uhpT Expression Research Articles

HptA Mutation May Mediate Fosfomycin Resistance in Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

Fosfomycin can be used alone or in combination to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. However, fosfomycin resistance has been observed in MRSA. In S. aureus, fosfomycin resistance is mediated by the fosfomycin-modifying enzyme FosB, or mutations in the target enzyme MurA. Mutations in the chromosomal glpT and uhpT genes, which encode fosfomycin transporters, also result in fosfomycin resistance. The three-component regulatory system HptRSA mediates the expression of uhpT and glpT in S. aureus. This study aimed to investigate the role of hptRSA mutation in fosfomycin resistance in MRSA clinical isolates. We found that hptRSA mutations were common in MRSA strains isolated from our hospital. Most mutations were amino acid substitutions and widely distributed in fosfomycin-sensitive and fosfomycin-resistant strains. However, HptA-truncated mutations were only found in fosB-negative fosfomycin-resistant strains with wild-type uhpT and glpT genes. Quantitative real-time PCR results showed that the transcription level of uhpT decreased by 13.7-25.6-fold in the HptA-truncated strains. Concordantly, the fosfomycin minimum inhibitory concentration (MIC) of HptA-truncated strains was 64-128 μg/mL, while SA240 was 2 μg/mL. The low transcription level of uhpT and high increase in MIC suggest that hptA mutation may lead to fosfomycin resistance in MRSA. We complemented hptA in one of the HptA-truncated clinical strains (SA179), showing reversal of fosfomycin resistance (from 128 to 32 μg/mL). Then we knocked out hptA in S. aureus Newman; fosfomycin MIC increased from 4 to 64 μg/mL, suggesting that HptA mutation may play an important role in fosfomycin resistance.

Distantly Related Homologue of UhpT in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative bacteria that affects patients in intensive care units and chronic respiratory disease patients. Compared to other bacteria, it has a wide genome (around 6.3-Mb) that supports its metabolic versatility and antimicrobial resistance. Fosfomycin (FF) is primarily used as an oral treatment for urinary tract infections (UTIs). FF diffuses inside the cell via glycerol-3-phosphate transporter (GlpT) PA, as well as in other bacteria. In other bacteria, such as E. coli, glucose-6-phosphate transporter (UhpT) functions as FF transporter. Since mutant GlpT leads to FF resistant PA, it is assumed that GlpT is the only FF transporter. However, it is also assumed that PA uses glucose-6-phosphate and, thus, homologous proteins of UhpT may be present in its genome. Here, we present an attempt to find a distant related homologue of UhpT in PA. A Hidden Markov Model (HMM) was created to seek for Major facilitator family (MFS) domain in 21 PA genomes of 14 CF patients annotated with prokka and the statistical analysis was performed (MCC: 0.84, ACC: 0.99). Then, the HMM was applied to PA genomes. Besides the actual GlpT, annotated as glpt_1, one more GlpT protein was found in 21 out of 21 genomes, annotated as glpt_2. Since glpt_2 clusters closer to UhpT than GlpT, glpt_2 was selected to build a model. Computing a structural superimposition, the model and the template of UhpT have 0.6 Å of RMSD. The model of glpt_2 has some characteristics that are fundamental to UhpT functions. The binding site, consisting of 2 arginines (Arg46 and Arg275) and Lys45, is totally conserved, as well as the topology of the structure. Asp90 is also conserved in glpt_2 model. No studies aimed at searching for distant related homologous of UhpT. Since the high genetic exchange and high mutational rate in bacteria, it is likely that PA has a UhpT-like protein in the PA genome. The binding site is superimposable to UhpT protein as well as the overall topology. In fact, the 12 TMs are completely comparable, suggesting a well-defined folding of the protein across the bilayer lipid membrane. To enforce our hypothesis, in all 21 PA genomes, we also found a protein annotated as membrane sensor protein UhpC, important for expression and function of UhpT in E. coli. Since PA strains are wild-type, we can assume that most of the PA have proteins like this. The presence of a homologue of UhpT suggests that this protein is conserved in PA genome.

Novel Chromosomal Mutations Responsible for Fosfomycin Resistance in Escherichia coli.

Fosfomycin resistance in Escherichia coli results from chromosomal mutations or acquisition of plasmid-mediated genes. Because these mechanisms may be absent in some resistant isolates, we aimed at decipher the genetic basis of fosfomycin resistance in E. coli. Different groups of isolates were studied: fosfomycin-resistant mutants selected in vitro from E. coli CFT073 (MIC = 1 mg/L) and two groups (wildtype and non-wildtype) of E. coli clinical isolates. Single-nucleotide allelic replacement was performed to confirm the implication of novel mutations into resistance. Induction of uhpT expression by glucose-6-phosphate (G6P) was assessed by RT-qPCR. The genome of all clinical isolates was sequenced by MiSeq (Illumina). Two first-step mutants were obtained in vitro from CFT073 (MICs, 128 mg/L) with single mutations: G469R in uhpB (M3); F384L in uhpC (M4). Second-step mutants (MICs, 256 mg/L) presented additional mutations: R282V in galU (M7 from M3); Q558∗ in lon (M8 from M4). Introduction of uhpB or uhpC mutations by site-directed mutagenesis conferred a 128-fold increase in fosfomycin MICs, whereas single mutations in galU or lon were only responsible for a 2-fold increase. Also, these mutations abolished the induction of uhpT expression by G6P. All 14 fosfomycin-susceptible clinical isolates (MICs, 0.5–8 mg/L) were devoid of any mutation. At least one genetic change was detected in all but one fosfomycin-resistant clinical isolates (MICs, 32 – >256 mg/L) including 8, 17, 18, 5, and 8 in uhpA, uhpB, uhpC, uhpT, and glpT genes, respectively. In conclusion, novel mutations in uhpB and uhpC are associated with fosfomycin resistance in E. coli clinical isolates.

Characterization of Fosfomycin and Nitrofurantoin Resistance Mechanisms in Escherichia coli Isolated in Clinical Urine Samples.

Fosfomycin and nitrofurantoin are antibiotics of choice to orally treat non-complicated urinary tract infections (UTIs) of community origin because they remain active against bacteria resistant to other antibiotics. However, epidemiologic surveillance studies have detected a reduced susceptibility to these drugs. The objective of this study was to determine possible mechanisms of resistance to these antibiotics in clinical isolates of fosfomycin- and/or nitrofurantoin-resistant UTI-producing Escherichia coli. We amplified and sequenced murA, glpT, uhpT, uhpA, ptsI, cyaA, nfsA, nfsB, and ribE genes, and screened plasmid-borne fosfomycin-resistance genes fosA3, fosA4, fosA5, fosA6, and fosC2 and nitrofurantoin-resistance genes oqxA and oqxB by polymerase chain reaction. Among 29 isolates studied, 22 were resistant to fosfomycin due to deletion of uhpT and/or uhpA genes, and 2 also possessed the fosA3 gene. Some modifications detected in sequences of NfsA (His11Tyr, Ser33Arg, Gln67Leu, Cys80Arg, Gly126Arg, Gly154Glu, Arg203Cys), NfsB (Gln44His, Phe84Ser, Arg107Cys, Gly192Ser, Arg207His), and RibE (Pro55His), and the production of truncated NfsA (Gln67 and Gln147) and NfsB (Glu54), were associated with nitrofurantoin resistance in 15/29 isolates; however, the presence of oqxAB plasmid genes was not detected in any isolate. Resistance to fosfomycin was associated with the absence of transporter UhpT expression and/or the presence of antibiotic-modifying enzymes encoded by fosA3 plasmid-mediated gene. Resistance to nitrofurantoin was associated with modifications of NfsA, NfsB, and RibE proteins. The emergence and spread of these resistance mechanisms, including transferable resistance, could compromise the future usefulness of fosfomycin and nitrofurantoin against UTIs. Furthermore, knowledge of the genetic mechanisms underlying resistance may lead to rapid DNA-based testing for resistance.

Cooperative Actions of CRP-cAMP and FNR Increase the Fosfomycin Susceptibility of Enterohaemorrhagic Escherichia coli (EHEC) by Elevating the Expression of glpT and uhpT under Anaerobic Conditions.

Bacterial infections to anaerobic site are often hard to be treated because the activity of most of antimicrobials decreases under anaerobic conditions. However, fosfomycin rather provides a greater activity under anaerobic conditions than aerobic conditions. Previously, we found that expression of glpT and uhpT, fosfomycin symporters in enterohaemorrhagic Escherichia coli (EHEC) was upregulated by FNR, a global regulator during the anaerobiosis of the bacterium, which led to increased uptake and susceptibility to this drug. In this study, we showed that expression of glpT and uhpT is induced by CRP-cAMP, the regulator complex under both aerobic and anaerobic conditions. The activity of CRP-cAMP in EHEC was elevated under anaerobic conditions because levels of both CRP and cAMP were higher in the cells when grown anaerobically than those when grown aerobically. Results of expression study using mutants indicated that CRP-cAMP is indispensable for expression of glpT but not uhpT—whereas that of uhpT requires UhpA that is the response regulator composing of two-component system with the sensor kinase, UhpB. The CRP-cAMP protein bound to a region that overlaps RNA polymerase binding site for glpT and region upstream of UhpA binding site for uhpT. FNR bound to a region further upstream of CRP-cAMP binding site on region upstream of the glpT gene. These combined results suggested that increased antibacterial activity of fosfomycin to EHEC under anaerobic conditions is due to activation of FNR and increment of CRP-cAMP activity. Then, FNR enhances the expression of glpT activated by CRP-cAMP while CRP-cAMP and FNR cooperatively aids the action of UhpA to express uhpT to maximum level.

Regulatory mechanism of the three-component system HptRSA in glucose-6-phosphate uptake in Staphylococcus aureus.

Glucose-6-phosphate (G6P) is a common alternative carbon source for various bacteria, and its uptake usually relies on the hexose phosphate antiporter UhpT. In the human pathogenic bacterium Staphylococcus aureus, the ability to utilize different nutrients, particularly alternative carbon source uptake in glucose-limiting conditions, is essential for its fitness in the host environment during the infectious process. It has been reported that G6P uptake in S. aureus is regulated by the three-component system HptRSA. When G6P is provided as the only carbon source, HptRSA could sense extracellular G6P and activate uhpT expression to facilitate G6P utilization. However, the regulatory mechanism of HptRSA is still unclear. In this study, we further investigated the HptRSA system in S. aureus. First, we confirmed that HptRSA is necessary for the normal growth of this pathogen in chemically defined medium with G6P supplementation, and we discovered that HptRSA could exclusively sense extracellular G6P compared to the other organophosphates we tested. Next, using isothermal titration calorimetry, we found that HptA could bind to G6P, suggesting that it may be the G6P sensor. After that experiment, using an electrophoresis mobility shift assay, we verified that the response regulator HptR could directly bind to the uhpT promoter and identified a putative binding site from -67 to -96-bp. Subsequently, we created different point mutations in the putative binding site and revealed that the entire 30-bp sequence is essential for HptR regulation. In summary, we unveiled the regulatory mechanism of the HptRSA system in S. aureus, HptA most likely functions as the G6P sensor, and HptR could implement its regulatory function by directly binding to a conserved, approximately 30-bp sequence in the uhpT promoter.

Elevated Expression of GlpT and UhpT via FNR Activation Contributes to Increased Fosfomycin Susceptibility in Escherichia coli under Anaerobic Conditions.

Because a shortage of new antimicrobial agents is a critical issue at present, and with the spread of multidrug-resistant (MDR) pathogens, the use of fosfomycin to treat infections is being revisited as a "last-resort option." This drug offers a particular benefit in that it is more effective against bacteria growing under oxygen-limited conditions, unlike other commonly used antimicrobials, such as fluoroquinolones and aminoglycosides. In this study, we showed that Escherichia coli strains, including enterohemorrhagic E. coli (EHEC), were more susceptible to fosfomycin when grown anaerobically than when grown aerobically, and we investigated how the activity of this drug was enhanced during anaerobic growth of E. coli. Our quantitative PCR analysis and a transport assay showed that E. coli cells grown under anaerobic conditions had higher levels of expression of glpT and uhpT, encoding proteins that transport fosfomycin into cells with their native substrates, i.e., glycerol-3-phosphate and glucose-6-phosphate, and led to increased intracellular accumulation of the drug. Elevation of expression of these genes during anaerobic growth requires FNR, a global transcriptional regulator that is activated under anaerobic conditions. Purified FNR bound to DNA fragments from regions upstream of glpT and uhpT, suggesting that it is an activator of expression of glpT and uhpT during anaerobic growth. We concluded that the increased antibacterial activity of fosfomycin toward E. coli under anaerobic conditions can be attributed to elevated expression of GlpT and UhpT following activation of FNR, leading to increased uptake of the drug.

Investigation of in vivo cross-talk between key two-component systems of Escherichia coli.

Intracellular signal transfer in bacteria is dominated by phosphoryl transfer between conserved transmitter and receiver domains in regulatory proteins of so-called two-component systems. Escherichia coli contains 30 such systems, which allow it to modulate gene expression, enzyme activity and the direction of flagellar rotation. The authors have investigated whether, and to what extent, these separate systems form (an) interacting network(s) in vivo, focussing on interactions between four major systems, involved in the responses to the availability of phosphorylated sugars (Uhp), phosphate (Pho), nitrogen (Ntr) and oxygen (Arc). Significant cross-talk was not detectable in wild-type cells. Decreasing expression levels of succinate dehydrogenase (reporting Arc activation), upon activation of the Pho system, appeared to be independent of signalling through PhoR. Cross-talk towards NtrC did occur, however, in a ntrB deletion strain, upon joint activation of Pho, Ntr and Uhp. UhpT expression was demonstrated when cells were grown on pyruvate, through non-cognate phosphorylation of UhpA by acetyl phosphate.

Expression of the pho regulon interferes with induction of the uhpT gene in Escherichia coli K-12.

The uptake of hexose 6-phosphates in Escherichia coli is mediated by the transporter UhpT, the synthesis of which is induced by the presence of glucose 6-phosphate (glucose 6-P) in the medium. Since this protein functions as an anion exchanger, it is generally assumed to be geared for the use of sugar phosphates as a carbon source. However, the question was unresolved whether this transporter can also provide the cells with glucose 6-P as a phosphate source. It is demonstrated in this work that UhpT-mediated glucose 6-P uptake does not allow the cells to grow on glucose 6-P as phosphate source. Hence, the expression of UhpT under phosphate limitation would not be particularly advantageous and some form of interaction between the uhp system and the Pi-limitation-inducible pho regulon, the products of which are involved in phosphate acquisition, may be anticipated. Indeed, the use of an uhpT-lacZ fusion revealed that much higher concentrations of the inducer glucose 6-P were required to elevate uhpT transcription when the pho regulon was expressed. This interference was the result of degradation of glucose 6-P by one of the products of the pho regulon, the periplasmic enzyme alkaline phosphatase. The specific form of interaction between the Pho and Uhp systems is designated inducer degradation.

Involvement of the amino-terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli.

The UhpA protein is required for expression of the sugar phosphate transporter UhpT in Escherichia coli and is regulated by phosphate transfer from the transmembrane UhpBC sensor kinase complex. UhpA action requires the sensor kinase complex and the site of phosphorylation, Asp-54, under normal conditions, but not when UhpA is overexpressed. Directed mutagenesis of the uhpA gene allowed examination of the role of several residues of UhpA in response to phosphorylation and in transcription activation. Residues Asp-9, Asp-54, and Lys-101 are highly conserved and required for function in other response regulators. Changes at any of these residues in UhpA resulted in complete loss of phosphorylation-dependent activity, but did not affect the high-level, constitutive, UhpBC-independent expression when the UhpA variants were overexpressed. Thus, these residues are important for the response to the phosphorylation pathway but not for transcription activation. Eight independent uhpA mutants selected for activity in the absence of UhpBC function carried the F17-->V or H170-->Y substitutions. Other substitutions for Phe-17 conferred various phenotypes, ranging from inducible to high-level constitutive behaviour. Residues in helix-1 flanking Phe-17 were converted to Ala or other residues. Alanine substitutions at Val-13, Arg-14, and Leu-20 resulted in complete loss of phosphorylation-dependent activation. Change of Gly-16 to Ala had no effect, but changes to other residues resulted in loss of function. Alanine substitutions at Phe-17 and at Gln-19 resulted in high-level constitutive expression, and changes at Ala-18 and Leu-21 had only modest effects. Most interesting was the L20-->A substitution, which conferred low uhpT expression when overexpressed and interfered with action of the wild-type chromosomal allele. The combination of the L20-->A change with changes at Phe-17, Asp-54 and His-170 indicated that the trans-dominant action of L20-->A occurred at several steps. The observations that UhpA can activate uhpT transcription in its unphosphorylated state are consistent with its occupancy of low-affinity binding sites necessary for promoter function. We propose that the effect of phosphorylation of UhpA is to enhance its oligomerization on the DNA surface to extend to the low-affinity sites, and that helix-1 participates in the process of oligomer formation.

Regulation of Escherichia coli adenylate cyclase activity during hexose phosphate transport.

In Escherichia coli, cAMP levels vary with the carbon source used in the culture medium. These levels are dependent on the cellular concentration of phosphorylated EnzymeIIAglc, a component of the glucose-phosphotransferase system, which activates adenylate cyclase (AC). When cells are grown on glucose 6-phosphate (Glc6P), the cAMP level is particularly low. In this study, we investigated the mechanism leading to the low cAMP level when Glc6P is used as the carbon source, i.e. the mechanism preventing the activation of AC by phosphorylated EnzymeIIAglc. Glc6P is transported via the Uhp system which is inducible by extracellular Glc6P. The Uhp system comprises a permease UhpT and three proteins UhpA, UhpB and UhpC which are necessary for uhpT gene transcription. Controlled expression of UhpT in the absence of the regulatory proteins (UhpA, UhpB and UhpC) allowed us to demonstrate that (i) the Uhp regulatory proteins do not prevent the activation of AC by direct interaction with EnzymeIIAglc and (ii) an increase in the amount of UhpT synthesized (corresponding to an increase in the amount of Glc6P transported) correlates with a decrease in the cAMP level. We present data indicating that Glc6P per se or its degradation is unlikely to be responsible for the low cAMP level. It is concluded that the level of cAMP in the cell is determined by the flux of Glc6P through UhpT.

Non-physiological expression of UhpT does not lead to uncontrolled leakage of sugar phosphates out of Escherichia coli cells

De-regulated expression of uhpT under control of the tac promoter, by increasing concentrations of isopropyl-thio-β- d-galactoside, progressively inhibited the growth rate of Escherichia coli cells, to such an extent that growth was fully inhibited at 1 mM of the inducer. Significantly, addition of glucose 6-phosphate to the growth medium of the cells did not protect against this inhibition. Furthermore, efflux of sugar phosphates from the cells did not take place under these conditions, unless protonophoric uncouplers were added. We therefore conclude that the regulation of uhpT expression i.e. via extracellular induction through a two-component system, did not evolve in order to prevent loss of essential metabolites from the cytoplasm under conditions when extracellular sugar phosphates are not available.

Non-physiological expression of UhpT does not lead to uncontrolled leakage of sugar phosphates out of Escherichia coli cells.

De-regulated expression of uhpT under control of the tac promoter, by increasing concentrations of isopropyl-thio-beta-D-galactoside, progressively inhibited the growth rate of Escherichia coli cells, to such an extent that growth was fully inhibited at 1 mM of the inducer. Significantly, additio of glucose 6-phosphate to the growth medium of the cells did not protect against this inhibition. Furthermore, efflux of sugar phosphates from the cells did not take place under these conditions, unless protonophoric uncouplers were added. We therefore conclude that the regulation of uhpT expression, i.e. via extracellular induction through a two-component system, did not evolve in order to prevent loss of essential metabolites from the cytoplasm under conditions when extracellular sugar phosphates are not available.

Transcription activation at the Escherichia coli uhpT promoter by the catabolite gene activator protein.

Transport and utilization of sugar phosphates in Escherichia coli depend on the transport protein encoded by the uhpT gene. Transmembrane induction of uhpT expression by external glucose 6-phosphate is positively regulated by the promoter-specific activator protein UhpA and the global regulator catabolite gene activator protein (CAP). Activation by UhpA requires a promoter element centered at -64 bp, relative to the start of transcription, and activation by CAP requires a DNA site centered at position -103.5. This DNA site binds the cyclic AMP-CAP complex in vitro, and its deletion from the promoter reduces transcription activity to 7 to 9% of the wild-type level. Ten uhpT promoter derivatives with altered spacing between the DNA site for CAP and the remainder of the promoter were constructed. Their transcription activities indicated that the action of CAP at this promoter is dependent on proper helical phasing of promoter elements, with CAP binding on the same face of the helix as RNA polymerase does. Five CAP mutants defective in transcription activation at class I and class II CAP-dependent promoters but not defective in DNA binding or DNA bending (positive control mutants) were tested for the ability to activate transcription. These CAPpc mutants exhibited little or no defect in transcription activation at uhpT, indicating that CAP action at uhpTp involves a different mechanism than that which is used for its action at other classes of CAP-dependent promoters.

Promoter elements required for positive control of transcription of the Escherichia coli uhpT gene.

The uhpABCT locus of Escherichia coli encodes the transport system which allows the cell to accumulate a variety of sugar phosphates in unaltered form. The expression of uhpT, the gene encoding the transport protein, is regulated by the uhpABC gene products. The UhpA protein is required for expression; its deduced amino acid sequence shows that it belongs to a subfamily of bacterial transcription regulators including NarL, DegU, and FixJ. Members of this subfamily have an amino-terminal phosphorylation domain characteristic of so-called two-component regulators, such as OmpR, CheY, PhoB, and NtrC, and a carboxyl-terminal domain conserved among many transcriptional activators, including LuxR and MalT. The major sequence elements in the uhpT promoter that are needed for uhpT expression were investigated. Northern (RNA) hybridization analysis showed that the uhpT transcript was only present in cells induced for UhpT transport activity. The start site of transcription was identified by primer extension. Comparison of the regions upstream of the uhpT transcription start site in E. coli and Salmonella typhimurium suggested the presence of four sequence elements that might be involved in promoter function: a typical -10 region, a short inverted repeat centered at -32, a long inverted repeat centered at -64, and a cyclic AMP receptor protein-binding sequence centered at -103. Deletion and linker substitution mutations in the promoter demonstrated that the presence of the cyclic AMP receptor protein-binding site resulted in about an eightfold increase in promoter activity and that the -64, -32, and -10 elements were essential for promoter function. In vivo titration of transcriptional activator UhpA by the intact or mutant promoters on multicopy plasmids identified the -64 element as the UhpA-binding site. The two halves of the -64 inverted repeat did not contribute equally to promoter function and did not have to be intact for UhpA titration. The sequence recognized by UhpA is predicted to be 5' -GGCAAAACNNNGAAA.

Role of uhp genes in expression of the Escherichia coli sugar-phosphate transport system.

The uhpABCT locus of Escherichia coli is responsible for expression of the sugar-phosphate transport system and its induction by external glucose 6-phosphate. Expression of uhpT-lacZ fusions depended on the function of uhpA, uhpB, and uhpC but not of uhpT. A plasmid carrying only uhpT conferred transport activity in a host strain deleted for the uhp region. Thus, uhpT encodes the polypeptide required for transport function, and the other three uhp genes regulate uhpT transcription. The presence of uhpA at elevated copy number resulted in a substantial increase in uhpT expression. This elevated expression was only about 50% of the level seen in induced haploid cells, and no further increase occurred after addition of inducer. Activation by multicopy uhpA was not affected by the status of uhpC but was decreased in the absence of uhpB, suggesting a role for UhpB in directly activating UhpA. Transcription of uhpA, monitored by expression of a uhpA-lacZ fusion, was not affected by either inducer or the presence of the wild-type uhpA allele. The presence of multiple copies of the uhpT promoter region reduced uhpT expression in strains with uhpA in single copy number but not in those with multiple copies, consistent with competition for the activator. Amino acid sequence comparisons showed that UhpA was homologous to a family of bacterial regulatory proteins, some of which act as transcriptional activators (OmpR, PhoB, NtrC, and DctD). The C-terminal portion of UhpB displayed matches to the corresponding portions of another family of proteins (EnvZ, PhoMR, NtrB, and DctB) that participate in regulation of gene expression in response to environmental factors.

Molecular cloning of the uhp region and evidence for a positive activator for expression of the hexose phosphate transport system of Escherichia coli.

The uhp locus of Escherichia coli contains genes for the sugar phosphate transport system (uhpT) and the regulatory system which allows its induction by external glucose 6-phosphate (uhpRA). The uhp region was cloned onto high-copy-number plasmids, both from Uhp(+) plasmids of the Clarke-Carbon collection and from genetically characterized specialized transducing phages carrying uhpT-lac operon fusions. Two Clarke-Carbon plasmids and their Uhp(+) subclones in pBR322 shared restriction sites defining the uhp region, but exhibited different regulation of Uhp expression and dependence on chromosomal uhp genotype. Plasmid pLC17-47 and derivatives conferred constitutive glucose 6-phosphate uptake activity in all strains, even those with complete deletions of uhp. These plasmids also rendered constitutive the expression of a chromosomal uhpT-lac operon fusion. Plasmid pLC40-33 conferred inducible Uhp expression, which required the presence of the uhpA(+) gene on the chromosome. The induced transport levels in all strains carrying these plasmids were not appreciably amplified over haploid levels. Similar behavior was seen with the cloned operon fusions. A fusion-bearing plasmid that carried an intact regulatory system (uhpR(+)A(+)) exhibited trans-dominant constitutive expression of beta-galactosidase, regardless of the chromosomal uhp genotype. In contrast, the cloned fusion carrying only uhpR(+) gave glucose 6-phosphate-inducible production of beta-galactosidase that was dependent on the presence of chromosomal uhpA(+). Expression of both fusions in the haploid state was inducible. From these results, it was concluded that the uhpA product is necessary for uhpT transcription and that elevated dosage of uhpA results in at least partially constitutive expression of uhpT. A tentative model for uhp regulation is presented.