pBR322 Derivative Research Articles

Design and Study of Properties of Avirulent Genetically Modified Vibrio cholerae Biovar El Tor Strain with Inactivated Genes of Thermolabile Hemolysin and Effective Expression of Cloned Cholera Toxin B-subunit Gene

The results of the construction of an avirulent strain of V. cholerae genovariant of the O1 serogroup of the El Tor biovar with efficient production of the cholera toxin (CT) B-subunit, which causes the formation of antitoxic immunity during cholera infection, are presented. In the beginning of the study, a modified P18899ΔCTXφchr:TnphoA (KmRHly–) strain was obtained via nondirectional transposon mutagenesis on the basis of a nontoxigenic strain of a P18899ΔCTXφHly+ genovariant. It lost the ability to produce thermolabile hemolysin, which is an additional toxin. The ctxB gene, which encodes biosynthesis of the CT B-subunit, was further introduced into cells of this strain. To accomplish this, the cointegrative recombinant plasmid pIEM3 was used. It was generated in the process of the fusion of a conjugative plasmid pIEM1 and a nonconjugative one, pCTΔ27, which is a derivative of pBR322 (which carries the cloned gene ctxB). Via restriction analysis, it was established that disjunction of a cointegrate occurred in vibrio cholera cells, followed by the preservation of a multicopy plasmid (pCTΔ27) only. As a result, avirulent clones of KmRTcR with a high production level (5–6 μg/mL) of secreted CT B-subunit were obtained, one of which (E99) was selected for further studies. Via real-time PCR, it was discovered that the expression of the ctxB plasmid gene in the cells of a designed strain of V. cholerae, E99, doesn’t depend on the activity of the key regulatory gene toxT, which located on the chromosome. Efficient expression of the ctxB gene in E99 cells allows their use to obtain CT B-subunit in order to produce cholera immunobiological drugs.

A simple technique for suppressor detection in Escherichia coli.

To study the viability of agyrA S83 stop mutation found in anEscherichia coli J53 ciprofloxacin-resistant strain (J53 CipR27), a pBR322 derivative was constructed with a TAG mutation in thebla gene knocking out ampicillin resistance. Ampicillin resistance was restored, suggesting that the strain contains tRNA suppressor activity able to suppress the UAG codongyrA and allow viability. The method was applied to 22 unique clinicalE. coli isolates, and all were found to have low-level suppressor activity.

Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli

Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 degrees C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ, a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor sigma32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 degrees C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ. Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ-independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.

An Escherichia coli biosensor capable of detecting both genotoxic and oxidative damage.

A two-plasmid dual reporter Escherichia coli biosensor was developed using the genes for bacterial bioluminescence and a mutant of the green fluorescent protein, GFPuv4. To achieve this, the two plasmids, which were derivatives of pBR322 and pACYC184, had compatible origins of replication and different antibiotic selection markers: ampicillin and tetracycline. The parent strains DK1 and ACRG43, each carrying a single plasmid with one of the fusion genes (strain DK1 harboring a fusion of the katG promoter to the lux operon while in ACRG43, the recA promoter was fused with the GFP gene), were responsive to oxidative and DNA damage, respectively, resulting in higher bioluminescence or fluorescence under the relevant toxic conditions. The responses of the dual sensor strain, DUAL22, to various toxicants, e.g., mitomycin C, N-methyl-N-nitro--nitrosoguanidine, hydrogen peroxide and cadmium chloride, were characterized and compared with the responses of the parent strains to the same chemicals. Finally, several chemical mixtures that cause various stress responses were tested to demonstrate the ability of this biosensor to detect specific stress responses within a multiple toxicity environment.

Development of an Escherichia coli whole cell biocatalyst for the production of l-amino acids

A whole cell biocatalyst for the enzymatic production of l-amino acids from hydantoins was created by coexpressing the genes encoding the l-hydantoinase, the l- N-carbamoylase and the hydantoin racemase from Arthrobacter aurescens in Escherichia coli. In order to construct a well balanced reaction system the enzymatic activity in the cells was varied by using vectors with different copy numbers for expression of the genes. Derivatives of pSC101, pACYC184 and pBR322 were employed for the various constructions and in one construct the hydantoinase gene was integrated into the E. coli chromosome. All constructs carried the E. coli rhamnose promoter system enabling gene expression control by transcriptional regulation. The productivity for l-tryptophan from the corresponding hydantoin was more than 6-fold higher than achieved with Arthrobacter aurescens.

Structure and function of a pyrimidine/purine-biased sequence from the 5'-flanking region of the basidiomycete Lentinus edodes gene priA.

The priA gene of the basidiomycete Lentinus edodes possesses a pyrimidine (CT)-rich stretch (26 bp) that includes a short (6-bp) repeat, the elements of which form a mirror repeat at and near the transcriptional initiation sites. A DNA fragment that included this sequence was inserted into pBR322, and the resulting plasmids were introduced into Escherichia coli. Analysis of the susceptibility of these pBR322 derivatives to cleavage by S1 nuclease, following isolation from E. coli, indicated the formation of an open, S1-sensitive structure within and just downstream of the CT/AG-biased sequence. Replacement of two dTMP residues in one of the repeat elements by dGMP resulted in the elimination of the S1-cleavable open structure from the plasmids. To analyze the effect of the CT/AG-biased sequence from priA in the basidiomycete Coprinus cinereus, the integrating vectors pLC2 and pLC2mutCT were used; these contained the wild-type priA promoter and the mutant priA promoter with the aforementioned mutation in the mirror repeat, respectively. The Streptomyces-derived bialaphos resistance gene (bar) was fused downstream of the promoters, and the resulting plasmids, pLC2-bar and pLC2mutCT-bar, were introduced into C. cinereus. Transformants carrying pLC2mutCT-bar grew significantly more slowly on bialaphos-containing agar plates and contained a noticeably lower level of the bar transcript when compared with the transformants obtained with pLC2-bar. These results suggest that an unusual structure induced by the CT/AG-biased sequence is required for efficient gene expression from the priA promoter.

The Monoclonal Antibody 1F6 Identifies a pH-dependent Conformational Change in the Hydrophilic NH2 Terminus of NhaA Na+/H+ Antiporter ofEscherichia coli

One of the most interesting properties of the NhaA Na(+)/H(+) antiporter of Escherichia coli is the strong regulation of its activity by pH. This regulation is accompanied by a conformational change that can be probed by digestion with trypsin and involves the hydrophilic loop connecting the transmembrane helices VIII-IX. In the present work we show that a monoclonal antibody (mAb), 1F6, recognizes yet another domain of NhaA in a pH-dependent manner. This antibody binds NhaA at pH 8.5 but not at pH 4.5, whereas two other mAbs bind to NhaA independently of pH. The epitope of mAb 1F6 was located at the NH(2) terminus of NhaA by probing proteolytic fragments in Western blot analysis and amino acid sequencing. The antibody bound to the peptide HLHRFFSS, starting at the third amino acid of NhaA. A synthetic peptide with this sequence was shown to bind mAb 1F6 both at acidic and alkaline pH suggesting that this peptide is accessible to mAb 1F6 in the native protein only at alkaline pH. Although slightly shifted to acidic pH, the pH profile of the binding of mAb 1F6 to the antiporter is similar to that of both the Na(+)/H(+) antiporter activity as well as to its sensitivity to trypsin. We thus suggest that these pH profiles reflect a pH-dependent conformational change, which leads to activation of the antiporter. Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activity as well as its sensitivity to trypsin, affects in a similar pattern the binding of mAb 1F6 to NhaA. Furthermore, the binding site of mAb 1F6 is involved in the functioning of the antiporter as follows: a double Cys replacement H3C/H5C causes an acidic shift by half a pH unit in the pH dependence of the antiporter; N-ethylmaleimide, which does not inhibit the wild-type protein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.

A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells.

A genetically controlled luminescent bacterial reporter assay, the SOS lux test, was developed for rapid detection of environmental genotoxins. The bioassay is based on the recombinant plasmid pPLS-1, which was constructed as a derivative of pBR322, carrying the promoterless luxCDABFE genes of Photobacterium leiognathi downstream of a truncated cda gene from ColD with a strong SOS promoter. E. coli recA+ strains containing this construction are inducible to high levels of light production in the presence of substances or agents that cause damage to the DNA of the cells. The light signal, reflecting the SOS-inducing potency, is recorded from the growing culture within 1 s, and the test results are available within 1 to 2 h. Induction of bioluminescence was demonstrated by treatment of E. coli C600(pPLS-1) with 6 genotoxic chemicals (mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, nalidixic acid, dimethylsulfate, hydrogen peroxide, and formaldehyde) and with UV and gamma radiation. A clear dose-response relationship was established for all eight genotoxins. The sensitivity of the SOS lux test is similar to that of other bioassays for genotoxicity or mutagenicity, such as the SOS chromotest, umu test, and Ames mutatest. These results indicate that the SOS lux test is potentially useful for the in situ and continuous detection of genotoxins.

Expression of the Escherichia coli bo-Type Ubiquinol Oxidase with a Chimeric Subunit II Having the CuA-Cytochrome c Domain from the Thermophilic Bacillus caa3-Type Cytochrome c Oxidase

The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.

Structure and function in Escherichia coli of plasmids containing pyrimidine/purine-biased stretch originated from the 5'-flanking region of the basidiomycete ras gene.

The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained. To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT[ras] and pBR-invCT[ras] were introduced into E. coli strains DM800 (deltatopA gyrB225) and JM109 (topA+ gyrA96). In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)coding strand and in pBR-invCT[ras] the complementary purine-rich sequence is on this strand. DNAs of pBR-CT[ras] and pBR-invCT[ras] isolated from DM800 were frequently cleaved with single-strand-specific S1 nuclease within the CT/AG sequence, showing the formation of extended open structure. Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT [ras] showed much higher levels of tetracycline resistance (Tcr), while both strains carrying pBR-invCT[ras] showed clearly lower levels of Tcr. pBR-CT [ras] and pBR-invCT [ras], however, conferred reduced activity of beta-lactamase on DM800 and JM109. pBR-CT [ras] derivatives lacking the counterpart of the mirror repeat did not form the S1-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tcr and beta-lactamase activity. The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.

A sensitive reporter gene system using bacterial luciferase based on a series of plasmid cloning vectors compatible with derivatives of pBR322

We present a series of plasmid cloning vectors that are derived from a mutant of pSC101 possessing an elevated number of copies per genome equivalent. These vectors are compatible with any plasmid replicating from a pBR322 origin and use spectinomycin and/or streptomycin as a selective marker. They can be used whenever the simultaneous presence of several plasmids in the cell is desired. We use this vector system for the constitutive expression of the genes that are responsible for the production of the aldehyde substrate of bacterial luciferase. Transcription from promoters carried on a second plasmid can thus be measured within the living cell over a range of 3 orders of magnitude using bacterial luciferase as a reporter gene.

Plasmid stability: comments on the dimer catastrophe hypothesis.

Using a derivative of the plasmid pBR322 we have tested the dimer catastrophe hypothesis of plasmid instability. Most of the theory was confirmed by our observations, but our data suggest that some of the quantitative aspects need modification. In a recF strain of Escherichia coli we estimated the difference in loss rate between the plasmid in the monomeric and the dimeric state to be a factor of 13-14 and the difference in the loss rate between the plasmid in the monomeric and the trimeric state to be a factor of 14-50. We were able to confirm that plasmid oligomers were heterogeneously distributed within a rec+ population, but we were unable to detect any pronounced difference in the level of growth inhibition exerted by the plasmid when in the monomeric, dimeric, or trimeric state. This leaves open the question as to whether runaway plasmid multimerization was prevented (i) by a small correlation between the inhibition of growth and the 'multimeric status' of the plasmid, (ii) by intramolecular homologous recombination, or (iii) whether the process of runaway multimerization is too slow to be recognized within the duration of the experiments, i.e. 200 generations of growth.

The ColE1 Unidirectional Origin Acts as a Polar Replication Fork Pausing Site

Co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogenes broad-host-range plasmid pSM19035 and Escherichia coli pPI21 are among the exceptions. pPI21, which is a derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. Analysis of pPI21 replication intermediates (RIs) by two-dimensional agarose gel electrophoresis and electron microscopy revealed the accumulation of a specific RI containing a single internal bubble. The data obtained demonstrated that initiation of DNA replication occurred at a single origin in pPI21. Progression of the replicating fork initiated at either of the two potential origins was transiently stalled at the other inversely oriented silent ColE1 origin of the plasmid. The accumulated RIs, containing an internal bubble, occurred as a series of stereoisomers with different numbers of knots in their replicated portion. These observations provide one of the first functional explanations for the disadvantage of head-to-head plasmid multimers with respect to head-to-tail ones.

Transcription-induced deletions in plasmid vectors: M13 DNA replication as a source of instability.

We have previously shown that concurrent progression of pBR322 replication and pTac-directed transcription in opposite orientations induces illegitimate recombination events. We tested here the effects of M13 rolling circle replication on the incidence of plasmid deletions. The progression of the M13 replication fork leads to an increase of more than 300-fold in the frequency of transcription-dependent deletion events. pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used. Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA replication, provided that the two proceed in opposite orientations. We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication leads to a fivefold decrease in plasmid copy number within 2 h, which is consistent with the proposal that deletions arise because replication fork progression is impeded. This decrease in parental plasmid copy number leads in turn to an enrichment in deleted plasmid forms. Our data confirm and extend the notion that simultaneous transcription and replication in opposite directions can efficiently promote deletion formation. In addition, this instability may be amplified when the rearranged molecules acquire a replicative advantage.

High Expression of Plasmid-Encoded Tetracycline Resistance Gene inE. coliCauses a Decrease in Membrane-Bound ATPase Activity

Expression of the plasmid pBR322-encoded tetracycline-resistant gene (tet) is known to cause other pleiotropic effects in addition to mediating the efflux of tetracycline from bacterial host cells. We have recently reported that expression of thetetgene in plasmid pKH47, a high-copy-number derivative of pBR322, causes growth inhibition ofEscherichia colicells harboring this plasmid. In this paper we report that reduced membrane-bound ATPase activity is found inE. colicells containing plasmid pKH47. This effect is dependent on the presence of an intacttetgene and reduces the ability of the cells to grow in a minimum medium containing succinate as the sole carbon source. The same effect is more dramatically observed in cells containing an unrelated plasmid in whichtetgene expression is under the control of thetacpromoter.

E. coliGrowth Inhibition by a High Copy Number Derivative of Plasmid pBR322

We have observed that plasmid pKH47, a pBR322-derivative containing a 100bp poly(dA)-poly(dT) insertion, causes growth inhibition of hostE. colicells harboring it. In this paper we show that this inhibitory effect is due to an increased copy number property of this plasmid, which in turn leads to an over expression of the plasmid-encodedtetgene. Our work also indicates that contrary to other pleiotropic effects caused by thetetgene product, which solely depend on the expression of the 5′ end of the gene, growth inhibition requires an intacttetgene. In addition we present the isolation of anE. colimutant that is refractive to the inhibitory effect of pKH47 and shares some properties with the parental bacteria containing plasmid pKH4.

Transcription‐induced deletions in Escherichia coli plasmids

Characterization of functions that render DNA susceptible to rearrangement is important for a better understanding of genome instability. In a previous work, we showed that sequences located downstream of a strong promoter are particularly prone to deletion. In this paper, the parameters that influence transcription-induced deletions were studied. pBR322 derivatives carrying the M13 (+) replication origin and a PTac-dependent transcription region were used. Deletion formation was analysed in the presence of the replication protein of M13, which introduces a nick at the phage replication origin, and in a rep- strain to avoid M13-driven replication. Our study showed that: (i) 4 h after induction of transcription, a few per cent of the plasmids have experienced a deletion; (ii) these deletions result in joining of the M13 replication origin to a nucleotide located in or downstream of the transcribed region; (iii) deletion formation strongly depends on the orientation of transcription, on promoter strength and transcript length, but is independent of translation; (iv) formation of transcription-induced supercoiling domains does not induce deletion formation. We propose that deletions in the transcribed region result from collisions between converging replication and transcription machineries.

Studies of the anaerobically induced promoter p nirB and the improved expression of bacterial antigens

The promoter of the Escherichia coli gene nirB is induced by both the presence of nitrite in the environment and by low oxygen tensions. It has been used to direct the high-level expression of heterologous proteins by E. coli strains in fermentors, and attenuated Salmonella strains expressing foreign proteins under nirB promoter (p nir) control have efficiently induced an immune response against these proteins. The genes encoding two different E. coli envelope proteins, the outer membrane protein LamB and the periplasmic protein in MalE, were placed under p nir control on pBR322 derivatives, and both proteins were expressed at high levels during anaerobic growth. Our results showed that the expression level of MalE was influenced by the distance between the p nir promoter and the Shine-Dalgarno sequence: the highest levels were obtained by the longest constructs made; p nirn directed a 4-fold increase in the level of MalE expression relative to the level reached by the previously described ptac-MalE expression vector. The best p r construct produced 25 mg of MalE protein per 5 × 10 11 bacteria, which represents over 20% of total cell protein. Overexpression of Male was well tolerated by E coli, even under strict anaerobic conditions; for LamB, optimal induction was achieved under partial anaerobiosis. A MalE-HIV1 hybrid protein (33 residues from the V3 loop of HIV1 gp160 inserted into site 133 of MalE) was also overexpressed at a similar yield under p nir control, without apparent degradation of the hybrid protein. Moreover, when expressed in attenuated aroA S. typhimurium strain SL3261, the plasmids carrying malE and malE-HIV genes were stable in vitro and in vivo.

High-efficiency transformation and gene inactivation in Streptococcus suis type 2

An efficient electrotransformation system for Streptococcus suis type 2 is described. It is demonstrated that vectors based on the broad-host-range plasmid pWVO1 replicate in S. suis type 2. Transformation efficiencies of about 10(7) transformants per micrograms of plasmid DNA could be obtained. Derivatives of plasmid pBR322 containing S. suis chromosomal DNA did not replicate but integrated into the chromosome. Southern hybridization analysis revealed that double as well as single cross-over integration events had occurred. Double cross-over events occurred at a frequency of about 15%. With these transformation and integration systems, recombinant DNA technology can now be applied to this important pathogenic species.

PBR322 and Other pSC101 Derivatives Have a Gene Fragment Related to the Mini-F Plasmid Resolvase

Sequence comparisons have shown that part of pBR322 and other pSC101 derivatives contain a remnant resolvase gene closely related to that of plasmid mini-F. This observation illuminates part of the history of these plasmids.