Search

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C. salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as

Background The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. Results We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not c

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 temperatur

Background The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Results Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect − mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L−1) and volumetric ectoine yields (up to 4.21 g L−1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic

Uso de la ectoína o sus derivados como agentes antiinflamatorios y/o antioxidantes en enfermedades causadas por la formación de agregados proteicos. La invención propone el uso de la ectoína y sus derivados como agentes antiagregantes y/o antioxidantes en células, preferiblemente neuronales. La ectoína y sus derivados se proponen así como compuestos de utilidad en la elaboración de medicamentos para la prevención y/o tratamiento de la muerte celular causada por daño oxidativo y/o la formación de agregados proteicos, preferiblemente inflamación en respuesta a dichos agregados, más preferiblemente para la prevención y/o tratamiento de enfermedades neurodegenerativas, tales como Alzheimer. La invención también proporciona las concentraciones óptimas de tales compuestos, así como una posible vía de administración.

La emergencia de una nueva enfermedad infecciosa es algo que sucede desde el principio de los tiempos y puede verse influida por numerosos factores que condicionan su aparición y la forma en la que afecta a la población. Para ilustrar cómo surgen estas nuevas enfermedades infecciosas, puede tomarse el ejemplo de la familia de los coronavirus. Estos virus de ARN, prácticamente desconocidos hasta hace poco tiempo, tienen una distribución global y suelen causar síntomas respiratorios leves en el ser humano. Sin embargo, hay dos excepciones que pueden poner en peligro la vida del enfermo: los virus SARS-CoV y MERS-CoV, causantes del SARS y el MERS, respectivamente. El SARS (Síndrome Respiratorio Agudo Severo), cuyo primer caso apareció en China en noviembre de 2002, tuvo un inicio explosivo pero de corta duración, no habiéndose dado más casos desde entonces. Eso no indica que no haya sido importante, ya que causó más de 800 muertes y 8.000 enfermos, siendo reconocida como la primera pandemia del siglo XXI y originando un cambio fundamental en la forma de llevar a cabo el control de las infecciones. El MERS (Síndrome Respiratorio de Oriente Medio) es aún más reciente, surgiendo en 2012, y con brotes de aparición periódica desde entonces. Constituye actualmente una gran amenaza sanitaria por su elevada tasa de mortalidad, cercana al 40%, y la gravedad de la sintomatología que origina. Pese a todo, la baja tasa de transmisibilidad del virus MERS-CoV hace que, en cinco años, el número total de enfermos sea cercano a los 1.800, con más de 600 fallecidos documentados. Estos dos virus, y la forma en la que se ha tratado su aparición tanto desde el campo científico y epidemiológico, constituyen un gran ejemplo a seguir en la forma de tratar y controlar las enfermedades infecciosas futuras que amenacen a nuestra sociedad.

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 M NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-13C]-, [2-13C]-, [6- 13C]-, and [U-13C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria

Hydroxyectoine overproduction by the natural producer Chromohalobacter salexigens is presented in this study. Genetically engineered strains were constructed that at low salinity coexpressed, in a vector derived from a native plasmid, the ectoine (ectABC) and hydroxyectoine (ectD) genes under the control of the ectA promoter, in a temperature-independent manner. Hy- droxyectoine production was further improved by increasing the copies of ectD and using a C. salexigens genetic background unable to synthesize ectoines.

Chromohalobacter salexigens is one of nine currently known species of the genus Chromohalobacter in the family Halomonadaceae. It is the most halotolerant of the so-called ‘moderately halophilic bacteria’ currently known and, due to its strong euryhaline phenotype, it is an established model organism for prokaryotic osmoadaptation. C. salexigens strain 1H11T and Halomonas elongata are the first and the second members of the family Halomonadaceae with a completely sequenced genome. The 3,696,649 bp long chromosome with a total of 3,319 protein-coding and 93 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2004

In this study, the connection between iron homeostasis and the osmostress response in the halophile Chromohalobacter salexigens was investigated. A decrease in the requirement for both iron and histidine and a lower level of siderophore synthesis were observed at high salinity, and these findings were correlated with a lower protein content in salt-stressed cells. A six-gene operon (cfuABC-fur-hisI-orf6 operon) located downstream of the ectABC ectoine synthesis genes was characterized. A fur strain (in which the ferric iron uptake regulator Fur was affected) had the Mn resistance phenotype typical of fur mutants, was deregulated for siderophore production, and displayed delayed growth under iron limitation conditions, indicating that fur encodes a functional iron regulator, hisI was essential for histidine synthesis, which in turn was necessary for siderophore production. Fur boxes were found in the promoters of the cfuABC-fur-hisI-orf6 and ectABC operons, suggesting that Fur directly interacts with DNA in these regions. Fur mediated the osmoregulated inhibition of cfuABC-fur-hisI-orf6 operon expression by iron and functioned as a positive regulator of the ectABC genes under high-salinity conditions, linking the salt stress response with iron homeostasis. Excess iron led to a higher cytoplasmic hydroxyectoine content, suggesting that hydroxyectoine protects against the oxidative stress caused by iron better than ectoine. This study provides the first evidence of involvement of the iron homeostasis regulator Fur as part of the complex circuit that controls the response to osmotic stress in halophilic bacteria

The halophilic bacterium Chromohalobacter salexigens synthesizes and accumulates compatible solutes in response to salt and temperature stress. 13C-nuclear magnetic resonance analysis of cells grown in minimal medium at the limiting temperature of 45°C revealed the presence of hydroxyectoine, ectoine, glutamate, trehalose (not present in cells grown at 37°C), and the ectoine precursor, N')'-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 tem- perature. 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°C the wild-type strain grew with 0.5 to 3.0 M NaCl, with optimal growth at 1.5 M NaCl, at 45°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°C in minimal medium with 1.5 to 2.5 M NaCl, but it did not affect growth at 37°C at any salinity tested. With 2.5 M NaCl, the ectD mutant synthesized 38% (at 37°C) and 15% (at 45°C) of the hydroxyectoine produced by the

Durante las dos últimas décadas, nuestro Equipo de Investigación se ha centrado en el estudio de los microorganismos halófilos, tanto extremos como moderados. En general, las bacterias halófilas moderadas, aquellas que crecen ópticamente en medios que contienen un 3-15% de sal, han sido objeto de un gran número de estudios taxonómicos, fisiológicos y ecológicos (Kushner y Kamekura, 1988,l Rodríguez-Valera, 1991; Vreeland y Hochstein, 1993; Ventosa y col., 1998b). Así, en nuestro laboratorio se ha descrito un elevado número de especies halófilas moderadas (Garcia y col., 1987; Ventosa y col., 1989a, 1989b, 1990, 1992; Márquez y col., 1990; Spring y col., 1996; Garabito y col., 1997) y se han realizado importantes aportaciones en cuanto a sus relaciones taxonómicas y filogenéticas (Ventosa y col., 1982, 1983, 1984; Márquez y col., 1987; Mellado y col., 1995a, 1996; Mota y col., 1997). Sin embargo, son muy escasos los estudios disponibles sobre la genética de este grupo de microorganismos halófilos, habiéndose obtenido la mayoría de los datos al respecto en los últimos años. Así, se han descrito diversos plásmidos autóctonos (Fernández-Castillo y col., 1992; Mellado y col., 1997; Llamas y col., 1997; Louis y Galinski, 1997), utilizándose algunos de ellos para la construcción de diversos vectores de clonación útiles para este grupo bacteriano (Vargas y col., 1995; Mellado y col., 1995c), y se han realizdo estudios de promotores en estas bacterias (Arvanitis y col., 1995; Tegos y col., 1997). A pesar de estos avances, los conocimientos que se poseen sobre los mecanismos de transferencia de material genético entre ellas, tanto en sus hábitats naturales como en condiciones de laboratorio, son muy escasos, lo que dificulta enormemente su manipulación genética con fines aplicados. A este respecto, es importante destacar el papel fundamental que juegan los compuestos antimicrobianos como marcadores en estudios de intercambio genético. En el caso de las bacterias halófilas mod

The moderately halophilic heterotrophic aerobic bacteria form a diverse group of microorganisms. The property of halophilism is widespread within the bacterial domain. Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and salted food products. Most species keep their intracellular ionic concentrations at low levels while synthesizing or accumulating organic solutes to provide osmotic equilibrium of the cytoplasm with the surrounding medium. Complex mechanisms of adjustment of the intracellular environments and the properties of the cytoplasmic membrane enable rapid adaptation to changes in the salt concentration of the environment. Approaches to the study of genetic processes have recently been developed for several moderate halophiles, opening the way toward an understanding of haloadaptation at the molecular level. The new information obtained is also expected to contribute to the development of novel biotechnological uses for these organisms.

Durante la última década, nuestro Equipo de Investigación ha realizado numerosos estudios encaminados a un mejor conocimiento de la biología de las bacterias halófilas moderadas, procariotas que conjuntamente con las arqueas aerobias halófilas extremas, son los microorganismos más representativos de los ambientes hipersalinos. En concreto, las bacterias halófilas moderadas predominan en medios con un 3-15% de sal, presentando un crecimiento óptimo a una concentración de un 10% de sal (Ventosa y col., 1998). Si bien la mayoría de estos estudios se han centrado en su taxonomía, filogenia, ecología y fisiología, solo hasta muy recientemente se han abordado estudios relativos a su genética, pudiéndose considerar estudios pioneros en dicho campo. Así dichos estudios han permitido el desarrollo de las herramientas necesarias para la manipulación genética de estos microorganismos extremófilos, habiéndose caracterizados a nivel molecular una serie de plásmidos nativos que han permitido la construcción de diversos vehículos de clonación, así como la optimización de los sistemas y condiciones de transferencia genética por conjugación entre bacterias halófilas moderadas y bacterias no halófilas. El estudio de la genética de estos microorganismos posee un gran interés aplicado, ya que dichas bacterias no sólo son importantes por el papel ecológico que desempeñan en los alimentos hipersalinos que habitan, sino también por su gran potencial biotecnológico. Además de producir exoenzimas de interés industrial del tipo de las amilasas, fosfatasas, nucleadas y proteasas, acumulan una serie de compuestos osmoprotectores o solutos compatibles (Ventosa y col., 1998). Estos solutos compatibles, que constituyen un mecanismo de defensa frente a la baja actividad e agua presente en este tipo de ambientes, han demostrado desempeñar una función estabilizadora y protectora de enzimas y células frente a elevadas salinidades, desnaturalización por calor, desecación y procesos de congelación/desc

A total of 232 metal-tolerant bacterial strains were isolated from water and sediment samples collected in different hypersaline environments located in Cádiz, Huelva and Morón de la Frontera (Spain). They were isolated on a medium containing mercury, chromium, cadmium, copper or zinc. These halotolerant isolates were analyzed by numerical taxonomy techniques by using the simple matching (SSM) and Jaccard (SJ) coefficients; clustering was achieved using the unweighted pair group method with averages (UPGMA) algorithm. At the 81% and 83% similarity level, different numbers of phenons were obtained for Gram-negative and Grampositive halotolerant microorganisms. Most of the 48 Gram-negative metal-tolerant strains studied were grouped into nine phenons, representing the genera Pseudoalteromonas, Alteromonas, Xanthomonas, Pseudomonas, Alcaligenes and Enterobacteria. The 72 Gram-positive metal-tolerant strains grouped into eight phenons, with only 15 strains left unassigned. Most of the isolates were assigned to the genus Bacillus (seven phenons), and one phenon comprised microorganisms with phenotypic characteristics similar to those of the genus Celullomonas.

The role of choline in osmoprotection in the moderate halophile Halomonas elongata has been examined. Transport and conversion of choline to betaine began immediately after addition of choline to the growth medium. Intracellular accumulation of betaine synthesized from choline was salt dependent up to 2.5 M NaCl. Oxidation of choline was enhanced at 2.0 M NaCl in the presence or absence of externally provided betaine. This indicates that the NaCl concentration in the growth medium has major effects on the choline-betaine pathway of H. elongata.

The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl–glucose–M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salin- ities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (Km = 3.06 J..M and Vmax = 9.96 nmol of betaine min mg of protein -1). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.

The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata. One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H. elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters. A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies. The ice nucleation gene was also expressed in H. elongata and V. eurihalina under the control of Pbla (the promoter of the (3-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis). One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subgla- ciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila. In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species.

The moderately halophilic bacterium Chromohalobacter marismortui contains a 17.5-kb narrow-host-range plasmid, pCM1, which shows interesting properties for the development of cloning vectors for the genetic manipulation of this important group of extremophiles. Plasmid pCM1 can stably replicate and is maintained in most gram-negative moderate halophiles tested. The replication origin has been identified and sequenced, and the minimal pCM1 replicon has been localized to a 1,600-bp region which includes two functionally discrete regions, the oriV region and the repA gene. oriV, located on a 700-bp fragment, contains four iterons 20 bp in length adjacent to a DnaA box that is dispensable but required for efficient replication of pCM1, and it requires trans-acting functions. The repA gene, which encodes a replication protein of 289 residues, is similar to the replication proteins of other gram-negative bacteria.

En relación a los microorganismos existentes en estanques de salinas marinas, se ha observado que la mayor parte de la población se compone de bacterias marinas en aquellos estanques que poseen hasta aproximadamente un 10% de sal; a medida que la concentración de sal aumenta, empiezan a predominar las bacterias halófilas moderadas y extremas, constituyendo porcentajes variables de la población m icrobiana, dependiendo del grado de salinidad existente (Rodríguez-Valera y col., 1985). La repercusión económica de los microorganismos halófilos ha cobrado un nuevo impulso gracias a que muchas salinas están siendo reconvertidas actualmente en sistemas de cultivo de peces y moluscos. Con ello se pretende la cría controlada de peces en estanques, lo cual supone una notable repercusión económica en las zonas donde se desarrolla estas actividades. Así la actividad acuícola en Andalucía ocupa, con distintos grados de identificación, el 90% de la superficie explotada. El policultivo extensivo de las especies propias de los esteros, como por ejemplo la dorada, se realiza en gran parte de las antiguas salinas existentes en la Región Suartlántica, no dedicándose éstas por tanto a la producción de sal y teniendo como objetivo el aprovechamiento acuícola. Los avances más significativos logrados en Andalucía, respecto a los cultivos marinos, incluyen las técnicas de producción de juveniles (alevines, postlarvas y semillas), de forma que se puede afirmar que Andalucía está a la cabeza de Europa y en disposición de exportar juveniles y las técnicas de obtención de los mismos; así como las técnicas de preengorde y engorde, especialmente en el cultivo de doradas (Sparus aurata), tanto el realizado en estanques de tierra, como en sistemas flotantes en el mar. La producción de doradas en 1992 y la positiva evolución a lo largo de estos últimos años, ha llevado a la dorada a constituirse en el principal producto de este tipo de cultivos piscícolas en Andalucía, ya que la cantidad obtenida a

A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonrrs halmphila, Deleya halophila and Vibrio costkola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coh'JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirmed by Southern hybridization. The plasmid carries genetic determinants that mediate resistance to kanamycin, tetracycline, and neomycin. This property, together with its relatively small size, its stability in E colicells, and the presence of unique restriction sites, makes pMHl a good candidate for the development of a cloning vector for moderate halophiles.

La norma morfogenética cortical de división en H. similisy H. muscorum es similar a la descrita para otros oxytriquidos aunque difieren en el origen del cirro bucal del protero y en el numero de primordios de la ciliación dorsal. La morfogénesis de regeneración subsiguiente al tratamiento con urea en estas dos especies y en G. steinii presenta algunas diferencias con respecto a la morfogénesis de división no totalmente explicables por la hipótesis de la coexistencia de los gradientes de actividad morfogena y de inhibición sino que hay que postular la existencia de un área refractaria en el extremo posterior del ciliado. Al igual que ocurre con otros ciliados en el ciclo celular de H. moscurum y G. steini no existe el periodo G2 micronuclear ni sincronía entre los periodos correspondientes de ambos tipos de núcleos.

The tolerance patterns, expressed as MICs, for 250 moderately halophilic eubacteria to 10 heavy metals were surveyed by using an agar dilution method. The moderate halophiles tested included 12 culture collection strains and fresh isolates representative of Deleya halophila (37 strains), Acinetobacter sp. (24 strains), Flavobacterium sp. (28 strains), and 149 moderately halophilic gram-positive cocci included in the genera Marinococcus, Sporosarcina, Micrococcus, and Staphylococcus. On the basis ofthe MICs, the collection strains showed, overall, similar responses to silver, cobalt, mercury, nickel, lead, and zinc. All were sensitive to silver, mercury, and zinc and tolerant of lead. The response to arsenate, cadmium, chromium, and copper was very heterogeneous. The metal susceptibility levels of the 238 freshly isolated strains were, in general, very heterogeneous among the four taxonomic groups as well as within the strains included in each group. The highest toxicities were found with mercury, silver, and zinc, while arsenate showed the lowest activity. All these strains were tolerant of nickel, lead, and chromium and sensitive to silver and mercury. Acinetobacter sp. strains were the most heavy-metal tolerant, with the mlijority of them showing tolerance of eight ditferent metal ions. In contrast, Flavobacterium sp. strains were the most metal sensitive. The inftuence of salinity and yeast extract concentrations of the culture medium on the toxicity of the heary metals tested for sorne representative strains was also studied. Lowering the salinity, in general, led to enhanced sensitivity to cadmium and, in sorne cases, to cobalt and copper. However, increasing the salinity resulted in only a slight decrease in the cadmium, copper, and nickel toxicities. Reduction in the yeast extract concentration resulted in an increased sensitivity to all metals, especially when this component was lowered to 0.01% (wt/vol). In contrast, a higher concentration only slightly les

Sixty-eight halobacteria, including both culture coUection strains and fresh isolates from widely dift'ering geographical areas, were tested for susceptibility to arsenate, cadmium, chromium, cobalt, copper, lead, mercury, nickel, silver, and zinc ions by an agar dilution technique. The culture collection strains showed dift'erent susceptibilities, clustering into five groups. Halobacterium medilerranei and Halobacterium volcanii were the most metal tolerant, whereas Haloarcula californiae and Haloarcula sinaiiensis had the highest susceptibilities of the culture collection strains. Dift'erent patterns of metal susceptibility were found for all the halobacteria tested, and there was a uniform susceptibility to mercury and silver. All strains tested were multiply metal tolerant.

The extremely halophilic archaebacterium Halococcus morrhuae CCM 537 was found to contain’’ a plasmid, pHM2, of 6.0 kb. A restriction map was constructed. Southern hybridization of pHM2 DNA to DNA extracted from other Halococcus sp. strains revealed the presence of a similar plasmid in one other strain. A plasmid of Halobacterium trapanicum shared common sequences with pHM2. Other plasmids from halobacteria displayed no homology with pHM2. The characterization of pHM2 may make it useful for the development of cloning vectors for extremely halophilic archaebacteria.

La régénération chez Histriculus similis a été étudiée en microscopie électronique. Les premiers signes du procesus de régénération s'observenl une heure apres le traitement par l'urée. Pendant la morphogenese corticale de régénération tanl du primordium oral que des primordiums de la ciliature parorale et des cirres, les états premiers sont constitués par des couples de cinétosomes, qui portent des microtubules postciliaires dans le cinétosome postérieur et des microtubules transverses a l'antérieur. L'orientation de ces couples dépend du primordium qu'ils vonl produire. Une fois que les couples de cinétosomes sont alignés, a Iieu une prolifération de nouveaux cinélosomes proches de ces couples. Plus tard il y a une fragmentation transversale de ces primordiums , a l'exception du primordium paroral qui se divise longitudinalement., The cortical morphogenesis of regeneration in Histriculus similis has been studied in this work at level of electronic microscopy. The first signs of the regeneration process become evident one hour after the treatment with urea. During the early steps of the cortical morphogenesis of regeneration, the oral primordium , paroral ciliation primordium and the ciliary streaks, are formed by pairs of kinetosomes. These pairs carry postciliary microtubules in the posterior kinetosome and transverse microtubules in the anterior one. The orientation of these pairs of kinetosomes depends on the type of primordium that is going to be formed. Once the pairs of kinetosomes are aligned, there is a proliferation of new kinetosomes in the vicinity of these pairs of kinetosomes. Afterwards there is a transversal fragmentation of the above mentioned primordia, with the exception of the paroral primordium that is longitudinally divided.

The role of light as an energy source for slightly aereated cultures of halobac­ teria was studied, using continuous cultures with low nutrient concentrations and a low oxygen supply. A series of experiments were carried out with non­ illuminated and differently illuminated cultures and with ditferent oxygen transfer rates. Under low oxygen availability, light proved to be a decisively important energy source that allowed the populations to reach higher growth rates and much higher population densities. Oxygen inftuenced the growth over only a minimal level, below which neither the illuminated nor the dark cultures were atfected by the oxygen transfer rate. From these results, it appears that the bacteriorhodop­ sin-mediated energy supply could have a very important role for the ecology of halobacteria in their microaerophilic habitats. In the illuminated cultures, cells that originated purple colonies on plates appeared. These cells, which could be bacteriorhodopsin-constitutive mutants, are now being studied.