Joaquín J. Nieto

Biotechnological applications and potentialities of halophilic microorganisms.

Halophilic microorganisms are found as normal inhabitants of highly saline environments and thus are considered extremophiles. They are mainly represented, but not exclusively, by the halobacteria (extremely halophilic aerobic Archaea), the moderate halophiles (Bacteria and some methanogens) and several eukaryotic algae. These extremophilic microorganisms are already used for some biotechnological processes, for example halobacteria are used for the production of bacteriorhodopsin, and the alga Dunaliella is used in the commercial production of β-carotene. Several other present or potential applications of halophiles are reviewed, including the production of polymers (polyhydroxyalcanoates and polysaccharides), enzymes, and compatible solutes, and the use of these extremophiles in enhanced oil recovery, cancer detection, drug screening and the biodegradation of residues and toxic compounds.

Ectoines in cell stress protection: uses and biotechnological production.

Microorganisms produce and accumulate compatible solutes aiming at protecting themselves from environmental stresses. Among them, the wide spread in nature ectoines are receiving increasing attention by the scientific community because of their multiple applications. In fact, increasing commercial demand has led to a multiplication of efforts in order to improve processes for their production. In this review, the importance of current and potential applications of ectoines as protecting agents for macromolecules, cells and tissues, together with their potential as therapeutic agents for certain diseases are analyzed and current theories for the understanding of the molecular basis of their biological activity are discussed. The genetic, biochemical and environmental determinants of ectoines biosynthesis by natural and engineered producers are described. The major limitations of current bioprocesses used for ectoines production are discussed, with emphasis on the different microorganisms, environments, molecular engineering and fermentation strategies used to optimize the production and recovery of ectoines. The combined application of both bioprocess and metabolic engineering strategies, allowing a deeper understanding of the main factors controlling the production process is also stated. Finally, this review aims to summarize and update the state of the art in ectoines uses and applications and industrial scale production using bacteria, emphasizing the importance of reactor design and operation strategies, together with the metabolic engineering aspects and the need for feedback between wet and in silico work to optimize bioproduction.

Characterization of the Genes for the Biosynthesis of the Compatible Solute Ectoine in the Moderately Halophilic Bacterium Halomonas elongata DSM 3043

The ectoine synthesis genes of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been precisely located in a 2.8-kb EcoRI region of a cosmid clone previously isolated (CANOVAS, D., VARGAS, C., IGLESIAS-GUERRA, F., CSONKA, L. N., RHODES, D., VENTOSA, A., NIETO, J. J.: Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes. J. Biol. Chem. 272, 25794-25801, 1997). This region was sequenced and three open reading frames were found corresponding to the genes ectA (encoding the diaminobutyric acid acetyl transferase), ectB (encoding the diaminobutyric acid aminotransferase) and ectC (encoding the ectoine synthase). These three genes were able to restore the salt tolerance of two H. elongata mutants defective in the synthesis of ectoine (strains CHR62 and CHR63). However, the H. elongata ectoine synthesis genes did not confer to Escherichia coli the ability to synthesize ectoine. Transposon insertion in the salt-sensitive mutant strain CHR63 was exactly mapped within the ectC gene. Moreover, sequences homologous to the H. elongata ect region have been found in a number of moderately halophilic bacteria belonging to the genera Halomonas and Chromohalobacter.

Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes Halomonas elongata DSM 3043 and ATCC 33174.

Two strains that were originally isolated and characterized as members of the moderately halophilic species Halomonas elongata, strains DSM 3043 (= 1H11) and ATCC 33174 (= 1H15), were studied in detail. Their complete 16S rRNA sequences were determined and, when compared to sequences available from the databases, they showed a close phylogenetic relationship to Chromohalobacter marismortui. In addition, DNA-DNA hybridization experiments showed that both strains are members of the same species, but their DNA relatedness to the type strains of Halomonas elongata, ATCC 33173T, and Chromohalobacter marismortui, ATCC 17056T, is very low. Phenotypically, the two strains showed very similar features, related to those of Chromohalobacter, but clear differences were found between these two strains and Chromohalobacter marismortui. On the basis of these data, it is proposed that Halomonas elongata DSM 3043 and ATCC 33174 should be included in a new species of the genus Chromohalobacter, Chromohalobacter salexigens sp. nov. The type strain is DSM 3043T (= ATCC BAA-138T = CECT 5384T = CCM4921T = CIP106854T = NCIMB 13768T).

Phylogenetic inferences and taxonomic consequences of 16S ribosomal DNA sequence comparison of Chromohalobacter marismortui, Volcaniella eurihalina, and Deleya salina and reclassification of V. eurihalina as Halomonas eurihalina comb. nov.

The phylogenetic positions of the moderately halophilic bacteria Chromohalobacter marismortui, Volcaniella eurihalina, and Deleya salina were determined by PCR amplification of rRNA genes and direct sequencing. The resulting data were compared with data for other bacteria obtained from 16S rRNA sequence databases. C. marismortui, V. eurihalina, and D. salina clustered phylogenetically within the gamma subclass of the Proteobacteria and are closely related to other species on the Halomonas-Deleya branch. C. marismortui belongs in the family Halomonadaceae and has the characteristic 16S rRNA signatures defined for this family, including the distinctive cytosine residue at position 486 found in all members of the Halomonadaceae. V. eurihalina is closely related to the type species of the genus Halomonas, Halomonas elongata, and we formally propose that V. eurihalina should be transferred to the genus Halomonas as Halomonas eurihalina comb. nov. The type strain of this species is strain F9-6 (= ATCC 49336). D. salina is not as closely related to other species belonging to the Halomonas-Deleya complex, but is more closely related to Halomonas elongata than to Deleya aquamarina, the type species of the genus Deleya. A polyphasic approach will be necessary to determine the natural taxonomic positions of the species belonging to the genera Halomonas and Deleya, as well as C. marismortui, V. eurihalina, Halovibrio variabilis, and Paracoccus halodenitrificans.

Isolation and Characterization of Salt-sensitive Mutants of the Moderate Halophile Halomonas elongata and Cloning of the Ectoine Synthesis Genes

The moderate halophile Halomonas elongata Deustche Sammlung für Mikroorganismen 3043 accumulated ectoine, hydroxyectoine, glutamate, and glutamine in response to osmotic stress (3 m NaCl). Two Tn1732-induced mutants, CHR62 and CHR63, that were severely affected in their salt tolerance were isolated. Mutant CHR62 could not grow above 0.75 m NaCl, and CHR63 did not grow above 1.5m NaCl. These mutants did not synthesize ectoine but accumulated ectoine precursors, as shown by 13C NMR and mass spectroscopy. Mutant CHR62 accumulated low levels of diaminobutyric acid, and mutant CHR63 accumulated high concentrations of N-γ-acetyldiaminobutyric acid. These results suggest that strain CHR62 could be defective in the gene for diaminobutyric acid acetyltransferase (ectB), and strain CHR63 could be defective in the gene for the ectoine synthase (ectC). Salt sensitivity of the mutants at 1.5–2.5 m NaCl could be partially corrected by cytoplasmic extracts of the wild-type strain, containing ectoine, and salt sensitivity of strain CHR62 could be partially repaired by the addition of extracts of strain CHR63, which contained N-γ-acetyldiaminobutyric acid. This is the first evidence for the role of N-γ-acetyldiaminobutyric acid as osmoprotectant. Finally, a cosmid from the H. elongata genomic library was isolated which complemented the Ect− phenotype of both mutants, indicating that it carried at least the genes ectB and ectC of the biosynthetic pathway of ectoine.

The ectD Gene, Which Is Involved in the Synthesis of the Compatible Solute Hydroxyectoine, Is Essential for Thermoprotection of the Halophilic Bacterium Chromohalobacter salexigens

The halophilic bacterium Chromohalobacter salexigens synthesizes and accumulates compatible solutes in response to salt and temperature stress. (13)C-nuclear magnetic resonance analysis of cells grown in minimal medium at the limiting temperature of 45 degrees C revealed the presence of hydroxyectoine, ectoine, glutamate, trehalose (not present in cells grown at 37 degrees C), and the ectoine precursor, Ngamma-acetyldiaminobutyric acid. High-performance liquid chromatography analyses showed that the levels of ectoine and hydroxyectoine were maximal during the stationary phase of growth. Accumulation of hydroxyectoine was up-regulated by salinity and temperature, whereas accumulation of ectoine was up-regulated by salinity and down-regulated by temperature. The ectD gene, which is involved in the conversion of ectoine to hydroxyectoine, was isolated as part of a DNA region that also contains a gene whose product belongs to the AraC-XylS family of transcriptional activators. Orthologs of ectD were found within the sequenced genomes of members of the proteobacteria, firmicutes, and actinobacteria, and their products were grouped into the ectoine hydroxylase subfamily, which was shown to belong to the superfamily of Fe(II)- and 2-oxoglutarate-dependent oxygenases. Analysis of the ectoine and hydroxyectoine contents of an ectABC ectD mutant strain fed with 1 mM ectoine or hydroxyectoine demonstrated that ectD is required for the main ectoine hydroxylase activity in C. salexigens. Although in minimal medium at 37 degrees C the wild-type strain grew with 0.5 to 3.0 M NaCl, with optimal growth at 1.5 M NaCl, at 45 degrees C it could not cope with the lowest (0.75 M NaCl) or the highest (3.0 M NaCl) salinity, and it grew optimally at 2.5 M NaCl. The ectD mutation caused a growth defect at 45 degrees C in minimal medium with 1.5 to 2.5 M NaCl, but it did not affect growth at 37 degrees C at any salinity tested. With 2.5 M NaCl, the ectD mutant synthesized 38% (at 37 degrees C) and 15% (at 45 degrees C) of the hydroxyectoine produced by the wild-type strain. All of these data reveal that hydroxyectoine synthesis mediated by the ectD gene is thermoregulated and essential for thermoprotection of C. salexigens.

Analysis of 16S rRNA gene sequences of Vibrio costicola strains: description of Salinivibrio costicola gen. nov., comb. nov.

The phylogenetic positions of six Vibrio costicola strains were determined by direct sequencing and analysis of their PCR-amplified 16S ribosomal DNAs. A comparative analysis of the sequence data revealed that the moderate halophile V. costicola forms a monophyletic branch that is distinct from other Vibrio species and from moderately halophilic species of other genera. These results complement phenotypic and genotypic data determined previously. The molecular evidence, together with several phenotypic differences, distinguishes V. costicola from species of the genus Vibrio and other species belonging to the gamma subclass of the Proteobacteria and indicates that V. costicola should be placed in a new and separate genus. The name Salinivibrio costicola gen. nov., comb. nov. is proposed for this bacterium. The guanine-plus-cytosine content of the DNA is 49.4 to 50.5 mol%. The type strain of S. costicola is strain NCIMB 701 (= ATCC 33508).

Unravelling the adaptation responses to osmotic and temperature stress in Chromohalobacter salexigens, a bacterium with broad salinity tolerance

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. Osmoprotection is achieved by the accumulation of compatible solutes either by transport (betaine, choline) or synthesis (mainly ectoine and hydroxyectoine). Ectoines can play additional roles as nutrients and, in the case of hydroxyectoine, in thermotolerance. A supplementary solute, trehalose, not present in cells grown at 37°C, is accumulated at higher temperatures, suggesting its involvement in the response to heat stress. Trehalose is also accumulated at 37°C in ectoine-deficient mutants, indicating that ectoines suppress trehalose synthesis in the wild-type strain. The genes for ectoine (ectABC) and hydroxyectoine (ectD, ectE) production are arranged in three different clusters within the C. salexigens chromosome. In order to cope with changing environment, C. salexigens regulates its cytoplasmic pool of ectoines by a number of mechanisms that we have started to elucidate. This is a highly complex process because (i) hydroxyectoine can be synthesized by other enzymes different to EctD (ii) ectoines can be catabolized to serve as nutrients, (iii) the involvement of several transcriptional regulators (σS, σ32, Fur, EctR) and hence different signal transduction pathways, and (iv) the existence of post-trancriptional control mechanisms. In this review we summarize our present knowledge on the physiology and genetics of the processes allowing C. salexigens to cope with osmotic stress and high temperature, with emphasis on the transcriptional regulation.

The α-amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization

Two types of Tn1732-induced mutants defective in extracellular amylase activity were isolated from the moderate halophile Halomonas meridiana DSM 5425. Type I mutants displayed amylase activity in the periplasm, and were unable to use any of the carbon sources tested, including starch and its hydrolysis product maltose. The type II mutant was affected in the gene responsible for the synthesis of the extracellular α-amylase. This gene (amyH) was isolated by functional complementation of mutant II and sequenced. The deduced protein (AmyH) showed a high degree of homology to a proposed family of α-amylases consisting of enzymes from Alteromonas (Pseudoalteromonas) haloplanktis, Thermomonospora curvata, streptomycetes, insects and mammals. AmyH contained the four highly conserved regions in amylases, as well as a high content of acidic amino acids. The amyH gene was functional in the moderate halophile Halomonas elongata and, when cloned in a multicopy vector, in Escherichia coli. AmyH is believed to be the first extracellular-amylase-encoding gene isolated from a moderate halophile, a group of extremophiles of great biotechnological potential. In addition, H. meridiana and H. elongata were able to secrete the thermostable α-amylase from Bacillus licheniformis, indicating that members of the genus Halomonas are good candidates for use as cell factories to produce heterologous extracellular enzymes.