Physiological role of the GlnK signal transduction protein of Escherichia coli : survival of nitrogen starvation

Escherichia coli contains two PII-like signal trans-duction proteins, PII and GlnK, involved in nitrogen assimilation. We examined the roles of PII and GlnK in controlling expression of glnALG , glnK and nac during the transition from growth on ammonia to nitrogen starvation and vice versa. The PII protein exclusively controlled glnALG expression in cells adapted to growth on ammonia, but was unable to limit nac and glnK expression under conditions of nitrogen starvation. Conversely, GlnK was unable to limit glnALG expression in cells adapted to growth on ammonia, but was required to limit expression of the glnK and nac promoters during nitrogen starvation. In the absence of GlnK, very high expression of the glnK and nac promoters occurred in nitrogen-starved cells, and the cells did not reduce glnK and nac expression when given ammonia. Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation. GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia. After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia. In contrast, wild-type cells that were starved immediately resumed rapid growth when fed ammonia. Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild-type cells. This complex phenotype resulted partly from the requirement for GlnK to regulate nac expression; deletion of nac restored wild-type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation. The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the glnK promoter in cells lacking GlnK restored the wild-type phenotypes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72950/1/j.1365-2958.2002.03153.x.pd

Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors.

The pluripotent nature of human embryonic stem cells (hESCs) makes them convenient for deriving therapeutically relevant cells. Here we show using Wnt reporter hESC lines that the cells are heterogeneous with respect to endogenous Wnt signalling activity. Moreover, the level of Wnt signalling activity in individual cells correlates with differences in clonogenic potential and lineage-specific differentiation propensity. The addition of Wnt protein or, conversely, a small-molecule Wnt inhibitor (IWP2) reduces heterogeneity, allowing stable expansion of Wnt(high) or Wnt(low) hESC populations, respectively. On differentiation, the Wnt(high) hESCs predominantly form endodermal and cardiac cells, whereas the Wnt(low) hESCs generate primarily neuroectodermal cells. Thus, heterogeneity with respect to endogenous Wnt signalling underlies much of the inefficiency in directing hESCs towards specific cell types. The relatively uniform differentiation potential of the Wnt(high) and Wnt(low) hESCs leads to faster and more efficient derivation of targeted cell types from these populations

Nac-mediated repression of the serA promoter of Escherichia coli

Escherichia coli and related bacteria contain two paralogous PII-like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK. Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon. Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR-type transcription factor that is part of the Ntr regulon. Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type. When expressed from high-copy plasmids, Nac imparts a growth defect to wild-type cells in an expression level-dependent manner. Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects. The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium. These observations suggest that high Nac expression results in a reduction in serine biosynthesis. β -Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10-fold in cells with high Nac expression. We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co-ordinated manner when cells become starved for nitrogen.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72401/1/j.1365-2958.2002.02994.x.pd

Two Classes of Dosage Compensation Complex Binding Elements along Caenorhabditis elegans X Chromosomes ▿ †

Dosage compensation equalizes X-linked gene products between the sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both X chromosomes in XX animals and halves the transcription from each. The DCC is recruited to the X chromosomes by a number of loci, rex sites, and is thought to spread from these sites by an unknown mechanism to cover the rest of the chromosome. Here we describe a novel class of DCC-binding elements that we propose serve as “way stations” for DCC binding and spreading. Both rex sites and way stations comprise strong foci of DCC binding on the native X chromosome. However, rex sites maintain their ability to bind large amounts of DCC even on X duplications detached from the native X, while way stations do not. These results suggest that two distinct classes of DCC-binding elements facilitate recruitment and spreading of the DCC along the X chromosome

Antagonism of PII signalling by the AmtB protein of Escherichia coli

Escherichia coli AmtB is a member of the MEP/Amt family of ammonia transporters found in archaea, eubacteria, fungi, plants and animals. In prokaryotes, AmtB homologues are co-transcribed with a PII paralogue, GlnK, in response to nitrogen limitation. Here, we show that AmtB antagonizes PII signalling through NRII and that co-expression of GlnK with AmtB overcomes this antagonism. In cells lacking GlnK, expression of AmtB during nitrogen starvation prevented deinduction of Ntr gene expression when a nitrogen source became available. The absence of AmtB in cells lacking GlnK allowed rapid reduction of Ntr gene expression during this transition, indicating that one function of GlnK is to prevent AmtB-mediated antagonism of PII signalling after nitrogen starvation. Other roles of GlnK in controlling Ntr gene expression and maintaining viability during nitrogen starvation were unaffected by AmtB. Expression of AmtB from a constitutive promoter under nitrogen-rich conditions induced full expression of glnALG and elevated expression of glnK in wild-type and glnK cells; thus, the ability of AmtB to raise Ntr gene expression did not require a factor found only in nitrogen-starved cells. Experiments with intact cells showed that AmtB acted downstream of a uridylyl transferase uridylyl-removing enzyme (UTase/UR) and upstream of NRII, suggesting that the target was PII. AmtB also slowed the deuridylylation of PII∼UMP upon ammonia addition, showing that multiple PII interactions were affected by AmtB. Our data are consistent with a hypothesis that AmtB interacts with PII and GlnK, and that co-transcription of glnK and amtB prevents titration of PII when AmtB is highly expressed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75322/1/j.1365-2958.2003.03479.x.pd

Context-Dependent Functions of the PII and GlnK Signal Transduction Proteins in Escherichia coli

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK

Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors.

The pluripotent nature of human embryonic stem cells (hESCs) makes them convenient for deriving therapeutically relevant cells. Here we show using Wnt reporter hESC lines that the cells are heterogeneous with respect to endogenous Wnt signalling activity. Moreover, the level of Wnt signalling activity in individual cells correlates with differences in clonogenic potential and lineage-specific differentiation propensity. The addition of Wnt protein or, conversely, a small-molecule Wnt inhibitor (IWP2) reduces heterogeneity, allowing stable expansion of Wnt(high) or Wnt(low) hESC populations, respectively. On differentiation, the Wnt(high) hESCs predominantly form endodermal and cardiac cells, whereas the Wnt(low) hESCs generate primarily neuroectodermal cells. Thus, heterogeneity with respect to endogenous Wnt signalling underlies much of the inefficiency in directing hESCs towards specific cell types. The relatively uniform differentiation potential of the Wnt(high) and Wnt(low) hESCs leads to faster and more efficient derivation of targeted cell types from these populations

Activation of the glnA, glnK, and nac Promoters as Escherichia coli Undergoes the Transition from Nitrogen Excess Growth to Nitrogen Starvation

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRI∼P (NtrC∼P). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration