Penelope I. Higgs

Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation.

BackgroundMyxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities.ResultsOur analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate.ConclusionsThese results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation.

Myxococcus xanthus developmental cell fate production: heterogeneous accumulation of developmental regulatory proteins and reexamination of the role of MazF in developmental lysis

Myxococcus xanthus undergoes a starvation-induced multicellular developmental program during which cells partition into three known fates: (i) aggregation into fruiting bodies followed by differentiation into spores, (ii) lysis, or (iii) differentiation into nonaggregating persister-like cells, termed peripheral rods. As a first step to characterize cell fate segregation, we enumerated total, aggregating, and nonaggregating cells throughout the developmental program. We demonstrate that both cell lysis and cell aggregation begin with similar timing at approximately 24 h after induction of development. Examination of several known regulatory proteins in the separated aggregated and nonaggregated cell fractions revealed previously unknown heterogeneity in the accumulation patterns of proteins involved in type IV pilus (T4P)-mediated motility (PilC and PilA) and regulation of development (MrpC, FruA, and C-signal). As part of our characterization of the cell lysis fate, we set out to investigate the unorthodox MazF-MrpC toxin-antitoxin system which was previously proposed to induce programmed cell death (PCD). We demonstrate that deletion of mazF in two different wild-type M. xanthus laboratory strains does not significantly reduce developmental cell lysis, suggesting that MazFs role in promoting PCD is an adaption to the mutant background strain used previously.

EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus

Myxococcus xanthus undergoes a complex starvation-induced developmental program that results in cells forming multicellular fruiting bodies by aggregating into mounds and then differentiating into spores. This developmental program requires at least 72 h and is mediated by a temporal cascade of gene regulators in response to intra- and extracellular signals. espA mutants, encoding an orphan hybrid histidine kinase, alter the timing of this developmental program, greatly accelerating developmental progression. Here, we characterized EspA and demonstrated that it autophosphorylates in vitro on the conserved histidine residue and then transfers the phosphoryl group to the conserved aspartate residue in the associated receiver domain. The conserved histidine and aspartate residues were both required for EspA function in vivo. Analysis of developmental gene expression and protein accumulation in espA mutants indicated that the expression of the A-signal-dependent spi gene was not affected but that the MrpC transcriptional regulator accumulated earlier, resulting in earlier expression of its target, the FruA transcriptional regulator. Early expression of FruA correlated with acceleration of both the aggregation and sporulation branches of the developmental program, as monitored by early methylation of the FrzCD chemosensory receptor and early expression of the sporulation-specific dev and Mxan_3227 (Omega7536) genes. These results show that EspA plays a key role in the timing of expression of genes necessary for progression of cells through the developmental program.

A Novel “Four-component” Two-component Signal Transduction Mechanism Regulates Developmental Progression in Myxococcus xanthus

Histidine-aspartate phosphorelays are employed by two-component signal transduction family proteins to mediate responses to specific signals or stimuli in microorganisms and plants. The RedCDEF proteins constitute a novel signaling system in which four two-component proteins comprising a histidine kinase, a histidine-kinase like protein, and two response regulators function together to regulate progression through the elaborate developmental program of Myxococcus xanthus. A combination of in vivo phenotypic analyses of in-frame deletions and non-functional point mutations in each gene as well as in vitro autophosphorylation and phosphotransfer analyses of recombinant proteins indicate that the RedC histidine kinase protein autophosphorylates and donates a phosphoryl group to the single domain response regulator, RedF, to repress progression through the developmental program. To relieve this developmental repression, RedC instead phosphorylates RedD, a dual receiver response regulator protein. Surprisingly, RedD transfers the phosphoryl group to the histidine kinase-like protein RedE, which itself appears to be incapable of autophosphorylation. Phosphorylation of RedE may render RedE accessible to RedF, where it removes the phosphoryl group from RedF-P, which is otherwise an unusually stable phosphoprotein. These analyses reveal a novel “four-component” signaling mechanism that has probably arisen to temporally coordinate signals controlling the developmental program in M. xanthus. The RedCDEF signaling system provides an important example of how the inherent plasticity and modularity of the basic two-component signaling domains comprise a highly adaptable framework well suited to expansion into complex signaling mechanisms.

Four Unusual Two-Component Signal Transduction Homologs, RedC to RedF, Are Necessary for Timely Development in Myxococcus xanthus

We identified a cluster of four two-component signal transduction genes that are necessary for proper progression of Myxococcus xanthus through development. redC to redF mutants developed and sporulated early, resulting in small, numerous, and disorganized fruiting bodies. Yeast two-hybrid analyses suggest that RedCDEF act in a single signaling pathway. The previously identified espA gene displays a phenotype similar to that of redCDEF. However, combined mutants defective in espA redCDEF exhibited a striking additive developmental phenotype, suggesting that EspA and RedC to RedF play independent roles in controlling developmental progression.

Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition.

Myxococcus xanthus is a Gram‐negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation‐independent remodelling of the rod‐shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co‐polymerase 2a (OPX/PCP‐2a) machinery. Conversion of the capsule‐like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo or two other genetic loci encoding homologues of polysaccharide synthesis enzymes fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel Gram‐negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition.

EspC Is Involved in Controlling the Timing of Development in Myxococcus xanthus

The espC null mutation caused accelerated aggregation and formation of tiny fruiting bodies surrounded by spores, which were also observed in the espA mutant and in CsgA-overproducing cells in Myxococcus xanthus. In addition, the espC mutant appeared to produce larger amounts of the complementary C-signal than the wild-type strain. These findings suggest that EspC is involved in controlling the timing of fruiting body development in M. xanthus.

Two Ser/Thr protein kinases essential for efficient aggregation and spore morphogenesis in Myxococcus xanthus

Myxococcus xanthus has a complex life cycle that involves vegetative growth and development. Previously, we described the espAB locus that is involved in timing events during the initial stages of fruiting body formation. Deletion of espA caused early aggregation and sporulation, whereas deletion of espB caused delayed aggregation and sporulation resulting in reduced spore yields. In this study, we describe two genes, pktA5 and pktB8, that flank the espAB locus and encode Ser/Thr protein kinase (STPK) homologues. Cells deficient in pktA5 or pktB8 formed translucent mounds and produced low spore yields, similar in many respects to espB mutants. Double mutant analysis revealed that espA was epistatic to pktA5 and pktB8 with respect to aggregation and fruiting body morphology, but that pktA5 and pktB8 were epistatic to espA with respect to sporulation efficiency. Expression profiles of pktA5–lacZ and pktB8–lacZ fusions and Western blot analysis showed that the STPKs are expressed under vegetative and developmental conditions. In vitro kinase assays demonstrated that the RD kinase, PktA5, autophosphorylated on threonine residue(s) and phosphorylated the artificial substrate, myelin basic protein. In contrast, autophosphorylation of the non‐RD kinase, PktB8, was not observed in vitro; however, the phenotype of a pktB8 kinase‐dead point mutant resembled the pktB8 deletion mutant, indicating that this residue was important for function and that it likely functions as a kinase in vivo. Immunoprecipitation of Tap‐tagged PktA5 and PktB8 revealed an interaction with EspA during development in M. xanthus. These results, taken together, suggest that PktA5 and PktB8 are STPKs that function during development by interacting with EspA and EspB to regulate M. xanthus development.

Synthesis and Assembly of a Novel Glycan Layer in Myxococcus xanthus Spores

Background: During Myxococcus xanthus sporulation, a rigid coat is assembled on the cell surface. Results: The coat consists of oligosaccharides (1:17 Glc:GalNAc) and glycine, which are absent or unprocessed in exo or nfs mutants. Conclusion: The spore coat glycan is secreted by Exo and rigidified by Nfs machineries. Significance: The spore coat is a novel de novo synthesized structural glycan layer. Myxococcus xanthus is a Gram-negative deltaproteobacterium that has evolved the ability to differentiate into metabolically quiescent spores that are resistant to heat and desiccation. An essential feature of the differentiation processes is the assembly of a rigid, cell wall-like spore coat on the surface of the outer membrane. In this study, we characterize the spore coat composition and describe the machinery necessary for secretion of spore coat material and its subsequent assembly into a stress-bearing matrix. Chemical analyses of isolated spore coat material indicate that the spore coat consists primarily of short 1–4- and 1–3-linked GalNAc polymers that lack significant glycosidic branching and may be connected by glycine peptides. We show that 1–4-linked glucose (Glc) is likely a minor component of the spore coat with the majority of the Glc arising from contamination with extracellular polysaccharides, O-antigen, or storage compounds. Neither of these structures is required for the formation of resistant spores. Our analyses indicate the GalNAc/Glc polymer and glycine are exported by the ExoA-I system, a Wzy-like polysaccharide synthesis and export machinery. Arrangement of the capsular-like polysaccharides into a rigid spore coat requires the NfsA–H proteins, members of which reside in either the cytoplasmic membrane (NfsD, -E, and -G) or outer membrane (NfsA, -B, and -C). The Nfs proteins function together to modulate the chain length of the surface polysaccharides, which is apparently necessary for their assembly into a stress-bearing matrix.

Intra- and Interprotein Phosphorylation between Two-hybrid Histidine Kinases Controls Myxococcus xanthus Developmental Progression

Background: His-Asp phosphorelay proteins can be components of complex signaling systems. Results: Two hybrid histidine kinases, EspA and EspC, form a signaling complex; EspA phosphorylates both receiver modules to regulate proteolytic turnover of a regulatory protein. Conclusion: Two hybrid histidine kinases are integrated by inter- and intra-histidine aspartate phosphotransfer. Significance: A novel His-Asp phosphorelay signal transduction mechanism was identified. Histidine-aspartate phosphorelay signaling systems are used to couple stimuli to cellular responses. A hallmark feature is the highly modular signal transmission modules that can form both simple “two-component” systems and sophisticated multicomponent systems that integrate stimuli over time and space to generate coordinated and fine-tuned responses. The deltaproteobacterium Myxococcus xanthus contains a large repertoire of signaling proteins, many of which regulate its multicellular developmental program. Here, we assign an orphan hybrid histidine protein kinase, EspC, to the Esp signaling system that negatively regulates progression through the M. xanthus developmental program. The Esp signal system consists of the hybrid histidine protein kinase, EspA, two serine/threonine protein kinases, and a putative transport protein. We demonstrate that EspC is an essential component of this system because ΔespA, ΔespC, and ΔespA ΔespC double mutants share an identical developmental phenotype. Neither substitution of the phosphoaccepting histidine residue nor deletion of the entire catalytic ATPase domain in EspC produces an in vivo mutant developmental phenotype. In contrast, substitution of the receiver phosphoaccepting residue yields the null phenotype. Although the EspC histidine kinase can efficiently autophosphorylate in vitro, it does not act as a phosphodonor to its own receiver domain. Our in vitro and in vivo analyses suggest the phosphodonor is instead the EspA histidine kinase. We propose EspA and EspC participate in a novel hybrid histidine protein kinase signaling mechanism involving both inter- and intraprotein phosphotransfer. The output of this signaling system appears to be the combined phosphorylated state of the EspA and EspC receiver modules. This system regulates the proteolytic turnover of MrpC, an important regulator of the developmental program.