Biomedical subjects

Inmaculada Llamas

Publications and source records attributed to Inmaculada Llamas.

16 recordsLinked to original sources

PubMed2006

Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis.

We studied exopolysaccharides (EPSs) produced by Halomonas ventosae and Halomonas anticariensis, two novel species of halophilic bacteria. Under optimum environmental and nutritional conditions, H. ventosae strains Al12(T) and Al16 excreted 28.35 mg and 28.95 mg of EPS per 100 ml of culture medium (34.55 and 38.6 mg of EPS per gram of dry cell weight) respectively. The molecular masses of the polymers were about 50 kDa and their main components were glucose, mannose and galactose. They had high protein fractions and showed emulsifying activity on several hydrophobic substrates. Under optimum environmental and nutritional conditions, H. anticariensis strains FP35(T) and FP36 excreted about 29.65 and 49.95 mg of EPS per 100 ml of culture medium (43.6 and 50.95 mg of EPS per gram of dry cell weight) respectively. The molecular masses of the polymers were about 20 and 46 kDa respectively and were composed mainly of glucose, mannose and galacturonic acid. All EPSs produced solutions of low viscosity and pseudoplastic behaviour. They also had a high capacity for binding cations and incorporated considerable quantities of sulphates, which is highly unusual in bacterial polysaccharides. All strains assayed formed biofilms both in polystyrene wells and borosilicate test tubes.

Biofilms↗

PubMed2006

Halomonas maura is a physiologically versatile bacterium of both ecological and biotechnological interest.

Halomonas maura is a bacterium of great metabolic versatility. We summarise in this work some of the properties that make it a very interesting microorganism both from an ecological and biotechnological point of view. It plays an active role in the nitrogen cycle, is capable of anaerobic respiration in the presence of nitrate and has recently been identified as a diazotrophic bacterium. Of equal interest is mauran, the exopolysaccharide produced by H. maura, which contributes to the formation of biofilms and thus affords the bacterium advantages in the colonisation of its saline niches. Mauran is highly viscous, shows thixotropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immunomodulator effect in medicine. All these attributes have prompted us to make further investigations into its molecular characteristics. To date we have described 15 open reading frames (ORF's) related to exopolysaccharide production, nitrogen fixation and nitrate reductase activity among others.

Biotechnology↗

PubMed2006

A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura.

The halophilic bacterium Halomonas maura is capable of anaerobic respiration on nitrates. By insertional mutagenesis with the minitransposon Tn-5 we obtained the mutant Tc62, which was incapable of anaerobic respiration on nitrates. An analysis of the regions adjacent to the transposon allowed us to characterize the membrane-bound anaerobic-respiratory nitrate reductase narGHJI gene cluster in H. maura. We identified consensus sequences for fumarate and nitrate reductase regulator (FNR)-like protein-binding sites in the promoter regions of the nar genes and consensus sequences corresponding to the NarL binding sites upstream of the nar genes. RT-PCR analysis showed that the narGHJI operon was expressed in response to anaerobic conditions when nitrate was available as electron acceptor. This membrane-bound nitrate reductase is the only enzyme responsible for anaerobic respiration on nitrate in H. maura. In this article we discuss the possible relationship between this enzyme and a dissimilatory nitrate-reduction-to-ammonia process (DNRA) in H. maura and its role in the colonization of the rhizosphere.

Anaerobiosis↗

PubMed2005

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas.

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35(T) were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C(4)-HL), N-hexanoyl homoserine lactone (C(6)-HL), N-octanoyl homoserine lactone (C(8)-HL) and N-dodecanoyl homoserine lactone (C(12)-HL). This study suggests that quorum sensing may also play an important role in extreme environments.

4-Butyrolactone↗

PubMed2005

The moderately halophilic bacterium Halomonas maura is a free-living diazotroph.

Halomonas maura is a moderately halophilic bacterium which lives in saline soils and synthesises an exopolysaccharide known as mauran. Strain S-31T grew in a nitrogen-free medium under an N2 atmosphere; the acetylene reduction assay proved positive under specific conditions. We identified the nifH gene in this strain by using degenerate oligonucleotides designed from highly preserved gene sequences obtained from the alignment of a large number of nifH sequences from different microorganisms. Our results lead us to conclude that H. maura is capable of fixing nitrogen under microaerobic conditions.

Base Sequence↗

PubMed2005

Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain.

Two Gram-positive, endospore-forming bacterial strains, CR-502T and CR-14b, which produce surfactant molecules are described. Phenotypic tests and phylogenetic analyses showed these strains to be members of the genus Bacillus and related to the species Bacillus atrophaeus, Bacillus mojavensis, Bacillus subtilis, Bacillus vallismortis and Bacillus amyloliquefaciens, although they differ from these species in a number of phenotypic characteristics. DNA-DNA hybridization confirmed that they show less than 20 % hybridization with the above-mentioned species and therefore represent a novel species of Bacillus. The DNA G+C content is 46.4 mol% in strain CR-502T and 46.1 mol% in strain CR-14b. The main fatty acids in strain CR-502T are 15 : 0 anteiso (32.70 %), 15 : 0 iso (29.86 %) and 16 : 0 (13.41 %). The main quinone in strain CR-502T is MK-7 (96.6 %). In the light of the polyphasic evidence gathered in this study, it is proposed that these strains be classified as a novel species of the genus Bacillus, with the name Bacillus velezensis sp. nov. The type strain (CR-502T=CECT 5686T=LMG 22478T) was isolated from a brackish water sample taken from the river Vélez at Torredelmar in Málaga, southern Spain.

Bacillus↗

PubMed2005

Bacillus axarquiensis sp. nov. and Bacillus malacitensis sp. nov., isolated from river-mouth sediments in southern Spain.

Two Gram-positive, rod-shaped, endospore-forming bacteria (strains CR-119(T) and CR-95(T)) were isolated from brackish sediments in the mouth of the river Velez in Malaga, southern Spain, and subjected to a polyphasic taxonomic study. Phenotypic tests showed that these strains were related to other Bacillus species at a similarity level of less than 87.6 %. Both strains are halotolerant, aerobic, chemoheterotrophic, motile with peritrichous flagella and biosurfactant producers. Their endospores are oval, subterminal and non-deforming structures. The predominant menaquinone in both strains is MK-7. The fatty-acid profiles of both strains contain large quantities of branched and saturated fatty acids. The major fatty acids (%) are 15 : 0 anteiso (32.4), 15 : 0 iso (16.8), 17 : 0 iso (13.4), 16 : 0 (11.5) and 17 : 0 anteiso (10.2) in strain CR-119(T) and 15 : 0 anteiso (37.5), 17 : 0 iso (16.0) and 17 : 0 anteiso (15.8) in strain CR-95(T). The G + C contents of strains CR-119(T) and CR-95(T) are 41.0 and 42.5 mol%, respectively. RAPD analysis confirmed the low degree of similarity between the two strains and also amongst other Bacillus species. 16S rRNA gene analysis of strain CR-119(T) showed the highest sequence similarity to be 97.4 %, with Bacillus mojavensis and Bacillus subtilis subsp. spizizenii. In the case of strain CR-95(T), the maximum similarity value was 99.5 %, with B. mojavensis. DNA-DNA hybridization of strains CR-119(T) and CR-95(T) with the above species produced values lower than 46.9 %. Therefore, on the basis of phenotypic characteristics, phylogenetic data and genomic distinctiveness, we conclude that these Bacillus strains merit classification as novel species, for which we propose the names Bacillus axarquiensis sp. nov. (type strain CR-119(T) = CECT 5688(T) = LMG 22476(T)) and Bacillus malacitensis sp. nov. (type strain CR-95(T) = CECT 5687(T) = LMG 22477(T)).

Aerobiosis↗

PubMed2005

Halomonas almeriensis sp. nov., a moderately halophilic, exopolysaccharide-producing bacterium from Cabo de Gata, Almería, south-east Spain.

Halomonas almeriensis sp. nov. is a Gram-negative non-motile rod that was isolated from a saltern in the Cabo de Gata-Níjar wildlife reserve in Almería, south-east Spain. It is moderately halophilic, capable of growth at concentrations of 5-25% w/v sea-salt mixture, the optimum being 7.5% w/v. It is chemo-organotrophic and strictly aerobic, produces catalase but not oxidase, does not produce acid from any sugar and does not synthesize hydrolytic enzymes. The most notable difference between this micro-organism and other Halomonas species is that it is very fastidious in its use of a carbon source. It forms mucoid colonies due to the production of an exopolysaccharide. Its G+C content is 63.5 mol%. A comparison of 16S rRNA gene sequences confirmed its relationship to Halomonas species. The most closely related species is Halomonas halmophila with 95.8% similarity between their 16S rRNA gene sequences. DNA-DNA hybridization with H. halmophila is 10.1%. Its major fatty acids are 18:1omega7c, 16:0, 16:1omega7c/15:0 iso 2-OH, 12:0 3-OH, 12:0, 11-methyl 18:1omega7c and 10:0. The proposed name is Halomonas almeriensis sp. nov., with strain M8(T) (=CECT 7050(T)=LMG 22904(T)) as the type strain.

Aerobiosis↗

PubMed2005

Alteromonas hispanica sp. nov., a polyunsaturated-fatty-acid-producing, halophilic bacterium isolated from Fuente de Piedra, southern Spain.

Strain F-32T, which produces exopolysaccharides and contains polyunsaturated fatty acids, was isolated from a hypersaline water sample collected from Fuente de Piedra (southern Spain). Phylogenetic analyses indicated conclusively that the strain in question belonged to the genus Alteromonas. Phenotypic tests showed that it could be assigned to the genus Alteromonas although it had a number of distinctive characteristics: it is moderately halophilic, growing best with 7.5-10 % w/v NaCl; it grows at 4 degrees C and produces H2S; it does not grow with d-cellobiose, d-fructose, d-galactose, d-glucose or lactose as sole sources of carbon and energy; and its fatty-acid profile is typical of Alteromonas but it also contains a large amount of an unusual acid with three double bonds [18 : 3omega6c (6, 9, 12); 5.01 %, w/v]. The major isoprenoid quinone is Q8. The DNA G+C content is 46.3 mol%. The phylogenetic, phenotypic and genetic properties of strain F-32T place it within a novel species, for which the name Alteromonas hispanica sp. nov. is proposed. The type strain is F-32T (=CECT 7067T=LMG 22958T).

Alteromonas↗

PubMed2005

Palleronia marisminoris gen. nov., sp. nov., a moderately halophilic, exopolysaccharide-producing bacterium belonging to the 'Alphaproteobacteria', isolated from a saline soil.

Strain B33(T) is a moderately halophilic, exopolysaccharide-producing, Gram-negative, non-motile rod isolated from a hypersaline soil bordering a saline saltern on the Mediterranean seaboard in Murcia (Spain). The bacterium is chemoheterotrophic and strictly aerobic. It contains a pink pigment but does not synthesize bacteriochlorophyll a. It requires 0.66 M Na+, 0.1 M Mg2+ and 0.1 M K+ for optimum growth. It does not produce acid from carbohydrates. It cannot grow with carbohydrates, organic acids, sugars, alcohols or amino acids as sole sources of carbon and energy. Its major fatty-acids are 18 : 1omega7c (68.9 %) and 19 : 0 cyclo omega8c (12.8 %). The sole respiratory lipoquinone found in strain B33T is ubiquinone-10. The DNA G+C content is 64.2 mol%. 16S rRNA gene sequence comparisons show that the isolate is a member of the Roseobacter clade within the class 'Alphaproteobacteria'. The similarity values with Roseivivax halodurans and Roseivivax halotolerans are 88.2 and 88.0 % respectively and 92.2 % with Salipiger mucosus. DNA-DNA hybridization values with these species are <30 %. In the light of the polyphasic evidence gathered in this study it is proposed that the isolate be classified as a novel genus and species with the name Palleronia marisminoris gen. nov., sp. nov. The proposed type strain is strain B33T (=CECT 7066T=LMG 22959T).

Bacteriochlorophyll A↗

PubMed2005

epsABCJ genes are involved in the biosynthesis of the exopolysaccharide mauran produced by Halomonas maura.

The moderately halophilic strain Halomonas maura S-30 produces a high-molecular-mass acidic polymer (4.7 x 10(6) Da) composed of repeating units of mannose, galactose, glucose and glucuronic acid. This exopolysaccharide (EPS), known as mauran, has interesting functional properties that make it suitable for use in many industrial fields. Analysis of the flanking regions of a mini-Tn5 insertion site in an EPS-deficient mutant of H. maura, strain TK71, led to the identification of five ORFs (epsABCDJ), which form part of a gene cluster (eps) with the same structural organization as others involved in the biosynthesis of group 1 capsules and some EPSs. Conserved genetic features were found such as JUMPstart and ops elements, which are characteristically located preceding the gene clusters for bacterial polysaccharides. On the basis of their amino-acid-sequence homologies, their putative hydropathy profiles and the effect of their mutations, it is predicted that EpsA (an exporter-protein homologue belonging to the OMA family) and EpsC (a chain-length-regulator homologue belonging to the PCP family) play a role in the assembly, polymerization and translocation of mauran. The possibility that mauran might be synthesized via a Wzy-like biosynthesis system, just as it is for many other polysaccharides, is also discussed. This hypothesis is supported by the fact that EpsJ is homologous with some members of the PST-exporter-protein family, which seems to function together with each OMA-PCP pair in polysaccharide transport in Gram-negative bacteria, transferring the assembled lipid-linked repeating units from the cytoplasmic membrane to the periplasmic space. Maximum induction of the eps genes is reached during stationary phase in the presence of 5 % (w/v) marine salts.

Amino Acid Sequence↗

PubMed2004

Halomonas ventosae sp. nov., a moderately halophilic, denitrifying, exopolysaccharide-producing bacterium.

Halomonas ventosae sp, nov. includes three moderately halophilic, exopolysaccharide-producing strains isolated from saline soils in Jaén (south-eastern Spain). These strains can grow anaerobically using either nitrate or nitrite as terminal electron acceptor and hydrolyse both tyrosine and phenylalanine. Their G+C content varies between 72.6 and 74.3 mol%. The affiliation of the isolates with the genus Halomonas was confirmed by 16S rRNA gene sequence comparison. DNA-DNA hybridization shows 70.4-82.7 % relatedness among the three strains. Nevertheless, their relatedness is less than 43 % compared to related reference strains. The proposed type strain for Halomonas ventosae is strain Al12(T) (=CECT 5797(T)=DSM 15911(T)). It grows best at 8 % (w/v) sea salts and requires the presence of Na(+). Its major fatty acids are 18 : 1 omega7c, 16 : 0, 16 : 1 omega7c, and 15 : 0 iso 2-OH. The predominant respiratory lipoquinone found in strain Al12(T) is ubiquinone with nine isoprene units (Q-9).

Base Composition↗

PubMed2004

Use of Sinorhizobium meliloti as an indicator for specific detection of long-chain N-acyl homoserine lactones.

Population-density-dependent gene expression in gram-negative bacteria involves the production of signal molecules characterized as N-acyl homoserine lactones (AHLs). The synthesis of AHLs by numerous microorganisms has been identified by using biosensor strains based on the Agrobacterium tumefaciens and Chromobacterium violaceum quorum-sensing systems. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti is rapidly becoming a model organism for the study of quorum sensing. This organism harbors at least three different quorum-sensing systems (Sin, Mel, and Tra), which play a role in its symbiotic relationship with its host plant, alfalfa. The Sin system is distinguished among them for the production of long-chain AHLs, including C(18)-HL, the longest AHL reported so far. In this work, we show that construction of a sinI::lacZ transcriptional fusion results in a strain that detects long-chain AHLs with exquisite sensitivity. Overexpression of the SinR regulator protein from a vector promoter increases its sensitivity without loss of specificity. We also show that the resulting indicator strain can recognize long-chain AHLs produced by unrelated bacteria such as Paracoccus denitrificans and Rhodobacter capsulatus. This S. meliloti indicator strain should serve as a tool for the specific detection of long-chain AHLs in new systems.

4-Butyrolactone↗

PubMed2003

Megaplasmids in Gram-negative, moderately halophilic bacteria.

We have discovered that many Halomonas species harbour large extrachromosomal DNA elements. Using currently available protocols it is technically very difficult to identify large plasmids in bacteria, and even more so when they are coated in mucous polysaccharide. We used culture conditions suitable for both halophilic and halophilic exopolysaccharide-producing bacteria and applied a modified gel electrophoresis method to locate and visualise the megaplasmids. Almost all the species of Halomonas studied harbour two plasmids of about 70 kb and 600 kb and some species carry other smaller extrachromosomal DNA elements. The common presence of these megaplasmids may well be related to the survival strategies of the bacteria in their special surroundings.

DNA, Bacterial↗

PubMed2003

Identification and characterization of the carAB genes responsible for encoding carbamoylphosphate synthetase in Halomonas eurihalina.

Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology. Strain F2-7 of H. eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain. By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS. S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines. We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.

Amino Acid Sequence↗

PubMed2002

Analysis of the genome of the moderate halophile Halomonas eurihalina.

We have studied the genomic organization of Halomonas eurihalina, a moderately halophilic bacterium that produces an anionic exopolysaccharide with a potentially wide range of applications in industry. To estimate the genome size of strain F2-7, large restriction fragments of genomic DNA were separated by pulsed-field gel electrophoresis. According to the average size of the resolved restriction fragments, the genome size of H. eurihalina strain F2-7 was estimated to be around 2500 kb. The physical map of the chromosome for the endonuclease SwaI has been constructed. The F2-7 strain has two plasmids, pVE1 and pVE2, and in this study we have isolated three new plasmids, pVE3, pVE4, and pVE5, of 5.3, 16, and 6.5 kb, respectively, from strains H-1, H-217, and H-236 of H. eurihalina. We have characterized these plasmids and constructed their physical maps. Curing experiments produced no evidence for the involvement of these plasmids in the expression of the mucous phenotype.

Blotting, Southern↗