Cell envelope proteins involved in the transport of maltose and sn-glycerol-3-phosphate in Escherichia coli

Two types of proteins are discussed in their role of facilitating the transport of maltose and sn-glycerol-3-phosphate in E. coli. The first protein is the receptor for phage δ, known to be an outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the δ receptor is to decrease the K_m of the transport system without influencing the V_max of substrate flux. The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn-glycerol-3-phosphate in whole cells but not in membrane vesicles. This protein has solely been identified by the use of a two-dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild-type and mutants defective in sn-glycerol-3-phosphate transport.     

Identification of sn-Glycerol-1-phosphate Dehydrogenase Activity from Genomic Information on a Hyperthermophilic Archaeon,Sulfolobus tokodaii Strain 7

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.     

Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC)

Summary An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).     

Characteristics of a Binding Protein-Dependent Transport System for sn-Glycerol-3-Phosphate in Escherichia coli That Is Part of the pho Regulon

The ugp-dependent transport system for sn-glycerol-3-phosphate has been characterized. The system is induced under conditions of phosphate starvation and in mutants that are constitutive for the pho regulon. The system does not operate in membrane vesicles and is highly sensitive toward osmotic shock. The participation of a periplasmic binding protein in the transport process can be deduced from the isolation of transport mutants that lack the binding protein. As with other binding protein-dependent transport systems, this protein appears to be necessary but not sufficient for transport activity. The isolation of mutants has become possible by selection for resistance against the toxic analog 3,4-dihydroxybutyl-1-phosphonate that is transported by the system. sn-Glycerol-3-phosphate transported via ugp cannot be used as the sole carbon source. Strains have been constructed that lack alkaline phosphatase and glycerol kinase. In addition, they are constitutive for the glp regulon and contain high levels of glycerol-3-phosphate dehydrogenase. Despite the fact that these strains exhibit high ugp-dependent transport activity for sn-glycerol-3-phosphate they are unable to grow on it as a sole source of carbon. However, when cells are grown on an alternate carbon source, ¹⁴C label from [¹⁴C]sn-glycerol-3-phosphate appears in phospholipids as well as in trichloroacetic acid-precipitable material. The incorporation of ¹⁴C label is strongly reduced when sn-glycerol-3-phosphate is the only carbon source. In the presence of an alternate carbon source, this inhibition is relieved, and sn-glycerol-3-phosphate transported by ugp can be used as the sole source of phosphate.     

Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44°C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42 543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.     

Co-Regulation in Escherichia coli of a Novel Transport System for sn-Glycerol-3-Phosphate and Outer Membrane Protein Ic (e, E) with Alkaline Phosphatase and Phosphate-Binding Protein

Mutants constitutive for the novel outer membrane protein Ic (e or E) contained a recently discovered binding protein for sn-glycerol-3-phosphate. The corresponding parental strains missing the outer membrane protein Ic (e, E) were negative or strongly reduced in the synthesis of the binding protein. In addition, strains that were previously isolated as mutants constitutive for the sn-glycerol-3-phosphate transport system (ugp⁺ mutants) and that produced the novel periplasmic proteins GP1 to GP4 also synthesized a new outer membrane protein with the same electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as protein Ic. Screening of different ugp⁺ mutants revealed the existence of three types in respect to the four novel periplasmic proteins GP1, -2, -3, and -4: (i) one containing all four proteins; (ii) one containing only proteins GP1, -2, and -3; (iii) one containing only proteins GP1, -2, and -4. In confirmation of the data presented in the accompanying paper by Tommassen and Lugtenberg (J. Bacteriol. 143:151–157, 1980), we found that purified GP1 is identical to alkaline phosphatase, whereas purified GP3 has binding activity of inorganic phosphate and is identical to the phosphate-binding protein. Moreover, growth conditions that lead in a wild-type strain to the derepression of alkaline phosphatase synthesis also derepressed the synthesis of the sn-glycerol-3-phosphate-binding protein as well as the corresponding transport system. Thus, the new sn-glycerol-3-phosphate transport system is part of the alkaline phosphatase regulatory system.     

Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary Using a novel positive selection method for G3P transport activity, phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.Abbreviations DHBP 3,4-dihydroxibutyl-1-phosphonate - G3P sn-glycerol-3-phosphate - G3PBP glycerol-3-phosphate binding protein - IPTG isopropyl--d-thiogalactopyranoside - XG 5-bromo-4-chloro-3-indolyl--d-galactopyranoside     

Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically.Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute.The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic- spectrophotometric procedure.An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.Abbreviations GPC sn-glycerol-3-phosphorylcholine - GPE sn-glycerol-3-phosphorylethanolamine - GPS sn-glycerol-3-phosphorylserine - GPA sn-glycerol-3-phosphoric acid - PDE glycerophosphorylcholine-phosphodiesterase - GPA-Ox glycerophosphate oxidase - Cho-Ox choline oxidase     

The utilisation of d-galactonate and d-2-oxo-3-deoxygalactonate by Escherichia coli K-12

1. Escherichia coli K-12 mutants unable to grow on d-galactonate have been isolated and found to be defective in either galactonate dehydratase, 2-oxo-3-deoxygalactonate 6-phosphate aldolase or devoid of both of these enzymes and of 2-oxo-3-deoxygalactonate kinase.
2. 2-Oxo-3-deoxygalactonate kinase and 2-oxo-3-deoxygalactonate 6-phosphate aldolase are still induced by galactonate in mutants lacking galactonate dehydratase, suggesting that galactonate rather than a catabolic product of galactonate is the inducer of the galactonate catabolic enzymes. Synthesis of the enzymes is subject to glucose catabolite repression.
3. Mutants defective in 2-oxo-3-deoxygalactonate 6-phosphate aldolase accumulate 2-oxo-3-deoxygalactonate 6-phosphate when exposed to galactonate and this compound causes general growth inhibition.
4. Secondary mutants that no longer show this inhibition fail to make 2-oxo-3-deoxygalactonate 6-phosphate due to additional defects in galactonate transport, galactonate dehydratase, 2-oxo-3-deoxygalactonate kinase or a putative promoter mutation that prevents formation of these enzymes.
5. A spontaneous mutant capable of growth on 2-oxo-3-deoxygalactonate has been isolated. It has two genetically distinct mutations. One permits constitutive formation of the galactonate catabolic enzymes and the other allows the uptake of 2-oxo-3-deoxygalactonate. Neither mutation on its own permitted growth on 2-oxo-3-deoxygalactonate.
6. Genes specifying the various galactonate catabolic enzymes have been located at min 81.7 on the E. coli K-12 linkage map and probably constitute an operon. The gene sequence in this region was shown to by: pyrE uhp dgo dnaA.

     

Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport

Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281–4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.     

Coupling of ethanol metabolism to lipid biosynthesis: labelling of the glycerol moieties of sn-glycerol-3-phosphate,a phosphatidic acid and a phosphatidylcholine in liver of rats given [1,1-2H2]ethanol

The mechanism behind ethanol-induced fatty liver was investigated by administration of [1,1-²H₂]ethanol to rats and analysis of intermediates in lipid biosynthesis. Phosphatidic acid and phosphatidylcholine were isolated by chromatography on a lipophilic anion exchanger and molecular species were isolated by high-performance liquid chromatography in a non-aqueous system. The glycerol moieties of palmitoyl-linoleoylphosphatidic acid, the corresponding phosphatidylcholine and free sn-glycerol-3-phosphate were analysed by GC/MS of methyl ester t-butyldimethylsilyl derivatives. The deuterium labelling in the glycerol moiety of the phosphatidic acid was 2–3-times higher than in free sn-glycerol-3-phosphate, indicating that a specific pool of sn-glycerol-3-phosphate was used for the synthesis of phosphatidic acid in liver. The results indicate that NADH formed during ethanol oxidation is used in the formation of a pool of sn-glycerol-3-phosphate that gives rise to triacylglycerol and possibly fatty liver.     

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D₂O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (R_i) of 3 the lamellar gel (L or L_c)-to-hexagonal type II (H_II) phase sequence is observed on increasing the temperature. For R_i3 the thermotropic sequence (L or L_c)-L-H_II is detected. On increasing hydration from R_i=3, the H_II phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and H_II phases are R_i 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the H_II phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - T_c lamellar gel-to-lamellar fluid transition temperature - T_h lamellar fluid-to-hexagonal transition temperature     

Isolation and mapping of Escherichia coli K12 mutants defective in Tn9 transposition

Summary Five mutants (called tnm) of Escherichia coli with impaired ability for transposition of Tn9 were isolated after treatment with ethyl methanesulfonate (EMS) or N-methyl-N-nitro-N-nitrosoguanidine (NG).The map locations of the tnm mutations were deterimined by a combination of Hfr matings, F episome complementation and P1 transductional mapping. The data obtained show that the five tnm mutations are located near 91 min on the Escherichia coli linkage map and are cotransducible with the metA marker with a frequency of 3%–4%. Introduction of F plasmids containing this region complements the Tnm^- phenotype for the two mutants tested i.e. tnm-1 and tnm-2 are recessive in tnm ⁺/tnm^-merodiploids.     

Mutants of Escherichia coli with temperature-sensitive lesions in membrane phospholipid synthesis: genetic analysis of glycerol-3-phosphate acyltransferase mutants

Summary Escherichia coli mutants possessing temperature-sensitive lesions in glycerol-3-phosphate acyltransferase, the enzyme catalysing the first step in phospholipid biosynthesis, have been characterized genetically. By recombinational and complementation tests, the mutants have been found to map in a single locus, called plsA, which is cotransducibile with the purE locus and lies between the purE and proC loci at minute 13 on the E. coli genetic map.     

Did Archaeal and Bacterial Cells Arise Independently from Noncellular Precursors? A Hypothesis Stating That the Advent of Membrane Phospholipid with Enantiomeric Glycerophosphate Backbones Caused the Separation of the Two Lines of Descent

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively. Received: 24 March 1997 / Accepted: 21 May 1997     

Mapping of chlorate-resistant mutants of Citrobacter freundii by deletion and complementation analysis

Summary From Citrobacter freundii mutants have been isolated, with deletions extending chl genes. 53% of these mutants mapped in the gal-bio region of the chromosome. Genetic mapping by three methods—deletion analysis, linkage analysis in crosses with C. freundii Hfr donors and complementation with Escherichia coli F factors—establishes the gene order: chl H-gal-chl D-hut-bio-uvr B-chl A-chl I-chl E In an other segment a gene order ilv-chl-pdx was found. Furthermore chl loci were found adjacent to 7 different chromosomal markers. C. freundii can form nitrate reductase A, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. These enzymes are not formed in most of the chlorate-resistant mutants. In some of these mutants the enzyme activities can be restored by complementation with F factors of E. coli.     

The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa

Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10⁻⁶. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10⁻¹¹). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.Pseudomonas aeruginosa is an opportunistic, life-threatening bacterial pathogen that especially affects critically ill patients in intensive care units or those suffering from chronic respiratory diseases such as cystic fibrosis (19, 40). Its 6.3-Mb genome supports its enormous metabolic versatility and, consequently, its adaptability to almost any challenging environment. One of the consequences of this versatility is the rapid adaptation to stressful environmental conditions, including starvation, desiccation, and antibiotic treatments (14, 40). Mutants resistant to one or several antibiotics will evolve during sufficiently prolonged treatments, this being a process facilitated by the presence of hypermutable alleles (31, 32). After years of treating cystic fibrosis patients with antibiotics, P. aeruginosa became unavoidably resistant to many or all of them (5). Multidrug-resistant strains of P. aeruginosa are an important problem for the treatment of nosocomial outbreaks and cystic fibrosis patients (27, 37). Currently, the treatment of multidrug-resistant P. aeruginosa requires the combination of various antimicrobial agents. Fosfomycin (Fos) has been reported to be effective in combination with other antipseudomonal agents (6, 29, 42, 44). The proportion of Fos-resistant (Fos-R) strains in clinical isolates of P. aeruginosa currently is not well known, and even the mechanisms that support Fos resistance in P. aeruginosa are not clear. Thus, the knowledge of the molecular bases involved in the development of spontaneous Fos resistance in P. aeruginosa is of particular interest.Fos is a unique broad-spectrum bactericidal antibiotic that is chemically unrelated to any other known antimicrobial agent used to treat urinary tract and gastrointestinal infections in humans (9, 35). It binds UDP-GlcNAc enol-pyruvyltransferase (MurA), acting as a phosphoenolpyruvate analogue and avoiding the formation of UDP-N-acetylglucosamine-3-O-enolpyruvate from UDP-N-acetylglucosamine and phosphoenolpyruvate (12, 33). Fos is taken up actively into bacterial cells via transport systems. In Escherichia coli, Fos is imported through two nutrient transport systems, the glycerol-3-phosphate (glycerol-3-P) transporter (GlpT) and glucose-6-phosphate (glucose-6-P) transporter (UhpT), to achieve its target and inhibits the initial step in cell wall synthesis (12, 17). The expression of these transport systems is induced by their substrates (glycerol-3-P and glucose-6P) and requires the presence of the cyclic AMP receptor protein (cAMP-CRP) complex (23, 30). Additionally, the high-level expression of UhpT requires the regulatory genes uhpA, uhpB, and uhpC (12, 30). Therefore, Fos-R strains isolated in E. coli contain mutations that prevent Fos transport using GlpT or UhpT (23, 30). Plasmid-encoded resistance also has been described previously (4, 41).In this paper, we describe the screening and analysis of Fos-R clones in a P. aeruginosa PA14 ordered insertional library (18). In addition, we studied the mutations responsible for the spontaneous resistance to Fos in P. aeruginosa PA14, the effect of these mutations on the in vitro growth rate, and the uniqueness of the mutational target.     

Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12. Identification of the pit+ gene product and physical mapping of the pit-gor region of the chromosome

Summary The pit ⁺ gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E. coli genes inserted in the cosmid vector pHC79. A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase. Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region. Subcloning and deletion analysis indicated that the entire pit ⁺ gene was located within a 2.2-kb Sal1-Ava1 fragment. The pit ⁺ gene product was identified by SDS-polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: (i) ³⁵S-methionine-labelling of minicells carrying pit ⁺ plasmids or plasmids from which all or part of the pit ⁺ gene was deleted. (ii) Overproduction of the Pit protein using a thermoinducible runaway replication plasmid.Complementation of the pit-1 mutant allele using a unit-copy-number pit ⁺ plasmid indicated that the pit-1 mutation is recessive.Strains carrying a multicopy pit ⁺ plasmid show a 10-fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of ³²Pi accumulation.Abbreviations kb kilobase-pairs - kdal kilodalton - Pi inorganic phosphate - G3P sn-glycerol-3-phosphate - LB Luria broth - Tc tetracycline - Cm chloramphenicol - Ap ampicillin - UV ultraviolet light - TE 10 mM Tris.HCl, pH 8.0, 1 mM EDTA - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid