Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation

The spoT gene of Escherichia coli encodes a guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) as well as an apparent guanosine 3',5'-bis(diphosphate) synthetase (designated PSII). To determine the regions of the SpoT protein that are required for these two competing activities, we analysed plasmid-borne deletion mutations for their ability to complement chromosomal mutations defective in each activity. We found that a region containing the first 203 amino acids of the 702-amino-acid SpoT protein was sufficient for ppGppase activity while an overlapping region containing residues 67-374 was sufficient for PSII activity. These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT protein. A ppGppase-defective delta 1-58 deletion mutant strain failed to synthesize ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild-type SpoT does not increase in response to nutrient limitation. Using a strain lacking PSII activity but retaining ppGppase activity, we determined the contribution of the RelA protein (ppGpp synthetase I, PSI) to ppGpp synthesis following glucose starvation. We found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation but that this source of synthesis is absent when amino acids are present during glucose starvation.     

Effect of ppGpp on Escherichia coli cyclopropane fatty acid synthesis is mediated through the RpoS sigma factor (sigmaS)

Strains of Escherichia coli carrying mutations at the relA locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase. RelA protein catalyzes the synthesis of guanosine-3',5'-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis. The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification. We report that the effect of RelA on CFA synthesis is indirect. In vitro and in vivo experiments show no direct interaction between ppGpp and CFA synthase activity. The relA effect is due to ppGpp-engendered stimulation of the synthesis of the alternative sigma factor, RpoS, which is required for function of one of the two promoters responsible for expression of CFA synthase.     

A role for polyamines in the control of ppGpp levels in Escherichia coli

As an approach to understand the involvement of polyamines in the variation of intracellular guanosine 5'-diphosphate 3'-diphosphate (ppGpp) levels, kinetic studies with several polyamine-requiring relA and spoT Escherichia coli mutants have been carried out. The accumulation and turnover of the nucleotide have been followed under conditions of aminoacid depletion or energy source starvation. The results obtained strongly suggest an important role of the polycations mainly in the degradation of ppGpp, and also in its synthesis mediated by ppGpp synthetase I (PSI).     

Diagnosis of the abdominal vascular system by means of electron-beam computed tomography (EBT)]

Pokeweed antiviral protein (PAP) from Phytolacca americana is a highly specific N-glycosidase removing adenine residues (A4324 in 28S rRNA and A2660 in 23S rRNA) from intact ribosomes of both eukaryotes and prokaryotes. Due to the ribosome impairing activity the gene coding for mature PAP has not been expressed so far in bacteria whereas the full-length gene (coding for the mature 262 amino acids plus two signal peptides of 22 and 29 amino acids at both N- and C-termini, respectively) has been expressed in Escherichia coli. In order to determine: 1) the size of the N-terminal region of PAP which is required for toxicity to E. coli; and 2) the location of the putative enzymatic active site of PAP, 5'-terminal progressive deletion of the PAP full-length gene was carried out and the truncated forms of the gene were cloned in a vector containing a strong constitutive promoter and a consensus Shine-Dalgarno ribosome binding site. The ribosome inactivation or toxicity of the PAP is used as a phenotype characterized by the absence of E. coli colonies, while the mutation of PAP open reading frames in the small number of survived clones is used as an indicator of the toxicity to E. coli cells. Results showed that the native full-length PAP gene was highly expressed and was not toxic to E. coli cells although in vitro ribosome inactivating activity assay indicated it was active. However, all of the N-terminal truncated forms (removal of seven to 107 codons) of the PAP gene were toxic to E. coli cells and were mutated into either out of frame, early termination codon or inactive form of PAP (i.e., clone PAP delta107). Deletion of more than 123 codons restored the correct gene sequence but resulted in the loss of the antiviral and ribosome inactivating activities and by the formation of a large number of clones. These results suggest that full-length PAP (with N- and C-terminal extensions) might be an inactive form of the enzyme in vivo presumably by inclusion body formation or other unknown mechanisms and is not toxic to E. coli cells. However, it is activated by at least seven codon deletions at the N-terminus. Deletions from seven through to 107 amino acids were lethal to the cells and only mutated forms (inactive) of the gene were obtained. But deletion of more than 123 amino acids resulted in the loss of enzymatic activity and made it possible to express the correct PAP gene in E. coli. Because deletion of Tyr94 and Val95, which are involved in the binding of the target adenine base, did not abolish the activity of PAP, it is concluded that the location previously proposed for PAP enzymatic active site should be reassessed.     

Lung and kidney cancer mortality associated with arsenic in drinking water in Córdoba, Argentina

We have deleted the chromosomal rpsA gene, encoding ribosomal protein S1, from an Escherichia coli strain carrying a plasmid where rpsA was controlled by the lac promoter and operator. This exogenous source of protein S1 was essential for growth. Thus we have verified the absolute requirement for protein S1. To see if translation of individual mRNAs differed in the requirements for protein S1, we removed the inducer and followed the time-course of the synthesis of several individual proteins and of total RNA, DNA and protein. Growth immediately shifted from being exponential to being linear, with a rate of protein synthesis defined by the pre-existing amount of protein S1. The expression pattern of the individual proteins indicated that the translation of all mRNAs was dependent on protein S1. Unexpectedly, we found that depletion for protein S1 for extended periods introduced a starvation for amino acids. Such starvation was indicated by an increased synthesis of ppGpp and could be reversed by addition of a mixture of all 20 amino acids. Measurements of the peptide chain elongation rate in vivo showed that ribosomes without protein S1 were unable to interfere with the peptide chain elongation rate of the active ribosomes and that, therefore, protein S1 was unable to diffuse from one ribosome to another during translation. We conclude that protein S1-deficient ribosomes are totally inactive in peptide chain elongation on most, if not all, naturally occurring E. coli mRNAs.     

Lack of production of (p)ppGpp in Halobacterium volcanii under conditions that are effective in the eubacteria

The stringent halobacterial strain Haloferax volcanii was subjected to a set of physiological conditions different from amino acid starvation that are known to cause production of guanosine polyphosphates [(p)pp Gpp] in eubacteria via the relA-independent (spoT) pathway. The conditions used were temperature upshift, treatment with cyanide, and total starvation. Under none of these conditions were detectable levels of (p)ppGpp observed. This result, in conjunction with our previous finding that (p)ppGpp synthesis does not occur under amino acid starvation, leads to the conclusion that in halobacteria both growth rate control and stringency are probably governed by mechanisms that operate in the absence of ppGpp. During exponential growth, a low level of phosphorylated compounds with electrophoretic mobilities similar, but not identical, to that of (p)ppGpp were observed. The intracellular concentration of these compounds increased considerably during the stationary phase of growth and with all of the treatments used. The compounds were identified as short-chain polyphosphates identical to those found under similar conditions in Saccharomyces cerevisiae.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.     

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro

It was demonstrated previously that replication of plasmids derived from bacteriophage lambda (so-called lambda plasmids) is inhibited in wild-type Escherichia coli cells starved for isoleucine and arginine whereas it proceeds under the same conditions in relA mutants. Since replication of other replicons during the stringent or relaxed response depends on the nature of the deprived amino acid, we investigated replication of lambda plasmids in E. coli relA+ and relA- strains starved for different amino acids. We found that replication of lambda plasmids is generally inhibited during the stringent, but not relaxed, response. Differences between cells starved for different amino acids, although reproducible, were not dramatic. Amino acid starvation was previously proposed as a method for amplification of lambda plasmid DNA in vivo. We found that during amino acid limitation lambda plasmids replicate more extensively in the relA mutants than during amino acid starvation. The efficiency of plasmid DNA amplification was found to be dependent on the kind of limited amino acid; in relA- bacteria limited for leucine we observed about 10-fold plasmid amplification. Some lambda plasmid replication was also found under these conditions in the relA+ host. The mechanism of the stringent control of lambda plasmid DNA replication has already been proposed. Here the possible mechanism of the regulation of lambda plasmid replication during amino acid limitation is presented.     

Treatment of root perforations with calcium hydroxide and SuperEBA cement: a clinical report

Complete nucleotide sequence of a plasmid isolated from Enterobacter agglomerans has been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring the cat gene (pBW4), which could be introduced into Escherichia coli cells, and demonstrating that pBW4 cannot replicate in the absence of the polA function and that its copy number is significantly decreased in the pcnB mutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starved relA mutants but not in relA+ bacteria. Inactivation of the putative rom gene by insertion of an amplicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number in E. coli-despite the fact that amino acid sequence of the putative RNA 1 modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derived rom-like coding sequence is also homologous to the rom-like gene present in the Proteus vulgaris plasmid pPvul. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) in E. coli, one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the new rom-like gene coding for a protein useful in investigation of RNA-protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.     

Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements and Escherichia coli mutant defective in the ATPase activity of SecA

The secA gene of Streptomyces lividans was cloned using as probe a 57-mer oligonucleotide based on conserved sequences of the Escherichia coli secA and the Bacillus subtilis div genes. It encodes a protein of 946 amino acids (aa) with a deduced M(r) of 106,079, with high similarity to all known SecA proteins. All the previously described conserved motifs of SecA proteins were conserved in the S. lividans protein. The secA gene of S. lividans restored sensitivity to sodium azide in E. coli SecA4 (AzR) a mutant with an azide-resistant (ATPase defective) SecA protein. However, it did not complement the temperature-sensitive mutation in E. coli MM52 (SecAts) (a conditional lethal mutant defective in protein translocation) allowing only poor growth at the nonpermissive temperature. secA homologous sequences were present in 11 different species of Streptomyces and Nocardia.     

Molecular cloning, sequencing, purification, and characterization of Pseudomonas aeruginosa ribosome recycling factor

Ribosome recycling factor (RRF) is required for release of 70S ribosomes from mRNA on reaching the termination codon for the next cycle of protein synthesis. The RRF-encoding gene (frr) of Pseudomonas aeruginosa PAO1 was functionally cloned by using a temperature-sensitive frr mutant of Escherichia coli and sequenced. The P. aeruginosa frr was mapped at 30 to 32 min of the P. aeruginosa chromosome. The deduced amino acid sequence of RRF showed a 64% identity to that of E. coli RRF. In an assay including E. coli polysome and elongation factor G, purified recombinant RRF of P. aeruginosa released monosomes from polysomes. This is the first case in which an RRF homologue was found to be active in heterogeneous ribosome recycling machinery. The genes for ribosomal protein S2 (rpsB), elongation factor Ts (tsf), and UMP kinase (pyrH) are located upstream of frr. The arrangement of the genes, rpsB-tsf-pyrH-frr, resembles those reported for E. coli and Bacillus subtilis. Even in the cyanobacterium genome, the arrangement pyrH-frr is conserved. Although RRF homologues are found in eukaryotic cells, phylogenetic analysis suggests that they were originally present within the members of the phylogenetic tree of prokaryotic RRF. This finding suggests that the ribosome recycling step catalyzed by RRF is specific for prokaryotic cells and that eukaryotic RRF is required for protein synthesis in organelles, which are believed to be phylogenetically originated from prokaryotes.     

Cytomegalovirus disease occurring before engraftment in marrow transplant recipients

We developed a novel approach for the high-level production of a microbial transglutaminase (TGase) from Streptoverticillium in E. coli. The direct expression of the TGase gene in E. coli cells did not cause overproduction, probably due to the harmful influence of TGase activity, which introduces covalent crosslinks between proteins. Therefore, we fused the chemically synthesized TGase gene coding for the entire 331 amino acid residues at the amino terminus to a bacteriophage T7 gene 10 leader peptide (260 amino acids) using an inducible expression vector. The TGase gene was expressed as inclusion bodies in the E. coli cytoplasm. Restoring 15 amino acid residues upstream of the amino terminus of the mature TGase by a two-step deletion of the fusion sequence facilitated solubilization and subsequent proteolytic cleavage, thus releasing mature TGase. Although the mature form had less TGase activity than native TGase, because of the poor refolding rate, these results suggest that this system is suitable for the efficient production of TGase.