Multicellular development in Myxococcus xanthus is stimulated by predator-prey interactions

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.     

MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the DeltamspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the DeltamspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the DeltamspA mutant [corrected] This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.     

Role of phase variation in the resistance of Myxococcus xanthus fruiting bodies to Caenorhabditis elegans predation

The phenomenon of phase variation between yellow and tan forms of Myxococcus xanthus has been recognized for several decades, but it is not known what role this variation may play in the ecology of myxobacteria. We confirm an earlier report that tan variants are disproportionately more numerous in the resulting spore population of a M. xanthus fruiting body than the tan vegetative cells that contributed to fruiting body formation. However, we found that tan cells may not require yellow cells for fruiting body formation or starvation-induced sporulation of tan cells. Here we report three differences between the yellow and tan variants that may play important roles in the soil ecology of M. xanthus. Specifically, the yellow variant is more capable of forming biofilms, is more sensitive to lysozyme, and is more resistant to ingestion by bacteriophagous nematodes. We also show that the myxobacterial fruiting body is more resistant to predation by worms than are dispersed M. xanthus cells.     

Autocide AMI rescues development in dsg mutants of Myxococcus xanthus

Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo- mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo- mutant classes, most dramatically in the dsg mutants.     

Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.     

Contribution of the cyclic nucleotide phosphodiesterases PdeA and PdeB to adaptation of Myxococcus xanthus cells to osmotic or high-temperature stress

A tBLASTn search of the Myxococcus xanthus genome database at The Institute for Genomic Research (TIGR) identified three genes (pdeA, pdeB, and pdeC) that encode proteins homologous to 3',5'-cyclic nucleotide phosphodiesterase. pdeA, pdeB, and pdeC mutants, constructed by replacing a part of the gene with the kanamycin or tetracycline resistance gene, showed normal growth, development, and germination under nonstress conditions. However, the spores of mutants, especially the pdeA and pdeB mutants, placed under osmotic stress germinated earlier than the wild-type spores. The phenotype was the opposite of that of the receptor-type adenylyl cyclase (cyaA or cyaB) mutant. Also, pdeA and pdeB mutants were found to have impaired growth under the condition of high-temperature stress. Intracellular cyclic AMP (cAMP) levels of pdeA or pdeB mutant cells under these stressful conditions were about 1.3-fold to 2.0-fold higher than those of wild-type cells. These results suggest that PdeA and PdeB may be involved in osmotic adaptation during spore germination and temperature adaptation during vegetative growth through the regulation of cAMP levels.     

Changes in cell surface hydrophobicity of Myxococcus xanthus are correlated with sporulation-related events in the developmental program.

Cell surface hydrophobicity was measured in the bacterium Myxococcus xanthus during vegetative growth, fruiting body formation, and glycerol-induced spore formation by the method of Rosenberg et al. (FEMS Microbiol. Lett. 9:29-33, 1980). A significant decrease in cell surface hydrophobicity was observed 12 to 36 h after fruiting body formation and 60 to 120 min after glycerol-induced sporulation. The hydrophilic shift was correlated with the ability of the cells to sporulate but not with their ability to aggregate. Sucrose gradient purification removed the hydrophilic substance from the fruiting body spores but not from the glycerol-induced spores. The change in cell surface hydrophobicity in M. xanthus should be a useful developmental marker.     

Effect of depletion of FtsY on spore morphology and the protein composition of the spore coat layer in Bacillus subtilis

Bacillus subtilis FtsY is a homolog of the alpha-subunit of mammalian signal recognition particle (SRP) receptor, and is essential for protein translocation and vegetative cell growth. An FtsY conditional null mutant (strain ISR39) can express ftsY during the vegetative stage but not during spore formation. Spores of ISR39 have the same resistance to heat and chloroform as the wild-type, while their resistance to lysozyme is reduced. Electron microscopy showed that the outer coat of spores was incompletely assembled. The coat protein profile of the ftsY mutant spores was different from that of wild-type spores. The amounts of CotA, and CotE were reduced in spore coat proteins of ftsY mutant spores and the molecular mass of CotB was reduced. In addition, CotA, CotB, and CotE are present in normal form at T(8) of sporulation in ftsY mutant cells. These results suggest that FtsY has a pivotal role in assembling coat proteins onto the coat layer during spore morphogenesis.     

Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light.

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

Effects of heat shock upon the expression of developmentally regulated genes in Myxococcus xanthus

Abstract The effects of heat shock upon the expression of several developmentally regulated genes of Myxococcus xanthus were examined. No effects were observed on levels or timing of developmentally regulated β-galactosidase expression in eight randomly selected Tn5lac insertion mutants. However, heat shock significantly affected the fruiting behavior of temperature-sensitive aggregation ( tag ) mutants of M. xanthus . The tag mutant phenotype exhibits the normal aggregation of cells to form fruiting bodies at temperatures < 34°C, but cells fail to aggregate at temperatures ⩾ 34°C. Heat shock administered to tag mutant strains prior to starvation prohibited fruiting body formation at permissive temperatures. Additionally, tag mutant strains were found to be extremely sensitive to killing at 40°C. Heat shock was also found to increase tagA and tagE expression by 22 and 47%, respectively. Mutations in tagA blocked heat shock induced expression of tagE .     

Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development.

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.     

Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development

Myxococcus xanthus is a Gram-negative bacterium with a complex life cycle that includes vegetative swarming on rich medium and, upon starvation, aggregation to form fruiting bodies containing spores. Both of these behaviours require multiple Ser/Thr protein kinases. In this paper, we report the first Ser/Thr protein phosphatase gene, pph1, from M. xanthus. DNA sequence analysis of pph1 indicates that it encodes a protein of 254 residues (Mr = 28 308) with strong homology to eukaryotic PP2C phosphatases and that it belongs to a new group of bacterial protein phosphatases that are distinct from bacterial PP2C phosphatases such as RsbU, RsbX and SpoIIE. Recombinant His-tagged Pph1 was purified from Escherichia coli and shown to have Mn2+ or Mg2+ dependent, okadaic acid-resistant phosphatase activity on a synthetic phosphorylated peptide, RRA(pT)VA, indicating that Pph1 is a PP2C phosphatase. Pph1-expression was observed under both vegetative and developmental conditions, but peaked during early aggregation. A pph1 null mutant showed defects during late vegetative growth, swarming and glycerol spore formation. Under starvation-induced developmental conditions, the mutant showed reduced aggregation and failure to form fruiting bodies with viable spores. Using the yeast two-hybrid system, we have observed a strong interaction between Pph1 and the M. xanthus protein kinase Pkn5, a negative effector of development. These results suggest a functional link between a Pkn2-type protein kinase and a PP2C phosphatase.     

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.     

Isolation and phenotypic characterization of Myxococcus xanthus mutants which are defective in sensing negative stimuli.

Myxococcus xanthus is a gram-negative gliding bacterium that exhibits a complex life cycle. Exposure of M. xanthus to chemicals like dimethyl sulfoxide (DMSO) at nondeleterious concentrations or the depletion of nutrients caused several negative responses by the cells. DMSO (> 0.1 M) or nutrient depletion triggered a repellent response: cell swarming was inhibited and FrzCD (a methyl-accepting chemotaxis protein) was demethylated; higher concentrations of DMSO (> 0.3 M) or prolonged starvation induced an additional response which involved cellular morphogenesis: DMSO caused cells to convert from rod-shaped vegetative cells to spherical, environmentally resistant "DMSO spores," and starvation induced myxospore formation in the fruiting bodies. In order to investigate the nature of these responses, we isolated a number of mutants defective in negative chemotaxis and/or sporulation. Characterization of these mutants indicated that negative chemotaxis plays an important role in colony swarming and in developmental aggregation. In addition, the results revealed some of the major interrelationships between the signal transduction pathways which respond to negative stimuli: (i) DMSO exposure and starvation were initially sensed by different systems, the neg system for DMSO and the stv system for starvation; (ii) the repellent response signals triggered by DMSO or starvation were then relayed by the frz signal transduction system; mutants defective in these responses showed altered FrzCD methylation patterns; and (iii) the morphogenesis signals in response to DMSO or starvation utilize a group of genes involved in sporulation (spo).     

Cloning and characterization of the socA locus which restores development to Myxococcus xanthus C-signaling mutants.

The csgA gene produces an intercellular signal during fruiting body formation of the myxobacterium Myxococcus xanthus. Sporulating pseudorevertants were isolated to allow us to understand the mechanism by which CsgA is perceived by cells and used to regulate developmental gene expression. Two strains, LS559 and LS560, which have closely linked transposon insertions, soc-559 (formerly csp-559) and soc-560 (formerly csp-560), respectively, regained all the developmental behaviors lost by the csgA mutation including the ability to ripple, form fruiting bodies, and sporulate. The sequence analysis of the socA locus revealed that there are three putative protein-coding regions, designated socA1, socA2, and socA3. The deduced amino acid sequence of socA1 exhibits characteristics of the short-chain alcohol dehydrogenase family. The deduced amino acid sequence of socA2 shares 48% identity with the frdD gene product of the frd operon in Proteus vulgaris which anchors fumarate reductase to the membrane. The deduced amino acid sequence of socA3 does not show homology to any known proteins. Genotypic complementation, Northern (RNA) blotting, DNA sequence analysis, and the pattern of gene expression all suggest that these three genes are polycistronic. Since the socA mutations effectively bypass CsgA, the question of why csgA is maintained in M. xanthus was examined by studying the long-term stability of socA spores. Unlike the wild type, socA mutant spores germinated on starvation agar. Transmission electron micrographs of spore thin sections revealed that germination is not due to an obvious structural deficiency of the socA spores. These results suggest that the ability of socA myxospores to survive long periods under unfavorable environmental conditions is severely comprised. Therefore, soxA appears to be essential for the development of M. xanthus.     

Phosphorylation and methylation of proteins during Myxococcus xanthus spore formation

Post-translational modification of proteins was examined during the life cycle of Myxococcus xanthus. A specific pattern of protein phosphorylation was observed in vegetative cells. When spore formation was induced by glycerol, significant changes in the pattern of protein phosphorylation were observed, including the phosphorylation of two membrane proteins. In in vitro experiments, the same membrane proteins were phosphorylated by ATP when the membrane preparation from cells treated with glycerol was used. Changes in the pattern of protein methylation were also observed during spore formation induced by glycerol or fruiting body formation. These results suggest that post-translational protein modification may be required for spore formation or fruiting body formation.     

FibA and PilA act cooperatively during fruiting body formation of Myxococcus xanthus

The extracellular matrix (ECM) of Myxococcus xanthus is essential for social (S-) motility and fruiting body formation. An ECM-bound protein, FibA, is homologous to M4 zinc metalloproteases and is important for stimulation by a phosphatidylethanolamine (PE) chemoattractant and for formation of discrete aggregation foci. In this work, we demonstrate that a correlation exists between a reduced ability to respond to PE and the observed defects in fruiting body morphogenesis. Furthermore, the fibA aggregation defect is accentuated by the absence of either PilA, the structural subunit of type IV pili, or DifD, a chemosensory response regulator. The inability to form fruiting bodies is not due to a loss of S-motility, but rather the loss of PilA and pili as pilT fibA mutants form fruiting bodies. The FibA active site residue E342 is important for fruiting body morphogenesis in the absence of PilA. Mutants exhibiting defects in fruiting body morphogenesis also produce fewer viable spores. It is proposed that FibA and PilA act as extracellular sensors for developmental signals.