Download Metabolism of Mollicutes: the Embden-Meyerhof

Journal of General Microbiology (1989), 135, 683-691.
Printed in Great Britain
68 3
Metabolism of Mollicutes: the Embden-Meyerhof-Parnas Pathway and the
Hexose Monophosphate Shunt
By D A V I D D E S A N T I S , ' V I C T O R V . T R Y O N * A N D J . D E N N I S P O L L A C K 1 *
Department of Medical Microbiology and Immunology, The Ohio State University, Columbus,
Ohio 43210, U S A
Department of Microbiology, University of Texas Health Science Center, San Antonio,
Texas 78284, USA
(Received I0 May 1988; revised 3 November 1988; accepted 9 November 1988)
Cell-free extracts of eighteen fermentative and nonfermentative Mollicutes were examined for
enzyme activities associated with the hexose monophosphate shunt (HMS) and EmbdenMeyerhof-Parnas (EMP) pathway. All Acholeplasma spp. had glucose-6-phosphate (G6P)
dehydrogenase (EC 1.1.1.49), 6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44) and
hexokinase (EC 2.7.1 . 1) activity, Of these three enzyme activities, hexokinase was also
detected in Mycoplasma sp. Let. 1 but in no other fermentative or nonfermentative Mycoplasma
spp. The Acholeplasma and fermentative Mycoplasma spp. possessed all other HMS and EMP
activities examined. All Acholeplasma spp. possessed a pyrophosphate (PPi)-dependent
phosphofructokinase (PFK) (EC 2.7.1 .90) while fermentative Mycoplasma spp. possessed an
ATP-dependent PFK (EC 2 . 7 , l . 11). Transaldolase (EC 2.2.1.2) activity was detected in
some, but not all Acholeplasma and fermentative Mycoplasma spp. 2-Deoxyribose-5-phosphate
aldolase (EC 4.1 .2.4) activity was present in all mollicute extracts tested except for
Mycoplasma gallisepticum and Mycoplasma sp. Let. 1. The two nonfermentative Mycoplasma
spp. lacked all enzyme activities of the HMS pathway except for ribulose-5-phosphate
epimerase activity, and of the EMP pathway only phosphoglucose isomerase and the enzymes
converting glyceraldehyde 3-phosphate (G3P) to phosphoenolpyruvate (PEP) were detected.
We believe that the three major observations of this study are : (1) all Mycoplasma spp. lack G6P
and 6PG dehydrogenase activities, suggesting a reduction in their NADPH pool, which may
relate to the lipid growth requirement of this genus; (2) the fermentative Mycoplasma spp. have
an ATP-dependent PFK activity, while the fermentative Acholeplasma spp. have a PPidependent PFK activity; and (3) the nonfermentative Mycoplasma spp. lack ATP and PPidependent PFK and fructose-176-bisphosphate
aldolase activities but, like the fermentative
Mollicutes, can convert three-carbon compounds, G3P to PEP through the three-carbon arm of
the EMP pathway.
INTRODUCTION
The smallest self-replicating cells are members of the prokaryotic class Mollicutes. Some of
these wall-less, cytochrome-less micro-organisms may have only about 400 cell proteins
(Kawauchi et al., 1982). Knowledge of these proteins may contribute not only to a description of
the metabolic capabilities and potential of these small cells, but may reflect the minimal number
of enzymic activities necessary for free-living cellular life.
~~
Abbreviations: 6PG, 6-phosphogluconate ; F6P, fructose 6-phosphate; G1 P, G6P, glucose 1- and glucose 6phosphate; G3P, glyceraldehyde 3-phosphate; EMP pathway, Embden-Meyerhof-Parnas pathway; HMS,
hexose monophosphate shunt; PFK, phosphofructokinase; PGI, phosphoglucose isomerase; (d)R1P, (d)RSP,
(deoxy)ribose 1- and (deoxy)ribose 5-phosphate.
0001-4889 0 1989 SGM
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
684
D . DESANTIS, V . V . TRYON AND J . D . P O L L A C K
The studies of Rodwell (1960), and then Mitchell & Finch (1977, 1979), Neale et al. (1983) and
Cocks et al. (1985) have demonstrated a connection between glycolysis and nucleic acid
metabolism in Mycoplasma mycoides subsp. mycoides. Cocks et al. (1985) found that the
mollicute Ureaplasrna urealyticum was similar in many, but not all, respects to M . mycoides
subsp. mycoides. Our own studies with other Mollicutes have indicated that there are significant
distinctions between the Acholeplasma and Mycoplasma genera, and also between species in the
Mycoplasma genus (McElwain & Pollack, 1987; Manolukas et al., 1988). The metabolic
differences detected between fermentative and nonfermentative Mycoplasma spp. may serve as
markers when investigating their phylogenetic relationships. To determine how general these
metabolic differences are within and between the genera, as well as to continue to catalogue the
putative essential proteins within the class, we studied eighteen Mollicutes from the genera
Acholeplasma and Mycoplasma for the presence of and differences in their Embden-MeyerhofParnas (EMP) pathway and hexose monophosphate shunt (HMS).
METHODS
Organisms andgrowth conditions. The organisms and media used in this study are listed in Table 1. All organisms
were grown statically at 37 "C for 18-72 h (mid-exponential phase) except for M . hyopneumoniae which was grown
with constant shaking at 36 "C for 3-5 d. M . pneumoniae and M . genitalium were grown attached to glass or plastic
tissue culture flasks under < 1 cm of medium.
Preparation of cell extracts. The cells were harvested and washed by centrifugation and then lysed in kappa
buffer by osmotic shock, or by explosive decompression in a Parr-Bomb (Pollack, 1975). The lysates were
centrifuged at 225000g for 1 h at 4 "C. The supernatant (cell-free extract) was used in all of our experiments. In
preliminary studies, A . laidlawii B-PG9 extracts were tested before and after dialysis in three to four changes of 200
vols 10 mM-HEPES/NaOH, pH 7.4, 1 mM-MgC1, at 4 "C. We assayed for 12 different enzyme activities and
detected essentially no difference in specific activities between dialysed and undialysed samples. Therefore, we
did not dialyse our extracts in subsequent experiments.
Enzyme assays. Cell-free extracts of each organism were tested for 21 enzyme activities associated with the HMS
and the EMP pathway. The reaction mixtures were monitored spectrophotometrically and contained 5 x lo-'1.5 x lo3 pg cell-free protein in a final volume of 1.0 ml. All reaction mixtures were buffered with 0.1 ml (10%
final volume) of buffer A (400 mM-HEPES/NaOH, pH 7.4, and 25 mM-MgC1,) or when indicated, with buffer B
(200 mM-imidazole/HCl,pH 7.5). All required enzyme additions were at 0.5 IU (International Unit), and cofactor
,
where indicated. In assays where no enzyme
and substrate concentrations ranged from 160-640 p ~ except
activity was detected, 0.1-5.0 IU of the commercially available purified enzyme being studied was added directly
to the apparently negative complete reaction mixture.
Protein concentrations in the cell-free extracts were determined by the manufacturer's micro-assay technique
using the Bio-Rad protein dye reagent concentrate with BSA as the standard. All other enzymes, cofactors and
reagents were obtained from Sigma.
Details ofindividual assays. (i) Hexokinase (EC 2.7.1 . l ) was assayed by the method of Chou & Wilson (1975).
(ii) Glucose-6-phosphate (G6P) dehydrogenase (EC 1 . 1 . 1 .49) was assayed by the method of Pollack et a/. (1965).
NAD could not substitute for NADP in this reaction using extracts from 10 different Mollicutes (Pollack et a/.,
1965). (iii) 6-Phosphogluconate (6PG) dehydrogenase (EC 1 . 1 .1.44) was assayed by the method of DeMoss
(1955). (iv) Ribulose-5-phosphate epimerase (EC 5.1.3.1) was assayed by the method of Horecker et a/. (1957);
(v) the reverse direction was assayed in a similar manner. (vi) Phosphoribose isomerase (EC 5.3.1 .6) was assayed
by the method of Horecker et a / . (1957); (vii) the reverse direction was assayed in a similar manner. (viii)
Transketolase (EC 2.2.1 . 1) was assayed by the method of Kochetov (1982). (ix) Transaldolase (EC 2.2.1.2) was
assayed by the method of Horecker & Smyrniotis (1955); (x) the reverse reaction was assayed by the method of
Venkataraman & Racker (1961). (xi) The other transketolase reaction was assayed as for reaction (viii), except
that the reaction was started with xylulose 5-phosphate and erythrose 4-phosphate. (xii) 2-Deoxyribose 5phosphate (dR5P) aldolase (EC 4.1.2.4) was assayed by the method of Racker (1955). (xiii) and (xiv)
Phosphoglucose isomerase (PGI) (EC 5.3.1.9) were assayed by the method of Gracy & Tilley (1975). (xv)
Phosphofructokinase (PFK), ATP-dependent (EC 2.7. I . 1 1) was assayed by the method of Ling eta/. (1953, and
PFK, PP,-dependent (EC 2.7.1 .90) was assayed by the method of Reeves et a/. (1982). Extracts of M . capricolum,
Mycuplasma sp. Let. 1 and M . huminis 1612 were also tested anaerobically in nitrogen for ATP- or PP,-dependent
PFK. (xvii) Aldolase (EC 4.1.2.13) was assayed by the method of Taylor (1955). (xviii) Glyceraldehyde-3phosphate (G3P) dehydrogenase (EC 1.2.1.12) was assayed by the method of Krebs (1955). (xix) 3Phosphoglycerate phosphokinase (EC 2.7.2.3)was assayed by the method of Bucher (1955). (xx) Phosphoglyceromutase (EC 2.7.5.3) was assayed by our modification of the enolase assay of Morse et a/. (1974). The reaction
mixture contained: cell extract, buffer B, 160 p ~ - N A D H2.5
, ~ M - K C104
~ , pM-MgCl,, 20 p ~ - A D P and
, 0.5-2.5
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
685
E M P pathway and H M S in Mollicutes
Table 1. Organisms and growth media
Organism*
A . axanthurn S-743 (f)
A . equifetale C112 (f)
A . laidlawii A-PG8 (f)
A . laidluwii B-PG9 (f)
M . bovigenitulium PG11 (n)
M . cupricolum 14 ( f )
M . gullisepticurn S6 (f)
M . genitulium G37 ( f )
M . hominis PG21 (n)
M . hominis Botte (n)
M . hominis 1184 (n)
M . hominis 1612 (n)
M . hominis 10144 (n)
M . hominis 13408 (n)
M . hominis 13428 (n)
M . hyopneumoniue J (f)
M . pneumoniae M 129 (f)
Mycoplasma sp. Let. 1 (f)
Mediumt
Origin
J . G . Tully, Mycoplasma Reference
Laboratory, NIAID, Frederick,
MD, USA
Edward-Hayflick
Laboratory stock
M. F. Barile, Division of Bacterial
Products, Center for Biologics,
FDA, Bethesda, MD, USA
Edward-Hayflick containing
1 % (v/v) fresh yeast extract
and 0.15 % (v/v)
arginine/HCl, pH 6-8-7.0
R. F. Ross and T. Young, Veterinary
Friis
Medical Research Laboratory,
Iowa State Univ., Ames, IA, USA
Laboratory Stock
N. L. Somerson, Dept. of Medical
Microbiology and Immunology,
Ohio State Univ., Columbus, OH,
USA
(%?
Serum
v/v)S
0.5-1.5
0.5-1 ' 5
0
0
3-5
3-5
3-5
20
10
10
10
10
10
10
10
25
20
3-5
* f, Fermentative;
n, nonfermentative (Tully & Razin, 1977).
see Friis (1975).
$ Heat-inactivated donor horse serum was used except for M . hyopneumoniue, where acid-adjusted swine serum
(Friis, 1975) was used.
t Edward-Hayflick, see Beaman & Pollack (1981); Friis,
IU each of enolase, pyruvate kinase and lactate dehydrogenase. The reaction was started with 3 m ~ - 3 phosphoglycerate. (xxi) Enolase (EC 4.2.1 . 1 1) was assayed by method (xx), except that enolase was omitted and
the reaction was started with 3 m~-2-phosphoglycerate.
Statistical analysis. We studied 2-16 different batches of each of the eighteen Mollicutes. All assays were done at
two to five concentrations of each batch of cell-free extract to determine that the observed activity was
proportional to the amount of extract added. The reaction rate was calculated from those periods of all trials where
the reaction was linear (zero-order), i.e. the substrate concentration was apparently not limiting. The reaction
rates calculated for each assay of the same strain of cells were pooled and averaged. The data are reported for each
strain as nmol product synthesized min-' (mg protein)-' (mean SD). (The SD is reported only when n is 2 3.)
RESULTS
Enzyme activities of the hexose monophosphate shunt
Enzyme activities of the HMS were found in cytoplasmic extracts of the three Acholeplasma
species (Table 2). These are the two dehydrogenase reactions in Fig. 1, block A, and the
remaining HMS activities in Fig. 1, block B. However, transaldolase activity was variously
detected (Table 2). Such variation, using crude mollicute preparations, was also reported by
Cocks et al. ( I 985). The six fermentative Mycoplasma species lacked the two dehydrogenases,
only possessing the HMS activities shown in Fig. 1, block B. The two nonfermentative
Mycoplasma species, M . hominis (all six strains studied) and M . bovigenitalium lacked all the
HMS activities, except for ribulose-5-phosphate 3-epimerase activity.
Enzyme activities of' the Embden-MeyerhojlParnas path way
Cytoplasmic extracts of the three Acholeplasrna species and the six fermentative Mycoplasma
species possessed all EMP reactions studied (Table 3 ; Fig. 1, blocks C and D). Extracts of these
fermentative Mollicutes are capable of synthesizing PEP from G6P via the EMP pathway. One
difference between the genera is that the Acholeplasrna species have a PP,-dependent PFK
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
Glucose
D
14
PI
+
D
ATPp
I
Phosphoenolpyruvate
22
2-Phosphoglycerate
Wl
3-Phosphoglycerate
12,
p
1.3-Bisphosphoglycerate
NADH
NAD++P,
f
-
,,,
,
I
J.1
91 1 1 0
Fructose 6-phosphate
+
Erythrose 4-phosphate
2
Glyceraldehyde 3-phosphate
+
Sedoheptulose 7-phosphate
1
8r
II -
1I
411 5
Ribose 5-phosphate
2-Deoxyribose 5-phosphate +-++-DNA
Acetaldehyde
II
6l t7
Xylulose 5-phosphate
-RNA
Fig. 1. Schematic diagram of the enzyme activities detected in cell-free extracts of Achofepfusmaand
Mycoplusma spp. The enzyme assays are described in Methods. The reaction nos refer to the same
enzyme reactions numbered and described in Tables 2 and 3. The lines with two diagonal slashes
represent reaction sequences presumed to be present, but not investigated in this study. Block A,
dehydrogenase activities detected in Acholepfusma spp. but not in Mycoplusmu spp. Block B, HMS
activities detected in fermentative but not in nonfermentative Mollicutes. Block C, These activities (1 5
and 17) were not detected in nonfermentative Mollicutes. Block D, These EMP activities were detected
in all the Mollicutes studied, except no. 19 in M . houigenitufium.
,
u
\
d
Dihydroxyacetone phosphate
171
Fructose 1,6-bisphosphate
ADP/P,
A T P P P ~ ~
k w
Fructose 6-phosphate
I
Glyceraldehyde 3-phosphate
c
131
Glucose 6-phosphat
.............
P
0
fl
r
r
0
V
U
2
Z
P
v1
m
U
U
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
Deoxyri bose-5-phosphate
aldolase
Transaldolase
Transketolase
Phosphoribose isomerase
Glucose 6-phosphate
dehydrogenase
6-Phosphogluconate
dehydrogenase
Ribulose-5-phosphate
epimerase
Deoxyri bose-5-phosphate
aldolase
Transaldolase
Transketolase
Phosphoribose isomerase
* 1.2
* 2.0
NA
2.0 f 1.4
1.0
0-79 & 0.34
12 f 1-9
34 f. 1.7
6.0 & 12
0.62 & 0.77
38 k 4.4
62 k 17
1.1
NA
6.6
+_
6.3
19 13
40 f. 18
31 k 14
100 ? 10
3.0 k 0.3
6.8 k 5.3
70
+_
NA
NA
38
160 f. 21
300 +_ 100
26 k 31
81 f. 68
46 k 15
57 f 12
4
5
6
7
8
11
9
10
12
NA
3
11 k 19
20 k 3.9
NA
NA
2
55 & 6.8
NA
160 k 46
3.3
6.6 f 1.6
3.2
6-9
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
11
NA
NA
NA
NA
NA
NA
5200 k 2700
740 +_ 25
359
ND
NA
ND
NA
ND
NA
ND
M . bocigenitulium
M . hominis
1612
NA
2.9 f 0.82
NA
2000 850
590 +_ 310
250
30
300
14
120 k 52
140 f 35
58 k 23
60 k 22
34 k 1.7
89 & 36
NA
NA
NA
NA
NA
NA
NA
NA
1950 f. 580
750 k 180
NA
NA
NA
NA
NA
NA
NA
*
NA
NA
NA
NA
NA
*
9.1 k 2.9
20 2-6
2.4 & 0.99
10 f. 5.6
2.0 k 1.2
2.0 0.21
NA
NA
490 f. 140
540 k 220
120 54
190 k 8.5
120 k 14
230 k 75
NA
NA
NA
NA
NA
NA
NA
M . hominis
1184
M . hominis
Botte
M . hominis
13408
NA
NA
41 & 3.0
NA
NA
NA
NA
NA
33 k 16
*
NA
2200 f 500
5100 f. 660
500 & 83
1800 & 260
600 k 96
690 & 160
NA
1.8 0.42
130 k 68
59 2 28
94 44
120 f. 21
5 6 k 12
110 & 18
3.6 f 4.6
1.8 k 2.2
4.4 5 0.93
100 k 18
510 k 180
370 & 230
560 2 79
110 & 25
330 k 44
0.67 f. 0.48
3.6 k 2.1
NA
1100 k 430
2100 k 1100
110 f. 22
280 k 51
18 f. 9.3
30 k 23
NA
40 k 16
27 5 3.0
12 k 2.7
M . hominis
Reaction
no.*
M . pneumoniae M . genitulium
PG-21
12
3
4
5
6
7
8
11
9
10
2
Not done.
No activity was ever detected; we could detect l m I U enzyme activity in samples purchased from Sigma.
* Corresponds to reaction no. in Fig. 1.
ND,
NA,
Glucose-6-phosphate
dehydrogenase
6-Phosphogluconate
dehy d rogenase
Ribulose-5-phosphate epimerase
SD).
Reaction
Mycoplusma sp .
no.*
A . laidluwii A A. laidluwii B A . axunthum A . equifetale
Let. 1
M . cupricolum M . gullisepticurn M . hyopneumoniue
Enzyme activity is expressed as nmol product synthesized min-' (mg protein)-' (mean 2
Table 2. Hexose monophosphate shunt activities of Mollicutes
r/l
ea
s
z
2
5
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
Aldolase
3-Phosphoglycerate kinase
P hosphoglyceromutase
Enolase
pp,
Phosphofructokinase : ATP
Hexokinase
Phosphoglucose isomerase
Aldolase
3-Phosphoglycerate kinase
Phosphoglyceromutase
Enolase
Phosphofructokinase : ATP
pp,
Hexokinase
Phosphoglucose isomerase
NA
NA
NA
NA
3.3 f 2.1
15
12
1.6
3.1
NA
NA
NA
NA
1.1
14
3.5
NA
4.0 f 3.3
2.6 f 0.97
NA
M.
hominis
13408
f 420
f 17
NDt
*
210 4 150
21 9.9
140 k 92
14 f 7-6
2.3
NA
NA
NA
NA
6.9
22
NA
M.
hominis
1184
NA
NA
2.2
M y coplasma
sp. Let. 1
65 k 22
20
1.8
NAS
NAS
NA
NA
13
57
NA
M.
hominis
1612
45 k 6.5 2800 f 440
39 f 23
40 & 12
540 k 100 1000 f 1300
260 + 180
NA
260 f 60
NAS
NA
1400 f 600
110 f 20
1.8 f 2-6
1100 + 490
7.8 f 4.2
360 f 170 830 f 380
1000 k 500
310 k 100
A.
equifetale
NA
NA
6.0
10
NA
M.
hominis
Botte
630 k 150
380 f 88
130 f 32
+ 1800 380 + 220
f 5.4
570
k 36
950
NA
190
27
140 k 92
A.
axanthum
16
ND
ND
NA
NA
NA
NA
NA
3.8
12 11
8.4 f 8-8
*
M.
hominis
13428
450 220
580 f 1.2
120 -t 32
150 f 100
NAf
NA
210 f 78
760 f 300
6.1 &- 44$$
NA
M.
capricolum
ND
ND
17 k 15
NA
NA
NA
NA
NA
9.7 f 8.6
14 & 9.7
M.
hominis
10144
62 k 25
160 f 100
410 k 300
10 f 7.1
NA
NA
160 k 65
130 f 98
240 f 4.8
NA
M.
gallisepticum
Not done.
No activity was ever detected; we could detect 1 mIU enzyme activity in samples purchased from Sigma
* Corresponds to reaction no. in Fig. 1.
Activity was reported by Pollack & Williams (1986).
2 Reaction done aerobically and anaerobically.
0 No activity detected aerobically.
ND,
NA,
1
13
14
15
15
16
17
19
20
21
M.
hominis
PG-2 1
Reaction
no.*
*
560
39
3300
9.8
51
NA
NA
700 f 310
630 -t 240
440 f 190
8100 f 5200
15 f 1.5
120 49
55 f 32
0.78 f 0-08
1800 k 2600
A.
laidlawii B
14 f 2.4
210 f 23
420 f 120
A.
laidlawii A
1
13
14
15
15
16
17
19
20
21
Reaction
no. *
Enzyme activity is expressed as nmol product synthesized min-' (mg protein)-' (mean k SD).
Table 3 . Embden-Meyerhof-Parnas path way activities of Mollicutes
f 1.8
k 2.4
f 1.0
f 1.5
28
19
NA
NA
NA
NA
NA
46
NA
ND
M.
bovigenitalium
5.4
2.7
11
5.9
NA
NA
11 f 2.8
25 f 7-3
NA
NA
f 29
f 13
f 33
2 61
110 & 25
55 f 14
130 f 49
110 k 7.5
NA
NA
140 f 50
9.1 f 6.7
60 f 7.5
NA
M.
genitalium
59
45
71
290
NA
NA
150 k 31
21 & 27
220 + 79
NA
M.
M.
hyopneumoniae pneumoniae
00
Q\
00
EMP pathway and H M S in Mollicutes
689
activity while the Mycoplusmu species possess an ATP-dependent PFK. We found that the PP,dependent PFK of A . axanthum, A . equfetale and A . laidlawii A has an orthophosphatedependent back-reaction, as reported for A . laidlawii B-PG9 and A . frorum L1 (Pollack &
Williams, 1986). Arsenate could substitute for P, in this back-reaction (unpublished data). We
detected G3P dehydrogenase activity in A . laidlawii B, M-vcoplasma sp. Let. 1, M . gallisepticum
and M . pneumoniae [1400 f 100, 7.5 & 6.7, 33, and 40 & 25 nmol product synthesized min-l
(mg protein)-' f SD, respectively]. Hexokinase activity was detected only in extracts from the
Acholeplasma species and Mycoplasma sp. Let. 1.
The nonfermentative Mycoplasma species, M . hominis and M . bouigenitalium, possessed
enzyme activities of the EMP pathway from G3P to PEP (Table 3; and Fig. 1, block D). The
extracts from these Mollicutes lacked PFK and aldolase activities; PGI activity was present in
both. dR5P aldolase activity, a reaction in which G3P is synthesized from the catabolism of
DNA, was detected in all Acholeplasrna and Mycoplasma species except Mycoplasma sp. Let. 1
and M . gallisepticum (Table 3).
DISCUSSION
Our assays are all capable of detecting 0.001 IU of activity of commercially available enzymes
in the presence of 70-80% (vlv) mollicute extract. Nevertheless, our assay conditions may not be
similarly suitable for the detection of all mollicute enzyme activities and because these assays
were done with crude cytoplasmic extracts competitive activities may be present. We therefore
emphasize that the rates presented in Tables 2 and 3 should only be viewed qualitatively.
Notwithstanding, the presence of G6P dehydrogenase in all Acholeplasma spp. but in no
Mycoplasma spp. confirms the studies of Pollack et al. (1965) and O'Brien et al. (1981). In this
work, we also found that Acholeplasma spp., but not Mycoplasma spp. have 6PG dehydrogenase
activity. These observations may be taxonomically useful and relate to the nutrition of these
organisms. Dehydrogenase activities are generally considered to be a major source of cellular
NADPH, required for lipid biosynthesis. The absence of these dehydrogenase activities in all
Mycoplasma spp. studied may be related to the greater growth-need for exogenous lipid by
members of this genus. The balance of the HMS activities were detected in the Acholeplasma
spp. and fermentative Mycoplasrna spp. The data suggest that these organisms have a
functional HMS that may act as a connecting path that permits an interchange of carbons from
the EMP pathway, DNA and RNA metabolism and aromatic amino acid biosynthesis.
Aromatic amino acid synthesis, the shikimate pathway, is present in A . laidlawii, but absent in
M . iowae and M . gallinarum (Berry et al., 1987) and M . mycoides Y (Rodwell & Mitchell, 1979).
The two nonfermentative Mycoplasma spp., including all five strains of M . hominis, lack all
HMS activities, except, inexplicably, ribulose-5-phosphate epimerase, which was found in every
one. This activity may be the result of some unrecognized nonspecific enzyme activity.
Except for PFK activity, our study of the EMP pathway of fermentative Mollicutes is
generally unremarkable (Table 3). Hexokinase activity has been reported in Acholeplasma spp.
and M . mycoides subsp. mycoides (Rodwell & Rodwell, 1954; Castrejon-Diez et al., 1963;
Lanham et al., 1980, Salih et al., 1983; Cocks et al., 1985). We did not detect hexokinase activity
in any fermentative Mollicutes. Our data suggest that hexokinase activity in cell-free extracts,
using glucose as substrate, may not be a reliable identifier of all fermentative Mollicutes.
Of special interest was the observation that the three fermentative Acholeplasma spp. required
PP,, but not ATP, as the phosphorus donor in the PFK reaction, while the six fermentative
Mycoplasma spp. required ATP, but apparently not PP,. The PP,-requiring PFK of A . laidlawii
B has been purified and characterized (Pollack & Williams, 1986). The PP; requirement of the
fermentative members of the Acholeplasma genus may have greater taxonomic value, after the
EMP pathway of the nonfermentative Acholeplasma parvum is further studied (Atobe et al.,
1983). We have suggested that the PP, requirement of Acholeplasma spp. may also have
phylogenetic significance (Pollack & Williams, 1986). We detected the latter half, the threecarbon arm of the EMP pathway (Fig. 1, block D), i.e. from G3P to PEP in all Mollicutes,
whether fermentative or nonfermentative. A more rigorous distinction between fermentative
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
690
D. DESANTIS, V . V . TRYON A N D J. D . POLLACK
and nonfermentative Mollicutes, albeit more technically difficult to achieve, should perhaps
include an estimation of the activity of the hexose portion of the EMP pathway, i.e. to determine
if P F K or aldolase activity is present. The absence of PFK and aldolase activities in
nonfermentative Mycoplusmu spp. and the presence of these activities in fermentative
Mycoplasmu and Acholeplasma spp. suggests that these enzymes may be useful objects of a
comparative study of the molecular divergence of enzymes and the phylogeny of these genera
(Fothergill-Gilmore, 1986).
All nonfermentative Mollicutes lack hexokinase, PFK and aldolase activities, but have PGI
activity. We are unable to explain this curious observation. We speculate that the PGI activity
may be the result of a relatively nonspecific isomerase of another metabolic pathway, or a
remnant of some phylogenetic progenitor of these nonfermentative Mycoplasma spp. In contrast
to our observation, O’Brien et al. (1981), studying isoenzyme expression in 22 species of
Mollicutes, reported PGI activity in twelve fermentative Mollicutes, but not in the seven
nonfermentative Mycoplasmu spp. studied. These authors studied five strains of M . horninis,
including strain PG21, which was included in our study. In strain 1105 they detected trace PGI
activity, in the other four strains, no activity. We found activity in all seven strains of M . horninis,
including PG21, and also in M . bocigenitalium. Besides the differences in extract preparation
and assay technique, O’Brien et al. (1981) reported using G1P as the PGI reaction substrate,
whereas we used G6P or F6P.
Our findings also emphasize the importance of dR5P aldolase activity in the metabolism of
Mollicutes. Through the action of the dR5P aldolase, first reported in Mollicutes by Neale et al.
(1983), D N A may serve as a carbon and energy source for the growth of some Mycoplasrna spp.
(Pollack & Hoffmann, 1982). Cocks et al. (1985) reported a mutase in two other mollicutes,
Ureaplusmu urealyticum and M . mvcoides. This mutase converts dR1 P from DNA to dR5P and
R1 P from RNA to R5P. The dR5P is acted upon by the dR5P aldolase to synthesize G3P which
enters the glycolytic path; the R5P enters the HMS directly. If the pathways are reversible, a
route to the synthesis of nucleic acid precursors from the EMP pathway may be established. We
believe the route to nucleic acid precursors also involves the dR1 P accepting purine-pyrimidine
phosphorylase that we have found in Mollicutes (McElwain & Pollack, 1987).
Although not every enzyme activity of the HMS and EMP pathway was detected in both
directions (Fig. 1) and in all Mollicutes tested, we believe that there is sufficient in uitro evidence
to suggest that these pathways may be present and perhaps function in the whole cell and that
they are linked to each other through F6P and G3P and in Acholeplasma spp. through G6P as
well.
We would like to thank T. Young for help in preparing M . hyopneumoniae cells and M. F. Barile and D. K. F.
Chandler for help in preparing M . horninis cells. We would also like to thank the Graduate School of The Ohio
State University for its support.
REFERENCES
ATOBE,H., WATABE,
J. & OGATA,M. (1983). Achofeplasma parzwm, a new species from horses. Znternational Journal of Systematic Bacteriology 33,344-349.
K . D. & POLLACK,
J. D. (1981). Adenylate
BEAMAN,
energy charge in Acholeplasma laidhwii. Journal of
Bacteriology 146, 1055-1058.
BEAMAN,
K . D. & POLLACK,
J . D. (1983). Synthesis of
adenylate nucleotides by Mollicutes (mycoplasmas).
Journal of General Microbiology 129, 3 103-3 1 10.
BERRY,
A., AHMAD,
S., LISS,A. & JENSEN,
R. A. (1987).
Enzymological features of aromatic amino acid
biosynthesis reflect the phylogeny of mycoplasmas.
Journal (if Grnrrul Microbiology 133, 2 147-2 1 54.
BUCHER,T. (1955). Phosphoglycerate kinase from
brewer’s yeast. Methods in Enzymology 1, 415-422.
J., FISHER,
T. N . & FISHER,E., JR
CASTREJON-DIEZ,
(1963). Glucose metabolism of two strains of
Mycoplasma laidlawii. Journal of Bacteriology 86,
627-636.
CHOU,A. C. &WILSON,J. E. (1975). Hexokinase from
rat brain. Methods in Enzymology 42, 20-25.
COCKS,B. G., BRAKE,F. A., MITCHELL,
A. & FINCH,
L. R. (1985). Enzymes of intermediary carbohydrate
metabolism in Ureaplasma urealyticum and Mycoplasma mycoides subsp. mycoides. Journal of General
Microbiology 131, 2129-2135.
DEMOS, R. D. (1955). Glucose-6-phosphate and 6phosphogluconate dehydrogenases from Leuconostoc
mesenteroides. Methods in Enzymology 1, 328-334.
FOTHERGILL-GILMORE,
L. A. (1986). Domains of
glycolytic enzymes. In Multidomain Proteins - Structure and Evolution, pp. 85-174. Edited by D. G .
Hardie & J . R. Coggins. Amsterdam: Elsevier
Science Publishers.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31
EMPpathway and H M S in Mollicutes
FRIIS,N. F. (1 975). Some recommendations concerning primary isolation of Mycoplasma suipneumoniae
and Mycoplasma jiocculare : a survey. Veterinaermedicin 27, 337-339.
B. E. (1975). Phosphoglucose
GRACY,R. W. & TILLEY,
isomerase of human erythrocytes and cardiac tissue.
Merhods in Enzymology 41, 392-400.
HORECKER,
B. L. & SMYRNIOTIS,
P. Z. (1955). Transaldolase. Methods in Enzymology 1, 381-383.
HORECKER,
B. L., HURWITZ,J. & STUMPF,F. K.
(1957). The enzymatic synthesis of ribulose-1,5diphosphate and xylulose-5-phosphate. Methods in
Enzymology 3, 193-195.
Y., Muro, A. & OSAWA,C. (1982). The
KAWAUCHI,
protein composition of Mycoplasma capricolum.
Molecular and General Genetics 188, 7-1 1.
KOCHETOV,
G. A. (1982). Transketolase from yeast.
Methods in Enzymology 90, 209-223.
KREBS, E. G. (1955). Glyceraldehyde-3-phosphate
dehydrogenase from yeast. Methods in Enzymology 1,
40741 I ,
LANHAM,S. M., LEMCKE,R. M., SCOTT,C. M. &
GRENDON,
J . M. (1980). Isoenzymes in two species of
Acholeplusma. Journal of General Microbiology 117,
19-31.
LING, K.-H., BYRNE,W. L. & LARDY,H. (1955).
Phosphohexokinase. Methods in Enzymology 1, 306310.
MCELWAIN,
M. C. & POLLACK,
J . D. (1987). Synthesis
of deoxyribomononucleotides in Mollicutes : dependence on deoxyribose-1 -phosphate and PP,. Journal
of Bacteriology 169, 3647-3653.
MANOLUKAS,
J. T., BARILE,
M. F., CHANDLER,
D. K. F.
& POLLACK,
J. D. (1988). Presence of anaplerotic
reactions and transamination, and the absence of the
tricarboxylic acid cycle in Mollicutes. Journal of
General Microbiology 134, 79 1-800.
MITCHELL,A. & FINCH,L. R. (1977). Pathways of
nucleotide biosynthesis in Mycoplasma mycoides
subsp. mycoides. Journal of Bacteriology 130, 10471054.
MITCHELL,A. & FINCH,L. R. (1979). Enzymes of
pyrimidine metabolism in Mycoplasma mycoides
subsp. mycoides. Journal of Bacteriology 137, 10731080.
MORSE,S. A., STEIN,S. & HINES,J. (1974). Glucose
metabolism in Neisseria gonorrheae. Journal of
Bacteriology 120, 702-7 14.
NEALE,G . A. M., MITCHELL,
A. & FINCH,L. R. (1983).
Pathways of pyrimidine deoxyribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides.
Journal of Bacteriology 154, 17-22.
69 1
O'BRIEN,S. J., SIMONSEN,
J . M . , GRABOWSKI,
M. W. &
BARILE,M. F. (1981). Analysis of multiple isozyme
expression among twenty-two species of Mycoplasma and Acholeplasma. Journal of' Bacteriology
146, 222-232.
POLLACK,J . D. (1975). Localization of reduced
nicotinamide adenine dinucleotide oxidase activity
in Acholeplasma and M-vcoplasma species. International Journal ofSystematic Bacteriology 25, 108-1 13.
POLLACK,
J. D. & HOFFMANN,
P. J . (1982). Properties
of the nucleases of Mollicutes. Journal ofBacteriology
152, 538-541.
POLLACK,J . D. & WILLIAMS,M. V. (1986). PP,dependent phosphofructotransferase (phosphofructokinase) activity in the Mollicute (Mycoplasma)
Acholeplasma laidlawii. Journal o f Bacteriology 165,
53-60.
POLLACK,
J. D., RAZIN,S. & CLEVERDON,
R. C. (1 965).
Localization of enzymes in Mycoplasma. Journal cf
Bacteriology 90,617 -622.
RACKER,E. (1955). Deoxyribose phosphate aldolase
(dr-aldolase). Methods in Enzymology 1, 384--386.
REEVES,R. E., LOBELLE-RICH,
P. & EUBANK,W. B.
(1982). 6-Phosphofructokinase (pyrophosphate)
from Entamoeba histolytica. Methods in Enzymology
90, 97-102.
RODWELL,
A. W. (1960). Nutrition and metabolism of
Mycoplasma mycoides, subsp. mycoides. Annals oj'the
New York Academy of'Sciences 79, 499-507.
RODWELL,A. W. & RODWELL,E. S. (1954). The
breakdown of carbohydrates by Asterococcus mycoides, the organisms of bovine pleuropneumonia.
Australian Journal of Biological Science 7, 18-30.
RODWELL,
A. W. & MITCHELL,
A. (1979). Nutrition,
growth and reproduction. In The Mycoplasmas, vol.
I , pp. 103--136. Edited by M. F. Barile & S. Razin.
London : Academic Press.
v. & E R N 0 , H. (1983).
SALIH, M. M., SIMONSEN,
Electrophoretic analysis of isozymes of Acholeplasrna
species. International Journal of Sj.stematic Bacteriology 33, 166-172.
TAYLOR,
J. F. (1955). Aldolase from muscle. Methods in
Enzymology I , 3 10-3 1 5.
TULLY,J. G. & RAZIN,S. (1977). The mollicutes. In
CRC Handbook of Microbiology, vol. I , pp. 405-459.
Edited by A . I. Laskin and H. A. Lechavalier.
Cleveland, Ohio : CRC Press.
VENKATARAMAN,
R. & RACKER,E. (1961). Mechanism
of action of transaldolase I. Crystallization and
properties of the yeast enzyme. Journal uf'BiologicaI
Chemistry 236, 1876-1882.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.27.170
On: Thu, 13 Oct 2016 05:55:31