Comparative Biochemistry of the Cell Envelopes of Photobacterium leiognathi and Escherichia coli

Abstract

Photobacterium leiognathi closely resembles Escherichia coli with respect to cell lysis by lysozyme, and the fractionation of outer and cytoplasmic membranes. The two organisms differ in their phospholipid contents and, more significantly, in outer membrane protein compositions. I N T R O D U C T I O NPhotobacterium leiognathi ( P . mandapamensis) is a luminescent marine bacterium found as the specific symbiont in the luminous organ of Leiognathid and Apogonid fish, as a marine intestinal commensal or free-living in sea water (Reichelt & Baumann, 1973;Nealson & Hastings, 1979). The genus shares many properties with the Enterobacteriaceae (Baumann & Baumann, 1977). In particular, electron microscopy of thin sections of this organism reveals a cell envelope which is morphologically very similar to terrestrial Gram-negative bacteria, with the characteristic membrane, cell wall, periplasmic space and outer membrane (Reichelt & Baumann, 1973). It would not be surprising if closer analysis of the cell envelope of P. leiognathi revealed considerable differences from typical Gram-negative bacteria, since the marine organism must cope with a constant high osmotic pressure. At the same time, there is some evidence for greater membrane permeability in this genus. We have observed that P. leiognathi has a relatively high sensitivity to a variety of antibiotics and chemical mutagens. In addition, the periplasmic b-lactamase of Photobacterium species does not generally display the 'crypticity' typical of this enzyme in most other Gram-negative bacteria (Richmond and Sykes, 1973), and usually taken as a measure of outer membrane impermeability to b-lactam antibiotics (K. Smith & S. Lamb, unpublished results). We have accordingly analysed various aspects of the biochemistry of the cell envelope of P. leiognathi with particular emphasis on the composition of the outer membrane, and using Escherichia coli for comparative purposes. METHODSGrowth qf bacteria. The maintenance and growth of Escherichiu coli ML308 225 have been described previously (Allen & Scott, 1979). Photobacterium Ieiognathi strain K-721 (Nealson & Hastings, 1977) was obtained from the Roche Research Institute of Marine Pharmacology in Sydney, and was grown in liquid cultures using minimal medium (Reichelt & Baumann, 1973) without CaClz and supplemented with 0.2% (w/v) yeast extract. This bacterial strain was maintained on plates made by adding 1.2% (w/v) agar to the liquid medium.Mfnihranpprepcrrcition. Membranes from E. coli ML308 225 in the late-exponential growth phase were prepared and fractionated by sucrose density gradient centrifugation as described previously (Allen &Scott, 1979). A 500 ml culture of P. leiognarhi in the late-exponential growth phase was harvested by centrifugation, resuspended in 20 ml deionized water and mixed with 2 mg egg white lysozyme (Sigma) in 20 ml 30 mM-Tris/HCI buffer pH 7.5. Following incubation at 37 "C for 15 min, the mixture was centrifuged for 3 x lo4 g-min to remove intact cells and then for 2 x lo6 g-min. The resulting...