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Previous Article | Next Article 
Journal of Bacteriology, December 1998, p. 6424-6428, Vol. 180, No. 23
Department of Microbiology and Molecular
Genetics, University of Texas Medical School, Houston, Texas 77030
Received 28 July 1998/Accepted 14 September 1998
FtsK is essential for Escherichia coli cell division.
We report that cells lacking the C terminus of FtsK are defective in chromosome segregation as well as septation, often exhibiting asymmetrically positioned nucleoids and large anucleate regions. Combining the corresponding truncated ftsK gene with a
mukB null mutation resulted in a synthetic lethal
phenotype. When the truncated ftsK was combined with a
minCDE deletion, chains of minicells were generated, many
of which contained DNA. These results suggest that the C terminus of
FtsK has an important role in chromosome partitioning.
FtsK was originally discovered to be
an essential cell division protein in Escherichia coli. A
mutation in the 5' end of the gene ftsK44 resulted in a
temperature-sensitive lethal phenotype, in which a blockage of a very
late stage of septation occurred, resulting in cell chains
(4). Recent FtsK depletion experiments have confirmed that
FtsK is essential for septation and have suggested that it may act
early (14). The C terminus of FtsK may be a separate
functional domain, because an N-terminal fragment of FtsK was
sufficient to complement the ftsK44 mutation (4,
18), to localize to the septum in a merodiploid (18),
and to restore septation in a strain that lacks the native
ftsK gene (7). Furthermore, the extreme C
terminus of FtsK is separated from the N terminus by a large
proline-glutamine-rich linker, and this C terminus is highly similar in
sequence to the C termini of Bacillus subtilis SpoIIIE and
other members of the SpoIIIE family (4). SpoIIIE of
B. subtilis is involved in the rescue of chromosomes that are bisected by asymmetric septa, which normally arise only during
sporulation (16). The C terminus of SpoIIIE is specifically involved in DNA recognition, and the N terminus is required for localization and anchoring of SpoIIIE to the septum (17).
SpoIIIE is essential for sporulation because of its rescue function but is dispensable for vegetative growth, presumably because the normal preseptational partitioning process pulls the chromosomes sufficiently apart. However, a role for SpoIIIE in vegetative cells of B. subtilis was revealed artificially by introducing a min
mutation; many of the resulting polar minicells in a spoIIIE
min mutant contained chromosomal DNA, suggesting that the rescue
function is needed whenever an asymmetric septum forms (12).
The similarity between FtsK and SpoIIIE suggested that FtsK had a
chromosome partitioning function in addition to its unique septation
function (4). However, several attempts to demonstrate such
a function in E. coli were unsuccessful. For example, the cell chains of the ftsK44 mutant appeared to have normal
chromosome segregation (4). A truncated FtsK that
inactivated the C terminus but left the N terminus intact also was
reported to exhibit normal chromosome segregation (6).
Interestingly, this mutant, ftsK1::cat, forms cell chains under certain conditions, indicating that the C
terminus also functions in late septation. In this paper, we show that
the ftsK1::cat mutant indeed exhibits
defects in chromosome segregation and provide supporting evidence by
combining ftsK1::cat with other
mutations that affect chromosome and septum placement.
Abnormal nucleoid segregation in the
ftsK1::cat mutant.
Although
previous studies of mutations in the N terminus and C terminus of FtsK
suggested that both domains function in septation but not in chromosome
segregation, the similarity between the C terminus of FtsK and members
of the SpoIIIE family prompted us to examine chromosome segregation in
more detail in the ftsK1::cat mutant.
This mutation was transduced from AD10 (6) into the wild-type E. coli strain MG1655 to make WM974 and into
strain MC1061 to make WM977. The tendency of
ftsK1::cat cells to form chains
facilitated the evaluation of nucleoid positioning patterns within
aligned cells. We found that despite the presence of many normal
nucleoid patterns, approximately 20% of individual cells and cells
within chains of WM974 (of 1,591 cells counted) in logarithmic growth
contained nucleoids at asymmetric positions, resulting in large cell
segments that lacked chromosomal DNA (Fig.
1A). In addition to this positioning
problem, about 1% of the cells were anucleate. Although this is a low
proportion of cells, it is significantly higher than the proportion in
wild-type cells in logarithmic growth (which exhibit <0.03% anucleate
cells) (10) and provides additional evidence for a
chromosome segregation defect. Interestingly, the
ftsK1::cat-mediated segregation
problems became more pronounced in cells within or entering stationary phase and was also more severe in WM977 (data not shown). For both the
mutant and wild-type strains, DAPI (4',6-diamidino-2-phenylindole) staining of live cells revealed nucleoid patterns similar to those of
fixed cells (data not shown).
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Role of the C Terminus of FtsK in Escherichia
coli Chromosome Segregation
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FIG. 1.
Localization of nucleoids and FtsZ in the
ftsK1::cat mutant. Strain WM974 was
grown in M9 medium supplemented with glucose and Casamino Acids (A to C
and G to L) or Luria-Bertani medium (E and F) at 37°C. The wild-type
parental control strain MG1655 was grown in Luria-Bertani medium at
37°C (D). Cells were grown to an optical density at 600 nm of
approximately 0.2, fixed, stained with 0.5 µg of DAPI per ml, and
visualized as described previously (13). Immunostaining with
affinity-purified anti-FtsZ was also described previously
(13). For panels D to F, cephalexin at 20 µg per ml was
added to the culture, which was grown for an additional 2 h prior
to fixation and DAPI staining. Panels A, D, E, F, G, and J show
overlays of phase contrast plus DAPI staining (pseudocolored red);
panels B, H, and K show overlays of phase contrast plus FtsZ
immunostaining (green); and panels C, I, and L show overlays of DAPI
(red) plus FtsZ immunostaining (green). Long arrows in panels A and B
point to chains of cells with segregation defects, while short arrows
in these panels highlight single cells with defects. Arrows in panels E
and F point to filaments with severe segregation defects. Arrows in
panel J highlight two cells, probably daughters, with misplaced
nucleoids; the short arrow in panel K points to the FtsZ ring in the
bottom cell that is off center relative to the cell and adjacent to the
nucleoid; and the long arrows in panels K and L point to the FtsZ ring
in the top cell that is asymmetric relative to the misplaced nucleoid
but nevertheless at the cell midpoint. Bar, 5 µm.
Lethality of an ftsK1::cat
mukB double mutant.
The results described above suggest
that the C terminus of FtsK is involved in chromosome positioning.
Nevertheless, this defect is not sufficiently severe to abolish
viability, presumably because more cells appear to be unaffected by the
mutation. Because a mukB null mutant has severe chromosome
partitioning defects and yet is viable at temperatures below 28°C
(10), we tested whether cells lacking both MukB and the FtsK
C terminus would be viable. We attempted to combine the
mukB::kan (10) and
ftsK1::cat mutations by phage P1
transduction. The ftsK and mukB genes are within
cotransduction distance on the chromosome, at 20.1 and 21.0 min,
respectively. Therefore, a small percentage of
ftsK1::cat recipients (AD10 and WM974)
receiving the mukB::kan donor allele would be expected to become ftsK+ and
chloramphenicol sensitive (Cms), and some of the
mukB::kan recipients (WM949, which
is mukB::kan in MG1655) receiving
the ftsK1::cat allele would be expected to become mukB+ and kanamycin sensitive
(Kms). However, when ftsK1::cat
recipients AD10 and WM974 were transduced with
mukB::kan and grown at 22°C, 246 of 246 of the combined Kmr transductants were Cms.
Similarly, when the mukB::kan recipient
WM949 was transduced with ftsK1::cat at
22°C, 200 of 200 of the Cmr transductants were
Kms. These results suggest that the double mutants were not viable.
mukB::kan allele, selecting for
Apr Kmr at 22°C. Distinct small- and
large-colony transductants were obtained; we surmised that if pAD12
could only partially rescue the lethality, then the small colonies
probably were enriched for the double mutants relative to the large
colonies. In support of this idea, 92 of 356, or 26%, of the
small-colony class were Apr Kmr
Cmr, while only 9 of 272, or 3%, of the large-colony class
were Apr Kmr Cmr. These results
indicated that the ftsK gene on pAD12 could partially rescue
the synthetic lethal phenotype conferred by the combined ftsK1::cat and
mukB::kan mutations.
Minicell chains in an ftsK1::cat
minB double mutant.
To examine the effects of loss of
ftsK function on formation of minicells, we combined a
deletion of the entire minB locus (genes minCDE)
with the ftsK1::cat mutation. The
minB deletion was derived from PB114
(minB::kan) (5). The double
minB::kan ftsK1::cat mutant
was constructed by sequentially transducing MG1655 to kanamycin and
chloramphenicol resistance producing strain WM975. The viability of
this strain indicated that the loss of minB had no
synthetic phenotype. As expected, WM975 exhibited a combination of
minicells and filaments typical of minB mutants. However, because the ftsK1::cat
mutation delayed cell separation, many minicells were found still
attached to cell poles or, more strikingly, to each other (Fig.
2A and D). The minicell chains were
therefore a convenient record of several sequential rounds of minicell
septation events, consistent with previous observations of sequential
minicell formation in microculture (2).
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DNA in minicells of the ftsK1::cat minB double mutant. Minicells generated by a min mutant (1) or by overproduction of FtsZ or FtsZ and FtsA (3, 15) normally do not contain chromosomal DNA. This is presumably because septation events leading to minicell formation normally occur away from nucleoid regions. However, the chromosome positioning defect of the ftsK1::cat mutant prompted us to test whether chromosomal DNA might be abnormally partitioned into minicells in WM975. Examination of minicells revealed that 28% of all attached minicells (of 177) were strongly stained for DNA. This is probably an underestimate, because minicells with weak DAPI staining were not scored. Examples of minicells with and without DNA are shown in Fig. 2B, C, E, and F. In general, minicells with DNA seemed to occur most frequently when they were adjacent to a nucleate region of the mother cells and rarely when adjacent to an anucleate region. This phenomenon of minicells containing DNA is precisely what was observed in vegetatively growing spoIIIE min mutants of B. subtilis (12), further supporting the idea that FtsK and SpoIIIE may have similar roles in chromosome dynamics.
Conclusions. Using several approaches, we have discovered a role for FtsK in chromosome segregation in E. coli. We have shown that about one-fifth of cells containing a truncated FtsK have obvious chromosome positioning defects. These defects were most easily observed in cell chains or in filaments, where the positioning problems were compounded over a longer distance. The abnormal state of the chromosomes in these cells correlated with the failure to form proper FtsZ ring structures, and when FtsZ rings did form, they often were located at one side of the misplaced nucleoid. The lack of FtsZ rings might be caused in part by induction of the SOS response or by blockage of the division site by the abnormal nucleoid.
The hypothesis that FtsK and MukB may function together to partition chromosomes is supported by the synthetic lethality of amukB
ftsK1::cat double mutant. The phenotype suggests
that MukB and the C terminus of FtsK have some redundant functions, allowing the single mutants to survive. We speculate that in the presence of functional MukB, chromosomes are properly condensed and are
often in positions sufficient for segregation of intact chromosomes to
daughter cells after cell division, even without the postulated
positioning function of FtsK. Likewise, in the absence of MukB,
chromosomes become disorganized, but FtsK may be able to position them
in enough cells to achieve viability at lower temperatures. In cells
lacking both the FtsK C terminus and MukB, on the other hand, the
positioning and condensation defects may be compounded sufficiently to
preclude viability of a colony.
In the absence of the SpoIIIE-like portion of FtsK, many minicells
contained fragments of the chromosome, just like in B. subtilis spoIIIE min double mutants. This and the aberrant
localization of nucleoids in cell chains are presumably due to trapping
of nucleoids by invaginating septa, which normally is prevented by the
C terminus of FtsK. The abnormal nucleoid localization in some of the
cephalexin-induced ftsK1::cat filaments might
also be explained if a subset of cells had their nucleoids trapped by
septa prior to addition of the drug. However, the role of FtsK in
E. coli must be somewhat different from that of
B. subtilis SpoIIIE because FtsK possesses a septation
function that has not been demonstrated for SpoIIIE. Further studies of
the role of FtsK in chromosome segregation should allow a clearer
picture of the mechanistic aspects of the process to emerge.
While this paper was under review, Liu et al. published a report
describing chromosome segregation defects associated with a C-terminal
deletion of FtsK that are very similar to the defects observed here
(8). They also reported that the SOS response is induced by
an FtsK deficiency, presumably because of DNA damage incurred during
nucleoid trapping by the septum in some cells. SOS induction of SulA
might explain why FtsZ rings are often lacking in cells with aberrantly
positioned DNA.
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ACKNOWLEDGMENTS |
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We are grateful to W. Cook and L. Rothfield for the PB114 strain and to A. Diez and T. Nyström for strain AD10 and the pAD12 plasmid.
This work was supported by National Science Foundation grant MCB-9513521. E.K.W. was also supported by the University of Texas-Houston Summer Research Program.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX 77030. Phone: (713) 500-5452. Fax: (713) 500-5499. E-mail: margolin{at}utmmg.med.uth.tmc.edu.
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