Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.     

Translocations in DICTYOSTELIUM DISCOIDEUM

Fourteen translocations of independent origin were identified in Dictyostelium discoideum on the basis of segregation anomalies of diploids heterozygous for these chromosome rearrangements, all of which led to the cosegregation of unlinked markers. Many of these translocations were discovered in strains mutagenized with MNNG or in strains carrying mutations affecting DNA repair; however, spontaneous translocations were also obtained. Haploid mitotic recombinants of the rearranged linkage groups were produced from diploids heterozygous for the translocations at frequencies of up to 5% of viable haploid segregants; this is at least a ten-fold higher frequency than that seen with diploids not heterozygous for translocations (approximately 0.1%). These haploid recombinants included both translocated and nontranslocated strains. The T354(II, VII) translocation and possibly the T357(IV, VII) translocation reduce the chromosome number to n = 6; haploids carrying 11 other translocations all have karyotypes with n = 7. Genetic characterization of the T357(IV, VII) translocation showed that the bwnA and whiC loci normally found on linkage group IV were physically linked to the linkage group VII loci couA, phgA, bsgB and cobA.     

Genetic analysis of Bacillus stearothermophilus by protoplast fusion.

Efficient and reliable protoplasting, regeneration, and fusion techniques were established for the prototrophic strain Bacillus stearothermophilus NUB36. Auxotrophic mutants were isolated, and protoplast fusion was used to construct isogenic mutant strains and for chromosomal mapping. Markers were mapped using two-, three-, and four-factor crosses. The order of the markers was hom-1-thr-1-his-1-(gly-1 or gly-2)-pur-1-pur-2. These markers may be analogous to hom, thrA, hisA, glyC, and purA markers on the Bacillus subtilis chromosome. No analogous pur-1 marker has been reported in B. subtilis. The relative order of three of the markers (hom-1-thr-1-gly-1) was independently confirmed by transduction.     

Correlation of the number of pineal “synaptic” ribbons and spherules with the level of serum melatonin over a 24-hour period in male rabbits

Summary Previous studies have shown that pineal synaptic ribbons and spherules may respond differently under normal and experimental conditions. It has been suggested that the increase in the number of ribbons may be a prerequisite for enhanced melatonin formation. In the present study, the number of ribbons and spherules as well as the level of serum melatonin were monitored over a 24-h period in the male rabbit, the pineal gland of which is known to contain many spherules. It was found that both the number of ribbons and the levels of serum melatonin show the typical nocturnal increase, exhibiting peaks at 02:00 and 06:00 h, respectively. There is a good correlation (R = 0.8) of the two parameters. The spherules, in contrast, show no statistically significant circadian changes in number and cannot be correlated with the levels of serum melatonin. It is concluded that ribbons and spherules may differ in function and that the ribbons may be somehow involved in the regulation of melatonin formation.Supported by the Deutsche Forschungsgemeinschaft (Grant Vo 135/7) within the project SPP Neuroendokrinologie     

Genetic Analysis of Two Mutations Affecting Thymidine Metabolism in Dictyostelium Discoideum

The tmpA600 mutation confers thymidylate synthase deficiency and thymidine auxotrophy to Dictyostelium discoideum. The tdrA600 mutation enhances transport of thymidine and thereby reduces the auxotrophic requirement of tmpA600 strains. The tmpA locus maps to linkage group III. The tdrA600 mutation is dominant and cosegregates with both linkage groups IV and VI, possibly because of a translocation between the two. The tdrA600 allele is sufficient to allow efficient incorporation of exogenous [3H]thymidine or [3H]uridine into TCA-precipitable material and to sensitize the cell to the nucleoside-analog inhibitor, 5-fluorodeoxyuridine. These properties make the tdrA mutation useful for studies requiring labelling of DNA or RNA in vivo.     

An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity.

Infectious retrovirus particles are derived from structural polyproteins which are cleaved by the viral proteinase (PR) during virion morphogenesis. Besides cleaving viral polyproteins, which is essential for infectivity, PR of human immunodeficiency virus (HIV) also cleaves cellular proteins and PR expression causes a pronounced cytotoxic effect. Retroviral PRs are aspartic proteases and contain two copies of the triplet Asp-Thr-Gly in the active center with the threonine adjacent to the catalytic aspartic acid presumed to have an important structural role. We have changed this threonine in HIV type 1 PR to a serine. The purified mutant enzyme had an approximately 5- to 10-fold lower activity against HIV type 1 polyprotein and peptide substrates compared with the wild-type enzyme. It did not induce toxicity on bacterial expression and yielded significantly reduced cleavage of cytoskeletal proteins in vitro. Cleavage of vimentin in mutant-infected T-cell lines was also markedly reduced. Mutant virus did, however, elicit productive infection of several T-cell lines and of primary human lymphocytes with no significant difference in polyprotein cleavage and with similar infection kinetics and titer compared with wild-type virus. The discrepancy between reduced processing in vitro and normal virion maturation can be explained by the observation that reduced activity was due to an increase in Km which may not be relevant at the high substrate concentration in the virus particle. This mutation enables us therefore to dissociate the essential function of PR in viral maturation from its cytotoxic effect.     

Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis

Eight strains of highly amylolytic, sporeforming bacilli (hereafter referred to as Bacillus amyloliquefaciens) were compared with respect to their taxonomic relationship to B. subtilis. The physiological-biochemical properties of these two groups of organisms showed that B. amyloliquefaciens differed from B. subtilis by their ability to grow in 10% NaCl, characteristic growth on potato plugs, increased production of alpha-amylase, and their ability to ferment lactose with the production of acid. The base compositions of the deoxyribonucleic acid (DNA) of the B. subtilis strains consistently fell in the range of 41.5 to 43.5% guanine + cytosine (G + C), whereas that of the B. amyloliquefaciens strains was in the 43.5 to 44.9% G + C range. Hybrid formation between B. subtilis W23 and B. amyloliquefaciens F DNA revealed only a 14.7 to 15.4% DNA homology between the two species. Transducing phage, SP-10, was able to propagate on B. subtilis W23 and B. amyloliquefaciens N, and would transduce B. subtilis 168 (indole(-)) and B. amyloliquefaciens N-10 (arginine(-)) to prototrophy with a frequency of 3.9 x 10(-4) and 2.4 x 10(-5) transductants per plaque-forming unit, respectively. Attempts to transduce between the two species were unsuccessful. These data show that Bacillus amyloliquefaciens is a valid species and should not be classified as a strain or variety of B. subtilis.     

Restriction and Modification of Bacteriophage in Bacillus stearothermophilus

Host-controlled restriction and modification of TP-1C phage and infectious phage DNA occurs in Bacillus stearothermophilus and is subject to control by TP-8 or TP-12 prophage.     

Factors Affecting Transfection in Bacillus stearothermophilus

The conditions for the infection of Bacillus stearothermophilus 4S with TP-1C phage deoxyribonucleic acid (DNA) are described. Cells from log-phase cultures are the most susceptible to phage DNA infection (transfection). A cellular component (competence factor) which enhances transfection is released into the culture medium during the transition period between the log and stationary phase of growth. Transfection is stimulated in the order of decreasing effectiveness, by Fe(3+), Mn(2+), and Mg(2+). The efficiency of transfection is the highest in cells growing at 60.5 C and does not occur in cells growing at 67 C although the cells are growing normally. A cellular component (competence factor) of this organism, which is released into the culture medium, advances by 40 min some step in the uptake of phage DNA.