Nutritional Downshift Research Articles

Molecular components of physiological stress responses in Escherichia coli.

In order to survive under and adapt to different conditions Escherichia coli has evolved elaborate systems that are able to sense and respond to environmental stimuli. Very often, different stresses act on a bacterium simultaneously and a variety of stresses have similar effects on cellular molecules and processes. Therefore, the various stress response systems have to interact (cross talk) with each other. A complex network of global regulatory systems with a multitude of molecular components ensures a coordinated and effective answer. Such regulatory components include DNA, mRNAs, sRNAs, proteins, such as DNA-and RNA binding proteins, alternative sigma factors and two-component systems, as well as small molecular weight molecules, as for example (p)ppGpp. These regulatory systems govern the expression of a plethora of further effectors that aim at maintaining stability of the cellular equilibrium under the various conditions. Using five of the most important stress response systems, we will discuss the roles and mechanisms of such regulatory and effector molecules in more detail. The heat shock response, controlled by the sigma factor sigma32, and the envelope stress response, controlled by the sigma factor sigmaE and the Cpx two-component system, both result in an increased expression of chaperones and proteases in response to misfolded proteins. The cold shock response governs expression of RNA chaperones and ribosomal factors, ensuring accurate translation at low temperatures. The general stress response depends on the sigma factor sigmaS, which controls the expression of more than 50 genes conferring resistance to many different stresses. The (p)ppGpp-dependent stringent response reduces the cellular protein synthesis capacity and controls further global responses upon nutritional downshift. A lot has been learned in recent years about the mechanisms of action of single components. However, the main challenge for the future is to achieve an understanding of the interactions of these components under different physiological conditions.

Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli.

Inorganic polyphosphate (polyP) kinase was studied for its roles in physiological responses to nutritional deprivation in Escherichia coli. A mutant lacking polyP kinase exhibited an extended lag phase of growth, when shifted from a rich to a minimal medium (nutritional downshift). Supplementation of amino acids to the minimal medium abolished the extended growth lag of the mutant. Levels of the stringent response factor, guanosine 5'-diphosphate 3'-diphosphate, increased in response to the nutritional downshift, but, unlike in the wild type, the levels were sustained in the mutant. These results suggested that the mutant was impaired in the induction of amino acid biosynthetic enzymes. The expression of an amino acid biosynthetic gene, hisG, was examined by using a transcriptional lacZ fusion. Although the mutant did not express the fusion in response to the nutritional downshift, Northern blot analysis revealed a significant increase of hisG-lacZ mRNA. Amino acids generated by intracellular protein degradation are very important for the synthesis of enzymes at the onset of starvation. In the wild type, the rate of protein degradation increased in response to the nutritional downshift whereas it did not in the mutant. Supplementation of amino acids at low concentrations to the minimal medium enabled the mutant to express the hisG-lacZ fusion. Thus, the impaired regulation of protein degradation results in the adaptation defect, suggesting that polyP kinase is required to stimulate protein degradation.

RIBOSOME SYNTHESIS IN TETRAHYMENA: A QUANTITATIVE ANALYSIS

We have performed a detailed quantitative analysis of the transcription and accumulation of ribosomal RNA and ribosomal protein mRNA in the ciliated protozoan Tetrahymena thermophila during changes in growth conditions, and found that: (1) nutritional downshifts lead to a rapid decrease in transcriptional activity whereas nutritional upshifts lead to rapid restoration of transcriptional activity, (2) starvation leads to decreased translation of ribosomal protein mRNA and (3) the rate of ribosomal protein mRNA degradation decreases after a nutritional upshift. We present evidence that the proximal promoters of two ribosomal protein genes and the ribosomal RNA gene compete for binding of nuclear factor(s) in vitro, suggesting that the coordinated regulation of these genes may involve a common set of transcriptional regulators.

Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase

Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47–52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures.

The Ste16 WD-repeat protein regulates cell-cycle progression under starvation through the Rum1 protein in Schizosaccharomyces pombe.

The haploid cells of the fission yeast, Schizosaccharomyces pombe, are arrested in the G1-phase by nitrogen starvation and are committed to sexual reproduction (mating and sporulation). We isolated the sterile mutants which were defective in G1 arrest following nitrogen starvation. Genetic analysis of these mutants defined a single locus designated as ste16. The nucleotide sequence revealed that ste16+ encodes an 82-kDa protein containing eight WD40-repeats in its carboxy terminal half. The ste16 disruptant was viable, but arrested the cell cycle in the G2-phase after the nutritional down-shift. When transferred to fresh growth medium, the G2-arrested ste16Delta haploids resumed the mitotic cycle from the S-phase, resulting in diploidization. This diploidization phenomenon was completely suppressed by the null mutation of rum1 encoding the inhibitor of Cdc2 kinase. As the Rum1 protein level was remarkably elevated in the ste16Delta, the Ste16 protein negatively controls the Rum1 level. The loss of function of ste16 disturbs the cell-cycle progression and impairs the mechanism for the maintenance of ploidy.

In vitro Tn7 mutagenesis of Haemophilus influenzae Rd and characterization of the role of atpA in transformation.

Haemophilus influenzae Rd is a gram-negative bacterium capable of natural DNA transformation. The competent state occurs naturally in late exponential growth or can be induced by a nutritional downshift or by transient anaerobiosis. The genes cya, crp, topA, and sxy (tfoX) are known to function in the regulation of competence development. The phosphoenolpyruvate:carbohydrate phosphotransferase system functions to maintain levels of cyclic AMP necessary for competence development but is not directly involved in regulation. The exact signal(s) for competence and the genes that mediate the signal(s) are still unknown. In an effort to find additional regulatory genes, H. influenzae Rd was mutated by using an in vitro Tn7 system and screened for mutants with a reduced ability to induce the competence-regulatory gene, comA. Insertions in atpA, a gene coding for the alpha subunit of the F1 cytoplasmic domain of the ATP synthase, reduce transformation frequencies about 20-fold and cause a significant reduction in expression of competence-regulatory genes, while the expression of constitutive competence genes is only minimally affected. In addition, we found that an insertion in atpB, which encodes the a subunit of the F0 membrane-spanning domain, has a similar effect on transformation frequencies.

Expression analysis of the ssgA gene product, associated with sporulation and cell division in Streptomyces griseus

The ssgA gene of Streptomyces griseus B2682, when present in high copy number, results in both suppression of sporulation and fragmented growth of mycelia. Western analysis with polyclonal antibodies against the gene product (SsgA) revealed a close correlation between SsgA accumulation and the onset of sporulation in wild-type cells. The protein was only detected in the cytoplasm. Certain developmental mutants of S. griseus (afs, reIC and brgA) which are defective in aerial mycelium formation in solid culture and submerged spore formation in liquid culture failed to accumulate SsgA. The SsgA protein appeared shortly (1 h) after nutritional shift-down of strain B2682 cells. afs mutant cells sporulated and expressed SsgA only when A-factor was present both before and after nutritional shift-down. Introduction of the ssgA gene in a low-copy-number vector into strain B2682 resulted in fivefold overexpression of SsgA, and was accompanied by fragmented growth of mycelia and suppression of submerged spore formation (in liquid culture) and aerial mycelium formation (in solid culture). Streptomycin production was not inhibited. In a control experiment, a nonfunctional ssgA gene possessing a frameshift mutation near its N-terminus had no effect on either growth or sporulation. It is proposed that the ssgA gene product plays a role in promoting the developmental process of S. griseus.

Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization.

We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl). Seedlings revealed organ-specific expression and differential gene regulation after salt stress. A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid exchanges at specific positions, and this suggested differences in the enzyme properties or the regulation. SAMS1 and SAMS2 are of type I and SAMS3 is of type II. The properties of the isoenzymes were compared after heterologous expression of the individual enzymes, but no significant differences were detected in a) optima for temperature (37 to 45 degrees C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, NH4+, Na+); c) K(m)s for ATP and L-methionine; d) inhibition by reaction products (S-adenosyl-L-methionine, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography). The results represent the first characterization of plant SAMS isoenzyme properties with individually expressed proteins. The possibility is discussed that the isoenzyme differences reflect specificities in the association with enzymes that use S-adenosyl-L-methionine.

Role of the two-component signal transduction and the phosphoenolpyruvate: carbohydrate phosphotransferase systems in competence development of Haemophilus influenzae Rd.

Haemophilus influenzae Rd becomes competent for transformation by nutritional downshift or transient anaerobic growth through a process that requires cyclic AMP receptor protein and adenylate cyclase. Insertion mutations in crr or ptsI of the phosphoenolpyruvate:carbohydrate phosphotransferase system lowered transformation frequencies, and the effect was reversed by the addition of cyclic AMP. However, insertions into H. influenzae homologs of two-component signal transduction genes had no effect on competence.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), guanosine 5'-diphosphate 3'-monophosphate (ppGp) and antibiotic production in Streptomyces clavuligerus

Streptomyces clavuligerus produced cephamycin C under all nutrient limitations investigated in batch culture, whilst clavulanic acid was produced under phosphate and carbon limitations. Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) was produced at very low levels in all fermentations, prior to the detection of cephamycin C in the fermentation broth. Guanosine 5′-diphosphate 3′-monophosphate (ppGp) was observed at high levels in all fermentations; it appeared following a downturn in nutrient levels and immediately prior to detection of isopenicillin N synthase (IPNS). ppGp was detected following nutritional shiftdown by amino acid depletion and it was not produced via degradation of ppGpp. The results point to a possible role for ppGp in the regulation of cephamycin production in S. clavuligerus.

The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control.

In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma hrdB, the principal sigma factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNA(Leu) for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis

To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.

Vitamin-B12-independent methionine synthase from a higher plant (Catharanthus roseus). Molecular characterization, regulation, heterologous expression, and enzyme properties.

Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in L-methionine biosynthesis, and it also serves to regenerate the methyl group of S-adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50% identify with the cobalamin-independent methionine synthase from Escherichia coli (MetE) and 41% identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require S-adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plants extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.

Three genes hrdB, hrdD and hrdT of Streptomyces griseus IMRU 3570, encoding sigma factor-like proteins, are differentially expressed under specific nutritional conditions

Three genes ( hrd) homologous to the rpoD gene of Escherichia coli, that encode sigma factor-like proteins, have been cloned from DNA of the candicidin-producing strain Streptomyces griseus IMRU 3570. They are located in different regions of the chromosome. Sequence analysis showed that the first one is analogous to the hrdB gene of S. coelicolor. The second showed high similarity to the hrdD gene of S. coelicolor and S. aureofaciens and is linked, as in S. coelicolor, to a N-acetyltransferase-encoding gene ( nat) distantly related to the pat and bar genes that encode resistance to bialafos. The third showed no close homology with other known hrd genes from actinomycetes and has been named hrdT. Functional domains in the three S. griseus Hrd proteins are highly conserved in relation to those of the σ 70 protein family. Northern analysis showed that hrdB is expressed as a 1.9-kb transcript during active growth in phosphate-rich medium, but it is less efficiently transcribed under sporulation conditions (phosphate-starved) or after a heat-shock treatment. Two other shorter transcripts of 1.2 and 0.7 kb were also detected with the same probe. The hrdD gene is transcribed as a single 1.1-kb transcript under sporulation conditions following nutritional shiftdown and, to a lower extent, during growth conditions in phosphate-rich medium. The hrdT gene is weakly transcribed (1.5-kb RNA) under all conditions tested. The hrd-encoded sigma factors probably recognize actinomycetes promoters ( SEP type) with E. coli-like consensus sequences.

Isolation of Mutants of Streptomyces griseus That Sporulate in Nutrient Rich Media: Cloning of DNA Fragments That Suppress the Mutations.

Streptomyces griseus does not sporulate when grown in a nutrient rich medium. Two mutants that sporulate in such media were isolated and characterized. The mutants sporulate at the same time as the wildtype when grown in a nutrient poor medium. The mutants and wildtype exhibit the same commitment to sporulation following a nutritional shiftdown. Six genomic DNA fragments of wildtype cells when transformed into the mutants caused these mutants to acquire the wildtype phenotype of not sporulating in rich growth media. Restriction analyses show that the six cloned DNA fragments are different. The six clones suppressed sporulation when transformed into Streptomyces coelicolor and Streptomyces lividans and secondary metabolite production was suppressed in S. coelicolor.

Regulation of stringent mitochondrial transcription in yeast following amino-acid deprivation

In the yeast Saccharomyces cerevisiae, a phenotypically identical stringent response is induced by either nutritional downshift or starvation for a required auxotrophic amino acid (aa); in each case, the response selectively includes transcriptional curtailment for the mitochondrial (mt) genome. We have shown previously that the downshift-induced mt stringent response is governed by changing cellular cyclic AMP (cAMP) levels, via a mt cAMP-dependent protein kinase. In contrast, we demonstrate here that cAMP levels are not altered in yeast following starvation for a required aa, and we use in vitro mt transcription assays with organelles from wild-type and mutant strains to confirm that the aa starvation-induced mt stringent response is not governed by cAMP. Rather, such stringent organellar transcriptional attenuation may result from altered availability of an unidentified small molecule which is probably a product of the cytoplasmic and/or mt protein synthesis systems.

The Stringent Response, ppGpp and Antibiotic Production in Streptomyces coelicolor A3(2).

The correlation between ppGpp synthesis and the onset of antibiotic production in Streptomyces coelicolor A3(2) is reviewed. S. coelicolor A3(2) produces at least four antibiotics, two of which, actinorhodin and undecylprodigiosin (whose synthesis is determined by the act and red genes, respectively), are pigmented compounds. Conditions were established that gave dispersed and rapid exponential growth in liquid culture. Nitrogen limitation resulted in a short transition phase, during which a peak of ppGpp was observed, followed by stationary phase. Undecylprodigiosin production commenced in transition phase, while actinorhodin synthesis was detected 5∼6 hr later. Whereas the level of act and red biosynthetic transcripts increased markedly during transition phase, the level of the primary bldA transcript, which encodes the only tRNA of S. coelicolor A3(2) that can efficiently translate the rare leucine codon UUA and which is required for both actinorhodin and undecylprodigiosin production, decreased during early exponential phase; this decrease corresponded to an increased signal for the 5’ end of the mature tRNA, which then remained constant throughout growth. Nutritional shiftdown of exponentially growing cultures evoked immediate synthesis of ppGpp and a severe reduction in growth rate; transcription of act genes occurred shortly after, and was followed by actinorhodin production. However, there was no immediate stimulatory effect on red gene transcription, and undecylprodigiosin production did not occur. Addition of serine hydroxamate, a seryl tRNA synthetase inhibitor, to exponentially growing cultures led to ppGpp accumulation, but to lower levels than after nutritional shiftdown, and did not elicit antibiotic production. Thus only a partial correlation was observed between ppGpp synthesis and the transcription of antibiotic biosynthetic genes. S. coelicolor A3(2) DNA that hybridised to the Escherichia coli (p)ppGpp synthetase gene (relA) was cloned, but it did not encode a relA homologue, and polyclonal antibodies against RelA failed to reveal a homologue in S. coelicolor A3(2) cell extracts. A relA homologue of Serratia marcescens was isolated using the same approach, but failed to hybridise to S. coelicolor A3(2) DNA.

Dimensional rearrangement of Escherichia coli B/r cells during a nutritional shift-down.

In a search for the mechanism underlying dimensional changes in bacteria, the glucose analogue methyl alpha-D-glucoside was used to effect a rapid reduction in the mass growth rate of Escherichia coli by competitively inhibiting glucose uptake, a so-called nutritional shift-down. The new steady-state cell mass and volume were reached after 1 h, during which the rate of cell division was maintained; rearrangement of the linear dimensions (cell length, diameter), however, required an additional 2 h and caused an undershoot in cell length, consistent with the view that E. coli is slow to modify its diameter. The results are compared with the overshoot in cell length that occurs following nutritional shift-up.

Transcription of the yeast mitochondrial genome requires cyclic AMP

Using various mutant strains and nutritional manipulations, we investigated a potential role for cyclic AMP (cAMP) in the regulation of mitochondrial (mt) gene expression in the yeast Saccharomyces cerevisiae. In RAS mutants known to have either abnormally low or high cellular levels of this nucleotide, we show that both mt transcription rate and overall mt transcript levels vary directly with cellular cAMP levels. We further show that nutritional downshift of actively growing cells causes a severe, rapid fall in cAMP levels, and that this fall is concomitant with the stringent mt transcriptional curtailment that we and others have previously shown to follow this nutritional manipulation. In in vitro mt transcription assays using intact organelles from downshifted and actively growing cells, stringently curtailed mt gene expression can be restored to 75% of control levels by addition of cAMP to the assay mix. Consistent with these observations a RAS2vall9 mutant strain, which cannot adjust cAMP levels in response to external stimuli, shows no mt stringent response following nutritional downshift. We also demonstrate a significant but transient increase in both mt transcript levels and mt transcription rate following shift of actively respiring wild-type cells to glucose-based medium, a manipulation known to cause a short-lived pulse of cAMP in yeast; similar manipulation of the RAS2vall9 mutant strain generates no such response. Taken together all these observations indicate that cellular cAMP levels are involved in the regulation of mt transcription in yeast. Moreover, the lack of a mt stringent transcriptional response following downshift in a strain in which the BCY1 gene had been insertionally inactivated suggests that cAMP may influence mt transcription via a mt cAMP-dependent protein kinase. These results link mt gene expression with mechanisms governing growth control and nutrient adaptation in yeast, and they provide a means by which mt gene expression might be coordinated with that of related nuclear genes.

Rate maintenance of cell division in Escherichia coli B/r: analysis of a simple nutritional shift-down.

A competitive (nonmetabolizable) inhibitor of glucose uptake, alpha-methylglucoside, was used to limit the growth of Escherichia coli. Cell division during such a nutritional shift-down was studied in batch cultures and with the "baby-machine" technique. Following a brief delay, the rate of division was maintained for 60 to 70 min in batch cultures and for an extended period in the baby machine. Decreases in cell size were due, in part, to a possible reduction in the mass per chromosome origin at the time of replication initiation and a shorter time interval between initiation and the subsequent division. These unusual findings suggest that this method for abrupt change in growth rate without modifying repression patterns is useful for studying the control of various aspects of the bacterial cell.