173 Biochimica et Biophysics Acta, 574 (1979) o Elsevier/North-Holland Biomedical Press

173-176

BBA Report BBA 51249

LYSOZYME

SENSITIVITY

LACTOBACILLUS

OF PANTOTHENATE-DEFICIENT GROWN WITH EXOGENOUS

PLANTARUM

FATTY

ACIDS

JOSEPH

T. HOLDEN,

WILLIAM

GRANT

and JOHN DEGROOT

Division of Neurosciences, (U.S.A.)

City of Hope National Medical Center, Duarte, CA 91010

(Received

1979)

February

19th,

Key words: Lysozyme sensitivity; Pantothenate medium; (Lactobacillus plantarum)

deficiency;

Lipid synthesis; Fatty acid

Summary A pantothenic acid deficiency in Lactobacillus pZantarum reduces lipid synthesis, prevents normal uptake and retention of extracellular amino acids and markedly increases sensitivity of these cells to lysozyme induced lysis. Pantothenatedeficient cells provided with exogenous fatty acids synthesize additional lipid and express nearly normal solute transport activities. The present study has shown that such cells retain a heightened sensitivity to lysozyme induced lysis. These observations indicate that the lysozyme sensitivity of pantothenate-deficient cells is not produced as an indirect effect of membrane lipid depletion, but represents an independent consequence of pantothenate insufficiency.

Growth of Lactobacillus planturum in a pantothenic acid limited medium produces several changes in cell structure and function. We have shown previously that such cells contain reduced amounts of lipid and that the uptake and retention of all amino acids is depressed, apparently because the membrane becomes hyperpermeable [ 1, 21. There are selective changes also in the initial uptake rates of several amino acid transport systems [3]. Although L. pluntarum is normally resistant to lysozyme (EC 3.2.1.17), pantothenatedeficient cells of this organism were found to be readily lysed by this enzyme, and to have a greatly increased capacity for binding this and other basic proteins [4] suggesting a change in cell wall structure. The permeability and transport changes associated with a pantothenate deficiency can be reversed in the continued absence of the vitamin by providing these cells

174

with any one of several fatty acids [ 5, 61. The intention of the present study was to determine whether the restoration of normal membrane function achieved in pantothenatedeficient cells by fatty acid supplementation is accompanied by reversal of the lysozyme sensitivity of the cell wall, or whether this property represents a separate facet of pantothenate deprivation unrelated to the cell membrane lipid and associated permeability changes. Pantothenatedeficient cells of L. plantarum 17-5 (ATCC 8014) were grown as described previously [ 1, 71 in a medium whose calcium pantothenate concentration was reduced from the normal level of 400 pg/l to 3.5--4.0 pg/l. Unsaturated fatty acids were added to the growth medium as ethanol solutions. The final fatty acid concentration was 0.1 mM. The final ethanol concentration was 0.95%. Serum albumin (essentially fatty acid free, fraction V, Sigma) was added at 2 mg/ml to disperse and detoxify the fatty acids. Pantothenate-limited cultures grown without fatty acid supplementation were harvested at 0.199-0.20 mg/ml (dry weight), whereas such cultures provided with unsaturated fatty acids attained cell densities of 0.4-0.5 mg/ml depending on the fatty acid used. The lysozyme sensitivity of washed cells as described previously [4] was measured at 37°C in 0.03 M Tris (pH 7.0) using lysozyme at 10 pg/ml, cells at 0.2 mg/ml and measuring the decline in absorbance at 640 nm. The fatty acid content of washed cells was determined after saponification with methanolic KOH using a calorimetric procedure essentially as described by Novak [ 81, but with a modification [ 91 of the cobalt reagent. The effect of incubation with lysozyme on L. plantarum grown under various conditions of pantothenate and fatty acid availability is illustrated in Fig. 1. Control, late exponential phase cells provided with an excess of this vitamin (complete) were insensitive to lysozyme. In contrast, cells cultured with grown-limiting levels of pantothenate were sensitive, as indicated by a progressive decline in cell suspension turbidity during incubation with this enzyme. Cells grown with inadequate amounts of pantothenate but with growth stimulatory amounts of cis-vaccenic acid were at least as sensitive to lysozyme as the pantothenate deficient, lipid deficient cells. cis-Vaccenate supplemented cells contain nearly normal amounts of lipid, transport amino acids at normal rates and retain large intracellular pools of exogenously accumulated glutamic acid [ 51 (Holden, J.T. et al., in preparation). Thus, reversal of the permeability and lipid content changes of pantothenate-deficient cells achieved by providing cis-vaccenic acid in the growth medium was not accompanied by a reduction in lysozyme sensitivity of these cells. As shown in Table I, pantothenatedeficient cells cultured with fatty acids of various chain lengths and degrees of unsaturation retained the marked lysozyme sensitivity characteristic of pantothenate-deficient cells. In several cases, (palmitoleic acid, elaidic acid) there was a suggestion of a slightly enhanced tendency towards cell lysis during lysozyme digestion. In all cases shown in Table I, the fatty acid and lipid contents of the pantothenatedeficient cells were markedly increased by providing fatty acid in the growth medium. Examination of extracted lipids by thin layer chromatography has shown that the supplied fatty acids were incorporated into all major lipids. Analysis of the fatty acid composition by gas chromatography

175 I

,

LOW PANT0 cis VACCENIC

I

+ ACID

I

I

/

30

60

90

TIME (mm) Fig. 1. L~soz~me sensitivity of pantothenate-sufficient and -deficient cells and pantothenate-deficient cells grown with cisvaccenic acid. Complete refers to cells grown in a medium containing 400 fig/l of pantothenic acid (panto) and no exogenous fatty acid. TABLE

I

LYSOZYME

SENSITIVITY

OF FATTY

ACID ENRICHED

PANTOTHENATE-DEFICIENT

L.

PLANTARUM

The fatty acid content of complete cells was 8.95 gmol/lOO mg. Reduction of initial absorbance after incubation with lysozyme (10 pg/ml) corrected for absorbance changes in the absence of lysozyme. Ceil type

Relative fatty acid content (90)

Absorbance %

for 60 min. Ail values were

reduction

Low Panto + F.A. Low Pant0 (%)

Complete Low pant0 Low Panto Low Panto Low Panto Low Panto Low Panto

+ + + + +

Palmitoleic acid (16:l) cis-Vaccenic acid (18:l) Oleic acid (18:l) Elaidic acid (18:lt) Linolelaidic acid (18:2t)

100 75 108 96 92 133 94

0 50 70 56 54 61 55

100 140 112 108 122 110

has shown that in some cases (elaidic acid, linolelaidic acid) the supplied fatty acid constituted 90% of the total lipid fatty acid (Holden, J.T. et al., in preparation). It is apparent, therefore, that providing pantothenate-deficient cells with preformed fatty acids, which increased the lipid content and promoted the restoration of normal, or nearly normal permeability properties, did not

176

have a comparable normalizing effect on the enhanced lysozyme sensitivity of pantothenate-deficient cells. It is likely, therefore, that the cell wall lysozyme sensitivity does not arise indirectly as a result of the reduced membrane lipid content, but originates instead in a change in cell wall structure produced directly by a deficiency of this vitamin. We have shown previously that a pantothenate deficiency reduced the cell wall 0-acyl content of L. plantarum [4] but that it did not significantly change the relative amounts of the major cell wall amino acid and carbohydrate components [lo]. The change in 0-acyl content could be caused by an expected reduction in the coenzyme A content of pantothenate-deficient cells, and a consequently reduced ability to carry out acetylation reactions. It has been shown previously that reduction of the cell wall 0-acyl content increases wall sensitivity to lysozyme [ll] , particularly in L. arabinosus (plantarum) [12] and L. fermenti [13]. These findings also suggest that not all CoA-dependent catalysts are equally impaired in such pantothenate-deprived cells. Although the acetylation of cell wall components and the de novo synthesis of fatty acids are markedly attenuated, these cells retain a diminished but functionally effective capacity to incorporate preformed fatty acids into lipids. The occurrence of additional changes in cell wall organization is suggested by the markedly enhanced binding of lysozyme and other positively charged proteins by cell walls from pantothenate-deficient cells [4] . This property also was not modified by fatty acid supplementation of pantothenate-deficient cells. Therefore, the results of the present study indicate that the modifications of cell wall structure which lead to enhanced binding of and digestion by lysozyme persist in pantothenatedeficient L.plantarum despite the restoration of an essentially normal cell membrane lipid composition. The authors are indebted to J. Easton, J. Bolen and W. Laffin for generous technical assistance during these studies. This investigation was supported by Public Health Service grant GM-20395 from the National Institute of General Medical Sciences, the Joseph Stefan Research Fund and by a Calbiochem summer fellowship to W.G. References 1

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Part VI,

Lysozyme sensitivity of pantothenate-deficient Lactobacillus plantarum grown with exogenous fatty acids.

173 Biochimica et Biophysics Acta, 574 (1979) o Elsevier/North-Holland Biomedical Press 173-176 BBA Report BBA 51249 LYSOZYME SENSITIVITY LACTOBA...
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