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PubMed1984

Isolation and characterization of rho mutants of Escherichia coli with increased transcription termination activities.

A novel type of rho mutants, rhos, with increased transcription termination activities have been isolated. A termination defective rho mutation rho-ts702 (formerly designated nitA702), which causes temperature-sensitive cell growth, was found to be dominant over the wild-type allele in relieving mutational polarity. The rhos mutations were derived as temperature-resistant revertants of rho-ts702 carried by lambda transducing phage. They exhibited dominance over rho-ts702 leading to restoration of polarity. When the rhos mutations were introduced into the Escherichia coli chromosome, they caused increased polarity in the trp and lac operons. The rhos mutants were classified into two groups in terms of their terminator specificity: The first group demonstrated increased termination efficiencies against all terminators tested, whereas the second exhibited various efficiencies, either more than or less than the normal level depending on the terminator. The cellular content of p protein in each rhos strain was significantly lower than that in the rho+ strain. Moreover, in an in vitro transcription system, purified ps proteins showed increased termination activities against the trpE pseudoterminators. These results indicate that the rhos phenotype is due to qualitative alterations, rather than quantitative increases, of the p protein. The reduced content of ps enforces the current notion that the rho gene is autogenously regulated by rho-dependent transcriptional attenuation.

Base Sequence↗
PubMed1991

Ethanol production by recombinant Escherichia coli carrying genes from Zymomonas mobilis.

Efficient utilization of lignocellulosic feedstocks offers an opportunity to reduce the cost of producing fuel ethanol. The fermentation performance characteristics of recombinant Escherichia coli ATCC 11303 carrying the "PET plasmid" (pLOI297) with the lac operon controlling the expression of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes cloned from Zymomonas mobilis CP4 (Alterthum & Ingram, 1989) were assessed in batch and continuous processes with sugar mixtures designed to mimic process streams from lignocellulosic hydrolysis systems. Growth was pseudoexponential at a rate (generation time) of 1.28 h at pH 6.8 and 1.61 h at pH 6.0. The molar growth yields for glucose and xylose were 17.28 and 7.65 g DW cell/mol, respectively (at pH 6.3 and 30 degrees C), suggesting that the net yield of ATP from xylose metabolism is only 50% compared to glucose. In pH-stat batch fermentations (Luria broth with 6% sugar, pH 6.3), glucose was converted to ethanol 4-6 times faster than xylose, but the glucose conversion rate was much less than can be achieved with comparable cell densities of Zymomonas. Sugar-to-ethanol conversion efficiencies in nutrient-rich, complex LB medium were near theoretical at 98 and 88% for glucose and xylose, respectively. The yield was 10-20% less in a defined-mineral-salts medium. Acetate at a concentration of 0.1M (present in lignocellulosic hydrolysates from thermochemical processing) inhibited glucose utilization (about 50%) much more than xylose, and caused a decrease in product yield of about 30% for both sugars. With phosphate-buffered media (pH 7), glucose was a preferred substrate in mixtures with a ratio of hexose to pentose of 2.3 to 1. Xylose was consumed after glucose, and the product yield was less (0.37 g/g). Under steady-state conditions of continuous culture, the specific productivity ranged from 0.76-1.24 g EtOH/g cell/h, and the maximum volumetric productivity, 2.5 g EtOH/L/h, was achieved with a rich complex LB medium (glucose) at pH 6.0 (30 degrees C) and ethanol at 1.63% (v/v). Growth and fermentation were poor in a buffered-wood (aspen) "hemicellulose hydrolysate" containing 4% xylose and 0.1M acetate with added thiamine and mineral salts.

Alcohol Dehydrogenase↗
PubMed1989

A microtiter plate-based system for the semiautomated growth and assay of bacterial cells for beta-galactosidase activity.

The introduction of automated pipetting devices, microtiter readers, and microcomputers makes it possible to significantly increase the number of enzyme assays which can be performed as part of the analysis of a biological process. A number of difficulties must be overcome in any such integrated approach based on the microtiter plate. Among these are cell lysis, temperature control, the conversion of microtiter reader optical density values to standard 1-cm path length values, and data management. The utility of such a scheme can be extended to gene regulation and bacterial genetics studies, if bacterial cell culture techniques can be incorporated into the scheme. This paper addresses these issues in the application of a semiautomated system to the study of the induction of the gyrA promoter by treatment (of a gyrA-lac operon fusion-containing strain) with a gyrase inhibitor. This system is specific to the requirements of our studies into the modulation of gene expression by DNA relaxation. The general approach, however, can be readily adapted to other studies.

Bacteria↗
PubMed1986

Reversion reactions of beta-galactosidase (Escherichia coli).

The reversion reactions of beta-galactosidase (Escherichia coli) produced beta-galactosyl-galactoses and beta-galactosyl-glucoses. About 10 beta-galactosyl-galactose and 10 beta-galactosyl-glucose gas-liquid chromatographic peaks were detected and it is thus very likely that every possible isomer of beta-galactosyl-galactose and beta-galactosyl-glucose was formed by the reversion reactions (taking into account both anomers for each isomer). The presence of lactose and allolactose among the beta-galactosyl-glucoses was confirmed with standards. An important finding relating to the role of allolactose as an inducer of the lac operon was that allolactose (beta-D-galactosyl-(1----6)-D-glucose) was the only disaccharide formed initially, and at equilibrium it was present in the largest amount (50%). Obviously the enzyme is specific in its ability to form allolactose, and allolactose is the most stable beta-galactosyl-glucose, both important inducer properties. The equilibrium constant (concentration of disaccharides divided by the concentration of reactants at equilibrium) of the reaction was about 9.5 mM-1. This is the first report of an equilibrium constant for the beta-galactosidase reaction. Of mechanistic significance is the fact that only three compounds were able to replace D-galactose as a reversion reactant. Two of these (L-arabinose and D-fucose) had alterations at carbon 6. The 6 position, therefore, is not essential for reactivity. The third compound was D-galactal. Any other sugars tested (even with very minor changes relative to D-galactose) did not react. Of special consequence is the 2 position. The results strongly suggest that there has to be either an equatorial hydroxyl at the 2 position of a sugar or a special reactivity (as with D-galactal) in order for the enzyme to catalyze the beta-galactosidase reaction.

Binding Sites↗
PubMed1983

Stoichiometry of the H+-ATPase of Escherichia coli cells during anaerobic growth.

The H+/ATP stoichiometry of the H+-ATPase was investigated in Escherichia coli cells growing under anaerobic conditions at pH 6 and 7. The protonmotive force was determined from the intracellular accumulation of benzoate and tetraphenylphosphonium ions, as well as the accumulation of lactose in this lac operon inducible, but beta-galactosidase negative strain. The phosphorylation potential was calculated from the cellular concentrations of ATP, ADP and inorganic phosphate. By comparing the phosphorylation potential and the proton motive force under these steady state conditions, the H+/ATP stoichiometry was determined to be 3, similar to the value previously found in the same cells growing under aerobic conditions.

Adenosine Diphosphate↗
PubMed1984

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy.

The effect of specific DNA binding of the cAMP . cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP . cAMP receptor protein complex induces a kink of 30 to 45 degrees in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.

Binding Sites↗
PubMed1987

Feedback regulation of rRNA synthesis in Escherichia coli. Requirement for initiation factor IF2.

It has been shown that the transcription of rRNA in Escherichia coli is feedback-regulated by its own transcription products through a negative feedback loop which appears to require the assembly of rRNA into complete ribosomes. In order to examine whether the feedback loop involves the ribosomes' main function, translation, we have constructed a strain in which the chromosomal copy of infB, encoding IF2, was placed under lac promoter/operator control, and the effects of limitation of translation initiation factor IF2 on the regulation were examined. By varying the concentration of a lac operon inducer, isopropyl thiogalactoside (IPTG), it was possible to vary the cellular concentration of IF2. Under the growth conditions used, decreasing the concentration of IF2 about twofold affected the growth rate only slightly, but further deprivation of IF2 resulted in a significant decrease in growth rate, an increase in RNA content and a large accumulation of non-translating ribosomes. These accumulated ribosomes were apparently unable to cause feedback regulation of rRNA synthesis in the absence of sufficient IF2. When a higher concentration of IPTG was added to these IF2-deficient cells, a rapid increase in the IF2 level and a significant decrease in the rate of RNA accumulation were observed before the new steady-state growth was attained. These results indicate that IF2 apparently is necessary for feedback regulation of stable RNA and imply that ribosomes must enter translation for feedback regulation to occur.

Centrifugation, Density Gradient↗
PubMed1987

Interaction between the cyclic AMP receptor protein and DNA. Conformational studies.

The binding of the cyclic adenosine 3',5' monophosphate receptor protein (CRP or CAP) of Escherichia coli to non-specific DNA and to a specific lac recognition sequence has been investigated by circular dichroism (c.d.) spectroscopy. The effect of cAMP and cGMP on the co-operative non-specific binding was also studied. For the non-specific binding in the absence of cAMP a c.d. change (decrease of the intensity of the positive band with a shift of its maximum to longer wavelength) indicates that the DNA undergoes a conformational change upon CRP binding. This change might reflect the formation of the solenoidal coil previously observed by electron microscopy. The amplitude of the c.d. change increases linearly with the degree of saturation of the DNA and does not depend on the size of the clusters of CRP bound. From the variation of the c.d. effect as a function of the ionic strength, the product K omega (K, the intrinsic binding constant and omega, the co-operativity parameter) could be determined. The number of ion pairs involved in complex formation between CRP and DNA was found to be six to seven. Experiments performed with several DNAs, including the alternating polymers poly[d(A-T)] and poly[d(G-C)], demonstrated that the conformational change does not depend on the DNA sequence. However, in the presence of cAMP the c.d. spectrum of the DNA shows only a small variation upon binding CRP. In contrast, in the presence of cGMP the conformational change of the DNA is similar to that observed when non-liganded CRP binds. For the specific lac operon binding, the c.d. change is different from those observed for non-specific binding in the presence or absence of cAMP. These results emphasize the high variability of the DNA structure upon binding the same protein.

Circular Dichroism↗
PubMed1991

Osmotic induction of gene osmC expression in Escherichia coli K12.

osmC, an osmotically inducible gene of Escherichia coli, was physically mapped on the bacterial chromosome, cloned on multicopy plasmids, and its product, OsmC, was identified as a 14 kDa protein in maxicells. The DNA sequence of the gene and its upstream region were determined. The sequence of an osmC-phoA gene fusion confirmed the osmC reading frame. A deletion of osmC from the E. coli chromosome was constructed by gene replacement, demonstrating that it is not an essential gene. The osmCp promoter region was subcloned and a lac operon fusion transcribed under osmCp control was constructed. The expression of this operon fusion demonstrated that osmC regulation occurs at the transcriptional level. S1 nuclease protection experiments and deletion analysis identified two overlapping promoters with transcription start sites separated by ten nucleotides. All the sequences necessary for osmotic regulation of both promoters are located within a 137 base-pair DNA fragment extending from position -95 to +42 with respect to the putative osmC translation start. Two deletions were obtained that abolish the functioning of the upstream promoter. Yet, under our experimental conditions, the subsequent expression of the osmC-lacZ fusion was equivalent to that obtained from the tandem promoters. Mutations leading to constitutive expression of osmC were selected. Two independent mutations were obtained, both affected osmZ, the gene encoding the histone-like protein H1.

Amino Acid Sequence↗
PubMed1985

The stability of bacteriophage T4 gene 32 mRNA: a 5' leader sequence that can stabilize mRNA transcripts.

In T4-infected cells, the gene 32 monocistronic mRNA is very stable. To study the molecular basis for this stability, we have constructed chimeric plasmids containing the monocistronic promoter and the gene 32 translation initiation sequence fused to either most of the E. coli lac operon or only a segment of the lacZ gene, followed by the gene 32 transcription terminator. The resulting hybrid transcripts are unstable in uninfected cells. In phage-infected cells, however, the hybrid mRNAs are at least as stable as gene 32 mRNA itself. Analysis of other plasmid constructs indicates that the sequences on the gene 32 mRNA from its 5' end to slightly beyond the initiation codon suffice to stabilize these hybrids. Studies with a series of deletions of the gene 32 leader sequence suggest that an RNA sequence near the gene 32 initiation codon is involved. Various models to explain this mRNA stabilization are discussed.

DNA Helicases↗
PubMed1992

Mutagenic effects of heavy ions in bacteria.

The peculiarities and mechanisms of the mutagenic action of gamma-rays and heavy ions on bacterial cells have been investigated. Direct mutations in the lac-operon of E. coli in wild type cells and repair deficient strains have been detected. Furthermore, the induction of revertants in Salmonella tester strains was measured. It was found that the mutation rate was a linear-quadratic function of dose in the case of both gamma-rays and heavy ions with LET up to 200 keV/micrometer. The relative biological effectiveness (RBE) increased with LET up to 20 keV/micrometer. Low mutation rates were observed in repair deficient mutants with a block of SOS-induction. The induction of SOS-repair by ionizing radiation has been investigated by means of the "SOS-chromotest" and lambda-prophage induction. It was shown that the intensity of the SOS-induction in E. coli increased with increasing LET up to 40-60 keV/micrometer.

Alpha Particles↗
PubMed1996

A grammar describing 'biological binding operators' to model gene regulation.

The study of the mechanisms involved in the regulation of protein synthesis has become sufficiently advanced that it is appropriate to think about a knowledge formalism. The objective of the syntactic grammar which we present in this article is a representation of these phenomena which take place in the context of the cell. The proposed model considers two types of objects: transcriptional units on DNA and regulatory or structural proteins which are synthesised, and which are, in the case of regulatory proteins, themselves destined to activate or repress other transcriptional units in a later phase. A transcriptional unit is described by the list of its active sites (operator, promoter, binding sites for transcription factors). A regulatory protein is described by the list of its active sites (binding domain, activation domain, binding domain for ligand). The DNA sites and the protein domains are the terminal symbols of the proposed grammar. The interaction of these proteins with the DNA, and in certain cases preliminary interactions between proteins, leads to one of two antagonistic actions: expression or repression of the transcriptional unit. These protein-protein and protein-DNA interactions are grouped into syntactic categories (induction, inhibition, initiation complex, repressor complex, activation complex) which are called biological binding operators. The expression/repression action are described by grammar rules which provide the chain of execution by biological binding operators for the four activable/repressible regulatory systems modulated by positive/negative co-factors. The object of this modelization is the observation of a cell in a given state for a given process which involves a cascade of genes. This grammar is implemented by a simulation program which allows the user to vary the initial state of the cell and also to change parameters related to time and quantity. This syntactic and generative grammar is independent of the specificity of each transcriptional unit. The simulation uses examples which may combine several regulatory systems: the lac operon, regulation of metallothionein, galactose catabolism in yeast, the tryptophan operon, and phage lysogenic/lytic cascades.

DNA, Bacterial↗
PubMed1990

Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa: use of a new broad-host-range promoter-probe plasmid.

The broad-host-range plasmid, pRW2, is a derivative of pRK 2501 carrying the Escherichia coli lac operon without a promoter, downstream of a polylinker sequence. We have cloned a number of DNA fragments carrying promoters into this plasmid and measured promoter activity in both E. coli and Pseudomonas aeruginosa. Promoters carrying consensus -10 and -35 sequences were active in both backgrounds and the dependence of activity on the nucleotide sequence of the 35 region was the same in both cases. We also measured the activity of two promoters at which transcription in E. coli was totally dependent on the E. coli activators CRP and FNR: both promoters were found to be active in P. aeruginosa.

Base Sequence↗
PubMed1986

Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA.

Plasmid DNA substrates were used to study ultraviolet (UV)-induced recombination events in Escherichia coli host cells. Plasmids derived from pBR322, containing all or part of the lac operon of E. coli, were irradiated with ultraviolet light before transformation into E. coli strains of different recA and lacY genotypes. Recombinational exchanges were identified by phenotypic changes in lactose utilization and were confirmed by restriction analysis of isolated plasmids. Ultraviolet-induced reciprocal plasmid-chromosome recombination occurred at a slightly higher frequency then non-reciprocal chromosome-to-plasmid recombination, and at a much higher frequency than non-reciprocal plasmid-to-chromosome recombination. These frequencies did not depend on segregative mechanisms. The asymmetry of non-reciprocal exchange was not due to the particular arrangement of wild-type and lacY1 alleles because the same results were observed when these were interchanged. The host recA gene was required for plasmid-chromosome recombination, and slightly enhanced plasmid survival. Evidence for plasmid replication prior to recombination was found in reciprocal recombinants, but rarely in the non-reciprocal recombinants analyzed. Irradiation of competent bacterial host cells prior to transformation did not effectively induce plasmid-chromosome recombination.

DNA, Bacterial↗
PubMed1987

Hybrid protein thymidine kinase gene fusions: plasmid vectors for the study of transcription and translation initiation signals.

The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins. The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites. DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli. These gene fusions were shown to complement an E. coli strain which is deficient in TK function. Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl. TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint. The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK. This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity. Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed.

Cloning, Molecular↗
PubMed1987

In vivo DNA cloning with a mini-Mu replicon cosmid and a helper lambda phage.

A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo. This mini-Mu element can be derepressed to transpose at a high frequency. DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage. The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini. Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences. These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process. Clones of particular genes were isolated by their ability to complement specific mutations. Both recA+ and recA- recipient cells can be used with equal efficiency. Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon. They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage. The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences. Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.

Bacteriophage lambda↗
PubMed1988

Transposition of lambda placMu is mediated by the A protein altered at its carboxy-terminal end.

Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.

Alleles↗
PubMed1990

A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for gram-negative bacteria.

A series of broad-host-range expression and lac fusion vectors, based on RSF1010 derivatives, was constructed. The expression vectors contain various promoters (pNm, plac, ptac and pS1) for expression of foreign genes. The efficiency of the promoters was determined in Escherichia coli, Rhizobium meliloti, Rhizobium leguminosarum and Pseudomonas putida by beta-galactosidase activity measurements. Of the promoters assayed in E. coli, the most effective is the tac promoter, whereas in soil bacteria the appropriate promoter for overexpression of foreign genes is the NmR promoter. The GmR gene, serving as a selectable marker for the plasmids, was efficiently expressed in R. meliloti as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thus, pGm was also used to construct an expression vector. The translational fusion vectors allow the identification and characterization of promoter-carrying cloned fragments on the translational level, whereas the transcriptional fusion vectors can be used to identify and to study promoters on cloned fragments. All lac fusion vectors contain the E. coli lacZ gene or the complete lac operon facilitating quantification of expression.

Base Sequence↗