The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: John R. Guest. Tel: +44 114 2224406. Fax: +44 114 2222787. e-mail: j.r.guest{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: pyruvate metabolism, pyruvate dehydrogenase complex, growth rate, biomass yield and energetics, acetyl-CoA synthetase, continuous culture
Abbreviations: ACK, acetokinase; ACS, acetyl-CoA synthetase; PDH, pyruvate dehydrogenase; PDH-E1p, E1 component of the pyruvate dehydrogenase complex; PDHC, pyruvate dehydrogenase complex; PFL, pyruvate formate-lyase; PoxB, pyruvate oxidase; PTA, phosphotransacetylase; ThDP, thiamin diphosphate
a Present address: Botany Department, Faculty of Sciences, Minia University, Minia, Egypt.
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INTRODUCTION |
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PoxB is a peripheral membrane flavoprotein which catalyses the decarboxylation of pyruvate to acetate and CO2 with the reduction of flavin adenine dinucleotide, FAD (Gennis & Hager, 1976 ). The enzyme contains four identical subunits (62 kDa) each with single tightly bound FAD and loosely bound thiamin diphosphate (ThDP) and Mg2+. PoxB is strongly activated by a variety of phospholipids which increase the maximum velocity about 20-fold and lower the concentration of pyruvate needed for enzyme saturation about 10-fold (Gennis & Hager, 1976
; Mather et al., 1982
). Moreover, this activation can be mimicked by limited chymotryptic proteolysis leading to the cleavage of a 3 kDa peptide from the C-terminus of the enzyme (Russell et al., 1977
; Recny et al., 1985
). The poxB gene has been cloned and sequenced (Grabau & Cronan, 1984
, 1986a
) and this has greatly facilitated recent studies on both the structurefunction relationships (including the lipid activation) of PoxB (Grabau et al., 1989
; Wang et al., 1991
; Chang & Cronan, 1997
) and on the transcriptional regulation of the poxB gene (Chang et al., 1994
). The regulatory studies indicated that PoxB activity and poxB-lacZ expression reach maximal values in early stationary phase and both are completely dependent on the rpoS-encoded sigma factor (RpoS,
38 or
S) that is required for transcribing many genes that are induced in stationary phase. PoxB was also shown to be expressed (albeit at a lower rate) and active, during anaerobic growth. It was suggested that PoxB might be important for survival during the transition between exponential and stationary phases and that it might serve as a source of acetyl units under microaerobic conditions where both PDHC and PFL would function poorly (Chang et al., 1994
).
The present physiological studies were initiated to (a) establish whether an up-regulated PoxB can replace the PDH complex in providing the acetyl units needed for good aerobic growth in unsupplemented medium, (b) assess the energetic consequences of such a replacement, and (c) determine whether and to what extent PoxB normally contributes to the aerobic growth efficiency of E. coli.
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METHODS |
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Inactivation of the poxB gene by kanR insertion.
A PoxB null strain (JRG3434, poxB::kanR) was constructed by selecting KnR transductants of W3110 with a P1vir lysate of YY877 (Chang et al., 1994 ) which has a kanR cassette inserted at the SalI site in the poxB structural gene (Fig. 1a
). The same lysate was used to select KnR transductants of JRG3456 and JRG4077 in order to construct strains lacking pyruvate dehydrogenase (E1p) and PoxB, JRG3931 (aceE::camR poxB::kanR) and JRG4077 (aceE::camR poxB::kanR lacIq). Phenotypic characterization confirmed that JRG3434 was Ace+ and retained PDHC and PDH-E1p activities whereas JRG3931 was Ace- and had neither PDHC nor PDH-E1p plus PoxB activities.
Selection of a PDH-negative PoxB-constitutive strain.
A PoxB-constitutive derivative of the PDH-E1p null strain, JRG3456 (aceE::camR), was selected by acetate-starvation of a glucose-limited chemostat culture and designated JRG3980 (aceE::camR poxBc); see Results. The strain was characterized by the possession of an Ace+ phenotype, being totally deficient in PDHC activity, and maintaining a constant PoxB activity at twice the normal stationary-phase level throughout the growth cycle.
Multicopy poxB plasmids.
Several multicopy plasmids encoding the poxB gene and a derivative (poxBc) lacking 23 C-terminal codons (Abdel-Hamid, 1999
), and others that express the two genes from the tac promoter (Ptac-poxB and Ptac-poxBc
), were constructed in addition to those already described. The 4·8 kb BglIISphI fragment of pCG5 was cloned in pBR322 and pSU38 to generate medium-copy and low-copy poxB+ plasmids (pGS1276 and pGS1277) having a smaller insert than pCG5 (Fig. 1
). An analogous pair of poxBc
plasmids (pGS1275 and pGS1279) was constructed in the same way except that the downstream 1·8 kb SalISphI pox'B segment was replaced by terminally deleted segments flanked at one end by engineered sites (0·7 kb; SalIBamHI or XbaI, respectively). Two high-copy expression plasmids (pGS927, Ptac-poxB; and pGS1197, Ptac-poxBc
) were constructed by combining the Ptac-poxB' segment of pGS859 (1·1 kb EcoRISphISalI; Fig. 1
) and either the 1·8 kb SalISphI pox'B segment from pCG5 or the 0·7 kb SalIBamHI pox'Bc
segment, in pUC18 and using TG1 as the host for transformation in order to repress the tac promoter.
Microbiological and analytical methods.
The rich medium used for routine subculture was L broth (Lennox, 1955 ) containing glucose (5·6 mM) and supplements of acetate (2 mM), chloramphenicol (15 µg ml-1) and kanamycin (50 µg ml-1), as required. The minimal media used for nutritional tests and for both batch and continuous culture have been described previously (Davé et al., 1995
). The batch and continuous culture media contained glucose (20 mM) as the major carbon source or growth-limiting substrate, and supplements of acetate (2 mM) and IPTG (generally 50 µM) where indicated. Batch and continuous cultures were grown in chemostat vessels (nominal volumes, 1 l; working volumes, 605 and 650 ml). The pH was maintained at 7·0±0·1 by automatic titration with KOH (2 M). Dissolved O2 tensions were monitored with a galvanic oxygen electrode, and kept above 50% air saturation by adjusting the stirring speed (500750 r.p.m.) at a constant air-flow of 600 ml min-1. All inocula were cultured overnight, washed, and resuspended in the corresponding experimental medium.
Batch cultures were used to determine doubling times and hence µmax by measuring the OD430 (Pye Unicam SP6-250 spectrophotometer) and then deriving the slopes of log OD430 versus time plots in the exponential region by regression analysis. Small samples (25 ml) were taken at specified times, centrifuged (4500 g for 15 min) at room temperature, and the culture supernatants stored at -20 °C prior to enzymic analysis of glucose (Bergmeyer & Bernt, 1974 ) and acetate (Acetic Acid Kit; Digen). Continuous cultures were established at steady-states over a range of different dilution rates from 0·05 to 0·25 h-1 and sampled for metabolic analysis. Substrate consumption, O2 consumption and CO2 production rates (qs, qO2, qCO2, respectively) were determined and the carbon conversion efficiencies (percentage of substrate carbon fluxed into biomass; numerically 695D.qS-1%, assuming that the carbon content of the biomass is 50%, w/w) and carbon balances [numerically (695D+16·7qCO2).qS-1%] were calculated as described by Brooke et al. (1989)
.
Cell-free extracts were prepared for enzyme assay using bacteria from samples (2050 ml) of batch and steady-state continuous cultures, harvested, washed and resuspended at 1 g per 4 ml cold potassium phosphate buffer (20 mM, pH 7·0) containing MgSO4 . 7H2O (5 mM), disodium EDTA (2 mM), PMSF (1 mM) and benzamidine hydrochloride (1 mM). Suspensions at 4 °C were disrupted by ultrasonic treatment (MSE disintegration 150 W) at 15 microns for three periods of 30 s with 30 s intervals for cooling. Intact bacteria and cell debris were sedimented (25000 g for 20 min at 4 °C) and the supernatants were maintained at 0 °C whilst measuring enzyme activities. Standard spectrophotometric procedures were used for measuring PDHC activity with 3-acetyl pyridine adenine dinucleotide as the electron acceptor (Russell & Guest, 1990 ) and PoxB (EC 1 . 2 . 2 . 2) activity with sodium ferricyanide as the electron acceptor (Chang & Cronan, 1986
). The PoxB assay detects the pyruvate dehydrogenase component of the PDH complex (PDH-E1p; EC 1 . 2 . 4 . 1), albeit sub-optimally, and the analogous ferricyanide-based assay for PDH-E1p (Russell & Guest, 1990
), used here only for strain characterization, likewise detects PoxB sub-optimally (Abdel-Hamid, 1999
). Acetyl-CoA synthetase (EC 6 . 2 . 1 . 1) and acetokinase (EC 2 . 7 . 2 . 1) activities were measured according to Brown et al. (1977)
. The reaction rates were proportional to the amounts of added cell-free extract and linear for at least 3 min. Specific activities are quoted as µmol of product formed or molar equivalent of electron acceptor reduced (mg protein)-1 h-1 at 37 °C and the protein concentrations of cell-free extracts were assayed by the Lowry method with BSA as standard.
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RESULTS |
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Functional replacement of the PDH complex by PoxB in an aceE null strain
Several approaches were used to investigate conditions under which PoxB might replace the function normally attributed to the PDH complex viz. supplying the acetyl units (acetate) needed to support good aerobic growth in unsupplemented glucose minimal medium.
PoxB expression from a Ptac-regulated poxB gene. The possibility that PoxB could replace the PDH complex for supporting good acetate-independent growth on glucose was first investigated by constructing a PDH-E1p null strain (JRG3445; aceE::camR Ptac-poxB+) in which the chromosomal poxB gene is expressed from the IPTG-inducible tac promoter rather than the natural promoter. Plate growth tests using glucose minimal medium supplemented with either 2 mM acetate or 50 µM IPTG showed that JRG3445 exhibits an Ace- phenotype which can be satisfied by either acetate or IPTG, whereas the PDH-E1p null strain (JRG3456; aceE::camR) responds to acetate but not IPTG, and the PoxB null strain (JRG3434; poxB::kanR) resembles the Ace+ parental strain (W3110) in neither requiring nor being affected by either supplement (Table 2). It was concluded that when induced throughout the growth cycle, PoxB can support good acetate-independent growth in the absence of the PDH complex. Further studies with the Ptac-poxB+ strain (JRG3445) in controlled batch culture showed that the maximum specific growth rate (µmax) increases with IPTG concentration to a maximum value of 0·58 h-1 (i.e. 60% of the parental rate) with 30 µM IPTG (Fig. 2
). Under these conditions the synthesis of PoxB and hence the metabolic flux to acetate (acetyl) is controlled via the lacI-encoded repressor and the concentration of exogenously supplied IPTG. It may be significant that the IPTG/PoxB-dependent growth rate fails to reach that of the parental strain, because in comparable experiments with a strain in which the entire pdh operon (Ppdh-pdhR-aceEF-lpdA) is replaced by an engineered Ptac-aceEF-lpdA operon, the highest IPTG/PDHC-dependent growth rate (observed at 25 µM IPTG) exceeded by 25% the highest growth rate both of the parental strain and of the acetate-supplemented engineered strain (Guest et al., 1996
).
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Growth profiles, substrate utilization and enzyme activities of isogenic strains
A detailed analysis of the effects of altering the capacity for aerobic pyruvate metabolism was made with controlled batch cultures of the parental (wild-type) strain and various isogenic mutants. The medium contained glucose as the major carbon source and a supplement of acetate (2 mM), added to permit comparisons between both Ace- and Ace+ strains. Growth profiles including patterns of glucose utilization and acetate production (Fig. 4ae
) and the corresponding specific-activity profiles of PoxB (and/or PDH-E1p) and of the PDH complex (Fig. 5a
e
), were constructed. Three distinct patterns were apparent from the former profiles (Fig. 4a
e
).
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The second pattern of growth and substrate utilization was observed with the PDH-E1p null strain, JRG3456 (aceE::camR poxB+) (Fig. 4c). This strain requires acetate as a growth supplement and, as expected, glucose and acetate were used simultaneously. However, soon after the acetate was exhausted (at 7 h, with almost half of the glucose remaining), the acetate concentration slowly returned to the initial level (Fig. 4c
). The PoxB specific activity remained relatively constant until 8 h when it started to increase, slowly at first and then rapidly, as the culture entered the stationary phase (Fig. 5c
). These observations indicate that PoxB is responsible for the accumulation of acetate by stationary-phase cultures of the PDH-E1p null strain. This conclusion was further supported by experiments with cultures of two strains lacking PoxB and PDH-E1p activities: JRG3445 (aceE::camR Ptac-poxB+) grown without IPTG (Fig. 4c'
); and JRG3931 (aceE::camR poxB::kanR) the double mutant (Fig. 4c'
). In both cases glucose and acetate were utilized simultaneously, there was no subsequent period of acetate accumulation, and growth stopped when the exogenous acetate was exhausted. These two strains have the unique property of not accumulating acetate in aerobic stationary-phase cultures.
The third pattern was observed with the PDH-E1p null strain containing the IPTG-regulated poxB gene (JRG3445; aceE::camR Ptac-poxB+) grown with the inducer, and the PDH-E1p null strain having a constitutive poxB gene (JRG3980; aceE::camR poxBc) (Fig. 4d, e
). Here the onset of acetate excretion was delayed relative to the aceE+ strains (first pattern) and then followed by the accumulation of high but stable rather than declining levels of acetate (13 and 10 mM, respectively), presumably due to the presence of higher PoxB activities. Indeed, the PoxB specific activities reached by the IPTG-induced and constitutive strains in stationary phase [60 and 1·2 µmol (mg protein)-1 h-1, respectively; Fig. 5d
, e
] were approximately 100- and 2-fold higher than those of strains expressing the poxB gene from the natural promoter [0·6 µmol (mg protein)-1 h-1; Fig. 5a
, c
]. The unchanging specific activity of PoxB exhibited by the constitutive strain is entirely consistent with the view that the poxB gene is expressed from a promoter that is no longer subject to stationary-phase (RpoS-dependent) control or to any other major regulatory constraint. It should be noted that for most of the growth cycle, the PoxB activity of the constitutive strain is at least 3·8-fold higher than that of the parental aceE::camR strain, before declining to 2·0-fold in stationary phase (Fig. 5c
, e
).
It is significant that when the substrate (glucose) was completely consumed, the extents of growth of the aceE+ strains (Fig. 4a, b
) were invariably greater than those of the aceE strains, irrespective of whether they are poxB+, poxB or poxBc (Fig. 4c
, c'
, e
). In contrast, poxB inactivation appeared to have little effect on the growth attained by aceE+ cultures (Fig. 4a
, b
), although it was subsequently found to lower the growth yield in continuous culture (see below; Table 5
). The effect of poxB inactivation on the extent of growth was more apparent in the aceE background (Fig. 4c
versus c'
, c'
), and reversed by IPTG-induced or constitutive poxB expression (Fig. 4d
, e
). The maximum growth rates (µmax) calculated from the growth curves of the controlled batch cultures containing glucose and acetate showed that growth rate is lowered when either of the aceE or poxB genes are inactivated (Table 2
). Inactivation of the aceE gene of W3110 lowered the rate by 33% (0·69 to 0·46 h-1) whereas inactivation of poxB caused a 16% reduction (0·69 to 0·58 h-1), and the combined effects of inactivating both genes was a 48% reduction (0·69 to 0·36 h-1) which is almost additive. Furthermore, induction of PoxB activity by adding IPTG to JRG3445 (aceE::camR Ptac-poxB+) restored the maximum growth rate to that of aceE strains containing either wild-type or constitutive poxB genes (0·46 h-1). These observations demonstrate that PoxB has a very significant effect on the growth rate of E. coli both in the presence and absence of PDHC (and this applies even when the anabolic function of the PDH complex is satisfied by the provision of an acetate supplement).
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The PoxB activities of the transformants were not amplified to the levels expected with multicopy plasmids, suggesting that the poxB gene is strongly regulated, or that the overproduction of PoxB (or the product of another cloned gene) is deleterious. Indeed, growth impairment by the poxB and poxBc plasmids (but not the vectors) was observed when transformants of JRG3456 (aceE::camR) were grown under permissive conditions, i.e. with exogenous acetate. Furthermore, two high-copy expression plasmids, pGS927 (Ptac-poxB) and pGS1197 (Ptac-poxBc
), not only failed to complement the Ace- phenotype of JRG3456 (aceE::camR) in the presence or absence of IPTG, they either impaired growth (pGS1197 Ptac-poxBc
) or completely inhibited growth (pGS927 Ptac-poxB) in the presence of added acetate, and both plasmids impaired the growth of W3110 under all conditions. The different degrees of toxicity suggest that although excess functional enzyme is more deleterious than the non-functional enzyme, there must be some other inhibitory factor. The two expression plasmids had the same growth inhibiting and non-complementing effects in the lacIq background of TG1 and two TG1 derivatives, JRG4076 (aceE::camR) and JRG4077 (aceE::camR poxB::kanR) which more tightly controls the tac promoter in multicopy situations. In fact TG1 had to be used in order to recover both Ptac-pox plasmids during their construction. Very high PoxB and PoxBc
activities in the range 480600 µmol (mg protein)-1 h-1 (i.e. 6001000-fold amplifications) were induced by IPTG in freshly transformed cultures. However, these plasmids proved to be unstable, their ability to overexpress the corresponding enzyme declining rapidly during propagation under all growth conditions. Such instability or host intolerance was only observed with transformed strains containing multicopy plasmids. It was therefore concluded that the continuous expression of a moderate level of PoxB activity from a single constitutive or tac regulated gene (3·8100-fold amplification) is tolerated and allows full complementation of the nutritional defect of PDH-deficient mutants. In contrast, the weak complementation exhibited by plasmid transformants probably represents a compromise between several factors where the provision of acetate is limited by strong temporal regulation of the natural gene and the elimination of deleterious multicopy plasmids.
Replacing the PDH complex by PoxB lowers growth efficiency and growth yield
Carbon (glucose)-limited chemostat cultures were used to make a detailed quantitative investigation of the energetic consequences of replacing the PDH complex by PoxB. Steady-state cultures of JRG3980 (aceE::camR poxBc) and JRG3445 (aceE::camR kanR-Ptac-poxB+) having constitutive and IPTG-inducible poxB genes (respectively) were established at four different dilution rates in the range 0·050·25 h-1 and compared with analogous cultures of the wild-type (W3110) and PDH-null (JRG3456; aceE::camR) strains. The rates of glucose and O2 utilization (qs and qO2, respectively), CO2 production (qCO2) and the biomass formed (x) were measured. These values were used to calculate the percentage carbon fluxed into biomass (i.e. carbon conversion efficiency) and the percentage carbon balance (see Methods) and representative results obtained at one dilution rate (D=0·15 h-1) are shown in Table 3. At all steady-states the substrate carbon was almost fully accounted for by the total amount of carbon in the biomass formed and the CO2 produced (9398%). The aceE::camR mutants with normal or constitutive poxB genes exhibited higher rates of glucose consumption and lower carbon conversion efficiencies than the parental strain (W3110), consistent with an impaired metabolism. A similar impairment was also evident from the low carbon conversion efficiency observed when the poxB gene was continuously expressed from the IPTG-inducible tac promoter, but it is not clear why the rate of glucose consumption was also low under these conditions. Overall, the carbon conversion efficiencies were lowered by 2939% as a consequence of inactivating the PDH complex.
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DISCUSSION |
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Here it was shown that PoxB is able to replace the PDH complex and support good acetate-independent growth: (i) when the natural poxB promoter is replaced by the IPTG-inducible tac promoter; (ii) when a strain having the natural promoter is grown at low growth rates in chemostat culture; and (iii) when PoxB activity is expressed constitutively. The acetate-independent but IPTG-dependent growth of JRG3445 (aceE::camR Ptac-poxB+) shows that PoxB can replace the PDH complex by oxidizing pyruvate to acetate at the rate needed to support good growth on glucose. The relationship between the maximum growth rate of JRG3445 and IPTG concentration further indicated that the growth-limiting factor under these conditions is the carbon flux through PoxB. Grabau & Cronan (1984) had earlier shown that poxB+ transformants of one out of three PDHC-deficient strains grew without acetate, but only at 20% of the rate observed with added acetate, despite being able to produce (at least in the stationary phase) as much or more PoxB activity than required to restore normal growth to the constitutive strain studied here. They suggested that the Ace- phenotype of strains containing PoxB but lacking PDHC activity could be due to the inefficiency of the pathways responsible for converting PoxB-derived acetate to acetyl-CoA, rather than to insufficient PoxB. However, the present demonstration that the Ptac-poxB+ strain (JRG3445) grows without acetate in the presence of IPTG suggests that growth is not limited by the acetate utilization pathways but by a combination of insufficient PoxB activity early in the growth cycle and the growth inhibitory effects of deleterious multicopy plasmids. This view is also supported by the acetate-independence of the poxBc strain (JRG3980) which synthesizes PoxB throughout the growth cycle. Moreover, a 3·8-fold amplification of PoxB activity early in the growth cycle is sufficient to raise the permitted growth rate from 0·05 h-1 to greater than 0·25 h-1.
Although the exact nature of the constitutive mutation was not defined, the simplest explanation is that the poxB promoter has lost its RpoS-dependence. The stability and facile selection of such mutants indicates that they are not hyper strains containing multiple poxB genes, nor are they likely to have arisen by the activation of a novel pyruvate oxidase or a cryptic gene, because no revertants of aceEF or poxB mutants have ever been reported to possess a novel activity, nor does the E. coli genome contain an obvious candidate. There is no evidence for factor-mediated positive or negative regulation of poxB, e.g. by the pyruvate-sensitive regulator (PdhR), and no PdhR-site is associated with the poxB gene. Moreover, regulator or operator mutations are unlikely to alter the stationary-phase dependence of poxB gene expression.
Studies with the poxB plasmids indicated that compared to the Ptac-poxB+ and poxBc strains the presence of multiple copies of the poxB region impairs growth and hence compromises the transformants ability to grow well without acetate. Here it was also shown that expressing poxB from the natural promoter can sustain acetate-independent aerobic growth in a PDH null strain, albeit at a low growth rate. Indeed, the specific activity of PoxB was at its maximum in the parental strain (W3110) during growth at a low dilution rate (0·05 h-1) in glucose-limited chemostat culture whereas PDHC activity was undetectable. These observations show that expression of the poxB gene from its RpoS (38)-dependent promoter can be induced at low growth (dilution) rates, a finding which is entirely consistent with those of Notley & Ferenci (1996)
, who have concluded that growth at low dilution rates in glucose-limited chemostat culture induces the expression of RpoS (
38)-dependent genes in E. coli.
The major physiological consequences stemming from the use of PoxB to support the growth of the various aceE::camR strains are lower maximum growth rates in batch cultures (33% decrease), lower maximum growth yields (925% decrease) and carbon conversion efficiencies (2939% decrease) in steady-state chemostat cultures, and correspondingly greater use of the carbon substrate for energy generation, compared to the parental strain. Thus, although PoxB can support aerobic growth of E. coli in the absence of the PDH complex it is energetically less efficient. This can to some extent be explained by the need for energy to convert the PoxB-derived acetate to acetyl-CoA. E. coli possesses two pathways for this conversion, the acetokinase-phosphotransacetylase (ACK-PTA) and the acetyl-CoA synthetase (ACS) pathways, both of which require ATP (Brown et al., 1977 ). When PoxB was inactivated the activities of ACK and ACS were both relatively low, 18 and 0·12 µmol (mg protein)-1 h-1, respectively. However with an active PoxB, the ACK activity increased 1·52·5-fold both in the presence or absence of the PDH complex, and the ACS activity likewise increased 2·5-fold in the presence of the PDH complex, but in the PDH-E1p null strains the increase was far greater, 1320-fold. This strongly suggests that ACS is induced to convert PoxB-derived acetate to acetyl-CoA for further metabolism. It may also be significant that the affinity of ACS for acetate is very much higher than that of ACK; Km=0·2 mM for ACS and 0·3 M for ACK (Brown et al., 1977
; Rose, 1962
).
Previous studies on the effects of PoxB deficiency had indicated that poxB mutants grew at normal rates (Chang & Cronan, 1983 ). In marked contrast, the poxB::kanR mutation of JRG3434 was here shown to lower the maximum growth rate by 16% relative to the parental strain. This discrepancy might have been due to the use of point mutants in the earlier study, some of which may retain some residual activity in vivo. The current findings were further supported by observing that the flux of carbon into biomass was 24% lower and the maximum yield (Ymax) 14% lower in the poxB mutant relative to the parental strain. These results clearly indicate that poxB contributes to the aerobic growth efficiency of E. coli. It is not known how this is achieved but PoxB may function as a safety valve to convert excess pyruvate to acetate rather than to acetyl-CoA, thus maintaining the intracellular CoA pool for other metabolic functions including the conversion of 2-oxoglutarate to succinyl-CoA and hence citric acid cycle activity. Likewise, the exclusive use of PDH complex may have a detrimental effect on cellular redox balance by lowering the NAD+/NADH ratio. It was anticipated that the involvement of PoxB in pyruvate oxidation would lower the growth efficiency rather than being beneficial. This is because direct coupling to the respiratory chain via the PoxB flavin cofactor would bypass the energy-conserving step associated with NADH oxidation as well as imposing the need to return acetate to the citric acid cycle via an ATP-dependent route. The energy conservation derived from NADH oxidation depends upon the extent to which each of the two NADH dehydrogenases (NADH:quinol oxidoreductases), NdhI and NdhII, are used. The first, NdhI or complex 1 (product of the nuo operon), is a proton-translocating enzyme complex whereas NdhII (the ndh gene product) is a non-proton-translocating flavoprotein (Gennis & Stewart, 1996
). Although both enzymes are expressed aerobically and repressed in fermentation, NdhI is essential for fumarate respiration and it is used preferentially during low aeration, in the late exponential and stationary phases of the growth cycle and during carbon limitation, when energy conservation is most needed, whereas NdhII is used preferentially during aerobic and nitrate respiration (Bongaerts et al., 1995
; Calhoun & Gennis, 1993
; Calhoun et al., 1993
; Green & Guest, 1994
; Tran et al., 1997
). Clearly the deficit in respiratory-chain derived energy will be directly related to the extent to which NdhI is used relative to NdhII (which is equivalent to PoxB) under the carbon-limiting growth employed in the work. But why does inactivation of the PoxB route impair the aerobic growth efficiency? It would appear that energy conservation per se is not a major factor. On the contrary, E. coli must derive some benefit from being able to convert pyruvate to acetyl-CoA by the seemingly wasteful route via acetate. It has been suggested that PoxB functions in the stationary-phase conversion of cell wall D-Ala-D-Ala dipeptide to acetate via pyruvate (Lessard et al., 1998
) but it seems unlikely that this could account for the physiological perturbations observed here. Although further studies are needed to elucidate exactly how PoxB contributes to the metabolic economy of E. coli, it is evident from the adverse energetic consequences of its inactivation that PoxB must perform a significant beneficial role in the aerobic growth efficiency of E. coli.
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ACKNOWLEDGEMENTS |
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Received 19 January 2001;
accepted 9 February 2001.