Levels Of Guanosine Tetraphosphate Research Articles

The Borrelia burgdorferi RelA/SpoT Homolog and Stringent Response Regulate Survival in the Tick Vector and Global Gene Expression during Starvation.

As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; rel Bbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.

Deciphering the Nature of the 30S:BipA Ribosome Complex

BipA is a member of the translational family of GTPases and is required for bacterial survival under various adverse environmental conditions. It is the only protein that differentially binds to two ribosomal particles. Under normal growth conditions when GTP levels are high, BipA associates with 70S ribosomes. However, during stress or other unfavorable environmental changes, BipA binds to 30S ribosomes. This association is driven by increased levels of guanosine tetraphosphate (ppGpp), an alarmone responsible for adaptation to adverse growth conditions in bacteria. The significance of this differential association is not understood. We identified a single site substitution in BipA that functions to suppress the cold sensitivity of an Escherichia coliinfA (initiation factor 1, IF1) deletion strain suggesting a role for BipA in translation initiation events. Biochemical and biophysical measurements of the GTPase activity of this protein reveal that it binds ppGpp with similar affinity as wild type BipA, binds GTP weakly and only associates with the 30S ribosomal particle. Coupled with our crystallographic data, these results indicate that this protein behaves similarly to the ppGpp-bound form of wild type BipA. These data suggest that BipA acts as mediator between the cellular environment and the ribosome, possibly modulating the translation initiation events in response to cell stress.

Activities of constitutive promoters in Escherichia coli

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+spoT+strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient ΔrelAΔspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the β-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage λ; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of alac deletion strain. The amount of 5′-terminal lacZ mRNA and of β-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combininglacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (ΔrelAΔspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity ofrrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+spoT+) relative to ppGpp-deficient (ΔrelAΔspoT) bacterial strains.

Guanosine Tetra- and Pentaphosphate Promote Accumulation of Inorganic Polyphosphate in Escherichia coli

High levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), generated in response to amino acid starvation in Escherichia coli, lead to massive accumulations of inorganic polyphosphate (polyP). Inasmuch as the activities of the principal enzymes that synthesize and degrade polyP fluctuate only slightly, the polyP accumulation can be attributed to a singular and profound inhibition by pppGpp and/or ppGpp of the hydrolytic breakdown of polyP by exopolyphosphatase, thereby blocking the dynamic turnover of polyP. The Ki values of 10 microM for pppGpp and 200 microM for ppGpp are far below the concentrations of these nucleotides in nutritionally stressed cells. In the complex metabolic network of pppGpp and ppGpp, the greater inhibitory effect of pppGpp (compared with ppGpp) leading to the accumulation of polyP, may have some significance in the relative roles played by these regulatory compounds.

The RNA Chain Elongation Rate of the λ Late mRNA Is Unaffected by High Levels of ppGpp in the Absence of Amino Acid Starvation

In this study, the effects of high levels of guanosine tetraphosphate (ppGpp) on the decay and RNA chain elongation kinetics of the bacteriophage lambda late transcript in Escherichia coli were examined in the absence of amino acid starvation. The accumulation, mRNA decay kinetics, and RNA chain elongation rate of the lambda late mRNA were determined after heat induction of lambdacI857 lysogens in the presence of high levels of ppGpp induced from a RelAalpha fragment-overproducing plasmid. The accumulation kinetics and elongation rate determinations of the late mRNA were made at long times after induction to allow a new steady state of transcriptional activities under conditions of elevated intracellular levels of ppGpp. The results indicate no prolonged or significant effect on either mRNA decay or the RNA chain elongation rate of the late mRNA as a result of elevated ppGpp levels. Surprisingly, the RNA chain elongation rate determinations indicate an RNA polymerase processivity of approximately 90-100 nucleotides/s for the lambda late transcript despite the presence of high levels of ppGpp. The results are discussed in terms of various models for regulation of stable and messenger RNA synthesis in E. coli.

Identification and characterization of a relA-dependent starvation-inducible locus (sin) in Salmonella typhimurium.

By use of Mu cts d1(Ap lac) phage, a strain of Salmonella typhimurium was isolated containing a Mu d insertion in a locus (sinA) which is induced during nicotinate, thiamine, purine, amino acid, phosphate, and carbon starvation conditions. Depending on the starvation condition, a 2- to 10-fold increase in beta-galactosidase activity was demonstrated. The sinA locus, which mapped at 32 U, became induced after a decline in growth rate due to starvation. The introduction of relA into the sinA-lac strain prevented induction by nicotinate starvation and partially prevented induction by phosphate starvation. The data suggest that sinA responds to changes in growth rate due to various nutrient starvation conditions and probably responds in part to changes in guanosine tetraphosphate levels.

RNA synthesis in Yersinia pestis during growth restriction in calcium-deficient medium.

Yersinia pestis requires 2.5 mM Ca(2+) for growth at 37 degrees C but not at 26 degrees C. After a shift from 26 to 37 degrees C in a Ca(2+)-deficient medium, an ordered series of metabolic alterations occur which result in transition from a growing cell to a viable but non-proliferating cell. The earliest known alteration in normal metabolism associated with this transition is a termination of net RNA synthesis. Competitive RNA/DNA hybridizations with uniformly labeled RNA and stable RNA competitor indicated identical mRNA to stable RNA ratios in growing cells and non-proliferating Ca(2+)-deprived cells. Similar hybridizations with pulse-labeled RNA demonstrated that growing cells synthesized 57% mRNA, 37% rRNA, and 5% tRNA, whereas Ca(2+)-deprived cells synthesized 95% mRNA, 4.7% rRNA, and 0.7% tRNA. After addition of radioactive uracil and rifampin to growing and Ca(2+)-deprived cells, decay of approximately 40 and 90% of the newly synthesized RNA was found for growing and Ca(2+)-deprived cells, respectively. The half-life of the mRNA was found to be 1.5 min for growing cells and 4.5 min for Ca(2+)-deprived cells. Y. pestis elicited increases in the levels of guanosine tetraphosphate and guanosine pentaphosphate in response to amino acid deprivation and yielded transient increases in the levels of these phosphorylated nucleotides after a shift from 26 to 37 degrees C. These increases were independent of Ca(2+) availability and preceded the alteration in RNA synthesis by more than 1 h. The levels of these phosphorylated nucleotides then stabilized at about 80 and 40 pmol for Ca(2+)-deprived and Ca(2+)-supplemented cultures, respectively, and did not increase further in the Ca(2+)-deprived culture at the time corresponding to the reduction in stable RNA synthesis. These findings indicate that the early lesion in RNA synthesis associated with the growth restriction of Ca(2+)-deprived Y. pestis reflects a block in stable RNA synthesis and that this effect is not mediated by guanosine tetraphosphate or guanosine pentaphosphate.

The relationships among RNA synthesis, RNA polymerase synthesis and guanosine tetraphosphate levels in Escherichia coli during nutritional shift-up

The relationships among the rate of RNA synthesis, RNA polymerase synthesis and activity, and guanosine tetraphosphate levels were investigated following nutritional shift-up in Escherichia coli. RNA synthesis continues at the preshift rate for 1.5 min after which an increase is observed that reaches a new steady-state rate at between 2 and 2.5 min. RNA polymerase activity measured in crude extracts increases immediately and by 10 min has increased 50%. RNA polymerase synthesis as measured by the synthesis of the β and β′ subunits lags for 2.5 min and then increases 75% by 10 min. Guanosine tetraphosphate levels decrease 50% by 3 min to levels characteristic of steady-state post-shift-up cells. The significance of these data to the regulation of RNA synthesis during shift-up is discussed.

The effect of essential amino acid deprivation on macromolecular synthesis and nucleotide pool sizes in Neurospora crassa

In cultures of Neurospora crassa (leu-4, R108) deprived of an essential amino acid, there is a decrease in the amount of 32PO 4 incorporated into the 1.0 m formic acid-soluble pool, nucleotides, DNA, and RNA and a decrease in glycogen synthetase activity compared to leucine-supplemented cultures. However, energy charge values and nucleoside triphosphate to diphosphate ratios in the leucine-deprived cells approximate the values determined in exponentially growing cells. Low levels of guanosine tetraphosphate are found in both leucine-deprived and leucine-supplemented cultures. Cyclic 3′,5′-adenosine monophosphate levels were determined during exponential and prestationary phase and in leucine-deprived cultures. In leucine-supplemented cultures, an increase in the intracellular cAMP level and a decrease in the extracellular level occurs concurrently with a decrease in energy charge (from 0.78 to 0.65) occurring as exponential cultures enter prestationary phase. The energy charge (0.75) and the intracellular cAMP level in deprived cultures are comparable to values in exponentially growing cultures. However, the total cAMP produced in leucine-supplemented cultures is approximately half that produced in amino acid-deprived cultures.

Inapparent correlation between guanosine tetraphosphate levels and RNA contents in Escherichia coli

We have reexamined the intracellular levels of guanosine 5′-diphosphate 3′-diphosphate in a prototrophic stringent strain of Escherichia coli during balanced growth at eight different growth rates. No regular inverse relationship between the basal levels of this nucleotide and the RNA content per unit DNA was observed. Thus, it is concluded that the cellular capacity to accumulate RNA is not directly related to the basal level of this nucleotide. It was also observed that significant quantities of this nucleotide accumulated extracellularly to varying degrees depending on the carbon source used.

On the Regulation of Guanosine Tetraphosphate Levels in Stringent and Relaxed Strains of Escherichia coli

We have examined the intracellular levels of the guanosine tetraphosphate, guanosine 5'-diphosphate 2'- or 3'-diphosphate (ppGpp), in an isogenic pair of stringent and relaxed strains of Escherichia coli. As previously observed, relaxed strains do not accumulate ppGpp during amino acid deprivation, while stringent strains do. However, under all other culture conditions examined, relaxed and stringent cells regulate their ppGpp levels similarly. During carbon deprivation or step-down transitions both strains accumulate ppGpp. Neither strain accumulates appreciable amounts during phosphate deprivation or after treatment with colicin E1. The basal levels of ppGpp observed in both stringent and relaxed strains during balanced growth are, in most cases, the same, and vary inversely with the growth rate and RNA content of the cells.