Uncovering the Secrets of Bacterial Division
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February 19, 1999
Uncovering the Secrets of Bacterial Division
To the untrained observer, the life of a
bacterium could seem utterly boring. Grow and divide, sense the
environment, and if things look good, grow and divide again. In
a bug's life, it appears, to divide or not to divide is the main
Jonathan Beckwith studies how bacterial cells split
by looking at a group of proteins that localize to the
future division site.
But cell division is no trivial matter. An essential decision in
the life of a bacterium like E. coli, division is allowed
only when all other systems are go--if food is plentiful and no
damage has been done to the bacterial DNA, among other things. Once
DNA replication has occurred, a septum forms at a specific place
and time, creating the constriction that will split the cell in
two. How this septum is formed and how its formation is regulated
have remained major questions for microbiologists. A trio of recent
articles from the laboratory of Jonathan Beckwith, American
Cancer Society professor of microbiology and molecular genetics,
provides new insights into the assembly of the septum and the key
players in this process.
Like puzzle pieces coming together to reveal a bigger picture,
the articles published in the January issues of the Journal of
Bacteriology and Molecular Microbiology present a complementary
analysis of three membrane proteins that localize at the division
septum--FtsL, FtsI, and FtsQ--and show that their recruitment to
the septum depends on a linear, ordered chain of events.
String of Cells
Back in the 1960s, a temperature-sensitive screen in the Pasteur
Institute led to the identification of E. coli mutants with
an unusual shape. Elongated and thin, these mutants were said to
"filament," and were termed Fts mutants (for temperature-sensitive
filamentation). The striking phenotype suggested genes involved
in cell division, because these filaments were, in fact, strings
of cells that failed to completely divide and remained attached
at the ends.
|Joseph Chen, Nienke Buddel meijer, and
Dana Boyd are members of the team of researchers in Beckwith's
lab studying the proteins that participate in septal ring
It later became clear that not all mutants that formed filaments
were defective in the process of septation. Mutations in genes involved
in other cellular processes that indirectly affect division can
also result in filaments. Localization of proteins at the division
septum therefore became an important criterion for identifying proteins
that play a role in septation.
For a long time, though, only one of the septal proteins,
FtsZ, was abundant enough to be clearly visualized forming a ring
at the future division site. Although formation of this "Z ring"
was thought to be essential for the recruitment of eight other proteins
to the division site (including FtsL, Fts I, and FtsQ), technical
difficulties made further studies more challenging.
"It seemed clear to everyone in the field that a lot of the
proteins involved in cell division must localize at the septum,"
says Beckwith. But immunolocalization of these proteins was not
always successful. The proteins are made in very small quantities,
which might explain why they were hard to detect.
"This is why Green Fluorescence Protein [GFP] fusions have
become incredibly useful for us," says Beckwith. The scientists
fused several of the elusive Fts proteins to GFP, which served as
a luminous tag revealing their location. They used a novel approach
to introduce a single copy of the GFP fusion construct into the
bacterial genome, thereby expressing the recombinant protein at
physiological levels. When the researchers looked at the localization
of GFP fusion versions of FtsI, FtsL, and FtsQ, they observed specific
and clear restriction of the proteins to the Z ring.
How They Get There--and When
With an effective way to visualize these proteins at the division
septum, the next goal was to understand how they got there. Experiments
in which the distinct domains in FtsI, FtsL, and FtsQ were replaced
with domains from an unrelated protein showed that each septal protein
has different requirements for proper localization to the Z ring.
For FtsQ, only one domain is needed, whereas FtsI requires all its
domains for localization to the ring.
Finally, Beckwith's group helped to elucidate a sequential relationship
between the septal proteins. Using the GFP fusions, they analyzed
how FtsI, FtsL, and FtsQ localized in different E. coli strains
that were deficient in other septal proteins. These experiments
allowed them to establish a linear order of appearance at the septum,
where the presence of one Fts protein is necessary for the localization
of the more downstream components. The reconstruction of this linear
pathway of septal ring assembly reflects work from his and other
labs, Beckwith says. "We expect that more proteins involved in this
pathway will be identified," adds graduate student Joseph Chen.
Not Just Bacteria
Bacterial cells are not the only ones dealing with the complex
issue of septation. Septal ring equivalents are present in eukaryotic
cells, such as the actomyosin ring in yeast and the contractile
ring in mammalian cells. How homologous these structures are, however,
remains unclear. For example, bacterial cells lack actin and myosin,
which are components of the rings of eukaryotic cells. The differences
between the septal ring in bacteria and the ring in eukaryotic cells
are currently being explored by several biotechnology companies,
Beckwith says. Because they are specific to bacteria, septal proteins
represent a potential target for antibiotic therapy, he adds.
--Sylvia Pagán Westphal
Inserting Genes the Easy Way
Life will become a little easier for scientists attempting
to introduce their gene of choice into the genome of E.
coli, thanks to Dana Boyd, lecturer on microbiology
and molecular genetics at HMS. While in Beckwith's lab,
he developed a novel way to transfer a single copy of a
gene to a specific site in the bacterium's chromosome. The
method is unique in that no cloning steps are required.
"Everyone starts with a plasmid expression system and then
sometimes realizes that things should be put in the chromosome,"
says Boyd. "But recloning everything into a special vector
is a lot of work, so it often does not get done." With the
new system, a derivative of bacteriophage lambda called
Lambda InCh (for "Integration into Chromosome"), a virus
can pick up a gene from the plasmid vector that a scientist
has been using all along. This is done by simply growing
the virus on bacteria containing the plasmid. The virus--now
carrying the new gene--is used to reinfect bacterial cells,
integrating into a specific site on the chromosome. In the
last step, sequences from the viral DNA are eliminated,
leaving a single, stably integrated copy of the gene in
the E. coli genome. Having just one extra copy also
allows scientists to be more confident in the interpretation
of their results."Because you now have a single copy, you
can measure the activity of a protein accurately, and you
can do experiments under physiological conditions," Boyd