JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 1120-1122
Vol. 140, No. 3
0021-9193/79/12-1120/03$02.00/0
Lipolytic Activity Copurified with the Outer Membrane of Serratia marcescens KLAUS B. HELLERt Lehrstuhl Biologie der Mikroorganismen, Ruhr- Universitat Bochum, D-4630 Bochum, Germany Received for publication 22 August 1979
Lipase, nuclease, and protease activities could be shown primarily with the purified outer membrane fraction from Serratia marcescens. These activities increased and decreased in the different compartments dependent on the growth phase of the cell culture. Penicillin-hydrolyzing activity was exclusively demonstrated with the outer membrane fraction. The cell envelope of the Enterobacteriaceae consists of two distinct membranes, a cytoplasmic or inner membrane and an outer membrane. The two are separated by a peptidoglycan layer. Methods have been developed to separate these membranes (9). The cytoplasmic membrane contains the protein systems responsible for active transport and energy-yielding processes. The biochemical properties of the outer membrane have been studied extensively (for review see reference 4). However, at present, only a few enzymatic activities are known in the outer membrane of gramnegative bacteria: a phospholipase A in Salmonella typhimurium (9) and in Klebsiella sp. 5426; a pulluanas in Klebsiella sp. 5426 (16); and a protease in Escherichia coli (6). A characteristic trait of many strains of Serratia marcescens, another gram-negative bacterium, is that they produce extracellular enzymes. Lipase (13), protease (1, 13), and nuclease (5, 8, 12, 13) activities have been investigated. The mechanism by which these enzymes are released from the cell is not yet known. We are interested in the importance of the cell envelope for their secretion. During the course of our experiments, lipase, protease, and nuclease activities were also detected in considerable amounts with the purified outer membrane fraction (Table 1). In the cytoplasmic membrane and the cytosol there was only negligible protease and nuclease activity, whereas distinct lipase could be measured in these cell compartments (for experimental details of membrane preparation see reference 14; cross-contamination of the membrane fractions was less than 5%, as determined by the activity of NADH oxidase [9] and the content of 2-keto3-deoxy-octulonate [9]). t Present address: Department of Physiology, Harvard Medical School, Boston, MA 02115.
In S. marcescens extracellular lipase was released into the medium during late logarithmic and early stationary growth phase (Fig. IA and B) without concomitant cell lysis (14). Increasing extracellular activity seemed to be accompanied by a decreasing activity of the lipase copurified with the outer membrane fraction (Fig. 1B and C). The maximal lipase activity detectable in the isolated outer membrane fraction was reached about 2 h later than that of the cytoplasmic membrane. Again, the increase of activity in one compartment (i.e., the outer membrane fraction) was accompanied by decreasing activity in another (i.e., the cytoplasmic membrane fraction) (Fig. 10). Similar results were obtained for the release of nuclease and protease activity from the outer membrane fraction. The location of these enzymes was also growth phase dependent. Enzyme release and membrane-associated activity were maximum during earlier growth phases (data not shown). With the enzymatic tests used in these experiments (14) one cannot discriminate whether only one protein accounts for the enzymatic activity under consideration. In fact, in the case of the proteolytic activity in the medium there is evidence that three different proteins are involved (S. Neumann, personal communication). However, recent results obtained with the extracellular lipase of S. marcescens showed that this enzymatic activity is due to only one single protein which is glycosylated and has an apparent molecular weight of about 500,000 (J. Sossinka, manuscript in preparation). The enzymatic activities of both the extracellular enzyme and the enzymatic activity copurified with the membrane fractions could be increased by Ca2" ions. On the other hand, the outer membrane-associated phospholipase A (9) does not hydrolyze the substrate emulsion for the lipase assay, so this 1120
NOTES
VOL. 140, 1979
1121
TABLE 1. Enzymatic activities in the cellular fractions of S. marcescensa Activity (U/mg of protein)
Lipase
15.0
Cytoplasmic brane 53.3
Nuclease
Vol. 140, No. 3
0021-9193/79/12-1120/03$02.00/0
Lipolytic Activity Copurified with the Outer Membrane of Serratia marcescens KLAUS B. HELLERt Lehrstuhl Biologie der Mikroorganismen, Ruhr- Universitat Bochum, D-4630 Bochum, Germany Received for publication 22 August 1979
Lipase, nuclease, and protease activities could be shown primarily with the purified outer membrane fraction from Serratia marcescens. These activities increased and decreased in the different compartments dependent on the growth phase of the cell culture. Penicillin-hydrolyzing activity was exclusively demonstrated with the outer membrane fraction. The cell envelope of the Enterobacteriaceae consists of two distinct membranes, a cytoplasmic or inner membrane and an outer membrane. The two are separated by a peptidoglycan layer. Methods have been developed to separate these membranes (9). The cytoplasmic membrane contains the protein systems responsible for active transport and energy-yielding processes. The biochemical properties of the outer membrane have been studied extensively (for review see reference 4). However, at present, only a few enzymatic activities are known in the outer membrane of gramnegative bacteria: a phospholipase A in Salmonella typhimurium (9) and in Klebsiella sp. 5426; a pulluanas in Klebsiella sp. 5426 (16); and a protease in Escherichia coli (6). A characteristic trait of many strains of Serratia marcescens, another gram-negative bacterium, is that they produce extracellular enzymes. Lipase (13), protease (1, 13), and nuclease (5, 8, 12, 13) activities have been investigated. The mechanism by which these enzymes are released from the cell is not yet known. We are interested in the importance of the cell envelope for their secretion. During the course of our experiments, lipase, protease, and nuclease activities were also detected in considerable amounts with the purified outer membrane fraction (Table 1). In the cytoplasmic membrane and the cytosol there was only negligible protease and nuclease activity, whereas distinct lipase could be measured in these cell compartments (for experimental details of membrane preparation see reference 14; cross-contamination of the membrane fractions was less than 5%, as determined by the activity of NADH oxidase [9] and the content of 2-keto3-deoxy-octulonate [9]). t Present address: Department of Physiology, Harvard Medical School, Boston, MA 02115.
In S. marcescens extracellular lipase was released into the medium during late logarithmic and early stationary growth phase (Fig. IA and B) without concomitant cell lysis (14). Increasing extracellular activity seemed to be accompanied by a decreasing activity of the lipase copurified with the outer membrane fraction (Fig. 1B and C). The maximal lipase activity detectable in the isolated outer membrane fraction was reached about 2 h later than that of the cytoplasmic membrane. Again, the increase of activity in one compartment (i.e., the outer membrane fraction) was accompanied by decreasing activity in another (i.e., the cytoplasmic membrane fraction) (Fig. 10). Similar results were obtained for the release of nuclease and protease activity from the outer membrane fraction. The location of these enzymes was also growth phase dependent. Enzyme release and membrane-associated activity were maximum during earlier growth phases (data not shown). With the enzymatic tests used in these experiments (14) one cannot discriminate whether only one protein accounts for the enzymatic activity under consideration. In fact, in the case of the proteolytic activity in the medium there is evidence that three different proteins are involved (S. Neumann, personal communication). However, recent results obtained with the extracellular lipase of S. marcescens showed that this enzymatic activity is due to only one single protein which is glycosylated and has an apparent molecular weight of about 500,000 (J. Sossinka, manuscript in preparation). The enzymatic activities of both the extracellular enzyme and the enzymatic activity copurified with the membrane fractions could be increased by Ca2" ions. On the other hand, the outer membrane-associated phospholipase A (9) does not hydrolyze the substrate emulsion for the lipase assay, so this 1120
NOTES
VOL. 140, 1979
1121
TABLE 1. Enzymatic activities in the cellular fractions of S. marcescensa Activity (U/mg of protein)
Lipase
15.0
Cytoplasmic brane 53.3
Nuclease
