A positive, genetic selection against the activity of the nitrogen regulatory (NTR) system was used to isolate insertion mutations affecting nitrogen regulation in Kiebsiella aerogenes. Two classes of mutation were obtained: those affecting the NTR system itself and leading to the loss of almost all nitrogen regulation, and those affecting the nac locus and leading to a loss of nitrogen regulation of a family of nitrogen-regulated enzymes. The set of these nac-dependent enzymes included histidase, glutamate dehydrogenase, glutamate synthase, proline oxidase, and urease. The enzymes shown to be nac independent included glutamine synthetase, asparaginase, tryptophan permease, nitrate reductase, the product of the nifLA operon, and perhaps nitrite reductase. The expression of the nac gene was itself highly nitrogen regulated, and this regulation was mediated by the NTR system. The loss of nitrogen regulation was found in each of the four insertion mutants studied, showing that loss of nitrogen regulation resulted from the absence of nac function rather than from an altered form of the nac gene product. Thus we propose two classes of nitrogen-regulated operons: in class I, the NTR system directly activates expression of the operon; in class II, the NTR system activates nac expression and the product(s) of the nac locus activates expression of the operon.The enteric bacterium Klebsiella aerogenes is capable of using a large number of compounds as its sole source of nitrogen. The formation of the enzymes needed to metabolize these compounds is in most, if not all, cases regulated by the availability of the preferred nitrogen source, ammonium (22). The formation of the enzymes needed for the conversion of poor nitrogen sources to either ammonia or glutamate is increased when ammonia is absent. Conversely, the formation of the two enzymes ultimately responsible for assimilating ammonia into glutamate (glutamate dehydrogenase and glutamate synthase) is repressed when ammonia is absent. As early as 1973, Prival et al. (33) recognized a role for the glnA locus in the genetic control of nitrogen-regulation in K. aerogenes. After a false start suggesting that the product of the glnA gene (glutamine synthetase) was itself the activator of transcription for nitrogen-regulated genes (23), it eventually became clear that the glutamine synthetase encoded by the glnA locus was physiologically important for nitrogen regulation because it synthesized the internal signal of nitrogen excess (glutamine or a product thereof) from ammonia, but that glnA was not directly involved in the genetic control of nitrogen regulation. The work from several groups (for reviews, see references 19,22 can cause the conversion of promoter-bound RNA polymerase containing the novel sigma subunit sigma-54, encoded by rpoN (instead of the normal sigma-70), from a closed complex to an open complex-at least in the case of the glnA promoter (32). Thus NTRC can activate transcription directly.This transcriptional activation was directly demonstrated in vitro ...

The transformation efficiency of BaciUlus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam-Dcm-Escherichia coli strain efficiently transformed several B. thuringiensis strains. B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.Bacillus thuringiensis is a gram-positive bacterium that produces insecticidal crystal proteins during sporulation. Crystal protein genes have been cloned and characterized in Escherichia coli, Bacillus subtilis, and Bacillus megaterium (for recent reviews, see references 14 and 17) because of the lack of an efficient transformation protocol for B. thuringiensis. Transformation procedures for B. thuringiensis protoplasts (1, 7) and vegetative cells (13) are inefficient, tedious, and time-consuming. Electroporation is an efficient means of introducing plasmid DNA into a number of different bacteria (26), and recently, B. thuringiensis transformation by electroporation has been described (2,5,16,20,25). Here, we report that DNA modifications are important for efficient transformation of B. thuringiensis.The electroporation procedure used for this work is an adaptation of the protocol for Streptococcus faecalis (lla). Stationary B. thuringiensis cultures grown overnight with shaking at 30°C in BHIG (brain heart infusion plus 0.5% [wt/vol] glycerol) were diluted 1:20 into BHIG and incubated for 1 h at 30°C with shaking. The cells were washed once in EB (0.625 M sucrose-1 mM MgCl2) and suspended in 1/2 volume of EB. A 0.8-ml volume of cells was mixed with less than 10 ,ul of DNA in a 0.4-cm cuvette, and the mixture was chilled on ice for 5 min. A 5-Ql resistor was set in series between the cuvette and a Bio-Rad Gene-Pulser. A single discharge (2,500 V, 25 ,uF) was used for electroporation. The cells were incubated on ice for 5 min, diluted into 1.6 ml of BHIG, and incubated with shaking at 30°C for 1 h. The transformation efficiency of the acrystalliferous B. thuringiensis subsp. kurstaki HD73-26 with pNN101 (24) DNA isolated from B. megaterium was 3 x 106 transformants per

The gene for glutamate dehydrogenase (gdhD) has been mapped in Klebsiella aerogenes by P1 transduction. It is linked to pyrF and trp with the order pyrF-trp-gdh. Complementation analysis using F' episomes from Escherichia coli suggests an analogous location in E. coli. Two mutants able to produce glutamate dehydrogenase in the presence of high levels of glutamine synthetase have been isolated. One, tightly linked to gdhD, shows normal repression control by glutamine synthetase but produces four times as much glutamate dehydrogenase activity as does the wild type under all conditions tested. The other revertant is not linked to gdhD or glnA.

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Bacillus thuringiensis 8-endotoxin (crystal protein) genes are normally expressed only during sporulation. It is possible to produce crystal protein during vegetative growth by placing B. thuringiensis crystal protein genes downstream of a strong vegetative promoter. By removing a possible transcriptional terminator of the tetracycline resistance gene of pBC16 and inserting a multiple cloning site, 8-endotoxin genes can be cloned downstream from the tetracycline resistance gene promoter. This construct allows for readthrough transcription from the strong vegetative promoter. Crystal protein is then produced during vegetative growth as well as during sporulation in both B. thuringiensis and Bacillus megaterium. This construct also allows for production of 8-endotoxin in B. thuringiensis strains that do not normally produce 8-endotoxin because of a defect in sporulation.