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At every cell cycle, Caulobacter crescentus undergoes an asymmetric cell division that produces two distinct progeny cells: a swarmer cell and a stalked cell (see Fig. 1). For 25 to 30% of the cell cycle, the swarmer cell is unable to replicate its DNA. After this period, the swarmer cell initiates DNA replication, sheds its flagellum, and forms a stalk at the pole that previously contained the flagellum (2). The progeny stalked cell initiates a new round of DNA replication, elongates, initiates cell division, and synthesizes a flagellum at the pole opposite the stalk. The stalk is a thin cylindrical extension of the cell wall and cell membranes. The synthesis of the stalk is a complex event that involves a temporally and spatially localized topological change in cell surface growth. Radiolabeling studies, investigation of the effect of penicillin on growing cells, and studies of the growth of the surface array are all consistent with the biosynthesis of the stalk being localized in a relatively sharp area at its base (20, 21).

Since there is a requirement for peptidoglycan synthesis in the synthesis of a stalk, penicillin-binding proteins (PBPs), which catalyze peptidoglycan cross-linking, are likely to be involved in stalk synthesis. Because -lactam antibiotics covalently bind to PBPs and inhibit their activity, they can be useful reagents for studying developmental changes in cellular morphology. One -lactam antibiotic, mecillinam, binds specifically to PBP 2 in Escherichia coli and causes cells to become spherical and eventually lyse (23). A mecillinam-resistant mutant of C. crescentus with short stalks was previously isolated, suggesting that the PBP target of mecillinam is involved in stalk synthesis (12). Unfortunately, this mutant was not analyzed genetically and it is not known whether the same mutation was responsible for the mecillinam resistance and for the defect in stalk synthesis.

Here, we present an analysis of the effect of mecillinam on stalk biosynthesis. Our results indicate that mecillinam inhibits stalk elongation and prevents normal stalk morphogenesis. We describe mecillinam-resistant mutants that are deficient in stalk biosynthesis and show that in these short-stalked mutants (Sks phenotype), the mutation affecting stalk synthesis is the same as the mutation conferring mecillinam resistance (Mec^r).

Mecillinam inhibits stalk elongation and morphogenesis. Mecillinam inhibits both stalk synthesis and cell division in C. crescentus (12, 13). Because stalk synthesis can be inhibited indirectly as a result of an inhibition of cell division (16), mecillinam could act directly and/or indirectly on stalk synthesis. To determine if mecillinam can inhibit stalk synthesis directly, we isolated swarmer cells by density gradient centrifugation (9) and examined the effect of mecillinam on cells before and after the initiation of stalk synthesis. Mecillinam was added to swarmer cells of strain NA1000bla (strains used in this study are described in Table 1) in peptone-yeast extract (PYE) medium (17), and the cells were monitored throughout the cell cycle (Fig. 1). Cells were analyzed by transmission electron microscopy with a JEOL model JEM-1010 electron microscope at 60 kV as described previously (4). When mecillinam was added at the swarmer cell stage (Fig. 1, T1), no stalks were visible even when the culture was examined the next day (Fig. 1B and C). Because stalk synthesis normally precedes the initiation of cell division during the cell cycle, this result indicates that mecillinam can inhibit stalk synthesis directly. In addition to its effect on stalk synthesis, mecillinam had a dramatic effect on the cell shape of C. crescentus. Cells grown with mecillinam were unable to complete cell division and became wider as they increased in mass (Fig. 1B and C), indicating that the target(s) of mecillinam plays a role in defining cell shape.

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Effect of mecillinam on stalk biosynthesis in synchronized cultures. Cultures of strains NA1000bla and YB1463 were synchronized, and mecillinam was added at a concentration of 20 µg/ml in PYE medium at different stages of the cell cycle. The cell cycle is shown diagramatically at the top of the figure. The wavy line at the pole of the swarmer cell represents the single polar flagellum. The stalk grows at the site previously occupied by the flagellum. The structures within cells represent the chromosomes. T1 and T2 indicate the times at which mecillinam was added. The electron micrographs show cells that are representative of the whole population. Cells were grown without mecillinam (A and D) or with mecillinam added at T1 (B, C, E, and F) or at T2 (G and H). Magnification, ×6,400 (all panels). The stalk appears short in panel A because this photo was taken at the end of a single cell cycle. In the following descriptions of individual panels, the times given are the time points after growth began when the photos were taken, unless otherwise indicated. (A) NA1000bla, no mecillinam, 130 min; (B) NA1000bla, 20 µg of mecillinam per ml, 130 min; (C) NA100bla, 20 µg of mecillinam per ml, 22 h; (D) YB1463, no mecillinam, 250 min; (E) YB1463, 20 µg of mecillinam per ml, 250 min; (F) YB1463, 20 µg of mecillinam per ml, 22 h; (G) YB1463, 20 µg of mecillinam per ml added at 100 min, photo taken at 250 min; (H) YB1463, 20 µg of mecillinam per ml added at 100 min, photo taken at 22 h. Arrows indicate an area of outgrowth that became visible at one pole of YB1463 cells and that was still present after 22 h (E and F) and a thickening at the base of the stalk of YB1463 cells that occurred when mecillinam was added at T2 (G and H). Negatives were scanned with a Umax Super Vista S-12 scanner by using Adobe Photoshop 3.0.5 software.

Isolation of mecillinam-resistant mutants with short stalks. Previously, a stalkless mecillinam-resistant mutant of C. crescentus was isolated; however, it was not known whether a single mutation caused both the mecillinam resistance and the stalkless phenotype (12). To determine if mutations can cause mecillinam resistance and loss of stalk synthesis simultaneously, we isolated and analyzed mecillinam-resistant mutants. Spontaneous mecillinam-resistant (Mec^r) mutants were isolated from strain NA1000bla at a frequency of 10⁶ to 10⁷. Screening Mec^r colonies by light microscopy indicated that 38 of 78 colonies made stalks that were much shorter than those of wild-type cells in PYE (Sks phenotype). The other mecillinam-resistant mutants had a wild-type appearance or were curly and/or elongated. Two mutants that had been isolated independently, YB1964 and YB1980, had the most severe stalk synthesis defect. Approximately 35% of dividing cells had no stalk, and for those cells that did have a stalk, the mean stalk length was 0.2 µm, whereas for strain NA1000bla, all dividing cells had stalks and their average length was 2 µm. Cells of both mutants were slightly wider and shorter than wild-type cells (compare the cells in Fig. 2A and C to the wild-type cells shown in Fig. 1A [all photos are at the same magnification]) and retained their shape when grown with mecillinam (Fig. 2B and D). We refer to the mutations in strains YB1964 and YB1980 as mec-1101 and mec-1165, respectively.

View larger version (70K): [in this window] [in a new window]   FIG. 2.   Phenotypes of mecillinam-resistant mutants grown in PYE medium alone (Mec) and PYE medium plus mecillinam (+Mec). Cells shown are representative of the whole population. (A) YB1964 (mec-1101), PYE medium; (B) YB1964 (mec-1101), PYE medium plus 5 µg of mecillinam per ml; (C) YB1980 (mec-1165), PYE medium; (D) YB1980 (mec-1165), PYE medium plus 3 µg of mecillinam per ml. Magnification, ×6,400 (all panels). Negatives were scanned with a Umax Super Vista S-12 scanner by using Adobe Photoshop 3.0.5 software.

The same mutations are responsible for mecillinam resistance and the stalk defects. To demonstrate that the mutations causing mecillinam resistance were also responsible for the short-stalk phenotype of YB1964 and YB1980, we attempted to backcross them to a wild-type background by using transduction (6) followed by selection for Mec^r. This was unsuccessful due to the high background level of spontaneous mecillinam resistance. We therefore used a pool of approximately 6,000 random mini-Tn5 lacZ2 (a gift from M. R. K. Alley) insertions made in strain NA1000 to link the transposon encoding kanamycin resistance to the loci conferring mecillinam resistance, as described previously (15). Insertion 1980 had a 37% cotransduction frequency with mec-1165, and insertion 1964 had a 9.5% cotransduction frequency with mec-1101. To determine if the mutation conferring mecillinam resistance was also causing the stalk defects observed in the mutants, we transduced the Kan^r transposon from Sks strains containing 1964 or 1980 linked to mec-1101 or mec-1165, respectively, into a wild-type background. All of the resulting Kan^r Mec^r transductants displayed the short-stalked phenotype (Fig. 3). Conversely, all the Kan^r Mec^s transductants had normal stalks (Fig. 3). When we transduced 1980 from a wild-type background into the Mec^r mutant YB1980 (mec-1165), all of the Kan^r transductants that were Mec^s had wild-type stalks. The reciprocal cross was not attempted with YB1964 (mec-1101) because of the presence of a second mutation contributing to Mec^r in this mutant. These results suggest that for both mutants, the same mutation confers mecillinam resistance and the short-stalk phenotype.

View larger version (61K): [in this window] [in a new window]   FIG. 3.   Phenotypes of Mecs and Mecr transductants from YB1995 (1964) and YB1993 (1980). Cells shown are representative of the whole population. (A) YB1461 (mec-1101) grown in PYE medium; (B) YB1462 (mec+) grown in PYE medium; (C) YB1461 (mec-1101) grown in PYE medium plus 3 µg of mecillinam per ml; (D) YB1462 (mec+) grown in PYE medium plus 3 µg of mecillinam per ml; (E) YB1998 (mec-1165) grown in PYE medium; (F) YB1999 (mec+) grown in PYE medium; (G) YB1998 (mec-1165) grown in PYE medium plus 3 µg of mecillinam per ml; (H) YB1999 (mec+) grown in PYE medium plus 3 µg of mecillinam per ml. Magnification, ×6,400 (all panels). Negatives were scanned with a Umax Super Vista S-12 scanner by using Adobe Photoshop 3.0.5 software.