Structural studies on the capsid of Caulobacter crescentus bacteriophage phiCbK.
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Biomedical subjects
Publications and source records attributed to N Agabian-Keshishian.
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Caulobacter crescentus goes through a series of morphological changes during its life cycle, including the coincident expression of synthesis of flagella, pili, and receptor sites for DNA bacteriophage. Upon transfer of a mixed population of cells to medium containing lactose as the sole carbon source, these changes were blocked for about 20 hr until beta-galactosidase activity became apparent. The addition of dibutyryl cyclic AMP to the blocked cultures brought about the resumption of cell differentiation, growth, and the appearance of beta-galactosidase activity within 1 hr. Unlike Escherichia coli, the intracellular and extracellular concentrations of cyclic AMP in C. crescentus did not vary under several growth conditions, including catabolite repression. It would appear, therefore, that although there is an effect of cyclic AMP on the induction of beta-galactosidase and differentiation in C. crescentus, regulation of these processes occurs without consistent changes in the cellular level of this nucleotide.
The foregoing studies are intended to define a differentiation process and to permit genetic access to the mechanisms that control this process. In order to elucidate the basic mechanisms whereby a cell dictates its own defined morphogenic changes, we have found it helpful to study an organism that can be manipulated both biochemically and genetically. We have attempted to develop the studies initiated by Poindexter,Stove and Stanier, and Schmidt and Stanier (16, 17, 20) with the Caulobacter genus so that these bacteria can serve as a model system for prokaryotic differentiation. The Caulobacter life cycle, defined in synchronously growing cultures, includes a sequential series of morphological changes that occur at specific times in the cycle and at specific locations in the cell. Six distinct cellular characteristics, which are peculiar to these bacteria, have been defined and include (i) the synthesis of a polar organelle which may be membranous (21-23), (ii) a satellite DNA in the stalked cell (26), (iii) pili to which RNA bacteriophage specifically adsorb (16, 33), (iv) a single polar flagellum(17), (v) a lipopolysaccharide phage receptor site (27), and (vi) new cell wall material at the flagellated pole of the cell giving rise to a stalk (19, 20). Cell division, essential for the viability of the organism, is dependent on the irreversible differentiation of a flagellated swarmer cell to a mature stalked cell. The specific features of the Caulobacter system which make it a system of choice for studies of the control of sequential events resulting in cellular differentiation can be summarized as follows. 1) Cell populations can be synchronized, and homogeneous populations at each stage in the differentiation cycle can thus be obtained. 2) A specific technique has been developed whereby the progress of the differentiation cycle can be accurately measured by adsorption of labeled RNA phage or penetration of labeled phage DNA into specific cell forms. This technique can be used to select for mutants blocked in the various stages of morphogenesis. 3) Temperature-sensitive mutants of Caulobacter that are restricted in macromolecular synthesis and development at elevated temperatures have been isolated. 4) Genetic exchange in the Calflobacter genus has been demonstrated and is now being defined. Two questions related to control processes can now readily be approached experimentally. (i) Is the temporal progression of events occurring during bacterial differentiation controlled by regulator gene products? (ii) Is the differentiation cycle like a biosynthetic pathway where one event must follow another? The availability of temperature-sensitive mutants blocked at various stages of development permits access to both questions. An interesting feature of the differentiation cycle is that the polar organelle may represent a special segregated unit which is operative in the control of the differentiation process. Perhaps the sequential morphogenic changes exhibited by Caulobacter are dependent on the initial synthesis of this organelle. Because the ultimate expression of cell changes are dependent on selective protein synthesis, specific messenger RNA production-either from DNA present in an organelle or from the chromosome-may prove to be a controlling factor in cell differentiation. We have begun studies with RNA polymerase purified from Caulobacter crescentus to determine whether cell factors or alterations in the enzyme structure serve to change the specificity of transcription during the cell cycle. Control of sequential cell changes at the level of transcription has long been postulated and has recently been substantiated in the case of Bacillus sporulation (6). The Caulobacter bacteria now present another system in which direct analysis of these control mechanisms is feasible.
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The bacterium Caulobacter crescentus carries out a distinct differentiation process during its life cycle. Cultures of the bacterium have been synchronized and an assay has been developed for monitoring the course of morphogenesis by the selective adsorption of radioactive RNA bacteriophage.
The Caulobacter crescentus bacteriophage phiCbK was studied with respect to the physical and chemical properties of both the phage and its deoxyribonucleic acid (DNA). Electron micrographs reveal the phage to be among the largest DNA bacteriophages reported, with head dimensions of 64 by 195 nm and a flexible tail 275 nm in length. The phage is composed of 57% DNA. This DNA is double-stranded as indicated by (i) the sharp increase in extinction coefficient over a narrow range of temperature increase, (ii) an increase in density in CsCl upon thermal denaturation, and (iii) the equivalence of guanine and cytosine as well as adenine and thymine, determined by chemical analysis. phiCbK DNA cosediments with Escherichia coli phage T2 DNA and has therefore been assigned an S(20,w) value of 63.5S. The size of the phage and its DNA and the percentage of DNA indicate that the phiCbK phage head is relatively loosely packed. The properties of the DNA from bacteriophage phiCbK are similar to those of host C. crescentus DNA with respect to buoyant density, thermal transition point, and guanine plus cytosine content.