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.Multicopy plasmids have greatly facilitated gene structurefunction studies. However, the use of such plasmids can lead to high-copy-number artifacts, especially in physiological studies. Thus, several methods have been developed for recombining genes on bacterial chromosomes in order to study their functions in single copies. Such methods are frequently used to construct novel Escherichia coli strains that stably express foreign genes for use in both basic research and biotechnology (5,18,27). However, the development of strains encoding complex metabolic or regulatory pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. To address these concerns, we have developed a series of plasmid-host systems for the introduction of multiple genes into the same cell in single copies. Our approach is based on genome targeting systems that utilize plasmids carrying a conditional-replication origin and a phage attachment (attP) site (17). We refer to our plasmids as CRIM (conditionalreplication, integration, and modular) plasmids. CRIM plasmids can be integrated into or retrieved from their bacterial attachment (attB) site by supplying phage integrase (Int) without or with excisionase (Xis) in trans.Advantages of our CRIM plasmid-host systems include the use of alternative attP and attB sites (for phages , HK022, 80, P21, and P22) and different selectable markers (for chloramphenicol, gentamicin, k...

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Iclaprim binds and inhibits bacterial DHFR in a similar manner to trimethoprim. However, the increased hydrophobic interactions between iclaprim and DHFR account for increased affinity and, unlike trimethoprim, enable iclaprim to inhibit even the resistant enzyme with nanomolar affinity, thus overcoming the mechanism of trimethoprim resistance. The increased antibacterial activity and lower propensity for resistance make iclaprim a clinically promising and useful inhibitor.

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl ␣-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate 3 ribose 1-phosphate 3 ribose 1,5-bisphosphate 3 PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine ؉ P i 3 guanine ؉ ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (COP) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5-XMP, and by cochromatography of the reaction product with authentic PRPP.NAD biosynthesis in Escherichia coli usually proceeds by consumption of 5-phospho-D-ribosyl ␣-1-diphosphate (PRPP). NAD is synthesized from aspartate and dihydroxyacetone phosphate. A de novo pathway and a number of salvage pathways for the reutilization of nicotinamide mononucleotide and nicotinamide exist, as shown in Fig. 1 (32). Two of the enzymatic reactions, the reactions catalyzed by quinolinate and nicotinate phosphoribosyltransferases, require PRPP. PRPPless mutants with a deletion of the prs gene, encoding PRPP synthase, consequently require NAD or nicotinamide mononucleotide. ⌬prs strains also require guanosine, uridine, histidine, and tryptophan, which are likewise synthesized with PRPP as an intermediate (14,15). Nevertheless, mutants that suppress the NAD requirement are easily obtained by selecting for growth of ⌬prs cells on medium lacking NAD. These mutants still require guanosine, uridine, histidine, and tryptophan. All such NAD-suppressed mutants were previously shown to have lesions in the pst-phoU operon (17), which leads to highlevel constitutive expression of genes belonging to the phosphate (Ph...

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. P vanH -, P vanRa -, P vanY -, and P vanRb -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 (VanY). In type B VRE, vanY is upstream of vanH, whereas in type A the corresponding vanY comes after vanX. The consequence of this reordering is that while the promoters controlling the twocomponent regulatory system genes are P vanRa and P vanRb , respectively, the promoters for the structural genes are instead P vanH and P vanY (Fig. 1). Most previous genetic and enzymatic studies trying to define the molecular logic of how these proteins function in VRE have focused on the type A system. Courvalin and colleagues (7) also have demonstrated that mutations of type B VanS b can result in teicoplanin-inducible resistance.To characterize the two-component VRE signal transducing proteins, we previously have studied VanS a and VanR a in vitro and in vivo by using Escherichia coli (8,9). We now report studies on the in vivo function and selectivity of VanS a and VanR a along with similar ones of VanS b and VanR b on expression from the type A and B promoters (Fig. 1). We examined VanR a and VanR b for transcriptional activation of its cognate and noncognate promoters. We tested the requirements of VanS a and VanS b for activation (phosphorylation) of VanR a and VanR b . Additionally, we tested for cross talk between these heterologous VRE signaling proteins, comparing interactions between them to those with the response regulator PhoB and the sensor kinase PhoR of the E. coli Pho regulon (10) as a basis for distinguishing specific and nonspecific (cross talk) interactions. Our results indicate that, unlike the VanH, VanA͞VanB and VanX enzymes required for vancomycin resistance, the type A and type B two-component regulatory systems evolved independently.

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 P vanR or P vanH promoter of the vanRSHAX gene cluster. These strains also express vanR and vanS behind the native P vanR promoter, the arabinose-inducible P araB promoter, or the rhamnose-inducible P rhaB 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 P vanH , but not P vanR , expression upon activation Vancomycin is a glycopeptide antibiotic that is currently used for the treatment of gram-positive bacterial infections, especially ones caused by methicillin-resistant Staphylococcus aureus species (49). Vancomycin acts by binding to the terminal D-Ala-D-Ala moieties of bacterial cell wall precursors and effectively inhibits the transpeptidation and transglycosylation steps of the cell wall assembly process, thereby rendering the cell susceptible to osmotic shock. Over the past 10 years, a number of Enterococcus strains with high-level inducible resistance to vancomycin have been identified (11), and the relative incidence of these strains has increased sharply in the last 3 years (39). High-level resistance to the antibiotic has been found to require five plasmid-borne genes : vanR, vanS, vanH, vanA, and vanX. VanR and VanS comprise a two-component regulatory system (2, 51) that regulates transcription of the genes responsible for conferring resistance: vanH, vanA, and vanX (52). Twocomponent regulatory systems are signal transduction pathways commonly used by prokaryotes to sense and adapt to stimuli in the environment; as many as 50 different ones may exist in a single bacterium such as Escherichia coli (29). Analogous signal transduction pathways have recently been identified in both eucaryotes (38) and archaea (32). These systems are characterized by a sensor histidine kinase (often a transmembrane signaling kinase such as VanS) that undergoes autophosphorylation on a conserved histidine residue. The phosphoryl group is then transferred to a conserved aspartate residue on the response regulator protein (in this case, VanR) that usually acts as a transcriptional activator. Like many signaling kinases, VanS has an N-terminal domain with two transmembrane segments flanking an extracellular domain that is believed to act as the signal sensing domain and a C-terminal cytoplasmic transmitter domain with autophosphorylation and phosphotransfer activities. Biochemical studies on the cytoplasmic domain of VanS (M95 to S384) have shown that it is readily autop...

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 arabinosedependent Lac ؉ mutants. Changes in the PhoB AR 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 P i signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriR R6K␥ 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.Protein-protein interactions are important in genetic regulatory responses in all cells. Identifying domains and amino acid residues that determine how a given protein recognizes an interacting partner protein(s) is therefore a fundamental problem. Many interacting proteins are members of gene families and yet interact only with specific proteins that are not (or only poorly) recognized by other members of the same family.

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 ParaB promoter or the rhamnose-regulated PrhaB promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of ParaB orPrhaB fusions and for recombination of them onto the E. coli chromosome at the araCBAD orrhaRSBAD locus, respectively. Using strains carrying such single-copy fusions, we showed that a PrhaB fusion is more tightly regulated than a ParaB fusion in that a PrhaB-phoR + fusion but not a ParaB-phoR + fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, bothParaB-phoB + andPrhaB-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 ΔphoU mutations. However, we unexpectedly found that such ΔphoU 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 inphoB, phoR, or a pst gene, making their study particularly difficult. Here we found that, by usingParaB-phoB +,PrhaB-phoB +, orPrhaB-phoR + fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ ΔphoU 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.