Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda boulv. 4,Riga LV-1586, Latvia1
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, S10 2TN Sheffield, UK2
Author for correspondence: Robert K. Poole. Tel: +44 114 222 4447. Fax: +44 114 272 8697. e-mail: r.poole{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: Zymomonas mobilis, cyanide sensitivity, respiratory protection, cytochromes, acetaldehyde
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INTRODUCTION |
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In order to gain more understanding of the role of respiration in aerobically growing Z. mobilis, we examined aerobic growth under conditions in which the respiration of cells is inhibited by submillimolar cyanide concentrations. Cyanide was chosen because it is one of the few water-soluble inhibitors able to cross the membranes of Z. mobilis, thus allowing it to be used in growing, intact cells. Furthermore, additional information might come from the fact that cyanide-sensitive and -resistant branches of bacterial respiratory chains often differ with respect to their energy-conserving efficiency (Poole, 2000 ; Poole & Cook, 2000
). Here we report the effects of cyanide on culture growth, respiration, acetaldehyde production and ethanol yield, aerobic ATP synthesis and cytochrome content in Z. mobilis.
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METHODS |
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Continuous cultivation.
Continuous cultivation was carried out in a Labfors fermenter (Infors), 800 ml working volume, at 30 °C, with aeration at 1 l min-1, and stirring at 410 r.p.m. The growth medium was the same as for batch cultivations, except that the glucose concentration was 25 g l-1. The flow rate (D) was set at 0·23 h-1. pH was maintained at 6·0 by automated additions of a 10% (w/v) KOH solution. Potassium cyanide solution (1 mM) was pumped into the culture separately from the growth medium (because cyanide is unstable in slightly acidic medium) at 10% of the medium flow rate (approx. 20 ml h-1), thus resulting in a continuous feed of 100 µM cyanide. Fresh cyanide stock solution was prepared every 4 h. Five fermenter working volumes were exchanged during transition between two steady states.
Preparation of cytoplasmic membranes.
Cells were disrupted by sonication (with a Soniprep ultrasonic processor) using seven periods of 1 min duration, each with 1 min intervals for cooling, if not stated otherwise. Subsequent removal of unbroken cells and pelleting of membranes by ultracentrifugation was done as described previously (Kalnenieks et al., 1993 ).
Cytochrome spectroscopy.
Room-temperature reduced minus oxidized difference spectra were taken using 1 ml samples of membrane suspension with small amounts of solid sodium dithionite as reductant and potassium ferricyanide as oxidant. Alternatively, no oxidant was added; these samples are named as prepared. Spectra were recorded using a custom-built SDB-4 dual-wavelength scanning spectrophotometer, as described previously (Kalnenieks et al., 1998 ; Eaves et al., 1998
).
Analytical methods.
Glucose was determined with a Sigma glucose oxidase kit, and ethanol with a Sigma alcohol dehydrogenase kit, following the manufacturers instructions. Oxygen consumption was measured at regular time intervals in 2 ml culture samples, which were rapidly transferred from the shaken flasks directly into a Clark electrode chamber (Rank Bros.) and their respiration monitored without external substrate addition. For non-growing cells, a small amount of suspension was added to buffer in the electrode chamber, to give about a 50-fold dilution. Potassium cyanide was added to the desired concentration and, after 510 min incubation, respiration was started by addition of ethanol at 5 g l-1 (final concentration). Acetaldehyde concentration was determined using the alcohol dehydrogenase reaction and monitoring absorbance change at 340 nm (Stanley et al., 1997 ). ATP in samples was assayed by the luciferinluciferase method, as described previously (Kalnenieks et al., 1995
). Protein concentration was determined according to Markwell et al. (1978)
. Cell concentration was determined as optical density at 550 nm in cells of 1 cm pathlength in a Jenway 6100 spectrophotometer, after appropriate dilution to maintain apparent absorbance readings below about 0·7. Dry cell mass of the suspensions was calculated by reference to a calibration curve. All experiments described are typical of several replicates. Standard errors of means are given as error bars in the figures.
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RESULTS |
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As anticipated by the scheme in Fig. 1, the high respiration rate of the aerobic control was accompanied by abundant accumulation of acetaldehyde in the culture medium (Fig. 5a
). The concentration of acetaldehyde reached more than 2 g l-1, which is known to cause severe inhibition of growth and metabolism in Z. mobilis (Wecker & Zall, 1987
). However, in a cyanide-treated culture with partially inhibited respiration, the generation of acetaldehyde started later and proceeded more slowly, so that during the cultivation acetaldehyde did not reach levels as high as those of the aerobic control (Fig. 5a
). Growth of Z. mobilis was sensitive to small changes in acetaldehyde concentration: addition of several small increments of acetaldehyde to one of the cyanide-treated cultures (Fig. 5c
, marked by the empty arrows) caused slight inhibition of growth relative to the culture that received only cyanide. Thus, stimulation of growth by cyanide was largely due to inhibition of acetaldehyde generation in the aerated cultures. Along with inhibition of acetaldehyde generation, cyanide increased ethanol yield (YEtOH) during exponential growth phase: for the control culture in Fig. 5
between the first and sixth hour of cultivation YEtOH was 0·26 g ethanol per g consumed glucose, while in the presence of 100 µM cyanide it reached 0·42 g per g glucose.
In exponential phase, molar growth yields (Yx/s), estimated from the data in Fig. 5 (between the first and sixth hour of growth) were low and did not correlate with the biomass densities reached in the stationary phase. Under our experimental conditions, in all cases the growth yields were below 10 g dry biomass per mol consumed glucose. The aerobic control culture showed the highest yield, 7·9 g dry wt per mol glucose. For the anaerobic control, the yield was 4·2 g dry wt per mol glucose, while the cyanide-treated aerobic cultures showed intermediate values, around 5·15·2 g dry wt per mol glucose.
Cyanide effect on aerobic growth and product formation in continuous culture
The parameters of chemostat cultivation with continuous feeding of 100 µM cyanide are presented in Table 1. Continuous culture in a fermenter offered two advantages over batch cultivation: (1) provision of a constant cyanide concentration and maintenance of growth parameters for prolonged periods; (2) vigorous gassing of the culture, efficiently removing acetaldehyde. Therefore, it was possible to study cyanide effects on an aerobic culture, which generated, but did not accumulate, volatile inhibitory by-products.
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ATP generation and kinetics of respiratory inhibition in cyanide-treated non-growing cells
As shown previously (Kalnenieks et al., 1993 ), non-growing cells of Z. mobilis are able to perform oxidative phosphorylation with ethanol as a substrate. Here, aerobic ethanol consumption in whole cells was used as a convenient experimental model for qualitative estimation of the ATP-generating capacity of the cyanide-sensitive respiration. We monitored the time course of ATP synthesis with ethanol as substrate in washed and concentrated cell suspensions obtained from aerobic exponential-phase cultures grown without cyanide addition (Fig. 6
). The time course of intracellular ATP concentration in a control suspension and in cells preincubated (510 min) with 100 µM cyanide is shown in the inset of Fig. 6(a)
. In cyanide-treated cells, intracellular ATP levels were raised within 10 s to a level about twofold higher than in control cells, but then declined. The columns in Fig. 6(a)
represent the ATP levels measured at 10 s in cells pretreated with various cyanide concentrations, relative to that of control cells. These data strongly indicate that the Z. mobilis cyanide-sensitive respiratory pathway is an energy-nongenerating bypass, since its inhibition causes electron flux through a pathway with an overall increase in ATP synthesis.
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Cyanide sensitivity of whole cells and cytoplasmic membranes
Whole cells and membrane preparations of Z. mobilis differed strikingly in their cyanide sensitivity (Fig. 6b). Even with 500 µM cyanide, the initial inhibitory effect upon membrane respiration with NADH was much weaker than that of 100 µM cyanide upon cells respiring ethanol. We hypothesized that some essential component of the cyanide-sensitive (rapidly inhibited) respiratory branch was either cytoplasmic, periplasmic or loosely bound to the cell membrane, and hence was easily lost in the process of membrane preparation. To test this, the supernatant fraction obtained after ultracentrifugation of the cell-free extract was concentrated 15-fold by ultrafiltration (molecular mass cut-off, 10 kDa) and examined spectroscopically. To avoid too vigorous a disruption of membranes, a cell-free extract of aerobic exponentially growing cells for this experiment was prepared by gentle sonication, i.e. four periods of 30 s with 1 min intervals for cooling.
In Fig. 7, dithionite minus as prepared difference spectra of membranes (A) and concentrated supernatant (B) are shown. Spectral features of a b-type haem were clearly present in the concentrated supernatant, namely an absorbance maximum at 559 nm in the
-region, and a Soret band at 427·5 nm. These signals are close to the respective absorbances in the membrane preparation. However, the signal of cytochrome d was evident only in the membrane difference spectrum. The prominent trough at 455 nm most probably corresponds to absorbance of an oxidized flavin moiety in the as prepared concentrated supernatant sample. In contrast to what has been shown for Z. mobilis membrane preparations (Kalnenieks et al., 1998
), as well as for the cytoplasmic flavohaemoglobin of E. coli (Ioannidis et al., 1992
), haem b in the Z. mobilis supernatant could not be reduced by NADH (not shown). This implies that a haem b-containing component in an intact cell might be loosely attached to the membrane and receive electrons from the electron transport chain, but not directly from NADH.
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DISCUSSION |
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Remarkably, cyanide stimulated the growth of Z. mobilis, which otherwise during aerobic batch cultivation became inhibited by accumulation of acetaldehyde. In shaken flasks, a large part of the generated acetaldehyde did not evaporate from the culture fluid and at the end of cultivation its concentration exceeded 2 g l-1. However, acetaldehyde accumulation in the medium seemed not to be the primary cause of growth inhibition in our experiments: we suggest that it was excess acetaldehyde generation inside the cells rather than its accumulation outside. This is supported by the observation that in a vigorously aerated continuous culture, in which acetaldehyde was efficiently gassed out, cyanide nevertheless stimulated growth. Apparently, in both modes of cultivation cyanide acted to prevent intracellular acetaldehyde generation/accumulation, shown by the increase of YEtOH and slowdown of glucose consumption (see Table 1 and the scheme in Fig. 1
).
An obvious conclusion from our data is that the high respiration rate in Z. mobilis is unnecessary for biomass growth: the rapid respiration does not supply any surplus energy for biosynthesis and growth, yet rapidly generates acetaldehyde (Fig. 1). The respiration might be also too rapid for the needs of respiratory protection, another vital physiological function of respiration in Z. mobilis proposed previously (Pankova et al., 1988
). Presumably, the increase of the lag phase at higher cyanide concentrations might be explained in part by the respiratory protection hypothesis, the lag being due to full inhibition of respiration. As seen in Fig. 3
, growth of the cyanide-treated cultures did not start while the specific respiration rate was close to zero. However, the cyanide-treated cultures then started exponential growth at a high specific rate when oxygen uptake rate was still well below half of that in the control culture (compare the growth and oxygen uptake rates between the fourth and seventh hour; Fig. 3
). The inhibitory effect of cyanide on the glucose consumption rate (explicitly seen in the continuous culture; Table 1
) also might contribute to the extension of the batch culture lag phase.
A cyanide-sensitive component of NADH oxidase activity, inhibited at around 20 µM cyanide, has been reported previously in membranes of aerobically grown Z. mobilis (Toh & Doelle, 1997 ), but no effect on membrane NADH oxidase activity could be seen at 20 µM cyanide in our experiments (not shown). This discrepancy might be explained by the different techniques of cell disruption used in each case. Toh & Doelle (1997)
disrupted cells with a French press, which might allow retention of a loosely membrane-bound component of the cyanide-sensitive branch. Sonication was used in our study. Preparation of cell-free extracts of glucose-grown E. coli by sonication can result in loss of dehydrogenases from the membrane into the soluble fraction with consequent loss of energy-transducing function (e.g. ATP-dependent NAD+ reduction) (Poole & Haddock, 1974
).
In the present whole-cell experiments, a large fraction of respiration was rapidly inhibited by low (e.g. 100 µM) cyanide concentrations (Fig. 6b). Its role in aerobic energetics of growing Z. mobilis cells is intriguing. Partial inhibition of cellular respiration by cyanide did not result in significant increase of biomass yield, contrary to what could be anticipated from our finding that the cyanide-sensitive pathway potentially was an energy-nongenerating bypass. However, respiratory inhibition by low cyanide concentrations was biphasic, with a rapid and a slower component (Fig. 6b
). It might therefore be essential to distinguish between cyanide-sensitive and rapidly inhibited by cyanide parts of Z. mobilis respiration. The former was inhibited during growth in the presence of low cyanide concentrations, while the latter was shown to be ATP-nongenerating in whole-cell experiments. The nature of this rapidly inhibited by cyanide, apparently energy-nongenerating, pathway of respiration in Z. mobilis cells and its possible relation to the haem b-containing compound found in the supernatant of cell-free extract needs to be established.
The physiological role of the rapid respiration in aerobically growing Z. mobilis cells is obviously other than ensuring biomass growth. It is tempting to think that the production of inhibitory metabolites, like acetaldehyde, is a competitive growth strategy of aerated Z. mobilis. Namely, Z. mobilis might prefer production of substances inhibitory for other bacteria at the expense of rapid growth of its own cell mass. Indeed, it is well established that Z. mobilis is inhibitory for other bacteria in interspecies conjugation experiments (Pappas et al., 1997 ), but this has been thought to be due to colicin production (Haffie et al., 1985
). For anaerobic cultures, the competitive growth strategy of Z. mobilis might be based in part on the reported very high specific rates of ethanol production together with an ethanol tolerance exceeding that of many other micro-organisms (Viikari, 1988
). A similar strategy for aerobic growth would then imply high, excessive respiration rates, and would lead to the observed low growth yields and self-inhibition.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 February 2000;
accepted 8 March 2000.