JOURNAL OF BACTERIOLOGY, June 1973, p. 1346-1350Copyright 0 1973 American Society for Microbiology

Vol. 114, No. 3Printed in U.S.A.

Internal Membrane Control in Azotobactervinelandii

JACK L. PATE, VINOD K. SHAH, AND WINSTON J. BRILL

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 22 February 1973

Azotobacter vinelandii was grown on N2, NH,+, or NO0-, and an internalmembrane network was observed by electron microscopy of thin sections of cells.Cells obtained in early exponential growth contained less internal membranethan did cells from cultures in late exponential growth. It seems likely that 02

has a role in regulating the amount of internal membrane structure.

When Azotobacter vinelandii is fixing N2, itsgrowth is inhibited by high concentrations of02, but this 02 sensitivity is not seen when thecells are growing in the presence of sufficientNH,+ to repress nitrogenase synthesis (2). Inhi-bition of growth by 02 has been attributed tothe extreme 02 lability of the protein compo-nents of nitrogenase (2). One hypothesis fornitrogenase protection in vivo is that nitroge-nase is conformationally protected from 02inactivation (2). When cells are broken with aFrench pressure cell, nitrogenase is quite 02insensitive until the components are separatedon a (diethylaminoethyl cellulose) column. Ithas been suggested that the conformation pro-tection was possibly mediated by reduced formof nicotinamide adenine dinucleotide dehydro-genase, which protects nitrogenase from 02 in-activation in vitro (15). Another protectivemechanism was hypothesized to be respiratoryprotection (2) by which 02 is reduced rapidly(presumably via the cytochromes) and therebyis unable to inactivate nitrogenase. In support ofthis hypothesis are the reports of Oppenheimand Marcus (9; J. Oppenheim and L. Marcus,Bacteriol. Proc., p. 63, 1970) and Hill et al. (5)who claim that an extensive internal membra-nous network is seen in electron micrographs ofsections of A. vinelandii that have been grownon N2 and that only slight quantities of internalmembrane, concentrated around the peripheryof the cell, are seen when cells are grown withexcess NH4+. This finding is not supported,however, by the work of Phillips and Johnson(10) who showed that the great O2 demand of A.vinelandii is independent of nitrogen source.This report contradicts previous work (5, 9; J.Oppenheim and L. Marcus, Bacteriol. Proc., p.63, 1970) and shows that the internal membrane

network is found in both N2- and NH4+-growncells.

MATERIALS AND METHODSThe strain used was A. vinelandii OP. Chemicals

and methods of culture and derepression have beendescribed previously (13, 14).

Cells were fixed with osmium tetroxide in Veronal-acetate buffer by the method of Kellenberger et al.(6). Fixed cells were dehydrated in a graded series ofethyl alcohol and propylene oxide and embedded inEpon 812 by the procedure of Luft (7). Thin sectionswere cut by using an ultramicrotome (Porter-BlumMT-2) and a diamond knife (E. I. DuPont de Ne-mours & Co. Inc.). Sections were collected on Parlo-dion and carbon-coated grids and stained for 10 minwith 2% aqueous uranyl acetate and with lead citratefor 10 min thereafter (11). Micrographs were madewith an electron microscope (Hitachi HUllE) at 75kV with a 50-Mm objective aperture. All figures arerepresentative of many fields.

RESULTSBy using techniques previously described (9,

13) we were unable to duplicate the results ofOppenheim and Marcus (9) and Hill, et al. (5)who found the extensive internal membranenetwork absent from cells that have been grow-ing on excess NH4+ or excess NaNO2. Themembrane network was present in thin sectionsof cells that had been grown on N2, ammoniumacetate, NH4Cl, and NaNO2 (Fig. 1). Bothcomponents of nitrogenase are completely re-pressed by the concentration of ammoniumacetate or NH4Cl used (13). It is unlikely,therefore, that these membranes have a solefunction of protecting nitrogenase from 02 inac-tivation.A condition that does seem to change the

amount of internal membrane material is the1346

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lIc , ifldFIG. 1. Sections of cells of A. vinelandii taken from exponentially growing cultures with different sources of

nitrogen. The cultures were harvested during exponential phase at a cell titer of 2 x 108/ml. The sources ofnitrogen were: la, air (N2); lb, 400 jg of nitrogen/ml as NH4 acetate, lc, 400 ug of nitrogen/ml as NH4CI; ld, 400,ug of nitrogen/ml as NaNO3. Arrows in la indicate internal membranes. The marker bar in la represents 1 um.All micrographs are at the same magnification.

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FIG. 2. Sections of cells of A. vinelandii grown on atmospheric nitrogen. The cultures were harvested atdifferent cell titers. Cell titers at time of harvest were 2.6 x 107/ml, 6 x 107/ml, and 1.7 x 108/ml for cells shownin 2a, 2b, and 2c, respectively.

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MEMBRANE CONTROL

cell population and rate of agitation of theculture, irrespective of nitrogen source. N2-grown cells (fig. 2a) were harvested at a low celldensity (2.6 x 107 cells/ml; dissolved 02 concen-tration was 6.5 ppm). The membrane networkseemed to be only at the periphery of the cells,but at a higher density of cells (6 x 107 cells/ml;dissolved 02 concentration was 3.2 ppm), themembranes seemed to become more predomi-nant (Fig. 2b). When cells reached a populationdensity of 1.7 x 108 Cells/ml, the dissolved 02

concentration was 0.6 ppm, and the extensivenetwork was observed (Fig. 2c). The same

phenomenon of membrane quantity varyingwith cell density also was seen with cells thathad been growing in a medium containingexcess NH4+. Cells grew at the same rate up tocell densities greater than 2 x 108 cells/ml. Atcell densities between 1 x 108 cells/ml and 2x 108 cells/ml, presence of the membrane net-work depended upon whether the growth flaskhad baffles and upon the speed of shaking. At a

given cell density, cells that were harvestedfrom a flask without baffles had a more exten-sive membrane network than did cells from a

flask with baffles.

DISCUSSIONAn indication that growth on N2 is not

required for formation of the internal mem-brane network has been reported by Zey et al.(P. Zey et al., Abstr. Ann. Meet. Amer. Soc.Microbiol., p. 155, 1972) who stated that thismembrane structure is found in cells grown on

N2 as well as on NH4+. However, they usedmethylamine to repress nitrogenase. Methyla-mine is capable of uncoupling phosphorylation(4) and does not repress nitrogenase synthesis(12). In support of the original (9) mpmbraneresults, Oppenheim and Marcus (J. Oppenheimand L. Marcus, Bacteriol. Proc., p. 148-149,1970) and Marcus and Kaneshiro (8) showedthat phospholipid content is greater in N2-grown cells than in NH4+-grown cells of A.vinelandii. However, Drozd et al. (3) claim tohave found no such differences in phospholipidcontent between cells of A. chroococcum thatare repressed or derepressed for nitrogenasesynthesis.The results presented in this paper indicate

that the internal membrane network might beproduced in response to 02 availability ratherthan to nitrogen source. The cells seem torespond to dissolved 02 concentration by syn-thesizing more membrane material when 02 islimiting. These membranes could function to

increase the surface area and could be able tosequester enough 02 to allow the bacteria toremain in the exponential-growth phase.A freshly inoculated culture of A. vinelandii

will have a shorter lag period if the culture is notshaken for several hours before it is placed onthe shaker (2). Ackrell and Jones (1) havereported that the length of the lag period isdirectly related to the rate of aeration of cells ofA. vinelandii. Our results offer a possible expla-nation for this phenomenon. Usually, an inocu-lum from a slant or turbid "overnight" cultureis used to inoculate fresh medium in which 02 isnot limiting. Cells that had been growing at ahigh density are suddenly diluted into freshmedium. These cells, therefore, are suddenly inan environment in which the surface area ofmembrane in contact with the dissolved 02 isgreater than that actually needed. Perhapsexcess respiration under these conditions has adetrimental effect on growth. Resumption ofgrowth might begin after these extra mem-branes are degraded.

ACKNOWLEDGMENTS

This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison, byNational Science Foundation grant GB36787, and by PublicHealth Service Grant GM-19859-01 from the National Insti-tute of General Medical Sciences.

LITERATURE CITED

1. Ackrell, B. A. C. and C. W. Jones. 1971. The respiratorysystem of Azotobacter vinelandii. 2. Oxygen effects.Eur. J. Biochem. 20:29-35.

2. Dalton, H., and J. R. Postgate. 1969. Effect of oxygen ongrowth of Azotobacter chroococcum in batch andcontinuous culture. J. Gen. Microbiol. 54:463-473.

3. Drozd, J. W., R. S. Tubb, and J. R. Postgate. 1972. Achemostat study of the effect of fixed nitrogen sourceson nitrogen fixation, membranes, and free amino acidsin Azotobacter chroococcum. J. Gen. Microbiol.73:221-232.

4. Good, N. E. 1960. Activation of the Hill reaction byamines. Biochim. Biophys. Acta 40:502-517.

5. Hill, S., J. W. Drozd, and J. R. Postgate. 1972. Environ-mental effects on the growth of nitrogen-fixing bacte-ria. J. Appl. Chem. Biotechnol. 22:541-558.

6. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Elec-tron microscope study of DNA-containing plasms. II.Vegetative and mature phage DNA as compared withnormal bacterial nucleoids in different physiologicalstudies. J. Biophys. Biochem. Cytol. 4:671-678.

7. Luft, J. H. 1961. Improvements in epoxy resin embeddingmethods. J. Biophys. Biochem. Cytol. 9:409-414.

8. Marcus, L., and T. Kaneshiro. 1972. Lipid composition ofAzotobacter vinelandii in which the internal mem-brane network is induced or repressed. Biochim. Bio-phys. Acta 288:296-303.

9. Oppenheim, J., and L. Marcus. 1970. Correlation ofultrastructure in Azotobacter vinelandii with nitrogensource for growth. J. Bacteriol. 101:286-291.

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10. Phillips, D. A. and M. J. Johnson. 1961. Aeration infermentations. J. Biochem. Microbiol. Technol. Eng.111:277-309.

11. Reynolds, E. S. 1963. The use of lead citrate at high pH asan electron-opaque stain in electron microscopy. J. CellBiol. 17:208-212.

12. St. John, R. T., and W. J. Brill. 1972. Inhibitory effect ofmethylalanine on glucose-grown Azotobacter vine-landii. Biochim. Biophys. Acta 261:63-69.

13. Shah, V. K., L. C. Davis, and W. J. Brill. 1972.Nitrogenase I. Repression and derepression of the

J. BACTERIOL.

Fe-Mo and Fe-proteins of nitrogenase in Azotobactervinelandii. Biochim. Biophys. Acta 256:498-511.

14. Shah, V. K., L. C. Davis, J. K. Gordon, W. H. Orme-Johnson, and W. J. Brill. 1973. Nitrogenase III.Nitrogenaseless mutants of Azotobacter vinelandii:activities, cross-reactions and EPR spectra. Biochim.Biophys. Acta 292:246-255.

15. Yates, M. G. 1970. Effect of non-haem iron proteins andcytochrome c from Azotobacter upon the activity andoxygen sensitivity of Azotobacter nitrogenase. FEBSLett. 8:281-285.

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