Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin.

Chimeric proteins exhibiting antigenic determinants of the heat-labile enterotoxin (LT) and heat-stable (STa) enterotoxins on the same molecule may provide a means to obtain immunoprophylactic and diagnostic reagents for Escherichia coli-caused diarrhea. We recently showed that fusion of two different lengths of the STa gene to the C end of the A-subunit of LT (LTA) results in LTA::STa fusion proteins as monitored by GM1-ELISA [Sanchez et al.: FEBS Lett. 208 (1986) 194-198]. Here we determine the approximate molecular size of the LTA::STa fusion proteins and provide further evidence of their hybrid nature by immunoblot analysis. Using this technique we also demonstrate that to obtain detectable amounts of these recombinant proteins it is essential to coexpress them with the respective B-subunit of LT (LTB). We propose that this dependence on coexpression reflects the association between the LTA::STa hybrids and LTB subunits. The resulting LTA::STa/LTB complexes were found in the E. coli periplasm. This indicated that the exported hybrids, once associated with LTB, were stabilized and formed molecules that behaved essentially as native LT. The protective effect exerted by the B-subunit might conceivably be extended to other LTA-derived hybrid proteins, thus allowing the fusion of other foreign peptides to LTA and their subsequent recovery in the same fashion.

New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin.

Two new cloning vectors (pBEU28 and pBEU50) with temperature-controlled runaway-replication properties are described. pBEU28 is similar to aphA+ (KanR) plasmid pBEU2 but lacks a 1.8-kb duplication which is responsible for plasmid instability. pBEU50 is an analog of pBR313 and pBR322 in that it carries bla+(AmpR), which can be used for selection, and tet+(TetR) which can be inactivated by cloning at HindIII and BamHI restriction sites. Sublethal concentrations of novobiocin were exploited to suppress runaway replication and to restore the viability of the plasmid carriers. By this method copB deletion mutants of two temperature-controlled, conditional runaway-replication plasmids were detected and isolated. The unconditional runaway-replication property of these plasmids leads us to hypothesize that there are at least two controls of plasmid R1 copy number and that the copB-dependent control is temperature-sensitive in the conditional runaway replication mutants. The novobiocin suppression of the runaway replication permitted us to clone dnaN+ on pBEU28 and to identify its presence at 42 degrees C with a dnaN59 transformation recipient which was temperature-sensitive due to a defect in the dnaN gene.

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products.

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30 degrees C these miniplasmids are present in 20--50 copies per cell of Escherichia coli, whereas at temperatures above 35 degrees C the plasmids replicate without copy number control during 2--3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plamid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded beta-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.

Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated, amplified expression of cloned adventitious genes in Escherichia coli.

We describe a two-plasmid system that utilizes the lacZ gene promoter and temperature-responsive plasmid replicons to accomplish closely regulated high-level expression of heterologous genes in Escherichia coli. One of the plasmids fails to replicate at 42 degrees C and contains a gene encoding the lac repressor; the second plasmid, which undergoes multicopy "runaway" replication at elevated temperatures, contains an adventitious gene under control of the operator-promoter system of the lacZ gene. Concurrent derepression of lac promoter function and amplification of copy number of the lac-controlled gene occurs when the temperature is elevated. We have used a structural gene encoding chloramphenicol acetyltransferase to demonstrate that the gene product under control of the lacZ promoter represents a major fraction of the total protein synthesized at 43 degrees C, whereas only minimal quantities of this enzyme are made at 30 degrees C. The system described allows the controlled expression of gene products that may have detrimental effects on cell growth, and provides a simple method for identifying radioactivity-labeled protein products of cloned genes in bacterial whole-cell extracts. The system also offers an alternative to intragenic temperature-sensitive mutations for studying the function of various enzymatic or regulatory proteins.

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion.

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.

An apoptotic response by J774 macrophage cells is common upon infection with diarrheagenic Escherichia coli.

Representative strains of the different diarrheagenic Escherichia coli virotypes were tested for their potential cytotoxicity in the J774 macrophage cell line. All the seven virotypes of E. coli were cytotoxic to J774 macrophages, and in most cases the bacteria induced an apoptotic response. With the exception of the enterotoxigenic E. coli (ETEC) strain, all the other six virotypes caused induction of apoptosis as evidenced by quantitative analysis of the characteristic DNA fragmentation at the individual cell level. These results suggest that apoptosis could be one of the mechanisms contributing to the diarrheal disease development.

Runaway-replication plasmids as tools to produce large quantities of proteins from cloned genes in bacteria.

Here we review the properties and uses of runaway-replication vectors, a class of versatile plasmids discovered and developed in Escherichia coli. They are based on the IncFII plasmid, R1, in which an antisense RNA (CopA RNA) negatively controls the formation of a protein that is rate-limiting for replication. The copy number of the plasmid is determined by the balance between the rates of formation of CopA RNA and RepA mRNA. A small increase in the rate of formation of the latter drastically reduces the rate of formation of CopA RNA due to convergent transcription, which may lead to a total loss of copy number control (runaway replication), resulting in massive DNA amplification, and plasmid copy numbers up to 1000 per genome. Since this amplification occurs in the presence of protein synthesis, the protein that is encoded by a cloned gene can also be amplified, and may constitute 10-50% of the total protein.

Preferential inhibition of plasmid replication in vivo by altered DNA gyrase activity in Escherichia coli.

The thermosensitive growth phenotype exerted by runaway-mutant plasmids was suppressed by sublethal doses of the DNA gyrase inhibitors novobiocin or nalidixic acid, although the latter drug was less efficient. A novobiocin-resistant gyrB mutant Escherichia coli strain prevented expression of the runaway phenotype at 37 to 42 degrees C in the absence of any drug.

Regulation of virulence-associated plasmid genes in enteroinvasive Escherichia coli.

The transposon TnphoA was used for construction of gene fusions and for studies of gene regulation in an enteroinvasive strain of Escherichia coli. Several plasmid-encoded virulence genes (e.g., the ipaB and virG operons) of such enteroinvasive strains are subject to coordinated thermoregulation involving both operon-specific (the VirB and VirF activators) and global regulators. The nucleoid-associated E. coli protein H-NS was shown to be a negative regulator as judged by studies using H-NS gene deletion mutations and by increasing the level of H-NS protein in the cells. An increased gene dosage of H-NS led to enhanced repression of the ipa and virG operons, particularly at low (30 degrees C) growth temperature. The cyclic AMP receptor protein complex, which is another global transcriptional regulator in E. coli, was not required for the regulation of ipa and virG expression. The virG operon was expressed in an activator-independent manner in cells lacking H-NS protein. We suggest that the role of the VirF activator is to counteract the silencing effect of H-NS.

Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE-dependent endonucleolytic processing.

Differential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities. As the result of a post-transcriptional processing event, a papA gene-specific mRNA product (mRNA-A) accumulates in large excess relative to the primary mRNA-BA transcript. Our results show that the processed product, mRNA-A, is a translationally active molecule and that it is generated from the mRNA-BA precursor by an RNaseE-dependent mechanism. The processing did not occur under non-permissive conditions in an E. coli rne mutant strain with a temperature-sensitive RNaseE. The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA. A comparison of nucleotide sequences of mRNA-BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites. Mutations abolishing the activity of either of two other endoribonucleases, RNaseIII and RNaseP, did not affect the pap mRNA processing event. However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E. coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency. Our findings are consistent with the idea that ams is related/allelic to rne. Absence of the processing event in the RNaseE mutant (rne-3071) strain led to a four-fold stabilization of the mRNA-BA primary transcript. We conclude that the RNaseE-dependent processing event is the rate-limiting step in the decay of the papB-coding part of the primary transcript and in the production of the stable mRNA-A product.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinated and differential expression of histone-like proteins in Escherichia coli: regulation and function of the H-NS analog StpA.

The histone-like protein H-NS has been shown to influence the regulation of gene expression at the transcriptional level in several Escherichia coli operons. We have examined the regulation of the stpA gene, which encodes a protein sharing 58% identity with H-NS, by mRNA analysis and by using stpA-lacZ operon fusions. The expression of stpA is temperature dependent, with 2-fold higher expression at 37 degrees C than at 26 degrees C. In addition, stpA expression is stimulated by the global regulator Lrp. In an hns mutant E.coli derivative stpA expression is derepressed, suggesting that regulation of the two genes is coupled. Overproduction of the StpA protein affects expression from at least four hns regulated operons (the papB, proU, bgl and hns operons), in both the presence and absence of H-NS. The construction of E.coli strains carrying mutations in both stpA and hns demonstrated that the absence of both proteins affects growth rate and viability of the cells. Our work establishes that E.coli can express two H-NS-like proteins with coordinated yet differential regulation. Evidently, these proteins have both overlapping and distinct functions in the cell, and they are both important for normal cell growth and gene control.

In vitro analysis of mRNA processing by RNase E in the pap operon of Escherichia coli.

Differential gene expression from operons encoding fimbrial adhesins in Escherichia coli involves processing and differential decay of polycistronic transcripts. Previous analyses of mRNA processing in vivo using ribonuclease mutants of E. coli have given different results with the different fimbrial gene systems tested. For the pap operon from uropathogenic E. coli, the results suggested that the mRNA processing is dependent on ribonuclease E (RNase E), whereas in other fimbrial operons with similar genetic organisation, the processing was concluded to be RNase E independent. We have developed an in vitro system allowing us to assess the cleavage of pap mRNA, to study the mRNA processing of a fimbrial operon in more detail, and to define the enzymatic activity and target. The results of this study establish that RNase E does indeed cleave the papBA intercistronic transcript. Analysis of the cleavage products reveals that in vitro RNase E can cleave the mRNA at other positions in addition to the site preferentially cleaved in vivo. The specificity of the cleavage pattern was assessed using transcripts derived from mutants with base substitutions near, or within, the major in vivo cleavage site. Such mutants have alternative cleavage sites. A common feature of the different cleavage sites is a high A/U nucleotide content, similar to other known RNase E cleavage sites. Features of the secondary structure of the papBA intercistronic mRNA were investigated using single-strand-specific and double-strand-specific nucleases. The secondary structure model derived from stability calculations and our results from the nuclease-probing experiments indicate that the positions subject to RNase E cleavage are mainly single stranded and flanked by more stable stem-loop structures. The results are consistent with the notion that an mRNA conformation exposing A/U-rich, non-paired regions constitutes the target, i.e. a flexible determinant, for processing by RNase E in the pap transcript. The findings are discussed in relation to the existence of a potential recognition site for RNase E and the analysis of RNase E cleavages in other RNA molecules.

Differential protease-mediated turnover of H-NS and StpA revealed by a mutation altering protein stability and stationary-phase survival of Escherichia coli.

The Escherichia coli proteins H-NS is recognized as an important component among the major nucleoid-associated proteins. In studies of E. coli strains with defects in H-NS, we discovered a mutant that phenotypically restored stationary-phase viability (Rsv) of such strains. The Rsv phenotype was the result of a mutation that led to severalfold higher levels of the functionally and structurally related StpA protein. This mutation was a base pair change in the stpA structural gene, and the amino acid substitution in the StpA protein altered its turnover properties, suggesting a role for this residue in a cleavage site for proteolysis. We determined the stability of the StpA and the H-NS proteins and found that the StpA protein was degraded relatively rapidly in strains lacking functional H-NS, whereas H-NS remained stable irrespective of the presence/absence of StpA. Using protease-deficient mutants, we obtained evidence that the Lon protease was responsible for the degradation of StpA. The differential turnover of the nucleoid-associated proteins is suggested to contribute to the regulation of their stoichiometry and ratio in terms of homo- and heteromer formation. We conclude that StpA, in contrast to H-NS, is present mainly in heteromeric form in E. coli.

Plasmid incompatibility and control of replication: copy mutants of the R-factor R1 in Escherichia coli K-12.

Plasmid incompatibility was studied in Escherichia coli K-12. By double-antibiotic selection, clones were constructed that carried the two R-factors R1 and R100, both belonging to the compatibility group FII. After release of the selection pressure, each of the two plasmids was lost at the same rate (8% per generation). Mutants of R-factor R1 showing an increased number of copies per chromosome (copy mutants) were tested for their incompatibility towards R-factor R100. The results indicate that plasmid incompatibility is quantitative and not just a qualitative property. All copy mutants studied affected incompatibility, and there were two classes of mutants: one increasing and one decreasing the incompatiblity exerted towards the test plasmid R100. Evidence is presented that incompatibility is related to the mechanisms that control replication. The implications of such a relation on proposed models for control of replication are discussed. The data do not support the hypothesis that plasmid incompatibility is due to competition for a replicational or segregational site.

Overproduction of the Escherichia coli recA protein without stimulation of its proteolytic activity.

Plasmid pBEU14, which carries the Escherichia coli recA+ gene and which can be amplified by manipulation of growth temperature, was constructed. When pBEU14 deoxyribonucleic acid was amplified, a high rate of synthesis and accumulation of recA protein resulted. Amplification of the recA gene and protein did not cause induction of prophage lambda, indicating that the proteolytic activity of the recA protein was not stimulated.