Bacterial signalling involving eukaryotic-type protein kinases

Protein Ser, Thr and Tyr kinases play essential roles in signal transduction in organisms ranging from yeast to mammals, where they regulate a variety of cellular activities. During the last few years, a number of genes that encode eukaryotic-type protein kinases have also been identified in four different bacterial species, suggesting that such enzymes are also widespread in prokaryotes. Although many of them have yet to be fully characterized, several studies indicate that eukaryotic-type protein kinases play important roles in regulating cellular activities of these bacteria, such as cell differentiation, pathogenicity and secondary metabolism. A model based on the possible coupling between two-component systems and eukaryotic-type protein kinases is proposed to explain the function of eukaryotic-type protein kinases in bacterial signalling in the light of studies in bacteria, as well as in plants and yeast. These two groups of eukaryotes possess signal-transduction pathways involving both two-component systems and eukaryotic protein kinases.     

Procaryotic genesis and evolution of chemosignaling systems of eukaryotes

The analysis of own and literature data accumulated in the last two decades allowed to check and confirm the author's hypothesis about the prokaryotic origin and endosymbiotic genesis of chemosignalling systems of higher eukaryotes. The comparison of structural-functional organization of these information systems and their components (receptors, GTP-binding proteins, enzymes with cyclase activity, protein kinases etc.) in bacteria and eukaryotes revealed a number of similar features giving evidence for their evolutionary relationship. The conclusion was made that eukaryotic signaling systems have prokaryotic roots. The systems of signal transduction revealed in unicellular eukaryotes according to their architecture and functional properties represent a transient stage in the evolution of chemosignalling systems from prokaryotes to higher eukaryotes. The spreading of signalling systems among three super kingdoms--Bacteria, Archaea and Eukarya occurred as a result of horizontal transfer of bacterial genes and co-evolution of signalling components.     

Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter

Localization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes. Caulobacter crescentus is a small, single-celled bacterium that presumably lacks intracellular organelles. Yet in Caulobacter, the subcellular distribution of several two-component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle. This cell cycle-dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria. Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle.     

Activation of 6-phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6-phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.     

Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression

Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles—chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.     

The Sensory Histidine Kinases TorS and EvgS Tend to Form Clusters in Escherichia coli Cells

Microorganisms use multiple two-component sensory systems to detect changes in their environment and elicit physiological responses. Despite their wide spread and importance, the intracellular organization of two-component sensory proteins in bacteria remains little investigated. A notable exception is the well-studied clustering of the chemoreceptor-kinase complexes that mediate chemotaxis behaviour. However, these chemosensory complexes differ fundamentally from other systems, both structurally and functionally. Therefore, studying the organization of typical sensory kinases in bacteria is essential for understanding the general role of receptor clustering in bacterial sensory signalling. Here, by studying mYFP-tagged sensory kinases in Escherichia coli, we show that the tagged TorS and EvgS sensors have a clear tendency for self-association and clustering. These sensors clustered even when expressed at a level of a few hundred copies per cell. Moreover, the mYFP-tagged response regulator TorR showed clear TorS-dependent clustering, indicating that untagged TorS sensors also tend to form clusters. We also provide evidence for the functionality of these tagged sensors. Experiments with truncated TorS or EvgS proteins suggested that clustering of EvgS sensors depends on the cytoplasmic part of the protein, whereas clustering of TorS sensors can be potentially mediated by the periplasmic/transmembrane domain. Overall, these findings support the notion that sensor clustering plays a role in bacterial sensory signalling beyond chemotaxis.     

Two-component signal transduction pathways in Arabidopsis

The two-component system, consisting of a histidine (His) protein kinase that senses a signal input and a response regulator that mediates the output, is an ancient and evolutionarily conserved signaling mechanism in prokaryotes and eukaryotes. The identification of 54 His protein kinases, His-containing phosphotransfer proteins, response regulators, and related proteins in Arabidopsis suggests an important role of two-component phosphorelay in plant signal transduction. Recent studies indicate that two-component elements are involved in plant hormone, stress, and light signaling. In this review, we present a genome analysis of the Arabidopsis two-component elements and summarize the major advances in our understanding of Arabidopsis two-component signaling.     

Quantitative analysis of signaling networks

The response of biological cells to environmental change is coordinated by protein-based signaling networks. These networks are to be found in both prokaryotes and eukaryotes. In eukaryotes, the signaling networks can be highly complex, some networks comprising of 60 or more proteins. The fundamental motif that has been found in all signaling networks is the protein phosphorylation/dephosphorylation cycle--the cascade cycle. At this time, the computational function of many of the signaling networks is poorly understood. However, it is clear that it is possible to construct a huge variety of control and computational circuits, both analog and digital from combinations of the cascade cycle. In this review, we will summarize the great versatility of the simple cascade cycle as a computational unit and towards the end give two examples, one prokaryotic chemotaxis circuit and the other, the eukaryotic MAPK cascade.     

Bacterial observations: a rudimentary form of intelligence?

Genome sequencing has revealed that signal transduction in bacteria makes use of a limited number of different devices, such as two-component systems, LuxI-LuxR quorum-sensing systems, phosphodiesterases, Ser-Thr (serine-threonine) kinases, OmpR-type regulators, and sigma factor-anti-sigma factor pathways. These systems use modular proteins with a large variety of input and output domains, yet strikingly conserved transmission domains. This conservation might lead to redundancy of output function, for example, via crosstalk (i.e. phosphoryl transfer from a non-cognate sensory kinase). The number of similar devices in a single cell, particularly of the two-component type, might amount to several dozen, and most of these operate in parallel. This could bestow bacteria with cellular intelligence if the network of two-component systems in a single cell fulfils the requirements of a neural network. Testing these ideas poses a great challenge for prokaryotic systems biology.     

Cell shape,division and development: the 2002 American Society for Microbiology (ASM) conference on prokaryotic development

In the last decade, the use of cytological techniques, together with the analysis of complete genomes, has dramatically advanced our understanding of bacterial development. Work on several well-developed model systems such as Bacillus subtilis, Caulobacter crescentus, Myxococcus xanthus and Streptomyces spp., has provided us with an in-depth understanding of processes such as sporulation, multicellular behaviour and the bacterial cell cycle. At the same time, these studies have revolutionized our view of the bacterial cell and shown it to be a highly complex entity with spatial and temporal organization. The recent American Society for Microbiology (ASM) conference on prokaryotic development demonstrated that several laboratories have now started to connect data obtained through functional genomic analysis with subcellular organization, thereby generating three-dimensional regulatory networks. This meeting report highlights new findings in the field, such as regulation of protein localization during sporulation and the cell cycle, control of cell-cell interaction and the initiation of cell division.     

Protein-protein interactions between two-component system transmitter and receiver domains of Myxococcus xanthus

We present a novel dataset assessing the specificity of protein-protein interactions between 69 transmitter and receiver domains from two-component system (TCS)-signalling pathways. TCS require a conserved protein-protein interaction between partner transmitter and receiver domains for signal transduction. The complex prokaryote Myxococcus xanthus possesses an unusually large number of TCS genes, many of which have no obvious interaction partners. Interactions between TCS domains of M. xanthus were assessed using a yeast two-hybrid assay, in which domains were expressed as both bait and prey translational fusions. LacZ production was monitored as an indicator of protein-protein interaction, and the strength of interactions classified as weak, medium or strong. Two-hundred and fifty-five transmitter-receiver domain interactions were observed (46 strong), allowing identification of potential signalling partners for individual M. xanthus TCS proteins. In addition, the dataset provides interesting 'meta' information. For instance, many strong interactions were identified between different transmitter domain pairs (34) and receiver domain pairs (23), suggesting a surprisingly large degree of heterodimerisation of these domains. Proteins in our dataset that exhibited similar 'profiles' of interactions, often shared a similar biological function, suggesting that interaction profiles can provide information on biological function, even considering sets of homologous domains.     

Agents of change – concepts in Mycobacterium tuberculosis Ser/Thr/Tyr phosphosignalling

The flow of information from the outside to the inside of bacterial cells is largely directed by protein kinases. In addition to histidine/aspartate phosphorelays of two‐component response regulators, recent work in Mycobacterium tuberculosis (Mtb) reinforces the idea that phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) is central to bacterial physiology and pathogenesis, and that the corresponding phosphosystems are highly similar to those in eukaryotes. In this way, eukaryotes are a useful guide to understanding Ser/Thr/Tyr phosphorylation (O‐phosphorylation) in prokaryotes such as Mtb. However, as novel functions and components of bacterial O‐phosphorylation are identified, distinct differences between pro‐ and eukaryotic phosphosignalling systems become apparent. The emerging picture of O‐phosphorylation in Mtb is complicated, goes beyond the eukaryotic paradigms, and shows the limitations of viewing bacterial phosphosignalling within the confines of the ‘eukaryotic‐like’ model. Here, we summarize recent findings about Ser/Thr and the recently discovered Tyr phosphorylation pathways in Mtb, highlight the similarities and differences between eukaryotic and prokaryotic O‐phosphorylation, and pose additional questions about signalling components, pathway organization, and ultimately, the cellular roles of O‐phosphorylation in Mtb physiology and pathogenesis.     

氧化葡萄糖酸杆菌中一种潜在的双组分系统蛋白的功能研究

氧化葡萄糖酸杆菌(Gluconobacteroxydans)基因组编码的蛋白质中,有相当数量的传感器激酶和反应调控蛋白组成了细菌的多个双组分信号转导系统(two-componentsignaltransduction systems, TCSs),这些系统能够介导细菌对外界环境变化做出反应。但目前对G. oxydans中潜在的双组分系统成员蛋白质结构和功能缺少必要的研究【。目的】研究菌株G. oxydans 621H中GOX0645基因序列所编码蛋白质的自磷酸化活性,探究其与细菌趋化性运动的关联,揭示其是否作为一种双组分系统成员蛋白在细胞内发挥作用。【方法】以菌株G. oxydans 621H基因组中一段可能编码双组分系统蛋白质的基因GOX0645为基础,通过生物信息学分析其保守结构域;采用体外化学发光实验证明其编码蛋白的自磷酸化活性;利用基因定点突变筛选出与自磷酸化活性相关的氨基酸位点;通过差速离心法寻找双组分蛋白的亚细胞定位;最后运用体内双分子荧光互补和体外生物大分子相互作用实验印证其与下游鞭毛马达蛋白之间的相互作用。【结果】生物信息学分析发现GOX0645编码蛋白同时具有组氨酸激...     

Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology

Tyrosine phosphorylation is a key device in numerous cellular functions in eukaryotes, but in bacteria this protein modification was largely ignored until the mid-1990s. The first conclusive evidence of bacterial tyrosine phosphorylation came only a decade ago. Since then, several tyrosine kinases exhibiting unexpected features have been identified in a variety of bacteria. These enzymes use homologues of Walker motifs of nucleotide-binding proteins for their catalytic mechanism, thus defining an idiosyncratic type of bacterial tyrosine kinases. Recently, bacterial tyrosine kinases have been found to phosphorylate an increasing list of endogenous protein substrates. This discovery contributes to the emerging picture that bacterial tyrosine phosphorylation is an important regulatory arsenal of bacterial physiology in addition to the classical serine/threonine kinases, and the 'two-component' and phosphotransferase systems.     

Identification of a putative eukaryotic-like protein kinase family in the developmental bacterium Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium which, upon starvation, undergoes a spectacular developmental cycle culminating in the formation of spore-filled fruiting bodies. We recently characterized a protein serine-threonine kinase (Pkn1) that is required for normal development (J. Munoz-Dorado, S. Inouye, and M. Inouye, Cell 67:995-1006, 1991). pkn1 was cloned by polymerase chain reaction amplification with primers designed from conserved sequences in eukaryotic protein kinases. In this study, a fragment of the pkn1 gene and an oligonucleotide corresponding to another highly conserved region were employed as probes for Southern blot analyses, which indicated that there are at least 26 putative kinase genes in M. xanthus. Most of the putative kinase genes were cloned, and complete or partial sequencing of eight clones revealed that they indeed contained highly conserved sequences present in eukaryotic kinases. These results suggest that complex kinase cascades similar to those described for eukaryotes might be involved in regulation of the M. xanthus life cycle.