Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the ^S subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon ( P_mcc-lac ), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P_mcc-lac expression upon severe glucose starvation occurred in rpoS ⁺ and rpoS ^– strains. In cells carrying the rpoD800 mutation that renders the ⁷⁰ subunit of RNA polymerase temperature-sensitive, an activation of P_mcc-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors —nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol—did not activate P_mcc-lac expression in cells at the exponential growth phase.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 48–52.Original Russian Text Copyright © 2005 by Veselovskii, Metlitskaya, Lipasova, Bass, Khmel.     

Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase.     

Catabolite repression of the lac operon: effect of operator-constitutive mutations

Summary We have constructed and tested three lac diploid strains in an attempt to show whether operator-constitutive mutations relieve catabolite repression of the lac operon. Each of these carries a different operator mutation on the chromosome, and all three have the genotype I⁺P⁺O^cZ⁺Y^-polar/Flac I⁺P⁺O⁺Z^delY⁺A⁺. When these strains were grown in medium containing glucose plus gluconate, synthesis of -galactosidase (directed by a gene cis to a mutant operator) and of thiogalactoside transacetylase (directed by a gene cis to an intact operator) suffered equal catabolite repression. We conclude that the operator-constitutive mutations have no effect on catabolite repression. Since it has been shown in analogous experiments that all promoter mutations tested do alleviate catabolite repression, these results are consistent with the view that the operator and promoter are functionally distinct.     

Expression of cho and melC operons by a Streptococcus thermophilus synthetic promoter in Escherichia coli

A 63-base-pair synthetic promoter, sP1, was synthesized on the basis of the nucleotide sequence of a putative Streptococcus thermophilus promoter. When inserted upstream from the Streptomyces cho operon in a recombinant plasmid, pUCO195P-36, sP1 activated the expression of the cho genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants. The sP1-driven cholesterol oxidase production in pUCO195P-36-transformed cells was estimated to be 40% of that produced by P_lac-mediated cho expression in a pUCO193-containing host. The recombinant pUCO195P-36 appeared to be segregationally less stable in E. coli DH5 than in HB101. Its non-expressing counterpart, pUCO195P-1, was stable in both E. coli strains. The activity of sP1 was further demonstrated in E. coli by the expression of a Streptomyces melC operon. When placed upstream from the test operon in the pMCU22aPa construct, sP1 activated the melC expression as shown by the production of tyrosinase at (3.0±0.3) × 10^–3 U/mg and (16.0±1.0) × 10^–3 U/mg protein equivalent of cell extract in the absence and presence of isopropyl -d-thio-galactopyranoside, respectively. The presence of a counter-oriented P_lac at the 3 end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.Mention of brand or firm names does not constitute an endoresement by the U.S. Department of Agriculture over others of a similar nature not mentioned     

Utilization of promoter regions of coliphage lambda in Bacillus subtilis

Summary In vivo studies with galactokinase monitoring system demonstrated that the coliphage lambda P_RP_L promoter regions could be utilized in B. subtilis. These promoter regions were preferentially utilized during the stationary growth phase of B. subtilis. However, these promoter regions were not controlled by the cI₈₅₇ gene at permissive or non-permissive temperature in B. subtilis, although the P _RM promoter was utilized in B. subtilis. S1-nuclease mapping suggests that B. subtilis RNA polymerase recognizes specific sequences in P _R promoter region that is not utilized in E. coli.     

Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

A new method of constructing a set of bacterial cell clones varying in the strength of a promoter upstream of the gene of interest was developed with the use of Escherichia coli MG1655 and lacZ as a reporter. The gist of it lies in constructing a set of DNA fragments with tac-like promoters by means of PCR with the consensus promoter P _tac and primers ensuring randomization of the four central nucleotides in the ?35 region. DNA fragments containing the tac-like promoters and a selective marker (Cm ^R) were used to replace lacI and the regulatory region of the lactose operon in E. coli MG1655. Direct LacZ activity assays with independent integrant clones revealed 14 new promoters (out of 4⁴ = 256 possible variants), whose strength varied by two orders of magnitude: LacZ activity in the corresponding strains gradually varied from 10² Miller units with the weakest promoter to 10⁴ Miller units with consensus P _tac Sequencing of the modified promoters showed that randomization of three positions in the ?35 region is sufficient for generating a representative promoter library, which reduces the number of possible variants from 256 to 64. The method of constructing a set of clones varying in expression of the gene or operon of interest is promising for modern metabolic engineering.     

The construction of lambda transducing phages containing deletions defining regulatory elements of the lac and trp operons in E. coli

Summary Two sets of plaque-forming transducing phages have been isolated which carry parts of the tryptophan (trp) and lactose (lac) operons. The ptrp-lac set of phages carry the trp and lac operons in the same orientation connected by deletions which enter the lac regulatory region from the i side. These deletions start at various sites in or near the trp operon and end either late in the lac i gene, within the promoter, between the promoter and the operator, within the operator, between the operator and the z gene, or very early in the z gene. Starting with one particular trp-lac fusion strain, a series of transducing phages were isolated which contain varying portions of the trp operon extending from the trp A gene towards the trp operator. The other set of phages, which are designated ptrp/lac, carry trp and lac in opposing orientations. These ptrp/lac phages contain deletions which remove all of the lac structural genes and end between the operator and the z gene.A preliminary report of a portion of this work was presented at the Thirteenth International Congress of Genetics, Berkeley, 1973.     

Evidence that the geneuvrB is indispensable for a polymerase I deficient strain ofEscherichia coli K-12

Summary The gene coding for bacteriophage Lambda repressor (cI gene) has been fused to the lac operon of Escherichia coli. In some of the fusions Lambda repressor synthesis can be controlled by the lac operator and promoter. Upon induction of the lac operon the amount of Lambda repressor is increased by a factor of 7 over that found in a single lysogen. In combination with the polarity suppressor suA the induction factor rises to 20. Transducing phages of one fusion were constructed. After thermal induction of this phage the final level of Lambda repressor was enhanced by a factor of 150.Abbreviations xgal 5-bromo-4-chloro-3-indolyl--D-galactoside - IPTG isopropyl-thio--D-galactoside     

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans

The -amylase gene (amy) from Streptomyces griseus IMRU 3570 and the -galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P₂ or P₃. The -amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum. Correspondence to: J. F. Martín     

New regulatory mutations affecting the expression of the threonine operon in Escherichia coli K-12

Summary The promoter of the threonine operon was joined to the structural genes of the lac operon in Escherichia coli K12. The synthesis of -galactosidase was thus repressed by threonine plus isoleucine in the fusion strains. To isolate mutations which affect the expression of the threonine operon, alterations in the level of expression of the lacZ gene were selected. A new type of regulatory mutation was discovered.This paper is part of a thesis presented by Isabelle Saint-Girons in partial fulfillment of the requirements for the Dr. Sc. degree from the University of Paris     

Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism

Summary A selection procedure using Mn²⁺ is described. A high percentage of the Mn²⁺ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::placMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of -galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur ⁺ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%–70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.     

Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

Gene <Emphasis Type="Italic">yddG</Emphasis> of <Emphasis Type="Italic">Escherichia coli</Emphasis> encoding the putative exporter of aromatic amino acids: Constitutive transcription and dependence of the expression on the cell growth rate

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of aromatic amino acids overproduction YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P _yddG promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with σ⁷⁰ or σ^S subunits. By constructing a gene of the hybrid protein YddG’-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of β-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity was about 3 to 4% of the LacZ level under induction of the lac operon in E. coli wild-type cells). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (>1 mM) in the culture medium.