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Abstract:
The Alphaproteobacteria show a remarkable diversity of cell
cycle-dependent developmental patterns, which are governed by the
conserved CtrA pathway. Its central component CtrA is a DNA-binding
response regulator that is controlled by a complex two-component
signaling network, mediating distinct transcriptional programs in the
two offspring. The CtrA pathway has been studied intensively and was
shown to consist of an upstream part that reads out the developmental
state of the cell and a downstream part that integrates the upstream
signals and mediates CtrA phosphorylation. However, the role of this
circuitry in bacterial diversification remains incompletely understood.
We have therefore investigated CtrA regulation in the morphologically
complex stalked budding alphaproteobacterium Hyphomonas neptunium.
Compared to relatives dividing by binary fission, H. neptunium shows
distinct changes in the role and regulation of various pathway
components. Most notably, the response regulator DivK, which normally
links the upstream and downstream parts of the CtrA pathway, is
dispensable, while downstream components such as the pseudokinase DivL,
the histidine kinase CckA, the phosphotransferase ChpT and CtrA are
essential. Moreover, CckA is compartmentalized to the nascent bud
without forming distinct polar complexes and CtrA is not regulated at
the level of protein abundance. We show that the downstream pathway
controls critical functions such as replication initiation, cell
division and motility. Quantification of the signal flow through
different nodes of the regulatory cascade revealed that the CtrA pathway
is a leaky pipeline and must involve thus-far unidentified factors.
Collectively, the quantitative system-level analysis of CtrA regulation
in H. neptunium points to a considerable evolutionary plasticity of cell
cycle regulation in alphaproteobacteria and leads to hypotheses that may
also hold in well-established model organisms such as Caulobacter
crescentus.
Author summary
Bacteria show a variety of morphologies and life cycles. This is
especially true for members of the Alphaproteobacteria, a bacterial
class of considerable ecological, medical, and biotechnological
importance. The alphaproteobacterial cell cycle is regulated by a
conserved regulatory pathway mediated by CtrA, a DNA-binding response
regulator that acts as a transcriptional regulator and repressor of
replication initiation. CtrA controls the expression of many genes with
critical roles in cell growth, division, and differentiation. The
contribution of changes in the CtrA regulatory network to the
diversification of alphaproteobacterial species is still incompletely
understood. Therefore, we comprehensively studied CtrA regulation in the
stalked budding bacterium Hyphomonas neptunium, a morphologically
complex species that multiplies by forming buds at the end of a
stalk-like cellular extension. Our results show that this distinct mode
of growth is accompanied by marked differences in the importance and
subcellular localization of several CtrA pathway components. Moreover,
quantitative analysis of the signal flow through the pathway indicates
that its different nodes are less tightly connected than previously
thought, suggesting the existence of so-far unidentified factors. Our
results indicate a considerable plasticity of the CtrA regulatory
network and reveal novel features that may also apply to other
alphaproteobacterial species.
