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Isolation of spontaneously derived mutants of Caulobacter crescentus.

Caulobacter crescentus has a penicillinase which precludes the use of penicillin for mutant enrichment. However, two other antibiotics, fosfomycin and D-cycloserine, can be enrich for C. crescentus mutants. In enrichment procedures for C. crescentus auxotrophs, spontaneously derived mutants occur at a frequency of 5-10% among the survivors of an enrichment procedure. Consequently, large numbers of mutants are readily obtained without any need for mutagenesis. These mutants are heterogeneous both with regard to the type of mutation and to the nutritional requirement. A similar procedure has been used to isolate temperature-sensitive mutants.

Anti-Bacterial Agents

Galactose catabolism in Caulobacter crescentus.

Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.

Aldehyde-Lyases

Effect of macromolecular synthesis on the coordinate morphogenesis of polar surface structures in Caulobacter crescentus.

The Caulobacter polar surface structures (flagella, pili, and the deoxyribonucleic acid phage phiCbK receptors), which are expressed at proximal sites of swarmer cells in a coordinate manner (Shapiro, Annu. Rev. Microbiol., 30:377-407, 1976) could be blocked by a single mutation. The mutant C. crescentus CB13 ple-801 did not form these surface structures when grown at 35 degrees C. Upon shift down to 25 degrees C, the mutant cells initiated the formation of the surface structures. When mitomycin C was added to the mutant culture upon shift down from 35 to 25 degrees C, phiCbK receptor formation was inhibited to a minimal level. Rifampin and chloramphenicol completely inhibited phiCbK receptor formation when added to the mutant culture upon shift down. Deoxyribonucleic acid as well as ribonucleic acid and protein synthesis seem to be required for the formation of phiCbK receptors. Penicillin V also inhibited phiCbK receptor formation, indicating the involvement of cell wall synthesis. When the mutant CB13 ple-801 cells were shifted down briefly from 35 to 25 degrees C and then shifted up to 35 degrees C, flagella and phiCbK receptors were formed even at 35 degrees C to different extents depending on how long the cells were incubated at 25 degrees C. This formation of the surface structures at 35 degrees C was inhibited by rifampin. From these results, it appears that translation, assembly, or localization processes for the formation of the surface structures are not temperature sensitive at 35 degrees C in the pleiotropic mutant CB13 ple-801. The syntheses of deoxyribonucleic acid and the cell wall do not appear to be temperature sensitive either, since the mutant grows normally at 35 degrees C. It is suggested that there exists a regulatory step that commits the cells to initiate the synthesis of requisite ribonucleic acid for the formation of the polar surface structures.

Adsorption

Selection for nonbuoyant morphological mutants of Caulobacter crescentus.

Morphological mutants of Caulobacter crescentus were isolated by selecting for cells that did not possess normal, buoyancy-conferring stalks. The most prevalent phenotype enriched by the selection was structurally deficient stalks (designated "Cds"), observable as crumpled or flattened stalks. The second phenotype ("Abs") was observed as spontaneous shedding of normal-appearing stalks. Third, one mutant ("Ecs") was isolated that sheds not only stalks, but also miniature stalked and nonstalked cells.

Bacteria

Ammonia assimilation and glutamate formation in Caulobacter crescentus.

In the dimorphic bacterium Caulobacter crescentus, ammonia assimilation occurs only via the combined action of the enzymes glutamine synthetase and glutamate synthase. Mutants auxotrophic for glutamate lacked glutamate synthase activity, and the mutations leading to the glutamate auxotrophy appeared to lie at two distinct genetic loci. Both glutamate synthase and glutamine synthetase activities were subject to regulation by repression. Glutamate synthase activity was highest in cultures grown in minimal medium with ammonia as sole nitrogen source and was about fivefold lower in rich broth. Glutamine synthetase activity was highest in cells grown with growth-rate-limiting amounts of ammonia as nitrogen source and was about fourfold lower in rich broth. In addition, glutamine synthetase activity appeared to be regulated by an adenylylation system like that described for Escherichia coli.

Ammonia

Membrane phospholipid composition of Caulobacter crescentus.

The phospholipid composition of Caulobacter crescentus CB13 and CB15 was determined. The acidic phospholipids, phosphatidylglycerol and cardiolipin, comprise approximately 87% of the total phospholipids. Neither phosphatidylethanolamine nor its precursor phosphatidylserine was detected. The outer and inner membranes of C. crescentus CB13 were separated, and phospholipid analysis revealed heterogeneity with respect to the relative amounts of phosphatidylglycerol and cardiolipin in the two membranes. As has been shown to be the case for other bacterial membranes, the concentration of cardiolipin increases and phosphatidylglycerol decreases as cell cultures enter stationary phase.

Bacteria

Reconstitution and purification of flagellar filaments from Caulobacter crescentus.

Filaments from isolated flagella of Caulobacter crescentus have been purified by successive dissociation and reconstitution. After the second and third reconstitutions from subunits in 0.8 M sodium citrate, filament preparations contained only two proteins, flagellin A (26,000 daltons) and flagellin B (28,000 daltons). There was some enrichment for flagellin A during reconstitution by this procedure, since isolated flagella contained flagellin A and flagellin B in a ratio of approximately 3.8:1 and filaments after the third reconstitution contained the two proteins in a ratio of 5.0:1.

Bacteria

Purification and characterization of a polyhook protein from Caulobacter crescentus.

A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 micrometers). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pI of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.

Amino Acids

Physical characterization of Caulobacter crescentus flagella.

Preparations of intact flagella isolated from Caulobacter crescentus CB13B1a were found to contain two protein species of apparent molecular weights 28,000 and 25,000. Both proteins cross-reacted completely with each other and with purified flagella in Ouchterlony double-immunodiffusion assays. The amino acid compositions of the isolated proteins were similar to one another but precluded any precursor-product relationship. Absence of both the 25,000- and 28,000-molecular-weight proteins from a number of nonmotile mutants and the simultaneous reappearance of these proteins in a motile revertant provide further evidence of the relationship of these two proteins to flagellar structure.

Amino Acids

Amino acid composition of peptidoglycan in Caulobacter crescentus.

Peptidoglycan of a gram-negative stalked bacterium, Caulobacter crescentus CB13, contained alanine, diaminopimelic acid, and glutamic acid, in molar ratios of 2 : 1 : 1. The amino acid compositions of peptidoglycans isolated from cultures enriched in swarmer and stalked cells, and from a stalk-less mutant were similar. This finding conflicts with a previous observation that swarmer peptidoglycan does not contain diaminopimelic acid (Goodwin and Shedlarski (1975) Arch. Biochem. Biophys. 170, 23-36). It appears that, despite the morphological differences, the Caulobacter cells all contain a similar peptidoglycan in the cell wall.

Alanine

Characterization of unstable poly (A)-RNA in Caulobacter crescentus.

A significant amount of poly(A)-RNA in Caulobacter crescentus is located on polysomes and the size distribution of this polysomal poly(A)-RNA is small compared to the total pulse-labeled RNA in these cells. These observations suggest that the poly (A)-RNA represents a subset of small messenger RNA molecules. Poly (A)-RNA, and presumably the poly (A) portion of these molecules is extremely unstable: as assayed by binding to oligo (dT)-cellulose the poly (A)-RNA turns over with a chemical half-life of 15--20 s compared to a half-life of approx. 2 min for total cellular messenger RNA. The presence of adenosine in hydrolysates of poly(A) tracts showed that these sequences are located at the 3'-OH end of the RNA. The ratios of AMP/adenosine in the samples confirmed that the length of the A-tracts is approx. 13-17 nucleotides (Ohta, N., Sanders, M. and Newton, A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2343--2346)

Bacteria

Requirement of a cell division step for stalk formation in Caulobacter crescentus.

Penicillin G at low concentrations blocked cell division in Caulobacter crescentus without inhibiting cell growth. The long filamentous cells formed after two to three generations under these conditions had a stalk at one pole and usually one or more flagella at the opposite pole. The failure of the filaments to form a second stalk at the flagellated pole indicates that stalk formation was dependent upon completion of a step that was also required for cell division. Two observations support this conclusion. (i) Penicillin did not stop the normal development of synchronous swarmer cells into stalked initiation and stalk elongation. (ii) When the action of penicillin was reversed by the addition of penicillinase to cultures of filaments, stalks were not formed at the nonstalked pole until after cell division had occurred; thus the normal order of development events was maintained: cell division leads to stalk formation. These results are consistent with a model in which the organization of the developmental program for stalk formation occurs before cell division as a consequence of steps that branch from the cell division pathway.

Bacteria

Isolation and characterization of Caulobacter crescentus flagellar hooks.

The basal hook structure of the flagellar organelle Caulobacter crescentus was isolated from release flagella. Hook preparations contained a single major proteins species of 73,000 molecular weight and proteins in smaller amounts that may be minor hook components. Hooks isolated from C. crescents CB13B1a and CB15 were immunologically cross-reactive.

Bacteria

Chromosome replication in Caulobacter crescentus growing in a nutrient broth.

The pattern of chromosome replication in the Caulobacter crescentus cell cycle was studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G1), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.

Bacteria

Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus.

To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.

Bacteria

Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells.

Envelope-associated nucleoids have been isolated from Caulobacter crescentus by using a modification of the procedure of T. Kornberg et al. (Proc. Natl. Acad. Sci. U.S.A. 71:3189-3193, 1974). The development of a Ludox density gradient procedure has permitted preparation of large quantities of synchronous cells. The sedimentation coefficients of the envelope-associated nucleoids of stalked and swarmer cells, prepared under conditions of equivalent cell lysis, were 3,000S and greater than 6,000S respectively. Small differences in the relative amounts of deoxyribonucleic acid, ribonucleic acid, and protein in stalked and swarmer cell envelope-associated nucleoids could not account for the large differences in sedimentation behavior. These characteristic sedimentation coefficients were retained in mixing experiments.

Bacteria

Rate of major protein synthesis during the cell cycle of Caulobacter crescentus.

The rate of major protein synthesis was examined during the synchronous differentiation of Caulobacter crescentus. Total cell proteins were pulse-labeled with [35S]methionine at different times in the swarmer cell cycle and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The rates of synthesis of total cell proteins and of about one-half of the individual major proteins examined increased through G1 and S periods but remained nearly constant during G2 period. The rates of synthesis of the other half of the individual major proteins either increased continuously throughout the swarmer cell cycle or doubled during S period. One stage-specific protein was also detected in late S period. For most of the major proteins examined, the rate of synthesis in the swarmer cell was less than that in the stalked cell. It seemed that, before the onset of G2 period, the Caulobacter cell was already able to synthesize each major protein at the additive rate of the two progeny cells. Compared to the stability of cellular proteins, the functional degradation rate of mRNA coding for individual major proteins was rapid, with half-lives of 0.4 to 5.8 min. It thus seems that the rate of major protein synthesis mainly reflects the transcriptional control of gene expression.

Bacteria