Operon Fusion Research Articles

Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series

A series of Escherichia coli-mycobacteria shuttle plasmids for the isolation and study of gene regulatory sequences was constructed. These pJEM vectors contain an efficient transcription terminator and multiple cloning sites and allow either operon or gene fusions to lacZ. By constructing operon fusions with pJEM15, we assessed various previously characterized mycobacterial promoters in the fast-growing species Mycobacterium smegmatis and the slow-growing species M. bovis BCG. Our results suggest that M. smegmatis and M. bovis BCG RNA polymerases do not share the same specificity. To isolate new mycobacterial promoters, an M. tuberculosis DNA library was generated, using pJEM13, and screened in M. smegmatis. Several Lac+ clones were isolated, and the beta-galactosidase activity was measured.

Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD, dmsABC, and narGHJI of Escherichia coli

The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine-N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHJI) operons in Escherichia coli encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO or TMAO, and nitrate, respectively. They are regulated in response to anaerobiosis and nitrate availability. To determine how each operon is regulated in response to changes in cell growth rate and in oxygen availability, expression of frdA-lacZ, dmsA-lacZ, and narG-lacZ fusion genes was examined during continuous culture. After a change in the cell growth rate, each anaerobic electron transport pathway operon fusion responded somewhat differently. Whereas frdA-lacZ expression increased by fivefold as the growth rate decreased from 0.60 to 0.12/hour during aerobic growth, little change was seen under anaerobic conditions. In contrast, growth rate-dependent expression of narG-lacZ expression occurred under anaerobic conditions but not under aerobic conditions. Finally, dmsA-lacZ expression did not vary greatly for any of the growth rates tested. When cells were shifted from aerobic to anaerobic growth conditions, expression of each fusion increased at a moderate rate and peaked or "overshot" before reaching a new equilibrium value. This "overshoot" phenomenon was independent of the fnr gene product, which functions as a transcriptional activator of each respiratory operon during anaerobic conditions. In contrast to the moderate rate of anaerobic induction seen for narG-lacZ expression, the addition of nitrate caused a rapid induction response. The cell appears to have many ways to adjust cell respiration in response to changes in cell growth conditions.

Regulation of the citrate synthase (gltA) gene of Escherichia coli in response to anaerobiosis and carbon supply: role of the arcA gene product

As an enzyme of the tricarboxylic acid cycle pathway, citrate synthase participates in the generation of a variety of cellular biosynthetic intermediates and in that of reduced purine nucleotides that are used in energy generation via electron transport-linked phosphorylation reactions. It catalyzes the condensation of oxaloacetate and acetyl coenzyme A to produce citrate plus coenzyme A. In Escherichia coli this enzyme is encoded by the gltA gene. To investigate how gltA expression is regulated, a gltA-lacZ operon fusion was constructed and analyzed following aerobic and anaerobic cell growth on various types of culture media. Under aerobic culture conditions, expression was elevated to a level twofold higher than that reached under anaerobic culture conditions. ArcA functions as a repressor of gltA expression under each set of conditions: in a delta arcA strain, gltA-lacZ expression was elevated to levels two- and eightfold higher than those seen in a wild-type strain under aerobic and anaerobic conditions, respectively. This control is independent of the fnr gene product, an alternative anaerobic gene regulator in E. coli. When the richness or type of carbon compound used for cell growth was varied, gltA-lacZ expression varied by 10- to 14-fold during aerobic and anaerobic growth. This regulation was independent of both the crp and fruR gene products, suggesting that another regulatory element in E. coli is responsible for the observed control. Finally, gltA-lacZ expression was shown to be inversely proportional to the cell growth rate. These findings indicate that the regulation of gltA gene expression is complex in meeting the differential needs of the cell for biosynthesis and energy generation under various cell culture conditions.

Characterization of the sigma 38-dependent expression of a core Escherichia coli starvation gene, pexB

A reverse genetics approach was used to clone a pex starvation gene that codes for an 18-kDa polypeptide, designated PexB. Single-copy pexB-lacZ operon fusions were constructed to study transcriptional regulation and the promoter region of this gene. The induction by carbon starvation or osmotic stress was transcriptional and controlled by sigma 38 but was independent of this sigma factor by the oxidative stress; presumably, it was sigma 70 mediated under the latter stress. During nitrogen starvation, the induction was controlled at the posttranscriptional level. The pexB upstream region contained 245 nucleotides within which sequences approximating the consensus for cyclic AMP receptor protein and integration host factor binding sites were discernible. Deletion of 164 bp of the upstream region, which included these consensus sequences, did not affect starvation-or osmotic stress-mediated induction of pexB but abolished its induction by oxidative stress. The same start site was used in transcription during carbon starvation, osmotic stress, or oxidative stress, suggesting that the pexB promoter can be recognized in vivo by both sigma 38 and sigma 70, depending, presumably, on the presence of appropriate transcriptional factors. The -10 and -35 regions of pexB resembled those of some but not all genes known to be controlled by sigma 38.

The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli.

The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer. Here we show that this 5' UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III.

Novel growth rate control of dam gene expression in Escherichia coli.

Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical sigma 70 promoter with 18 bp spacing between the -10 and -35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of beta-galactosidase activity at all growth rates tested. In contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-4 confirming the different modes of regulation. The cde-4::miniTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene.

Use of an operon fusion to induce expression and crystallisation of a Bacillus thuringiensis delta-endotoxin encoded by a cryptic gene.

A delta-endotoxin gene previously cloned from Bacillus thuringiensis subsp. galleriae has been shown by a combination of restriction mapping and DNA sequence analysis to be a cryIIB clone; in common with other cryIIB genes it was found to lack a functional promoter. Addition of a promoter resulted in expression of the gene in Bacillus thuringiensis but did not result in the formation of the crystalline inclusions normally associated with such toxins. Inclusion formation was only observed when the gene was incorporated into an operon containing a gene known to be involved in the crystallisation of another delta-endotoxin.

Genetic selection for mutations that impair the co-operative binding of lambda repressor.

Bacteriophage lambda repressor binds co-operatively to adjacent pairs of DNA target sites. A novel combination of positive genetic selections, involving two different operon fusions derived from P22 challenge phages, was used to isolate mutant lambda repressors that have lost the ability to bind co-operatively to tandem sites yet retain the ability to bind a strong, single site. These cb (co-operative binding) mutations result in 10 different amino acid changes, which define eight residues in the carboxyl-terminus of repressor. Because challenge phage derivatives may be applied to study essentially any specific protein-DNA interaction, analogous combinations of genetic selections may be used to explore the ways that a variety of proteins interact to assemble regulatory complexes.

Regulatory circuits involved with pH-regulated gene expression in Salmonella typhimurium

Salmonella typhimurium encounters a variety of acid conditions during both its natural and pathogenic existence. The ability of this organism to respond transcriptionally to low pH is an area of active interest but little knowledge. As part of an ongoing investigation of low-pH adaptation, 18 pH-controlled lacZ operon fusions in Salmonella typhimurium have been identified (15 in this study) and categorized into at least 11 different loci. They include iroA (at 57 min), aciA (99 min), aciB (90-93 min), aciD (ompC, 45 min), aciJ, aciK (33-36 min), aniC (93 min), anil (33-36 min), hyd (59 min), cadA (54 min) and aniG (63 min). All but two were induced by low pH. One of the exceptions, the iron-regulated iroA locus, was induced at high pH. The unusual aciA locus was induced by low pH under semiaerobic conditions but high pH under aerobic conditions. Most of the other aci genes were expressed best under anaerobic conditions. Many of these genes exhibited strict co-inducer requirements for small molecules to be expressed in minimal medium. These included iron for iroA, tyrosine for aniC, I and aciK, mannose for aniG, formate for hyd, lysine for cadA, and unknown components of complex medium for aciA, aciB and aciD. Six regulatory circuits were revealed involving at least five regulatory loci (fur, oxrG, earAB, earC and ompR). As part of the adaptive response to low pH, S. typhimurium will induce an acid protection system called the acid tolerance response (ATR). As has been shown for fur mutations, the oxrG regulatory mutation interfered with the normal induction of this system.

Regulation of EcoRII methyltransferase: effect of mutations on gene expression and in vitro binding to the promoter region.

EcoRII Methyltransferase (M.EcoRII) which methylates the second C in the sequence CCWGG (W = A/T) is autogenously regulated by binding to the 5' regulatory region of its gene. DNase I footprinting experiments demonstrated that purified M.EcoRII protected a 47-49 bp region of DNA immediately upstream of the ecoRIIM coding region. We have studied this interaction with mutants of the enzyme, in vitro by DNA binding and in vivo by investigating the repression in trans of expression of beta-galactosidase from an ecoRIIM-lacZ operon fusion. Two catalytically active mutants failed to repress expression of the fusion whereas catalytically inactive mutants had repressor activity. However, with one of the catalytically inactive mutants, C186S, in which the catalytic Cys was replaced with Ser, and which bound unmethylated CCWGG sequences, repression could only be demonstrated when those sequences in cellular DNA were methylated by supplying a cloned dcm gene in trans. In vitro binding of the DNA fragment containing the ecoRIIM regulatory region was detected only with the mutants that showed repressor activity, including C186S. Results indicate that down-regulation of the gene in vivo and binding to the promoter in vitro are not dependent on the catalytic properties of M.EcoRII. Mobility shift experiments with C186S also revealed that it could bind either the promoter or unmethylated CCWGG sites, but not both. We conclude that the concentration of unmethylated CCWGG sites controls expression from the ecoRIIM promoter.

The expression and regulation of hsdK genes after conjugative transfer.

The type I restriction and modification genes of Escherichia coli can be transferred to other non-modified strains by conjugation without killing the recipient, implying that the restriction function must be regulated. In this study, two isogenic F' plasmids (r+K and r-K) served as donors in quantitative conjugation experiments with various restriction-deficient strains of E. coli and Salmonella typhimurium. Conjugation studies with hsd::lacZ operon fusions in F' plasmids indicate that both of the hsdK promoters, p(res) and pmod, express simultaneously following conjugative transfer. Thus these genes do not appear to be regulated at the transcriptional level. A spontaneous mutant of E. coli C was discovered that is presumably killed upon conjugative transfer of the hsdK genes (defined as a Crc- phenotype). The gene that is defective in the mutant was tentatively designated hsdC (control). Hfr gene replacement studies led to the localization of the putative hsdC gene between 6 and 16 min on the E. coli genetic map.

An Imbalance of HU Synthesis Induces Mucoidy in Escherichia coli

Mutations in a number of loci, including the lon gene, dramatically increase the production of colanic acid capsular polysaccharide and render Escherichia coli K-12 mucoid. The lon gene, which encodes an ATP-dependenty protease, is localized at ten minutes on the E. coli map and is very closely linked to the hupB gene coding for one of the two subunits of the histone-like protein HU. Surprisingly the introduction of a multi-copy plasmid carrying either the hupB or hupA gene into a wild-type E. coli strain, results in the overproduction of one of the HU subunits and repression of the synthesis of the other without changing the overall concentration of HU, also renders the cells mucoid. As in a lon strain, the transcription of the cps genes, the structural genes for the synthesis of colanic acid, is induced dramatically. Protease Lon negatively regulates cps genes by destabilizing RcsA, a positive regulator of capsule synthesis. Regulation of HU synthesis does not affect the steady state level of Lon, as judged by Western blotting. The UV sensitivity of the hup transformed lon + bacteria is identical to the lon + parental strain, suggesting that Lon activity for the degradation of SulA in these cells is normal. Using lac operon fusions to cps gene promoters and to the rcsA promoter we show that the deregulation of HU synthesis does not by pass the positive regulatory action of RcsA and RcsB for the expression of cps genes but functions by stimulating RcsA synthesis.

Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene.

The Haemophilus influenzae rec-1+ protein plays a central role in DNA metabolism, participating in general homologous recombination, recombinational (postreplication) DNA repair, and prophage induction. Although many H. influenzae rec-1 mutants have been phenotypically characterized, little is known about the rec-1+ gene at the molecular level. In this study, we present the genetic organization of the rec-1+ locus, the DNA sequence of rec-1+, and studies of the transcriptional regulation of rec-1+ during cellular assault by DNA-damaging agents and during the induction of competence for genetic transformation. Although little is known about promoter structure in H. influenzae, we identified a potential rec-1+ promoter that is identical in 11 of 12 positions to the bacterial sigma 70-dependent promoter consensus sequence. Results from a primer extension analysis revealed that the start site of rec-1+ transcription is centered 6 nucleotides downstream of this promoter. We identified potential DNA binding sites in the rec-1+ gene for LexA, integration host factor, and cyclic AMP receptor protein. We obtained evidence that at least one of the proposed cyclic AMP receptor protein binding sites is active in modulating rec-1+ transcription. This finding makes rec-1+ control circuitry novel among recA+ homologs. Two H. influenzae DNA uptake sequences that may function as a transcription termination signal were identified in inverted orientations at the end of the rec-1+ coding sequence. In addition, we report the first use of the Escherichia coli lacZ operon fusion technique in H. influenzae to study the transcriptional control of rec-1+. Our results indicate that rec-1+ is transcriptionally induced about threefold during DNA-damaging events. Furthermore, we show that rec-1+ can substitute for recA+ in E. coli to modulate SOS induction of dinB1 expression. Surprisingly, although 5% of the H. influenzae genome is in the form of single-stranded DNA during competence for genetic transformation, an event that could be a potent SOS-inducing signal, we failed to detect significant changes in rec-1+ transcription during the induction of genetic competence.

Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein?

The regulon controlled by the leucine-responsive regulatory protein (Lrp) of Escherichia coli consists of over 40 genes and proteins whose expression is regulated, either positively or negatively, by Lrp. The gltBDF operon, encoding glutamate synthase, was originally identified as a member of the Lrp regulon through a two-dimensional electrophoretic analysis of polypeptides from isogenic strains containing or lacking a functional Lrp protein. We have now demonstrated that Lrp regulates the transcription of gltBDF::lacZ operon fusions. Relative to expression in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium, gltBDF::lacZ expression in an lrp+ strain is repressed 2.2-fold in the presence of 10 mM exogenous leucine and 16-fold in Luria broth. Repression of gltBDF::lacZ expression by leucine or Luria broth is not seen for an isogenic strain containing a Tn10 insertion in lrp, and expression of gltBDF::lacZ is 44-fold lower than in the lrp+ strain when both are grown in glucose minimal MOPS medium. Lrp binds specifically to DNA fragments containing the gltBDF promoter region. Saturating levels of leucine do not abolish binding of Lrp upstream of gltBDF but merely increase its apparent dissociation constant from 2.0 to 6.9 nM. Electrophoretic analysis of the Lrp regulon established that target proteins differ greatly in the degree to which the effect of Lrp on their expression is antagonized by leucine. On the basis of our present results, we present a model for positive regulation of target genes by Lrp. Insensitivity to leucine would be expected when the effective intracellular concentration of Lrp is high relative to the affinity of Lrp binding sites required for transcription of the target gene. At lower concentrations of Lrp, transcription of the target gene should be sensitive to leucine. This model suggests that regulation of the concentration of active Lrp is critical to control of the Lrp regulon.

Isolation of electric pulse-induced Escherichia coli genes by operon fusion techniques

A search of electric-pulse induced genes in Escherichia coli was carried out by operon fusion techniques with a hybrid bacteriophage Mu, which creates transcriptional fusions of the structural gene of β-galactosidase to the host. Among two hundred transductants tested, nine colonies showed higher expression of more than twofold when they were treated with electric pulses. An electric-pulse stimulated transductant was not stimulated by UV irradiation, which is known to induce an SOS response. Conversely, strain PQ37, which has an operon fusion in one of the SOS genes, did not respond to an electric pulse treatment. A possibility of the presence of the electric-pulse stimulated genes, which were not induced by DNA damages, was suggested.

Cloning of the phs genetic locus from Salmonella typhimurium and a role for a phs product in its own induction.

The Salmonella typhimurium phs chromosomal locus essential for the reduction of thiosulfate to hydrogen sulfide was cloned, and some features of its regulation were examined. The phs locus conferred H2S production on Escherichia coli, suggesting that it contains the structural gene for thiosulfate reductase. H2S production by the E. coli host was, as in S. typhimurium, suppressed by nitrate or glucose in the growth medium. The presence of plasmid-borne phs genes in a S. typhimurium chl+ host containing a chromosomal phs::lacZ operon fusion was found to significantly increase the relative induction efficiency of beta-galactosidase by thiosulfate. These results are consistent with a model for phs regulation in which the true inducer is not thiosulfate per se and in which the action of a phs-encoded molybdoprotein, possibly the reductase itself, converts thiosulfate into a compound that resembles the true inducer more closely than does thiosulfate.

An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium

The 8th step in the 10-step heme biosynthetic pathway of Salmonella typhimurium is the oxidation of coproporphyrinogen III to protoporphyrinogen IX. On the basis of genetic studies, we have suggested that this reaction may be catalyzed by either of two different enzymes, an oxygen-dependent one encoded by hemF or an oxygen-independent enzyme encoded by hemN. Here, we report the cloning of the S. typhimurium hemF gene and its DNA sequence. The predicted amino acid sequence of the HemF protein is 44% identical to that of the coproporphyrinogen oxidase encoded by the yeast HEM13 gene. The wild-type S. typhimurium strain LT-2 produces an oxygen-dependent coproporphyrinogen oxidase activity detectable in crude extracts, which is not found in hemF mutants and is overproduced in strains carrying the hemF gene on a multicopy plasmid. the hemF gene is the second gene in an operon with an upstream gene with an unknown function, whose amino acid sequence suggests a relation to amidases involved in cell wall synthesis or remodeling. The upstream gene and hemF are cotranscribed from a promoter which was mapped by primer extension. A weaker, hemF-specific promoter is inferred from the behavior of an omega-Cm insertion mutation in the upstream gene. Although this insertion decreases expression of beta-galactosidase about 7.5-fold when placed upstream of a hemF-lacZ operon fusion, it still allows sufficient HemF expression from an otherwise wild-type construct to confer a Hem+ phenotype. The hemF operon is transcribed clockwise with respect to the genetic map.

Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation.

Mutations in the structural gene (hns) for the Escherichia coli nucleoid-associated DNA-binding protein H-NS cause highly pleiotropic effects on gene expression, site-specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA. We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation. A set of hns-lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome. Quantification of beta-galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10-fold in stationary phase compared with exponentially growing cells. Immunological detection of the H-NS protein in growing and stationary phase cells supported the genetic data and showed that H-NS synthesis varies with growth phase. In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase. Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns-lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product. Autoregulation of hns expression is particularly pronounced in log phase. Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter. In order to study the interaction of H-NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H-NS. DNA gel retardation assays showed that the H-NS protein can preferentially interact with a restriction fragment carrying the hns promoter. This restriction fragment showed features of curved DNA as judged by two-dimensional polyacrylamide gel electrophoresis performed at 4 degrees C and 60 degrees C.

Streptococcus mutans fructosyltransferase (ftf) and glucosyltransferase (gtfBC) operon fusion strains in continuous culture.

Three glucosyltransferases (GTFs), which catalyze the formation of water-insoluble adherent glucans, and fructosyltransferase (FTF), which synthesizes fructans, are believed to contribute to the pathogenic potential of Streptococcus mutans. Study of the regulation of expression of GTF and FTF has been difficult because of the complexity and number of exoenzymes produced by this bacterium. By using continuous chemostat culture to control environmental conditions, chloramphenicol acetyltransferase (CAT) operon fusions were utilized to measure transcriptional activity of the ftf and gtfBC gene promoters. Expression of these operon fusions was differentially regulated in response to culture pH and growth rate and during transition states between growth domains. Furthermore, the addition of sucrose to steady-state cultures resulted in significant increases in CAT specific activities for both fusions. In a few cases, GTF and FTF enzyme specific activities did not parallel those of the corresponding CAT fusion activities; this lack of correspondence was likely due to posttranscriptional events controlling enzyme secretion and enzyme activity, as well as to the differential expression of dextranase(s) and fructan hydrolase by S. mutans. These results clearly demonstrate that the extracellular polymer synthesis machinery of S. mutans is regulated in a complex manner. The use of operon fusions in combination with chemostat culture is a viable approach to analyzing gene expression in S. mutans and will be helpful in defining the molecular mechanisms underlying regulation of expression of virulence attributes under conditions that may more closely mimic those in dental plaque.

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth.

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.