Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria.

We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of Escherichia coli and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision ("curing"), or retrieval of the CRIM plasmids.

In vivo characterization of the type A and B vancomycin-resistant enterococci (VRE) VanRS two-component systems in Escherichia coli: a nonpathogenic model for studying the VRE signal transduction pathways.

Escherichia coli reporter strains modeling the high-level type A and B vancomycin resistances of Enterococcus faecium BM4147 and Ent. faecalis have been developed to study the respective VanR-VanS two-component regulatory systems. PvanH-, PvanRa-, PvanY-, and PvanRb-lacZ fusions report on expression from the vancomycin-resistant enterococci promoters of the type A vanRSHAXYZ and type B vanRSYWHBX gene clusters. These strains also express from single-copy chromosomal genes vanRa, vanRb, or vanRSb behind their respective promoter (PvanRa or PvanRb) or vanSa or vanSb behind the rhamnose-inducible PrhaB. Results show that activation (phosphorylation) of the response regulator VanRa by its sensor kinase VanSa leads to transcriptional activation of both PvanH and PvanRa. Additionally, VanRb activates its cognate promoters PvanY and PvanRb, although this occurs only in the absence of VanSb and presumably is caused by VanRb phosphorylation by an unidentified endogenous E. coli kinase. Thus, VanSb interferes with activation of VanRb, probably by acting as a phospho-VanRb phosphatase. Although both VanRa and VanRb activate their cognate promoters, neither activates the heterologous PvanR, PvanH, or PvanY, arguing against the interchangeability of type A and B two-component regulatory switches in vancomycin-resistant enterococci. VanRa also is activated by the nonpartner kinase PhoR. Because this occurs in the absence of its inducing signal (Pi limitation), PhoR autophosphorylation apparently is regulated in vivo. Furthermore, the activation of VanRa caused by cross talk from PhoR in the absence of a signal allows distinction of cross talk from crossregulation as the latter, but not the former, responds to environmental cues.

Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon.

Escherichia coli genes regulated by environmental inorganic phosphate (Pi) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the Pi-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated P(araB) promoter or the rhamnose-regulated P(rhaB) promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of P(araB) or P(rhaB) fusions and for recombination of them onto the E. coli chromosome at the araCBAD or rhaRSBAD locus, respectively. Using strains carrying such single-copy fusions, we showed that a P(rhaB) fusion is more tightly regulated than a P(araB) fusion in that a P(rhaB)-phoR+ fusion but not a P(araB)-phoR+ fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, both P(araB)-phoB+ and P(rhaB)-phoB+ fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise delta phoU mutations. However, we unexpectedly found that such delta phoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797-6809, 1993). They also readily give rise to compensatory mutants with lesions in phoB, phoR, or a pst gene, making their study particularly difficult. Here we found that, by using P(araB)-phoB+, P(rhaB)-phoB+, or P(rhaB)-phoR+ fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ delta phoU mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.

Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190.

Guanine or hypoxanthine, physiological corepressors of the Escherichia coli purine repressor (PurR), promote formation of the ternary PurR-corepressor-operator DNA complex that functions to repress pur operon gene expression. Structure-based predictions on the importance of Arg190 in determining 6-oxopurine specificity and corepressor binding affinity were tested by mutagenesis, analysis of in vivo function, and in vitro corepressor binding measurements. Replacements of Arg190 with Ala or Gln resulted in functional repressors in which binding of guanine and hypoxanthine was retained but specificity was relaxed to permit binding of adenine. X-ray structures were determined for ternary complexes of mutant repressors with purines (adenine, guanine, hypoxanthine, and 6-methylpurine) and operator DNA. These structures indicate that R190A binds guanine, hypoxanthine, and adenine with nearly equal, albeit reduced, affinity in large part because of a newly made compensatory hydrogen bond between the rotated hydroxyl side chain of Ser124 and the exocyclic 6 positions of the purines. Through direct and water-mediated contacts, the R190Q protein binds adenine with a nearly 75-fold higher affinity than the wild type repressor while maintaining wild type affinity for guanine and hypoxanthine. The results establish at the atomic level the basis for the critical role of Arg190 in the recognition of the exocyclic 6 position of its purine corepressors and the successful redesign of corepressor specificity.

Transcriptional regulation of the Enterococcus faecium BM4147 vancomycin resistance gene cluster by the VanS-VanR two-component regulatory system in Escherichia coli K-12.

An Escherichia coli K-12 model system was developed for studying the VanS-VanR two-component regulatory system required for high-level inducible vancomycin resistance in Enterococcus faecium BM4147. Our model system is based on the use of reporter strains with lacZ transcriptional and translational fusions to the PvanR or PvanH promoter of the vanRSHAX gene cluster. These strains also express vanR and vanS behind the native PvanR promoter, the arabinose-inducible ParaB promoter, or the rhamnose-inducible PrhaB promoter. Our reporter strains have the respective fusions stably recombined onto the chromosome in single copy, thereby avoiding aberrant regulatory effects that may occur with plasmid-bearing strains. They were constructed by using allele replacement methods or a conditionally replicative attP plasmid. Using these reporter strains, we demonstrated that (i) the response regulator VanR activates PvanH, but not PvanR, expression upon activation (phosphorylation) by the partner kinase VanS, the noncognate kinase PhoR, or acetyl phosphate, indicating that phospho-VanR (P-VanR) is a transcriptional activator; (ii) VanS interferes with activation of VanR by PhoR or acetyl phosphate, indicating that VanS also acts as a P-VanR phosphatase; and (iii) the conserved, phosphate-accepting histidine (H164) of VanS is required for activation (phosphorylation) of VanR but not for deactivation (dephosphorylation) of P-VanR. Similar reporter strains may be useful in new studies on these and other interactions of the VanS-VanR system (and other systems), screening for inhibitors of these interactions, and deciphering the molecular logic of the signal(s) responsible for activation of the VanS-VanR system in vivo. Advantages of using an E. coli model system for in vivo studies on VanS and VanR are also discussed.

Altered recognition mutants of the response regulator PhoB: a new genetic strategy for studying protein-protein interactions.

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a "patch" near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and P1 signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6K gamma attP "genome targeting" suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.

Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria.

We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6K gamma DNA replication origin (oriR(R6K gamma) so they replicate only in bacteria supplying the pi replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of pi. They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria. Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA. Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker. One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants. Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones. Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker. Plasmid-free segregants with an allele replacement can be subsequently selected as TetS or SucR recombinants. A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.

Immune response against the L-lactate dehydrogenase of Mycoplasma hyopneumoniae in enzootic pneumonia of swine.

The L-lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae, formerly named protein P36, belongs to the predominant immunogenic proteins in pigs which were naturally or experimentally infected with M. hyopneumoniae. The antigenic reaction against M. hyopneumoniae LDH has been shown to be species specific. Recombinant M. hyopneumoniae LDH, which was genetically engineered to contain six histidine residues at its C-terminal end, was expressed in E. coli and purified to a high degree using Ni-chelate affinity chromatography. The genetically engineered LDH still showed the same biochemical activity and immunological specificity as the wild-type LDH and was used as an antigen for a M. hyopneumoniae LDH ELISA. Using this assay, we showed that pigs experimentally infected with M. hyopneumoniae raised antibodies against LDH in two steps. An early, relatively weak anti-LDH response was detected between 5 to 10 weeks post-infection when clinical signs and lung lesions occur. This first minor raise of anti-LDH antibodies occurred simultaneously with the strong appearance of antibodies against an antigen consisting of membrane proteins of M. hyopneumoniae prepared with Tween 20 extraction. A second, strong raise in anti-LDH antibodies was observed from the twelfth week after infection, at a time when the disease signs and the infectious agent disappeared. The high anti-LDH titer persisted until 21 weeks post-infection, in contrast to the antibody titer against the membrane proteins which started to decrease after its peak at 12 weeks post-infection. A LDH-ELISA may also be useful for detecting past infections.

Sequence analysis and transcription of the apxI operon (hemolysin I) from Actinobacillus pleuropneumoniae.

The DNA sequence of the entire apxI operon from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 has been determined. This 8292-bp fragment of the chromosomal DNA contains four open reading frames (ORFs) of the strongly hemolytic ApxI toxin. These ORFs correspond to the genes apxIC, apxIA, apxIB and apxID, encoding the activator, the structural toxin protein and the two secretion proteins, respectively. Each of the four ORFs is preceded by a consensus sequence for a putative ribosome-binding site (RBS). The region upstream from apxIC contains several sites that could act as promoters. The transcription start point (tsp) of the apxI operon in A. pleuropneumoniae has been determined by primer extension analysis and was found to be located 133-bp upstream from the translation start codon. The tsp is preceded by sequences matching the -10 and -35 consensus sequence of promoters from Escherichia coli. This is the first promoter identified in A. pleuropneumoniae. The same tsp was used when the expression of apxI was induced by a high concentration of free Ca2+ in the growth medium, as well as when the expression of apxI was not induced by growing the cells in medium depleted of free Ca2+ ions. However, the signal strength of the primer extension was approximately tenfold stronger in Ca(2+)-grown cells. The leader sequence of the transcript is unusually long and very A+U rich (75% A+U).

DNA sequence determination and biochemical analysis of the immunogenic protein P36, the lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae.

The DNA sequence of the gene encoding the early and specific immunogenic protein P36 of Mycoplasma hyopneumoniae has been determined. Comparison of the DNA sequence and the deduced amino acid sequence of P36 with known genes and proteins in data banks indicated that P36 is a L-lactate dehydrogenase (LDH) (EC 1.1.1.27). Biochemical analysis of protein P36 expressed from the cloned gene in Escherichia coli confirmed that P36 has L-lactate dehydrogenase activity. Protein P36 of M. hyopneumoniae therefore is termed LDH and its gene ldh. M. hyopneumoniae LDH was shown to contain the typical domains of LDH of other bacterial species. Immunologically however, we have shown that polyclonal antibodies against M. hyopneumoniae LDH do not cross-react with related LDH and show high specificity for M. hyopneumoniae. The ldh gene is preceded by several typical -10 sequences found in promoters of prokaryotes, but lacks the -35 sequence. Sequences rich in A+T, however, precede the -10 boxes, suggesting that factors involved in transcription initiation and their regulation may be different in M. hyopneumoniae compared to other bacterial species, but the putative ribosome binding site seems to be conserved.

Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.

Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.

Use of antibodies against the P36 protein of Mycoplasma hyopneumoniae for the identification of M. hyopneumoniae strains.

Mycoplasma hyopneumoniae, the principal aetiological agent of porcine enzootic pneumonia, synthesizes a 36 kDa protein (P36) which is an early and strong immunogenic factor in experimentally and naturally infected swine. Polyclonal antibodies were made against the recombinant P36 protein in rabbits and used for the identification of M. hyopneumoniae by the immunoblot technique. The proteins from the M. hyopneumoniae reference strains and from 13 M. hyopneumoniae field strains isolated from naturally infected pigs in Switzerland, Hungary, France and Canada were analysed by the immunoblot technique using anti-P36 antibodies. All 13 field strains and the three reference J strains of M. hyopneumoniae, received from different collections and laboratories, exhibited a strong reaction with a protein of 36 kDa indicating that the P36 protein is a common M. hyopneumoniae antigen. None of the different porcine Mycoplasma species including M. flocculare, M. hyorhinis, M. hyosynoviae, A. axanthum, A. laidlawii and A. granularum showed any reaction on the immunoblot with the anti-P36 antibodies. In addition, we have found no reaction with anti-P36 antibodies using 47 different Mycoplasma or Acholeplasma species isolated from human, mice, rat, poultry, ruminant, dog and cat. In conclusion we have shown that P36 is a protein that is a common antigen of M. hyopneumoniae strains and is not found in other Mycoplasma or Acholeplasma species tested. Because of its high specificity, P36 protein, or antibodies made against this protein can be used for the identification of M. hyopneumoniae strains.