genes affected by sequence revision. The genome has been subjected to major reannotation in order to substantially increase the annotation quality.

Neutrons describe ectoine effects on water H-bonding and hydration around a soluble protein and a cell membrane.

Understanding adaptation to extreme environments remains a challenge of high biotechnological potential for fundamental molecular biology. The cytosol of many microorganisms, isolated from saline environments, reversibly accumulates molar concentrations of the osmolyte ectoine to counterbalance fluctuating external salt concentrations. Although they have been studied extensively by thermodynamic and spectroscopic methods, direct experimental structural data have, so far, been lacking on ectoine-water-protein interactions. In this paper, in vivo deuterium labeling, small angle neutron scattering, neutron membrane diffraction and inelastic scattering are combined with neutron liquids diffraction to characterize the extreme ectoine-containing solvent and its effects on purple membrane of H. salinarum and E. coli maltose binding protein. The data reveal that ectoine is excluded from the hydration layer at the membrane surface and does not affect membrane molecular dynamics, and prove a previous hypothesis that ectoine is excluded from a monolayer of dense hydration water around the soluble protein. Neutron liquids diffraction to atomic resolution shows how ectoine enhances the remarkable properties of H-bonds in water-properties that are essential for the proper organization, stabilization and dynamics of biological structures.

Potential applications of stress solutes from extremophiles in protein folding diseases and healthcare.

Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as Alzheimer's, Parkinson's and prion diseases. Drugs available on the market reduce the symptoms, but they are not a cure. Therefore, it is urgent to identify promising targets and develop effective drugs. Preservation of protein native conformation and/or inhibition of protein aggregation seem pertinent targets for drug development. Several studies have shown that organic solutes, produced by extremophilic microorganisms in response to osmotic and/or heat stress, prevent denaturation and aggregation of model proteins. Among these stress solutes, mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, diglycerol phosphate and ectoine are effective in preventing amyloid formation by Alzheimer's Aß peptide and/or α-synuclein in vitro. Moreover, mannosylglycerate is a potent inhibitor of Aß and α-synuclein aggregation in living cells, and mannosylglyceramide and ectoine inhibit aggregation and reduce prion peptide-induced toxicity in human cells. This review focuses on the efficacy of stress solutes from hyper/thermophiles and ectoines to prevent amyloid formation in vitro and in vivo and their potential application in drug development against protein misfolding diseases. Current and envisaged applications of these extremolytes in neurodegenerative diseases and healthcare will also be addressed.

A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581 T.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581(T) thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4,061,296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-L-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production.

PrfA protein of Bacillus species: prediction and demonstration of endonuclease activity on DNA.

The prfA gene product of Gram-positive bacteria is unusual in being implicated in several cellular processes; cell wall synthesis, chromosome segregation, and DNA recombination and repair. However, no homology of PrfA with other proteins has been evident. Here we report a structural relationship between PrfA and the restriction enzyme PvuII, and thereby produce models that predict that PrfA binds DNA. Indeed, wild-type Bacillus stearothermophilus PrfA, but not a catalytic site mutant, nicked one strand of supercoiled plasmid templates leaving 5'-phosphate and 3'-hydroxyl termini. This activity, much lower on linear or relaxed circular double-stranded DNA or on single-stranded DNA, is consistent with a role for this protein in chromosome segregation, DNA recombination, or DNA repair.

Base-change mutations induced by various treatments of Bacillus subtilis spores with and without DNA protective small, acid-soluble spore proteins.

Previous work has shown that spores of wild-type Bacillus subtilis are more resistant to killing by dry and wet heat, low vacuum lyophilization and hydrogen peroxide than are spores lacking the majority of their DNA protective alpha/beta-type small, acid-soluble spore proteins (SASP) (termed alpha(-)beta(-) spores). These four treatments kill alpha(-)beta(-) spores in large part by DNA damage with accompanying mutagenesis, but only dry heat kills wild-type spores by DNA damage and mutagenesis. DNA sequence analysis of nalidixic acid-resistant (nal(r)) mutants generated by these treatments has now shown that the nal(r) mutations are base changes in the gyrA gene that encodes one subunit of DNA gyrase. Analysis of the DNA sequence of the gyrA gene in a large number of nal(r) mutants also indicates that: (1) base changes induced by hydrogen peroxide and wet heat in alpha(-)beta(-) spores are similar to those in spontaneous nal(r) mutants with only a few notable differences; (2) base changes induced by dry heat in wild-type spores and low vacuum lyophilization of alpha(-)beta(-) spores are similar, and include a high level of a tandem base change seen previously only in spores treated with very high vacuum and (3) base changes induced by lyophilization and dry heat are very different from those in spontaneous mutants in wild-type and alpha(-)beta(-) spores, which exhibit only one significant difference. While the initial DNA damage generated in spores by dry heat, lyophilization or high vacuum is almost certainly different than that generated by hydrogen peroxide or wet heat, the precise nature of the DNA damage remains to be determined.

Localization of the cortex lytic enzyme CwlJ in spores of Bacillus subtilis.

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.