Feb 25, 1984 - Mycoplasma mycoides subsp. mycoides has been shown to possess an unusual ... One of the biosynthetic simplifications in Mycoplasma.
JOURNAL
OF
BACTERIOLOGY, June 1984,
p.
Vol. 158, No. 3
943-947
0021-9193/84/060943-05$02.00/0 Copyright © 1984, American Society for Microbiology
Uptake and Utilization of Deoxynucleoside 5'-Monophosphates by Mycoplasma mycoides subsp. mycoides GEOFFREY A. M. NEALE,* ALANA MITCHELL, AND LLOYD R. FINCH The Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia Received 27 Decemnber 1983/Accepted 25 February 1984
Mycoplasma mycoides subsp. mycoides has been shown to possess an unusual capacity for the uptake and utilization of exogenous deoxyribonucleoside 5'-monophosphates intact without prior dephosphorylation. In this study, it was found that once inside the cell, deoxyribonucleoside 5'-monophosphates were rapidly phosphorylated to the triphosphate level and incorporated into DNA. Catabolism of deoxyribonucleoside 5'-monophosphates was also observed. Competition studies indicated that a single uptake system with a higher affinity for deoxyribonucleotides mediates the uptake of nucleoside 5'-monophosphates.
Mycoplasmas are the smallest self-replicating organisms capable of growth outside host cells and are characterized by their lack of a cell wall. In keeping with their small dimensions, the genome of mycoplasmas is about one-fifth the size of that of Escherichia coli (7). Their complex requirements for growth in vitro (10) suggest that this economy in genome size may be related to a metabolic simplification through the genetic loss of many biochemical functions. One of the biosynthetic simplifications in Mycoplasma mycoides subsp. mycoides is the absence of de novo synthesis of nucleotides (3). Salvage pathways for nucleotide synthesis from the precursors uracil, guanine, and thymine have been defined in this organism (3, 5). Results from one of these studies (5) have given some evidence for the uptake and utilization of intact deoxyribonucleoside 5'-monophosphates (dNMP) as precursors for the synthesis of DNA. To obtain more direct evidence for these phenomena, we incubated cells of M. mycoides with [ot-32P]dNMP and then isolated deoxyribonucleotides and DNA for counting. To investigate the mechanism of this nucleotide salvage more closely, we followed the uptake of labeled dNMP into cells of M. mycoides subsp. mycoides and partially characterized the uptake process by examining the effect of possible competitors on the system. MATERIALS AND METHODS Organism and culture medium. Cultures of M. mycoides subsp. mycoides were grown in medium C4 described by Rodwell (11) and modified as described previously (4). In all experiments, the medium contained adenine, guanine, uracil, and thymine. Chemicals and radiochemicals. [U-14C]dCMP (465 Ci/mol), [U-14C]dTMP (528 Ci/mol), [U-14C]dAMP (518 Ci/mol), [U14C]dGMP (517 Ci/mol), [U-14C]CMP (507 Ci/mol), [a32P]dATP, and [cx-32P]dCTP were obtained from Amersham Corp. Cytidine and thymine were from Merck & Co., Inc., and all other bases, deoxyribonucleosides, deoxyribonucleotides, and 5-fluoro-2'-deoxyuridine 5'-monophosphate (5-fluoro-dUMP) were purchased from Sigma Chemical Co. Apyrase grade 1 and 2-(N-morpholino)ethanesulfonic acid (MES) were also supplied by Sigma. Preparation and purification of [a-32P]dNMP. [oa32P]dNMP were prepared from approximately 4 nmol of [a32P]deoxynucleoside triphosphates (dNTP) by incubation with 24 ,ug of apyrase for 5 min at 37°C in 20 ,ul of 30 mM
MES buffer (pH 6.5) containing 10 mM Pi. The digestion was stopped by heating at 100°C for 10 min, and the [a-32P]dNMP were separated from their respective triphosphates, diphosphates, and Pi by ascending chromatography on Whatman DEAE-cellulose paper (1.2 by 10 cm) with 1 M HCOOH. After location by autoradiography, the [a-32P]dNMP were eluted from the cellulose paper and lyophilized. Before incubation with cells, the [o-32P]dNMP were redissolved in 10 ,ul of 0.2 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (pH 7.8). Labeling of cellular dNTP and DNA with [Ia-32P]dNMP. At time zero, 0.2 ml of logarithmic-phase cultures (approximately 16 ,ug of cellular protein) was mixed with a radioactive dNMP at a final concentration of approximately 5 FLM and maintained at 37°C. After 1 min, 0.1 ml of cell suspension was removed for the extraction and chromatographic isolation of dNTP (5, 8). After an additional 4 min, the remaining cells were treated for the extraction and isolation of dNMP from DNA (5). Measurement of uptake of dNMP into whole cells. At time zero, 0.5 ml of logarithmic-phase cultures (approximately 40 ,ug of cellular protein) was added to the radioactive dNMP substrate or, where indicated, to the substrate mixed with a possible competitor. Uptake of the labeled substrate into the cell was measured by sampling at 10-s intervals over a period of 40 s. Samples of 50 ,ul of the cell suspension were taken into 2 ml of mycoplasma wash buffer (4) at 37°C above a Millipore filter (pore size, 0.22 p.m) in a filtration apparatus. The suspensions were filtered immediately under suction, and the retained cells were washed with an additional 2 ml of the wash buffer (37°C). The filtration time of the diluted sample and of the wash was 1 to 2 s. The wash buffer contained unlabeled dNMP at 1 mM. The filters were dried for 30 to 45 min at 70°C before counting by liquid scintillation spectrometry. RESULTS Fate of [a-32P]dNMP added to cultures of M. mycoides. To confirm previous indications (5) that dNMP from the extracellular medium are used in the cell for the synthesis of DNA, in preliminary experiments the fate of 32P-labeled dNMP added to cultures has been examined. Cells were incubated briefly with [a-32P]dAMP or [_-32P]dCMP and sampled for the extraction and isolation of deoxyribonucleotides (8) and for the isolation of dNMP from DNA (5). In Fig. 1 is shown an autoradiograph of the 32P-labeled derivatives
* Corresponding author. 943
944
NEALE, MITCHELL, AND FINCH
J. BACTERIOL.
TABLE 2. 32P radioactivity associated with deoxyribonucleotides isolated from DNA' Deoxyribonucleotide added to cells
5 cm
7 -o
I%
f- J 5
%_, f _.
-
%
:6
- 2-1 _3
I_,
I
1
- --------- 1st DIM. FIG. 1. Autoradiograph of a two-dimensional chromatogram of acid-soluble derivatives extracted from cells of M. mycoides incubated with [a-32P]dAMP. Spot 1, origin; spot 2, dGTP; spot 3, dATP; spot 4, dCTP; spot 5, dTTP; spot 6, dADP; spot 7, unidentified; spot 8, Pi; spot 9, dAMP. Dim, Dimension.
extracted from the culture incubated with [ao-32P]dAMP and separated by thin-layer chromatography after treatment with sodium periodate to destroy ribonucleotides. Of the dNTP, appreciable radioactivity was detected only in dATP (Table 1). Appreciable labeling also occurred for dADP. Major amounts of label were found in Pi and at the origin at which oligonucleotide fragments and oxidation products of ribonucleotides remained. An unidentified spot (Fig. 1, number 7) TABLE 1. 32P radioactivity associated with deoxyribonucleotides extracted from M. mycoidesa Sp act (pmol of 32p/g of protein) for the following dNMP of exNucleotide Spot nob tracted compound added to cells 1 2 3 4 S 6 7 8 9
Origin dGTP dATP dCTP dTTP dADP Unidentified
Pi
[a--32PjdAMP
[a-32P]dCMP
1.27 0.008 0.390 0.016 0.012
0.446
