Genetic control of recombination exchange frequency in Escherichia coli K-12.

The frequency of recombination exchanges per unit length of DNA (Freuld) can be estimated by measuring the scale of the genetic map that is the mean statistical distance between two neighboring crossovers. The scales appear to be equal for the alternative pathways of recombination, RecBCD (wild-type cells) or RecF (recBC- sbcB- sbcC- genotypes). The absolute value of the scale depends on specific experimental conditions. recR, recQ, ruv, recJ and recN genes of the RecF pathway of recombination (recBC- sbcBC- cell genotypes) do not appear to be silent in wild-type cells where the RecBCD pathway predominates. On the contrary, these genes are responsible for the Freuld. The list recF504::Kmr greater than recQ61::Tn3 greater than ruv-54 greater than recJ284::Tn10 shows decreasing efficiency in inhibiting recombination exchanges by these mutations. The recN264 mutation gives a small, but opposite effect of increasing the frequency of recombination exchanges. The effect of the recF and recQ mutations appears to be additive, but that is not the case in combinations of ruv-54 with recF504::Kmr or recQ61::Tn3.     

Genetic basis of minicell formation in Escherichia coli K-12.

Hfr- and P1-mediated genetic transfer experiments failed to confirm the presence of a " minA " gene in Escherichia coli K-12, leading to the conclusion that mutation at a single locus, the minB locus, is sufficient to cause minicell production in this species.     

Genetic study of Escherichia coli K-12 mutations that affect the transposition process

Results of genetic analysis of bacterial tnm mutations influencing transposition of Tn9 are presented. Five independent tnm mutations were mapped at 90,5 min of the E. coli genetic map. The tnm mutations were 3,5 and 46,5% contransducible with metA and malB markers, respectively. Two tnm mutations tested were recessive in tnm+/tnm- merodiploids. The effect of tnm mutations on other transposons--Tn10, Tn601, Tn3 and Tn5 was examined. It was shown that tnm1 and tnm2 mutations reduced the frequency of transposition of Tn10, Tn3, Tn5 and Tn601 from the genome of phage lambda and inhibited intracellular development of the infecting Mu phage. The latter effect was probably due to the inhibition of Mu integration into bacterial chromosome. The tnm3 mutation affected the transposition of Tn9 only.     

Small genes/gene-products in Escherichia coli K-12

Forty-two protein spots of observed M_r 6–15 kDa were resolved by two-dimensional gel electrophoresis, stained by Coomassie blue and subjected to Edman microsequencing. All of the proteins could be related back to their encoding open reading frames, thereby vindicating the bioinformatic tools currently utilised in their identification. However, only 14/42 gene-products were expressed as annotated. Translation was confirmed for 14 open reading frames with no attributed function (EcoGene Y-entries), while N-terminal sequence allowed the start codon to be accurately annotated for the genes yjgF, yccU, yqiC, ynfD, and yeeX. The methionine start codon was cleaved in 11 gene-products (AtpE, Hns, RpoZ, RplL, CspC, YccJ, YggX, YjgF, HimA, InfA, RpsQ) and a further five showed loss of a signal peptide (PspE, HdeB, HdeA, YnfD, YkfE). Internal (Tig, AtpA, TufA) and N-terminal fragmentation (CspD, RpsF, AtcU) of much larger proteins was also detected, which may have resulted from physiological or translational processes. M_r and pI isoforms were detected respectively for PtsH and GatB, each being phosphoproteins, as well as RplY which manifested differences with respect to predicted M_r and pI. In addition, YjgF was shown to belong to a small gene family of unknown function with ancient conserved regions across procaryotes and eucaryotes. YgiN was revealed to have a paralogue and orthologues in Bacillus subtilis, Synechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoea, and Rhodococcus erythropolis. Orthologues are also reported for YihD, YccU and YeeX. Of the 14 Y-genes, only YkfE possessed no detectable orthologues. These results highlight the need to complement genomic analysis with detailed proteomics in order to gain a better understanding of cellular molecular biology, while the confirmation of the open reading frame start codon using Edman degradation protein microsequencing has yet to be superseded by recent advances in mass spectrometry.     

Osmoregulation of alkaline phosphatase synthesis in Escherichia coli K-12.

Alkaline phosphatase, the phoA product, is synthesized constitutively in phoR mutants. This constitutive synthesis, which is independent of phosphate control, varies with changes in the osmolarity of the growth medium; phoA expression increases with increasing osmolarity. Maximum expression of the osmoregulated genes phoA, ompC, and ompF was achieved by osmotic manipulation of minimal medium; complex media repressed their expression.     

Nitrate-inducible formate dehydrogenase in Escherichia coli K-12. I. Nucleotide sequence of the fdnGHI operon and evidence that opal (UGA) encodes selenocysteine.

The fdnGHI operon of Escherichia coli encodes nitrate-inducible formate dehydrogenase. We report here the entire nucleotide sequence of fdnGHI. The sequence contains three open reading frames of sizes appropriate to encode the three subunits of formate dehydrogenase-N. fdnG contains an in-frame UGA codon that specifies selenocysteine incorporation, and the predicted amino acid sequence of FdnG shows similarity to two other bacterial formate dehydrogenase enzymes. FdnH contains 4 cysteine clusters typical of those found in iron-sulfur proteins. FdnG also contains a cysteine cluster. Evidence from sequence and spectral analyses suggest that FdnI encodes cytochrome bFdn556. Implications for the membrane topology of formate dehydrogenase-N and its mechanism of proton translocation are discussed.     

Novel Escherichia coli K-12 mutants impaired in S-adenosylmethionine synthesis.

S-Adenosylmethionine (AdoMet) plays a myriad of roles in cellular metabolism. One of the many roles of AdoMet in Escherichia coli and Salmonella typhimurium is as a corepressor of genes encoding enzymes of methionine biosynthesis. To investigate the metabolic effects of large reductions in intracellular AdoMet concentrations in growing cells, we constructed and examined mutants of E. coli which are conditionally defective in AdoMet synthesis. Temperature-sensitive mutants in metK, the structural gene for the S-adenosylmethionine synthetase (AdoMet synthetase) expressed in minimal medium, were constructed by in vitro mutagenesis of a plasmid-borne copy of metK. By homologous recombination, the chromosomal copy was replaced with the mutated metK gene. Both heat- and cold-sensitive mutants were examined. At the nonpermissive temperature, two such mutants had 200-fold-reduced intracellular AdoMet levels and required either methionine or vitamin B12 for growth. In the presence of methionine or vitamin B12, the mutants grew at normal rates even though the AdoMet levels remained 0.5% of wild type. A third mutant when placed at nonpermissive temperature had less than 0.2% of the normal AdoMet level and did not grow on minimal medium even in the presence of methionine or vitamin B12. All of these mutants grew normally on yeast-extract-based medium in which an alternate form of S-adenosylmethionine synthetase was expressed.     

Genetic control of the resistance to phage C1 of Escherichia coli K-12.

Escherichia coli K-12 lytic phage C1 was earlier isolated in our laboratory. Its adsorption is controlled by at least three bacterial genes: dcrA, dcrB, and btuB. Our results provide evidence that the dcrA gene located at 60 min on the E. coli genetic map is identical to the sdaC gene. This gene product is an inner membrane protein recently identified as a putative specific serine transporter. The dcrB gene, located at 76.5 min, encodes a 20-kDa processed periplasmic protein, as determined by maxicell analysis, and corresponds to a recently determined open reading frame with a previously unknown function. The btuB gene product is known to be an outer membrane receptor protein responsible for adsorption of BF23 phage and vitamin B12 uptake. According to our data the DcrA and DcrB proteins are not involved in these processes. However, the DcrA protein probably participates in some cell division steps.     

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Genetic analysis of the Escherichia coli K-12 srl region.

Specialized transducing lambda derivatives, deletion mapping, and Plkc transductional crosses have been used to analyze the genetic organization and regulation of the srl genes. Transducing phages obtained from a secondary site lambda insertion in srlA are of two types: lambdapsrlC1 and lambdaprecA are substituted in the b2 region of the lambda chromosome (galtype) and carry the srlC gene but not srlD; lambdapsrlD is substituted in the early region of the phage deoxyribonucleic acid (biotype) and carries the srlD gene but not srlC. The lambdapsrlC1 phage, which lysogenizes at attlambda, complements srlC mutants in trans, indicating that this gene codes for a diffusable positive regulatory element. The srl genes have been ordered relative to the cysC, recA, and alaS genes by two- and three-factor P1kc crosses. The order, cysC...srlD-srlA-srlC-recA-alaS, has been obtained. The srlA and srlD genes comprise an operon with srlD operator distal. From the secondary site lysogen, it has been possible to obtain deletion mutants of this region that are sensitive to ultraviolet light and are recombination deficient. Genetic evidence suggests that these deletions extend from srl into the recA gene.     

Genetic characterization of the metK locus in Escherichia coli K-12.

Three independently isolated metK mutants have been shown to have leisions lying between speB and glc near 57 min on the Escherichia coli chromosome. Two deletions result in a lack of the metC gene product but neither extends into the metK glc region. The three metK mutations are recessive to the wild-type allele carried on the KLF16 episome.