The cryptic β-glucoside GFB (bglGFB) operon in Escherichia coli (E. coli) can be activated by mutations arising under starvation conditions in the presence of an aromatic β-glucoside. This may involve the insertion of an insertion sequence (IS) element into a “stress-induced DNA duplex destabilization” (SIDD) region upstream of the operon promoter, although other types of mutations can also activate the bgl operon. Here, we show that increased expression of the bglG gene, encoding a well-characterized transcriptional antiterminator, dramatically increases the frequency of both IS-mediated and IS-independent Bgl+ mutations occurring on salicin- and arbutin-containing agar plates. Both mutation rates increased with increasing levels of bglG expression but IS-mediated mutations were more prevalent at lower BglG levels. Mutations depended on the presence of both BglG and an aromatic β-glucoside, and bglG expression did not influence IS insertion in other IS-activated operons tested. The N-terminal mRNA-binding domain of BglG was essential for mutational activation, and alteration of BglG’s binding site in the mRNA nearly abolished Bgl+ mutant appearances. Increased bglG expression promoted residual bgl operon expression in parallel with the increases in mutation rates. Possible mechanisms are proposed explaining how BglG enhances the frequencies of bgl operon activating mutations.
Using reporter gene (lacZ) transcriptional fusions, we examined the transcriptional dependencies of the bgl promoter (Pbgl) and the entire operon regulatory region (Pbgl-bglG) on eight transcription factors as well as the inducer, salicin, and an IS5 insertion upstream of Pbgl. Crp-cAMP is the primary activator of both Pbgl and the bgl operon, while H-NS is a strong dominant operon repressor but only a weak repressor of Pbgl. H-NS may exert its repressive effect by looping the DNA at two binding sites. StpA is a relatively weak repressor in the absence of H-NS, while Fis also has a weak repressive effect. Salicin has no effect on Pbgl activity but causes a 30-fold induction of bgl operon expression. Induction depends on the activity of the BglF transporter/kinase. IS5 insertion has only a moderate effect on Pbgl but causes a much greater activation of the bgl operon expression by preventing the full repressive effects of H-NS and StpA. While several other transcription factors (BglJ, RcsB, and LeuO) have been reported to influence bgl operon transcription when overexpressed, they had little or no effect when present at wild type levels. These results indicate the important transcriptional regulatory mechanisms operative on the bgl operon in E. coli.
Two recent reports described the isolation of derivatives of a Rhizobium trifolii strain that had gained the ability to nodulate Glycine max and Vigna radiata and that had demonstrated altered patterns of carbon source utilization, free-living nitrogen fixation, and hydrogen uptake. More extensive characterization of these strains now supports the conclusion that these strains are R. japonicum and are not derived from the putative parent R. trifolii.Two recent communications (1, 2) described derivatives of Rhizobium trifolii, strain T1, that appeared to have acquired several unusual properties simultaneously through a single-step selection for decreased assimilation of ammonium. Because these derivatives were also found to be resistant to the antimetabolite L-methionine-D,L-sulfoximine, additional isolates were selected as being resistant to this compound. These new isolates were also initially characterized as defective in ammonium assimilation, but in fact they lacked none of the enzymes of this pathway-namely, glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.4). These strains also grew much more slowly than the putative parent strain DT6, an isolate of R. trifolii T1, and metabolized a different range of carbon compounds. Furthermore, these isolates could reduce dinitrogen to ammonium (fix nitrogen) in appropriate culture conditions (2) even though neither R. trifolii nor any other fast-growingRhizobium has yet been found to do so.Subsequently, these same isolates were reported to form fully effective N2-fixing nodules on both Glycine max and Vigna radiata. This report was highly promising because R. japonicum strains are normally specific for the legume hosts with which they form effective symbioses (3), although some Rhizobium strains exhibit broader specificity. The previous report suggested a means of selecting Rhizobium derivatives with a greatly altered host range, and it offered a potential insight into the factors involved in the determination of specificity for both nodulation and nitrogen fixation.The potential significance of these observations prompted us to further examine strains DT71, DT72, DT125, DT128, DT129, and DT130 (1, 2) by various procedures: DNA equilibrium density centrifugation, phage susceptibility, carbon source utilization, immunofluorescence, immunodiffusion, and nodulation specificity. We show that these isolates behaved similarly to each other, but differed from the presumed parental strain, R. trifolii DT6, in all of these tests. At the same time, they showed a strong similarity with the R. japonicum strain 3I-lb-110 (USDA 110). Table 1.) Methods of culture and derivation of R. trifolii T1, its derivatives, and R. japonicum 3I-lb-110 (USDA 110) have been described (1, 2, 4-6). Strain DT71 was isolated-after selection for ammonium-growth-defective derivatives of DT6. Strain DT72 is a spontaneous rifr mutant of DT71. DT125 was isolated as a methionine sulfoximine-resistant (Msxr) derivative of DT6. DT128, DT129, a...
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