Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression.

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.     

Selected amplification of the cell division genes ftsQ-ftsA-ftsZ in Escherichia coli

Rapidly growing Escherichia coli is unable to divide in the presence of the antibiotic mecillinam, whose direct target is penicillin-binding protein 2 (PBP2), responsible for the elongation of the cylindrical portion of the cell wall. Division can be restored in the absence of PBP2 activity by increasing the concentration of the cell division proteins FtsQ, FtsA, and FtsZ. We tried to identify regulators of the ftsQ-ftsA-ftsZ operon among mecillinam-resistant mutants, which include strains overexpressing these genes. By insertional mutagenesis with mini-Tn10 elements, we selected for insertions that conferred mecillinam resistance. Among 15 such mutants, 7 suppressed the thermosensitivity of the ftsZ84(Ts) mutant, strongly suggesting that they had increased FtsZ activity. In all 7 cases, however, the mutants resulted from a duplication of the ftsQAZ region. These duplications seemed to result from multiple events, suggesting that no simple insertional inactivation can result in a mutant with sufficiently amplified ftsQAZ expression to confer mecillinam resistance. The structure of the duplications suggests a general method for constructing directed duplications of precise sequences.     

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp.

The Escherichia coli strain known as GC2553, FB8, UTH1038, or K12S (Luria), considered an F- lambda- wild-type strain, is shown here to carry a cryptic mutation, ftsR1, causing nonlethal filamentation during exponential growth in Luria-Bertani (LB) broth at 42 degrees C and the inability to grow in salt-free LB broth at 42 degrees C. The ftsR1 mutation is completely suppressed in genetic backgrounds which increase RelA-dependent synthesis of the nucleotide ppGpp, i.e., argS201 (Mecr) and alaS21 (Mecr) mutations, affecting aminoacyl-tRNA synthetases, or the presence of a plac-relA' plasmid. These backgrounds also confer resistance in LB broth to the beta-lactam mecillinam, an antibiotic which specifically inhibits penicillin-binding protein 2 and, in wild-type cells, causes an indirect block in cell division. Furthermore, the ftsR1 mutant (but not an isogenic ftsR+ strain) is sensitive to mecillinam in minimal glucose medium at 37 degrees C. Since the division block caused by mecillinam can be overcome by overproduction of the cell division protein FtsZ, we tested the effect of plasmid pZAQ (carrying the ftsZ, ftsA, and ftsQ genes) on the ftsR1 mutant; it suppressed the filamentation in LB broth and the mecillinam sensitivity on minimal glucose medium at 37 degrees C but not the growth defect in salt-free LB broth at 42 degrees C. Genetic analysis indicated that the full phenotype of the ftsR1 mutant is due to a single mutation in the rpoB gene (90 min), coding for the beta subunit of RNA polymerase; we call this allele rpoB369(Fts). We propose that the rpoB369(Fts) mutation alters the specificity of the polymerase and that the mutant enzyme can recover normal activity in the presence of high salt concentrations or via interaction with the nucleotide ppGpp.     

High temperature induction of a stringent response in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli

The cellular concentrations of ppGpp in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli were examined, since the thermosensitive RNA synthesis of these mutants is relaxed by an additional mutation in the relA gene. The results showed that ppGpp accumulated extensively in the dnaK(Ts) and dnaJ(Ts) mutants after a temperature shift up, reaching levels of 5 mM and 0.5 mM, respectively. This unusual accumulation of ppGpp was suppressed by the relA1 mutation, implying that it results from induction of a stringent response in these mutants at a nonpermissive temperature.     

High-level resistance to mecillinam produced by inactivation of soluble lytic transglycosylase in Salmonella enterica serovar Typhimurium

By screening for high-level mecillinam resistant derivatives of a low-level resistant strain (cysB403 galE1922 relA21::Tn10) of Salmonella enterica serovar Typhimurium, a MudJ insertion in the gene for soluble lytic transglycosylase (slt) was isolated. This insertion (slt-1::MudJ) increased the resistance to mecillinam of cysB and cysE strains (MIC: about 20-40 microg mL(-1)) to a strikingly high level (MIC: 160 microg mL(-1)). As in Escherichia coli K-12, the slt mutation slightly increased the sensitivity of the wild type and of several strains that carried mutations that did not increase mecillinam resistance. All the strains acquired a spherical cell shape when treated with mecillinam. The effect of slt-1::MudJ was limited to mecillinam, the response to several other antibiotics remaining unaltered by the insertion. The results presented in this paper demonstrate that soluble lytic transglycosylase performs an important role in the response to mecillinam, which only becomes evident when failure of CysB/CysE function causes medium-level resistance. The results also suggest that soluble lytic transglycosylase interacts with, and is partially inhibited by normal lipopolysaccharide.     

Escherichia coli mutant containing a large deletion from relA to argA.

A mutant of Escherichia coli has been isolated that contains a large deletion (about 3 X 10(7) daltons of deoxyribonucleic acid) encompassing argA, fuc, and relA. This mutant strain (AA-787) is also cold sensitive for growth at 18 degrees C. Strain AA-787 was obtained fortuitously as a cold-sensitive pseudorevertant of a strain having a heat-sensitive peptidyl-transfer ribonucleic acid hydrolase. Genetic analysis, using transduction and interrupted mating, showed the cold sensitivity mutation to be located adjacent to relA. Further analysis demonstrated loss of relA, fuc, and argA gene functions but retention of eno and recB, closely linked genes adjacent to relA and argA, respectively. Unusually high cotransduction of flanking markers (cysC and thyA) indicated loss of approximately 1 min of the E. coli genetic map in strain AA-787. Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) was synthetized in mutant strain AA-787 at basal levels, and ppGpp synthesis was stimulated by carbon-source downshift. No ppGpp synthesis could be obtained using ribosomes isolated from strain AA-787. These findings, taken together, show that deletion of relA in E. coli does not completely abolish ppGpp synthesis and suggests that another enzyme system must also be responsible for ppGpp synthesis.     

Nonsense and insertion mutants in the relA gene of E. coli: cloning relA.

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.     

Iron limitation induces SpoT-dependent accumulation of ppGpp in Escherichia coli

In Escherichia coli the beta-lactam mecillinam specifically inhibits penicillin-binding protein 2 (PBP2), a peptidoglycan transpeptidase essential for maintaining rod shape. We have previously shown that PBP2 inactivation results in a cell division block and that an increased concentration of the nucleotide ppGpp, effector of the RelA-dependent stringent response, confers mecillinam resistance and allows cells to divide as spheres in the absence of PBP2 activity. In this study we have characterized an insertion mutation which confers mecillinam resistance in wild-type and DeltarelA strains but not in DeltarelADeltaspoT strains, devoid of ppGpp. The mutant has an insertion in the fes gene, coding for enterochelin esterase. This cytoplasmic enzyme hydrolyses enterochelin-Fe(3+) complexes, making the scavenged iron available to the cells. We show that inactivation of the fes gene causes iron limitation on rich medium plates and a parallel SpoT-dependent increase of the ppGpp pool, as judged by the induction of the iron-regulated fiu::lacZ fusion and the repression of the stringently controlled P1(rrnB)::lacZ fusion respectively. We further show, by direct ppGpp assays, that iron starvation in liquid medium produces a SpoT-dependent increase of the ppGpp pool, strongly suggesting a role for iron in the balance of the two activities of SpoT, synthesis and hydrolysis of (p)ppGpp. Finally, we present evidence that ppGpp exerts direct or indirect positive control on iron uptake, suggesting a simple homeostatic regulatory circuit: iron limitation leads to an increased ppGpp pool, which increases the expression of iron uptake genes, thereby alleviating the limitation.     

Control mechanism of the Escherichia coli K-12 cell cycle is triggered by the cyclic AMP-cyclic AMP receptor protein complex.

The role of cyclic AMP (cAMP) in the cell cycle of Escherichia coli K-12 was studied in three mutant strains. One was KI1812, in which the cya promoter is replaced by the lacUV5 promoter. In KI1812, isopropyl-beta-D-thiogalactopyranoside induced the synthesis of cya mRNA, and at the same time cell division was inhibited and short filaments containing multiple nuclei were formed. The other strains were constructed as double mutants (NC6707 cya sulB [ftsZ(Ts)] and TR3318 crp sulB [ftsZ(Ts)]). In both double mutants, filamentation was repressed at 42 degrees C, but it was induced again by addition of cAMP in strain NC6707 and introduction of pHA7 containing wild-type crp in TR3318. These results indicate that lateral wall synthesis in the E. coli cell cycle is triggered by the cAMP-cAMP receptor protein complex.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression

Combinatorial overexpression of aromatic amino acid biosynthesis (AAAB) genes in the L-tyrosine producing Escherichia coli strains T1 and T2 was employed to search for AAAB reactions limiting L-tyrosine production. All AAAB genes except aroG and tyrA, which were substituted by their feedback resistant derivatives in the host strains, were cloned and overexpressed. A total of 72 different strains overexpressing various AAAB gene combinations were generated and from those strains with improved phenotype, enzymatic bottlenecks of the AAAB pathway could be inferred. The two major gene overexpression targets for increased L-tyrosine production in E. coli were ydiB and aroK, coding for a shikimate dehydrogenase and a shikimate kinase, respectively, and the combination of ydiB and aroK for overexpression resulted in the best L-tyrosine producing strains in this study, yielding 45% for strain T1 and 26% for strain T2, respectively, higher L-tyrosine titers. Interestingly, overexpression studies with combinations of more than one gene revealed that new gene targets could be identified when overexpessed together with other genes but not alone as single gene overexpression. For example, tyrB encoding the last enzyme of the AAAB pathway, an aromatic amino acid transaminase, improved L-tyrosine production significantly when co-overexpressed together with ydiB or aroK, but not when overexpressed alone. It is also noteworthy that E. coli T1, which generally yielded less L-tyrosine, was amenable to greater improvements than strain T2, i.e. E. coli T1 exhibited generally more space for phenotype improvement.     

Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli

The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation. The stringent response is thought to be mediated by ppGpp. Mutations in relA are known to result in pleiotropic phenotypes. We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41 degrees C. The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M. The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42 degrees C, a temperature that is normally nonlethal. The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities. The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis. This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone. The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the beta subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations. Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance.     

Role of peptide chain elongation factor G in guanosine 5'-diphosphate 3'-diphosphate synthesis.

In a wild-type strain (relA+) of Escherichia coli, starvation of amino acid led to an immediate cessation of the synthesis of stable ribonucleic acids, together with the accumulation of an unusual nucleotide, guanosine 5'-diphosphate 3'-diphosphate, commonly known as ppGpp. This compound also accumulated during heat shock. When temperature-sensitive protein synthesis elongation factor G (EF-G) was introduced into E. coli NF859, a relA+ strain, the synthesis of ppGpp was reduced to approximately one-half that of wild-type EF-G+ cells at a nonpermissive temperature of 40 degrees C. Furthermore, fusidic acid, an inhibitor of protein synthesis which specifically inactivates EF-G, prevented any accumulation of ppGpp during the heat shock. We suggest that a functional EF-G protein is necessary for ppGpp accumulation under temperature shift conditions, possibly by mediating changes in the function of another protein, the relA gene product. However, EF-G is probably not required for the synthesis of ppGpp during the stringent response, since its inactivation did not prevent ppGpp accumulation during amino acid starvation.     

Isolation and characterization of ftsZ alleles that affect septal morphology.

The ftsZ gene encodes an essential cell division protein that specifically localizes to the septum of dividing cells. In this study we characterized the effects of the ftsZ2(Rsa) mutation on cell physiology. We found that this mutation caused an altered cell morphology that included minicell formation and an increased average cell length. In addition, this mutation caused a temperature-dependent effect on cell lysis. During this investigation we fortuitously isolated a novel temperature-sensitive ftsZ mutation that consisted of a 6-codon insertion near the 5' end of the gene. This mutation, designated ftsZ26(Ts), caused an altered polar morphology at the permissive temperature and blocked cell division at the nonpermissive temperature. The altered polar morphology resulted from cell division and correlated with an altered geometry of the FtsZ ring. An intragenic cold-sensitive suppressor of ftsZ26(Ts) that caused cell lysis at the nonpermissive temperature was isolated. These results support the hypothesis that the FtsZ ring determines the division site and interacts with the septal biosynthetic machinery.