Dynamic spatial regulation in the bacterial cell.

When thinking about what the future holds, it is most rational to begin with the questions that can now be articulated and then take a leap of faith into the realm of possible answers to these questions. An overriding theme, of course, is that we are now in a position to build a complete system of information flow within the context of a living chemical machine.The discovery of multiple two component signal transduction proteins (30–50 pairs of sensor histidine kinases and response regulators per bacterial cell, sometimes grouped in extensive phosphorelays) has provided the machinery that links the perception of spatial and temporal perturbations to such varied cellular responses as transcriptional regulation, replication initiation, and chemotaxis. The activity of these two component phosphotransfer proteins are, in turn, regulated by where they are transiently located in the cell and by when they are present during the cell cycle. The challenge will be to integrate the multiple signal transduction pathways that control the ground state cell cycle circuitry with the use of other signaling pathways, such as those mediated by quorum sensing, to interpret extracellular signals. The identification of the full complement of signal proteins for each bacterial species (taking advantage of bacterial genome sequences), their dynamic positioning, and their function will provide a comprehensive network of regulatory information flow in free-living cells.Dynamic changes in subcellular localization has emerged as a common feature of regulatory and structural proteins in bacteria. As we have seen, examples include kinases and regulators of the two-component family of signal transduction proteins, proteins that mediate cytokinesis and chromosome segregation, proteins that dictate proper placement of the division septum, proteins that form the replication machine, and multiple transcription factors, proteases, and phosphatases that control cell cycle, cell differentiation, and morphogenesis. Clearly then, where proteins are deployed in cells and when they change their subcellular addresses is a pervasive and basic theme of the prokaryotic genetic network, indicating that in the future the bacterial cell will increasingly be understood in terms of the dynamic spatial behavior of proteins.In this process, bacterial regulatory pathways will take on the attributes of three-dimensional chess. Many of the regulatory pathways we have become accustomed to view as planar must in the future incorporate dynamic changes in cellular addresses. A closer look at the integration of spatial and temporal information and the regulatory circuitry raises multiple questions. For example, how do morphological changes in regions of the cell membrane and the assembly of the division septum control subsequent cellular responses such as the differential presentation of kinases, phosphatases, and proteases at specific cellular sites? How do these changes lead to consequent changes in transcriptional activity? We speculate that yet another controlled spatial parameter involves nucleoid organization and the requisite positioning of critical genes at specific regions of the cell cycle (pole versus midcell) and at specific times in the cell cycle. Thus, the three-dimensional organization of the nucleoid and its dynamic rearrangement may prove to be a contributing factor to the cellular regulatory circuitry.Turning to more mechanistic questions, how do proteins move to targeted destinations in the bacterial cell? Are there dedicated motor proteins? Can mechanisms that use diffusion alone account for the dynamic movement of multiple proteins in traffic patterns that are reminiscent of those in midtown Manhattan. The availability of full genome sequences and global screens for genes that affect movement will allow the identification of the genes dedicated to protein movement.Future work will reveal unique structures of cell membrane domains, such as those at the poles and at potential division sites that are used to position (anchor) functional machines, including the replication complex, the cell division apparatus, flagella, and the chemotaxis complex. These and other multiprotein complexes that convert chemical energy to mechanical energy and signal information flow from one part of the cell to another will be targeted to differentiated membrane domains. One of these might be a remnant of the division plane that, after cell division, remains in place and serves as a topological memory.In this context, an important objective will be to understand why protein complexes in the small bacterial cell need to be transiently moved to specific cellular addresses. For example, why should the histidine kinases that direct polar flagellar biogenesis be located at the site of flagellar assembly? Diffusion of molecules within the bacterial cell ensures that proteins could be fully dispersed everywhere in the cell within milliseconds. Clearly, mechanisms will be revealed that prevent free diffusion for some groups of proteins. Why and how have they evolved?To approach an understanding of the complete cellular machine, the subroutines that have specific cellular tasks will be defined, and then, in turn, these subroutines will be connected into the larger network that incorporates temporal and spatial constraints. Critical nodes in regulatory networks will be identified that direct subsequent events in defined pathways. A hallmark of bacterial cells is their ability to make choices in their metabolic responses to environmental fluctuations. Choices at these critical nodes will dictate progression down one pathway of a regulatory circuit rather than another. It is conceivable that these choices will be influenced by the spatial deposition of regulatory complexes during specific stages of the cell cycle.Although many of the questions raised here will be answered by investigating individual cases, we will do so within a larger context. We will no longer proceed solely at the pace of gene by gene, enzymatic reaction by enzymatic reaction, or even by the definition of each cellular machine, but rather we are now in the position to integrate the complete set of cellular reactions into the system that embodies a living entity. The value of analyzing organisms with a small genome (1.5–5.0 Mb) is the capacity to identify and manipulate all of the machinery that runs the cell's physiology and to allow us to view the process as if we were system engineers. Having full genome sequences lays the groundwork for the more difficult challenge, now within the scope of reality, of defining the complete genetic circuitry that runs the daily business of the bacterial cell. The identification of the genes, and establishing the connectivity among the genes, that run the cell cycle and govern cell specialization is just the first step. The subsequent integration of the time and the location of protein function during the cell cycle, the generation and maintenance of asymmetry, and responses to exogenous factors adds a critical layer of complexity to bacterial control mechanisms.