Axial filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase

Abstract

When chloramphenicol was added to a culture of Bacillus subtilis in early exponential growth, microscopic observation of cells stained by 4',6-diamidino-2-phenylindole showed nucleoids that had changed in appearance from irregular spheres and dumbbells to large, brightly stained spheres and ovals. In contrast, the addition of chloramphenicol to cultures in mid-and late exponential growth showed cells with elongated nucleoids whose frequency and length increased as the culture approached stationary phase. The kinetics of nucleoid elongation after the addition of chloramphenicol to exponential-phase cultures was complex. Immediately after treatment, the rate of nucleoid elongation was very rapid. The nucleoid then elongated steadily for about 4 min, after which the rate of elongation decreased considerably. Nucleoids of cells treated with 6-(p-hydroxyphenylazo)-uracil (an inhibitor of DNA synthesis) exhibited the immediate rapid elongation upon chloramphenicol treatment but not the subsequent changes. These observations suggest that axial filament formation during stationary phase (stage I of sporulation) in the absence of chloramphenicol results from changes in nucleoid structure that are initiated earlier, during exponential growth.Many investigators have sought to describe the arrangement of the nucleoplasm in the procaryotic cell. The subject has remained elusive, partly because the nucleoid undergoes rearrangement during the most gentle manipulations (e.g., rapid filtration and temperature changes, etc. [8]). Thus, after decades of effort, only a few noncontroversial statements can be made: the nucleoid of the exponential-phase cell is localized primarily in the center of the cell but does extend to the periphery of the cytoplasm (2,7,8,12,13); in stationary-phase cells, the nucleoid becomes confined to the center, and this results in a decrease in the density of this portion of the cell (3,6,21).Recently there has been a resurgence of interest in nucleoid structure. In part, this has resulted from the development of cryofixation techniques for electron microscopy (13), the availability of DNA-specific fluorescent stains such as 4',6-diamidino-2-phenylindole (DAPI) (11,17,19,25), and a rich supply of mutants whose phenotypes include characteristics that could be, or are, related to chromosome structure and function. In this light we have developed a method which uses image analysis to characterize the nucleoid in terms of size, shape, and distribution in a cell as the cell proceeds from exponential-to stationary-phase growth. The distinct internal organization of Bacillus subtilis as it undergoes symmetric and asymmetric division offers a model system for exploring nucleoid structure and function during exponential growth and post-exponential-phase differentiation.MATERIALS AND METHODS Growth conditions. B. subtilis BR151 trpC2 lys-3 metB5 (source, F. E. Young) was used for this study. Bacteria were grown in modified Schaeffer's sporulation medium (26). Cultures were grown aerobically (medium-to-flask volume ...

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis

“…Cells of this type were therefore assigned to a class called the preseptation class. The occurrence of an elongated, more diffuse nucleoid during stationary phase has been reported previously (6), and this form may be related to the axial filament seen by electron microscopy (36). The last class of cells observed were sporulating organisms, and in this class we included cells with a visible asymmetric septum or two nucleoids, one small and highly condensed (the prespore) and one larger and much less condensed (the mother cell).…”

“…1). These preparations could also be stained with 4Ј,6-diamidino-2-phenylindole (DAPI) (Sigma) and the DNA could be viewed by fluorescence microscopy, giving images similar to those reported previously for glutaraldehyde-fixed cells (6,41).…”

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…Despite their name, none of the classic spo0 mutations clearly prevented axial filament formation (228): spo0H mutants formed axial filaments (29,94), whereas spo0A, spo0B, and spo0F mutants underwent an additional symmetric division during sporulation, and it was not clear if that was preceded by axial filament formation (29,53). Some insight into the process was derived from analysis of the SMC (structural maintenance of chromosomes) protein, required for chromosome compaction and partitioning (25,176).…”

“…The vegetative cell is designated stage 0. Formation of an axial filament of chromatin, where two copies of the chromosome condense and elongate to form a filament that stretches across the long axis of the cell, is defined as stage I (21,29,308). Subsequently, the cell divides at a subpolar site, resulting in the formation of two unequally sized daughter cells.…”

“…The first is the formation of an axial filament of chromatin, in which both chromosomes in the predivisional cell elongate into a filament that stretches the length of the long axis of the cell. This structure was characterized by electron microscopy (78,188,252) and later by fluorescence microscopy (29,300).…”