ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.     

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

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 sugar analysis of murein sacculi revealed a simple A1gamma 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 greater than or equal to 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 thi s 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.     

Two outer membrane proteins are required for maximal type I secretion of the Caulobacter crescentus S-layer protein

Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaF(a) and rsaF(b) were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The rsaF(a) gene is located several kilobases downstream of the other transporter genes, while rsaF(b) is completely unlinked. An rsaF(a) knockout had approximately 56% secretion compared to wild-type levels, while the rsaF(b) knockout reduced secretion levels to approximately 79%. When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of approximately 9% remained inside the cell, suggesting posttranslational autoregulation. Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion. However, overexpression of rsaF(a) (with normal rsaF(b) levels) in concert with overexpression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system. This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system.     

An essential tyrosine phosphatase homolog regulates cell separation, outer membrane integrity, and morphology in Caulobacter crescentus

Although reversible phosphorylation on tyrosine residues regulates the activity of many eukaryotic proteins, there are few examples of this type of regulation in bacteria. We have identified the first essential tyrosine phosphatase homolog in a bacterium, Caulobacter crescentus CtpA. ctpA mutants with altered active-site residues are nonviable, and depletion of CtpA yields chains of cells with blebbed outer membranes, linked by unresolved peptidoglycan. CtpA overexpression reduces cell curvature in a manner similar to deleting the intermediate filament protein crescentin, but it does not disrupt crescentin localization or membrane attachment. Although it has no obvious signal sequence or transmembrane-spanning domains, CtpA associates with the Caulobacter inner membrane. Immunolocalization experiments suggest that CtpA accumulates at the division site during the last quarter of the cell cycle. We propose that CtpA dephosphorylates one or more proteins involved in peptidoglycan biosynthesis or remodeling, which in turn affect cell separation, cell envelope integrity, and vibrioid morphology.     

The Caulobacter crescentus outer membrane protein Omp58 (RsaF) is not required for paracrystalline S-layer secretion

To identify the outer membrane protein component of the Caulobacter crescentus CB2 surface-layer export machinery we used the Serratia marcescens LipD protein to find homologs in the CB2 genome. From two homologous sequences found, one encodes a putative OMP with a predicted molecular mass of 57.5 kDa, termed Omp58 (formerly RsaF). Comparison of membrane protein profiles revealed a protein with an appropriate molecular mass present in wild-type, but not CB2 omp58::kanamycin, a mutant strain with an inactivated omp58 gene. Disruption of omp58 did not affect surface-layer production, suggesting that Omp58 is not involved in surface-layer protein secretion and, thus, may not be the outer membrane protein component of the C. crescentus surface-layer export system.     

Murein hydrolases of Caulobacter crescentus

Caulobacter crescentus was found to exhibit a similar autolytic response to a variety of factors affecting the structure of the cell envelope and interfering with murein synthesis as several other species of bacteria. Autolysis was accompanied by the hydrolysis of murein with the release of soluble degradation products. Several murein hydrolases with different bond specificity were found and except for the absence of DD-carboxypeptidase and LD-carboxypeptidase activities the make-up of these enzymes resembled that of the well studied bacterium Escherichia coli.     

Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry

Attempts at protein profiling in the alkaline pH region using isoelectric focusing have often proved difficult, greatly limiting the scope of proteome analysis. We investigated several parameters using custom pH 8-11 immobilized pH gradients to separate a Caulobacter crescentus membrane preparation. These included sample application, quenching endoosomotic flow and gel matrix composition. Among these factors, the sample application position was the predominant parameter to affect two-dimensional gel quality. Separated proteins were silver stained and profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The use of a prototype MALDI-Q-Tof mass spectrometer assisted identification of several proteins by providing highly informative peptide fragmentation data from the sample digests. Thirty-two unique alkaline proteins were identified in this study, which complements our previously described C. crescentus membrane proteome. Our experiments point towards new options for proteomic researchers aiming to both extend the scope of analysis, and simplify methods of identifying proteins with high confidence.     

Synthesis and Structure of Caulobacter crescentus Flagella

During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. The released flagellum is composed of a filament, hook, and rod. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. By pulsing synchronized cultures with (14)C-amino acids it has been demonstrated that the synthesis of flagellin occurs approximately 30 to 40 min before cell division. Flagellin, therefore, is synthesized at a discrete time in the cell cycle and is assembled into flagella at a specific site on the cell. A mutant of C. crescentus that fails to synthesize flagellin has been isolated.     

Utilization of histidine by Caulobacter crescentus

Caulobacter crescentus has an inducible pathway which is responsible for the degradation of histidine. Induction of this pathway occurs in the presence of both glucose and ammonia. Growth yield experiments indicate that only two of the three available nitrogens are used for growth suggesting that formamide may be produced as a waste product. However, formamide was not detected in the culture fluid and formate was formed instead. These results suggest that histidine may be degraded in a novel pathway which results in the production of 1 mol each of ammonia, glutamate and formate per mol of histidine. The third nitrogen from histidine appears to be sequestered in some kind of secondary metabolite.     

Fluctuation analysis of Caulobacter crescentus adhesion

The aquatic bacterium Caulobacter crescentus divides asymmetrically to a flagellated swarmer cell and a cell with a stalk. At the end of the stalk is an adhesive organelle known as the holdfast, which the stalked cell uses to attach to a solid surface. Often there are two or more cells with their stalks attached to the same holdfast. By analyzing the fluctuations in the stalk angle for a pair of cells attached to a single holdfast, we determine the elastic stiffness of the holdfast. We model the holdfast as three torsional springs in series and find that the effective torsional spring constant for the holdfast is of the order of (10(-17)-10(-18)) Nm, with unequal spring constants. The asymmetry suggests the sequence in which the cells attach to each other, and in some cases suggests that strong crosslinks form between the stalks as they make a shared holdfast.     

Transport across the bacterial outer membrane

Diffusion of small molecules across the outer membrane of gram-negative bacteria may occur through protein channels and through lipid bilayer domains. Among protein channels, many examples of trimeric porins, which produce water-filled diffusion channels, are known. Although the channels are nonspecific, the diffusion rates of solutes are often drastically affected by their gross physicochemical properties, such as size, charge, or lipophilicity, because the channel has a dimension not too different from that of the diffusing solutes. In the last few years, the structures of three such porins have been solved by X-ray crystallography. It is now known that a monomer unit traverses the membrane 16 times as -strands, and one of the external loop folds back into the channel to produce a narrow constriction. Most of the static properties of the channel, such as the pore size and the position of the amino acids that produce the constriction, can now be explained by the three-dimensional structure. Controversy, however, still surrounds the issue of whether there are dynamic modulation of the channel properties in response to pH, ionic strength, or membrane potential, and of whether such responses are physiological. More recently, two examples of monomeric porins have been identified. These porins allow a very slow diffusion of solutes, but the reason for this low permeability is still unclear. Finally, channels with specific binding sites facilitate the diffusion of specific classes of nutrients, often those compounds that are too large to penetrate rapidly through the porin channels. Lipid bilayers in the outer membrane were shown to be perhaps 50- to 100-fold less permeable to uncharged, lipophilic molecules in comparison with the bilayers made of the usual glycerophospholipids. This is caused by the presence of a lipopolysaccharide leaflet in the bilayer, and more specifically, by the presence of a larger number of fatty acids in each lipid molecule, and by the absence of unsaturated fatty acids in the lipopolysaccharide structure.     

Intermediate filament-like cytoskeleton of Caulobacter crescentus

Eukaryotic cytoskeleton consists of three main types of filaments: actin microfilaments, microtubules and intermediate filaments (IFs). Actin and tubulin-like proteins are also found in bacteria where they perform diverse cytoskeletal functions. IFs, however, are considered to be a characteristic constituent of metazoan cells only, where they (among other functions) are involved in determination and maintenance of cell shape and cellular integrity. Surprisingly, a coiled coil-rich protein called crescentin was recently shown to play a key role in determining the complex curved and helical cell shapes of the bacterium Caulobacter crescentus, and to exhibit several characteristic properties of animal IF proteins. First, the arrangement of the coiled coil domains of crescentin closely resembles the tripartite molecular architecture of IF proteins. Second, crescentin also possesses the defining biochemical property of IF proteins to assemble into 10-nm-wide filaments in vitro without cofactors. Furthermore, crescentin forms a higher-order helical structure in vivo, which is localized asymmetrically along the concave side of the cell. In close association with the cell membrane, the crescentin structure promotes the helical growth of the cell and thereby determines a curved or a helical shape, depending on the length of the cell. The unexpected finding of an IF-like element in a bacterium raises several interesting questions concerning, for example, the molecular mechanisms whereby complex and asymmetric cell shapes are generated by different bacteria, or the functional and evolutionary relatedness of crescentin to animal IF proteins.     

Recombination deficient mutant of Caulobacter crescentus

Summary A recombination-deficient (Rec^-) strain of Caulobacter crescentus has been isolated from a collection of mutants sensitive to ultraviolet irradiation. The Rec^- mutant fails to give recombinants following Cr30-mediated generalized transduction or following RP4-mediated conjugation. The recombination frequency in the Rec^- strain is at least 5000-fold lower than in the wild type strains. The Rec^- mutant is indistinguishable from wild type in terms of morphology, growth rate, viability, and phage sensitivities, differing only in properties known to be associated with recA-type mutations in other organisms: recombination frequency, ultraviolet sensitivity, and Weigle reactivation. The map location of the rec-526 allele has not been identified, but rec-526 can be cotransferred with the fla-169 mutation by RP4-mediated conjugation at low frequency. This apparent linkage has been used to move the rec mutation to other strains. The Rec^- mutant resembles recA strains of other organisms and provides a healthy strain severely deficient in recombination for use in complementation and cloning studies involving C. crescentus.     

Characterization of the SOS regulon of Caulobacter crescentus

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN₇GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.     

Transport of iron across the outer membrane

Summary The TonB protein is involved in energy-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, ortonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in thefhuA gene are suppressed by point mutations in thetonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins. A physical interaction of these proteins with the TonB protein is suggested by TonB stabilization through ExbB and TolQR. We propose a permanent or reversible complex in the cytoplasmic membrane composed of the TonB protein and the ExbBD/TolQR proteins through which TonB is energized.     

The organization of the Caulobacter crescentus flagellar filament

The structural organization of the flagellar filament of Caulobacter crescentus, as revealed by immunoelectron microscopy, shows five antigenically distinct regions within the hook-filament complex. The first region is the hook. The second region is adjacent to the hook and is approximately 10 nm in length. On the basis of its location in the hook-filament complex, this region may contain hook-associated proteins. Next to this is the third region, which is approximately 60 nm in length. Antibody decoration experiments using mutant strains with deletions of the structural gene for the 29 x 10(3) Mr flagellin (flgJ) showed that the presence of this region is correlated with the expression of the 29 x 10(3) Mr flagellin gene. The next region (region IV), of length approximately 1 to 2 microns, appears to contain the 27.5 x 10(3) Mr flagellin, but at its distal end includes, in gradually increasing amounts, the 25 x 10(3) Mr flagellin. The rest of the filament (region V) is made up predominantly, if not completely, of the 25 x 10(3) Mr flagellin. Except for the hook, there are no morphological features that would otherwise distinguish these regions. A functional flagellum, having the wild-type length and morphology, is assembled by mutant strains deficient in the 29 x 10(3) Mr flagellin and 27.5 x 10(3) Mr flagellin.