Photorhabdus lux-operon heat shock-like regulation.

For decades, transcription of Photorhabdus luminescens lux-operon was considered being constitutive. Therefore, this lux-operon has been used for measurements in non-specific bacterial luminescent biosensors. Here, the expression of Photorhabdus lux-operon under high temperature was studied. The expression was researched in the natural strain Photorhabdus temperata and in the heterologous system of Escherichia coli. P. temperata FV2201 bacterium was isolated from soil in the Moscow region (growth optimum 28 °C). We showed that its luminescence significantly increases when the temperature rises to 34 °C. The increase in luminescence is associated with an increase in the transcription of luxCDABE genes, which was confirmed by RT-PCR. The promoter of the lux-operon of the related bacterium P. luminescens ZM1 from the forests of Moldova, being cloned in the heterologous system of E. coli, is activated when the temperature rises from room temperature to 42 °C. When heat shock is caused by ethanol addition, transcription of lux-operon increases only in the natural strain of P. temperata, but not in the heterologous system of E. coli cells. In addition, the activation of the lux-operon of P. luminescens persists in E. coli strains deficient in both the rpoH and rpoE genes. These results indicate the presence of sigma 32 and sigma 24 independent heat-shock-like mechanism of regulation of the lux-operon of P. luminescens in the heterologous E. coli system.

[Antibiotics produced by Photorhabdus luminescens ZMI, a symbiont of entomopathogenic nematodes].

A novel strain of Photorhabdus luminescens ZMI isolated from nematode larvae Heterorhabditis sp. was shown to produce antibiotic complexes with antibacterial and antifungal activities. The antibiotic complexes secreted extracellularly and intracellularly were separated into individual components. Comparison of their properties with the databases for biologically active compounds suggested that component A was identical to 3,5-dihydroxy-4-isopropylstilbene, components B and H belonged to anthraquinone derivatives, component C secreted only extracellularly was likely a novel antibiotic.

[Sequencing and comparative analysis of the lux operon of Photorhabdus luminescens strain ZM1: ERIC elements as putative recombination spots]. / Opredelenie i sravnitel'nyi analiz nukleotidnoi posledovatel'nosti lux-operona Photorhabdus luminescens, shtamm ZM1. ERIC-élementy kak predpolagaemye "goriachie" tochki rekombinatsii.

The EcoRI chromosomal fragment (6782 bp) containing the lux operon of Photorhabdus luminescens was cloned in pUC18 and completely sequenced. Enteric repetitive intergenic consensus (ERIC), an imperfect palindrome (125-127 bp) characteristics for Enterobacteriaceae genomes, was found in three sites. Strain Zm1 proved to differ in ERIC number and location from strains Hb, Hm, and Hw. Nucleotide substitution analysis showed that luxC and luxB, which are more than 1 kb away from ERIC, are similar to the corresponding Hb genes, whereas luxD, luxA, and luxE, which are close to ERIC, are intermediate between their Hb and Hw counterparts. The Hb/Hw nucleotide substitution ratio was 1:1 in regions adjacent to ERIC. Hence ERIC were assumed to play a role of recombination hot spots in the bacterial genome.

[Photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain in the presence of photodithazine]. / Fotoindutsirovannoe podavlenie bioliuminestsentsii genno-inzhenernogo shtamma bakterii Escherichia coli TG1 (pXen7) v prisutstvii fotoditazina.

The photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain was studied in the presence of the photosensitizer photodithazine, a glucosamine salt of chlorin e6. The photosensitized quenching of the bioluminescence was found to correlate with the colony-forming ability of the strain. The data obtained are discussed from the standpoint of using biosensor luminescent bacterial systems for the assessment of the efficiency of photosensitizers in antimicrobial photochemotherapy.

[Cloning and expression of the lux-operon of Photorhabdus luminescens, strain Zm1: nucleotide sequence of luxAB genes and basic properties of luciferase]. / Klonirovanie i ékspressiia lux-operona Photorhabdus luminescens, shtamm Zm1: nukleotidnaia posledovatel'nost' genov luxAB i osnovye kharakteristiki liutsiferazy.

A chromosomal fragment of bacteria Photorhabdus luminescence Zm1, which contains the lux operon, was cloned into the vector pUC18. The hybrid clone containing plasmid pXen7 with the EcoRI fragment approximately 7-kb was shown to manifest a high level of bioluminescence. By subcloning and restriction analysis of the EcoRI fragment, the location of luxCDABE genes relative to restriction sites was determined. The nucleotide sequence of the DNA fragment containing the luxA and luxB genes encoding alpha- and beta-subunits of luciferase was determined. A comparison with the nucleotide sequences of luxAB genes in Hm and Hw strains of Ph. luminescence revealed 94.5 and 89.7% homology, respectively. The enterobacterial repetitive intergenic sequence (ERIC) of 126 bp typical for Hw strains was identified in the spacer between the luxD and luxA genes. The lux operon of Zm1 is assumed to emerge through recombination between Hm and Hw strains. Luciferase of Ph. luminescence was shown to possess a high thermal stability: its activity decreased by a factor of 10 at 44 degrees C for 30 min, whereas luciferases of marine bacteria Vibrio fischeri and Vibrio harveyi were inactivated by one order of magnitude at 44 degrees C for 1 and 6 min, respectively. The lux genes of Ph. luminescence are suggested for use in gene engineering and biotechnology.

[FMN-reductase from Escherichia coli and its effect on the activity of luciferase from marine bacterium Vibrio fischeri]. / FMN-reduktaza iz Escherichia coli i ee vliianie na aktivnost' liutsiferazy iz morskikh bakterii Vibrio fischeri.

Interactions of luciferases isolated from Vibrio fischeri 6 and Escherichia coli JM109(pF3) (bearing cloned V. fischeri luxAB genes) with FMN reductase isolated from E. coli JM109 were studied. FMN reductase formed a stable complex with luciferase, suggesting similar properties of the FMN reductases in the taxonomically close families Vibrionaceae and Enterobacteriaceae.

[The radiobiological characteristics of the P+ variant of Bacillus brevis var. G.--B. in relation to gramicidin S synthesis]. / Radiobiologicheskie osobennosti P+-varianta Bacillus brevis var. G.--B.v sviazi s sintezom gramitsidina S.

The radiosensitivity of P(+) variant Bacillus brevis var. G.-B. cells cultured under condition of normal and inhibited gramicidin S synthesis, antibiotically high-active strain and high radioresistant cells has been studied. It has been shown that the radioresistance of bacterial cells correlates, in general, with their antibiotic activity: the antibiotic superproduced is more radioresistant than P(+) variant, the inhibition of antibiotic synthesis by beta-phenil-beta-alanin rises a little the sensitivity of P(+) variant cells. But the radioresistant fraction of P(+) variant contains the lower antibiotic amount than the whole population. It has been concluded that the radioprotective action of gramicidin S can not be the only reason of the above-mentioned differences in radiosensitivity.

[Various characteristics of lipid metabolism of Bacillus brevis var. G.-B]. / Nekotorye osobennosti lipidnogo obmena Bacillus brevis var. G.-B.

Some characteristic features of the lipid metabolism of Bacillus brevis var. G.-B. natural variants and Bacillus brevis mutant 101 were studied. The authors found that upon submerged cultivation gramicidine S-producing P+-variant and B. brevis mutant 101 synthesized higher amounts of tocopherols as compared to other colonial-morphological variants. The highest tocopherol content was observed in P+-variant, whose cells contained the highest amount of total lipids as compared to other gramicidine S-producers.

[Biological properties of a Bacillus brevis 101 mutant]. / Biologicheskie osobennosti mutanta Bacillus brevis 101.

Some features of the Bacillus brevis 101 mutant producing the antibiotic gramicidin S are described. The mutant is very close to the initial P+-variant of Bacillus brevis var. G-B by cultural, physiological and biochemical characteristics. The most typical features of Bacillus brevis 101 are high antibiotic activity (up to 2 g/l) and the specific phenotype of the colonies. The phenotypical features of Bacillus brevis 101 are dependent on the conditions of its cultivation. On minimal media rich in organics a change of the culture correlated with a lower antibiotic activity.

[An oligosporogenic mutant of Bacillus brevis]. / Oligosporogennyi mutant Bacillus brevis.

The treatment of Bacillus brevis var. G-B by chemical mutagens yielded a mutant similar in the major characteristics to the parent strain of the P+-variant, but producing less spores and synthesizing more gramicidin S. The mutant can be stored in the freeze-dried state.

[Structural study of the basal bodies of the flagella of Bacillus brevis var. G.-B. P+]. / Izuchenie struktury bazal'nykh tel zgutikov Bacillus brevis var. G.-B. P+.

The structural organisation of the flagellum basal body was studied in Bacillus brevis var. G.-B. P+ by electron microscopy. It was compared with that of Escherichia coli MS 1350. The basal body of a B. brevis flagellum contains, in addition to two pairs of rings on a rod, another ring-like structure (d = 13.6 nm, h = 4.3 nm) which we referred to as a "collar". The collar makes the basal body of B. brevis different from that of B. subtilis, another Gram-positive bacterium. The collar seems to fasten the flagellum of B. brevis to the cell wall. We have concluded that the basal body can differ not merely among bacterial systematic groups, but also among bacteria belonging to one and the same genus. The role of individual elements in the structure of the basal body of bacterial flagella is discussed.

[Lyophilization of the spores and vegetative cells of Bacillus brevis var. G-B.--producer of gramicidin S]. / Liofilizatsiia spor i vegetativnykh kletok Bacillus brevis var. G-B.--produtsenta gramitsidina S.

Viability, antibiotic properties and variation of 4 variants of Bac. brevis var. G.-B. were studied after lyophilization and storage for a year in the lyophilized state. It was shown that the spores and vegetative cells of S and P- variants not synthesizing gramicidin S were somewhat more stable than the spores and cells of R and P+ variants producing the antibiotic. The latter dissociated by 10 per cent towards the cells producing and not producing gramicidin. The developmental rate of the lyophilized vegetative cells was higher than that of the lyophilized spores. Under analogous cultivation conditions they produced higher amounts of the biomass and antibiotic. The lyophilization method described may be recommended for the maintenance of viability and stability of the spores and vegetative cells of Bacillus brevis var. G.-B. producing gramicidin S.

[Lipid and gramicidin C biosynthesis in Bacillus brevis during stab cultivation]. / Biosintez lipidov i gramitsidina C u Bacillus brevis v usloviiakh glubinnogo kul'tivirovaniia.

The content of lipids was studied in the gramicidin producing variants of Bacillus brevis var. G.-B. in the process of submerged cultivation. The greatest accumulation of lipids preceded the highest content of gramicidin C in the producing cells. The interrelation between the synthesis of lipids and that of gramicidin C is discussed.

[Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B]. / Rol' gramitsidina S v sporoobrazovanii R+ i R- variantov Bacillus brevis var. G.-B.

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.

[Some properties of 1 P+f bacteriophage for Bacillus brevis var. G.-B and its nucleic acid]. / Nekotorye svoistva bakteriofaga 1 P+f Bacillus brevis var. G.-B. i ego nukleinovoi kisloty

The 1 P+f phage, a virulent mutant of the moderate P+ phage for Bac. brevis var. G.-B., consists of a hexagonal head (90x90 nm) and a long non-contractile tail (340 nm). This phage is characterized by a relatively long latent period (90-110 min) and a low yield (40-50 particles per cell). The 1P+f phage is quite stable at pH values from 1 to 11, insensitive to osmotic shock, treatment with chloroform and acridine orange. The sensitivity of the phage to thermal treatment and UV-radiation has been studied. The nucleic acid of the P+f phage is double-stranded DNA of AT-type (GC equals 34.5 mole %) which contains 5-methylcytosine (0.18 mole %) and N6-methyladenine (0.32 mole%). The level of methylation of cytosine and adenine residues in DNA of the 1 P+f phage does not depend on the host studied (Bac. brevis, P- and S variants). The specificity of methylation of cytosine residues in the S and P- cells appears to be the same. DNA of the 1 P+f phage strongly differs from DNA of the host in nucleotide composition (GC equals 45.7 mole %). Nevertheless, phage DNA is very similar to DNA from Bac. subtilis in the character of pyrimidine distribution (the amount of different pyrimidine isopliths). This may testify to a somewhat common character of the nucleotide sequence organization in DNA of the phage and its host.