Substrate utilization by Legionella cells after cryopreservation in phosphate buffer

Abstract

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloroacetic acid-insoluble fractions from "4C-labeled substrates were determined. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCI or NaCI reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for a-ketoglutarate, succinate, and 'yaminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. a-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source. * Corresponding author. Laboratories, Richmond, Calif.), as specified by the manufacturer. Before the Bio-Rad reagent was added, the cells

Substrate utilization by Campylobacter jejuni and Campylobacter coli

“…The advantages of studying the metabolic activities of campylobacters by the method described in this paper are flexibility and sensitivity. A large number of substrates can be used singly or in combination with unlabeled substrates (23). With due caution, this method can be applied to formate as the substrate.…”

“…The protein content of the cell preparations was determined by the Bio-Rad protein assay procedure (2), as described previously (23).…”

“…These compounds were diluted with their unlabeled counterparts to provide 0.05 ,uCi per tube and a 0.02 M concentration unless otherwise indicated. The formation of CO2 was determined, as previously described (23), with tubes (16 by 100 mm) containing final volumes of 0.4 ml. The tubes were closed with rubber stoppers from which were suspended plastic cups containing filter paper wicks.…”

The life stage‐specific pathometabolism of Legionella pneumophila

“…Early (radiotracer) experiments revealed that amino acids serve as the main carbon and energy source of L. pneumophila when growing in vitro in different media . From these experiments, Ser, Thr, Glu and Tyr were identified as preferred substrates in vitro , and Cys, Gln, Ser, Met, Val, Glu, Tyr, Thr and Arg were shown to support growth in vivo . In addition, the identification of high activity of Glu‐Asp transaminase in lysates of in vitro grown bacteria suggested that this activity is involved in the linkage of the TCA cycle to gluconeogenesis and anabolic pathways .…”

“…Various experimental studies and the analysis of various genome sequences revealed that L. pneumophila is auxotrophic for Arg, Cys, Ile, Leu, Met, Thr and Val, and the amino acids Arg, Cys, Ile, Leu, Met, (Phe), Ser, Thr, (Tyr) and Val are essential for growth . Cysteine is absolutely required for the growth of legionellae, and media must be supplemented with cysteine to support growth of L. pneumophila , but this is not true for L. oakridgensis , a less virulent Legionella species .…”