Evidence that nitrogen regulatory gene ntrC of Salmonella typhimurium is transcribed from the glnA promoter as well as from a separate ntr promoter

The frizzy (frz) mutants of Myxococcus xanthus are unable to form fruiting bodies. Instead of forming discrete mounds, these strains aggregate as ifiaments which have a circular and tangled appearance. Mutations leading to this phenotype have been mapped to five complementation groups, frzA, frzB, fzCD, frzE, andfrzF. All have been found to be involved in the control of directional movement of the bacteria and, except forfrzB, to be homologous to the chemotaxis genes of enteric bacteria. In this report we present a study of the regulation of expression of the first four genes of the frz gene cluster (frzA, frzB, frzCD, and frzE) by using TnS-Lac transcriptional fusions as reporters of gene expression. We found that these frz genes are developmentally regulated, with their transcription peaking at about the time of early mound formation (12 to 18 h). Analysis of FrzCD expression by enzyme-linked immunosorbent assay showed a 10-fold greater induction at 15 h of development over the level of vegetative cell expression. Northern blot hybridization analysis suggested that the frz genes were arranged as an operon. To test this hypothesis, double mutants were constructed which contained TnS-132 either upstream or downstream of the reporter TnS-lac. The expression of thefrz genes in the double mutants was consistent with the hypothesis that the first four genes (frz4, frzB, frzCD, andfrzE) are organized as an operon with an internal promoter. Insertion mutations in frzCD lowered gene expression whether they were upstream or downstream of the reporter TnS-lac, suggesting that the FrzCD protein regulates transcription of the entire operon from a promoter upstream offrzA. Evidence is presented suggesting that FrzE is required for induction of transcription as well. Whenfrz mutations were placed in strains that were unable to aggregate (tag), thefrz genes were expressed at an elevated level on fruiting agar; this high level of expression was maintained for several days. These results suggest that the tag gene products interact with the frz functions.Myxococcus xanthus is a gram-negative, rod-shaped soil bacterium that moves by gliding motility. It exhibits a complex life cycle (36,40). When grown vegetatively on a solid surface, cells travel in large groups referred to as swarms or hunting groups which feed on other microorganisms or organic material in their path. In the absence of nutrients, these swarms aggregate to form moundlike structures in which the cells differentiate into myxospores (34). These mounds of spores are called fruiting bodies. When nutrients are replenished, the spores germinate in numbers large enough to create a new hunting group. We have been studying a group of mutants that exhibit an aberrant pattern of aggregation during development. These strains fail to form discrete mounds but, rather, assemble into complex multicellular filaments which have a circular and tangled appearance termed frizzy (Frz) (39). These strains, however, are capable of forming myxospores. Previous work in our laboratory h...

Section: Discussionsupporting

confidence: 71%

“…The proteins transcribed from the internal promoter, FrzCD and FrzE, are hypothesized to act as transcriptional activators of an upstream promoter of the gene cluster. The pattern of regulation shows similarities to those of other two-component regulatory systems of sensory transduction in bacteria (24,32).…”

mentioning

confidence: 51%

Evidence that the Myxococcus xanthus frz genes are developmentally regulated

Weinberg

Zusman

1989

“…An ntrC null allele (ntrC10::Tn5) was introduced by P1 transduction into a wild-type E. coli strain carrying the ⌽(nifHЈ-ЈlacZ) fusion (see Materials and Methods), and as expected, the resulting strain (NCM1850) could not utilize arginine as the sole nitrogen source (Aut Ϫ phenotype). (Although the pathway for arginine catabolism has not been defined, growth on arginine as a glnG and glnL in E. coli are referred to as ntrC and ntrB, respectively, in other organisms, and we employ the latter terminology in the text, figures, and Tables 2 to 7 the sole nitrogen source has been used as being diagnostic of the amount and activity of NtrC [33,34,40,51].) Surprisingly, in the ntrC strain carrying the nifLA plasmid (NCM1851), the differential rate of ␤-galactosidase synthesis from the nifH promoter was only about 8 U/ml/OD 600 under derepressing conditions, which is more than 200-fold lower than that in the wild-type strain (Table 3).…”

Section: Resultsmentioning

confidence: 99%

NtrC is required for control of Klebsiella pneumoniae NifL activity

Soupène

Kustu

1997

In response to molecular oxygen and/or fixed nitrogen, the product of the Klebsiella pneumoniae nitrogen fixation L (nifL) gene inhibits NifA-mediated transcriptional activation. Nitrogen regulation of NifL function occurs at two levels: transcription of the nifLA operon is regulated by the general Ntr system, and the activity of NifL is controlled by an unknown mechanism. We have studied the regulation of NifL activity in Escherichia coli and Salmonella typhimurium by monitoring its inhibition of NifA-mediated expression of a K. pneumoniae ⌽(nifH-lacZ) fusion. The activity of the NifL protein transcribed from the tac promoter is regulated well in response to changes of oxygen and/or nitrogen status, indicating that no nif-or K. pneumoniae-specific product is required. Unexpectedly, strains carrying ntrC (glnG) null alleles failed to release NifL inhibition, despite the fact that synthesis of NifL was no longer under Ntr control. Additional evidence indicated that it is indeed the transcriptional activation capacity of NtrC, rather than its repression capacity, that is needed, and hence it is a plausible hypothesis that NtrC activates transcription of a gene(s) whose product(s) in turn functions to relieve NifL inhibition under nitrogen-limiting conditions.In diazotrophs that are members of the proteobacteria, transcription of the nitrogen fixation nif genes is mediated by the nif-specific activator protein NifA, a member of the 54

“…In wild-type strains of enteric bacteria, NRI controls the transcription of ginG, which codes for NRI (1,15,16,22,23). To analyze the effect of changes in the concentration of NRI on the expression of the glnALG operon, we removed control of glnG expression from the gInALG operon.…”

Section: Resultsmentioning

confidence: 99%

“…1). NRI represses transcription from the glnL promoter (1,9,15,22,24,35), because a high-affinity NRI-binding site overlaps the RNA polymerase-binding site (36). Therefore, ,B-galactosidase activity from these cells is a measure of the specific binding of NR, to DNA.…”

Section: Resultsmentioning

confidence: 99%

Role of nitrogen regulator I (NtrC), the transcriptional activator of glnA in enteric bacteria, in reducing expression of glnA during nitrogen-limited growth

Shiau

Schneider

et al. 1992

During nitrogen-limited growth, transcription of ginA, which codes for glutamine synthetase, requires o54-RNA polymerase and the phosphorylated form of nitrogen regulator I (NRI; also called NtrC). In cells in which the lac promoter controlled expression of the gene coding for NRI, increasing the intracellular concentration of NRI lowered the level of glutamine synthetase. The reduction in glutamine synthetase does not appear to result from the NRI-dependent sequestering of any protein that affects transcription of ginA. Our results also suggest that the negative effect of a high concentration of NRI on ginA expression is a major determinant of the level of glutamine synthetase activity in nitrogen-limited cells of a wild-type strain. We propose that the inhibition results from an impairment of the interaction between NRI-phosphate and RNA polymerase that stimulates ginA transcription. We discuss a model that can account for this reduction in glutamine synthetase.Transcription of g1nA, which codes for glutamine synthetase, is initiated from the glnAp2 promoter in Escherichia coli and other enteric bacteria during nitrogen-limited growth (5, 10, 25). The initiation of transcription from glnAp2 requires a54-RNA polymerase (10, 11) and the phosphorylated form of nitrogen regulator I (NRI; also called NtrC) (13,20 We recently verified that a high concentration of NR, inhibits the synthesis of glutamine synthetase (4, 31); workers in another laboratory observed a similar phenomenon in an in vitro system (37). We analyzed the mechanism of the diminished transcription with a high concentration of NRI because it might be a manifestation of systems controlled either by phosphorylated proteins or by proteins that can activate from a distance. We conclude that in cells with a high concentration of NRI neither titration for any essential component nor the presence of low-affinity binding sites near the promoter can account for the lower level of glutamine synthetase. We also conclude that this reciprocal relationship is a major determinant of the level of glutamine synthetase in nitrogen-starved cells of a wild-type strain.(This work was submitted in partial fulfillment of the requirements for the degree of