JOURNAL OF BACTERIOLOGY, Apr. 1972, p. 363-367 Copyright 0 1972 American Society for Microbiology Vol. 110, No. 1 Printed in U.S.A. Effect of Growth Conditions on Morphology of Hydrogenomonas facilis and on Yield of a Phospholipoprotein JOHN HEPTINSTALL,I HARRY G. RITTENHOUSE, BRUCE A. McFADDEN, AND LEWIS K. SHUMWAY Departments of Chemistry and Botany and the Genetics Program, Washington State University, Pullman, Washington 99163 Received for publication 4 January 1972 Hydrogenomonas facilis grown heterotrophically on fructose with very low aeration eventually ceased to divide and produced elongated forms. Short forms were obtained from fructose-grown long forms by increasing the availa- bility of oxygen to the organisms. A phospholipoprotein, the protein moiety of which is known to be present in the cell envelope, precipitated upon lowering the ionic strength of extracts from cells in the earlier stages of elongation (i.e., in the middle and late log phase of growth). The maximal yield of the protein moiety of the phospholipoprotein precipitate (i.e., grams of protein/grams of soluble protein x 100) was 2%. Poly-f.-hydroxybutyric acid accumulated as growth on fructose progressed, the accumulation being more marked with lower aeration. The soluble fraction derived from Hydrogen- omonas facilis, after heterotrophic growth on fructose with slow shaking, yields a quasi- crystalline phospholipoprotein upon lowering the ionic strength (7). The extremely hydro- phobic protein moiety of this product appears to be homogeneous and has a molecular weight of 15,000 daltons (7, 10). It is readily detect- able by immunological techniques in the cell envelope and is present in the cell wall (10). In recent work, it has been found that greatly elongated cells of H. facilis obtained after growth with extremely slow shaking did not yield phospholipoprotein after dialysis of extracts. Consequently, we now describe a sub- sequent, systematic study of the recovery of phospholipoprotein and morphological varia- tion of H. facilis. MATERIALS AND METHODS The origin, maintenance, culture, and turbidity measurements of H. facilis were as described by Kuehn and McFadden (6). Cultures grown in 300 ml of fructose medium were transferred to 6-liter Erlen- meyer flasks containing sufficient fructose medium to yield the final volumes specified. Aeration rates were measured by the sulfite oxidation method (2). The two extremes of aeration employed were 0.16 'Present address: Department of Biochemistry, Univer- sity of Leicester, Leicester, England. mmole of 02 per liter per hr (5.4-liter culture shaken at 120 rev/min) and 2.48 mmoles of 02 per liter per hr (4.1-liter culture shaken at 200 rev/min). The method of Kuehn et al. (7) was employed for preparation of phospholipoprotein, the final product being washed five times with water. Per cent yields are expressed as the mass ratio of phospholipo- protein per soluble protein x 100 (7). Dry weight de- terminations of phospholipoprotein were performed after the product was dried in vacuo over NaOH for two days at 60 C. The amino acid composition of various samples of phospholipoprotein was deter- mined after a 48-hr hydrolysis (7). RESULTS AND DISCUSSION In four replicate isolations from portions of the same batch of H. facilis cells, the following per cent yields of phospholipoprotein were obtained: 1.86, 1.62, 1.81, and 1.93. Thus, the reproducibility of recovery is ±4%. Experiments in which the organisms were grown in a volume of 5.4 liters of fructose me- dium at very low shake rates (110 to 140 rev/min) showed that the phospholipoprotein, if present, did not precipitate during dialysis of the extracts (Fig. 1). Cells from the sta- tionary phase of this culture were unbranched and considerably longer (about 12 ,um) than the rods present at the beginning of growth (about 2 ltm) and lacked motility, but the colo- 363 on June 22, 2018 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACTERIOLOGY, Apr. 1972, p. 363-367Copyright 0 1972 American Society for Microbiology
Vol. 110, No. 1Printed in U.S.A.
Effect of Growth Conditions on Morphology ofHydrogenomonas facilis and on Yield of a
PhospholipoproteinJOHN HEPTINSTALL,I HARRY G. RITTENHOUSE, BRUCE A. McFADDEN, AND LEWIS K.
SHUMWAYDepartments of Chemistry and Botany and the Genetics Program, Washington State University, Pullman,
Washington 99163
Received for publication 4 January 1972
Hydrogenomonas facilis grown heterotrophically on fructose with very lowaeration eventually ceased to divide and produced elongated forms. Shortforms were obtained from fructose-grown long forms by increasing the availa-bility of oxygen to the organisms. A phospholipoprotein, the protein moiety ofwhich is known to be present in the cell envelope, precipitated upon loweringthe ionic strength of extracts from cells in the earlier stages of elongation (i.e.,in the middle and late log phase of growth). The maximal yield of the proteinmoiety of the phospholipoprotein precipitate (i.e., grams of protein/grams ofsoluble protein x 100) was 2%. Poly-f.-hydroxybutyric acid accumulated asgrowth on fructose progressed, the accumulation being more marked with loweraeration.
The soluble fraction derived from Hydrogen-omonas facilis, after heterotrophic growth onfructose with slow shaking, yields a quasi-crystalline phospholipoprotein upon loweringthe ionic strength (7). The extremely hydro-phobic protein moiety of this product appearsto be homogeneous and has a molecular weightof 15,000 daltons (7, 10). It is readily detect-able by immunological techniques in the cellenvelope and is present in the cell wall (10).
In recent work, it has been found thatgreatly elongated cells of H. facilis obtainedafter growth with extremely slow shaking didnot yield phospholipoprotein after dialysis ofextracts. Consequently, we now describe a sub-sequent, systematic study of the recovery ofphospholipoprotein and morphological varia-tion of H. facilis.
MATERIALS AND METHODSThe origin, maintenance, culture, and turbidity
measurements of H. facilis were as described byKuehn and McFadden (6). Cultures grown in 300 mlof fructose medium were transferred to 6-liter Erlen-meyer flasks containing sufficient fructose mediumto yield the final volumes specified. Aeration rateswere measured by the sulfite oxidation method (2).The two extremes of aeration employed were 0.16
'Present address: Department of Biochemistry, Univer-sity of Leicester, Leicester, England.
mmole of 02 per liter per hr (5.4-liter culture shakenat 120 rev/min) and 2.48 mmoles of 02 per liter perhr (4.1-liter culture shaken at 200 rev/min).
The method of Kuehn et al. (7) was employed forpreparation of phospholipoprotein, the final productbeing washed five times with water. Per cent yieldsare expressed as the mass ratio of phospholipo-protein per soluble protein x 100 (7). Dry weight de-terminations of phospholipoprotein were performedafter the product was dried in vacuo over NaOH fortwo days at 60 C. The amino acid composition ofvarious samples of phospholipoprotein was deter-mined after a 48-hr hydrolysis (7).
RESULTS AND DISCUSSIONIn four replicate isolations from portions of
the same batch of H. facilis cells, the followingper cent yields of phospholipoprotein wereobtained: 1.86, 1.62, 1.81, and 1.93. Thus, thereproducibility of recovery is ±4%.Experiments in which the organisms were
grown in a volume of 5.4 liters of fructose me-dium at very low shake rates (110 to 140rev/min) showed that the phospholipoprotein,if present, did not precipitate during dialysisof the extracts (Fig. 1). Cells from the sta-tionary phase of this culture were unbranchedand considerably longer (about 12 ,um) thanthe rods present at the beginning of growth(about 2 ltm) and lacked motility, but the colo-
FIG. 1. The effect of varying the degree of aera-tion of cultures of Hydrogenomonas facilis on theyield of phospholipoprotein. Organisms were grownin varying volumes of fructose medium at shake ratesof either 110 to 140 rev/min (0), 120 to 150 rev/min(A), or 140 rev/min (-). Phospholipoprotein yieldwas determined after dialysis of extracts of culturesharvested at Klett values of 200 X 20.
nial morphology representative of cellsthroughout growth was invariant. The elonga-tion may have been due to the very low aera-tion rate, which was 0.16 mmoles of 02 perliter per hr at 120 rev per min, or to the de-crease in the pH of the medium to 6.4. Thatlow aeration was the cause was demonstratedby increasing the aeration of a culture of longforms with a resultant yield of short forms re-gardless of the initial pH of the mediumwithin limits of 6.0 to 7.2.
Although it appears that increasing aerationincreases the yield of phospholipoprotein (Fig.1), it should be emphasized that the cultureswere not harvested at exactly the same pointin the growth cycle. To examine the variationof yield during the growth cycle, organismswere grown in replicate 4.1-liter batches offructose medium shaken at 140 rev/min, andthe yield of phospholipoprotein was deter-mined throughout growth. It can be seen fromFig. 2 that the maximum yield was obtainedduring the middle to late log phase of growth.Examination of cells with the light microscopeduring growth under these conditions revealed
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FIG. 2. Recovery of phospholipoprotein (U) at dif-ferent stages in the growth cycle (0) of Hydrogeno-monas facilis. Growth of a 4.1-liter culture shaken at140 rev/min is represented (0). Similar cultureswere harvested at various times and the yields ofphospholipoprotein (U) and PHB (0) determined.For determinations of PHB content, culture samples(10 to 50 ml) were removed at various times, and theorganisms were collected by filtration on membranefilters (Millipore Corp.). PHB-containing inclusionswere extracted by the method of Williamson andWilkinson (16) with 25 ml of commercial liquidbleach (6% NaOCI content). The insoluble granuleswere filtered and treated with 10 ml of hot chloro-form, and the small amount of insoluble materialwas removed by filtration. After evaporation down toabout 1 ml, the PHB was precipitated from hot chlo-roform with 2.5 ml of ether, redissolved in I ml ofhot chloroform, and precipitated with 2.5 ml of coldwater. Finally, the precipitate was dissolved in 1 mlof hot chloroform and precipitated with 2.5 ml ofether before drying at 110 C. Crotonic acid produc-tion arising after addition of concentrated H2SO4was determined spectrophotometrically (8). Alterna-tively, the polymer was determined gravimetrically(16).
that some elongation was occurring. Electronmicrographs representing sectioned cells aftergrowth for 12 (early log phase) and 74 (earlystationary phase) hr are shown in Fig. 3a andb, respectively. Measurement with the electronmicroscope of the length of cells shadowedwith platinum-palladium alloy (not shown)after 12 hr of growth gave values ranging from1.76 to 2.94 Am for 50 organisms. In contrast,analogous measurements after 70 hr of growthgave a mean value of 5.49 gm for 50 organisms,with extreme values of 9.7 and 2.35 ,m. The
FIG. 3. (a) Sectioned preparation of Hydrogenomonas facilis in the early log phase of growth in 4.1 liters offructose medium shaken at 140 rev/min. The bar represents 1 Am. Samples were fixed in ice-cold 6% glutar-aldehyde in 0.2 M phosphate (pH 7.0) for 90 min, washed in buffer, and then postfixed in 1% OsO, in thesame buffer for another 90 min. After fixation, the specimens were washed in buffer, dehydrated in a gradedseries of ethanol and propylene oxide, and embedded in araldite 6005. Thin sections were stained with 1%aqueous Ba(MnO4)2 for 10 to 15 min and examined in an electron microscope (Zeiss EM 9A). (b) As Fig. 3a,but the sample was taken from the culture in the early stationary phase ofgrowth.
most striking difference noted between theshorter- and longer-sectioned cells (compareFig. 3a and b) was the predominance of largeelectron-lucid bodies in the latter. Sections ofcells from the mid-log phase were intermediatein this regard and at higher magnification re-vealed no. difference in envelope structure. Theappearance of these bodies could be correlatedwith an increased poly-,8-hydroxybutyrate(PHB) accumulation to a maximum value of42.6 mg/100 ml of the late log-phase culture(36% of the dry weight) in the culture shakenat 140 rev/min (Fig. 2). PHB accumulationduring growth at a higher shake rate of 200rev/min was reduced to a maximal value of28.3 mg of polymer per 100 ml of culture (20%of the dry weight).The phospholipoprotein could be obtained in
good yield from cultures grown at 140 rev/minon fructose through seven serial transfers. Cul-tures grown on 33 mM D-galactose, disodiumsuccinate, or L-glutamate gave 45, 25, and 30%yields, respectively, compared with that aftergrowth on fructose. Despite the variation inyield of the phospholipoprotein from H. facilisgrown under different conditions, the proteinmoiety was found, by amino acid analysis, to re-semble that demonstrated by Kuehn et al. (7).Comparison of the analysis with that of theoriginal isolate was made using the deviationfunction of Harris et al. (5). Values in therange of 0.028 to 0.041 established that thesamples were similar or identical. A similarcomparison for an unrelated protein, cyto-chrome c from Pseudomonas fluorescens (1),gave a value of 0.110. Protein accounted forabout 40% by weight of the phospholipoproteincomplex in all cases. Therefore the proteincomponent at the highest yield represents 2%of the total soluble protein of the 105,000 x gsupernatant fraction. The highest yield of phos-pholipoprotein was obtained with a buffer oflow ionic strength (7) for both extraction and di-alysis.The present study is of significance because
recovery of the phospholipoprotein in optimalyields should open the way to elucidation ofthe primary structure of the protein moiety.Antiserum to the phospholipoprotein, whichcontains no carbohydrate (7), agglutinates in-tact cells of H. facilis, and it has been estab-lished that the immune response is to the pro-tein. The protein is detectable in the cell enve-lope (14). Possibly analogous proteins havebeen recently found in several gram-negativebacteria (11, 12). In the present investigation,the highest yield of the phospholipoproteinfrom H. facilis was obtained at one interme-
diate shake rate from fructose-grown cells inthe mid-log phase of growth. Ultrastructuralstudies of cells from early log, mid-log, andearly stationary phase revealed no obvious dif-ferences in envelope structure. Greatly elon-gated cells obtained from the early stationaryphase after growth with slow shaking had ahigh PHB content and yielded little phospholi-poprotein. However, a detailed study of PHBcontent and phospholipoprotein recovery re-vealed no simple inverse correlation betweenthese two substances.
It is well known that the phospholipid com-position of normally growing bacteria varieswith the growth cycle (3, 4, 9). Similar changeshave also been demonstrated during elonga-tions of Escherichia coli (14). Moreover, limita-tion of oxygen has been found to give rise to anincrease in the cytochrome content of H. fa-cilis (6) and Haemophilus parainfluenzae (13),with associated modifications in phospholipidmetabolism in the latter organism (15). Sincephospholipid accounts for 50 to 70% of thequasi-crystalline product from H. facilis, thevariation in yield may be merely a reflection ofthe phospholipid composition of the cells.
ACKNOWLEDGMENTSWe thank A. Denend for conducting the analyses for
poly- t-hydroxybutryate.This investigation was supported by Public Health
Service predoctoral fellowship GM 43746 from the NationalInstitutes of Health to H.R. and by Public Health Serviceresearch grant AM 14400 from the National Institute of Ar-thritis and Metabolic Diseases, by Career DevelopmentAward 2-K3-AI-5,268, and by Graduate School ResearchFunds from the Public Health Service Biomedical SupportGrant Award to B.A.M.
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