Vol. 127, No. 1

JOURNAL OF BACTERIOLOGY, JUlY 1976, p. 168-178 Copyright © 1976 American Society for Microbiology

Printed in U.SA.

Lipids of Branhamella catarrhalis and Neisseria gonorrhoeae J. L. BEEBE'* AND T. J. WLODKOWSKI Department of Microbiology, Cornell University Medical College, New York, New York 10021

Received for publication 16 March 1976

Three strains of Branhamella catarrhalis and three strains of Neisseria gonorrhoeae were analyzed with regard to their phospholipid and neutral lipid composition. B. catarrhalis (ATCC 23246) contained 5.12 ± 0.34% lipid, determined gravimetrically, compared to 8.56 ± 0.15% and 9.73 ± 0.06% for two strains of N. gonorrhoeae. Cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine were identified in extracts of both species. In addition, B. catarrhalis contained small amounts of phosphatidylcholine, and N. gonorrhoeae contained small amounts of lyso-phosphatidylethanolamine, which accumulated with autolysis accompanying late cell culture growth. The kinetics of change of relative amounts of phospholipids in both species were measured and found to differ substantially. Neutral lipid accounted for 30.4% of the total lipid of B. catarrhalis (ATCC 23246) and 7.6% of the total lipid ofN. gonorrhoeae NYH 002. Hydrocarbons, triglycerides, free fatty acids, coenzyme Q, diglycerides, and free hydroxy fatty acids were identified in the neutral lipid fraction of both species. The three strains of N. gonorrhoeae, sensitive, intermediate, and resistant to penicillin, exhibited no significant difference in the composition or metabolism of phospholipid.

Until recent years, the Neisseria have received little attention with regard to their lipid composition (16). This situation is being rapidly rectified, as a number of reports have begun to appear in the literature that have contributed significantly to our knowledge of this genus, especially with regard to N. gonorrhoeae. Moss et al. (24) have reported a definitive analysis of the fatty acid composition of N. meningitidis and N. gonorrhoeae. Subsequently, Lambert et al. (18) described the fatty acid composition of a number of nonpathogenic Neisseria species. Noteworthy in these reports is the observation of substantial amounts of hydroxy fatty acids in the extracts of all species examined. Regarding the phospholipids of Neisseria, little has been reported until recently. In the 1940's Stokinger et al. (29) examined N. gonorrhoeae lipids and reported the presence of phosphatidylcholine (PC) and sphingomyelin. Sud and Feingold (30) recently described an analysis of the lipid composition of N. gonorrhoeae and reported the presence of PC as well as cardiolipin (CL), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG). Walstad et al. (32) reported the identity of inhibitory substances produced by various strains of gonococci and included in their analysis an exami' Present address: Clinical Microbiology Service, Columbia-Presbyterian Medical Center, New York, N.Y. 10032.

nation of the lipid composition of N. gonorrhoeae. They did not detect PC, but they did find large amounts of lyso-Pe. We present here a definitive analysis of the phospholipid and neutral lipid composition of Branhamella catarrhalis and N. gonorrhoeae. The strains of N. gonorrhoeae employed exhibited varying levels of resistance to penicillin, and the existence of correlative elements of lipid composition to antibiotic resistance was also evaluated. (This work was reported in part previously [Bull. N.Y. Acad. Med. Abstr. 51:1148, 1975] and was presented at the Annual Meeting of the American Society for Microbiology, Chicago, Ill., 12 to 17 May 1974.) MATERIALS AND METHODS Cultures. The strains of B. catarrhalis employed were ATCC strains 23246, 8176 and 8193; the latter two were generously supplied by R. Weaver of the Center for Disease Control, Atlanta, Ga. The strains of N. gonorrhoeae employed were a strain supplied by R. Weaver designated F19 and clinical isolates of the New York Hospital Microbiology Laboratory that were designated NYH 002 and NYH 006. After reception the strains were examined for colonial and microscopic morphology, Gram stain, oxidase test, and fermentation of appropriate sugars. Cultures were maintained for short duration by daily (N. gonorrhoeae) or biweekly transfer (B. catarrhalis) on GC medium base agar plates supplemented with 1% supplement B (Difco; GCBDS) incubated at 35 C 168

VOL. 127, 1976

LIPIDS OF B. CATARRHALIS AND N. GONORRHOEAE

in a candle jar. Strains were maintained over long periods by freezing suspensions of cells in 1% peptone-20% glycerol at -60 C as suggested by Ward and Watt (33). Cultivation of cells. GCBDS plates were inoculated by loop and incubated for 18 to 24 h in a candle jar at 35 C. Subinoculations were made from these plates to either solid medium (GCBDS plates) or liquid medium. The liquid media employed in this study were designated (i) LGCBDS (which consisted of: proteose peptone no. 3 [Difco], 15 g; K2HPO4, 4 g; KIH2PO4, 1.0 g; soluble starch, 1.0 g, and NaCl, 5 g; diluted to 1 liter with distilled water) and (ii) LGCBDS(P10) (which has the same formulation as LGCBDS with the exception of the phosphates, which are reduced 10-fold to: K2HPO4, 0.4 g; and KH2PO4, 0.1 g). Cultivation and harvest of cells on agar plates were accomplished as follows. Plates were inoculatd by swab from a seed plate and incubated in candle jars at 35 C for 18 h. Growth was removed from the plates by a gentle stream of distilled water, harvested by centrifugation at 1,500 x g for 15 min in an International model CS centrifuge, washed, and recentrifuged. Cultivation of cells on liquid media was accomplished by loop inoculation of 50 ml of medium and incubation at 35 C in an American Optical reciprocal water bath shaker at 90 cycles/min for 12 to 18 h. Suspensions of cells with an optical density at 600 nm of 1.0 were used to inoculate 100-ml portions of medium in 250-ml Erlenmeyer flasks that were incubated under the conditions described. Isotopic labeling of cellular lipids. Carrier-free H332PO4 (New England Nuclear, Boston, Mass.) was added at a rate of 0.5 to 1.0 ,uCi/ml of culture medium to label cellular phospholipid during growth. [U-14C]glucose (200 Ci/mol) was added to culture medium Lt a rate of 0.5 ,uCi/ml to label all cellular lipids. In experiments using [U-_4C]glucose to label cellular lipid, unlabeled D-glucose was added to culture medium as a sterile solution to a concentration of 0.1%. Lipid extraction and purification. Lipid was extracted from cells by the extraction method of Folch et al. (8), and nonlipid contaminants were removed by the method of Wells and Dittmer (34) modified as follows. Chloroform-methanol, 2:1 (vol/vol), was added to cells at a ratio of 100 ml per g of cells (wet weight). The mixture was stirred mechanically at room temperature under nitrogen gas for 3 h. The extract was filtered through Whatman no. 42 filter paper, reduced to dryness with a rotary evaporator in a 45 C water bath, and suspended in chloroformmethanol-water, 60:30:4.5 (vol/vol/vol). The solution was loaded onto a 5-g Sephadex G25 column (1 by 10 cm) previously equilibrated with chloroform-methanol-water, 60:30:4.5 (vol/vol/vol), and eluted with chloroform-methanol, 2:1 (vol/vol). Additional chloroform and water were added to the eluate in a separatory funnel. The mixture was shaken, the chloroform phase was drawn off, and the water phase was reextracted with an additional volume of chloroform. The chloroform phases were pooled, reduced by rotary evaporator to a viscuous oil, dried in vacuo over P205 for 24 h, and weighed. Samples of

169

purified lipid were dissolved in small volumes of chloroform-methanol, 2:1 (vol/vol), and stored at 0 C. The percentage of lipid was determined by calculation from weights of lipid extracts dried in vacuo and accompanying cell samples dried at 80 C overnight in aluminum weighing dishes. Fractionation and isolation of individual lipids. Silicic acid (Mallinckrodt, 100 mesh) was treated to remove fines, acetone washed, dried and activated by heating at 110 C overnight, and stored in a desiccator over CaCl2 (11). Typically a 10-g column (1 by 20 cm) fitted with a Teflon stopcock was prepared and washed with several bed volumes of chloroform. A sample of purified lipid was dissolved in chloroform and loaded on the column on the top of which a perforated Teflon disk was placed to prevent disturbance of the column bed. The lipids were eluted in discontinuous fashion by the addition of 100-ml portions of the following solvent mixtures: chloroform, chloroform-methanol, 94:6 (vol/vol); chloroform-methanol, 4:1 (vol/vol); chloroform-methanol, 2:1 (vol/vol); and chloroform-methanol, 1:1 (vol/vol). Flow rates of 1.0 to 2.0 ml per min permitted satisfactory fractionation. As a rule, no more than 5 mg of lipid per g of silicate was loaded on the column in a fractionation. Fractions were collected in 50-ml portions once it had been established that smaller fractions offered no particular advantage to separation of the various lipids. The individual fractions were reduced to dryness and either stored at 0 C or subjected to thin-layer chromatography (TLC) to isolate pure samples of the individual lipids. TLC and paper chromatography. Pure samples of the individual lipids were obtained by chromatography of the silicic acid column fractions on Silica Gel G thin-layer plates (Analtech; 20 by 20 cm, 250 jum) in one of the following solvent systems: (i) chloroform-methanol-water-acetic acid, 65:25:4:1 (vol/vol/ vol/vol), or (ii) chloroform-methanol-7 N ammonia, 60:35:5, (vol/vol/vol). Rf values for the pure lipids were also obtained using these systems, and twodimensional separation of whole cell lipid was accomplished by using system (ii) in the first direction and, after drying, system (i) at right angles to the first. Lipids were visualized by exposure to iodine vapor (2) or by spraying dried plates with 50% sulfuric acid and heating at 150 C for 30 min (2). Chromatography of neutral lipid was performed by a two stage thin-layer system using successively (i) isopropyl ether-acetic acid, 96:4 (vol/vol), for 12 cm with drying of the plate followed by (ii) petroleum ether-ethyl ether-acetic acid, 90;10:1 (vol/vol/ vol), for 17 cm. Quantitation of the neutral lipids was performed as described previously (3). The purified lipids were chromatographed on silicic acid-impregnated paper (Whatman SG81) in ascending fashion using the following solvent systems: (i) diisobutyl ketone-glacial acetic acid-water, 40:20:3 (vol/vol/vol); and (ii) diisobutyl ketone-glacial acetic acid-water, 40:25:5 (vol/vol/vol) (22). Samples of the individual lipids were deacylated by the alkaline methanolysis procedure of White (35), and the water-soluble products of the hydrolysis were chromatographed on Whatman no. 1

170

BEEBE AND WLODKOWSKI

paper in an ascending fashion using the following solvent systems: (i) methanol-formic acid-water, 80:13:7 (vol/vol/vol) (7); (ii) n-butanol-propionic acid-water, 142:71:100 (vol/vol/vol) (4); and (iii) phenol-water, 100:38 (wt/vol) (6). Visualization on thin-layer plates was accomplished by sulfuric acid, charring iodine vapor, ninhydrin, or a-naphthol reagent for glycolipids (17). Paper chromatographs were examined for phosphorus (2), choline (26), phospholipid by rhodamine 6G and ninhydrin (2), and vicinal hydroxyls by periodate-Schiff (31). Lipid quantitation. Lipid extracted from cells labeled with either H332P04 or [U-C'4]glucose was subjected to two-dimensional TLC on Silica Gel G plates. Dried plates were exposed to Kodak RP14 X-ray film for periods of 12 to 96 h and developed to locate labeled lipids. Areas of silica gel containing labeled lipid localized by radioautography or by iodine vapor were removed from the plate and eluted with chloroform-methanol, 1:1 (vol/vol), bye the method described by Ames (1). Eluates were dried in scintillation vials, and scintillant [consisting of 5-phenyloxazole, 4 g; 2,2-para-(phenylenebis)-5-phenyloxazole, 50 mg; and toluene, 1 liter] was added. Activity of samples was measured with a Packard model 3003 Tri-Carb liquid scintillation spectrometer. Counting efficiencies were 96% for 32p and 80% for 14C. Activities were corrected for quenching by the method of channel ratios. Removal of salt forms. Samples of lipid were dissolved in chloroform-methanol, 1:1 (vol/vol), and partitioned against 0.9 volume of aqueous 0.1 N HCl. The chloroform phases was withdrawn, and the aqueous phase was reextracted with two additional volumes of chloroform. The chloroform phases were pooled and neutralized with methanolic 0.2 N NH40H. When reduced in volume this preparation was free of salt forms of phospholipids detectable by the chromatographic systems described. Chemical analyses. Protein was determined by the method of Lowry et al. (21) using bovine serum albumin as a standard. Ultraviolet absorption spectra of neutral lipids was determined with chloroform solutions in a Beckman model DU spectrophotometer using 1-cm quartz cuvettes. Determination of MIC. Minimal inhibitory concentrations (MICs) for a variety of antibiotics were determined by the plate-dilution technique as described by Gavan et al. (9) and using a Steers replicator (28). The following antibiotics were tested: ampicillin, sodium cloxacillin, kanamycin sulfate, sodium methicillin, sodium oxacillin, and tetracycline hydrochloride (generously supplied by Bristol Laboratories, East Syracuse, N.Y.); sulfadiazine, sulfamerazine, sulfamethazine, neomycin sulfate, phenoxymethyl penicillin, erythromycin, cephaloridine, and cephalothin (gifts of Eli Lilly & Co.); sodium nafcillin and potassium penicillin G (gifts of Wyeth Laboratories, Philadelphia, Pa.); and naladixic acid (a gift of Sterling Drug, Inc., Rensselaer, N.Y). Chemicals. The chemicals which were employed are as follows: cardiolipin (Nutritional Biochemicals Co., Cleveland, Ohio); phosphatidic acid, PG, PE, and PC (Supelco, Inc., Bellafonte, Pa.); dimethyl-

J. BACTERIOL.

PE (Mann Biochemicals); monomethyl-PE (isolated by extraction of whole cells of Thiobacillus thiooxidans and purified by TLC). [U-_4C]glucose (International Chemical and Nuclear, Inc., Waltham, Mass.; and carrier-free [32P]phosphoric acid (New England Nuclear Corp., Boston, Mass.). Chloroform, methanol, acetone, and phenol were redistilled before use in chromatography or lipid extraction.

RESULTS Lipid content. Cultivation of B. catarrhalis ATCC 23246 on GCBDS agar plates for 18 h yielded cells containing 5.12 + 0.34% lipid by weight. In marked contrast, cells of N. gonorrhoeae NYH 022 and NYH 006 contained, respectively, 8.56 + 0.15% and 9.73 ± 0.06% lipid. Each figure represents the average of three trials. Cultivation of these strains on LGCBDS medium yielded cells with similar lipid contents.

When samples of purified whole cell lipid extracts were subjected to two-dimensional TLC and visualized by acid charring, the results shown in Fig. 1 were obtained. Figure 1A depicts the appearance of a typical chromato-

graph of B. catarrhalis whole cell lipid. The lipids visualized were assigned the arbitrary numerical designation shown. The lipid designated as 3 was not always observed and is represented by a broken line circle. The appearance of an extract of N. gonorrhoeae is shown in Fig. 1B. The individual lipids were assigned the letter designations shown. Fractions obtained from silicic acid column chromatography of lipid extracts were subjected to two-dimensional TLC to evaluate efficiency of separation. Lipid 1 from B. catarrhalis and lipid A from N. gonorrhoeae were effectively eluted by chloroform from silicic acid. Lipids 2, 3, 4, B, and C were eluted by chloroform-methanol, 94:6 (vol/vol). Lipids 6, 8, E, and G predominated in eluates of chloroform-methanol, 4:1 (vol/vol), and lipids 5 and D were eluted most effectively by chloroformmethanol, 2:1 (vol/vol). Elution with chloroform-methanol, 1:1 (vol/vol), removed the remaining lipids, 7 and F, from the column. Pure samples of individual lipids were obtained by repeated TLC of the silicic acid fractions in different TLC systems. Analytical paper chromatography and TLC were performed on purified samples of each lipid and appropriate standards. Table 1 summarizes the data obtained from these studies. A sample of each lipid was deacylated, and the water-soluble products of hydrolysis were chromatographed in three different systems using Whatman no. 1 paper and visualized by ammonium molybdate reagent for phosphorus

LIPIDS OF B. CATARRHALIS AND N. GONORRHOEAE

VOL. 127, 1976

A 2 3 1

1

7

(2)

8

(1)

B B

F

G

FIG. 1. Two-dimensional TLC of whole cell lipid extract of B. catarrhalis ATCC 23246 (A) and N. gonorrhoeae NYH 002 (B). Solvent systems used were (1) chloroform-methanol-7 N ammonia, 60:35:5 (vollvol/vol), in the first direction and (2) chloroform-methanol-water-acetic acid, 65:25:4:1 (vollvol/vollvol), in the second direction.

171

graph with an authentic standard. Lipid 3 is phosphatidic acid; lipids 5 and D are PE; and lipids 6 and E are PG. Lipids 7 and F appeared to be identical in all respects to PG, with the exception of their behavior on thin-layer systems, where 7 and F had dissimilar Rf values and frequently showed contamination with very small amounts of PG. It was observed that treatment of whole cell lipid extracts with acidified solvent designed to remove salt forms of lipid resulted in the apparent disappearance of lipids 7 and F from the chromatograph. On this basis it is believed that lipids 7 and F are salt forms of PG. Lipid 8 obtained from B. catarrhalis is identified as PC on the basis of chromatographic mobility on paper and thin-layer systems, a characteristic yellow color produced by staining of the lipid by rhodamine 6G, and a positive Dragendorf test. Lipid G was identified as lysoPE on the basis of a positive ninhydrin test of whole and deacylated forms, characteristic pink staining reaction with rhodamine 6G, and R, values on paper and thin-layer systems compared to an authentic standard. Lipids 2 and B, which have nearly identical chromatographic mobility on both paper and thin layer, were not identified. They contain no phosphate, free amino groups, or choline groups. These lipids gave negative reactions for both the periodate test for vicinal hydroxyl and the a-naphthol test, indicating the absence of a carbohydrate moiety. No identifiable products were obtained by deacylation, and no characteristic color was obtained by staining the lipids with rhodamine 6G. In addition to the lipids identified, small amounts of other lipid appeared irregularly on chromatographs and were assumed to be lysoforms on the basis of chromatographic mobility. Lipid quantitation. Lipids of the various strains of B. catarrhalis and N. gonorrhoeae were quantitated on the basis of incorporated '4C and 32P labels after 18 h growth at 35 C (Table 3). It was determined that a 10-fold reduction of the amount of phosphate in LGCBDS medium had no appreciable effect on growth kinetics or cell yield and resulted in a 3.4-fold increase in the amount of radioactivity incorporated into lipid. LGCBDS(P10) medium was used in all 32P-labeled lipid quantitation stud-

radioautography. Authentic lipid samples deacylated, and the water-soluble phosphoesters were employed as standards (Table 2). On the basis of the data obtained, the lipids were identified as follows. Lipids 1 and A are ies. neutral lipid on the basis of their mobility and Although B. catarrhalis will not ferment glulack of reactivity to the various reagents. Lip- cose, i.e., take up and metabolize the sugar ids 4 and C are cardiolipin on the basis of their with production and excretion of acid products, chromatographic mobility on paper and thin- the species will utilize the monosaccharide, and layer systems and their ability to co-chromato- ['4C]glucose label is incorporated into all the or by were

172

BEEBE AND WLODKOWSKI

J. BACTERIOL.

TABLE 1. Characteristics of the lipids of B. catarrhalis and N. gonorrhoeae Qualitative testsd TLCb PChc Lipida

1 A NL

R, 1 0.95 0.95 0.95

Rf 2 0.95 0.95 0.95

Rf 3 0.95 0.95 0.95

Rf 4 0.95 0.95 0.95

2 B

0.65 0.65

0.90 0.90

0.30 0.30

0.41 0.41

3 PA

0.65 0.66

0.78 0.78

0.45 0.45

0.54 0.55

+ +

-

-

-

-

4

C CL

0.85 0.85 0.85

0.85 0.85 0.85

0.60 0.59 0.60

0.67 0.62 0.67

+ + +

5 D PE

0.55 0.42 0.55

0.63 0.45 0.63

0.35 0.49 0.35

0.48 0.53 0.48

6 7 E F PG

0.65 0.27 0.60 0.30 0.65

0.52 0.40 0.35 0.25 0.54

0.30 0.30 0.35 0.48 0.30

8 PC

0.25 0.28

0.35 0.30

0.18 0.18

G

0.20 0.22

0.20

Lyso-PE

0.22

DPE MPE

0.63 0.43

0.45 0.50

0.16 0.30

P

I04

Nin

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

R6G Brown Brown Brown

Chol

Gly

-

-

-

-

-

-

Blue Blue

-

-

-

-

Blue Blue

-

-

-

-

-

-

-

-

-

-

Blue Blue Blue

+ + +

-

+ + +

Pink Pink Pink

-

-

-

-

-

-

0.36 0.35 0.41 0.56 0.36

+ + + + +

+ + + + +

-

-

-

-

-

-

Blue Blue Blue Blue Blue

-

-

0.45 0.45

+ +

-

-

Yellow Yellow

+ +

-

-

0.18

0.22

0.19

+ +

-

0.16

-

+ +

Pink Pink

_ _

0.39 0.45

+ +

-

+

+

Pink Pink

_

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

a Lipid standards were: cardiolipin (CL); phosphatidic acid (PA); phosphatidylglycerol (PG); phosphatidylethanolamine (PE); monomethylphosphatidylethanolamine (MPE); dimethylphosphatidylethanolamine (DPE); phosphatidylcholine (PC); neutral lipid (NL); and lyso-phosphatidylethanolamine (lyso-PE). b Rf values were obtained from TLC using chloroform-methanol-7 N ammonia, 60:35:5 (vol/vol/vol), for Rf 1 and chloroform-methanol-water-acetic acid, 65:25:4:1 (vol/vol/vol/vol) for Rf 2. e Rf values were obtained from paper chromatographs (PCh) using diisobutylketone-acetic acid-water, 40:20:3 (vol/vol/vol), for Rf 3 and diisobutyl ketone-acetic acid-water, 40:25:5 (vol/vol/vol), for Rf 4. d The qualitative tests were conducted on the chromatograms to detect phosphate (P); vicinal hydroxyls by periodate-Schiff (IO4); free amino groups by ninhydrin (Nin); phospholipid by rhodamine 6G (R6G); choline (Chol) by Dragendorf; and glycolipids (Gly) by a-naphthol reaction. These data were obtained from treatment of paper chromatograms, except for the a-naphthol reaction, which was performed on TLC plates.

various lipids present in the organism. N. gonorrhoeae readily incorporates ['4C]glucose label into lipid. Approximately 1.0 and 0.5% of available label is present in cells of B. catarrhalis and N. gonorrhoeae, respectively, after 18 h of growth in LBCBDS(P10) medium. Approximately 1 to 3% of cellular label is lipid extractable. About 30% of cellular lipid of B. catarrhalis ATCC 23246 is neutral lipid on the basis of incorporated carbon label (Table 3). This contrasts markedly with N. gonorrhoeae, which has less than 8% of incorporated activity as neutral

lipid. There is reasonable agreement between the lipid composition obtained using 14C and 32P regarding quantitation of individual phospholipid ofB. catarrhalis ATCC 23246 and N. gonorrhoeae NYH 002. B. catarrhalis ATCC 23246 contains nearly equal amounts of PG and PE, whether calculated on the basis of incorporated carbon or phosphorus. This strain contains small amounts of CL, PC, and the unidentified lipid 2. PE and PG predominate in strains ATCC 8176 and ATCC 8193 as well, although the amounts may vary considerably. The difference in the amounts of CL is of doubtful signifi-

VOL. 127, 1976

LIPIDS OF B. CATARRHALIS AND N. GONORRHOEAE

TABLE 2. Chromatographic mobility of the deacylated lipids of B. catarrhalis, N. gonorrhoeae, and authentic standards R,b

Lipid

Methanolformic acidwater

Butanol-propionic acidwater

Phenol-water

GP

0.66 0.66

0.19 0.19

0.17 0.17

4 C

GPGPG

0.45 0.44 0.42

0.04 0.05 0.05

0.11 0.11 0.10

6 E 7 F GPG

0.34 0.35 0.34 0.34 0.34

0.17 0.14 0.16 0.16 0.17

0.40 0.39 0.40 0.40 0.40

5 D G GPE

0.46 0.45 0.46 0.46

0.23 0.23 0.24 0.23

0.60 0.60 0.60 0.60

8 GPC

0.61 0.62

0.25 0.29

0.66 0.69

3

173

cance if one takes into account the amounts of molar phosphate present in each lipid. A small amount of PA is present in strain 8193, and small amounts (0.6 to 4.0%) of the salt form of PG are obtained from each strain. The results indicate that N. gonorrhoeae differs substantially from B. catarrhalis. PE is overwhelmingly the predominant lipid of N. gonorrhoeae, accounting for 66 to 82% of the total phospholipid, depending on the strain. This is more than twice the contribution of PE in B. catarrhalis. PG and CL are present in smaller quantities, and PC was not detected in N. gonorrhoeae. N. gonorrhoeae contains easily detected and reproducible levels of lyso-PE, which accumulates with culture age. The amount of lyso-PE can be as great as 15% of the total phospholipid (strain F19, Table 3). Small amounts of PG salt forms are present in all strains. PA was not detected. Strain NYH 002 contained a small amount (0.5%) of unidentified lipid B. Effect of culture age on phospholipid composition. When samples of cells of N. catarrhalis ATCC 23246 and N. gonorrhoeae NYH 002 grown on LGCBDS(P10) medium containing [32Plphosphoric acid were extracted and analyzed for lipid, the results shown in Fig. 2 were seen. By mid-log phase of growth of B. catarrhalis, PE accounted for 50% of the total phospholipid, and PG accounted for less than 30%. Subsequently PE declined and PG increased, until there were nearly equal amounts at 24 h of growth. Cardiolipin declined slightly during this period. There was little discernible change in the amount of PC and PG salt form. A totally different situation was obtained in

a Standards were ca-glycerol phosphate (GP) and glycerylphosphorylethanolamine (GPE) (commercial products); 1,3-diglycerylphosphorylglycerol (GPGPG), glycerylphosphorylglycerol (GPG), and glycerylphosphorylcholine (obtained by mild alkaline methanolysis of diacyl lipid standards [351). b Rf values were determined for methanol-formic acid-water, 80:13:7 (vol/vol/vol); n-butanol-propionic acid-water, 142:71:100 (vol/vol/vol); and phenolwater, 100:38 (wt/vol). TABLE 3. Lipid content of B. catarrhalis and N. gonorrhoeae

Total incorporated lipid radioactivity (%) a from strain: Lipid

ATCC 23246 14Cbf

32p

ATCC 8176

ATCC 8193

32p

32p

NYH 002 14C

NYH

F19

32p

32p

006

32p

NL 30.4 7.6 PA 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 CL 6.6 17.5 20.4 29.1 4.7 8.6 5.9 6.0 PE 25.2 38.1 23.1 43.7 71.6 74.2 82.2 66.9 PG 32.9 39.2 48.6 25.9 15.4 9.1 15.4 11.9 PC 3.2 3.0 4.2 0.6 0.0 0.0 0.0 0.0 0.0 Lyso-PE 0.0 0.0 0.4 0.0 1.8 2.2 14.9 PG (salt form) 0.8 1.8 4.0 0.6 0.4 1.1 0.4 1.3 Unidentified 2.0 0.5 a Values are percentages of total lipid radioactivity of 18-h-old cells using either [U-_4C]glucose or H332PO4 as precursor cultivated in LGCBDS(P10) medium at 35 C on a water bath shaker. The medium contained 0.1% 1-glucose added as sterile solution in the experiments using [U-'4C]glucose labeling. Values are an average of three separate trials. b Isotope used.

174

BEEBE AND WLODKOWSKI

J . BACTERIOL .

1.S E -

" F

I-49 I-

z

t 0

I"

TIME, HOURS

B s0

0'~~~---O--,,o PS 7.

1-~~~~~~ /~~~~

E

'9

2

30 .

,.

o// -

/ I-

U

4L26

and CL increased threefold in the percentage of phospholipid. Lyso-PE was present in very small amounts until 24 h, when a significant increase was apparent. Incorporation of ['4Clcholine. To evaluate the possible contribution of medium components to cellular PC, the rates of uptake and incorporation of [methyl-14C]choline (specific activity, 50 Ci/mol) by B. catarrhalis and N. gonorrhoeae were measured. Cells were cultivaied for 4 h on LGCBDS(P10) containing 3 x 10-3 M choline chlori4e. Rates of uptake of choline (measured as moles per minute/milligram ofprotein x 10-s) were determined. The concentration of unlabeled choline was 10-4 M in the uptake experiments. Rates of uptake of ['4C]choline of 0.070 for B. catarrhalis ATCC 23246 and 0.034 for N. gonorrhoeae were obtained. The rates of uptake of tL_U-'4C]leucine measured as a control were 0.054 and 1.470, respectively, for the two strains. The results indicate choline is actively taken up by both strains. To establish possible incorporation of choline into PC, lipid was extracted from cells incubated for 18 h with [methyl-'4C]choline (0.5 ,uCi/ml of medium). Less than 0.001% of available label was recovered as extractable lipid. The label was found in all lipids approximately in proportion to the molar contribution of lipid as determined by ["4C]glucose on 32P label experiments. Neutral lipids. Purified neutral lipid from each strain was subjected to a two-phase onedimensional TLC separation and then visual-

*.S F (A

z

l

w

a

A'

4

z

us1

0

I

CL

I I

S

r6

I

12

4 Is

.PGS

24

TIME, HOURS

FIG. 2. Effect of culture age on the relative phospholipid composition of B. catarrhalis ATCC 23246 (A) and N. gonorrhoeae NYH 002 (B). Cultures in 100-mi volumes of LGCBDS(P10) medium with H332PO4 added at 1.0 ,uCi/mi were analyzed for lipid content at 6-h intervals for 24 h after inoculation. Cultures were incubated at 35 C on a reciprocating water bath shaker (90 cycles/min). The lipids measured were PE (0), PG (@), CL (0), PC (U), PG (salt form) (A), and lyso-PE (V). Optical density is indicated by broken line.

alterations of N. gonorrhoeae lipid during growth. PE remained largely unchanged during growth. PG declined by approximately 40%,

ORIGIW

FIG. 3. One-dimensional TLC of the neutral lipid fraction of Neisseria. (A) Pattern of neutral lipid separation in the two-phase system employed; (B) densitometric tracing (Photovolt model 52-C with Varicord model 42B recorder-integrator) of the acidcharr-ed plate.

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LIPIDS OF B. CATARRHALIS AND N. GONORRHOEAE

ized by acid charring (Fig. 3B). When the plate was subjected to densitometric quantitation, the tracing shown in Fig. 3A was obtained. The neutral lipid components were identified where possible by comparison of Rf values to that of authentic standards. Table 4 summarizes the data obtained for the various neutral lipid components, including chromatographic mobility and quantity in relation to total neutral lipid. Hydrocarbons accounted for 50% of the total neutral lipids in strains ofboth species. Triglycerides were present in extracts of two of three strains of each species. Free fatty acids were detected in all strains, and small amounts of hydroxy fatty acids were present in B. catarrhalis ATCC 8176. On occasion, very faint spots, which may be hydroxy fatty acids, were observed on TLC of the' other strains of B. catarrhalis and N. gonorrhoeae. Diglycerides were present in all strains, accounting for 10 to 18% of the neutral lipid. The chromatographic system separates the 1,3-isomer (5.5 to 11.1%) from the 1,2-isomer (3.5 to 7.4%). Coenzyme Q. Neutral lipid 4 accounted for 20 to 27% of the neutral lipid of the strains tested. It appeared as a bright orange-yellow spot and fluoresced a dark blue color under ultraviolet light on developed but uncharred plates. It was eluted from silica gel and samples subjected to spectrophotometer analysis. It did not adsorb in the visible range but exhibited the ultraviolet absorption spectrum shown in Fig. 4. The absorption maximum of 275 nm agrees with published reports describing this compound (20). Addition of NaBH4 to a chloroform solution of this lipid resulted in a rapid decolorization and a loss of peak absorption at 275 nm. On the basis of these data, neutral lipid 4 is coenzyme Q.

175

Antibiotic susceptibilities of N. gonorrhoeae strains. Table 5 presents the MICs for a number of antibiotics determined by the plate dilution technique for three strains of N. gonorrhoeae. Strain NYH 002 had an MIC of 0.08 for penicillin G and was susceptible to that antibiotic. Strains NHY 006 and F19, with MICs of 0.24 and 1.66, were intermediately resistant and resistant, respectively. In general, F19 had 0.5

z

0

310

zsu z70 WAVELENGTH,nm 4. Ultraviolet absorption spectrum

2su

of neutral FIG. lipid 4. The orange band obtained from TLC plates was eluted from the silica gel and dissolved in chloroform, and the absorbance in the range of 250 to 310 nm was measured using a Beckman model DU spectrophotometer using a 1-cm quartz cuvette.

TABLE 4. Quantitation of the neutral lipids of B. catarrhalis and N. gonorrhoeae % Neutral lipida from strain:

Lipidb

N. gonorrhoeae

B. catarrhalis

Rf 23246

8176

8193

002

006

F19

33.5 37.2 44.5 42.9 50.6 0.85 41.5 Hydrocarbons 1.3 0.0 0.8 0.0 0.5 0.71 3.8 Triglycerides 8.9 11.1 13.6 11.5 6.5 0.65 5.6 Free fatty acids 27.4 21.9 21.6 19.9 23.1 0.59 25.2 Coenzyme Q 12.0 11.5 12.2 9.1 11.1 0.50 10.3 Unknown 11.1 10.5 6.1 6.6 5.5 0.41 6.2 Diglycerides (1,3-isomer) 7.2 4.1 6.3 7.4 3.5 4.7 0.35 Diglycerides (1,2-isomer) 0.0 0.0 0.0 0.0 2.1 0.0 0.28 Free hydroxy fatty acids a Percentages were determined densitometrically on silica gel thin-layer plates developed with isopropyl ether-glacial acetic acid, 96:4 (vol/vol), for 12 cm, followed by drying and development with petroleum ether-ethyl etier-acetic acid, 90:10:1 (vol/vol/vol), for 17 cm. b Identity of each component except coenzyme Q was based on R, value and comparison to standards: palmitic, palmitoleic and oleic acids (0.64 to 0.66), triolein and tripalmitin (0.70 to 0.72), diolein (0.41 and 0.35), monoolein (0.11), 1,2-hydroxystearate (0.36), 2-hydroxypalmitate (0.23), and methyl hexanoate (0.68).

176

J. BACTERIOL.

BEEBE AND WLODKOWSKI

TABLE 5. MICs of N. gonorrhoeae strains MIC (Jg/ml) with strain:

Antimicrobial

agenta

002

006

F19

PenG 0.08 0.24 1.66 0.24 Amp 0.08 0.40 Naf 2.5 2.5 5.0 PenV 0.63 2.5 5.0 Clox 2.5 0.03 10.0 0.02 Ox 5.0 10.0 Meth 5.0 0.03 10.0 0.16 0.03 5.0 Cept 10.0 1.25 5.0 Cepd Ery 0.03 0.04 0.8 Tet 0.24 0.48 0.8 10.0 Str 10.0 10.0 Rif 0.24 0.06 0.63 0.24 1.8 0.8 Cap Kan 10.0 10.0 Neo 60.0 20.0 80.0 Smer 3.8 12.5 25.0 1.56 8.0 25.0 Sdia 12.5 50.0 80.0 Smet Nal 1.56 0.78 0.39 a PenG, Potassium penicillin G; Amp, ampicillin; NaF, nafcillin; PenV, phenoxymethyl penicillin; Clox, sodium cloxacillin; Ox, sodium oxacillin; Meth, sodium methicillin; Cept, cephalothin; Cepd, cephaloridine; Ery, erythromycin; Tet, tetracycline hydrochloride; Str, streptomycin sulfate; Rif, rifampicin; Cap, chloramphenicol; Kan, kanamycin sulfate; Neo, neomycin sulfate; Smer, sulfamerazine; Sdia, sulfadiazine; Smet, sulfamethazine; Nal, nalidixic acid.

MICs greater than that of NYH 006 and 002 with regard to the penicillins and the cephalosporins. There was also a gradation of resistance among the three strains with respect to several other antibiotics. This gradation in resistance is observed for phenoxymethyl penicillin, tetracycline, sulfamerazine, sulfadiazine, and sulfamethazine.

DISCUSSION A body of evidence has accumulated that indicates that the genus Neisseria is not genetically homogeneous. Lambert et al. (18) documented the fatty acid composition of many strains of nonpathogenic Neisseria and showed that N. catarrhalis, N. ovis, N. caviae, and one strain of N. cinerea form a group with a fatty acid content distinct from all other species tested. This observation has been substantiated by other investigators (5, 36). Holten (13-15) examined certain enzymes of many Neisseria species and showed that those ofN. catarrhalis, N. ovis and N. caviae were dissimilar from the other species of Neisseria to a degree that suggests that these species constitute a separate

genus. These and other data have led to the renaming of N. catarrhalis as Branhamella catarrhalis (19). The results described here-a twofold difference in total lipid content between B. catarrhalis and N. gonorrhoeae, large differences in the kinetics of change in the amounts of PE, PG, and CL, and differences in the amounts of these lipids present at any given stage of growth cycle, especially a two- to threefold difference in PE content -lend support to this action. The single most remarkable difference observed in comparison of the lipid composition of these two species is the presence of PC in B. catarrhalis. PC has been shown to be present in a number of other bacteria, such as the order Pseudomonadales and the family Rhizobiaceae of the order Eubacteriales (10, 16). Its presence in B. catarrhalis may indicate phylogenetic proximity of the species to the latter groups. Both groups also possess hydroxy fatty acids. PC has been reported to be present in N. gonorrhoeae (9, 30) but was not detected in extracts of the three strains of N. gonorrhoeae examined in this study. The methylated PE derivatives, monomethyl- and dimethyl-PE, are also a common feature of the lipids of bacteria that contain PC (10), because PC is synthesized by three successive methylations of PE, and the mono- and dimethyl intermediates often accumulate to substantial levels. Despite careful examination of lipid extracts of B. catarrhalis, neither lipid was detected. To exclude the possibility that PC may be a nonspecific medium contaminant, uninoculated medium was extracted, examined, and found to be free of this lipid. Labeling experiments showed that very little ['4C]choline is incorporated into lipid and is diluted among all lipid species not preferentially incorporated into PC. This suggests a synthesis of PC in B. catarrhalis by sequential methylation of PE requiring S-adenosyl methionine, not a direct incorporation of choline. Snipes et al. (27) described the active uptake of [3H]choline by the marine pseudomonad BAL-31, but the organism failed to incorporate the label into lipid. There is a threefold difference in the neutral lipid content between B. catarrhalis and N. gonorrhoeae, although there is no significant difference in the types and amounts of neutral

lipids. We agree with the observation of others (12, 23) regarding autolysis of N. gonorrhoeae. Autolysis occurred most rapidly with strain F19 in LGCBDS medium with a glucose concentration

VOL. 127, 1976

LIPIDS OF B. CATARRHALIS AND N. GONORRHOEAE

of 0.1%, coincidental with an increase of lysoPE in the lipid extracts of this strain. Autolysis and the increase in lyso-PE content also occurred with strains 002 and 006, but 2 to 4 h later. These results suggest that the process of autolysis includes lipolytic activity, resulting in the deacylation of PE to lyso-PE and free fatty acids. Walstad et al. (32) identified lysoPE and free fatty acids in N. gonorrhoeae as inhibitory substances that had previously been suspected to be bacteriocins. Our data also support their report of free fatty acids in lipid extracts of N. gonorrhoeae. We did not observe the amounts of free fatty acids observed by those investigators, but free fatty acids may accumulate to greater levels as autolysis proceeds in cultures older than 18 h. We expect that free fatty acids and lyso-PE could be extracted in significant quantities from cell-free culture filtrates after 18 to 24 h of growth as a direct result of autolysis and dissolution of cellular components into the external medium. A large part of the interest in gonococcal lipids is due to possible correlating features to antibiotic resistance, chiefly penicillin resistance. This effort has evolved into a search of structural features of cells of resistant strains of N. gonorrhoeae to which resistance could be ascribed. As yet there have been no reports of beta-lactamase or any other penicillin-degrading enzyme activity in resistant strains of N. gonorrhoeae. The results of this study indicate that there are no significant qualitative or quantitative differences in the phospholipid or neutral lipid composition of N. gonorrhoeae that correlate with penicillin resistance. This supports the conclusions of Sud and Feingold (30), who found no evidence of correlates of penicillin resistance in the phospholipid and fatty acid composition of N. gonorrhoeae. The MICs of the three strains we examined show broadly increasing resistance involving not only beta-lactam antibiotics but tetracycline and sulfa drugs as well. The biochemical determinant of resistance appears to be nonlipoidal and nonspecific. Rodriguez and Saz (25) have provided some evidence that the mechanism of penicillin resistance involves an increased binding of penicillin to the cell wall of resistant strains of N. gonorrhoeae. It is not clear whether this binding is associated with the outer membrane, the plasma membrane, or both. ACKNOWLEDGMENTS This investigation was supported by Public Health Service General Research Support grants RR-05396-09 and RR05396-011 from the National Institutes of Health, grant U2462 from the New York City Health Research Council, and

177

by grants from Hoffman-LaRoche, Inc. and the Weld Fund. I am grateful to L. B. Senterfit for the procurement of the clinical isolates ofN. gonorrhoeae and to W. O'Leary for his comments and suggestions regarding this investigation. The technical assistance of Margret Yamamoto is greatly appreciated. LITERATURE CITED 1. Ames, G. F. 1968. Lipids of Salmonella typhimurium and Escherichia coli: structure and metabolism. J. Bacteriol. 95:833-843. 2. Ansell, G. B., and J. N. Hawthorne. 1964. Phospholipids-chemistry, metabolism and function, p. 411-419. Elsevier Publishing Co., Amsterdam. 3. Beebe, J. L., and W. W. Umbreit. 1971. Extracellular lipid of Thiobacillus thiooxidans. J. Bacteriol. 108: 612-614. 4. Benson, A. A., and B. Maruo. 1958. Plant phospholipids. I. Identification of the phosphatidyl glycerols. Biochim. Biophys. Acta 27:189-195. 5. Brooks, J. B., D. S. Kellogg, L. Thacker, and E. M. Turner. 1972. Analysis by gas chromatography of hydroxy acids produced by several species of Neisseria. Can. J. Microbiol. 18:157-168. 6. Card, G. L., C. D. Georgi, and W. E. Militzer. 1969. Phospholipids from Bacillus stearothermophilus. J. Bacteriol. 97:186-192. 7: Dawson, R. M. C. 1960. A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochem. J. 75:45-53. 8. Folch, J., L. Ascoli, M. Lees, J. A. Meath, and F. N. Lebaron. 1951. Preparation of lipide extracts from brain tissue. J. Biol. Chem. 191:833. 9. Gavan, T. L., E. L. Cheattle, and H. W. McFadden, Jr. 1971. Antimicrobial susceptibility testing. American Society of Clinical Pathologists, Chicago. 10. Goldfine, H. 1972. Comparative aspects of bacterial lipids, p. 1-58. In A. H. Rose and D. W. Tempest (ed.) Advances in microbial physiology, vol. 8. Academic Press Inc., New York. 11. Hanahan, D. J., J. C. Dittmer, and E. Warashina. 1957. A column chromatographic separation of classes of phospholipids. J. Biol. Chem. 228:685-700. 12. Hebeler, R. H., and F. F. Young. 1975. Autolysis of

Neisseria gonorrhoeae. J. Bacteriol. 122:385-392.

13. Holten, E. 1973. Glutamate dehydrogenases in genus Neisseria. Acta Pathol. Microbiol. Scand. Sect. B 810:49-58. 14. Holten, E. 1974. Glucokinase and glucose-6-phosphate dehydrogenase in Neisseria. Acta Pathol. Microbiol. Scand. Sect. B 82:201-206. 15. Holten, E. 1974. 6-Phosphogluconate dehydrogenase and enzymes of the Entner-Doudoroff pathway in Neiseria. Acta. Pathol. Microbiol. Scand. Sect. B 82:207-213. 16. Ikawa, M. 1967. Bacterial phosphatides and natural relationships. Bacteriol. Rev. 31:54-64. 17. Jacin, H., and A. R. Mishkin. 1965. Separation of carbohydrates on borate-impregnated silica gel G plates. J. Chromatogr. 18:170-173. 18. Lambert, M. A., D. G. Hollis, C. W. Moss, R. E. Weaver, and M. L. Thomas. 1971. Cellular fatty acids of nonpathogenic Neisseria. Can. J. Microbiol. 17:1491-1502. 19. Lennette, E. H., E. H. Spaulding, and J. P. Truant. (ed.). 1974. Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 20. Lester, R. L., Y. Hatefi, C. Widmer, and F. L. Crane. 1959. Studies on the electron transport system. XX. Chemical and physical properties of the coenzyme Q family of compounds. Biochim. Biophys. Acta 33:169185.

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21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Pr6tein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 22. Marinetti, G. V. 1964. Chromatographic analysis of polar lipids on silicic acid impregnated paper, p. 339377. In A. J. James and L. J. Morris (ed.), New biochemical separations. D. Van Nostrand Co., Ltd., London. 23. Morse, S. A., and L. Bartenstein. 1974. Factors affecting autolysis of Neisseria gonorrhoeae. Proc. Soc. Exp. Biol. Med. 145:1418-1421. 24. Moss, C. W., D. S. Kellogg, Jr., D. C. Farshy, M. A. Lambert, and J. D. Thayer. 1970. Cellular fatty acids of pathogenic Neisseria. J. Bacteriol. 104:63-68. 25. Rodriguez, W., and A. K. Saz. 1975. Possible mechanism of decreased susceptibility of Neisseria gonorrhoeae to penicillin. Antimicrob. Agents Chemother. 7:788-792. 26. Skipski, V. P., and M. Barclay. 1969. This layer chromatography of lipids, p. 530-597. In J. M. Lowenstein (ed.), Methods in enzymology, vol. XIV. Academic Press Inc., New York. 27. Snipes, W., A. Keith, and P. Wanda. 1974. Active transport of choline by a marine pseudomonad. J. Bacteriol. 120:197-202. 28. Steers, E., F. Foltz, B. J. Graves, and J. Riden. 1959. An inocula replicating apparatus for routine testing of bacteria susceptibility to antibiotics. Antibiot. Chemother. 9:307-311.

J. BACTZRIOL. 29. Stokinger, H. E., H. Ackerman, and C. M. Carpenter. 1944. Studies on the gonococcus. I. Constituents ofthe cell. J. Bacteriol. 47:129-139. 30. Sud, I. J., and D. S. Feingold. 1975. Phospholipids and fatty acids of Neisseria gonorrhoeae. J. Bacteriol. 124:713-717. 31. Vorbeck, M. L., and G. V. Marinetti. 1965. Separation of glycosyl diglycerides from phosphatides using silicic acid column chromatography. J. Lipid Res. 6:36. 32. Walstad, D., R. C. Reitz, and P. F. Sparling. 1974. Growth inhibition among strains of Neisseria gonorrhoeae due to production of inhibitory free fatty acids and lyso-phosphatidylethanolamine: absence of bacteriocins. Infect. Immun. 10:481-488. 33. Ward, M. E., and P. J. Watt. 1971. The preservation of gonococci in liquid nitrogen. J. Clin. Pathol. 24:122123. 34. Wells, M. A., and J. C. Dittmer. 1963. The use of Sephadex for the removal of nonlipid contaminants from lipid extracts. Biochemistry 2:1259-1263. 35. White, D. C. 1968. Lipid composition of the electron transport membrane ofHaemophiius parainfluenzae. J. Bacteriol. 96:1159-1170. 36. Yamakawa, T., and N. Ueta. 1964. Gas chromatographic studies of microbial components. I. Carbohydrates and fatty acid constitution of Neisseria. Jpn. J. Exp. Med. 34:361-374.

Lipids of Branhamella catarrhalis and Neisseria gonorrhoeae.

Vol. 127, No. 1 JOURNAL OF BACTERIOLOGY, JUlY 1976, p. 168-178 Copyright © 1976 American Society for Microbiology Printed in U.SA. Lipids of Branha...
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