Previous work has indicated that nitrogen regulatory genes ntrB and ntrC of Salmonella typhimurium are closely linked to glnA, the structural gene encoding glutamine synthetase; proceeding clockwise the order of genes in the 86 U region of the map is polA...ntrC ntrB glnA glnA promoter...rha. To study ntrC transcription we have constructed operon fusions of ntrC to lacZ using the Casadaban Mu d1 (Apr lac) phage so that we can measure beta-galactosidase activity as a reflection of ntrC transcription and we have introduced into fusion strains promoter constitutive mutations at glnA [glnAp(Con)]. The glnAp(Con) mutations, which elevate glnA expression in fusion strains, also elevate beta-galactosidase activity, indicating that ntrC is cotranscribed with glnA. Consistent with this interpretation, polar insertion mutations in glnA decrease beta-galactosidase activity of fusion strains carrying glnAp(Con) mutations. However, glnA insertions do not eliminate beta-galactosidase activity of glnAp(Con) ntrC::Mu d1 strains and they have little effect on beta-galactosidase activity of the original ntrC::Mu d1 fusion strains. The latter results confirm that ntrC can also be transcribed from an ntr promoter downstream of glnA. Polar insertion mutations in ntrB eliminate beta-galactosidase activity of both the original fusion strains and fusion strains carrying glnA(Con) mutations, indicating that the ntr promoter lies between glnA and ntrB.
In enteric bacteria products of nitrogen regulatory genes ntrA, ntrB and ntrC are known to regulate transcription both positively and negatively at glnA, the structural gene encoding glutamine synthetase [L-glutamate:ammonia-ligase (ADP-forming), EC 6.3.1.2]. We have characterized two types of cis-acting mutations in the glnA promoter-regulatory region. One type, which we have called promoter Up [glnAp (Up)], elevates transcription of glnA to high levels without need for ntr-mediated activation but leaves expression sensitive to ntr-mediated repression. The other type renders glnA transcription insensitive to repression but leaves it normally responsive to activation. Properties of the two types of promoter-regulatory mutations suggest that sites for ntr-mediated activation of glnA transcription are functionally distinct from sites for ntr-mediated repression.
Secondary mutants able to utilize
d
-histidine,
dhu
, were isolated in histidine auxotrophs of
Salmonella typhimurium
. Mutations of one class (
dhuA
) are closely linked with the
hisP
locus which codes for a component of histidine permease. The specific activity of
l
-histidine permeation was estimated as increased two- to seven-fold in
dhuA
mutants. The
dhuB
mutants which have not been mapped also had elevated specific activity of
l
-histidine permeation. The uptake of
d
-histidine, barely detectable in the parental strains, was prominent in
dhuA
mutants and showed an apparent Michaelis constant about 1,000-fold higher than that observed with
l
-histidine. No change was detected in the kinetics of
l
-histidine permeation.
d
- and
l
-histidine competed in the uptake process. Tertiary mutants which lost the ability to grow on
d
-histidine were isolated by ampicillin counter-selection in
dhuA his
−
strains. All of them mapped in the
dhuA hisP
region. Most of them had all known properties of
hisP
mutants. It is inferred from these data that the
dhuA
mutations increase synthesis of components critical to
d
- and
l
-histidine permeation.
Using the Casadaban Mu d1 phage (Casadaban and Cohen 1979) we fused cis-acting regulatory sites for the Salmonella typhimurium glnA gene, the structural gene encoding glutamine synthetase, to lacZ so that transcription of lacZ was controlled by the glnA promoter-operator. Activities of beta-galactosidase in two glnA::Mu d1 fusion strains were high, approximately 25% and 125% the induced level of beta-galactosidase when transcription of lacZ is under control of the lac promoter, indicating that glutamine synthetase is not required to activate transcription of its own structural gene. Introduction of nitrogen regulatory mutations ntrA::Tn10 or ntrC::Tn10 into fusion strains resulted in greatly decreased synthesis of beta-galactosidase indicating that the positive regulatory factors encoded by ntrA and ntrC activate glnA expression at the level of transcription. Comparison of beta-galactosidase activities in fusion strains with those in fusions carrying ntrC or ntrA mutations indicated that: 1) the magnitude of activation of glnA expression is at least 43-fold; 2) the magnitude of repression is approximately 13-fold and repression occurs at the level of transcription; 3) the degree of modulation of glnA expression by ntr products is at least 560-fold (13 X 43); and 4) glutamine synthetase is not required for repression of transcription of its own structural gene. In contrast to strains carrying non-polar mutations in glnA, strains carrying glnA insertion mutations, including glnA::Mu d1 fusions, are apparently defective in activating expression of some nitrogen controlled genes other than glnA. Defects cannot be accounted for by the absence of glutamine synthetase protein or catalytic activity; they appear to be due to decreased expression of nitrogen regulatory genes ntrB and/or ntrC, which are adjacent to glnA.
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