Caulobacter crescentus cell envelope: effect of growth conditions on murein and outer membrane protein composition

Abstract

The murein and membrane protein compositions of Caulobacter crescentus strains CB13B1a and CB15 have been characterized, and the influence on cell envelope constituents of culture conditions which affect morphogenesis have been studied. Amino acid and amino sugar analysis of murein sacculi revealed a simple AlY murein configuration typical of gram-negative bacteria. The membranes of C. crescentus had low levels of 2-keto-3-deoxyoctonate relative to enteric bacteria, in addition to the absence of lipid A components (Shapiro et al., Science 173: 884-892, 1971; Chow and Schmidt, J. Gen. Microbiol. 83:369-373, 1974). Nevertheless, C. crescentus membranes could be fractionated into inner and outer membrane components by sucrose density gradient centrifugation procedures developed for Escherichia coli. The proteins of the outer membrane were distributed between three major (I, II, and III) and two minor (IV and V) protein classes. Class I proteins were -74,000 daltons and constituted the primary proteins of the outer membrane. Class I proteins were separated into approximately 50 polypeptides by two-dimensional gel electrophoresis; the protein composition of this class was affected by culture conditions in both CB13B1a and CB15. Class II (47,000 to 39,000 daltons) and III (20,000 to 11,500 daltons) proteins differed in each strain in composition and response to culture conditions. prepared by adaptation of the procedures of Schwarz et al. (19). Late exponential-phase cells (optical density at 660 nm = 0.8 to 1.0) were collected by centrifugation at 12,000 rpm for 15 min in a Sorvall GSA rotor and stored as frozen cell pellets. Cells (0.5 to 2.0 g) were thawed and suspended in 8 to 12 ml 0.1 M MgSO4 containing 50 ,Lg of DNase I (Worthington) per ml. Acid-washed glass beads (0.17-mm diameter) were added to the cell suspension (1 g/ml) and mixed 987 on July 31, 2020 by guest

Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus

“…Results of NADH dehydrogenase assays on the membrane fraction with potassium ferricyanide as the electron acceptor showed that indeed fraction III exhibited a very high NADH dehydrogenase activity. Succinate dehydrogenase activity has also been used as a marker of the cytoplasmic membrane (1,5,11,17,18,22,23,35,44). As shown in Table 2, this activity appeared to be significantly enriched in fraction I.…”

“…Fraction II, which proved to be unresolved cytoplasmic and outer membrane, contained approximately 15% of the total recovered membrane protein. KDO, an eight-carbon sugar specific to LPS, has been used in numerous studies as the definitive marker of the outer membrane (1,5,22,23,35,39,40,44). KDO was present in M. xanthus in concentrations of approximately 5 x 10-12 ,mol/cell.…”

Heat modifiability and detergent solubility of outer membrane proteins of Rhodopseudomonas sphaeroides

“…Although the outer membrane of Escherichia coli has been well studied and maniy observations about it are applicable to the membranes of other organisms, it is becoming clear that the outer membranes of free-living organisms may be more complex than the outer membranes of enterics. For example, the Caulobacter crescentus outer membrane has a major membrane protein but also has a preponderance of high-molecular-weight polypeptides (1). The Pseudomonas aeruginosa outer membrane also has a greater variety of proteins, but only a few minor proteins are above a molecular weight of 50,000 (12,14).…”

Expression in Caulobacter crescentus of the phosphate-starvation-inducible porin OprP of Pseudomonas aeruginosa

“…We have not been able to reliably separate the proteins of the inner and outer membranes of Caulobacter by standard methods involving sucrose density gradient centrifugation or differential detergent solubility of membranes (Hancock and Nikaido, unpublished data; Walker and Smit, unpublished data). Despite reports by others of successful segregation of Caulobacter membranes [26,27], in our experience the methods yield membrane fractions with nearly identical protein profiles. Surface labeling with porin P antibody gave negative results, yet 221 was not definitive, since the antibodies used in this study are unable to label P. aeruginosa cells grown under phosphate limitation with rough or smooth LPS.…”