A specific cyclic guanosine 3':5'-monophosphate-binding protein in Caulobacter crescentus.

A binding protein specific for cyclic guanosine 3':5'-monophosphate (cyclic GMP) has been partially purified from extracts of the eubacterium Caulobacter crescentus and resolved from cyclic adenosine 3':5'-monophosphate (cyclic AMP)-binding activity. Binding of cyclic GMP is not affected by the addition of cyclic AMP or 5'-GMP, but is inhibited about 50 percent by a 50-fold molar excess of dibutyryl cyclic GMP or cyclic hypoxanthine 3':5'-monophosphate. The apparent dissociation constant for the cyclic GMP-binding protein complex is 1.1 X 10(-6) M.     

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.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

Synchronous cell differentiation in Caulobacter crescentus.

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Stalkless mutants of Caulobacter crescentus.

A stalk, a single falgellum, several pili, and deoxyribonucleic acid (DNA) phage receptors are polar surface structures expressed at a defined time in the Caulobacter crescentus cell cycle. When mutants were isolated as DNA phage phiCbK-resistant or ribonucleic acid (RNA) phage phiCp2-resistant, as well as nonmotile, strains, 5 out of 30 such mutant isolates were found not to possess stalks, but did possess inactive flagella. These stalkless mutants were resistant simultaneously to both DNA and RNA phages and did not possess pili and DNA pendent stalkless mutants. All motile revertants simultaneously regained the capacity to form stalks and susceptibility to DNA and RNA phages. It is suggested that a single mutation pleiotropically affects stalk formation, flagella motility, and coordinate polar morphogenesis of pili and DNA phage receptors. The stalkless mutants grew at a generation time similar to that of the wild-type strain at 30 degrees C. Cell size and morphology of a stalkless mutant, C. crescentus CB13 pdr-819, were also similar to those of the wild-type strain, except for the absence of a stalk. In addition, the CB13 pdr-819 predivisional cells were partitioned into smaller and larger portions, indicating asymmetrical cell division, as in the wild-type strain. From these results, it is suggested that swarmer cells undergo transition to cells of a stalked-cell nature without stalk formation and that the cell cycle of the stalkless mutant proceeds in an ordered sequence similar to that defining the wild-type cell cycle.     

RNA-controlled regulation in Caulobacter crescentus

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Pathway of glucose catabolism in Caulobacter crescentus.

Glucose when present as a sole organic carbon source in a mineral salts medium is dissimilated by Caulobacter crescentus ATCC 15252 (strain CB-2) by the Entner-Doudoroff pathway throughout the culture cycle (exponential, transition, and stationary phase). Most of the available glucose that is present at the onset of exponential growth is assimilated by the cells during the transition phase or the period associated with stalk cell development. Swarmer cell development is minimized during this phase. During this same period the pH drops from 6.1 to 4.9 as a result of an abundant excretion of acetic acid. Simultaneously, poly-beta-hydroxybutyrate accumulates within the cells at an accelerated rate. An NADP-dependent glyceraldehyde-3-phosphate dehydrogenase is also present throughout the culture cycle which subsumes the presence of the subsequent enzymes of the Embden-Meyerhof-Parnas pathway in pyruvate formation. An operative tricarboxylic acid cycle is associated with cells throughout the culture cycle.     

Transposon mutagenesis in Caulobacter crescentus

Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.     

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.     

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.     

Synchronous Cell Differentiation in Caulobacter crescentus

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Phospholipids of the differentiating bacterium Caulobacter crescentus.

The phospholipid composition of the stalked and swarmer cell types of the differentiating, Gram-negative bacterium Caulobacter crescentus was determined. The phospholipid composition of the stalked cell type was 86.5% phosphatidylglycerol, 10.4% lysylphosphatidylglycerol, and 3.0% cardiolipin; that of the swarmer cell type was 84.1, 11.4, and 4.4% respectively. Phosphatidylethanolamine, which is a major phospholipid component of most Gram-negative bacteria, was totally absent.     

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.