Unicellular organisms use a variety of mechanisms to co-ordinate activity within a community and accomplish complex multicellular processes. Because some of the processes that are exhibited by one species can be physiologically incompatible, it raises the question of how entry into these different pathways is regulated. In the Gram-positive bacterium Bacillus subtilis, genetic competence, swarming motility, biofilm formation, complex colony architecture and protease production are all regulated by the response regulator DegU. DegU appears to integrate environmental signals and co-ordinate multicellular behaviours that are subsequently manifested at different levels of DegU phosphorylation. Data are presented which indicate that: (i) swarming motility is activated by very low levels of DegU approximately P that can be generated independently from its cognate sensor kinase DegS; (ii) complex colony architecture is activated by low levels of DegU approximately P that are produced in a DegS-dependent manner to activate transcription of yvcA, a novel gene required for complex colony architecture; and (iii) high levels of DegU approximately P inhibit complex colony architecture and swarming motility but are required prior to the activation of exoprotease production. A model is proposed to explain why such a system may have evolved within B. subtilis to control these multicellular processes through a single regulator.

Biofilm formation is an example of a multicellular process which depends on cooperative behavior and differentiation within a bacterial population. Our findings indicate that there is a complex feedback loop that maintains the stoichiometry of the extracellular matrix and other proteins required for complex colony development by Bacillus subtilis. Analysis of the transcriptional regulation of two DegU-activated genes that are required for complex colony development by B. subtilis revealed additional involvement of global regulators that are central to controlling biofilm formation. Activation of transcription from both the yvcA and yuaB promoters requires DegUϳphosphate, but transcription is inhibited by direct AbrB binding to the promoter regions. Inhibition of transcription by AbrB is relieved when Spo0Aϳphosphate is generated due to its known role in inhibiting abrB expression. Deletion of SinR, a key coordinator of motility and biofilm formation, enhanced transcription from both loci; however, no evidence of a direct interaction with SinR for either the yvcA or yuaB promoter regions was observed. The enhanced transcription in the sinR mutant background was subsequently demonstrated to be dependent on biosynthesis of the polysaccharide component that forms the major constituent of the B. subtilis biofilm matrix. Together, these findings indicate that a genetic network dependent on activation of both DegU and Spo0A controls complex colony development by B. subtilis.

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The UhpABC regulatory system in enterobacteria controls the expression of the hexose phosphate transporter UhpT. Signaling is initiated through sensing of extracellular glucose 6-phosphate by membrane-bound UhpC, which in turn modulates the histidine-protein kinase UhpB. Together with the cytoplasmic response regulator UhpA, they constitute a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Activated (i.e., phosphorylated) UhpA binds to the promoter region of uhpT, resulting in initiation of transcription. We have investigated the contribution of transmembrane signaling (through UhpBC) and intracellular activation (through UhpA) to the overall Uhp response (UhpT expression) in vivo. UhpA activation could be made independent of transmembrane signaling when ⌬uhpBC cells were grown on pyruvate. Bacteria must be able to accurately respond to a large number of extracellular signals in their continuously changing surroundings. Therefore, they have evolved sophisticated sensory mechanisms coupled to intracellular signal transduction pathways. The majority of these prokaryotic intracellular signaling routes are based on the reversible phosphorylation of sensor kinases and response regulators in so-called two-component regulatory systems (23,28,30). According to this concept, sensory input affects autophosphorylation on a conserved histidine residue in the transmitter module of the kinase. The phosphoryl group is subsequently transferred to a conserved aspartate residue in the (amino-terminal) receiver domain of the cognate response regulator. This covalent modification elicits a molecular switch, and above a certain threshold amount of phosphorylated regulator, a specific response is turned on, which is in most cases gene transcription via (enhanced) affinity of the carboxyl-terminal output domain of the regulator for the target promoter sequence.To provoke a cellular response, the environmental physical or chemical stimulus has to cross the membrane. Therefore, in most cases the first step in a signal transduction cascade is the activation of a membrane-bound protein. In order to be able to quantitatively study specific transmembrane (receptor) signaling leading to cellular activation, the first (ligand-induced) step and the subsequent response should be well defined and straightforward to quantify. However, despite the huge number of characterized signaling pathways, such examples are not abundant.An exception is the Uhp regulatory system in Escherichia coli (for a review, see reference 13). This signaling pathway is triggered by external glucose 6-phosphate, which is recognized by the membrane-bound receptor protein UhpC. UhpC interacts with a second membrane-bound protein, UhpB, the kinase/phosphatase of the Uhp two-component system. Sensing of glucose 6-phosphate is supposed to cause a conformational change in the supposed UhpBC complex, leading to histidine autophosphorylation in the (cytoplasmic) transmitter domain of UhpB. Upon phosphoryl transfer, the response regulator UhpA (pre...

Expression of the UhpT sugar-phosphate transporter in Escherichia coli is regulated at the transcriptional level via the UhpABC signalling cascade. Sensing of extracellular glucose 6-phosphate (G6P), by membrane-bound UhpC, modulates a second membrane-bound protein, UhpB, resulting in autophosphorylation of a conserved histidine residue in the cytoplasmic (transmitter) domain of the latter. Subsequently, this phosphoryl group is transferred to a conserved aspartate residue in the response-regulator UhpA, which then initiates uhpT transcription, via binding to the uhpT promoter region. This study demonstrates the hypothesized transmembrane signal transfer in an ISO membrane set-up, i.e. in a suspension of UhpBC-enriched membrane vesicles, UhpB autophosphorylation is stimulated, in the presence of [γ-32 P]ATP, upon intra-vesicular sensing of G6P by UhpC. Subsequently, upon addition of UhpA, very rapid and transient UhpA phosphorylation takes place. When P " UhpA is added to G6P-induced UhpBC-enriched membrane vesicles, rapid UhpA dephosphorylation occurs. So, in the G6P-activated state, UhpB phosphatase activity dominates over kinase activity, even in the presence of saturating amounts of G6P. This may imply that maximal in vivo P " UhpA levels are low and/or that, to keep sufficient P " UhpA accumulated to induce uhpT transcription, the uhpT promoter DNA itself is involved in stabilization/sequestration of P " UhpA.