Aims: The aim of this study was to determine the diversity of moderately halophilic bacteria with hydrolase activities. Methods and Results: Screening bacteria from different hypersaline environments in South Spain led to the isolation of a total of 122 moderately halophilic bacteria able to produce different hydrolases (amylases, DNases, lipases, proteases and pullulanases). These bacteria are able to grow optimally in media with 5-15% salts and in most cases up to 20-25% salts. In contrast to strains belonging to previously described species, that showed very little hydrolase activities, environmental isolates produced a great variety of hydrolases. These strains were identified as members of the genera: Salinivibrio (55 strains), Halomonas (25 strains), Chromohalobacter (two strains), Bacillus-Salibacillus (29 strains), Salinicoccus (two strains) and Marinococcus (one strain), as well as eight nonidentified isolates. Conclusions: Moderately halophilic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases and pullulanases. Significance and Impact of the Study: Although most culture collection strains are not able to produce hydrolases, it has been shown that environmental isolates can produce these potentially biotechnological important enzymes.

A total of 26 proteolytic moderate halophiles were isolated and characterized. Most isolates were members of the genus Salinivibrio (16 strains), while others were identified as Bacillus (4 strains), Salinicoccus (2 strains), or members of the gamma-Proteobacteria (4 strains). Strain CP76 was selected as the best producer of an extracellular protease, designated CP1, and was used for further studies. Sequence analysis of the 16S rRNA gene in addition to phenotypic tests led to the placement of this organism in the genus Pseudoalteromonas. Maximal protease production was detected at the end of the exponential growth phase. This CP1 protease was purified and biochemically characterized, showing optimal activity at 55 degrees C, pH 8.5, and high tolerance to a wide range of NaCl concentrations (0 to 4 M NaCl). The most interesting features of this enzyme are its moderate thermoactivity, its activity at a range of pH values (6-10), and, especially, its salt tolerance (optimal activity at 7.5% total salts). The purified protease has a molecular mass of 38 kDa, and the N-terminal amino acid sequence determined showed similarity to metalloproteases previously described. The protease activity was strongly inhibited by EDTA, PMSF, and Pefabloc. No significant inhibition was detected with E-64, bestatin, chymostatin, or leupeptin. These results suggest that Pseudoalteromonas sp. strain CP76 produces an extracellular metalloprotease moderately thermotolerant and stable at high salt concentrations.

A group of moderately halophilic bacteria able to degrade aromatic organic compounds contaminating hypersaline habitats in southern Spain have been isolated and characterized. The taxonomic position of these strains was determined using phenotypic, phylogenetic and genotypic methods. The G+C content of their DNA ranged from 61?0 to 62?9 mol%. DNA-DNA hybridization studies showed that they constitute a genospecies, having DNA-DNA hybridization values of 90-100 %. Analysis of the complete 16S rRNA gene sequence revealed a high level of similarity with members of the genus Halomonas, sharing 98 % 16S rRNA gene sequence similarity with the type strains of Halomonas salina and Halomonas halophila. However, phenotypic differences and the low level of DNA-DNA hybridization suggest the placement of these strains as a novel species within the genus Halomonas. The name Halomonas organivorans sp. nov. is proposed, with strain G-16.1 T (=CECT 5995 T =CCM 7142 T ) as the type strain. This novel species of Halomonas is characterized by its ability to use a wide range of organic compounds (benzoic acid, p-hydroxybenzoic acid, cinnamic acid, salicylic acid, phenylacetic acid, phenylpropionic acid, phenol, p-coumaric acid, ferulic acid and p-aminosalicylic acid), and it could be useful for the decontamination of polluted saline habitats.

There is growing interest in the development and optimization of bioremediation processes to deal with environments with high salinity that are contaminated with aromatic compounds. To estimate the diversity of moderately halophilic bacteria that could be used in such processes, enrichments were performed based on growth with a variety of aromatic compounds including phenol as a model pollutant. A group of bacteria that were able to grow over a wide range of salt concentrations were isolated, with the majority of these assigned to the genus Halomonas using phenotypic features and 16S rRNA sequences comparison. PCR amplification with degenerate primers revealed the presence in these isolates of genes encoding ring-cleaving enzymes in the beta-ketoadipate pathway for aromatic catabolism: catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. Furthermore, the activity of these two enzymes was detected in the newly described species Halomonas organivorans. Together, these studies indicate that moderately halophilic bacteria have the potential to catabolize aromatic compounds in environments with high salinity.

In the course of a screening programme in hypersaline habitats of southern Spain to isolate halophilic bacteria that are able to produce different extracellular enzymes, a novel, moderately halophilic bacterium (strain SM19 T ) that displays lipolytic activity has been isolated and characterized. Strain SM19T is a Gram-negative rod that grows optimally in culture media that contain 7?5 % NaCl. The DNA G+C content was 57?0 mol%. According to phenotypic and genotypic data, this strain was assigned to the genus Marinobacter. However, 16S rDNA sequence similarity between strain SM19 T and species of the genus Marinobacter was <96?7 %; this value is sufficiently low to propose its designation as a novel species. In addition, DNA-DNA hybridization with reference strains of close phylogenetic relatives was between 11 and 19 %. On the basis of these data, the inclusion of strain SM19 T in the genus Marinobacter as a novel species is proposed, with the name Marinobacter lipolyticus sp. nov. The type strain of the novel species is SM19 T (=DSM 15157Isolation and characterization of extracellular hydrolytic enzymes that show optimal activity at different salt concentrations constitutes an interesting research field with potential biotechnological applications. Availability of such enzymes would facilitate different industrial processes that require activity at high salt concentrations.Moderately halophilic bacteria have adapted to live in a wide range of salt concentrations (3-15 % NaCl) and constitute an interesting group of micro-organisms that could be used as a source of such salt-adapted enzymes (Ventosa et al., 1998). A screening programme to detect hydrolytic activities, such as protease, amylase, cellulase, pullulanase and lipase, has recently been performed in different hypersaline environments of southern Spain (Sánchez-Porro et al., 2003). This study revealed a wide diversity of moderately halophilic bacteria with the potential to hydrolyse a range of structurally non-related polymers. Among the isolates, 207 moderately halophilic bacteria that display lipolytic activity have been detected. Applications of lipolytic enzymes, which comprise mainly esterases and lipases, are widely found in the food, detergents, pharmaceutical and chemical industries (Jaeger et al., 1999; Pandey et al., 1999). A large number of microbial lipolytic enzymes has been identified and characterized to date; however, no lipolytic enzymes have so far been characterized from moderate halophiles. Taxonomic/phylogenetic studies performed on these halophilic, lipolytic enzyme producers accommodate them within different genera, such as Salinivibrio, Halomonas, Chromohalobacter, Salibacillus, Marinococcus and Marinobacter. In this study, the most promising isolate among the lipolytic producers, strain SM19 T , was assigned to the genus Marinobacter. This genus was proposed by Gauthier et al. (1992), belongs to the c-subclass of the class Proteobacteria and, at the time of writing, includes two species with validly published names: Marinobacter...

The genetic organization of the left edge (ty/€DHFJ region) of the tylosin biosynthetic gene cluster from Streptomyces fradiae has been determined. Sequence analysis of a 12.9 kb region has revealed the presence of 11 ORFs, 10 of them belonging to the biosynthetic cluster. The putative functions of the proteins encoded by these genes are as follows: peptidase (ORF1, d d d ) , tylosin resistance determinant (ORFZ, tlrB), glycosyltransferase (ORF3, ty//U), methyltransferase (ORF4, tyl€), ketoreductase (ORF5, tylD), ferredoxin (ORF6, tylH2), cytochrome P450 (ORF7, tylH7), methyltransferase (ORF8, tylF), epimerase (ORF9, tylJ), acyl-CoA oxidase (ORF10, tylP) and receptor of regulatory factors (ORF11, tylQ). The functional identification of the genes in the proposed tylosin biosynthetic pathway has been deduced by database searches and previous genetic complementation studies performed with tylosin idiotrophic mutants blocked a t various stages in tylosin biosynthesis. The tlrB gene has been shown to be useful as a tylosin resistance marker in Streptomyces liwidans, Streptomyces parvulus and Streptomyces coelicolor and the effect of tylF on macrocin depletion has been confirmed. A pathway for the biosynthesis of 6-deoxy-~-allose, the unmethylated mycinose precursor, involving the genes tylD, tylJ and tylN is proposed.

The genetic organization of the region upstream of the car gene of the clavulanic acid biosynthetic gene cluster of Streptomyces clavuligerus has been determined. Sequence analysis of a 121 kb region revealed the presence of 10 ORFs whose putative functions, according to database searches, are discussed. Three co-transcriptional units are proposed : ORF10-11, ORF12-13 and ORF15-16-17-18. Potential transcriptional terminators were identified downstream of ORF11 (fd ) and ORF15. Targeted disruption of ORF10 (cyp) gave rise to transformants unable to produce clavulanic acid, but with a considerably higher production of cephamycin C. Transformants inactivated at ORF14 had a remarkably lower production of clavulanic acid and similar production of cephamycin C. Significant improvements of clavulanic acid production, associated with a drop in cephamycin C biosynthesis, were obtained with transformants of S. clavuligerus harbouring multiple copies of plasmids carrying different constructions from the ORF10-14 region. This information can be used to guide strain improvement programs, blending random mutagenesis and molecular cloning, to optimize the yield of clavulanic acid.

The phylogenetic positions of six Ebrio 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 K costicola forms a monophyletic branch that is distinct from other Ebrio 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 I/. costicofu from species of the genus Kbrio and other species belonging to the gamma subclass of the Proteobacteria and indicates that K costicofu should be placed in a new and separate genus. The name Salinivibrio costicofu 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).Vibrio costicola is a moderately halophilic bacterium that was originally isolated from salted foods (29) and grows optimally in media containing 10% (wt/vol) salts (7). This species has been isolated frequently from salted meats and brines (8,28), and it has been determined only recently that V. costicola is a normal inhabitant of hypersaline environments (7,19,32). In fact, V. costicola is the predominant organism in saltern pond waters that have intermediate salt concentrations (concentrations between 10 and 15%) (26). V. costicola has been also isolated from other saline habitats, such as saline soils, but in lower proportions (24). Until recently, only a few moderately halophilic species had been described, and V. costicola was widely used by researchers as a model for the study of the physiological features of obligate halophiles. Thus, in many respects, I ? costicola has been considered the representative model for studies of moderately halophilic bacteria and has been used as the moderately halophilic organism in which osmoregulatory and other physiological mechanisms have been studied (15,16).A recent study of isolates obtained from hypersaline habitats and four strains isolated from cured meats demonstrated that V. costicola is widely distributed in hypersaline aquatic environments, and an emended description of this species was proposed by Garcia et al. (7). In addition, DNA-DNA homology studies revealed the genotypic homogeneity of representative strains of this species (ll), and this species was shown to be not closely related to other Vibrio species (3, 11). The data in these studies suggested that I/: costicola should be placed in a separate genus (3).Recently, Kita-Tsukamoto et al. (14) compared partial 16s rRNA sequences (approximately 600 nucleotides) of a large number of Vibrio species, and the resulting data also demonstrated that K costicola is not related to other Vibrio species or related organisms. While the manuscript of this paper was being prepared, Ruimy et al. (27) analysis of these authors also demonstrated that V. costicola clusters outside the main bo...