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D R Zusman

Publications and source records attributed to D R Zusman.

At least 19 recordsLinked to original sources
PubMed1992

Determinants of an unusually stable mRNA in the bacterium Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium which has a complex life cycle that includes development (fruiting body formation). The gene for myxobacterial haemagglutinin, mbhA, is developmentally regulated and highly expressed. In this report we show that the mbhA mRNA is exceptionally stable for a prokaryotic organism, exhibiting a chemical half life (t1/2) of 150 min at 18 h of development. The mbhA mRNA was not stable in vegetatively growing cells nor was it stable when expressed in Escherichia coli. We have used site-directed mutagenesis of the mbhA gene to analyse some of the determinants which mediate the stability of the mbhA transcript. Sequences within the 3'-untranslated region (3'-UTR) were found to be crucial for mRNA stability. This region of mRNA can potentially form an extremely stable stem-loop structure immediately adjacent to the translational stop codon. A deletion within this region caused a 10-fold increase in the decay rate of the transcript. Furthermore, conditions which were associated with reduced mbhA translation or mutations that caused premature termination of translation drastically reduced mRNA stability even in the presence of the wild type 3'-UTR. These results suggest that a significant aspect of mbhA mRNA stability involves a synergistic interaction of the translational machinery with sequence elements within the 3'-UTR.

Bacterial Proteins↗
PubMed1992

Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior.

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.

Bacterial Proteins↗
PubMed1991

Sensory transduction in the gliding bacterium Myxococcus xanthus.

Sensory transduction in the gliding bacterium Myxococcus xanthus is mediated by the frz genes. These genes are homologous to the chemotaxis genes of enteric bacteria and control the rate of cell reversal during gliding. Sensory transduction is hypothesized to involve the recognition of substances present in the medium at the cell surface and the subsequent stimulation of a cytoplasmic methyl-accepting protein, FrzCD. Phosphorylation of FrzE is also involved in the sensory transduction pathway. Despite the similarities between the chemotaxis proteins of enteric bacteria and M. xanthus Frz proteins, fundamental differences exist between these different bacteria in terms of the ability of cells to recognize and respond to substances in their environment. The mechanism of directional switching and the nature of the gliding motor remain obscure. It is hoped that the study of the interaction of the Frz proteins will allow greater understanding of these problems.

Cell Movement↗
PubMed1991

Development in Myxococcus xanthus involves differentiation into two cell types, peripheral rods and spores.

Myxococcus xanthus, a gram-negative bacterium, has a complex life cycle. In response to starvation, most cells in a population participate in the formation of multicellular aggregates (i.e., fruiting bodies) in which cells differentiate into spores. However, some cells do not enter aggregates. In this and the two accompanying reports, the biology and physiology of these nonaggregated cells is examined. A technique to separate aggregated cells from nonaggregated cells was developed; then differentiating cells at stages throughout the course of development were isolated. In this report we (i) describe peripheral rods, those cells which remain outside aggregates after aggregation has ceased in the rest of the population; (ii) document the occurrence of peripheral rods in several wild-type strains; and (iii) characterize the expression of developmentally regulated genes in both aggregated and nonaggregated cells. These studies have shown that myxobacterial hemagglutinin, protein S (Tps), protein S1 (Ops), protein C, and several phosphatase activities are expressed in cell-type-specific patterns. These data demonstrate that peripheral rods constitute a cell type distinct from either vegetatively growing cells or spores. The description of a second, late developmental cell type (in addition to spores) opens an entirely new line of investigation in M. xanthus, i.e., the regulation of the differentiation of vegetatively growing cells into two cell types that differ significantly in biology, shape, and localization within the population.

Bacterial Outer Membrane Proteins↗
PubMed1991

Analysis of Myxococcus xanthus cell types by two-dimensional polyacrylamide gel electrophoresis.

Myxococcus xanthus is a gram-negative, soil-dwelling bacterium that undergoes development in response to depletion of nutrients. Whereas most cells aggregate into multicellular mounds in which they differentiate into spores, 10 to 20% of the developing cells remain outside fruiting bodies as peripheral rods. We used two-dimensional polyacrylamide gel electrophoresis to analyze the global expression of polypeptides in cells taken from six stages in the life cycle: vegetatively growing cells, cells 15 h after the induction of development, peripheral rods, prespores (sonication-sensitive, aggregated cells), fruiting-body spores (sonication-resistant, aggregated cells) 96 h after the induction of development, and glycerol-induced spores 15 h after induction. Seven hundred sixty-one discrete sample spots (SSPs) were identified among the six gels. Comparisons among the samples revealed that each sample had some unique SSPs, ranging from 0.3% of the 15-h developing cell SSPs to 17.9% of 96-h peripheral rod SSPs. Sixty-eight SSPs were ubiquitously distributed, but the relative amounts of these SSPs varied among the samples. Statistical analyses of the distribution and relative quantities of the SSPs indicate that, within a confidence level of greater than 99.99%, peripheral rods are significantly different from vegetatively growing cells, 15-h developing cells, prespores, fruiting-body spores, and glycerol-induced spores. In fact, among the six samples studied, only 15-h developing cells and glycerol-induced spores were similar to each other within a confidence level of P greater than or equal to 0.05. These results are consistent with the description of peripheral rods as a distinct developmental cell type.

Bacterial Proteins↗
PubMed1991

Behavior of peripheral rods and their role in the life cycle of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium with a complex life cycle including a developmental phase in which cells aggregate and sporulate in response to starvation. In previous papers, we have described a heretofore unsuspected layer of complexity in the development of M. xanthus: vegetatively growing cells differentiate into two cell types during development. In addition to the differentiation of spores within fruiting bodies, a second cell type, peripheral rods, arises outside fruiting bodies. The pattern of expression of proteins in peripheral rods is different from that of either vegetatively growing cells or spores, and peripheral rods express a number of recognized developmental markers. In this report, we examine four aspects of the biology of peripheral rods: (i) the influence of nutrients on the proportion of peripheral rods in a population of developing cells, (ii) the capacity of peripheral rods to recapitulate development, (iii) the development of peripheral rods on conditioned medium, and (iv) the ability of peripheral rods to resume growth on low amounts of exogenously added nutrients. The results of these studies suggest that peripheral rods play a significant role in the life cycle of M. xanthus by allowing the exploitation of low amounts or transient influxes of nutrients without the investment of energy in spore germination. The differentiation of vegetatively growing cells into two cell types that differ significantly in biology, shape, and localization within the population has been incorporated into a model of the life cycle of M. xanthus.

Cell Adhesion↗
PubMed1991

Myxococcus xanthus protein C is a major spore surface protein.

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.

Antigens, Surface↗
PubMed1991

Transcription of the myxobacterial hemagglutinin gene is mediated by a sigma 54-like promoter and a cis-acting upstream regulatory region of DNA.

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation of the fruiting bacterium Myxococcus xanthus. In this study, DNA sequences mediating the transcriptional regulation of mbhA have been identified. Examination of nucleotide sequences upstream of the start site for mbhA transcription has indicated a region of DNA that bears strong homology to the consensus sequence for promoters recognized by the sigma 54 holoenzyme form of RNA polymerase of Escherichia coli and other eubacteria. Deletion of this sequence completely abolished mbhA transcription. Additionally, a cis-acting DNA element, affecting the efficiency of mbhA transcription, has been mapped within a region of DNA 89 to 276 nucleotides upstream of the sigma 54-like sequence. Transposon insertions, mapping within the cis element, drastically reduced mbhA transcriptional activity. These observations suggest that transcription of mbhA requires a productive interaction between a form of RNA polymerase that recognizes a sigma 54-like sequence and a transcriptional activator that binds to DNA sequences upstream of the mbhA promoter.

Base Sequence↗
PubMed1979

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↗
PubMed1979

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↗
PubMed1979

Rifampin-resistant mutants of Myxococcus xanthus defective in development.

Rifampin, an antibiotic which is known to bind to and inhibit RNA polymerase, was used to probe the molecular regulation of development in Myxococcus xanthus. Rifampin-resistant mutants were screened for defects in fruiting-body formation. About 20% of the isolates in the initial screenings showed major defects in developmental aggregation or sporulation. Eleven independent mutants with wild-type growth rates and stable phenotypes were analyzed by transduction. In these strains, the rifampin-resistant and nonfruiting phenotypes showed cotransduction frequencies equal to or greater than 99.0 to 99.9%. The RNA polymerase activities were resistant to rifampin in vitro, indicating that the RNA polymerase is altered in these strains. Although their fruiting phenotypes are heterogeneous, these strains can be divided into two classes based on the level of aggregation. The results suggest that RNA polymerase plays a significant role in the regulation of development in M. xanthus since mutations which cause no apparent changes in vegetative growth result in striking defects in fruiting-body formation.

DNA-Directed RNA Polymerases↗
PubMed1979

Myxococcus xanthus mutants with temperature-sensitive, stage-specific defects: evidence for independent pathways in development.

Fruiting-body formation in the bacterium Myxococcus xanthus consists of a temporal sequence of cellular aggregation and sporulation. To examine the developmental stages more closely, we established synchronous and reproducible conditions for fruiting-body formation. Mutants that are temperature sensitive for fruiting-body formation were isolated and analyzed under these conditions. The terminal morphologies of the mutant strains at the nonpermissive temperature were found to resemble intermediate stages of fruiting-body formation and therefore were grouped in the following phenotypic classes: (i) rough mutants, which show no aggregation; (ii) swirl mutants, which show defective aggregation; (iii) flat-mound mutants and translucent-mound mutants, mutants which aggregate but show very low levels of sporulation. The mutants were characterized by temperature-shift experiments and found to exhibit discrete and reproducible temperature-sensitive periods. The ends of the temperature-sensitive periods in the various mutants covered a broad range of the developmental cycle. No correlation was found between the terminal morphologies at the restrictive temperature and the timing of the temperature-sensitive periods. However, the terminal morphologies correlated well with sporulation. The rough and swirl mutants produced normal numbers of myxospores at 34 degrees C even though they failed to aggregate. In contrast, the flat-mound and translucent-mound mutants, which aggregate normally, produced very few spores. The translucent-mound mutants were also temperature sensitive for induction of glycerol spores. The results indicate that both aggregation and sporulation are initiated early in the developmental cycle and that these processes are largely independent of each other.

Glycerol↗
PubMed1978

Chromosome replication in Myxococcus xanthus.

The rates of DNA synthesis during the cell-division cycle were measured in Myxococcus xanthus growing in three different media permitting a twofold variation in doubling time. In all three media, simple DNA cycles were observed. Synthesis of DNA occurred during 85% of the cell-division cycle, independent of generation time, from 5 to 11 h. Cells were observed to contain one bacterial nucleoid at birth that later divided synchronously midway through the cell cycle. Nucleoid segregation appeared to begin before chromosome replication was completed. The DNA content of exponential-phase bacteria was determined to be about 20 +/- 3 X 10(-9) microgram per cell; newborn bacteria contained about 14 +/- 2 X 10(-9) microgram of DNA per cell. Exponential-phase bacteria showed about a 50% increase in DNA in the presence of chloramphenicol (50 microgram/ml). The number of randomly segregating chromosomes present in exponential-phase bacteria was determined by following the fate of prelabeled DNA during outgrowth in nonradioactive media. The results are consistent with a model in which cells are born with exactly one complete unreplicated chromosome. The molecular weight of such a chromosome is about 8.4 +/- 1.2 X 10(9).

Cell Cycle↗
PubMed1978

Changes in cyclic AMP levels during development in Myxococcus xanthus.

Cyclic AMP levels doubled in Myxococcus xanthus under conditions in which cells aggregate and form fruiting bodies. In liquid medium, glycerol- or dimethyl sulfoxide-induced sporulating cultures exhibited a sharp but transient rise in cyclic AMP concentration after 45 min.

3',5'-Cyclic-AMP Phosphodiesterases↗