Operon Fusion Research Articles

Molecular genetic analysis of virulence in Mannheimia (pasteurella) haemolytica.

Mannheimia haemolytica (previously known as Pasteurella haemolytica) is a weakly hemolytic, gram-negative coccobacillus that is an opportunistic pathogen of cattle, sheep and other ruminants. In stressed, immunocompromised animals, the organism causes a fibrinous, necrotic pneumonia, commonly called "shipping fever". In the United States, economic losses due to shipping fever pneumonia surpass the combined cost of all other diseases of cattle. M. haemolytica, which is a member of the family Pasteurelleaceae, includes twelve serotypes (A1, A2, A5-A9, A12-14, A16 and A17) based on capsular antigen typing. Worldwide, serotypes A1 and A2 predominate, though all serotypes can cause disease. Serotype A1 causes pasteurellosis in cattle and has been the subject extensive study, while serotype A2 causes disease in sheep and is less-well characterized. Potential virulence factors of M. haemolytica have been identified and characterized by gene cloning and DNA sequence analysis. These factors include a ruminant-specific leukotoxin, an anti-phagocytic capsule, lipopolysaccharide, iron-regulated outer membrane proteins, lipoproteins, a sialoglycoprotease, a neuraminidase and two potential immunoglobulin proteases. Unlike the well-characterized leukotoxin, little is known about the expression of these other virulence factors. Attempts to dissect the mechanisms of M. haemolytica pathogenesis have been hindered by the lack of a robust genetic system for mutation of the organism, though new tools for genetic manipulation of M. haemolytica have been developed. Expression plasmids and operon fusion plasmids have been created and a series of antibiotic resistance cassettes useful for site-specific recombination have been constructed. It is anticipated that use of these tools for gene expression and mutagenesis, in combination with the soon to be released genomic sequence of a serotype A1 organism, will aid in understanding the molecular mechanisms of pathogenesis of M. haemolytica and will help to drive development of new vaccines to prevent shipping fever.

Molecular characterization of the mycobacterial SenX3-RegX3 two-component system: evidence for autoregulation.

Environmental regulation of bacterial gene expression is often mediated by two-component signal transduction systems, which are themselves tightly regulated. The response regulator RegX3 and the cytoplasmic portion of the histidine kinase SenX3 from Mycobacterium bovis BCG were overproduced in Escherichia coli and purified as N-terminally (His)(6)-tagged proteins. Phosphorylation assays demonstrated autophosphorylation of the cytoplasmic portion of SenX3 and a phosphotransfer from SenX3 to RegX3, involving conserved histidine and aspartate residues, respectively. In addition, as shown by electrophoretic mobility shift assays, (His)(6)RegX3 was able to specifically bind to the promoter region of the senX3-regX3 operon. Furthermore, operon fusion analyses indicated that the overexpression of the senX3-regX3 operon increases the activity of the senX3 promoter in Mycobacterium smegmatis. Together, these results indicate that the mycobacterial SenX3-RegX3 two-component system is positively autoregulated.

A portion of the nucleotide sequence corresponding to the N-terminal coding region of livJ is essential for its transcriptional regulation

We investigated the regulation of the livJ and livKHMGF operons, which are involved in branched-chain amino acid high-affinity transport in Salmonella typhimurium. When livJ was fused to lacZ at the second codon of livJ to make a livJ–lacZ protein fusion, expression from the livJ promoter was not repressed even under repressing growth conditions; however, expression of an analogous construct of livK–lacZ was repressed. When livJ was fused to lacZ at the twelfth codon of livJ, the expression level under unrepressing growth conditions was elevated, resulting in apparent repressibility of the livJ–lacZ protein fusion. Expression from the livJ–lacZ operon fusion, in which livJ was fused to lacZ 159 bp downstream from the A of the start codon of livJ, was relatively normal under unrepressing growth conditions. Deletion analysis and site-directed base-substitution analysis strongly suggested that cis-acting element for regulation of livJ transcription, 5′-GGCAGGATGTATCG-3′, starting at +21 and ending at +34 downstream from the A of the start codon of livJ, was present in the N-terminal coding region of livJ.

The use of operon and gene fusions in studies of regulation of the ibpAibpB operon

The use of operon and gene fusions in studies of regulation of the <i>ibpAibpB</i> operon

Detection and quantification of tetracyclines by whole cell biosensors

Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, Ptet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing beta-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different gram-negative bacteria by conjugation. An Escherichia coli strain containing the Ptet-lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors.

Detection and quantification of tetracyclines by whole cell biosensors

Three different mini-Tn 5 plasmids, containing a tetracycline-inducible promoter, P tet and a regulatory gene, tetR, in operon fusions with a reporter gene system ( lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing β-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different Gram-negative bacteria by conjugation. An Escherichia coli strain containing the P tet - lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors.

Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response.

The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP-CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the sigmaE stress response. Because the sigmaE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.

Construction and application of mycobacterial reporter transposons

The transposon Tn 5367, which is a derivative of the mycobacterial insertion sequence IS 1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions. A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional ( lacZ), or translational (‵ aph) fusions. Mutants containing lacZ produced varying intensities of blue colour on indicator media. This reporter activity could be used as a quantitative measure of promoter strength. Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the ‵ aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions. Finally, inclusion of the R6Kγ origin within Tn 5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin. This study demonstrates that transcriptional and translational reporter derivatives of Tn 5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria.

Immuno-capture differential display method (IDDM) for the detection of environmentally induced promoters in rhizobacteria

A rapid immunological method for trapping and selection of functionally regulated prokaryotic promoters is described. The method is based on application of a novel mini-Tn 5 derived promoter probe (pUTTKZY-promoterless lacZY as a reporter and kanamycin resistance) to mutagenise a plant growth promoting fluorescent pseudomonad, Pseudomonas fluorescens 54/96. The transposon allows selection of operon fusion mutants ( lacZY +) directly on media containing lactose as a sole carbon source as well as selection for kanamycin and lacZ (β-galactosidase) expression on X-gal indicator media. We have extended the technique to target the surface expression of the induced lactose permease gene ( lacY) from mutagenised libraries and the immuno-capture of bacteria with magnetic beads and anti-LacY monospecifc antisera. The benefits of the lacZY reporter are that a library can be rapidly generated and screened in vitro to isolate non-expressed mutants for further in situ screening. Here we demonstrate the development and utility of the technique and its potential as a differential display method for the isolation of promoters that direct regulated gene expression in the phytosphere, or under other imposed conditions.

The induction of SOS function in Escherichia coli K-12/PQ37 by 4-nitroquinoline oxide (4-NQO) and fecapentaenes-12 and -14 is bile salt sensitive: implications for colon carcinogenesis

The response of Escherichia coli to genotoxic agents involves the triggering of a complex system of genes known as the SOS response. In E. coli PQ37, a test organism used for the assessment of genotoxicity, lacZ, the β-galactosidase gene is placed under the control of sfiA, one of the SOS genes through an operon fusion. The induction of β-galactosidase activity, when the organism is exposed to genotoxic agents, is an indirect measure of the genotoxic activity of the test compound. Incubation of E. coli PQ37 with either 4-nitroquinoline oxide (4-NQO) or one of the fecal mutagens, fecapentaene-12 or -14 (F-12 or F-14) in the presence of sodium taurocholate or sodium deoxycholate resulted in a significant enhancement of induction of β-galactosidase activity. The molecular mechanisms of 4-NQO-induced mutagenesis in E. coli are similar to those of the effects of UV light in which both replication-dependent and repair-dependent pathways of mutagenesis exist. Since E. coli PQ37 is excision-repair-deficient, alternate pathways are involved in this system. Bile salts by themselves do not trigger the SOS response, and hence their role in enhancing the SOS-inducing potency of mutagens may involve the potentiation of the cleavage-inactivation of lexA (repressor of SOS) by the protein product of the SOS-controlled gene, recA. The potentiating effect of bile salts on the fecal mutagens, F-12 and F-14, has implications in their suspected role in colon carcinogenesis associated with high-fat, low-fiber diets.

Characterization of two novel regulatory genes affecting Salmonella invasion gene expression.

A Salmonella typhimurium chromosomal deletion removing approximately 19 kb of DNA at centisome 65 reduces invasion of cultured epithelial cells as well as the expression of lacZY operon fusions to several genes required for the invasive phenotype. As the deleted region contains no genes previously known to affect Salmonella invasion, we investigated the roles of individual genes in the deleted region using a combination of cloning, complementation and directed mutation. We find that the deletion includes two unrelated regulatory genes. One is the Salmonella homologue of Escherichia coli barA (airS ), which encodes a member of the multistep phosphorelay subgroup of two-component sensor kinases. The action of BarA is coupled to that of SirA, a member of the phosphorylated response regulator family of proteins, and includes both HilA-dependent and HilA-independent components. The other regulatory gene removed by the deletion is the Salmonella homologue of E. coli csrB, which specifies a regulatory RNA implicated in controlling specific message turnover in E. coli. These results identify a protein that is likely to play a key role in the environmental control of Salmonella invasion gene expression, and they also suggest that transcriptional control of invasion genes could be subject to refinement at the level of message turnover.

PpdD type IV pilin of Escherichia coli K-12 can Be assembled into pili in Pseudomonas aeruginosa.

Escherichia coli K-12 possesses at least 16 chromosomal genes related to genes involved in the formation of type IV pili in other gram-negative bacteria. However, E. coli K-12 does not produce type IV pili when grown under standard laboratory conditions. The results of reverse transcription-PCR, operon fusion analysis, and immunoblotting demonstrated that several of the putative E. coli piliation genes are expressed at very low levels. Increasing the level of expression of the major pilin gene (ppdD) and the linked assembly genes hofB and hofC (homologues of the Pseudomonas aeruginosa type IV pilus assembly genes pilB and pilC) did not lead to pilus production. However, expression of the ppdD gene in P. aeruginosa led to assembly of PpdD into pili that were recognized by antibodies directed against the PpdD protein. Assembly of PpdD into pili in P. aeruginosa was dependent on the expression of the pilB and pilC genes and independent of expression of the P. aeruginosa pilin structural gene pilA.

2] A practical guide to the construction and use of lac fusions in Escherichia coli

Publisher Summary This chapter discusses the construction and use of lac fusions in Escherichia coli. It discusses the applications of β-galactosidase. β-galactosidase is known as the best transcriptional reporter. Using appropriate media, mutations that increase or decrease the expression of an operon fusion of interest can be isolated easily. Conversely, screening pools of random LacZ chromosomal insertions can identify targets of a regulator (either transcriptional or translational). β-galactosidase is also useful in the study of translational regulation. The chapter presents the protocols for making specific or random lac fusions (both transcriptional and translational). Transcriptional fusions are also known as promoter or operon fusions, whereas translational fusions are alternatively referred to as gene or protein fusions. Two classes of LacZ fusions cover most uses: fusions created to specific cis-acting regulatory regions (including inflame protein fusions) and fusions created by random chromosomal insertion of the lac operon. The chapter also presents a protocol for creating a fusion of a specific DNA fragment to the lac operon and the subsequent isolation of the fusion in single copy on the bacterial chromosome for analysis.

General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5al.

Klebsiella oxytoca can assimilate nitrate and nitrite by using enzymes encoded by the nasFEDCBA operon. Expression of the nasF operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of nasR gene expression. We constructed strains bearing single-copy Phi(nasR-lacZ) operon fusions within the chromosomal rhaBAD-rhaSR locus. The expression of DeltarhaBS::[Phi(nasR-lacZ)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both ntrC and rpoN null mutants, indicating that Ntr control of nasR gene expression requires the NtrC and sigma(N) (sigma(54)) proteins. Sequence inspection of the nasR control region reveals an apparent sigma(N)-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the sigma(N)-dependent nasR promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of beta-galactosidase expression indistinguishable from Phi(nasR-lacZ) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of nasR transcription. Together, these results indicate that nasR gene expression does not require specific upstream NtrC-binding sequences, as previously noted for argT gene expression in Salmonella typhimurium (G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107-117, 1988).

DNA region responsible for transcriptional regulation of the Escherichia coli penicillin amidase ( pac) gene by CRP and PAA

Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene ( pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene ( lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.

D-Allose catabolism of Escherichia coli: involvement of alsI and regulation of als regulon expression by allose and ribose.

Genes involved in allose utilization of Escherichia coli K-12 are organized in at least two operons, alsRBACE and alsI, located next to each other on the chromosome but divergently transcribed. Mutants defective in alsI (allose 6-phosphate isomerase gene) and alsE (allulose 6-phosphate epimerase gene) were Als(-). Transcription of the two allose operons, measured as beta-galactosidase activity specified by alsI-lacZ(+) or alsE-lacZ(+) operon fusions, was induced by allose. Ribose also caused derepression of expression of the regulon under conditions in which ribose phosphate catabolism was impaired.

Expression of the Escherichia coli NRZ nitrate reductase is highly growth phase dependent and is controlled by RpoS, the alternative vegetative sigma factor.

In the absence of oxygen, many bacteria preferentially use nitrate as a terminal electron acceptor for anaerobic respiration. In Escherichia coli, there are two membrane-bound, differentially regulated nitrate reductases. While the physiological basis for this metabolic redundancy is not completely understood, during exponential growth, synthesis of NRA is greatly induced by anaerobiosis plus nitrate, whereas NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. In the course of identifying genes controlled by the stationary phase regulatory factor RpoS (sigmas), we found that the expression of NRZ is induced during entry into stationary phase and highly dependent on this alternative sigma factor. Expression studies, using operon fusions and nitrate reductase assays, revealed that the NRZ operon is controlled mainly at the level of transcription and is induced 10-fold at the onset of stationary phase in rich media. Consistent with previous reports of RpoS expression, the RpoS dependency of NRZ in minimal media was very high (several hundredfold). We also observed a fivefold stationary phase induction of NRZ in an rpoS background, indicating that other regulatory factors, besides RpoS, are probably involved in transcriptional control of NRZ. The RpoS dependence of NRZ expression was confirmed by Northern analyses using RNA extracted from wild-type and rpoS- strains sampled in exponential and stationary phase. In toto, these data indicate that RpoS-mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress-associated conditions.

Monitoring GFP‐operon fusion protein expression during high cell density cultivation of Escherichia coli using an on‐line optical sensor

Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L−1 DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the pBAD promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 54–64, 1999.

Monitoring GFP-operon fusion protein expression during high cell density cultivation ofEscherichia coli using an on-line optical sensor

Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L(-1) DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity.

RsmC of the soft-rotting bacterium Erwinia carotovora subsp. carotovora negatively controls extracellular enzyme and harpin(Ecc) production and virulence by modulating levels of regulatory RNA (rsmB) and RNA-binding protein (RsmA).

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin(Ecc) (the elicitor of hypersensitive reaction) in Erwinia carotovora subsp. carotovora is regulated by RsmA, an RNA-binding protein, and rsmB, a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219-234, 1998). We have cloned and characterized a novel regulatory gene, rsmC, that activates RsmA production and represses extracellular enzyme and harpin(Ecc) production, rsmB transcription, and virulence in E. carotovora subsp. carotovora. In an rsmC knockout mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosomal copy of the wild-type rsmA(+) allele, the basal levels of Pel, Peh, Cel, Prt, and harpin(Ecc) as well as the amounts of rsmB, pel-1, peh-1, celV, and hrpN(Ecc) transcripts are high, whereas the levels of rsmA transcripts and RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fusion is lower in the RsmC(-) mutant than in the RsmC(+) parent. Conversely, the expression of an rsmB-lacZ operon fusion is higher in the RsmC(-) mutant than in the RsmC(+) parent. These observations establish that RsmC negatively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC(-) mutant, an RsmA(-) mutant, and an RsmA(-) RsmC(-) double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on rsmA and rsmB. Exoprotein production by the RsmC(-) mutant is partially dependent on the quorum sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of rsmC sequences in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora. These findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a functional homolog of RsmC.