Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex.

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane ß-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.

Recombinant DNA technology and DNA sequencing.

DNA present in all our cells acts as a template by which cells are built. The human genome project, reading the code of the DNA within our cells, completed in 2003, is undoubtedly one of the great achievements of modern bioscience. Our ability to achieve this and to further understand and manipulate DNA has been tightly linked to our understanding of the bacterial and viral world. Outside of the science, the ability to understand and manipulate this code has far-reaching implications for society. In this article, we explore some of the basic techniques that enable us to read, copy and manipulate DNA sequences alongside a brief consideration of some of the implications for society.

Essential chemistry for biochemists.

Within every living organism, countless reactions occur every second. These reactions typically occur more rapidly and with greater efficiency than would be possible under the same conditions in the chemical laboratory, and while using only the subset of elements that are readily available in nature. Despite these apparent differences between life and the laboratory, biological reactions are governed by the same rules as any other chemical reaction. Thus, a firm understanding of the fundamentals of chemistry is invaluable in biochemistry. There are entire textbooks devoted to the application of chemical principles in biological systems and so it is not possible to cover all of the relevant topics in depth in this short article. The aim is instead to provide a brief overview of those areas in chemistry that are most relevant to biochemistry. We summarize the basic principles, give examples of how these principles are applied in biological systems and suggest further reading on individual topics.

Positioning of chemosensory proteins and FtsZ through the Rhodobacter sphaeroides cell cycle.

Bacterial chemotaxis depends on signalling through large protein complexes. Each cell must inherit a complex on division, suggesting some co-ordination with cell division. In Escherichia coli the membrane-spanning chemosensory complexes are polar and new static complexes form at pre-cytokinetic sites, ensuring positioning at the new pole after division and suggesting a role for the bacterial cytoskeleton. Rhodobacter sphaeroides has both membrane-associated and cytoplasmic, chromosome-associated chemosensory complexes. We followed the relative positions of the two chemosensory complexes, FtsZ and MreB in aerobic and in photoheterotrophic R. sphaeroides cells using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which FtsZ extends circumferentially to form the Z-ring. Membrane-associated chemosensory proteins form a number of dynamic unit-clusters with mature clusters containing about 1000 CheW(3) proteins. Individual clusters diffuse randomly within the membrane, accumulating at new poles after division but not colocalizing with either FtsZ or MreB. The cytoplasmic complex colocalizes with FtsZ at midcells in new-born cells. Before cytokinesis one complex moves to a daughter cell, followed by the second moving to the other cell. These data indicate that two homologous complexes use different mechanisms to ensure partitioning, and neither complex utilizes FtsZ or MreB for positioning.

Bacterial cell identification in differential interference contrast microscopy images.

BACKGROUND: Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. RESULTS: We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. CONCLUSIONS: Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes.

Recent data have shown that plasmid partitioning Par-like systems are used by some bacterial cells to control localization of protein complexes. Here we demonstrate that one of these homologs, PpfA, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. Using fluorescent microscopy and point mutations, we show dynamic chromosome binding and Walker-type ATPase activity are essential for cluster segregation. The N-terminal domain of a cytoplasmic chemoreceptor encoded next to ppfA is also required for segregation, probably functioning as a ParB analog to control PpfA ATPase activity. An orphan ParA involved in segregating protein clusters therefore uses a similar mechanism to plasmid-segregating ParA/B systems and requires a partner protein for function. Given the large number of genomes that encode orphan ParAs, this may be a common mechanism regulating segregation of proteins and protein complexes.

Feedback control architecture and the bacterial chemotaxis network.

Bacteria move towards favourable and away from toxic environments by changing their swimming pattern. This response is regulated by the chemotaxis signalling pathway, which has an important feature: it uses feedback to 'reset' (adapt) the bacterial sensing ability, which allows the bacteria to sense a range of background environmental changes. The role of this feedback has been studied extensively in the simple chemotaxis pathway of Escherichia coli. However it has been recently found that the majority of bacteria have multiple chemotaxis homologues of the E. coli proteins, resulting in more complex pathways. In this paper we investigate the configuration and role of feedback in Rhodobacter sphaeroides, a bacterium containing multiple homologues of the chemotaxis proteins found in E. coli. Multiple proteins could produce different possible feedback configurations, each having different chemotactic performance qualities and levels of robustness to variations and uncertainties in biological parameters and to intracellular noise. We develop four models corresponding to different feedback configurations. Using a series of carefully designed experiments we discriminate between these models and invalidate three of them. When these models are examined in terms of robustness to noise and parametric uncertainties, we find that the non-invalidated model is superior to the others. Moreover, it has a 'cascade control' feedback architecture which is used extensively in engineering to improve system performance, including robustness. Given that the majority of bacteria are known to have multiple chemotaxis pathways, in this paper we show that some feedback architectures allow them to have better performance than others. In particular, cascade control may be an important feature in achieving robust functionality in more complex signalling pathways and in improving their performance.

Adaptation and control circuits in bacterial chemotaxis.

Bacteria are capable of sensing and responding to changes in their environment. One of the ways they do this is via chemotaxis, regulating swimming behaviour. The chemotaxis pathway senses chemoattractant gradients and uses a feedback loop to change the bacterial swimming pattern; this feedback loop differs in detail between species. In the present article, we summarize the current understanding of the regulatory mechanisms in three species and how these pathways can be viewed and analysed through the ideas of feedback control systems engineering.

A model invalidation-based approach for elucidating biological signalling pathways, applied to the chemotaxis pathway in R. sphaeroides.

BACKGROUND: Developing methods for understanding the connectivity of signalling pathways is a major challenge in biological research. For this purpose, mathematical models are routinely developed based on experimental observations, which also allow the prediction of the system behaviour under different experimental conditions. Often, however, the same experimental data can be represented by several competing network models. RESULTS: In this paper, we developed a novel mathematical model/experiment design cycle to help determine the probable network connectivity by iteratively invalidating models corresponding to competing signalling pathways. To do this, we systematically design experiments in silico that discriminate best between models of the competing signalling pathways. The method determines the inputs and parameter perturbations that will differentiate best between model outputs, corresponding to what can be measured/observed experimentally. We applied our method to the unknown connectivities in the chemotaxis pathway of the bacterium Rhodobacter sphaeroides. We first developed several models of R. sphaeroides chemotaxis corresponding to different signalling networks, all of which are biologically plausible. Parameters in these models were fitted so that they all represented wild type data equally well. The models were then compared to current mutant data and some were invalidated. To discriminate between the remaining models we used ideas from control systems theory to determine efficiently in silico an input profile that would result in the biggest difference in model outputs. However, when we applied this input to the models, we found it to be insufficient for discrimination in silico. Thus, to achieve better discrimination, we determined the best change in initial conditions (total protein concentrations) as well as the best change in the input profile. The designed experiments were then performed on live cells and the resulting data used to invalidate all but one of the remaining candidate models. CONCLUSION: We successfully applied our method to chemotaxis in R. sphaeroides and the results from the experiments designed using this methodology allowed us to invalidate all but one of the proposed network models. The methodology we present is general and can be applied to a range of other biological networks.

A bifunctional kinase-phosphatase in bacterial chemotaxis.

Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA(3), is not only a phosphodonor for the principal CheY protein, CheY(6), but is also is a specific phosphatase for CheY(6)-P. This phosphatase activity accelerates CheY(6)-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA(3) possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA(3) is phosphorylated by CheA(4), and it subsequently acts as a phosphodonor for the response regulators. A CheA(3) mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA(3) is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA(3) lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases.

Two chemosensory operons of Rhodobacter sphaeroides are regulated independently by sigma 28 and sigma 54.

Rhodobacter sphaeroides has a complex chemosensory system, with several loci encoding multiple homologues of the components required for chemosensing in Escherichia coli. The operons cheOp2 and cheOp3 each encode complete pathways, and both are essential for chemosensing. The components of cheOp2 are predominantly localized to the cell pole, whereas those encoded by cheOp3 are predominantly targeted to a discrete cluster in the cytoplasm. Here we show that the expression of the two pathways is regulated independently. Overlapping promoters recognized by sigma(28) and sigma(70) RNAP holoenzyme transcribe cheOp2, whereas cheOp3 is regulated by one of the four sigma(54) homologues, RpoN3. The different regulation of these operons may reflect the need for balancing responses to extra- and intracellular signals under different growth conditions.