groE Promoter Research Articles

Francisella tularensis FTL_0580 N-Terminal Domain Enhances Fluorescence Protein Expression

Francisella tularensis is a gram-negative, intracellular pathogen capable of establishing a lethal infection at a dose of <10 CFU. Fluorescence microscopy is a vital tool used to study F. tularensis host-pathogen interactions. However, F. tularensis is only weakly fluorescent when expressing fluorescent proteins such as emGFP and BFP using previously described molecular tools. In contrast, our laboratory previously described robust expression of tdTomato while under the control of the Francisella glucose-repressible promoter (FGRp; plasmid construct pTC3D). However, F. tularensis bacteria were unable to produce substantial expression of tdTomato or other fluorescent proteins, such as emGFP, while genes encoding these proteins were under the control of the robust groE promoter (groEp). Therefore, we analyzed the DNA upstream of tdtomato in pTC3D and identified an in-frame N-terminal fusion of the first 36 codons of FTL_0580 (580N), followed by code for a three amino linker domain (PAT), and subsequently the start codon of tdTomato potentially producing a stable hybrid protein. The previously identified FGRp alone could not produce robust expression of fluorescent proteins. Moreover, a frame-shift disrupting the coding sequence of 580N resulted in poor fluorescent protein expression. Inclusion of 580N in-frame controlling expression of Emerald GFP, emGFP, under the control of FGRp or groEp, however, resulted in robust fluorescence. We hypothesize that 580N stabilizes the fluorescent protein to which it is fused within F. tularensis. Application of this new biotechnology will expand our ability to investigate F. tularensis through the use of fluorescence microscopy.

Francisella tularensis expressing TC3D Leader Peptide and Promotor from tdtomato Enhances Fluorescence

Francisella tularensis is a gram-negative, intracellular pathogen capable of causing infection at a low dose of <10 CFU. For this reason, this bacterium has been classified as a Category A Bioterrorism Agent by the Center for Disease Control (CDC). Fluorescence microscopy is a valuable tool that investigators can utilize to study the many interactions between F. tularensis and its host cells. However, in our hands, F. tularensis is only weakly fluorescent when expressing certain fluorescent proteins such as emGFP and BFP using previously described molecular tools. On the other hand, our laboratory previously described robust expression of tdTomato while under the control of the Francisella glucose-repressible promoter (FGRp; this plasmid construct was referred to as pTC3D). However, F. tularensis bacteria were unable to produce robust expression of tdTomato or other fluorescent proteins, such as emGFP, while these genes were under the control of the groE promoter (which is much more robust). Therefore, we analyzed the DNA upstream of tdtomato in pTC3D. Here, we identified an in-frame N-terminal leader peptide that is likely translated with tdTomato potentially producing a stable fusion. The previously identified FGRp sequence alone could not produce robust expression of fluorescent proteins. Moreover, a frame-shift disrupting the coding sequence of the leader peptide resulted in poor fluorescent protein expression. Inclusion of the leader peptide in-frame controlling expression of Emerald GFP, emGFP, under the control of the FGRp, however, resulted in robust green fluorescence suggesting that this domain can be used to stabilize fluorescent proteins in F. tularensis. Application of this new biotechnology will expand our ability to investigate F. tularensis through fluorescence microscopy.

Construction of pTM series plasmids for gene expression in Brucella species

Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp.

Development of versatile shuttle vectors for Deinococcus grandis

To develop new shuttle vectors for Deinococcus species, the nucleotide sequence of the small cryptic plasmid pUE30 from Deinococcus radiopugnans ATCC19172 was determined. The 2467-bp plasmid possesses two open reading frames, one encoding 88 amino acid residues (Orf1) and the other encoding 501 amino acid residues (Orf2). The predicted amino acid sequence encoded by Orf1 exhibits similarity to the N-terminal regions of replication proteins encoded by repABC-type plasmids of a-proteobacteria . On the other hand, the predicted amino acid sequence encoded by Orf2 exhibits similarity to replication proteins encoded by plasmids of D. radiodurans SARK and Thermus species. Hybrid plasmids consisting of pUE30 and pKatCAT5, which replicates in E. coli with a chloramphenicol resistance determinant, were shown to autonomously replicate in D. grandis ATCC43672. Deletion analysis revealed that Orf2 was necessary for replication of the plasmids in D. grandis. On the other hand, a DNA fragment encompassing the Orf1-coding region was involved in the instability of the plasmid in D. grandis. An expression plasmid that possesses the D. radiodurans minimal groE promoter was constructed, and a firefly luciferase gene was successfully expressed in D. grandis. The D. grandis host-vector system developed in this study should prove useful in the bioremediation of radioactive waste and for the investigation of DNA repair mechanisms.

Mycobacterium tuberculosis groEpromoter controls the expression of the bicistronicgroESL1operon and shows differential regulation under stress conditions

Heat shock promoters of mycobacteria are strong promoters that become rapidly upregulated during macrophage infection and thus serve as valuable candidates for expressing foreign antigens in recombinant BCG vaccine. In the present study, a new heat shock promoter controlling the expression of the groESL1 operon was identified and characterized. Mycobacterium tuberculosis groESL1 operon codes for the immunodominant 10 kDa (Rv3418c, GroES/Cpn10/Hsp10) and 60 kDa (Rv3417c, GroEL1/Cpn60.1/Hsp60) heat shock proteins. The basal promoter region was 115 bp, while enhanced activity was seen only with a 277-bp fragment. No promoter element was seen in the groES-groEL1 intergenic region. This operon codes for a bicistronic mRNA transcript as determined by reverse transcriptase-PCR and Northern blot analysis. Primer extension analysis identified two transcriptional start sites (TSSs) TSS1 (-236) and TSS2 (-171), out of which one (TSS2) was heat inducible. The groE promoter was more active than the groEL2 promoter in Mycobacterium smegmatis. Further, it was found to be differentially regulated under stress conditions, while the groEL2 promoter was constitutive.

Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ 32 , Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

The heat shock sigma factor (sigma(32) in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of sigma(32) is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, sigma(70). This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing sigma(32), implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in sigma(32) impedes recovery from heat shock (exposure to 42 degrees C), and it also leads to overexpression at lower temperatures (30 degrees C) of the Flu protein, which is associated with biofilm formation.

Mutational analysis of Escherichia coli heat shock transcription factor sigma 32 reveals similarities with sigma 70 in recognition of the -35 promoter element and differences in promoter DNA melting and -10 recognition.

Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor sigma32 rise greatly, resulting in the increased formation of the sigma32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on sigma32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving sigma32 expression from one plasmid and a beta-galactosidase reporter gene driven by the sigma32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on sigma32 activity. It was found that the recognition of the -35 region involves similar amino acid residues in regions 4.2 of E. coli sigma32 and sigma70. Three conserved amino acids in region 2.3 of sigma32 were found to be only marginally important in determining activity in vivo. Differences between sigma32 and sigma70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis

Two plasmid-based expression vectors have been constructed where one allows intracellular production of recombinant proteins while the second directs the proteins into the culture medium. Both vectors use the strong promoter preceding the groESL operon (codes for the essential heat shock proteins GroES and GroEL) of Bacillus subtilis fused to the lac operator allowing their induction by addition of ITPG. While the background level of expression of these expression cassettes is very low in the absence of the inducer, an induction factor of about 1300 was measured. When the genes htpG and pbpE (coding for a heat shock protein and a penicillin-binding protein, respectively) were fused to the groE promoter, the amount of recombinant protein produced after addition of IPTG represented 10 and 13%, respectively, of the total cellular protein. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding an α-amylase from Bacillus amyloliquefasciens was fused to the groE promoter. High-level secretion of amyQ α-amylase and cellulase A and B of Clostridium thermocellum was demonstrated.

Improved Expression Vector for Brucella Species

We constructed an improved vector pNSGroE for gene expression stud-ies in Brucella spp. It is derived from the broad host range cloning vector pBBR1MCS (1). This new plasmid has several advantages over pBBR1MCS or its derivatives pBBGroE (2): (i) it is smaller in size, 2.9 kb; (ii) it expresses proteins as His-tagged fusion for easy detection and purification; (iii) it car-ries the groE promoter for constitutive expression that is enhanced under con-ditions of stress in vitro and in vivo; and (iv) it has higher levels of heterologous protein expression. Our expression studies in Brucella abortus strain RB51 indicated that the level of heterologous protein expression is higher with pNS-GroE compared to pBBGroE vector. We have also demonstrated the ability of the new vector to express heterolo-gous fusion proteins stably in Brucella species. The smaller size of the new vector was achieved by combining only the very essential elements for replica-tion and expression [minimum origin of replication, promoter, antibiotic selection marker, and multiple cloning sites (MCS)] and removing all nones-sential elements in the original vector pBBR1MCS or pBBGroE (lacZ gene, mobilization gene, and 1 kb upstream of the groE promoter). The vector was constructed by excision of a 185-bp fragment containing the MCS of pRSETA vector (Invitrogen, Carlsbad, CA, USA) using XbaI and HindIII. This fragment was cloned into XbaI and HindIII sites of pGEM11 vector (Promega, Madison, WI, USA) to form construct A. The groE promoter of Brucella spp. was amplified from the genomic DNA of B. abortus strain 2308 using Platinum

The importance of a proper helical structure in the promoter-10 binding region to Bacillus subtilis sigma A structure and function.

Two Bacillus subtilis sigA mutants with amino acid substitutions tending to disrupt the structure of the promoter -10 binding helix of B. subtilis sigma A factor were constructed. B. subtilis DB1001 which contained an A197P substitution was very sensitive to temperature elevation. B. subtilis DB1002 had a T199G substitution and was low in growth potential at the elevated temperature. Degradation of sigma A in B. subtilis DB1001 (t(1/2)=63.2 min) and DB1002 (t(1/2)=186.0 min) occurred readily even at 37 degrees C; however, sigma A in B. subtilis DB2 (wild-type) was fairly stable at the same temperature. The activities of both DB1001 and DB1002 sigma A factors on groE promoter (sigma A-type) were lower than those of the wild-type counterpart at both permissive and restrictive temperatures. The failure of a higher sigma A concentration to suppress the Ts phenotype of DB1001 indicated that the temperature sensitivity of B. subtilis DB1001 was due to altered function, rather than insufficient concentration, of sigma A in the cells. Taken together, our results suggest that the helicity of the promoter -10 binding helix is essential to the packing interaction in the hydrophobic core region of sigma A, which helps to maintain the stable and functional sigma A structure.

Three disparately regulated genes for sigma 32-like transcription factors in Bradyrhizobium japonicum.

Bradyrhizobium japonicum possesses a subclass of heat-shock genes whose members are transcribed from a sigma 32 consensus promoter. Having identified previously one gene (rpoH1) encoding a sigma 32-like RNA polymerase transcription factor, we report here the characterization of two additional rpoH-like genes (rpoH2 and rpoH3). B. japonicum thus represents the first example of an organism possessing an rpoH multigene family. All three rpoH genes encode functional proteins that are able to initiate transcription from the Escherichia coli groE promoter. Each rpoH gene is apparently regulated by a different mechanism. Although both rpoH1 and rpoH2 are transcribed from sigma 70-type promoters, transcription of the rpoH1 operon was found to be heat inducible by an unknown mechanism, whereas the level of rpoH2 mRNA decreased after heat shock. At extreme temperatures (48 degrees C), rpoH2 was transcribed from a second promoter that resembled the E. coli sigma E-type promoter. The rpoH3 gene was found to be associated with two upstream genes, ragA and ragB, coding for a classical two-component regulatory system. Transcription initiated from a promoter that mapped in front of the putative response regulator gene ragA, suggesting that ragA, ragB and rpoH3 are organized in an operon. The ragA promoter was similar to a sigma 32 consensus promoter. The three B. japonicum rpoH genes also varied in their significance to support growth of the organism. While the rpoH2 gene could not be eliminated by mutation, knock-out mutants of rpoH1 and/ or rpoH3 were readily obtained and shown to be indistinguishable from the wild type under aerobic growth conditions or during root-nodule symbiosis. We conclude that rpoH2 is essential for the synthesis of cellular proteins under physiological growth conditions, whereas rpoH1, and probably also rpoH3, are involved in their synthesis during the stress response.

Influence of the linking number of template DNA on sigma 32-dependent transcription initiation in vitro of the Escherichia coli groE gene.

Plasmids carrying the groE promoter with a superhelical density of 0 to -0.091 were used for in vitro transcription with RNA polymerase holoenzyme containing sigma 32. The promoter activity of the groE gene in vitro on the plasmid DNA was decreased to 100-fold when the superhelical density of the template DNA decreased from -0.059 to 0. As the superhelical density of DNA inside the cells was estimated to be approximately -0.025, it seems likely that the increase in the linking number of template DNA caused by stress which induces heat shock response may not stimulate the groE promoter activity in vivo on the chromosomal DNA. At higher concentrations of salt, the dependence of the groE promoter activity on negative supercoiling was even more apparent. Since melting of the DNA duplex is inhibited by high salt concentrations, DNA supercoiling may facilitate melting of the groE promoter by an RNA polymerase holoenzyme containing sigma 32.

Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE).

To study the regulatory mechanism controlling the heat-inducible expression of Bacillus subtilis groE, two regulatory elements, the sigma A-like promoter and the inverted repeat (IR [CIRCE]) in the control region, were characterized. The groE promoter was shown to be transcribed by the major RNA polymerase under both heat shock and non-heat shock conditions. The IR was found to have two functions. (i) It ensures the fast turnover of the groE transcript, and (ii) it serves as an operator. This IR acts as a negative heat shock regulatory element, since deletion of this sequence resulted in high-level expression of groE even at 37 degrees C. Although this IR is present in the 5' untranslated region of the groE transcript, groE transcripts under heat shock and non-heat shock conditions showed similar in vivo half-lives of 5 min. This rapid turnover at 37 degrees C requires the presence of the IR. Without the IR, the groE transcript showed a longer half-life of 17 min. Increasing the distance between the groE transcription start site and the IR systematically by inserting nucleotide sequences from 5 to 21 bp in length resulted in a gradual abolition of the negative regulatory effect mediated by the IR. This effect was not due to a significant change in transcript stability or the transcription start site and is consistent with the model that this IR serves as an operator.

Expression of LacZ from the htrA, nirB and groE promoters in a Salmonella vaccine strain: Influence of growth in mammalian cells

Attenuated Salmonella strains are currently being evaluated as live vectors for the delivery of heterologous antigens to the mammalian mucosal and systemic immune systems. An approach to improving the stability of heterologous antigen expression during vaccination is to drive expression of the foreign protein from promoters e.g. nirB, that become activated when Salmonella enter the host. Salmonella strains were constructed that harboured similar multicopy plasmids encoding the lacZ gene. In each strain, lacZ expression was driven from either the nirB, htrA or groE promoters. Expression of LacZ increased in all vaccine strains as they were shifted from conditions of low to high temperature. In addition, expression of lacZ driven from the htrA and nirB promoters significantly increased when the Salmonella entered eukaryotic cells, including macrophages. Expression of lacZ from the groE promoter was significantly elevated in macrophages but not in cells derived from epithelia. These promoters may be useful for optimising heterologous antigen expression within immune cells of the host.

Expression of LacZ from the htrA, nirB and groE promoters in a Salmonella vaccine strain: influence of growth in mammalian cells.

Attenuated Salmonella strains are currently being evaluated as live vectors for the delivery of heterologous antigens to the mammalian mucosal and systemic immune systems. An approach to improving the stability of heterologous antigen expression during vaccination is to drive expression of the foreign protein from promoters, e.g. nirB, that become activated when Salmonella enter the host. Salmonella strains were constructed that harboured similar multicopy plasmids encoding the lacZ gene. In each strain, lacZ expression was driven from either the nirB, htrA or groE promoters. Expression of LacZ increased in all vaccine strains as they were shifted from conditions of low to high temperature. In addition, expression of lacZ driven from the htrA and nirB promoters significantly increased when the Salmonella entered eukaryotic cells, including macrophages. Expression of lacZ from the groE promoter was significantly elevated in macrophages but not in cells derived from epithelia. These promoters may be useful for optimising heterologous antigen expression within immune cells of the host.

Nucleotide sequence of the Escherichia coli groE promoter

The promoter region of the Escherichia coli groE operon was cloned using the polymerase chain reaction (PCR). The 118-bp nucleotide sequence of the cloned groE promoter was determined on both strands of DNA. Two bp were found between the −10 and −35 regions of this promoter that have not been reported in previous publications of this sequence.

Transcriptional analysis of groEL genes in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor A3(2), synthesis of the groES, groES-groEL1 and groEL2 transcripts is induced either by heat shock or by undefined physiological stress signals present at a certain stage of growth. Under all conditions tested, transcription of groES and groES-groEL1 originated from a unique start site upstream of groES, whereas transcription of groEL2 originated from a unique site upstream of groEL2. RNA polymerase isolated either from heat-shocked or control mycelia allowed in vitro transcription from the P1 promoter of groES/EL1 and the P2 promoter of groEL2. The fact that these two RNA polymerase preparations both initiated transcription with equal efficiency from the same sites suggested that a heat shock-specific sigma factor is not responsible for the temperature-induced transcription of groE genes. Instead, regulation of these genes from vegetative-type promoters may be effected by a DNA-binding protein observed in gel retardation assays, which recognizes a motif found in the groE and dnaK promoter regions of many prokaryotic genes.

Development of RNA polymerase-promoter contacts during open complex formation

We have charted the movements of E σ 32 RNA polymerase at the heat-shock promoter P groE throughout open complex formation, using hydroxyl radical footprinting. In combination with methylation protection and DNase I experiments, these data suggest the following model for open complex formation. E σ 32 initially anchors itself in the upstream region of the promoter forming the first closed complex, RP C1; in this complex the enzyme makes backbone contacts in the −35 region of the promoter that are maintained throughout open complex formation. An isomerization follows resulting in a second closed complex, RP C2; in this complex the enzyme makes base-specific and backbone contacts in the −10 region that are almost identical to those found in the open complex. Thus, at the groE promoter, upstream contacts are established in RP c1 and downstream contacts in RP c2. A similar pattern of backbone contacts was obtained for E σ 32 bound in the open complex at two additional heat-shock promoters, suggesting that the overall topology of holoenzyme in the open complex is similar regardless of sequence variations in the promoter.

Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor σ32 at the groE promoter

The interaction of Eσ32 with the groE promoter at temperatures between 0 °C and 37 °C was studied using DNase I footprinting and dimethyl sulfate methylation. Three distinct complexes were observed. At 0 °C Eσ32 fully protected sequences between −60 and −5 from DNase I digestion on the top (non-template) strand of the promoter. At 16 °C the majority of the Eσ32 promoter complexes had a DNase I footprint almost identical with that seen at 37 °C, protecting the DNA from about −60 to +20; however, little DNA strand separation had occurred, and the changes in sensitivity of guanine residues to dimethyl sulfate methylation caused by Eσ32 differed from those seen at 37 °C. DNA strand separation, and changes in the pattern of protections from and enhancements of methylation by dimethyl sulfate to those characteristic of the open complex, occurred at temperatures between 16 °C and 27 °C. It is plausible to assume that these temperature-dependent isomerizations are analogous to the time-dependent sequence of intermediates on the pathway to open complex formation at 37 °C. Therefore we propose that the formation of an open complex by Eσ32 at the groE promoter involves three classes of steps: Eσ32 initially binds to the promoter in a closed complex (RPC1) in which the enzyme interacts with a smaller region of the DNA than in the open complex. Eσ32 then isomerizes to form a second closed complex (RPC2) in which the enzyme interacts with the same region of the DNA as in the open complex. Finally, a process of local DNA denaturation (strand opening) leads to formation of the open complex (RPO).

Interaction of Escherichia coli RNA polymerase holoenzyme containing σ32 with heat shock promoters: DNase I footprinting and methylation protection

The DNase I protection pattern of Eσ32 was assayed on three heat shock promoters, the Eσ32 promoter for the groESL operon, P2 of the dnaKJ operon, and rpoD PHS, the Eσ32 promoter upstream from rpoD. Eσ32 protected each of these promoters from DNase I digestion from around −60 to around +20. Protection from dimethyl sulfate methylation was assayed at the groE promoter. Eσ32 binding altered the sensitivity to methylation of bases in the vicinity of both the −10 and −35 regions.The DNase I footprints for the Eσ32 promoters were very similar to the DNase I footprint of Eσ70 on the lacUV5 promoter. After analyzing the DNase I footprints by taking into account the contacts predicted to be made by DNase I, it appeared that Eσ32, like Eσ70, contacts the DNA primarily on one face of the helix in the −35 region and on both faces in the −10 region.