A novel autoregulatory loop between the Gcn2-Atf4 pathway and (L)-Proline [corrected] metabolism controls stem cell identity.

Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity.

The Rhizobium GstI protein reduces the NH4+ assimilation capacity of Rhizobium leguminosarum.

We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs. In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.

Functional characterization of an ammonium transporter gene from Lotus japonicus.

NH(4)(+) is the main product of symbiotic nitrogen fixation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction. We report the cloning and characterization of the first member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus. Sequence analysis reveals a close relationship to plant transporters of the AMT1 family. The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast. LjAMT1;1 is transcribed in roots, leaves and nodules of L. japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH(4)(+) by the plant cells.

The Rhizobium etli argC gene is essential for Arginine biosynthesis and nodulation of Phaseolus vulgaris.

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.

Inhibition of glutamine synthetase II expression by the product of the gstI gene.

We report the identification of a previously unrecognized gene that is involved in the regulation of the Rhizobium leguminosarum glnII (glutamine synthetase II) gene. This gene, which is situated immediately upstream of glnII, was identified by means of a deletion/complementation analysis performed in the heterologous background of Klebsiella pneumoniae. It has been designated gstI (glutamine synthetase translational Inhibitor) because, when a complete version of gstI is present, it is possible to detect glnII-specific mRNA, but neither GSII activity nor GSII protein. The gstI gene encodes a small (63 amino acids) protein, which acts in cis or in trans with respect to glnII and is transcribed divergently with respect to glnII from a promoter that was found to be strongly repressed by the nitrogen transcriptional regulator NtrC. A mutated version of GstI lacking the last 14 amino acids completely lost its capacity to repress glnII expression. Our results indicate that gstI mediates the translation inhibition of glnII mRNA and, based on in silico analyses, a mechanism for GstI action is proposed.

Nodule invasion and symbiosome differentiation during Rhizobium etli-Phaseolus vulgaris symbiosis.

By means of a detailed ultrastructural analysis of nodules induced by Rhizobium etli on the roots of Phaseolus vulgaris, we observe that the development of host-invaded cells is not synchronous. An accumulation of mitochondria was found in freshly invaded host cells, containing only a few symbiosomes (SBs) that are released from highly branched intracellular ramification of the infection threads. Moreover, besides the fusion between the SB membrane with host secretory vesicles, we observe also a great number of fusions between the outer leaflets of adjoining SB membranes, thus resulting in structures that resemble the tight junction network (zona occludens with a five-layered structure) of epithelian cells. This process was found to be induced strongly and earlier both in the invaded host cells of ineffective nodules (elicited by Fix- mutant strains of R. etli) and in the older (senescence) invaded cells of effective nodules, whereas bacteroid division is seldom if ever observed. Our observations strongly suggest that multiple-occupancy SBs also arise by fusion of single-occupancy SBs and the physiological consequence of this process is discussed.

The Rhizobium etli trpB gene is essential for an effective symbiotic interaction with Phaseolus vulgaris.

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.

The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus vulgaris.

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.

The Rhizobium etli amtB gene coding for an NH4+ transporter is down-regulated early during bacteroid differentiation.

During development of root nodules, Rhizobium bacteria differentiate inside the invaded plant cells into N2-fixing bacteroids. Terminally differentiated bacteroids are unable to grow using the ammonia (NH3) produced therein by the nitrogenase complex. Therefore, the nitrogen assimilation activities of bacteroids, including the ammonium (NH4+) uptake activity, are expected to be repressed during symbiosis. By sequence homology the R. etli amtB (ammonium transport) gene was cloned and sequenced. As previously shown for its counterpart in other organisms, the R. etli amtB gene product mediates the transport of NH4+. The amtB gene is cotranscribed with the glnK gene (coding for a PII-like protein) from a nitrogen-regulated sigma 54-dependent promoter, which requires the transcriptional activator NtrC. Expression of the glnKamtB operon was found to be activated under nitrogen-limiting, free-living conditions, but down-regulated just when bacteria are released from the infection threads and before transcription of the nitrogenase genes. Our data suggest that the uncoupling between N2-fixation and NH3 assimilation observed in symbiosomes is generated by a transcriptional regulatory mechanism(s) beginning with the inactivation of NtrC in younger bacteroids.

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation.

By its inability to grow on sulfate as the sole sulfur source, a mutant strain (CTNUX8) of Rhizobium etli carrying Tn5 was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a cysG (siroheme synthetase)-homologous gene. By RNase protection assays, it was established that the cysG-like gene had a basal level of expression in thiosulfate- or cysteine-grown cells, which was induced when sulfate or methionine was used. Unlike its wild-type parent (strain CE3), the mutant strain, CTNUX8, was also unable to grow on nitrate as the sole nitrogen source and was unable to induce a high level of nitrite reductase. Despite its pleiotropic phenotype, strain CTNUX8 was able to induce pink, effective (N2-fixing) nodules on the roots of Phaseolus vulgaris plants. However, mixed inoculation experiments showed that strain CTNUX8 is significantly different from the wild type in its ability to nodulate. Our data support the notion that sulfate (or sulfite) is the sulfur source of R. etli in the rhizosphere, while cysteine, methionine, or glutathione is supplied by the root cells to bacteria growing inside the plant.

Cloning and transcriptional analysis of the lipA (lipoic acid synthetase) gene from Rhizobium etli.

We report here the isolation of a Rhizobium etli gene involved in lipoic acid metabolism, the lipA gene, which complements a lipA mutant strain of Escherichia coli. A promoter region (lipAp) was mapped immediately upstream of lipA and two in vivo transcription initiation sites were identified, preceded by sequences showing some homology to the -10/-35 promoter consensus sequences. The activity of the lipAp was found not to be regulated either by the carbon source or by the addition of lipoic acid. Moreover, quantitative analysis of the lipA transcript by RNase protection assays indicated its down-regulation during entry into stationary phase.

Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated.

Rhizobium bacteria fix atmospheric nitrogen during symbiosis with legume plants only after bacterial division is arrested. The role of the major vegetative sigma factor, SigA, utilized by Rhizobium bacteria during symbiosis is unknown. By using PCR technology, a portion of the sigA gene corresponding to domain II was directly amplified from Rhizobium etli total DNA by using two primers designed in accordance with the published sequence of sigA from Agrobacterium tumefaciens. The amplified fragment was cloned and used as a hybridization probe for cloning of the R. etli sigA gene. Sequencing data revealed an open reading frame of 2,055 bp showing extensive similarity to various vegetative sigma factors. The 5' end of the sigA transcript was determined and revealed a long, seemingly untranslated region of 170 nucleotides. Quantitative analysis of the sigA transcript by RNase protection and by primer extension assays indicated its down-regulation during entry into the stationary phase. On the basis of the structures of various vegetative sigma factors and considering previous information on heterologous expression, we speculate on the function of domain I of vegetative sigma factors.

In vitro characterization of the ORF1-ntrBC promoter of Rhizobium etli.

Rhizobium sigma vegetative-dependent promoters are different from those of enteric bacteria and have never been characterized before. We report here the biochemical characterization of the ORF1-ntrBC promoter of Rhizobium etli. The minimal promoter region was located by means of a transcriptional fusion and further characterized by in vitro transcription and gel retardation experiments. Oligonucleotides used as DNA competitors in runoff transcription experiments allowed the precise localisation of the promoter region. Protein extracts from an ntrC+, but not from an ntrC- strain, inhibited in vitro transcription. The NtrC protein was found to bind specifically to the promoter, where an NtrC binding site overlapping the transcription initiation site, is present.

DNA binding activity of NtrC from Rhizobium grown on different nitrogen sources.

The DNA-binding activity of the NtrC protein can be demonstrated in gel retardation assays with concentrated protein extracts of Rhizobium etli. Using extracts from either the wild type or a ntrC mutant strain and an antiserum raised against the NtrC protein, we demonstrate specific binding of NtrC to the upstream regulatory region of the glnII gene, where two putative NtrC-binding sites are present. KNO3-grown bacteria contain less NtrC protein and more NtrC-binding activity than NH4Cl-grown bacteria, thus showing that with this protocol it is possible to detect changes in NtrC-binding activity. The advantages of this assay system in comparison with that using pure proteins is discussed.

Regulation of nitrogen metabolism is altered in a glnB mutant strain of Rhizobium leguminosarum.

We isolated a Rhizobium leguminosarum mutant strain altered in the glnB gene. This event, which has never been described in the Rhizobiaceae, is rare in comparison to mutants isolated in the contiguous gene, glnA. The glnB mutation removes the glnBA promoter but in vivo does not prevent glnA expression from its own promoter, which is not nitrogen regulated. The glnB mutant strain does not grow on nitrate as a sole nitrogen source and it is Nod+, Fix+. Two -24/-12 promoters, for the glnII and glnBA genes, are constitutively expressed in the glnB mutant, while two -35/-10-like promoters for glnA and ntrBC are unaffected. We propose that the glnB gene product, the PII protein, plays a negative role in the ability of NtrC to activate transcription from its target promoters and a positive role in the mechanism of nitrate utilization.

Uridylylation of the PII protein in Rhizobium leguminosarum.

Permeabilization with cetyl trimethyl ammonium bromide was used to study the post-translational modification of the PII protein in Rhizobium leguminosarum. Upon incubation with radioactive UTP a single band was obtained after SDS-PAGE and autoradiography. RNase resistance and snake venom phosphodiesterase sensitivity showed that radioactivity was bound through a phosphodiester bond to a protein which was absorbed by an antiserum specific for the PII protein. Uridylylation of the PII protein was shown to be dependent on the modifications of the glutamine/alpha-ketoglutarate ratio.

The ntrBC genes of Rhizobium leguminosarum are part of a complex operon subject to negative regulation.

We report here that ntrB and ntrC genes of Rhizobium leguminosarum biovar phaseoli are cotranscribed with an open reading frame (called ORF1) of unknown function. The promoter region of the ORF1-ntrB-ntrC operon was mapped immediately upstream of ORF1 and two in vivo transcription initiation sites were identified, both preceded by -35/-10 promoter consensus sequences. Some major aspects differentiate R. leguminosarum from the enteric nitrogen regulatory system: the ntrBC genes are cotranscribed with ORF1 which is homologous to an ORF located upstream of ntrBC of R. capsulatus and to the ORF1 located upstream of the fis gene of Escherichia coli; ntrBC are not transcribed from a -24/-12 promoter and are only autogenously repressed. Moreover, the intracellular concentration of the NtrC protein increases when the bacterium is grown on ammonium salts, while under the same conditions the promoter of one of its target genes, glnII, is 12 times less active.

Activation of the Rhizobium leguminosarum glnII gene by NtrC is dependent on upstream DNA sequences.

The cloning and sequence determination is reported of the DNA region of Rhizobium leguminosarum coding for glutamine synthetase II (GSII). An open reading frame (ORF) encoding 326 amino acids was defined as the glnII gene on the basis of its similarity to other glnII genes and the ability of a DNA fragment carrying this ORF to complement the glutamine auxotrophy of a Klebsiella pneumoniae glnA mutant. We find that the glnII gene in R. leguminosarum is transcribed as a monocistronic unit from a single promoter, which shows structural features characteristic of rpoN (ntrA)-dependent promoters. In K. pneumoniae, such promoters require the ntrC and rpoN (ntrA) gene products for transcription. The intracellular level of glnII mRNA changes when R. leguminosarum is grown on different nitrogen sources, as expected for regulation by the nitrogen regulatory system. Promoter deletion analysis has shown that an extensive upstream DNA sequence (316 bp) is essential for in vivo activation of the glnII promoter in different biovars of R. leguminosarum. This DNA region requires a wild-type ntrC gene for activity and includes two conserved putative NtrC-binding site sequences. The results conclusively show that transcription from the R. leguminosarum glnII promoter is fully dependent on positive control by NtrC protein and on an upstream activator sequence (UAS).

Mitochondrial activity and heat-shock response during morphogenesis in the pathogenic fungus Histoplasma capsulatum.

Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms. In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 degrees C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium- to yeast-phase transition. The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature. We have found that by inducing thermotolerance, i.e., an initial incubation at 34 degrees C, the thermosensitive attenuated Downs strain of H. capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures. Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 degrees C proceeds at rates comparable to those of the virulent strains.