Mutation spoT of Escherichia coli increases expression of the histidine operon deleted for the attenuator

Abstract

F'-episomes carrying the Salmonella typhimurium wild-type or attenuatordeleted histidine (his) operons were introduced into Escherichia coli strains containing relA or spoT single and double mutations known to affect guanosine 3'-diphosphate 5'-diphosphate (ppGpp) and guanosine 3'-triphosphate 5'-diphosphate (pppGpp) levels. Expression of the his operon and expression of the gene for 6-phosphogluconate dehydrogenase (gnd) were measured during balanced growth in amino acid-rich and minimal media. The data were consistent with the interpretation that ppGpp is a positive effector of his operon expression, whereas pppGpp is not an essential effector. The conclusion that his operon expression is maximally stimulated at a lower than maximum intracellular ppGpp concentration was further confirmed. Neither ppGpp nor pppGpp appeared to influence gnd gene expression. The metabolic regulation of the E. coli his operon was found to be similar to the ppGpp-mediated metabolic regulation of the S. typhimurium his operon.Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been proposed as a positive effector of Salmonella typhimurium histidine (his) operon transcription both in vitro (21) and in vivo (21,26). The ppGpp-mediated effect on his operon expression in vivo has been termed "metabolic regulation" (21, 26). It has been suggested that ppGpp increases the mRNA initiation frequency from the his operon primary promoter in vivo by possibly acting as a direct effector of RNA polymerase (26).The conclusion that ppGpp acts as an in vivo effector has relied on a positive correlation between his operon expression and ppGpp levels for bacteria grown exponentially in amino acidrich medium versus minimal medium (26). The sampling showing this correlation was limited. Additional experiments suggesting ppGpp as an in vivo effector have been completed under conditions of unbalanced growth in strains which retained his operon attenuated control (hisO+) (21, 26). Therefore, although ppGpp clearly acts in vitro to stimulate his operon transcription (21), the evidence implicating ppGpp as an in vivo effector of his operon expression has been indirect.In this paper, we further examine the role of ppGpp as an in vivo positive effector of his operon expression in relA and spoT mutants of t Contribution no. Escherichia coli. The results were consistent with ppGpp as a positive effector of his operon expression during balanced growth. In contrast, guanosine 5'-triphosphate 3'-diphosphate (pppGpp) did not appear to play a necessary role in his operon metabolic regulation. MATERIALS AND METHODSBacterial strains and phage. The strains used or constructed in this study are given in Table 1. Auxotrophic markers were confirmed by growth tests. The presence or absence of the relA mutation in derivatives of strains WI, W2, W1l, and W21 was checked by a sensitivity test to the amino acid analogs ,Bchloroalanine, 2-thiazolealanine, and serine hydroxamate (J. A. Gallant, personal communication;21). In particular, strain W2 derivatives (relA+ spoT), which t...

In vivo role of the relA+ gene in regulation of the lac operon

“…This set of results with the lac operon in vivo and in vitro is consistent with a number of recent reports that identify specific genes (trp, his, ilv, ara) for which ppGpp may be a positive regulator (13,14,23,30,32,34). The evidence suggests that ppGpp can stimulate expression of both biosynthetic and catabolic enzymes; thus a simple nutritional shift, single amino acid limitation, can potentially have very broad consequences for the control of cellular gene activity through the positive and negative actions of this one regulatory nucleotide.…”

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression

“…The spoTI mutation caused elevated basal levels of ppGpp in cells growing in amino acid-supplemented minimal medium ( Table 2). Considering the data of Winkler et al (36,37) and the supportive evidence presented above, we ex- pected that the spoTI mutation would cause an elevation of the histidine biosynthetic enzymes during steady-state growth in the presence of amino acids. We monitored histidinol dehydrogenase (EC 1.1.1.23) activity, the final step in histidine biosynthesis, as a measure of his operon expression.…”

“…In vitro and in vivo studies have indicated that the his operon of Salmonella typhimurium is positively regulated by guanosine 5'-diphosphate-3'-diphosphate (ppGpp) (31,36,37). Stephens et al (31) have demonstrated that ppGpp is required for maximal in vitro expression by a mechanism which is independent of attenuator control.…”

The effect of the stringent response on mutation rates in Escherichia coli K‐12

“…As discussed earlier, the stringent response is characterized by a stimulation in the synthesis of his, trp, and a number of other messenger RNAs at the level of transcription initiation. Expression of the his operon has been correlated with ppGpp levels both in vivo (Rudd et al, 1985;Winkler et al, 1979) and in vitro (Riggs et al, 1986;Shand et al, 1989;Stephens et al, 1975); the range of ppGpp-dependent amino acid operon expression may be quite widespread (Xiao et al, 1991). The stimulating effect of ppGpp on transcription is specific for promoters in a number of amino acid biosynthetic genes and catabolic genes that have unfavourable sequences for initiating transcription in the absence of ppGpp, which apparently modifies the conformation of RNA polymerase and increases open complex formation and transcription.…”