Analysis of the expression from Rhizobium meliloti fix-promoters in other Rhizobium backgrounds.

Using translational fusions to lacZ, we have measured expression from the promoters of Rhizobium meliloti regulatory genes, nifA and fixK, and structural genes, nifH and fixA, in other fast-growing rhizobia whose nitrogen fixation regulation is less known. Neither nifA nor fixK promoters were activated under both free-living microaerobic and symbiotic conditions, except in R. tropici, where clear symbiotic activation of either nifA or fixK expression could be observed. Both nifH and fixA promoters showed strong heterologous activation during symbiosis and weak activation under free-living nitrogen starvation conditions. Only when the nifH promoter was in R. tropici and R. leguminosarum bv. phaseoli, was clear induction observed in the microaerobic free-living state. Deletion analysis of these promoters suggested that a NifA binding site (UAS) was needed for full heterologous activation of nifHp, either in microaerobiosis or symbiosis. In contrast, the UAS region seemed to be unnecessary for fixA activation. However, a region containing a potential integration host factor (IHF) binding site was observed to be needed for complete heterologous symbiotic induction from fixAp. The moderate induction observed in nitrogen-free medium only required the sigma 54 holoenzyme recognition sequence; this may be indicative of the existence of non-specific activation by NtrC-like proteins. Our results suggest possible common and different features in the control mechanisms of the nitrogen fixation gene expression among Rhizobium species.

Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring.

A system for stable tagging of gram-negative bacteria with the firefly luciferase gene, luc, is described. A previously constructed fusion constitutively expressing luc from the lambdap(R) promoter was used. Stable integration into the bacterial genome was achieved by use of mini-Tn5 delivery vectors. The procedure developed was applied for tagging of representative gram-negative bacteria, such as Escherichia coli, Rhizobium meliloti, Pseudomonas putida, and Agrobacterium tumefaciens. The system permitted the detection of tagged R. meliloti in the presence of more than 10 CFU per plate without the use of any selective markers (such as antibiotic resistance genes). No significant differences in growth rates or soil survival were found between the marked strain and the wild-type strain. Studies of bioluminescent R. meliloti also revealed a good correlation between cell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive method for the detection and enumeration of bacteria in the environment.

Characterization of a plasmid from moderately halophilic eubacteria.

A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonas halmophila, Deleya halophila and Vibrio costicola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coli 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. coli cells, and the presence of unique restriction sites, makes pMH1 a good candidate for the development of a cloning vector for moderate halophiles.

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters.

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH-luc fusion to be observed by a dark-adapted eye and photographed.

Relationships among Vibrio costicola strains assessed by DNA-DNA hybridization.

Deoxyribonucleic acid (DNA)-DNA hybridization was used to determine the relationships among 14 strains of Vibrio costicola isolated from cured meats or hypersaline waters. Our results indicate that the strains investigated should be considered members of the same genomic species, with homologies of 60% or higher with the V. costicola strains used as reference. Low DNA relatedness was found with other marine vibrios or moderately halophilic bacteria tested. The guanine-plus-cytosine base contents of DNAs from the 14 V. costicola strains were 47.6 to 51.1 mol%.

Survey of metal tolerance in moderately halophilic eubacteria.

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 of the 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 majority of them showing tolerance of eight different metal ions. In contrast, Flavobacterium sp. strains were the most metal sensitive. The influence of salinity and yeast extract concentrations of the culture medium on the toxicity of the heavy metals tested for some representative strains was also studied. Lowering the salinity, in general, led to enhanced sensitivity to cadmium and, in some cases, to cobalt and copper. However, increasing the salinity resulted in only a slight decrease in the cadmium, copper, and nickel toxicities.(ABSTRACT TRUNCATED AT 250 WORDS)

Susceptibility of halobacteria to heavy metals.

Sixty-eight halobacteria, including both culture collection strains and fresh isolates from widely differing 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 different susceptibilities, clustering into five groups. Halobacterium mediterranei and Halobacterium volcanii were the most metal tolerant, whereas Haloarcula californiae and Haloarcula sinaiiensis had the highest susceptibilities of the culture collection strains. Different 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.

Accumulation of Poly (beta-Hydroxybutyrate) by Halobacteria.

Some species of extremely halophilic archaebacteria, Halobacteriaceae, have been shown to accumulate large amounts of poly (beta-hydroxybutyrate) under conditions of nitrogen limitation and abundant carbon source. The production of poly (beta-hydroxybutyrate), at least in large quantities, was restricted to two carbohydrate-utilizing species, Halobacterium mediterranei and H. volcanii. In addition to the nutrients in the media, the salt concentration also influenced poly (beta-hydroxybutyrate) accumulation, which was greater at lower salt concentrations. The possible application of these microorganisms for the production of biodegradable plastics is discussed.

Light as an energy source in continuous cultures of bacteriorhodopsin-containing halobacteria.

The role of light as an energy source for slightly aereated cultures of halobacteria 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 different 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 influenced the growth over only a minimal level, below which neither the illuminated nor the dark cultures were affected by the oxygen transfer rate. From these results, it appears that the bacteriorhodopsin-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.

Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations.

Solar salterns, based on a multi-pond system, give a discontinuous gradient of salt concentrations. The heterotrophic bacterial populations of ponds containing from 10% salt to saturation have been studied. Saltern samples were spread on agar plates containing different media for halophilic bacteria and one medium made with water of the pond plus nutrients. Replica plating was done to determine the salt range for growth of the colonies. We studied 150 strains to determine the salt spectra of growth, the morphology, and nutrient requirements. The following conclusions were reached: (a) In salt concentrations above 10% (total salts), most bacteria are halophilic and few are halotolerant; (b) the two types of halophilic bacteria, moderate and extreme, show different distributions; in these ponds a narrow overlap exists between 25% and 32% salts with moderate halophiles predominating below this interval and extreme halophiles above it; (c) the populations of moderate halophiles are highly heterogeneous, and the salt concentration of their habitat affects their taxonomic composition, salt range for growth, and nutrient requirements. The population composition of extreme halophiles is less affected by the salt concentrations at which these bacteria are found.

Behaviour of mixed populations of halophilic bacteria in continuous cultures.

Two main physiological groups of bacteria are known which are adapted to high saline environments, moderate and extreme halophiles. In order to clarify some aspects of the competition between these two groups in their natural habitats, continuous cultures were used to provide a changing spectrum of conditions of salt concentration, temperature, and nutrient concentration (dilution rate). The effects of these parameters on natural solar saltern populations were studied. Complex media were used to increase the range of competing microorganisms. Nineteen strains of halophilic bacteria were isolated and studied with respect to their growth response at different salt concentrations. The temperature seemed to be the decisive factor within the range of salt concentrations studied (20-30%, w/v), the moderate halophiles being favored by low temperatures. Within this group, motile, gram-negative rods, and spiral forms were the predominant morphological types. In general, microorganisms that showed high growth rates in batch cultures predominated in continuous cultures with high dilution rates (high nutrient concentrations); those that grew slowly in batch cultures predominated in cultures with low dilution rates.

Isolation of extreme halophiles from seawater.

Extreme halophilic bacteria were isolated from the ocean off the coast of Spain. All were gram-negative cocci. One isolate was compared to Halococcus sp. NCMB 757 and was found to have similar characteristics.

Relationship between amino acid production and phosphate-dissolving capacity of bacteria.

The ability of soil bacteria to produce amino acids (alanine, aspartic acid, leucine, arginine, glutamic acid, and lysine) was related to the ability to dissolve inorganic phosphate. With the exception of lysine, amino acid production increased with increasing ability to dissolve phosphate.