Mutants of the Clo DF13 plasmid inEscherichia coli with a decreased bacteriocinogenic activity

Summary Three Clo DF13 mutant plasmids (designated asclp03, clp05 andclp21) that show a decreased cloacin activity were isolated. The decreased cloacin activity was not due to a reduced number of Clo DF13 copies per cell. The cloacins produced by theclp03 and theclp21 mutant plasmids have a strongly decreased killing activityin vivo in comparison with the wild type cloacin and the cloacin of theclp05 mutant plasmid. Furthermore no lacunae could be observed fromclp03 orclp21 harbouring strains, while strains harbouring theclp05 plasmid showed a 50–100 times decreased frequency of lacunae. In addition theclp05 mutant showed a decreased rate of RNA synthesis inclp05 harbouringEscherichia coli minicells. No complementation between the three mutant plasmids was observed. We suggest that theclp03 andclp21 mutations are located in the gene coding for the cloacin. Since the cloacin produced by theclp05 mutant plasmid has retained all the known wild type cloacin activities, the reduced inhibition zone in the stab test is probably caused by a mutation affecting the expression of the cloacin gene. The nature of this mutation is discussed.     

An unusual suicidal interaction inEscherichia coli involving nucleoid protein H-NS

A conditional-lethal mutation (rpoB364) mapping to the gene that encodes the β-subunit of RNA polymerase was obtained inEscherichia coli. This mutation caused cell filamentation at the restrictive growth temperature and partial derepression of the osmotically regulatedproU operon at the permissive growth temperature. Even under the latter condition, transformants of therpoB364 mutant strain carrying the plasmid vector pACYC184, but not those carrying otherpolA-dependent multicopy plasmids such as pACYC177 or pBR322, were killed in early stationary phase; one class of suppressor mutants isolated as survivors within these transformant colonies were further derepressed forproU-lac expression, and the mutation in each of several independent clones of this class was mapped tohns, the gene that encodes the protein H-NS of theE. coli nucleoid. Thehns mutations did not suppress the conditional-lethal growth phenotype of therpoB364 mutant itself. On the other hand, intracellular overproduction of guanosine 3’, 5’-bispyrophosphate (ppGpp) in therpoB364 strain alleviated both the growth inhibition at the restrictive temperature and the pACYC184-mediated stationary-phase lethality. Upon subcloning into pUC19 or into pACYC177, a 105-bpXbal-HindIII fragment from pACYC184 was shown to be sufficient to confer therpoB364 hns ⁺-dependent lethal phenotype. We suggest that the level in stationary-phase cultures of a gene product(s) that interacts with the pACYC184 DNA fragment is altered in therpoB364 hns+derivative (compared to that inrpoB+ orrpoB364 hns strains) and that this results in cell suicide.     

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein

We previously described a dominant negative secY ^-d 1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY ^-d 1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240→Asp). The secY249 mutation (Ala249→Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity␣to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a “hyper reversion” with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY^-d1. When the secY ^-d 1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY ^-d 1–241) showed an increased ability to interfere with protein export. On the basis of our model for SecY^-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex. Received: 20 October 1997 / Accepted: 1 December 1997     

Emergence of a mutagenic ochre suppressor mutation under lactose selection in appm mutant ofEscherichia coli harbouring the F′lacZU118 episome

WhenEscherichia coli harbouring theppm (earlier calledadi) mutation and the F′lacZU118 episome is subjected to lactose selection in the presence of suboptimal concentrations of glycerol, Lac⁺ colonies emerge after 5–6 days. They are shown to harbour an ochre suppressor mutation at 15.15 min. Inactivation ofrecA results in approximately four-fold reduction in the response. In theppm — ochre suppressor double mutant background the leakiness of thelacZ allele carried by F′ CC105 is enhanced, suggesting misreading of a valine codon (GUG) as glutamic acid codon (GAG). This is accompanied by reversion of thelacZ mutation tolacZ ⁺ (GTG → GAG). In LB medium both the leakiness and reversion are inhibited by streptomycin. Inactivation ofrecA did not affect leakiness but abolished reversion. These data are discussed in relation to the importance of allele leakiness and restricted growth in stationary-phase (adaptive) mutagenesis.     

In vivo cloning of a carboxy-terminalrpoB allele which confers altered transcriptional properties

A 3′-terminal mutation of the gene encoding the β subunit ofEscherichia coli RNA polymerase was isolated using anin vivo polA(Ts) technique. Cloning of the allele was monitored by virtue of the fact that the deletion Δ(rpoB) 1570-1 resulted in an altered-size restriction fragment. DNA sequencing confirmed the predicted nature and location of the mutation: Δ(rpoB) 1570-1 involved an in-frame deletion of 186 bp (62 codons) encoding amino acid residues 967–1028. The phenotype conferred by Δ(rpoB) 1570-1 is discussed with respect to conserved domains within the β polypeptide. Dedicated to Dr. J. Spížek on the occasion of his 60th birthday     

A new mutation inEscherichia coli K12,isfA, which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

The paromomycin resistance mutation (parr-454) in the 15 S rRNA gene of the yeast Saccharomyces cerevisiae is involved in ribosomal frameshifting

Summary The leaky expression of the yeast mitochondrial geneoxi1, containing a frameshift mutation (+1), is caused by natural frameshift suppression, as shown previously (Fox and Weiss-Brummer 1980). A drastic decrease in the natural level of frameshifting is found in the presence of thepar ^r-454 mutation, localized at the 3′ end of the 15 S rRNA gene. This mutation causes resistance to the antibiotic paronomycin in the yeast strains D273-10B and KL14-4A (Li et al. 1982; Tabak et al. 1982). The results of this study imply that in the yeast strain 777-3A this mutation alone is sufficient for restriction of the level of natural frameshifting but is insufficient to confer resistance to paromomycin. A second mutation, arising spontaneously with a frequency of 10⁻⁴ leads, in combination with thepar ^r-454 mutation, to full paromomycin resistance in strain 777-3A.     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.     

The “Sardinian”HLA-A30,B18,DR3,DQw2 haplotype constantly lacks the21-OHA andC4B genes. Is it an ancestral haplotype without duplication?

TheC4 and21-OH loci of the class III HLA have been studied by specific DNA probes and the restriction enzymeTaq I in 24 unrelated Sardinian individuals selected from completely HLA-typed families. All 24 individuals had theHLA extended haplotypeA30,Cw5,B18, BfF1,DR3,DRw52,DQw2, named “Sardinian” in the present paper because of its frquency of 15% in the Sardinian population. Eighteen of these were homozygous for the entire haplotype, and six were heterozygous at theA locus and blank (or homozygous) at all the other loci. In all completely homozygous cells and in four heterozygous cells at theA locus, the restriction fragments of the21-OHA (3.2 kb) andC4B (5.8 kb or 5.4 kb) genes were absent, and the fragments of theC4A (7.0 kb) and21-OHB (3.7 kb) genes were present. It is suggested that the “Sardinian” haplotype is an ancestral haplotype without duplication of theC4 and21-OH genes, practically always identical in its structure, also in unrelated individuals. The diversity of this haplotype in the class III region (about 30 kb less) may be at least partially responsible for its misalignment with most haplotypes, which have duplicatedC4 and21-OH genes, and therefore also for its decreased probability to recombine. This can help explain its high stability and frequency in the Sardinian population. The same conclusion can be suggested for the Caucasian extended haplotypeA1,B8,DR3 that always seems to lack theC4A and21-OHA genes.     

Known mutation (A3072G) in intron 3 of theIGF2 gene is associated with growth and carcass composition in Polish pig breeds

IGF2 is one of the genes that control muscle development. Moreover,IGF2 is imprinted, as only the paternal allele is expressed in the offspring. Using real-time PCR forIGF2 genotyping (Carrodegous et al. 2005), we evaluated the frequency of theIGF2 A3072G mutation (Van Laere et al. 2003) in pigs: Polish Landrace (PL,N = 271) and Large White (LW,N = 267). Our results are consistent with previous reports, showing that theA allele is common in breeds subjected to strong selection for lean meat content (A allele frequency was 0.79 in LW and 0.69 in PL). Moreover, we compared body composition, growth performance and meat quality traits in pigs carrying opposite genotypes (A/A andG/G) inthe IGF2 gene. The association study revealed that theA allele increases the weight of loin (WL) (additive gene effect = 450±50 g in LW and 213±64g in PL), weight of ham (WH) (544±48 g in LW and 302±72 g in PL), loin eye area (LEA) (4.9±0.46 cm² in LW and 2.1 ±0.95 cm² in PL), carcass meat percentage (CP) (3.12±0.27% in LW and 1.89±0.47% in PL), and decreases average backfat thickness (ABF) (−0.2±0.036 cm in LW and −0.2±0.048 cm in PL). Additionally, in PL, theA allele increases the weight of tenderloin (WT) (11±0.01 g), average daily gain (ADG) (30.7±17.29 g), and decreases feed intake (F) (−121±45 g) and days of feeding (DF) (−3.5±2.08 days). No significant effects were observed for meat quality traits. Our results suggest that selection based on theIGF2 mutation in Poland may be very useful in PL and LW pigs, where theG allele is still relatively frequent.     

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% betweenMyc-1.2 andGpt-1, 30.2% betweenGpt-1 andGdc-1, and 9.4% betweenGdc-1 andHox-3.1. The following order of these genes is proposed,Myc-1.2—Gpt-1—Gdc-1—Hox-3.1. All laboratory strains carry theHox-3.1 ^a andMyc-1.2 ^a alleles. Among strains of wild origin,domesticus strains carry only theHox-3.1 ^a andMyc-1.2 ^a alleles, as do the laboratory strains. One strain ofbrevirostris carries theHox-3.1 ^a andMyc-1.2 ^b alleles. Other wild subspecies from Europe and Asia,M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai carry theHox-3.1 ^b andMyc-1.2 ^b alleles.     

Mapping of mutation tsm-8 with respect to transfer RNA genes on the mitochondrial DNA of Saccharomyces cerevisiae.

Summary A precise localization of thetsm-8 mutation in relation to the transfer RNA genes has been attempted by rho^- deletion analysis. The data show that thetsm-8 mutation is in close proximity to the isoleucyl transfer RNA gene. However, it is not yet possible to decide whether thetsm-8 mutation is within this transfer RNA gene.