Abstract

Defined deletion mutants of Escherichia coli defective for the synthesis of pyruvate formate-lyase (PFL) or pyruvate dehydrogenase (PDH) were analysed in regards their growth in batch culture and their enzyme levels under fermentative and nitrate respiratory conditions. A pfl mutant proved not to be completely auxotrophic for acetate when grown anaerobically in glucose minimal medium. In contrast, a pfl aceEF double mutant exhibited an absolute requirement for acetate, indicating that PDH is the source of acetyl-CoA in the pfl mutant. Growth of both pfl and aceEF single mutants under nitrate respiratory conditions was essentially indistinguishable from the wild-type. Thus, either PFL or PDH can be used to catabolise pyruvate in nitrate-respiring cells. The activities of PFL and PDH measured after growth with nitrate are commensurate with this proposal.

References

1
Knappe
J.
Sawers
G.
(
1990
)
A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli
FEMS Microbiol. Rev.
 ,
75
,
383
398
.
2
Stewart
V.
(
1988
)
Nitrate respiration in relation to facultative metabolism in enterobacteria
Microbiol. Rev.
 ,
52
,
190
232
.
3
Sawers
G.
Böck
A.
(
1988
)
Anaerobic regulation of pyruvate formate-lyase from Escherichia coli
J. Bacteriol.
 ,
170
,
5330
5336
.
4
Guest
J.R.
Angier
S.J.
Russell
G.C.
(
1989
)
Structure, expression, and protein engineering of the pyruvate dehydrogenase complex of Escherichia coli
Ann. N.Y. Acad. Sci.
 ,
573
,
76
99
.
5
Spencer
M.E.
Guest
J.R.
(
1985
)
Transcription analysis of the sucAB, aceEF and lpd genes of Escherichia coli
Mol. Gen. Genet.
 ,
200
,
145
154
.
6
Pascal
M.C.
Chippaux
M.
Abou-Jaoude
A.
Blaschkowski
H.P.
Knappe
J.
(
1981
)
Mutants of Escherichia coli K12 with defects in anaerobic pyruvate metabolism
J. Gen. Microbiol.
 ,
124
,
35
42
.
7
Casadaban
M.J.
Cohen
S.N.
(
1979
)
Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences
 
2nd ed.
,
76
, In
Proc. Natl. Acad. Sci. USA
 , pp
4530
4533
.
8
Sirko
A.
Zehelein
E.
Freundlich
M.
Sawers
G.
(
1993
)
Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli
J. Bacteriol.
 ,
175
,
5769
5777
.
9
Sauter
M.
Sawers
R.G.
(
1990
)
Transcriptional analysis of the genes encoding pyruvate formate-lyase-activating enzyme of Escherichia coli
Mol. Microbiol.
 ,
4
,
355
363
.
10
Christiansen
L.
Pedersen
S.
(
1981
)
Cloning, restriction endonuclease mapping and posttranslational regulation of rpsA, the structural gene for ribosomal protein S1
Mol. Gen. Genet.
 ,
181
,
548
551
.
11
Hamilton
C.M.
Aldea
M.
Washburn
B.K.
Babitzke
P.
Kushner
S.R.
(
1989
)
New method for generating deletions and gene replacements in Escherichia coli
J. Bacteriol.
 ,
171
,
4617
4622
.
12
Lowry
O.H.
Rosebrough
N.J.
Farr
A.L.
Randall
R.J.
(
1951
)
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
 ,
193
,
265
275
.
13
Knappe
J.
Blaschkowski
H.P.
(
1975
)
Pyruvate formate-lyase from Escherichia coli and its activation system
Methods Enzymol.
 ,
41
,
508
518
.
14
Guest
J.R.
Creaghan
I.T.
(
1973
)
Gene-protein relationships of the α-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and characterisation of lipoamide dehydrogenase mutants
J. Gen. Microbiol.
 ,
75
,
197
210
.
15
Sawers
R.G.
Ballantine
S.P.
Boxer
D.H.
(
1985
)
Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme
J. Bacteriol.
 ,
164
,
1324
1331
.
16
Gennis
R.B.
Hager
L.P.
(
1976
)
Pyruvate oxidase
2nd ed.
(
Martonosi
A.
, Ed) Vol.
2
, In
The Enzymes of Biological Membranes
 , pp
493
504
Plenum
,
New York, NY
.
17
Snoep
J.L.
Teixeira de Mattos
M.J.
Postma
P.W.
Neijssel
O.M.
(
1990
)
Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775
Arch. Microbiol.
 ,
154
,
50
55
.