Biomedical subjects

D R Zusman

Publications and source records attributed to D R Zusman.

At least 55 records · Page 3Linked to original sources

PubMed1990

Purification and characterization of the Myxococcus xanthus FrzE protein shows that it has autophosphorylation activity.

Myxococcus xanthus exhibits multicellular interactions during vegetative growth and fruiting body formation. Gliding motility is needed for these interactions. The frizzy (frz) genes are required to control directed motility. FrzE is homologous to both CheA and CheY from Salmonella typhimurium. We used polyclonal antiserum raised against a fusion protein to detect FrzE in M. xanthus extracts by Western immunoblot analysis. FrzE was clearly present during vegetative growth and at much lower levels during development. A recombinant FrzE protein was overproduced in Escherichia coli, purified from inclusion bodies, and renatured. FrzE was autophosphorylated when it was incubated in the presence of [gamma-32P]ATP and MnCl2. Chemical analyses of the phosphorylated FrzE protein indicated that it contained an acylphosphate; probably phosphoaspartate. FrzE was phosphorylated in an intramolecular reaction. Based on these observations, we propose a model of the mechanism of FrzE phosphorylation in which autophosphorylation initially occurs at a conserved histidine residue within the "CheA" domain and then, via an intramolecular transphosphorylation, is transferred to a conserved aspartate residue within the "CheY" domain.

Adenosine Triphosphate↗

PubMed1990

Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and germination.

Acid Phosphatase↗

PubMed1990

Genetic analysis of tag mutants of Myxococcus xanthus provides evidence for two developmental aggregation systems.

Temperature-dependent aggregation mutants (tag) of the myxobacterium Myxococcus xanthus aggregated into mounds and developed into fruiting bodies normally at 28 degrees C; however, they failed to form mounds at 34 degrees C. The timing of sporulation was unaffected by the mutations, and normal numbers of spores were produced at both permissive and restrictive temperatures. This class of mutations was originally identified through screening of ethyl methanesulfonate (EMS)-generated mutations. Subsequent work identified a linked insertion of transposon Tn5, which was used to map the EMS-generated mutations to four loci. In this paper, we describe the cloning of the tag loci and the use of transposon mutagenesis to further analyze the tag loci. Nine tag complementation groups spanning 8.5 kilobase pairs of DNA were identified through mapping of 28 independent Tn5 insertions. All insertion and deletion mutants had the same phenotype as the EMS mutants: they were temperature sensitive for mound formation. This result suggests that M. xanthus has at least two sets of genes for developmental aggregation. The tag genes constitute one set of these genes; they are required for normal development at 34 degrees C but are not required for normal development at 28 degrees C.

Coliphages↗

PubMed1990

Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD.

Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation). The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies. Individual frz cells cannot control the frequency at which they reverse direction while gliding. Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins. In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase. We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli. Thus, although M. xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria.

Amino Acid Sequence↗

PubMed1989

"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↗

PubMed1989

Patterns of cellular interactions during fruiting-body formation in Myxococcus xanthus.

Aggregation and mound formation during development of the myxobacterium Myxococcus xanthus were examined by scanning electron microscopy and light microscopy. Several complex patterns of multicellular associations were observed. These observations imply that complex, organized cell-cell interactions occur during the process of development. Examination of sliced aggregates revealed that, contrary to common perception, the process of sporulation commenced during mound formation rather than after the completion of mound morphogenesis. The morphogenesis of M. xanthus fruiting bodies is compared with the morphogenesis of fruiting bodies of other members of the Myxobacteriales previously described in the literature.

Microscopy, Electron, Scanning↗

PubMed1989

Evidence that the Myxococcus xanthus frz genes are developmentally regulated.

The frizzy (frz) mutants of Myxococcus xanthus are unable to form fruiting bodies. Instead of forming discrete mounds, these strains aggregate as filaments which have a circular and tangled appearance. Mutations leading to this phenotype have been mapped to five complementation groups, frzA, frzB, frzCD, frzE, and frzF. All have been found to be involved in the control of directional movement of the bacteria and, except for frzB, to be homologous to the chemotaxis genes of enteric bacteria. In this report we present a study of the regulation of expression of the first four genes of the frz gene cluster (frzA, frzB, frzCD, and frzE) by using Tn5-lac transcriptional fusions as reporters of gene expression. We found that these frz genes are developmentally regulated, with their transcription peaking at about the time of early mound formation (12 to 18 h). Analysis of FrzCD expression by enzyme-linked immunosorbent assay showed a 10-fold greater induction at 15 h of development over the level of vegetative cell expression. Northern blot hybridization analysis suggested that the frz genes were arranged as an operon. To test this hypothesis, double mutants were constructed which contained Tn5-132 either upstream or downstream of the reporter Tn5-lac. The expression of the frz genes in the double mutants was consistent with the hypothesis that the first four genes (frzA, frzB, frzCD, and frzE) are organized as an operon with an internal promoter. Insertion mutations in frzCD lowered gene expression whether they were upstream or downstream of the reporter Tn5-lac, suggesting that the FrzCD protein regulates transcription of the entire operon from a promoter upstream of frzA. Evidence is presented suggesting that FrzE is required for induction of transcription as well. When frz mutations were placed in strains that were unable to aggregate (tag), the frz genes were expressed at an elevated level on fruiting agar; this high level of expression was maintained for several days. These results suggest that the tag gene products interact with the frz functions.

DNA Transposable Elements↗

PubMed1989

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.

Disaccharides↗

PubMed1988

Reexamination of the role of autolysis in the development of Myxococcus xanthus.

It has been widely reported that 80 to 90% of the cell population undergoes autolysis during sporulation in Myxococcus xanthus. A re-evaluation of the techniques used to measure autolysis in M. xanthus showed that the methods previously used to draw this conclusion are subject to artifacts, which result in a substantial underestimation of the number of cells present during development. We found that at least 80% of the cells that enter development survive throughout fruiting body formation. The cell loss that did occur appeared to be gradual over a period of at least 7 days. Our results suggest that autolysis is not an obligate stage in the development of M. xanthus. The data also showed that sporulating cells pass through a prespore stage in which they become osmotically and physically fragile and therefore difficult to harvest intact. The fragility was correlated with the change from a rod to a spherical shape. As the prespores differentiated into refractile spores, they lost fragility and became amenable to harvesting by standard protocols.

Bacteriolysis↗

PubMed1987

Cloning of the gene for myxobacterial hemagglutinin and isolation and analysis of structural gene mutations.

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation in the bacterium Myxococcus xanthus. It has been shown that this lectin is targeted to the cell surface and periplasmic space of developmental cells, suggesting that it may play a role in cell-cell recognition or agglutination. We have cloned the structural gene for MBHA by using synthetic deoxyoligonucleotides containing sequences deduced from the amino acid sequence of MBHA and have used the cloned gene to construct strains of M. xanthus that cannot synthesize MBHA. We found that although the MBHA-deficient strains are delayed in their developmental time course, they are otherwise able to aggregate and sporulate normally. Our results suggest that MBHA may function to increase the efficiency of fruiting-body formation but is not a critical component of cellular aggregation.

Cloning, Molecular↗

PubMed1986

Nucleotide sequence of the myxobacterial hemagglutinin gene contains four homologous domains.

Myxococcus xanthus, a Gram-negative bacterium, has a complex life cycle that includes fruiting-body formation, a primitive form of multicellular development. Myxobacterial hemagglutinin (MBHA) is a lectin that is induced during the aggregation phase of fruiting-body formation. We have cloned the gene for MBHA and determined its sequence by the dideoxy chain-termination technique. The sequence data show the probable sites for translational initiation and termination and suggest that MBHA does not contain a cleaved leader signal peptide. The DNA sequence shows four strong internal homologies. The deduced amino acid sequence shows that the protein (Mr 27,920) consists of four highly conserved domains each consisting of 67 amino acids. Thus MBHA is physically multivalent in structure, a requirement for all hemagglutinins.

Amino Acid Sequence↗

PubMed1986

Analysis of the products of the Myxococcus xanthus frz genes.

The frizzy (frz) genes of Myxococcus xanthus control the ability of cells to reverse direction of gliding motility. The orientation of the frz genes was studied by isolating transcriptional fusions with the transposon derivative Tn5-lac. The frz genes were then cloned in the proper orientation in an expression vector. By using maxicell experiments, we were able to identify several labeled bands which were plasmid encoded. To identify the labeled proteins and their respective genes, we constructed deletion plasmids in which various regions of the insert DNA had been removed. The plasmid-encoded proteins were then labeled in maxicell experiments, and the bands which correspond to the frzCD, frzE, and frzF gene products were identified. The sizes of the gene products agreed with the genetic and physical map of the cloned DNA.

Bacterial Proteins↗

PubMed1986

Genetic analysis of Myxococcus xanthus and isolation of gene replacements after transduction under conditions of limited homology.

Genetic analysis of Myxococcus xanthus is greatly facilitated by the ability to introduce cloned DNA into M. xanthus to generate gene replacement and merodiploid strains. However, gene replacement strains are difficult to obtain when the region(s) of homology between the cloned DNA and the M. xanthus chromosome is limited (less than 1 kilobase). We found that gene replacements can be obtained at an increased frequency by a two-step procedure involving the use of bacteriophage P1 to isolate merodiploid strains followed by generalized transduction to another M. xanthus strain by using phage Mx4.

Anti-Bacterial Agents↗

PubMed1986

Combined heart-lung transplantation for end-stage Eisenmenger's syndrome.

Between May, 1981, and December, 1984, thirteen combined heart-lung transplants were performed in 12 patients for the treatment of Eisenmenger's syndrome. The age range of the recipients was 22 to 42 years. Two patients had undergone previous open cardiac operations; in addition, one had had closure of a persistent ductus arteriosus, one an open lung biopsy, one a pulmonary artery banding, and one patient received a second heart-lung transplant after 3 years. Four recipients died before hospital discharge, one at operation and three at 4, 10, and 33 days after operation. Early symptomatic results and cardiopulmonary function were excellent in all of the survivors. Two patients died 14 and 15 months after transplantation of accelerated graft arteriosclerosis and respiratory failure, respectively, and six remain alive 7 to 44 months after transplantation. Four of these surviving patients and the two patients who died late subsequently had major pulmonary complications. Symptoms included progressive breathlessness, cough (often productive), and fever with physical signs of diffuse crepitations and expiratory rhonchi. Serial pulmonary function tests showed progressive obstructive physiology in all six patients with superimposed restrictive defects in four. Histologic examination of tissue from open lung biopsy or autopsy displayed bronchiolitis obliterans in five of these patients, one of whom required retransplantation. It is possible that these late changes are the result of rejection, since similar changes in one other patient have now been reversed with augmented immunosuppression. Further understanding of the causes and manifestations of late pulmonary deterioration should improve the late functional results of this operation for Eisenmenger's syndrome.

Adult↗

PubMed1986

Naturally corrected double-outlet right ventricle with biventricular outflow tract obstruction.

A 5-year-old girl had presyncopal episodes associated with exertion. Diagnostic evaluation revealed a combination of congenital abnormalities: double-outlet right ventricle with apparent "natural correction" of a large subaortic ventricular septal defect and severe biventricular outflow tract obstruction. Intraoperative and postoperative studies confirmed satisfactory relief of the biventricular outflow tract obstruction.

Child, Preschool↗

PubMed1986

Timing of abnormal interventricular septal motion after cardiopulmonary bypass operations. Lack of injury proved by preoperative, intraoperative, and postoperative echocardiography.

Abnormal interventricular septal motion after cardiopulmonary bypass is a widely known occurrence. The cause and exact timing of this phenomenon remain unclear. We have studied 21 patients prospectively with preoperative, intraoperative, and postoperative two-dimensional and M-mode echocardiograms. Intraoperative studies were obtained with the pericardium closed and open and after completion of procedures performed with cardiopulmonary bypass. Fourteen patients had coronary artery bypass graft operations alone. Six patients had valve replacement with or without coronary bypass, and one patient had removal of a left atrial myxoma. All patients had normal interventricular septal motion before the operation, and none had abnormal septal motion intraoperatively. Four to eight days postoperatively, the septum still thickened normally in all patients, with five patients having normal, nine patients abnormal, and seven patients paradoxical interventricular septal motion. Studies in 11 patients 1 to 4 months postoperatively showed no change from the early postoperative study. The pericardium was left open postoperatively in all patients. Six patients were studied a few hours after sternal closure and all had abnormal interventricular septal motion. We conclude that abnormal interventricular septal motion after cardiac operations is not due to injury of the septum, adhesion formation, or loss of pericardial constraint. Closure of the chest wall itself, with the pericardium left open, is associated with this abnormality.

Cardiomyopathies↗

PubMed1985

Cloning and complementation analysis of the "Frizzy" genes of Myxococcus xanthus.

Fruiting-body formation in Myxococcus xanthus involves the aggregation of cells into raised mounds, where they sporulate. "Frizzy" mutants fail to aggregate into mounds, but rather aggregate into "frizzy" filaments (D.R. Zusman 1982). The frizzy mutations (frz) were found to be genetically linked. The region of DNA carrying the frz genes was cloned in Escherichia coli by selecting for the kanamycin resistance element present on a transposon Tn5 insertion linked to the frz genes. Phage P1 mediated transduction of the cloned DNA into M. xanthus frizzy mutants showed that the cloned DNA could complement the frz mutations. The cloned DNA was analyzed by isolating and characterizing new Tn5 insertions at short intervals within the M. xanthus DNA and by constructing in vitro deletions. The mutated DNA was then transduced into M. xanthus where the cloned DNA became integrated into the bacterial chromosome as gene replacements or as merodiploids. The gene replacement strains allowed us to define the limits of the frz region, since Tn5 insertions in the frz genes resulted in the frizzy phenotype. The merodiploid strains allowed us to perform complementation analyses. Using appropriate crosses, we were able to identify 5-6 frz complementation groups on 7.5 kb of cloned DNA. One of the complementation groups was separated from the others by 1.4 kb of DNA, whereas the others were contiguous. The different frz loci behave as separate transcriptional groups although interactions between some of the gene products are indicated.

Bacteria↗

PubMed1985

"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↗