Two new mutant loci (smhB and lytD) in Escherichia coli which confer temperature-sensitive growth and lysis phenotypes

A temperature-sensitive mutation in the murH gene of Escherichia coli confers a lysis phenotype at the restrictive temperature. An extragenic suppressor of murH apparently representing a new locus at 12.5 min on the linkage map and designated smhB is described. The smhB mutation by itself also conferred a temperature-sensitive lysis phenotype. A mutation in another new locus designated lytD which arose spontaneously in the smhB mutant was mapped close to smhB at 12.7 min on the linkage map. The lytD mutation by itself conferred a temperature-sensitive lysis phenotype indistinguishable from that of the murH mutant. Thus, the suppression of lysis in the smhB murH and the smhB lytD double mutants suggests a mechanism involving the reciprocal suppression of the two individual lysis-causing mutant alleles. The suppressor activity of smhB was apparently relatively specific in that smhB failed to prevent lysis induced by either mutational (murE or murF) or antibiotic-induced blocks in peptidoglycan synthesis. This suggests that murH, smhB, and lytD may be functionally related.     

murH, a new genetic locus in Escherichia coli involved in cell wall peptidoglycan biosynthesis.

A temperature-sensitive mutant of Escherichia coli defective in peptidoglycan synthesis was characterized. The incorporation of radiolabeled meso-diaminopimelate into peptidoglycan by the mutant was inhibited at the restrictive growth temperature, resulting in autolysis. The defective step appeared to be part of the terminal stage in peptidoglycan synthesis involving the incorporation of disaccharide peptide units into the wall peptidoglycan. The mutation was assigned to a new locus, designated murH, at 99.2 min on the E. coli linkage map.     

A novel rho promoter::Tn10 mutation suppresses and ftsQ1(Ts) missense mutation in an essential Escherichia coli cell division gene by a mechanism not involving polarity suppression.

An extragenic suppressor of the Escherichia coli cell division gene ftsQ1(Ts) was isolated. The suppressor is a Tn10 insertion into the -35 promoter consensus sequence of the rho gene, designated rho promoter::Tn10. The ftsQ1(Ts) mutation was also suppressed by the rho-4 mutant allele. The rho promoter::Tn10 strain does not exhibit rho mutant polarity suppressor phenotypes. In addition, overexpression of the ftsQ1(Ts) mutation does not reverse temperature sensitivity. Furthermore, DNA sequence analysis of the ftsQ1(Ts) allele revealed that the salt-remediable, temperature-sensitive phenotype arose from a single missense mutation. The most striking phenotype of the rho promoter::Tn10 mutant strain is an increase in the level of negative supercoiling. On the basis of these observations, we conclude that the ftsQ1(Ts) mutation may be suppressed by a change in supercoiling.     

Phenotypic revertant mutations of a new OmpR2 mutant (V203Q) of Escherichia coli lie in the envZ gene, which encodes the OmpR kinase.

The Escherichia coli ompR2 allele ompR472 contains a valine-to-methionine point mutation at position 203, resulting in an OmpF-constitutive OmpC- outer membrane phenotype. In the present study, OmpR residue V-203 was replaced with glutamine (V203Q mutation), resulting in the same outer membrane phenotype. However, unlike the OmpFc OmpC- phenotype conferred by the OmpR(V203M) mutant protein, the OmpFc OmpC- phenotype produced by the OmpR(V203Q) mutation was suppressed by the envZ11(T247R) allele. Additional suppressors of OmpR(V203Q) were isolated by random mutagenesis. All suppressor mutations were found in the envZ gene and conferred an OmpC+ OmpF- phenotype in the presence of the wild-type ompR. These envZ11-like mutations mapped to a region different from those previously reported and were incapable of suppressing the ompR(V203M) allele. Our results indicate that while methionine or glutamine replacements could cause similar effects on OmpF and OmpC expression, they conferred different abilities on the mutant proteins to be suppressed by envZ.     

Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation.

We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain. Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C. However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed. This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C. The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E. coli chromosome). DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide. Gene disruption experiments indicated that dksA is not an essential gene.     

pH-sensitive CDP-diglyceride synthetase mutants of Escherichia coli: phenotypic suppression by mutations at a second site.

In Escherichia coli, mutations which lower the level of CDP-diglyceride synthetase are designated cds and map at min 4. The cds-8 mutation resulted in strikingly defective enzyme activity and also rendered cells pH sensitive for growth. Both the inhibition of growth and the massive accumulation of phosphatidic acid which occur in a cds-8 mutant at pH 8 were suppressed by mutations at a second locus, designated cdsS, which mapped between argG and gltB near min 68. The cdsS3 mutation by itself did not affect CDP-diglyceride synthetase activity in wild-type cells, but it caused a twofold stimulation of the residual activity present in strains harboring cds-8. Both the insensitivity to pH and the twofold stimulation of residual activity were lost by introduction of an F' strain carrying cdsS+ into a recA1 cds-8 cdsS3 host. When a culture of a cds-8 cdsS+ strain was shifted to pH 8, the residual specific activity of synthetase dropped by 75% within 100 min. In a cds-8 cdsS3 double mutant under the same conditions, the activity declined appreciably less, about to the level found in the cds-8 cdsS+ strain under permissive conditions (pH 6). Thus, it appears that mutations in the cdsS gene suppress the pH sensitivity of cds mutants by inhibiting the decay of residual CDP-diglyceride synthetase activity at the nonpermissive pH. The cdsS locus appears to be distinct from any known nonsense or missense suppressor.     

Identification of new cell division genes in Escherichia coli by using extragenic suppressors.

To facilitate the analysis of the cell division control apparatus in Escherichia coli, we studied extragenic suppressor mutations of a previously characterized temperature-sensitive division mutation, ftsM1. Cells of strain GD40 which harbor this mutation were spread on agar plates and incubated at 42 degrees C, and the surviving cells were analyzed for the presence of a suppressor mutation. One group of suppressed mutants had acquired a new mutation which, by conjugation, was found to be located in the 30- to 40-min region of the E. coli genetic map. The other group comprised revertants carrying a suppressor which appeared to map between thr and leu. This suppressor gene, called sftA, was cloned with a mini-Mu-derived in vivo cloning system by selection for suppression of temperature sensitivity in GD40 cells. Subsequent subcloning of a fragment of the chromosomal DNA from the mini-Mu plasmid into pBR325 resulted in the delineation of the suppressor gene on a 1.8-kilobase XhoI-PvuI fragment. A strain, CV514, which does not express the temperature sensitivity phenotype of the ftsM1 mutation, was found to harbor a natural suppressor of this mutation. UV sensitivity, another known phenotype of the ftsM1 mutation, was also corrected by the presence of the sftA suppressor in the cell. Thus, the characterization of extragenic suppressors may allow the identification of new genes involved in the control of cell division.     

Gene Interactions Affecting Muscle Organization in CAENORHABDITIS ELEGANS

Revertants of unc-15(e73)I, a paralyzed mutant with an altered muscle paramyosin, include six dominant and two recessive intragenic unc-15 revertants, two new alleles of the previously identified suppressor gene, sup-3 V, and a new suppressor designated sup-19(m210)V. The recessive intragenic unc-15 revertants exhibit novel alterations in paramyosin paracrystal structure and distribution, and these alterations are modified by interaction with unc-82(e1220)IV, another mutation that affects paramyosin. A strain containing both unc-15 and a mutation in sup-3 V that restores movement was mutagenized, and paralyzed mutants resembling unc-15 were isolated. Twenty mutations that interfere with suppression were divided into three classes (nonmuscle, sus-1, and mutations within sup-3) based on phenotype, genetic map position and dominance. The nonmuscle mutations include dumpy and uncoordinated types that have no obvious direct effect on muscle organization. Two recessive mutations define a new gene, sus-1 III. These mutations modify the unc-15(e73) phenotype to produce a severely paralyzed, dystrophic double mutant that is not suppressed by sup-3. Five semidominant, intragenic sup-3 antisuppressor mutations, one of which occurred spontaneously, restore the wild-type sup-3 phenotype of nonsuppression. However, reversion of these mutants generated no new suppressor alleles of sup-3, suggesting that the sup-3 antisuppressor alleles are not wild type but may be null alleles.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.     

Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli.

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.     

Physiological properties of cold-sensitive suppressor mutations of a temperature-sensitive dnaZ mutant of Escherichia coli

Suppressors of a temperature-sensitive dnaZ polymerization mutant of Escherichia coli have been identified by selecting temperature-insensitive revertants. Those suppressed strains which concomitantly became cold sensitive were chosen for further study. Intragenic suppressor mutations, which caused cold-sensitive defects in DNA polymerization, were located in dnaZ by transduction with lambda dnaZ+ phages. Extragenic suppressor mutations were mapped within the initiation gene dnaA. These suppressor-containing strains were defective in initiation at low temperature as determined by measurements of DNA synthesis in vivo and in toluene-treated cells. The occurrence of suppressor mutations of dnaZ(Ts) within the dnaA gene is considered evidence that the dnaA and dnaZ products interact in vivo. A second indication of a dnaA-dnaZ protein-protein interaction was provided by the observation that the introduction of additional copies of the dnaZ+ gene into a strain carrying the dnaA suppressor mutation was lethal [whether the strain was dnaZ+ or dnaZ(Ts)].     

The phenotypic suppression of a mutation in the gene rplX for ribosomal protein L24 by mutations affecting the lon gene product for protease LA in Escherichia coli K12

Summary A suppressor mutation of a temperature-sensitive mutant of ribosomal protein L24 (rplX19) was mapped close to the lon gene by genetic analysis and was shown to affect protease LA. The degradation and the synthesis rates of individual ribosomal proteins were determined. Proteins L24, L14, L15 and L27 were found to be degraded faster in the original rplX19 mutant than in the rplX19 mutant containing the suppressor mutation. Other ribosomal proteins were either weakly or not at all degraded in both mutants. Temperature-sensitive growth was also suppressed by the overproduction of mutant protein L24 from a plasmid. Our results suggest that (1) either free ribosomal proteins or proteins bound to abortive assembly precursors are highly susceptible to the lon gene product and (2) the mutationally altered protein L24 can still function at the nonpermissive growth temperature of the mutant, if it is present in sufficient amounts.     

Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome.     

Intragenic suppression in the uvrD gene of Escherichia coli. I. Temperature-sensitive uvrD mutations

A temperature-sensitive uvrD mutant, HD323 uvrD4, was isolated from the uvrD mutant HD4 uvrD3. The temperature sensitivity of the uvrD4 gene product was reversible. The suppressor mutation uvrD44 which rendered the uvrD3 mutant temperature-sensitive could be separated from the uvrD3 mutation by replacing the PstI fragment, which encodes the C-terminal half of the UvrD protein. The uvrD44 mutation was found to make host bacteria lethal at non-permissive temperatures only when cloned on a low copy vector pMF3. The nucleotide sequence of the uvrD3 and uvrD4 mutant genes was determined. The nucleotide change found in the uvrD3 at +1235, GAA to AAA, only alters the amino acid sequence from Glu at 387 to Lys. The uvrD44 has another nucleotide change at +1859, GAA to AAA (Glu at 595 to Lys), which is considered to be the suppressor mutation uvrD44.     

Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli

We have previously described a temperature-sensitive mutant, ts215, which is defective in protein secretion. Complementation studies indicated that the mutation was located at the distal part of the spc ribosomal protein operon and the gene secY is required for efficient protein secretion. We now report a more complete genetic and biochemical analysis of the ts215 mutant. These studies revealed that the ts215 mutant has an amber mutation in the gene rp10 for ribosomal protein L15, which is located upstream and adjacent to secY. The amber mutation exerts a polar effect on secY causing a defect in protein secretion. These conclusions were supported by the following observations. The mutant strain carries a phi 80 prophage containing a temperature-sensitive suppressor, supFts6. The strain contains decreased amounts of L15 and is suppressible by a temperature-independent nonsense suppressor. In addition, L15 contains an extra tyrosine residue when suppressed by supF. DNA sequence analysis revealed the presence of a single base change in rp10 resulting in an amber codon at the 38th codon of L15. The mutant phenotype is complemented by a plasmid carrying only the secY gene under lac promoter control. The mutant cells complemented by secY can grow and synthesize proteins at normal rates and abundances at 42 degrees C, despite the fact that their ribosomes contain barely detectable levels of L15. These results indicate that ribosomal protein L15 is dispensable for protein synthesis and cell growth. In contrast, the decreased level of expression of the secY gene leads to defective protein secretion and defective cell growth.     

Interaction between two regulatory proteins in osmoregulatory expression of ompF and ompC genes in Escherichia coli: a novel ompR mutation suppresses pleiotropic defects caused by an envZ mutation.

The ompR and envZ genes, which together constitute the ompB operon, are involved in osmoregulatory expression of the OmpF and OmpC proteins, major outer membrane proteins of Escherichia coli. The envZ11 mutation results in the OmpF- OmpC-constitutive phenotype. A mutant which suppressed defects caused by the envZ11 mutation was isolated. The suppressor mutation also suppressed the LamB- PhoA- phenotype caused by the envZ11 mutation. The mutation occurred in the ompR gene and hence was termed ompR77. The ompR77 mutation alone produced no obvious phenotype. Functioning of the ompR77 allele remained envZ gene dependent. Although the ompR77 mutation suppressed the envZ11 mutation, it did not suppress a mutation that occurred in another position within the envZ gene (envZ160). These results indicate that OmpR and EnvZ, two regulatory proteins, functionally interact with each other.