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Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.

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In their natural environments, moderately halophilic bacteria are confronted not only with high salinities but also with low oxygen tensions due to the high salinities. The growth of H. halophilus is strictly aerobic. To analyze the dependence of respiration on the NaCl concentration and oxygen availability of the medium, resting cell experiments were performed. The respiration rates were dependent on the NaCl concentration of the growth medium, as well as on the NaCl concentration of the assay buffer, indicating regulation on the transcriptional and the activity level. Respiration was accompanied by the generation of an electrochemical proton potential (Δµ(H+)) across the cytoplasmic membrane whose magnitude was dependent on the external pH. Genes encoding proteins involved in respiration and Δµ(H+) generation, such as a noncoupled NADH dehydrogenase (NDH-2), complex II, and complex III, were identified in the genome. In addition, genes encoding five different terminal oxidases are present. Inhibitor profiling revealed the presence of NDH-2 and complex III, but the nature of the oxidases could not be resolved using this approach. Expression analysis demonstrated that all the different terminal oxidases were indeed expressed, but by far the most prominent was cta, encoding cytochrome caa3 oxidase. The expression of all of the different oxidase genes increased at high NaCl concentrations, and the transcript levels of cta and qox (encoding cytochrome aa3 oxidase) also increased at low oxygen concentrations. These data culminate in a model of the composition and variation of the respiratory chain of H. halophilus.

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The lipid composition of Halobacillus halophilus was investigated by combined thin-layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the total lipid extract. Main polar lipids were found to be sulfoquinovosyldiacylglycerol and phosphatidylglycerol, while cardiolipin was a minor lipid together with phosphatidic acid, alanyl-phosphatidylglycerol and two not yet fully identified lipid components. In addition the analyses of residual lipids, associated with denatured proteins after the lipid extraction, revealed the presence of significant amounts of cardiolipin, indicating that it is a not readily extractable phospholipid. Post decay source mass spectrometry analyses allowed the determination of acyl chains of main lipid components. On increasing the culture medium salinity, an increase in the shorter chains and the presence of chain unsaturations were observed. These changes in the lipid core structures might compensate for the increase in packing and rigidity of phospholipid and sulfoglycolipid polar heads in high-salt medium, therefore contributing to the homeostasis of membrane fluidity and permeability in salt stress conditions.

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A procedure for markerless mutagenesis gene deletions was developed for the moderately halophilic model strain Halobacillus halophilus. Gene transfer was achieved by protoplast fusion and allelic replacement by a two-step procedure. In the first step the non-replicating plasmid integrated over the upstream or the downstream region of the target gene or operon into the chromosome to obtain single-crossover mutants. When cells were grown under non-selective conditions a second homologous recombination happened (segregation). This resulted in either the wild-type or the mutated allele. The method was used to delete the proHJA operon from H. halophilus. The mutant still produced proline and thus was not proline auxotroph but it completely lost the ability to produce proline as a compatible solute. However, growth was not impaired and the loss of the solute proline was compensated for by an increase in glutamate, glutamine and ectoine concentration. Expressions of the genes encoding the biosynthesis enzymes of theses solutes were upregulated and the activity of the key enzyme in glutamine biosynthesis, the glutamine synthetase, was increased. A model for the proline biosynthesis in the ΔproHJA mutant is discussed.

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The amino acid proline is not only synthesized as a compatible solute but also used as a carbon and energy source by the moderately halophilic bacterium Halobacillus halophilus. Growth on proline was not affected by the salinity of the medium. Proline was degraded by proline dehydrogenase (ProDH) and Δ(1) -pyrroline-5-carboxylate dehydrogenase (P5CDH) to glutamate via Δ(1) -pyrroline-5-carboxylate. The basic biochemical parameters for ProDH and P5CDH activities were obtained for both in cell free extracts. The encoding genes were identified. H. halophilus has two isogenes each for prodh and p5cdh. prodh2 and p5cdh2 form an operon (put operon) whose mRNA is highly abundant in proline-grown cells. Expression of the put operon was also upregulated by salinity and late growth phase in glucose-grown cells. Similarly, ProDH and P5CDH activities increased in late exponential growth phase. This observation is in line with the previous notion that the compatible solute proline is degraded in stationary phase (in glucose grown cultures).

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We performed the chemical mutagenesis of Halobacillus halophilus (the producer of a C(30) carotenoid, methyl glucosyl-3,4-dehydro-apo-8'-lycopenoate) to isolate novel carotenoids that are biosynthetic intermediates of methyl glucosyl-3,4-dehydro-apo-8'-lycopenoate. As a result, we isolated two novel C(30) carotenoids, hydroxy-3,4-dehydro-apo-8'-lycopene and methyl hydroxy-3,4-dehydro-apo-8'-lycopenoate, which were biosynthesized through a novel 8'-apo C(30) pathway. These carotenoids showed antioxidative activity in the (1)O(2) suppression model.

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Inhibitor studies and mutant analysis revealed a C(30) pathway via 4,4'-diapophytoene and 4,4'-diaponeurosporene to 4,4'-diaponeursoporene-4-oic acid esters related to staphyloxanthin in Halobacillus halophilus. Six genes may be involved in this biosynthetic pathway and could be found in two adjacent gene clusters. Two genes of this pathway could be functionally assigned by functional pathway complementation as a 4,4'-diapophytoene synthase and a 4,4'-diapophytoene desaturase gene. These genes were organized in two operons together with two putative oxidase genes, a glycosylase and an acyl transferase ortholog. Pigment mutants were obtained by chemical mutagenesis. Carotenoid analysis showed that a white mutant accumulated 4,4'-diapophytoene due to a block in desaturation. In a yellow mutant carotenogenesis was blocked at the stage of 4,4'-diaponeurosporene and in an orange mutant at the stage of 4,4'-diaponeurosporene-4-oic acid. The protective function of these pigments could be demonstrated for H. halophilus after inhibition of carotenoid synthesis by initiation of oxidative stress. A degree of oxidative stress which still allowed 50% growth of carotenogenic cells resulted in the death of the cells devoid of colored carotenoids.

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