Genome prediction of PhoB regulated promoters in Sinorhizobium meliloti and twelve proteobacteria

In proteobacteria, genes whose expression is modulated in response to the external concentration of inorganic phosphate are often regulated by the PhoB protein which binds to a conserved motif (Pho box) within their promoter regions. Using a position weight matrix algorithm derived from known Pho box sequences, we identified 96 putative Pho regulon members whose promoter regions contained one or more Pho boxs in the Sinorhizobium meliloti genome. Expression of these genes was examined through assays of reporter gene fusions and through comparison with published microarray data. Of 96 genes, 31 were induced and 3 were repressed by Pi starvation in a PhoB dependent manner. Novel Pho regulon members included several genes of unknown function. Comparative analysis across 12 proteobacterial genomes revealed highly conserved Pho regulon members including genes involved in Pi metabolism (pstS, phnC and ppdK). Genes with no obvious association with Pi metabolism were predicted to be Pho regulon members in S.meliloti and multiple organisms. These included smc01605 and smc04317 which are annotated as substrate binding proteins of iron transporters and katA encoding catalase. This data suggests that the Pho regulon overlaps and interacts with several other control circuits, such as the oxidative stress response and iron homeostasis.     

phoR1, a gene encoding a new histidine protein kinase Myxococcus xanthus

A soil bacterium able to undergo multicellular development and a coordinated gliding in swarms, requires an accurate regulatory network of phosphorelay proteins. Inorganic phosphate is a limiting nutrient in soil and its importance in regulation is critical. As a step towards studying phosphate regulation and its influence in the developmental process in this bacterium, we screened a Myxococcus xanthus library for clones with phosphatase activity, and found four different ones. The deduced sequence of one of the cloned inserts is similar to that of the classic transmembrane histidine protein kinase of the sensor family of the two-component signal transduction systems with a high sequence similarity to the sensor kinase in the Pho regulon of Bacillus subtilis PhoR. This gene has been named phoR1 and its deduced amino acid sequence consists of 455 residues with a predicted molecular mass of 48.5 kDa. The M. xanthus PhoR1 deduced sequence contains all the characteristic histidine protein kinase motifs in the same order and with the same spacing. A hydropathy profile indicates two membrane-spanning segments located at the extreme N-terminus, according to the putative sensor role of this domain. A gene-disrupted mutant is unable to produce normal mature fruiting bodies and produces fewer spores. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inhibition of Development of Myxococcus xanthus by Eukaryotic Protein Kinase Inhibitors

Myxococcus xanthus is a social bacterium that lives in the soil and undergoes spectacular development to form multicellular fruiting bodies. It contains a large family of eukaryote-like serine/threonine protein kinases. We found that a number of inhibitors for eukaryotic protein serine, threonine, and tyrosine kinases could inhibit the development and sporulation of M. xanthus to various degrees. These results suggest that serine/threonine and tyrosine phosphorylation may be involved in development of M. xanthus. None of the inhibitors tested had any effect on vegetative growth of M. xanthus. Most of them seemed to act during the early stages of development. However, the expression of a very early development-specific gene, Ω4521, was not significantly affected by the inhibitors. The patterns of protein phosphorylation during development were also not significantly altered by the inhibitors, suggesting that the targets of the inhibitors are minor or unstable phosphoproteins but play key roles in fruiting-body formation in M. xanthus.     

Two Systems for Conditional Gene Expression in Myxococcus xanthus Inducible by Isopropyl-β-d-Thiogalactopyranoside or Vanillate

Conditional expression of a gene is a powerful tool to study its function and is typically achieved by placing the gene under the control of an inducible promoter. There is, however, a dearth of such inducible systems in Myxococcus xanthus, a well-studied prokaryotic model for multicellular development, cell differentiation, motility, and light response and a promising source of secondary metabolites. The few available systems have limitations, and exogenously based ones are unavailable. Here, we describe two new, versatile inducible systems for conditional expression of genes in M. xanthus. One employs isopropyl-β-d-thiogalactopyranoside (IPTG) as an inducer and is inspired by those successfully applied in some other bacteria. The other requires vanillate as an inducer and is based on the system developed originally for Caulobacter crescentus and recently adapted for mammalian cells. Both systems are robust, with essentially no expression in the absence of an inducer. Depending on the inducer and the amounts added, expression levels can be modulated such that either system can conditionally express genes, including ones that are essential and are required at high levels such as ftsZ. The two systems operate during vegetative growth as well as during M. xanthus development. Moreover, they can be used to simultaneously induce expression of distinct genes within the same cell. The conditional expression systems we describe substantially expand the genetic tool kit available for studying M. xanthus gene function and cellular biology.     

A Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in Myxococcus xanthus

Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.     

Genome-Wide Analysis of the Pho Regulon in a pstCA Mutant of Citrobacter rodentium

The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s) because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein) and the tad focus (which encodes type IVb pili in Yersinia enterocolitica). Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized virulence determinants of C. rodentium, namely, DegP and NleG8.     

The Phosphatomes of the Multicellular Myxobacteria Myxococcus xanthus and Sorangium cellulosum in Comparison with Other Prokaryotic Genomes

Background

Analysis of the complete genomes from the multicellular myxobacteria Myxococcus xanthus and Sorangium cellulosum identified the highest number of eukaryotic-like protein kinases (ELKs) compared to all other genomes analyzed. High numbers of protein phosphatases (PPs) could therefore be anticipated, as reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes.

Methodology

Here we report an intensive analysis of the phosphatomes of M. xanthus and S. cellulosum in which we constructed phylogenetic trees to position these sequences relative to PPs from other prokaryotic organisms.

Principal Findings

Predominant observations were: (i) M. xanthus and S. cellulosum possess predominantly Ser/Thr PPs; (ii) S. cellulosum encodes the highest number of PP2c-type phosphatases so far reported for a prokaryotic organism; (iii) in contrast to M. xanthus only S. cellulosum encodes high numbers of SpoIIE-like PPs; (iv) there is a significant lack of synteny among M. xanthus and S. cellulosum, and (v) the degree of co-organization between kinase and phosphatase genes is extremely low in these myxobacterial genomes.

Conclusions

We conclude that there has been a greater expansion of ELKs than PPs in multicellular myxobacteria.     

Genetics of gliding motility and development inMyxococcus xanthus

Successful development in multicellular eukaryotes requires cell-cell communication and the coordinated spatial and temporal movements of cells. The complex array of networks required to bring eukaryotic development to fruition can be modeled by the development of the simpler prokaryoteMyxococcus xanthus. As part of its life cycle,M. xanthus forms multicellular fruiting bodies containing differentiated cells. Analysis of the genes essential forM. xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state. Development inM. xanthus is induced by starvation, and early events in development suggest that signaling, stages have evolved to monitor the metabolic state of the developing cell. In the absence of these signals, which include amino acids, α-keto acids, and other intermediary metabolites, the ability of cells to differentiate into myxospores is impaired. Mutations that block genes controlling gliding, motility disrupt the morphogenesis of fruiting bodies and sporogenesis in surprising ways. In this review, we present data that encourage future genetic and biochemical studies of the relationships between motility, cell-cell signaling, and development inM. xanthus.     

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.     

Growth of Myxococcus xanthus in Continuous-Flow-Cell Bioreactors as a Method for Studying Development

Nutrient sensors and developmental timers are two classes of genes vital to the establishment of early development in the social soil bacterium Myxococcus xanthus. The products of these genes trigger and regulate the earliest events that drive the colony from a vegetative state to aggregates, which ultimately leads to the formation of fruiting bodies and the cellular differentiation of the individual cells. In order to more accurately identify the genes and pathways involved in the initiation of this multicellular developmental program in M. xanthus, we adapted a method of growing vegetative populations within a constant controllable environment by using flow cell bioreactors, or flow cells. By establishing an M. xanthus community within a flow cell, we are able to test developmental responses to changes in the environment with fewer concerns for effects due to nutrient depletion or bacterial waste production. This approach allows for greater sensitivity in investigating communal environmental responses, such as nutrient sensing. To demonstrate the versatility of our growth environment, we carried out time-lapse confocal laser scanning microscopy to visualize M. xanthus biofilm growth and fruiting body development, as well as fluorescence staining of exopolysaccharides deposited by biofilms. We also employed the flow cells in a nutrient titration to determine the minimum concentration required to sustain vegetative growth. Our data show that by using a flow cell, M. xanthus can be held in a vegetative growth state at low nutrient concentrations for long periods, and then, by slightly decreasing the nutrient concentration, cells can be allowed to initiate the developmental program.