The FtsH protease is involved in development, stress response and heat shock control in Caulobacter crescentus.

The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism. In C. crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase. Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics. Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response. Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH. In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor sigma32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C. crescentus heat shock response. In agreement with this, transcription of the heat-induced sigma32-dependent gene dnaK was derepressed at normal temperature when FtsH was absent. In contrast, the groEL gene, which is controlled in response to heat stress by both sigma32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant. Finally, FtsH is involved in C. crescentus development and cell cycle control. ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype. In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions. Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C. crescentus.

Proteomic analysis of the bacterial cell cycle.

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control.

Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae.

BACKGROUND: Porins are channel-forming membrane proteins that confer solute permeability to the outer membrane of Gram-negative bacteria. In Escherichia coli, major nonspecific porins are matrix porin (OmpF) and osmoporin (OmpC), which show high sequence homology. In response to high osmolarity of the medium, OmpC is expressed at the expense of OmpF porin. Here, we study osmoporin of the pathogenic Klebsiella pneumoniae (OmpK36), which shares 87% sequence identity with E. coliOmpC in an attempt to establish why osmoporin is best suited to function at high osmotic pressure. RESULTS: The crystal structure of OmpK36 has been determined to a resolution of 3.2 A by molecular replacement with the model of OmpF. The structure of OmpK36 closely resembles that of the search model. The homotrimeric structure is composed of three hollow 16-stranded antiparallel beta barrels, each delimiting a separate pore. Most insertions and deletions with respect to OmpF are found in the loops that protrude towards the cell exterior. A characteristic ten-residue insertion in loop 4 contributes to the subunit interface. At the pore constriction, the replacement of an alanine by a tyrosine residue does not alter the pore profile of OmpK36 in comparison with OmpF because of the different course of the mainchain. Functionally, as characterized in lipid bilayers and liposomes, OmpK36 resembles OmpC with decreased conductance and increased cation selectivity in comparison with OmpF. CONCLUSIONS: The osmoporin structure suggests that not an altered pore size but an increase in charge density is the basis for the distinct physico-chemical properties of this porin that are relevant for its preferential expression at high osmotic strength.

Detergent binding in trigonal crystals of OmpF porin from Escherichia coli.

The structure of the detergent, ocytyl hydroxyethylsufoxide (C8(HE)SO), bound to the OmpF porin from E coli (in the trigonal crystal form) has been determined by neutron crystallography. Due to a dynamic exchange of detergent molecules with their environment they are not ordered on an atomic scale. The structure reported here is therefore at a resolution of approximately 16 A. The X-ray crystallographically determined structure of the protein provides a starting point for the neutron analysis in which the detergent is visualized primarily thanks to its high contrast against D2O. The structure shows the detergent to be located mainly in two areas. It forms toroidal annuli around each OmpF trimer, these annuli fusing to form a detergent belt surrounding a solvent filled column traversing the crystal. Those areas of the protein to which the detergent binds are formed almost exclusively of hydrophobic residues and form a band about 30 A high around the trimer. Its upper and lower bounds are defined by two bands of aromatic residues, tyrosines pointing away from the detergent belt and interacting with the polar headgroups while phenylalanines point inwards. This strongly suggests that the same areas define, in vivo, the location at which protein interacts with lipid. The hydrophobic moiety of detergent is also found mediating the hydrophobic protein-protein interactions at the interface between two trimers on the crystallographic two-fold axis.

Lipidic Cubic Phases: New Matrices for the Three-Dimensional Crystallization of Membrane Proteins.

The major constraint in attaining high resolution structures of membrane proteins by X-ray crystallography is the growth of well-ordered three-dimensional crystals. To enable such crystallizations, we have used lipidic cubic phases consisting of monoglycerides and water. Bacteriorhodopsin and lysozyme, as paradigms of membrane and soluble proteins, nucleate and grow to well-ordered crystals that diffract X-rays isotropically in all three dimensions to 2.0 Å. We envisage bacteriorhodopsin to partition into, and diffuse within, the bilayer of a lipidic cubic matrix, while the polar lysozyme resides in the aqueous compartment thereof. The phenomenology of bicontinuous cubic phases, consisting of curved bilayers whose structures follow infinitely periodic minimal surfaces (IPMS), is presented. Detailed prescriptions of the preparation of lipidic cubic phase matrices are given and their potential for the crystallization of other biological macromolecules is discussed. Copyright 1998 Academic Press.

X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases.

Lipidic cubic phases provide a continuous three-dimensional bilayer matrix that facilitates nucleation and growth of bacteriorhodopsin microcrystals. The crystals diffract x-rays isotropically to 2.0 angstroms. The structure of this light-driven proton pump was solved at a resolution of 2.5 angstroms by molecular replacement, using previous results from electron crystallographic studies as a model. The earlier structure was generally confirmed, but several differences were found, including loop conformations and side chain residues. Eight water molecules are now identified experimentally in the proton pathway. These findings reveal the constituents of the proton translocation pathway in the ground state.

Structural and functional characterization of OmpF porin mutants selected for larger pore size. I. Crystallographic analysis.

OmpF porin is a nonspecific pore protein from the outer membrane of Escherichia coli. Previously, a set of mutants was selected that allow the passage of long maltodextrins that do not translocate through the wild-type pore. Here, we describe the crystal structures of four point mutants and one deletion mutant from this set; their functional characterization is reported in the accompanying paper (Saint, N., Lou, K.-L., Widmer, C., Luckey, M., Schirmer, T., Rosenbusch, J. P. (1996) J. Biol. Chem. 271, 20676-20680). All mutations have a local effect on the structure of the pore constriction and result in a larger pore cross-section. Substitution of each of the three closely packed arginine residues at the pore constriction (Arg-42, Arg-82, and Arg-132) by shorter uncharged residues causes rearrangement of the adjacent basic residues. This demonstrates mutual stabilization of these residues in the wild-type porin. Deletion of six residues from the internal loop (Delta109-114) results in disorder of seven adjacent residues but does not alter the structure of the beta-barrel framework. Thus, the large hollow beta-barrel motif can be regarded as an autonomous structure.

The structure of OmpF porin in a tetragonal crystal form.

BACKGROUND: OmpF porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules, such as nutrients and waste products, across the outer membrane of Escherichia coli. Very few membrane proteins have been crystallized in three dimensions, yet this stable protein can be obtained in several crystal forms. Comparison of the structures of the same membrane protein in two different packing environments is of major interest, because it allows us to explore the integrity of the structure outside the natural membrane environment. RESULTS: The structure of OmpF porin in a tetragonal crystal form with two trimers per asymmetric unit has been determined at 3.2 A resolution and compared with that obtained previously in a trigonal crystal form. The lattice contacts involve only polar atoms, whereas extensive hydrophobic protein-protein interactions were found in the trigonal lattice. The trimer structure is virtually identical in both. CONCLUSIONS: Our comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints and, thus, presumably bears close resemblance to the in vivo structure. The tetragonal crystal structure has provided the starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article.

A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding.

A recombinant plasmid containing ompK36, the gene coding for the Klebsiella pneumoniae outer membrane protein OmpK36, was constructed by transposon mutagenesis and subcloning. Clones were identified in a cosmid library in Escherichia coli on the basis of their reaction with antiserum against the OmpK36 protein and by the presence in gel electrophoretic analysis of a band in E. coli outer membranes migrating with a mobility corresponding to 36 kDa. The ompK36-encoded protein exhibited characteristic properties of porins, such as heat modifiability and resistance to trypsin. The sequence of the gene revealed that OmpK36 is a close relative of the enterobacterial porin family, with a high degree of homology with E. coli OmpC, PhoE, and OmpF. On the basis of the structures of OmpF and PhoE porins, determined previously by X-ray analysis, it appears likely that the three-dimensional structure of OmpK36 also contains the motif of a 16-stranded beta-barrel, with long loops on one end and short turns on the other. Like the OmpC porin from E. coli, OmpK36 contains a long insertion in loop 4. The results of a binding study of complement component C1q to OmpK36 and the analysis of the OmpK36 model suggest that C1q binding sites are covered by the lipopolysaccharide core in the native porin.

Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli.

A strain of Escherichia coli, selected on the basis of its resistance to colicin N, reveals distinct structural and functional alterations in unspecific OmpF porin. A single mutation [Gly-119-->Asp (G119D)] was identified in the internal loop L3 that contributes critically to the formation of the construction inside the lumen of the pore. X-ray structure analysis to a resolution of 3.0 A reveals a locally altered peptide backbone, with the side chain of residue Asp-119 protruding into the channel, causing the area of the constriction (7 x 11 A in the wild type) to be subdivided into two intercommunicating subcompartments of 3-4 A in diameter. The functional consequences of this structural modification consist of a reduction of the channel conductance by about one-third, of altered ion selectivity and voltage gating, and of a decrease of permeation rates of various sugars by factors of 2-12. The structural modification of the mutant protein affects neither the beta-barrel structure nor those regions of the molecule that are exposed at the cell surface. Considering the colicin resistance of the mutant, it is inferred that in vivo, colicin N traverses the outer membrane through the porin channel or that the dynamics of the exposed loops are affected in the mutant such that these may impede the binding of the toxin.

Crystal structures explain functional properties of two E. coli porins.

Porins form aqueous channels that aid the diffusion of small hydrophilic molecules across the outer membrane of Gram-negative bacteria. The crystal structures of matrix porin and phosphoporin both reveal trimers of identical subunits, each subunit consisting of a 16-stranded anti-parallel beta-barrel containing a pore. A long loop inside the barrel contributes to a constriction of the channel where the charge distribution affects ion selectivity. The structures explain at the molecular level functional characteristics and their alterations by known mutations.

Trigonal crystals of porin from Escherichia coli.

Trigonal crystals of the integral membrane protein porin from Escherichia coli have been grown and characterized. They belong to space group P321 with unit cell constants a = b = LL8.4, c = 52.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals grow as well-defined hexagonal prisms to a size of 0.25 mm in all dimensions, and diffract to 2.7 A. The molecular symmetry coincides with 3-fold crystallographic symmetry, giving two trimers per unit cell (1 monomer/asymmetric unit). This corresponds to VM = 2.9 A3/Da. Native X-ray data to 3.0 A resolution have been collected on a FAST area detector and a search for heavy atom derivatives is underway.

Purification and characterization of the ninth component of rabbit complement.

A method of purification of C9 from rabbit serum is described. The three-step procedure, consisting of anion exchange chromatography, gel-filtration and isoelectric focusing yielded a homogeneous, single band protein as judged by SDS-PAGE. With regard to its physicochemical properties, rabbit C9 resembled C9 purified from human or guinea-pig serum.