Original Article: Kanda K., Tatsuta T., Suzuki T., Taguchi F., Naito K., Inagaki Y., Toyoda K., Shiraishi T., Ichinose Y. (2011) Two flagellar stators and their roles in motility and virulence in Pseudomonas syringae pv. Tabaci 6605. Molecular genetics and genomics 285(2): 163-174
Post By: Wamiah Chowdhury
In the non-microbial world, bacteria are always seen as the
bad guys. They are never considered to be as complex as the eukaryotes, and
many people believe that they are only good at causing diseases. But when one
starts to dig deeper into the microbial world, it is hard not to appreciate the
world of bacteria. After all, they are one of the few organisms capable of surviving
in any situation. In my opinion, their complexity as single-celled organisms
will always surpass the complexity of eukaryotic cells. Take for example, the bacterial
appendage flagellum. Flagellum is responsible for locomotion in many bacteria. It
allows a bacterium to either move towards a favorable condition or escape a
stressful situation. The basic components of a flagellum include a filament, which
is made up of the protein Flagellin. The filament is joined to a motor via a
hook. The motor contains stator proteins, which pump ions to generate torque.
The torque then allows the filament to rotate. Sounds simple right? Well,
nothing about bacteria is ever simple. This “simple” structure of flagellum is
made up of at least 30 different kinds of proteins, with each protein having up
to a thousand copies within the flagellum. Imagine all that protein in an
organism that is not even visible to the naked eye!
Figure 1: Flagellar components of Salmonella
enterica serovar Typhimurium. The figure shows the different components
that make up a bacterial flagellum. Each of these proteins have its own
function, making the flagella one of the most complex bacterial structure1.
Although flagellum is primarily associated with movement, it
can play important roles in other functions of a bacterium. In the bacteria Pseudomonas syringae, genes that encode
for motor proteins of the flagellum were also found to play a role in virulence
(the ability to cause diseases in other organisms). P. syringae are gram-negative, rod shaped bacteria that cause
diseases in plants. Each strain of this bacteria cause disease in specific
plants, which greatly effects the agricultural industry, making it extremely
important to study the organism. Previous research had shown that deletion of the gene responsible
for flagellin production caused the bacteria to lose its virulence, however, it
was not known why. A study conducted by Kanda et al3. showed that
virulence was also lost when genes coding for stator proteins in the motor of
the flagella were deleted. They saw that the deletion caused the bacteria to
stop producing quorum-sensing molecules. Quorum sensing is a phenomenon where
certain bacteria produce chemical signaling molecules for other bacteria to
take up, resulting in changes in gene expression2. Quorum sensing
can be used for virulence, colony formation, symbiosis, antibiotic production,
motility etc2. Kanda et al. speculated that the loss of motility due
to gene deletion of the stator protein caused a change in the production of
AHLs (quorum sensing molecules) through a cell-density interaction, which
resulted in the bacteria losing its virulence. Since P. syringae cause plant
diseases and negatively affect agriculture of tobacco and tomato, finding a way
to disrupt its virulence will greatly benefit the agricultural industry. With
less pathogenic bacteria around, agricultural production will greatly increase,
bringing down the price of food (and making everyone happy). Antibiotics
targeting the stator proteins can potentially make the bacteria non-pathogenic,
which is why the work done by Kanda et al. is important.
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| FUN FACT: Pseudomonas syringae plays a role in rain and snow formation. They are used in making artificial snow! |
The stator of the motor proteins in P. syringae is made up of two complexes: MotA/MotB and MotC/MotD.
Although the amino acid sequence of MotC/D showed it to be a Na+ driven
complex rather than a typical proton driven complex (which generates proton
motive force as an energy source), experiments with phenamil (sodium-motor
specific inhibitor) showed that the presence or absence of Na+ had
no effect on the mobility of the bacteria, indicating that the MotC/D stator
was not in fact dependent on sodium. However, experiments with proton-motor
specific inhibitor (CCCP) showed that the motility of the bacteria was greatly
reduced in presence of the compound. This showed that both the MotA/B and
MotC/D complexes use proton gradient as their energy source.
The research group made three mutant strains of the bacteria
to better understand the role of the stator proteins in motility and virulence.
ΔmotAB mutant
lacked the gene encoding for stator proteins MotA and MotB, and therefore did
not have the MotA/B complex. ΔmotCD mutant did not have the
genes required for the production of MotC and MotD proteins, and therefore
lacked Mot C/D complex. ΔmotABCD mutant had no genes for
any of the Mot protein production, and as a result, did not have any of the
stator complex. The researchers found that the mutant strains of the bacteria
could not swim as well as their wild type counterparts. The research group also
noticed, through microscopy, that all the mutants had higher number of flagella
compared to wild-type strain. They also discovered that the production of
Flagellin protein was high in the ΔmotCD and ΔmotABCD
strains. Although they did not have a clear answer as to why the amount of
Flagellin protein was higher in bacteria with defective stators, they
speculated that the over production of Flagellin was possibly an attempt by the
bacteria to compensate for the loss of mobility.
Figure 2: (a) The number of flagella is higher in the mutant
strains compared to wild-type strains, as seen by electron microscopy. (b)
Statistical analysis of the number of flagella per cell of the mutants shown as
histograms. (c) Western blot analysis of the strains show that the flagellin
production is upregulated in the ΔmotCD and ΔmotABCD
strains.
The research group then carried out experiments to see the
virulence power of the mutant strains. They found that the wild-type bacteria
and ΔmotAB
bacteria
could infect tobacco leaves, however, ΔmotCD and ΔmotABCD lost
their virulence.
Figure 3: Virulence on tobacco plant. Tobacco leaves grown at 26°C with a 16-h photoperiod for 2 mol were inoculated with each bacterium at 2 9 108 cfu/ml by the spray method. Leaves were photographed 14 days after inoculation. (a) Wild-type strain sprayed in the tobacco leaves (b)ΔmotAB strain sprayed in the tobacco leaves (c)ΔmotCD strain sprayed in the tobacco leaves (d)ΔmotABCD strain sprayed in the tobacco leaves.
They also noticed that in the ΔmotCD and ΔmotABCD
strains, AHLs (quorum-sensing molecules) were produced in tiny amounts, whereas
ΔmotAB
strain could produce AHLs in the amount similar to that produced by wild-type.
Surprisingly, when genes coding for AHLs protein productions
were deleted, the bacteria were still able to form normal flagella, showing
that AHLs production required motility but not vice versa. The researchers
explained the observations through cell-density dependent pathogenesis. When
the bacteria infect a plant cell, they stop moving and form microcolonies using
quorum sensing. Once a suitable population size is reached, production of more
AHLs might cause reduction in bacterial division, and therefore the colonizing
bacteria become immotile and stop producing AHLs. This mechanism can
potentially explain why the loss of motility in the strains caused loss of AHLs
production. The researchers also suggested that AHL production might require
high proton concentration, and therefore loss of stator proteins, which
regulate proton gradient, might be the reason for the loss of AHLs production
in ΔmotCD and ΔmotABCD
strains
They conclude with the hypothesis that without proper AHLs production, bacteria
cannot form a pathogenic colony, meaning that enough of them do not come
together without AHLs production, to infect the plant cells.
Although the researchers did a great job in linking motility
with virulence, a few questions were left unanswered. For instance, it was not
discussed why the production of Flagellin was higher in the ΔmotCD and ΔmotABCD
strains, whereas the production was downregulated in the ΔmotAB strain.
Moreover, the two strains with more flagellin production seemed to have
complete loss of virulence, compared to the ΔmotAB strain. Does this mean
that Flagellin production might actually be the one that regulates AHLs
production? The research group had previously seen that deletion of the
flagellin gene also resulted in loss of AHLs production and virulence, which is
contradictory to the observations discussed in the paper. Is there a
possibility that some other protein of the flagellum is actually responsible
for AHLs production and virulence? There seems to be a missing link between
stator proteins, Flagellin proteins and AHLs production, and the pathway needs
to be elucidated. The data shown in the paper also suggests that loss of MotC/D
complex might actually be more harmful for the bacteria, since they lose their
mobility and virulence more than strains lacking MotA/B. The researchers make
no comment about that. Future studies should be targeted towards better
understanding the separate roles of Mot A/B and Mot C/D complexes in AHLs
production and virulence.
Since it is not known how motility is affecting AHLs
production and virulence, further work needs to be done to better understand
the mechanism. Future experiments could include constructing more mutants of
varied combinations, such as ΔmotAD or ΔmotBC, to
see if any of these can still infect plants, or whether loss of virulence is
only related to ΔmotCD strain. Another
experiment that could be done would be to look at genes that are upregulated
for flagellar production in ΔmotCD and ΔmotABCD,
which
could provide answers to reduction of AHLs production and virulence. All in
all, this paper provides a gateway to defeating virulence of Pseudomonas syringae.
References:
1) Chevance F.F.V and Hughes K.T (2008). Coordinating
assembly of a bacterial macromolecular machine. Nature Reviews Microbiology 6:455-465
2) Miller M.B and Bassler BL (2001). Quorum sensing in
bacteria. Annual Review of Microbiology
55:165-99
3) Kanda K., Tatsuta T., Suzuki T., Taguchi F., Naito K.,
Inagaki Y., Toyoda K., Shiraishi T., Ichinose Y. (2011) Two flagellar stators
and their roles in motility and virulence in Pseudomonas syringae pv. Tabaci 6605. Molecular genetics and genomics 285(2): 163-174
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