FEMS Microbiology Letters 2 (1977) 285-288 © CopyrightFederationof EuropeanMicrobiological Societies Published by Eltevier/Nortll-Honand Biomedical Preu ~ : .... CELL ENVELOPE OF PARACOCCU$ OENITRIFICANS: OUTER MEMBRANE PERMEABILITY TO LYSOZYME AND tlYDROPHOBIC ANTIBIOIJCS BRIAN I. WILKINSON Department~of Medicineand Microbiology.MayoMemorialBuilding, University of Minnesota. Minneapoliso MN 55455. U.SA. Received 23 September 1977 285 1. Introduction Although Paracoccus denitrifkans, like other Gram.negative bacteria, possesses an outer membrane [1 ] the organism is susceptible to lysozyme [2] with- out the prior ethylenediamine tetraacetate (EDTA) treatment normally required for Gram-negative spe- cies [3]. Lipopolysaccharlde (LPS)-defective entero- bacterial mutants are often susceptible to ly~zyme as well as to several antibiotics, dyes and detergents (see ref. 4). I found P. denitrificans to be highly suscepti- ble to "hydrophobic" antibiotics [4], dyes and deter- gents. LPS markers, 2-deto-3-deoxyoctonic acid (KDO) and 3-hydroxy fatty acids, were found in analyses of isolated envelop Cs. Low amounts of enve- lope carbohydrate might indicate long polysaccharide chains are lacking from the LPS of the organism. The results are discussed in terms of Nikaido's [4] propo- sals on outer membrane architecture. Much current interest in the organism has arisen from the proposal of an ancestral resembling P. deni- ttiflcans as the source of the mitochondrial inner membrane [5]. 2. Materials and Methods 2.1. Organism and cultural conditions P. denli,lflcans (ATCC 17~543)was grown in *.he complex medium described by Scholes and Smith [2] at 30°C except that lacto-Peptone (Difco) was replaced by Phytonr ~eptone (BBL, CockeysviUe, Maryland, U.S.A.). ~xponential phase organisms (,4 about 1.0 at 625 nm) were used. 2.2 Determination of the minimum inhibitory con- centration (MIC) o f antibiotics, dyes and detergents Antibiotic MIC value~ after 24 h at 30~C were ~ determined using the microtitre [6]. A 1-100 dilu- tion of organisms was used as the inoculum. Dye and detergent MICs were determined by tube dilution. 2.3. Preparation o f envelopes Organisms were ruptured ~n the French Pressure Cell (American Instrument Inc., Silver Springs, Mary- land, U.S.A.). Intact cells were removed by centrio fuging (500Og for 10 min at 2°C) and the supema- tant was centrifuged (27 578 g for 30 rain at 2°C) to deposit the envelopes. Envelopes were treated with RNAase and DNAase, washed five times in cold water and lyophilized. 2.4. Chemical analyses o f the envelopes ~otein was estimated by the method of Lowry et al. [7] modified by including 1 drop of 10% (w/v) s~dium dodecyl sulfate in the assay to aid solubiliza- tion of preparations. Total carbohydrate was esti- mated by the method of Dubois et al. [8]. KDO was esthnated by the method of Weisbach and Hmwitz [9] as modified by Osbom [10]. l.ipids were extract- ed by a modified E~gh and Dyer [I 1] method. Fatty acids were released by saponification and extracted and methylated as descn~oedby White and Cox [12]. Methyl esters were treated with trifhmracetic anhydride (TFA) as described by Moss et al. [13] to aid in identifying hydroxy fatty acids. Fa~y acid methyl esters were chromatographed on a Packa~ 409 gas liquid chromatograph (Packard Instnm~nt Co., Illinois, U.S.A.) on a 3% Silar 5CP (10 ft X ~ in) column at 200°C. Downloaded from https://academic.oup.com/femsle/article-abstract/2/5/285/500664 by guest on 26 May 2020