Diaminopimelic Acid and Lysine Auxotrophs of Pseudomonas aeruginosa 8602

Clarkson, Carolyn E.; Meadow, Pauline M.

doi:10.1099/00221287-66-2-161

Cited by 13 publications

(10 citation statements)

References 21 publications

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“…All cultures were grown in 1 1 minimal medium with 0.5 "/b (w/v) trisodium citrate. 2H20 as carbon source (Clarkson & Meadow, 1971) in 5 1 conical flasks at 37 "C with shaking. They were harvested in mid-exponential phase 1.0) by centrifugation (10000 g, 10 min), washed with 50 mM-Tris/HCl, 7 mM-2-mercaptoethano1, pH 8 (TM buffer) and stored as a frozen pellet until required.…”

Section: E T H O D Smentioning

confidence: 99%

Biosynthesis of the Core Part of the Lipopolysaccharide of Pseudomonas aeruginosa

Asonganyi

Meadow

1980

Microbiology

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The incorporation of rhamnose and glucose into the core part of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa was studied using enzyme preparations from strain PAC1 R and LPS-defective mutants derived from it. Crude membrane preparations from the LPS-defective mutant PAC556 transferred rhamnose from dTDP-L-[14C]rhamnose to material insoluble in trichloroacetic acid. The preparations contained both transferase enzyme and acceptor, the former being destroyed by heating. Between 60 and 70% of the radioactive rhamnose transferred to the membranes was extractable by aqueous phenol and non-diffusible. The material extracted did not move in any of the chromatography solvents tested and contained rhamnose as the sole radioactive component. Soluble dTDP-L-rhamnose-LPS rhamnosyltransferase was obtained from the parent strain PAC 1 R by ammonium sulphate precipitation of a 105000 g supernatant fraction from broken bacteria. It was most active at pH 8 with 5 mM-MgC1, and required heat-treated membranes of PAC556 as acceptor. This mutant, whose LPS lacks both 0-antigenic side-chains and rhamnose in the core, was shown to lack either the epimerase or the NADP-dependent oxidoreductase used to synthesize dTDPrhamnose. After preincubation with soluble transferase and UDPglucose, heated membranes of mutant strains PAC6 I 1, PAC6 12 and PAC605 could also act as acceptors for rhamnose. These mutants all lacked some or all of the glucose as well as the rhamnose from the core of their LPS and the experiments thus provided confirmation that rhamnose was the terminal hexose of the core in P. aeruginosa PAC1.

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Section: E T H O D Smentioning

confidence: 99%

Biosynthesis of the Core Part of the Lipopolysaccharide of Pseudomonas aeruginosa

Asonganyi

Meadow

1980

Microbiology

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“…Diaminopimelate epimerase is not entirely absent, and the mutant can make enough meso-Dap to support growth when this small amount of meso-Dap is used only for peptidoglycan synthesis, and is not needed to serve as a precursor of lysine as well (Clarkson & Meadow, 1971). Mutant PAC7 excreted Dap (solely LL-isomer; about 100pg ml-l) after growth in minimal medium (carbon source succinate) plus L-lysine (40 mg 1-l) at 37 "C (Clarkson & Meadow, 1971).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, the introduction into P A C~ of a further mutation causing inability to form lysine (as a result of the loss of diaminopimelate decarboxylase), might increase the yield of LL-Dap. However, Clarkson & Meadow (1971) found that mutants of P. aeruginosa 8602 that lacked diaminopimelate decarboxylase had excreted only relatively small amounts of Dap (about 7 mg 1-l) by the beginning of the stationary phase of growth. Furthermore, if conversion of rneso-Dap to lysine could be 260…”

Section: Discussionmentioning

confidence: 97%

“…Lysine synthesis (from succinate) in PAC7 is not completely blocked, and this may be a reason for the relatively low excretion of Dap. Both diaminopimelate epimerase (0.00 I unit/mg protein) and diaminopimelate decarboxylase (0.005 unit/mg protein) were present in an extract of P A C~ (Clarkson & Meadow, 1971). Lysine causes a feedback inhibition of Dap biosynthesis (see Clarke & Ornston, 1975), and excretion of Dap by the mutant begins only when lysine is exhausted from the medium.…”

Section: Discussionmentioning

confidence: 98%

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Use of Auxotrophic Mutants to Isolate LL- or DD-Isomers of 2,6-Diaminopimelic Acid

Saleh¹,

White²

1976

Journal of General Microbiology

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S U M M A R YPseudomonas aeruginosa PAC7 (a mutant deficient in diaminopimelate epimerase), excreted diaminopimelate (solely LL-isomer) after growth in a minimal medium plus lysine with succinate as carbon source. More diaminopimelate was excreted when bacteria were transferred at the end of the exponential phase of growth into fresh minimal medium without lysine but supplemented with pyruvate and additional (NH,),SO,. The excreted LL-isomer was isolated from the culture filtrate by ionexchange chromatography and purified by crystallization ( I -7 g/9 1 culture).A diaminopimelate-requiring mutant of Bacillus megaterium NCIB758 I grew on LL-and/or meso-diaminopimelate but not on the DD-isomer. This mutant was used to isolate the DD-isomer from a mixture of synthetic LL-and DD-diaminopimelate. It was grown in a minimal medium containing glycerol as carbon source and LLplus DD-diaminopimelate at a growth-limiting concentration (300 mg 1-l) ; when growth stopped, the DD-diaminopimelate that remained in the culture was isolated and crystallized (I -0 g/I I I culture). I N T R O D U C T I O N2,6-Diaminopimelic acid (Dap) exists in three stereoisomeric forms : the LL-, DD-and meso-isomers (Work, 1963). Authentic samples of each are required in studies of the isomeric composition of Dap in walls and spores of bacteria.Diaminopimelate synthesized chemically is a mixture of all three isomers : about 50 % meso-, 25 % LL-and 25 % DD-isomer (see later). Diaminopimelate can also be isolated from the culture filtrates of lysine-requiring mutants of Escherichia coli (Work, 1963) ; such Dap is a mixture of about 75 % meso-and 25 % LL-isomers (White & Kelly, 1965). The mesoDap can be separated from the other two isomers by crystallization because it is less soluble in ethanol/water. From the meso-plus LL-Dap mixture obtained by excretion (i.e. 'fermentation Dap '), pure LL-Dap can be recovered after precipitation of meso-Dap. Until recently, commercial Dap was 'fermentation material ' ; but at present, only synthetic Dap can be bought. Removing the meso-isomer from synthetic Dap leaves a mixture of the LL-and DD-isomers, which are difficult to separate. Work et al. (1955) separated the three isomers by preparing their diamides, treating these with a stereospecific amidase (which deaminated groups in the L-configuration only), and then separating the mixture of free LL-Dap, DD-Dap diamide and meso-Dap (D-)monoamide on an ion-exchange column.Free DD-and meso-Dap were regenerated by acid hydrolysis of the amides. A later modification of this method was described by Wade et al. (1957). These methods are relatively laborious and result in low yields of the pure isomers.In this paper, we describe the isolation of LL-Dap from cultures of a mutant of Pseudomonas aeruginosa (called PAC7) which excretes solely the LL-isomer (Clarkson & Meadow,

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“…The method of pyocine typing described by Gillies and Govan was used. In addition to the tryptone soya blood agar medium used by these authors, various other media were tested-a minimal medium (Clarkson and Meadow, 1971) incorporating 0.1 per cent. of glucose, sodium glutamate or sodium succinate as sole carbon source, and also a very simple medium containing L-asparagine (Georgia and Poe,193 1).…”

Section: Methodsmentioning

confidence: 99%

Pyocine Typing of Mucoid Strains of Pseudomonas Aeruginosa Isolated from Children with Cystic Fibrosis

Williams

Govan

1973

Journal of Medical Microbiology

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Diaminopimelic Acid and Lysine Auxotrophs of Pseudomonas aeruginosa 8602

Cited by 13 publications

References 21 publications

Biosynthesis of the Core Part of the Lipopolysaccharide of Pseudomonas aeruginosa

Biosynthesis of the Core Part of the Lipopolysaccharide of Pseudomonas aeruginosa

Use of Auxotrophic Mutants to Isolate LL- or DD-Isomers of 2,6-Diaminopimelic Acid

Pyocine Typing of Mucoid Strains of Pseudomonas Aeruginosa Isolated from Children with Cystic Fibrosis

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