Operon Fusion Research Articles

Linkage between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli.

The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Phi(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.

Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea.

A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a Delta hpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of Delta proC Delta hpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of beta-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina: A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed approximately 300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.

Thermoregulation of the Escherichia coli O157:H7 pO157 ecf operon and lipid A myristoyl transferase activity involves intrinsically curved DNA.

Escherichia coli O157:H7 survives in diverse environments from the ruminant gastrointestinal tract to cool nutrient-dilute water. We hypothesized that the gene regulation required for this flexibility includes intrinsically curved DNA that responds to environmental changes. Three intrinsically curved DNAs were cloned from the E. coli O157:H7 virulence plasmid (pO157), sequenced and designated Bent 1 through Bent 3 (BNT1, BNT2 and BNT3). Compared to BNT1 and BNT3, BNT2 had characteristics typical of intrinsically curved DNA including electrophoretic gel retardation at 4 degrees C, six partially phased adenine:thymine tracts and transcriptional activation. BNT2::lacZ operon fusions showed that BNT2 activated transcription at 24 degrees C compared to 37 degrees C and was partially repressed by a bacterial nucleoid-associated protein H-NS. BNT2 regulated the E. coli attaching and effacing gene-positive conserved fragments 1-4 (ecf1-4) that are conserved in Shiga toxin-producing E. coli associated with human disease. Experimental analyses showed that ecf1-4 formed an operon. ecf1, 2 and 3 encoded putative proteins associated with bacterial surface polysaccharide biosynthesis and invasion and ecf4 complemented a chromosomal deletion of lpxM encoding lipid A myristoyl transferase. Mass spectrometric analysis of lipid A from ecf and lpxM single and double mutants showed that myristoylation was altered at lower temperature.

Mu dI1(Ap lac) mutagenesis of Yersinia pestis plasmid pFra and identification of temperature-regulated loci associated with virulence

The F1 capsule of Yersinia pestis, encoded by the 100 kb plasmid pFra, is often assumed to be essential for full virulence of Y. pestis. However, virulent strains of Y. pestis that are F1 − and either pFra + or pFra − have been reported. To assess the role of pFra-encoded factors in virulence, mutants in pFra with insertions of the defective transposing bacteriophage Mu dl(Ap lac) were obtained, by using the wild type (wt) and the pLcr-cured derivative of strain C092. Mutants that exhibited temperature regulation of lactose fermentation and retarded electrophoretic mobility of pFra were selected. A total of 15 insertion mutants were isolated in the wt strain (12 of which had a single insertion in the genome, in pFra); and 24 mutants in the isogenic pLcr − derivative. Four of the pLcr + mutants, and none of the pLcr − mutants, were F1 −. All F1 − mutants were decreased in virulence for mice compared to the wt parent; and five of the F1 + mutants also were significantly attenuated in mice. Fusion end-joints of insert DNA were cloned into Escherichia coli by using pMLB524, a vector for rescuing operon fusions of lacZ. Recombinants were obtained which contained pFra inserts ranging from <2 kb to approximately 36 kb, and the insertions occurred at several sites on pFra. All of the four F1 − mutants tested mapped within the F1 capsule operon ( caf1). The remaining five attenuated mutants sequenced were F1 + and mapped outside of but near the operon. Sequencing and complete analysis of the pFra insertions mutants could facilitate identification of new potential virulence factors.

Global regulation of the Salmonella enterica serovar typhimurium major porin, OmpD.

The OmpD porin is the most abundant outer membrane protein in Salmonella enterica serovar Typhimurium and represents about 1% of total cell protein. Unlike the case with the less abundant OmpC and OmpF porins, the stoichiometry of OmpD in the outer membrane does not change in response to changes in osmolarity. The abundance of OmpD increases in response to anaerobiosis and decreases in response to low pH, conditions encountered by serovar Typhimurium during the infection of its murine host. By constructing an operon fusion of the lacZY genes with the ompD promoter, we show that the abundance of OmpD in the outer membrane is regulated primarily at the level of transcription and is subject to catabolite repression. In response to anaerobiosis, the abundance of OmpD in the outer membrane also appears to be controlled posttranscriptionally by a function dependent on Fnr.

Regulation of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli

The expressions of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli were compared under a variety of growth conditions and in several genetic backgrounds. LacZ operon fusions were constructed and were inserted in single copies into strain MC4100 and into its fnr, arcA or hemA carrying derivatives. During aerobic growth the expressions of both ubiD and ubiX depended on the carbon source: succinate>glycerol>glucose. Mutations in fnr, arcA or hemA increased the expressions of both genes. During anaerobic growth in LB medium glucose strongly inhibited the expression of ubiD but not of ubiX.

Dual Control by Regulators, GntH and GntR, of the GntII Genes for Gluconate Metabolism in Escherichia coli

Escherichia coli possesses two systems, GntI and GntII, for gluconate uptake and catabolism, whose genes are regulated by GntR as a repressor and GntH as an activator, respectively. Additionally, GntH exerts negative control of the GntI genes via the same binding element as that of GntR. We thus examined whether GntR involves regulation of the GntII genes or not. This regulation and the control by GntH were examined by using single-copy lacZ operon fusions and by RT-PCR, suggesting positive and negative regulation by GntR and positive regulation by GntH. Moreover, the introduction of mutations into possible GntR-binding elements revealed that both regulators share at least one of the elements. The results presented allow us to speculate that GntR initiates expression of the GntII genes, followed by their large induction by GntH when cells were grown in gluconate minimum medium. As in the case of the GntI genes, such a cross-regulation between the GntI and GntII via the two regulators may be important for cells to grow with gluconate.

First step towards a quantitative model describing Czc-mediated heavy metal resistance in Ralstonia metallidurans.

Quantitative models were derived to explain heavy metal resistance in Ralstonia metallidurans. A deltaczcA deletion of the gene for the central component of the Co2+/Zn2+/Cd2+ efflux system CzcCBA combined with the expression level of czcCBA as studied with a phi(czcC-lacZ-czcBA) operon fusion demonstrated that CzcCBA was the only prerequisite for resistance to Co2+/Zn2+/Cd2+ at concentrations of 200 microM and above. The cellular content of the CzcA protein (resistance nodulation cell division protein family RND) determined by Western blot was used to model the CzcCBA expression level as the response to various metal concentrations. These data and experimentally determined uptake velocities were used to derive a flow equilibrium model that describes the cytoplasmic content c(i) of the cells as an interaction between cation uptake and CzcCBA-mediated efflux. Alternatively, binding of heavy metals to inactivated R. metallidurans cells was described with a modified Freundlich's equation. The metal content of growing R. metallidurans cells was determined and compared to the predictions of both models. High amounts of zinc precipitates. exclusively formed by living cells, prevented a model validation for zinc. An additional net efflux activity let to lower amounts of cell-bound Co2+ than predicted. The flow equilibrium model described cadmium resistance sufficiently for R. metallidurans growing in the presence of 0.2-1 mM Cd2+. Description of cadmium resistance in early stationary cells requires the binding model in addition to the flow equilibrium model. Thus, it was possible to simulate physiological events in growing cells by quantitative models that are derived from the biochemical data of the interacting transport proteins.

Regulation of polyphenol oxidase activities and melanin synthesis in Marinomonas mediterranea: identification of ppoS, a gene encoding a sensor histidine kinase.

Marinomonas mediterranea is a melanogenic marine bacterium that expresses two different polyphenol oxidases. One of them is a multipotent laccase able to oxidize a wide range of substrates. The second enzyme is an SDS-activated tyrosinase. Using transposon mutagenesis, a mutant affected in the regulation of both polyphenol oxidase activities and melanogenesis has been isolated. The sequencing of the gene disrupted by the mini-Tn10 transposon in this mutant indicates that it encodes a hybrid sensor kinase. This sensor kinase shows three phosphorylated conserved domains: the transmitter domain containing a histidine site typical of sensor kinases, a receiver domain with an aspartate residue and an additional phosphotransferase domain with a second conserved histidine. This structural organization is characteristic of kinases participating in a phosphorelay system. Northern blot and lacZ operon fusions indicate that the multipotent laccase activity is regulated not only by PpoS but also by growth phase at the transcriptional level. These results suggest that PPO activities and melanin synthesis play a role in the adaptive response of M. mediterranea to stressful environmental conditions.

Small broad-host-range lacZ operon fusion vector with low background activity.

Small broad-host-range lacZ operon fusion vector with low background activity.

Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements.

The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.

Modeling of inducer exclusion and catabolite repression based on a PTS-dependent sucrose and non-PTS-dependent glycerol transport systems in Escherichia coli K-12 and its experimental verification

We used genetically engineered sucrose positive Escherichia coli K-12 derivatives as a model system for the modeling and experimental verification of regulatory processes in bacteria. These cells take up and metabolize sucrose by the phospho enolpyruvate (PEP)-dependent sucrose phosphotransferase system (Scr-PTS). Expression of the scr genes, which cluster in two different operons ( scrYAB and scrK), is negatively controlled by the ScrR repressor. Additionally, expression of the scrYAB operon, but not of the scrK operon is positively controlled by the cAMP–CRP complex. Modeling of sucrose transport and metabolism through the Scr-system and of the scr gene expression has been performed using a modular and object-orientated new approach. To verify the model and identify important model parameters we measured in a first set of experiments induction kinetics of the scr genes after growth on glycerol using strains with single copy lacZ operon fusions in the scrK or scrY genes, respectively. In a second set of experiments an additional copy of the complete scr-regulon was integrated into the chromosome to construct diplogenotic strains. Differences were observed in the induction kinetics of the cAMP–CRP-dependent scrY operon compared to the cAMP–CRP independent scrK operon as well as between the single copy and the corresponding diplogenotic strains.

Use of operon fusions in Mannheimia haemolytica to identify environmental and cis-acting regulators of leukotoxin transcription.

The leukotoxin of Mannheimia haemolytica is an important virulence factor that contributes to much of the pathology observed in the lungs of animals with bovine shipping fever pneumonia. We believe that identification of factors that regulate leukotoxin expression may provide insight into M. haemolytica pathogenicity. The DNA sequence upstream of the leukotoxin operon is divergently shared by P(lapT), which transcribes an arginine permease gene. The intergenic region contains several elements that are potential sites for transcriptional modulation of the promoters. We have developed plasmid-borne chloramphenicol acetyltransferase (cat) operon fusions, as well as lktC::cat chromosomal fusions, to study transcription initiation in M. haemolytica. Using these genetic tools, we have identified cis-acting sequences and environmental conditions that modulate transcription of the leukotoxin and lapT promoters. By deletion analysis, promoters were shown to rely on sequences upstream of their -10 and -35 regions for full activity. Direct repeats of the sequence TGT-N(11)-ACA and a static bend region caused by phased adenine tracts were necessary for full activation of P(lkt). A computer-generated model of the promoter's structure shows how DNA bending brings the repeat sequences within close proximity to the P(lkt) RNA polymerase, and we hypothesize that these repeats are a binding site for an activator of leukotoxin transcription. The lktC::cat operon fusion was also used to demonstrate that, like that of other RTX toxins, leukotoxin transcription is environmentally regulated. Roles for iron deprivation and temperature change were identified.

Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions.

It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.

Singlet molecular oxygen triggers the soxRS regulon of Escherichia coli.

The electronically excited molecular oxygen (singlet oxygen, 1O2) can be detrimental to cells in several ways, although recent reports indicate that it may play a role as an intercellular signal in eukaryotes. Here we present evidence that 1O2, generated by thermodissociation of disodium 3,3'-(1,4-naphthylidene) diproprionate endoperoxide, activates transcription of genes of the soxRS regulon, and that this induction is paralleled by induction of a soxS'::lacZ operon fusion. The inductions were dependent on a functional soxR gene. These data imply that protective responses, such as induction of the soxRS regulon, may be triggered by diverse environmental oxidative stresses, and that 1O2 may also function as a signal molecule in prokaryotes.

Generic model control of induced protein expression in high cell density cultivation of Escherichia coli using on-line GFP-fusion monitoring

A model-based control algorithm (generic model control) is presented for fed-batch cultivation of recombinant Escherichia coli producing either transcriptional or translational fusion products. With the recent development of translational and operon fusions using green fluorescent protein (GFP) [Albano et al. (1998) Biotechnol Prog 14:351–354] along with an on-line GFP sensor [Randers-Eichhorn et al. (1997) Biotechnol Bioeng 55:921–926], real-time measurements of foreign protein level are now possible. A mathematical model is presented that is both accurate and simple so as to ensure that all state variables remain observable during cultivation. A balance between model accuracy and mathematical tractability was obtained to facilitate the formulation of the control algorithm. Generic model control (GMC) is a process model-based control algorithm incorporating a process model directly within the control structure. GMC was desirable since linearization of the process model was not necessary and robust performance could be obtained despite process disturbances or plant/model mismatch. Furthermore, a time-delay compensator was built into the control law to account for the observed 90-min lag associated with GFP fluorescence. The feasibility of the GMC algorithm was demonstrated by simulations.

MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium.

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli chi7122 conferred mannose-resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain chi7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low-copy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature- and RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions.

Transcription of the ybcZ--ylcA ylcBCD--ybdE region of the Escherichia coli K38 chromosome was analysed by Northern RNA--DNA hybridization, RT-PCR and primer extension. Transcription of a dicistronic ybcZ--ylcA mRNA and a tetracistronic ylcBCD--ybdE mRNA was induced by silver and was initiated from the sigma-70 promoters ylcAp and ylcBp. Expression of beta-galactosidase activity from a Phi(ylcBp--lacZ) operon fusion was also induced by Ag(+) and Cu(2+), but not by Zn(2+). In-frame deletion of ybdE from the chromosome yielded a silver-sensitive E. coli mutant strain which did not differ in its copper resistance from its wild-type strain. On the other hand, deletion of the copA gene for the copper-exporting P-type ATPase CopA resulted in copper sensitivity, but not in silver sensitivity. A Delta ybdE Delta copA double mutant strain behaved towards copper as the Delta copA strain and towards silver as the Delta ybdE strain. Thus, in E. coli, the YlcBCD--YbdE system may be involved in silver- but not in copper resistance, and CopA may be involved in copper- but not in silver resistance.

Degenerative minimalism in the genome of a psyllid endosymbiont.

Psyllids, like aphids, feed on plant phloem sap and are obligately associated with prokaryotic endosymbionts acquired through vertical transmission from an ancestral infection. We have sequenced 37 kb of DNA of the genome of Carsonella ruddii, the endosymbiont of psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the aphid endosymbiont Buchnera aphidicola. Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion, and lack of the usual promoter sequences upstream of 16S rDNA. These features suggest the synthesis of long mRNAs and translational coupling. The most extreme instances of base compositional bias occur in the genes encoding proteins that have less highly conserved amino acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10%. The shift in base composition has a large effect on proteins: in polypeptides of C. ruddii, half of the residues consist of five amino acids with codons low in guanine plus cytosine. Furthermore, the proteins of C. ruddii are reduced in size, with an average of about 9% fewer amino acids than in homologous proteins of related bacteria. These observations suggest that the C. ruddii genome is not subject to constraints that limit the evolution of other known eubacteria.

Activation of the Escherichia coli SoxRS-regulon by nitric oxide and its physiological donors.

Activation of the Escherichia coli SoxRS-regulon by nitric oxide (NO) and its physiological donors (S-nitrosothiol (GS-NO) and dinitrosyl iron complexes with glutathione (DNIC(glu)) and cysteine (DNIC(cys)) ligands) has been studied. To elucidate the molecular mechanisms of signal transduction via nitrosylation of Fe-S-centers in SoxR, the ability of pure NO and NO-producing agents to activate the SoxRS-regulon in E. coli cells bearing a soxS::lacZ operon (promoter) fusion has been compared. EPR spectroscopy of whole cells has been used to monitor the formation of inducible protein-DNIC complexes. DNIC(cys), GS-NO, and pure NO appeared to be potent inducers of soxS expression, whereas DNIC(glu) was considerably less efficient. Thus, lower in vitro stability of DNIC(cys) was in contrast with its higher biological activity. Pretreatment of the cells with o-phenanthroline, a chelating agent for iron, prevented soxS expression by GS-NO. Treatment of intact E. coli cells with DNIC, GS-NO, and NO at equimolar concentration 150 microM resulted in formation of a single EPR-detectable DNIC-type signal with g = 2.03. The initial stage in the SoxR transcription activity is supposed to include two steps: first, DNIC primers are formed from exogenous NO and free iron, and then these DNIC disintegrate SoxR [2Fe-2S] clusters and thus activate SoxRS-regulon transcription.