Mapping of a genetic locus that affects glycerol 3-phosphate transport in Bacillus subtilis.

Two types of fosfomycin-resistant mutants of Bacillus subtilis were isolated. Mutants of the first type (GlpT mutants) were resistant to at least 200 microgram of fosfomycin per ml and failed to take up exogenous glycerol 3-phosphate. Mutants of the second type were resistant to lower concentrations of fosfomycin and transported glycerol-3-phosphate as efficiently as wild-type bacteria. The glpT mutations, but not the mutations in the second type of fosfomycin-resistant mutants, map in the cysA-aroI region of the B. subtilis chromosome.     

sn-Glycerol-3-phosphate transport in Salmonella typhimurium

Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased.     

A second transport system for sn-glycerol-3-phosphate in Escherichia coli.

Strains containing phage Mucts inserted into glpT were isolated as fosfomycin-resistant clones. These mutants did not transport sn-glycerol-3-phosphate, and they lacked GLPT, a protein previously shown to be a product of the glpT operon. By plating these mutants on sn-glycerol-3-phosphate at 43 degrees C, we isolated revertants that regained the capacity to grow on G3P. Most of these revertants did not map in glpT and did not regain GLPT. These revertants exhibited a highly efficient uptake system for sn-glycerol-3-phosphate within an apparent Km of 5 micron. In addition, three new proteins (GP 1, 2, and 3) appeared in the periplasm of these revertants. None of these proteins were antigentically related to GLPT. However, like GLPT, GP1 exhibits abnormal behavior on sodium dodecyl sulfate-polyacrylamide gels. GP 2 is an efficient binding protein. The new uptake system showed different characteristics than the system that is coded for by the glpT operon. It was inhibited neither by phosphate nor fosfomycin. So far, none of the systems that transport organic acids in Escherichia coli could be implicated in the new sn-glycerol-3-phosphate uptake activity. The mutation ugp+, which was responsible for the appearance of the new transport system and the appearance of GP 1, 2, and 3 in the periplasm was cotransducible with araD by phage P1 transduction and was recessive in merodiploids.     

Map locations and functions of Salmonella typhimurium men genes

Menaquinone (men) mutants of Salmonella typhimurium isolated on the basis of their inability to produce trimethylamine were characterized with respect to mutation site, the ability to cross-feed each other and be cross-fed by known Escherichia coli men mutants, and response to intermediates of the menaquinone biosynthetic pathway. Cross-feeding tests were based on the requirement of menaquinone for hydrogen sulfide production. Genotypes corresponding to the menA, B, C, D, and possibly E genes described in E. coli were all identified. Additional studies of deletions in the menBCD area revealed that this cluster lies between ack/pta and glpT, as in E. coli. The ack and pta mutants were also defective in the production of trimethylamine and failed to produce gas in the absence of added formate.     

Mutants of Escherichia coli K-12 Blocked in the Final Reaction of Ubiquinone Biosynthesis: Characterization and Genetic Analysis

The ubiquinone precursor 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1, 4-benzoquinone was isolated from two ubiquinone-deficient mutants of Escherichia coli and identified by nuclear magnetic resonance and mass spectrometry. The results of genetic analysis of the mutants indicate that each mutant carries a mutation in a gene designated ubiG which was located, by cotransduction with the nalA and glpT genes, at minute 42 on the E. coli chromosome.     

Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants.

Fifteen independent menaquinone biosynthesis mutants (men) of Escherichia coli K12, selected for their inability to use fumarate as terminal electron acceptor, were investigated. Two nutritionally distinct groups were detected. The major group (13 mutants) responded to 1,4-dihydroxy-2-naphthoate (DHN), 2-succinylbenzoate (SB) and its dilactone, whereas the minor group (2 mutants) only responded to DHN. DHN was at least five times more effective than SB but it inhibited growth at concentrations greater than 10 microM. For anaerobic growth on glucose minimal medium the auxotrophs responded to much lower concentrations of DHN and SB and these intermediates could be replaced by uracil. Anaerobic growth tests showed that glycerol, formate and H2 are good substrates for E. coli when fumarate is the ultimate electron acceptor but growth with lactate or with fumarate alone is poor. All 15 men mutations were located between glpT and purF at approximately 49 min in the E. coli linkage map. Cotransduction frequencies with relevant markers were: nalA (21%), glpT (35%) and purF (15%). The presence of at least three genetically distinct classes (menC and menD, SB-requirers; menB, DHN-requirers) was indicated using abortive transduction as a complementation test and three-factor genetic analysis. The relative orientation nalA...menC-(D,B)...purF was indicated. Fluoroacetate-resistant mutants were isolated and four different classes were identified: ack, lacking acetate kinase; pta, lacking phosphotransacetylase; facA, lacking both of these activities; and facB, which retained both of these enzyme activities. Some of the pta mutants and all of the facA mutants failed to grow on media containing fumarate as terminal electron acceptor or anaerobically on glucose minimal medium. All four types had genetic lesions clustered between the men and purF sites. Average cotransduction frequencies with relevant markers were: nalA (4%), men (27 to 35%) and purF (71 to 80%).     

Menaquinone biosynthesis: mutants of Escherichia coli K-12 requiring 2-succinylbenzoate.

Two independent mutants of Escherichia coli K-12, selected for their inability to grow anaerobically with fumarate as the terminal electron acceptor, were shown to be deficient in menaquinone biosynthesis. In both cases, exogenously supplied 2-succinylbenzoate promoted normal anaerobic growth on a lactate plus fumarate medium. Anaerobic growth of the mutants on glucose minimal medium was impaired but could be restored to normal by adding either uracil or 2-succinylbenzoate. The addition of 2-succinylbenzoate (but not uracil) permitted the synthesis of menaquinone and demethylmenaquinone by both mutants. The menaquinone content of the parental strain grown on lactate plus fumarate was three times greater than observed after growth on glucose. Transduction studies with phage P1 showed that the two mutations are very closely linked and probably affect the same gene, menC, which is cotransducible with nalA (23%), glpT (51%), and purF (8 to 14%). The gene order nalA-nrdA-glpTA-menC-purF was indicated. The results were consistent with 2-succinylbenzoate being an intermediate in menaquinone biosynthesis and show that the gene designated menC (located at 48.65 min of the E. coli chromosome) is involved in the conversion of chorismate to 2-succinylbenzoate. It was also concluded that menaquinone is essential for electron transport to fumarate in E. coli.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

Properties of cysK mutants of Escherichia coli K12.

Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed.     

In vitro and in vivo functional activity of Chlamydia MurA,a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance

Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.     

Use of Escherichia coli operon-fusion strains for the study of glycerol 3-phosphate transport activity.

Strains of Escherichia coli K-12 deleted in the native lac operon and bearing both a wild-type glpT operon encoding for sn-glycerol 3-phosphate (G3P) transport and a hybrid operon in which glpT operator and promoter regions are fused to the lacZ gene were constructed. In strains with such a hybrid operon, beta-galactosidase and beta-galactoside permease become inducible by G3P. In these mutants the function and maturation of the glpT-coded proteins should be distinguishable from the level of gene expression, since the beta-galactosidase activity can serve as an index of the latter. With the aid of such mutants, it was shown that: (i) the expressions of the two neighboring operons, glpT and glpA (encoding anaerobic G3P dehydrogenase), are not coordinate; (ii) upon induction, the appearance of the cytoplasmic beta-galactosidase activity preceded that of methyl-beta-D-thiogalactoside transport activity (requiring only a cytoplasmic membrane protein) by about 4 min and that of G3P transport activity (requiring both a cytoplasmic membrane protein and a periplasmic protein) by about 9 min; and (iii) when cells grown at several temperatures from 24 to 42 degrees C were measured for G3P transport activity at 30 degrees C, the activity increased with the growth temperature, indicating that, within the range studied, the rate of transport increases with the fluidity of membrane phospholipids.     

Inhibition of lateral wall elongation by mecillinam stimulates cell division in certain cell division conditional mutants of Escherichia coli.

The effect of mecillinam, a beta-lactam antibiotic that specifically binds penicillin-binding protein 2 of Escherichia coli, causes transition from rod to coccal shape, and inhibits cell division in sensitive cells, has been tested on three different E. coli temperature-sensitive cell division mutants. At the nonpermissive temperature, the antibiotic allows an increase in cell number for strains BUG6 and AX655 but not for AX621. In strain AX655, the cell division stimulation was observed only if the antibiotic was added immediately after shifting to the nonpermissive temperature, whereas in BUG6, the rise in cell number was observed also when mecillinam was added after 90 min of incubation at the nonpermissive temperature. In all cases, cell division began occurring 30 min after addition of the antibiotic. Mecillinam had no effect on division of dnaA, dnaB temperature-sensitive mutants or on division of BUG6 derivatives made resistant to this antibiotic. Other beta-lactam antibiotics such as penicillin, ampicillin, cephalexin, and piperacillin and non beta-lactam antibiotics such as fosfomycin, teichomycin, and vancomycin that inhibit cell wall synthesis did not show any effect on cell division for any of the mutants. The response of the three cell division mutants to mecillinam is interpreted in terms of a recently proposed model for shape regulation in bacteria.     

Fosfomycin causes transient lysis in Escherichia coli strains carrying fosfomycin-resistance plasmids

Escherichia coli cells carrying fosfomycin-resistance plasmids show high levels of resistance towards this drug. However, the plasmid-carrying strains exhibited a transient lytic phase induced by fosfomycin when grown in rich liquid media. This lytic phase was not observed if the cells were grown in liquid minimal media. Fosfomycin-induced lysis depended on the accumulation of drug inside the bacteria, presumably as a result of the saturation of the fosfomycin modification system. Growth recovery after lysis was not due to drug inactivation in the culture medium and could be explained by selection of mutants showing impaired fosfomycin transport when high concentrations of fosfomycin were used. However, there was no selection of mutants with low drug concentrations.     

Roles for menaquinone and the two trimethylamine oxide (TMAO) reductases in TMAO respiration in Salmonella typhimurium: Mu d(Apr lac) insertion mutations in men and tor.

Three groups of mutants defective in trimethylamine oxide (TMAO) reduction were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). Mutants were identified by their acidic reaction on a modified MacConkey-TMAO medium. Group I consisted of pleiotropic chlorate-resistant mutants which were devoid of TMAO reductase activity. None expressed the lac operon. Group II mutants were partially defective in TMAO reductase. Electrophoretic studies revealed that they lacked the inducible TMAO reductase, but retained the constitutive activity. The genotypic designation tor was suggested for these mutants. The tor mutation in one was located between 80 and 83 U on the S. typhimurium chromosome. Expression of the lac operon in these mutants was not affected by air, TMAO, or nitrate. Group III mutants reduced little or no TMAO in vivo, but their extracts retained full capacity to reduce it with methyl viologen. These mutants also failed to produce hydrogen sulfide from thiosulfate and could not grow anaerobically on glycerol-fumarate. Two subgroups were distinguished. Vitamin K5 restored wild-type phenotype in subgroup IIIa only; vitamin K1 restored wild-type phenotype in both IIIa and IIIb isolates. The genotypic designation men (menaquinone) was suggested for group III isolates. The mutation in IIIa mutants was cotransducible with glpT, which corresponds to the menBCD site in Escherichia coli. That in IIIb mutants was cotransducible with glpK, which corresponds to the menA site in E. coli. Expression of the lac operon in IIIa, but not IIIb, mutants was repressed by air. An additional mutant group isolated on the same medium consisted of strains defective in formate hydrogenlyase.     

A second target of the antimalarial and antibacterial agent fosmidomycin revealed by cellular metabolic profiling

Antimicrobial drug resistance is an urgent problem in the control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies for the treatment of malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and Escherichia coli bacteria: in both organisms, metabolic profiling demonstrated a block of isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent for blocking non-mevalonate isoprenoid metabolism and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.     

Isolation of Escherichia coli K-12 mutants that affect the development of phage phi m173 (imm phi 80)

Escherichia coli mutants which block the development of a number of lambdoid phages, particularly, phi m173 and phi 80 were selected. These mutants have different phenotypes, being resistant to different groups of lambdoid phages. There are also differences between new mutants and gro mutants of E. coli studied earlier. The results obtained support the suggestion that no replication of different lambdoid phages takes place in these mutants.     

Antibiotic resistant mutants of Escherichia coli K12 show increases in heterologous gene expression

Using a variety of antibiotics, it was found that nine separate isolates of spontaneous antibiotic resistant mutants of Escherichia coli K12 pPSX-vioABCDE overproduce the anti-tumour antibiotic violacein. Subsequent analysis showed that seven of these mutations occurred on the plasmid pPSX-vioABCDE. The other two overproducing strains carried spontaneous chromosomal mutations to lincomycin and kanamycin. The kanamycin resistant mutant of E. coli K12 DH10B (AA23) and a lincomycin resistant mutant of E. coli K12 LE392 (AA24) increased the synthesis of violacein. The plasmid pPSX-vioABCDE opv-1 contains a violacein over-production (opv-1) mutation which when introduced into either E. coli K12 AA23 or AA24, resulted in a hyper-production of violacein. Remarkably, E. coli K12 AA23 pPSX-vioABCDE opv-1 produced 41 times the normal level of violacein. In addition, both E. coli K12 AA23 and E. coli K12 AA24 demonstrated an increase in expression of an alpha amylase gene from Streptomyces lividans and the urease gene cluster from Klebsiella oxytoca. These results suggest that selection of antibiotic resistant mutants can increase heterologous gene expression in E. coli K12. Additionally, the increased expression is a general effect applicable to genes and gene clusters cloned into E. coli K12 from both Gram-positive and Gram-negative bacteria.     

Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli.

Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon. Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts. Other mutations exhibited no apparent effect of GLPT. Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA. Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT. This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport. Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far. However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate.