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Monoclonal antibodies against cell-surface antigens of developing cells of Myxococcus xanthus.

Monoclonal antibodies (MCA) have been developed against cell surface antigens (CSA) of Myxococcus xanthus undergoing fruiting body formation. Three of these antibodies are directed against CSA which increase during development, six against CSA which decrease and three against CSA which show no change during development. Western-type immunoblots have been done to determine the molecular weights of the CSA to which the various MCA bind. Various applications of these MCA to the study of myxobacterial cell interactions are discussed.

Animals↗

Myxococcus xanthus spore coat protein S may have a similar structure to vertebrate lens beta gamma-crystallins.

The Gram-negative bacterium Myxococcus xanthus has a complex life cycle during which large amounts of a protein of relative molecular mass (Mr) 19,000, known as protein S, are assembled into a spore surface coat by a process that specifically requires calcium ions. The gene for protein S has been cloned and the DNA sequence shows that the gene product is composed of four internally repeated homologous sequences, each 40 amino acids long. Although protein S resembles calmodulin both in its internally duplicated structure and its ability to bind calcium, it apparently has a beta-sheet secondary structure rather than the helix-loop-helix motifs that characterize the calmodulin family. We now show that protein S has a striking homology with the beta- and gamma-crystallins of the vertebrate eye lens which are beta-sheet proteins with internally duplicated structures. This implies that the beta- and gamma-crystallins evolved from already existing proteins, whose ancestors occurred in the prokaryotes. The biological function of protein S, as a closely packed, stable protein in a relatively dehydrated environment, has implications for the functions of crystallins, which are found closely packed in the lens fibre cells, where their stability is essential for maintenance of transparency.

Amino Acid Sequence↗

Regulation of development in Myxococcus xanthus: effect of 3':5'-cyclic AMP, ADP, and nutrition.

An assay was developed to study the regulation of fruiting in Myxococcus xanthus. The nucleotides, adenosine 3':5'-cyclic monophosphate (cyclic AMP) and adenosine diphosphate (ADP), were found to greatly stimulate fruiting under the assay conditions. Very sharp concentration optima were observed. Even under conditions of starvation, these nucleotides greatly increased the number of aggregation sites. Nutrition was found to influence fruiting body morphology. The effect of amino acids on the nucleotide stimulation of fruiting was studied under our assay conditions. L-Methionine and L-isoleucine (1 mM) completely blocked either L-threonine or D,L-diaminopimelic acid synergistically enhanced the amount of fruiting in the presence of these nucleotides. The data presented suggest the existence of differentiation-related regulatory compounds in M. xanthus.

Adenosine Diphosphate↗

Biosynthesis and self-assembly of protein S, a development-specific protein of Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium that has a complex life cycle including a temporal sequence of cellular aggregation, mound formation, and myxosporulation. During development, protein S (molecuar weight 23,000) is induced and accumulates in very large amounts. Protein S was found in the soluble fraction of early developmental extracts and in the insoluble fraction in later extracts. This insoluble form of protein S can be solubilized by the addition of 1 M NaCl at 0 degrees C to extracts from aggregated cells (mound stage) or by the addition of 1 M NaCl at 30 degrees C to mature spores. Salt extraction (1 M NaCl) of protein S from mature spores was partially inhibited by the addition of Mg(2+) and almost completely inhibited by the addition of Ca(2+). The viability of spores was not changed by a salt extraction that removed their protein S. Examination of thin sections of mature spores and extracted spores by electron microscopy suggested that the protein S-deficient spores lacked a spore surface coat about 300 A thick. Purified protein S will spontaneously self-assemble onto protein S-deficient spores after removal of the NaCl by dialysis or by addition of 10 mM Ca(2+) to undialyzed samples. Glycerol-induced spores did not contain protein S and did not serve as primers for assembly of protein S. Quantitation of the self-assembly process showed almost stoichiometric binding of protein S to the protein S-deficient spores until saturation at 3.3 x 10(6) molecules per spore, a value 1.35 times higher than the normal level of proteins S found in mature spores. Protein S in the "reconstituted" spores was as protease resistant and sonication resistant as the protein S of native spores. Electron microscopy of the reconstituted spores revealed the assembly of new material on the spore surface. Adjacent spores were sometimes observed to be fused to each other through a common protein S layer. These results suggest that protein S serves a function in spore-spore interaction in the fruiting body.

Bacteria↗

Myxobacterial hemagglutinin: a development-specific lectin of Myxococcus xanthus.

Fruiting body formation in the bacterium Myxococcus xanthus consists of a temporal sequence of cellular aggregation and sporulation. During the period of cellular aggregation, a major new development-specific protein that has lectin-like activity is synthesized. This protein, called myxobacterial hemagglutinin (MBHA), was able to agglutinate sheep or guinea pig erythrocytes but not horse, ox, chicken, or human erythrocytes. MBHA was undetectable in extracts of vegetative cells, cells starved in liquid buffer, or in glycerol-induced cells. However, cells starved on a fruiting medium produced large amounts of MBHA (about 5% of protein synthesis), starting at about 6-8 hr of development. The protein accumulated in the soluble fraction of cells, reaching a peak of 1-2% of total protein at about the time when aggregation was completed. At later times the amount of MBHA present in the soluble fraction declined although synthesis continued. The hemagglutinating activity of MBHA could not be inhibited with simple sugars or aminosugars but could be inhibited with fetuin, a fetal calf serum glycoprotein. The O-glycosidically linked trisaccharide glycopeptide of fetuin was shown to be inhibitory by itself. The penultimate galactose of this glycopeptide was directly implicated in the inhibitory activity, because the inhibition by asialofetuin was reduced to 1/60th by periodate oxidation and to 1/15th after beta-galactosidase treatment. MBHA is an abundant biochemical marker of development in M. xanthus. The fact that it is a lectin suggests that it may play a role in cell-cell recognition or agglutination.

Binding, Competitive↗

Structural similarities between the development-specific protein S from a gram-negative bacterium, Myxococcus xanthus, and calmodulin.

During differentiation of Myxococcus xanthus, a large amount of protein S is produced and assembled on the surface of the myxospore by a process that specifically requires Ca2+. The gene for protein S has been cloned, and two tandemly repeated homologous genes have been found to be within a short distance of each other in the M. xanthus chromosome. We determined the DNA sequence of 3,692 bp encompassing both genes and deduced the amino acid sequences of the two gene products. The gene 1 (upstream) product and the gene 2 (downstream) product show extensive amino acid sequence homology (88%). However, from their structures, protein S was found to be produced from gene 2, indicating that gene 2 is specifically turned on during differentiation. The structure of protein S shows striking similarities with calmodulin: protein S is composed of four internally homologous domains. In particular, the first and the third domains, consisting of 38 residues each, show a high level of homology (79%), and the second and the fourth domains, consisting of 40 residues each, show homology of 65%. In the first and the third domains, there is a common sequence of nine residues, Glu (or Asp)-Asn-Asn-Thr-Ile-Ser-Ser-Val-Lys, which is highly homologous to one of the proposed Ca2+-binding sequences in bovine brain calmodulin, Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys.

Amino Acid Sequence↗

Developmental cell interactions in Myxococcus xanthus and the spoC locus.

The product(s) of the Myxococcus xanthus spoC locus is required for two multicellular activities in fruiting body development, rippling and sporulation. Ripples, which are formed early in development, are spatially separated ridges of cells that move synchronously. Myxospores are heat-resistant resting cells that are formed near the end of the developmental process. To investigate the function of spoC, it was cloned in an Escherichia coli plasmid, then transferred to M. xanthus by specialized transduction with coliphage P1. The plasmid, which cannot replicate in M. xanthus, integrated into the M. xanthus chromosome, producing two copies of the spoC locus in tandem. Cells containing one copy of a mutant allele and one copy of the wild-type allele displayed the wild-type phenotype. Cells containing two different mutant alleles failed to ripple or sporulate, implying that all four independent spoC mutations are in the same gene or unit of transcription. Homozygous mutant duplications arose from constructions in which DNA from a spo(+) donor was transduced into a spoC recipient, or vice versa, at an average frequency of 14%, indicating that gene conversion was a frequent event.

Journal Article↗

Evidence for long-lived mRNA during fruiting body formation in myxococcus xanthus.

Half-lives of Myxococcus xanthus mRNA were determined by inhibiting RNA polymerase with rifampin and then measuring the rate of [35S]methionine incorporation into protein. Vegetative cells resuspended in clone fruiting liquid culture showed an average mRNA half-life of approximately 3.5 min. Developmental cells (24 or 48 hr) exhibited biphasic decay curves with apparent mRNA half-lives of approximately 3.5 min and 20-30 min. The more-stable mRNA species accounted for about 30% of all mRNA present in 24-hr cells and as much as 40% of all mRNA of 48-hr cells. Analysis of the 35S-labeled proteins by NaDodSO4/polyacrylamide gel electrophoresis showed that only 5-10 major polypeptides are synthesized after 30 min of rifampin treatment. One of these is protein S, the spore surface coat protein, because immunoprecipitation of the 35S-labeled proteins with antisera specific for protein S showed continued synthesis of protein S in the presence of rifampin. The half-life for its mRNA was calculated to be 15-30 min. RNA from vegetative and developing cells was pulse labeled with 32PO4 followed by rifampin treatment. Analysis of the labeled RNA on 3% NaDodSO4/polyacrylamide gels showed 5-10 long-lived mRNA bands in developing cells. These results show that there are several abundant mRNA species synthesized developmentally that are exceptionally long lived. The fact that the majority of the mRNA species show the shorter half-life suggests that developing cells retain the normal mechanism for mRNA degradation.

Bacterial Proteins↗

"Frizzy" genes of Myxococcus xanthus are involved in control of frequency of reversal of gliding motility.

Myxococcus xanthus, a Gram-negative bacterium, has a complex life cycle that includes fruiting body formation. Frizzy (frz) mutants are unable to aggregate normally, instead forming frizzy filamentous aggregates. We have found that these mutants are defective in the control of cell reversal during gliding motility. Wild-type cells reverse their direction of gliding about every 6.8 min; net movement occurs since the interval between reversals can vary widely. The frzA-C, -E and -F mutants reverse their direction of movement very rarely, about once every 2 hr. These mutants cannot aggregate normally and give rise to frizzy filamentous colonies on fruiting agar or motility agar. In contrast, frzD mutants reverse their direction of movement very frequently, about once every 2.2 min; individual cells show little net movement and form smooth-edged "nonmotile" type colonies. Genetic analysis of the frzD locus shows that mutations in this locus can be dominant to the wild-type allele and that its gene product(s) must interact with the other frz gene products. Our results suggest that the frz genes are part of a system responsible for directed movement of this organism.

Chromosome Mapping↗

Inhibition of development in Myxococcus xanthus by monoclonal antibody 1604.

Monoclonal antibody (mAb) 1604 is directed against a cell surface antigen of Myxococcus xanthus. Purified antibody 1604 inhibited development of M. xanthus under conditions of submerged culture procedure otherwise leading to fruiting body formation. Intact molecules of mAb 1604, as well as its Fab fragments, inhibited developmental aggregation, autolysis, fruiting body formation, and sporulation. The addition of relatively small amounts of antibody every 4 hr was much more effective than a single large dose given at the onset of development. The inhibitory action of mAb 1604 on development was reversible after prolonged incubation of the antibody with cells; this was probably due to proteolytic degradation of the antibody. The effect of mAb 1604 on submerged bacterial development was neutralized by affinity-purified 1604 cell surface antigen. Another antibody, mAb 2788, directed against an M. xanthus cell surface antigen, did not block development. These data suggest that 1604 cell surface antigens is involved in contact-mediated cell interactions in M. xanthus.

Antibodies, Bacterial↗

Light induction of gene expression in Myxococcus xanthus.

The synthesis of carotenoids by Myxococcus xanthus requires illumination with blue light. Mutations at two loci (carA and carR) remove the blue-light requirement and cause constitutive production of carotenoids. Mutations at a different locus (carB) prevent carotenogenesis in both wild-type and constitutive mutant strains. We describe here three independent car mutations induced by insertion of Tn5 lac, a transposon that carries a transcriptional probe for exogenous promoters. All three transposon insertions block carotenogenesis even in constitutive mutant strains. One insertion is in a previously unknown car gene and the other two are in the carB locus. One of the carB insertions expresses beta-galactosidase at very low levels in the dark but is strongly activated by light. When this Tn5 lac insertion is introduced in carA or carR mutants it expresses beta-galactosidase in dark- as well as light-grown cells. We conclude that carotenogenesis in M. xanthus is activated at the level of transcription by a light-induced mechanism in which the carA and the carR loci (or their gene products) take part. The potential usefulness of M. xanthus as a simple and sensitive tool for studies in photobiology is discussed.

Journal Article↗

"Frizzy" aggregation genes of the gliding bacterium Myxococcus xanthus show sequence similarities to the chemotaxis genes of enteric bacteria.

The frz genes of Myxococcus xanthus are necessary for proper aggregation of cells to form fruiting bodies. Mutations in the frz genes affect the frequency with which individual cells reverse their direction of movement. We have subcloned and determined the nucleotide sequence of three of the frz genes. From the sequence we predict three open reading frames corresponding to frzA, frzB, and frzCD. The putative FrzA protein (17,094 Da) exhibits 28.1% amino acid identity with the CheW protein of Salmonella typhimurium. The putative FrzCD protein (43,571 Da) contains a region of about 250 amino acids which is similar to the C-terminal portions of the methyl-accepting chemotaxis receptor proteins of the enteric bacteria. FrzCD also contains a region with potentially significant similarity to the DNA-binding region of the Bacillus subtilis sigma 43. The putative FrzB protein (12,066 Da) shares no significant identity with known chemotaxis proteins. The sequence similarities between the putative Frz proteins and the chemotaxis proteins of the enteric bacteria strongly support the hypothesis that the frz genes define a system of signal transduction analogous to the enterobacterial chemotaxis systems.

Amino Acid Sequence↗

Purification and properties of Myxococcus xanthus C-factor, an intercellular signaling protein.

C-factor, a Myxococcus xanthus protein that restores the developmental defects of a class of nonautonomous mutants resulting from mutation of the csgA gene, has been purified approximately 1000-fold from starved wild-type cells. The monomeric form of C-factor is a single polypeptide with a molecular mass of 17 kDa that can be solubilized by detergent from membrane components. Characterization by gel filtration and denaturing gel electrophoresis suggests that biologically active C-factor is a dimer composed of two 17-kDa monomers. Antibodies against a form of the M. xanthus csgA gene product overexpressed in Escherichia coli react with purified C-factor.

Bacterial Proteins↗

FrzE of Myxococcus xanthus is homologous to both CheA and CheY of Salmonella typhimurium.

Myxococcus xanthus exhibits multicellular development. The "frizzy" (frz) mutants are unable to complete the developmental pathway. Instead of forming fruiting bodies, these mutants form tangled filaments of cells. We have previously shown that four of the frz gene products are homologous to enteric chemotaxis proteins and have proposed that the frz genes constitute a signal-transduction pathway that controls the frequency at which cells reverse their gliding direction. We show here that frzE encodes a protein with a calculated molecular mass of 83 kDa. FrzE is homologous to both CheA and CheY of Salmonella typhimurium, which are members of a family of "two-component response regulators." It is thought that the modulator components autophosphorylate and transfer a phosphate group to their cognate effector components. FrzE contains an unusual (alanine plus proline)-rich region that might constitute a flexible hinge facilitating phosphate transfer between functional domains. We suggest that FrzE is a second messenger that relays information between the signaling protein FrzCD and the gliding motor.

Alanine↗

Transposon tagging of genes for cell-cell interactions in Myxococcus xanthus.

The prokaryote Myxococcus xanthus is a model for cell interactions important in multicellular behavior. We used the transposon TnphoA to specifically identify genes for cell-surface factors involved in cell interactions. From a library of 10,700 insertions of TnphoA, we isolated 36 that produced alkaline phosphatase activity. Three TnphoA insertions tagged cell motility genes, called cgl, which control the adventurous movement of cells. The products of the tagged cgl genes could function in trans upon other cells and were localized primarily in the cell envelope and extracellular space, consistent with TnphoA tagging genes for extracellular factors controlling motility.

Alkaline Phosphatase↗

Two independent retrons with highly diverse reverse transcriptases in Myxococcus xanthus.

A reverse transcriptase (RT) was recently found in Myxococcus xanthus, a Gram-negative soil bacterium. This RT has been shown to be associated with a chromosomal region designated a retron responsible for the synthesis of a peculiar extrachromosomal DNA called msDNA (multicopy single-stranded DNA). We demonstrate that M. xanthus contains two independent, unlinked retrons, one for the synthesis of msDNA-Mx162 and the other for msDNA-Mx65. The structural analysis of the retron for msDNA-Mx65 revealed that the coding regions for msdRNA (msr) and msDNA (msd), and an open reading frame (ORF) downstream of msr are arranged in the same manner as found for the Mx162 retron. The ORF encodes a polypeptide of 427 amino acid residues. The amino-terminal domain (residues 1-138) shows no striking similarity to these proteins presently available in the data bases including the msDNA-Mx162 ORF, while the sequence from residues 139-394 can be aligned with various known RT sequences and has 47% identity with the RT domain of the msDNA-Mx162 ORF. On the basis of these findings, possible origins of two highly diverse retrons on the M. xanthus chromosome are discussed.

Amino Acid Sequence↗

Loss of social behaviors by myxococcus xanthus during evolution in an unstructured habitat.

Social behaviors are often targets of natural selection among higher organisms, but quantifying the effects of such selection is difficult. We have used the bacterium Myxococcus xanthus as a model system for studying the evolution of social interactions. Changes in the social behaviors of 12 M. xanthus populations were quantified after 1,000 generations of evolution in a liquid habitat, in which interactions among individuals were continually hindered by shaking and low cell densities. Derived lineages were compared with their ancestors with respect to maximum growth rate, motility rates on hard and soft agar, fruiting body formation ability, and sporulation frequency during starvation. Improved performance in the liquid selective regime among evolved lines was usually associated with significant reductions in all of the major social behaviors of M. xanthus. Maintenance of functional social behaviors is apparently detrimental to fitness under asocial growth conditions.

Journal Article↗

Recovery of quenched radioactivity from thin-layer chromatographic plates. An improved assay for cGMP phosphodiesterase in Myxococcus xanthus.

Ammonium bicarbonate was found useful in extracting a variety of radiolabeled compounds from thin-layer chromatographic plates. The technique significantly increased the sensitivity of the assay for cyclic nucleotide phosphodiesterases. This method was used to show unequivocally, the presence of cGMP phosphodiesterase in vegetative cells of Myxococcus xanthus.

3',5'-Cyclic-AMP Phosphodiesterases↗