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.     

Transport and localization of protein S, a spore coat protein, during fruiting body formation by Myxococcus xanthus.

Protein S, the most abundant soluble protein synthesized by Myxococcus xanthus FB during early fruiting body formation, accumulates in the soluble fraction of developing cells, reaching a peak at about 24 h; at late stages of fruiting body formation, protein S is found on the surface of spores (M. Inouye et al. Proc. Natl. Acad. Sci. U.S.A. 76:209-213, 1979). In this study, the transport and localization of protein S were investigated. Cells were fractionated to give osmotic shock, membrane, cytoplasmic, and spore fractions. The various fractions were then analyzed for protein S. Protein S was first detected in the cytoplasmic fraction at about 3 to 6 h of development. However, transport of protein S through the cytoplasmic membrane was not observed until 15 to 18 h of development. Thus, protein S is unusual among translocated proteins in that it accumulates as a soluble cytoplasmic protein before translocation. Biosynthesis of protein S ceased after 48 h; by 72 h, protein S was only found on the surface of spores. Pulse-chase experiments were performed to determine the transport kinetics of protein S. The results showed that in 24-h developing cells, the transport of protein S across the cytoplasmic membrane was rapid, occurring in less than 2 min. However, transport across the outer membrane was slow, requiring 10 to 15 min. Pulses of 15 s with [35S]methionine failed to reveal any short-lived precursor form in immunoprecipitated material separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing also failed to detect any precursor form of protein S. Thus, protein S appears to be translocated in the absence of a cleaved signal peptide.     

Protein U, a late-developmental spore coat protein of Myxococcus xanthus, is a secretory protein.

Protein U is a spore coat protein produced at the late stage of development of Myxococcus xanthus. This protein was isolated from developmental cells, and its amino-terminal sequence was determined. On the basis of this sequence, the gene for protein U (pru) was cloned and its DNA sequence was determined, revealing an open reading frame of 179 codons. The product from this open reading frame has a typical signal peptide of 25 amino acid residues at the amino terminal end, followed by protein U of 154 residues. This result indicates that protein U is produced as a secretory precursor, pro-protein U, which is then secreted across the membrane to assemble on the spore surface. This is in sharp contrast to protein S, a major spore coat protein produced early in development, which has no signal peptide, indicating that there are two distinct pathways for trafficking of spore coat proteins during the differentiation of M. xanthus.     

Two homologous genes coding for spore-specific proteins are expressed at different times during development of Myxococcus xanthus.

The ops and tps genes of Myxococcus xanthus have ca. 90% DNA and amino acid sequence homology and are in the same orientation separated by a spacer region of only 1.4 kilobases. The products of the two genes were found to cross-react immunologically, and both were capable of Ca2+-dependent self-assembly on the surface of myxospores. However, the ops and tps genes were expressed very differently during the developmental cycle of M. xanthus. The tps gene is induced early during fruiting body formation on a solid surface, and its product, protein S, is made in large quantities (up to 15% of total protein synthesis). When the cells turn into myxospores, protein S is assembled on the outer surface of the spore. We have now also found it in much smaller quantities inside the spores. The ops gene, on the other hand, appears to be induced later in development, after the cells have sporulated, since the ops gene product was found only inside the spores. When an ops gene under the control of a tps gene promoter was inserted into a wild-type strain, the ops gene product was synthesized at the same time as protein S and assembled onto the spore surface.     

Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.     

Characterization of calcium-binding sites in development-specific protein S of Myxococcus xanthus using site-specific mutagenesis

Protein S, the most abundant protein synthesized during development of the Gram-negative bacterium Myxococcus xanthus, assembles on the surface of the spores. It can be dissociated from the spores using divalent metal chelators and will reassemble on the spores in the presence of calcium. The amino acid sequence of protein S contains regions which have homology to the calcium-binding sites of calmodulin. Protein S was found to bind 2 mol of calcium/mol of protein with Kd values of 27 and 76 microM. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin (Ser40----Arg,Ser129----Arg), and a double mutant was also constructed. Each mutant gene was then transduced into the genome of a M. xanthus strain from which the wild-type genes had been deleted. All three mutants produced protein S normally during development. One of the mutants (Ser129----Arg) had normal amounts of protein S on its spores, whereas the other (Ser40----Arg) bound much less and the double mutant had virtually none. Analysis of the calcium binding affinities of the purified proteins showed that [Arg40]protein S and [Arg40, Arg129]protein S did not bind detectable quantities of calcium, whereas [Arg129]protein S bound less calcium than the wild-type protein and with a reduced affinity.     

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.     

Development-specific protein S of Myxococcus xanthus: purification and characterization.

Protein S, a development-specific protein of Myxococcus xanthus, was purified from the cells of a late stage of development and crystallized. Its circular dichroism spectra indicated that protein S had a high content of beta-structure in both the presence and absence of calcium ion, which is required for self-assembly of protein S on the myxospore surface. Its amino and carboxyl terminal sequences were determined to be alanine-aspartic acid-isoleucine-glycine-valine-alanine-methionine-asparagine-asparagine-aspartic acid-threonine-serine-serine and isoleucine-arginine (isoleucine, serine), respectively. When protein S (molecular weight, 23,000) was digested with trypsin, a trypsin-resistant core of 10,000 molecular weight was obtained. The core peptide was purified, and its amino acid composition was compared with that of protein S. The core peptide was capable of self-assembly on the spore surface in the presence of calcium ion and competed with protein S for binding on the spore surface. The ratio of affinity to the spore surface for protein S to that for the core peptide was 1.55.     

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

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.     

Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S

The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at Mr approximately 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/10(5) cells per 24 h and contain 44 fmol/10(5) cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor Va inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by greater than 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 microM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.     

Trehalose accumulation in vegetative cells and spores of Myxococcus xanthus.

The disaccharide trehalose is found in the spores and cysts of a variety of organisms. We analyzed developing cells of Myxococcus xanthus for trehalose accumulation. Vegetative cells grown in media with low osmotic strengths contained less than 5 micrograms of trehalose per mg of protein. Spores formed in fruiting bodies accumulated up to 1,100 micrograms of trehalose per mg of protein. Spores formed in liquid culture following the addition of glycerol contained up to 300 micrograms of trehalose per mg of protein. The trehalose contents of both spore types decreased rapidly during the early stages of germination. Trehalase activity was not detected in extracts of dormant or germinating spores. Trehalose accumulation in M. xanthus was also associated with elevated osmotic strength. Vegetative cells accumulated up to 214 micrograms of trehalose per mg of protein when grown in media containing elevated levels of solutes.     

The spore wall and polar tube proteins of the microsporidian Nosema grylli: the major spore wall protein is released before spore extrusion

Incubation of Nosema grylli spores in alkaline--saline solution (10 mM KOH, 170 mM KCl) leads to solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year) in water or 0.02% sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting. IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40 under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral solution leads to their discharge. Subsequent wash of discharged spores with 1-3% SDS, 9 M urea and treatment by 100% 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular weights about 46, 34, 21 and 15 kDa.     

Patterns of Protein Production in Myxococcus xanthus During Spore Formation Induced by Glycerol, Dimethyl Sulfoxide, and Phenethyl Alcohol

Spore formation of Myxococcus xanthus can occur not only on agar plates during fruiting body formation, but also in a liquid culture by simply adding glycerol, dimethyl sulfoxide, or phenethyl alcohol to the culture. This chemically-induced spore formation occurs synchronously and much faster than that occurring during fruiting body formation. Dramatic changes in patterns of protein synthesis were observed during chemically-induced spore formation, as had previously been observed during fruiting body formation (Inouye et al., Dev. Biol. 68:579-591, 1979). However, the production of protein S, one of the major development-specific proteins during fruiting body formation, was not detected at all, although protein U, another development-specific protein, was produced in a late stage of spore formation as in the case of fruiting body formation. This indicates that the control of the gene expression during chemically-induced spore formation is significantly different from that during fruiting body formation. It was also found that during spore formation, every cell seems to have a potential to form a spore regardless of its age, since smaller cells as well as larger cells separated by sucrose density gradient centrifugation could equally form spores upon the addition of glycerol. Patterns of protein synthesis were almost identical for all the three chemicals. However, the final yield of spores was significantly different depending upon the chemicals used. When phenethyl alcohol was added with glycerol or dimethyl sulfoxide, the final yields were determined by the multiple effect of the two chemicals added. This suggests that although these chemicals are able to induce the gene functions required for spore formation, they may have inhibitory effects on some of the gene functions or the processes of spore formation.     

Identification of a Nosema bombycis (Microsporidia) spore wall protein corresponding to spore phagocytosis

Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.     

In vitro recognition of a 190 kda putative attachment receptor from the cuticle of Meloidogyne javanica by Pasteuria penetrans spore extract

Studies were undertaken to develop an improved understanding of the mechanism by which spores of Pasteuria penetrans bind to the cuticle of susceptible nematode hosts. A polyclonal antibody recognized an antigenic ladder at Mr ～ 41 kDa when Meloidogyne javanica cuticle extracts were electrophoresed in the presence of SDS and blotted on to a nitrocellulose membrane. Digestion of the cuticle extracts prior to electrophoresis with Proteinase K and lipase showed the antigenic ladder to be a protein. Attempts to block the antigenic ladder with N‐acetylglucosamine in the form of chitobiose did not prevent the binding of the antibody and the chitobiose could not be detected binding to the antigenic ladder using the lectin wheat germ agglutinin (WGA). Blots probed with Pasteuria spore extract and visualized with a polyclonal antibody to the spore showed that the spore extract did not bind to the antigenic ladder but did bind to a protein with Mr ～ 190 kDa and a much weaker band at ～ 45 kDa. An antibody to the lectin WGA (anti‐WGA) recognized the ～ 190 kDa polypeptide but this was not blocked by chitobiose. Concanavalin A also recognized the ～ 190 kDa protein and the presence of inhibitory sugars prohibited Con A from binding, showing the protein to be glycosylated. It is conjectured that the ～ 190 kDa glycoprotein present in the cuticle extract is a binding receptor involved in the attachment of Pasteuria spores to the nematode cuticle.     

Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor

Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.     

Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.