Frozen motion of gliding bacteria outlines inherent features of the motility apparatus.

High-resolution data of actively gliding wild-type bacteria of four different species and of four different gliding mutants of Myxococcus xanthus were obtained from scanning electron micrographs. By shock freezing and freeze drying, motility-associated surface patterns could be fixed and consequently distinct intermediate states of motion could be observed for the first time. It is shown that these topographic patterns are immediately lost when gliding motility is stopped by blocking the respiratory chain with potassium cyanide or sodium azide. From the surface topography, the mode of action of the gliding apparatus of all four bacterial species examined can be described as a twisted circularly closed 'band'. During gliding, groups of nodes of the supertwisted apparatus show evidence of travelling like waves along the trichomes. However, the spacing between the nodes is not constant but varies within a certain range. This indicates that they are flexibly modulated as a consequence of the gliding state of the individual trichome.

New lessons for combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of Stigmatella aurantiaca DW4/3-1.

The biosynthetic mta gene cluster responsible for myxothiazol formation from the fruiting body forming myxobacterium Stigmatella aurantiaca DW4/3-1 was sequenced and analyzed. Myxothiazol, an inhibitor of the electron transport via the bc(1)-complex of the respiratory chain, is biosynthesized by a unique combination of several polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS), which are activated by the 4'-phosphopantetheinyl transferase MtaA. Genomic replacement of a fragment of mtaB and insertion of a kanamycin resistance gene into mtaA both impaired myxothiazol synthesis. Genes mtaC and mtaD encode the enzymes for bis-thiazol(ine) formation and chain extension on one pure NRPS (MtaC) and on a unique combination of PKS and NRPS (MtaD). The genes mtaE and mtaF encode PKSs including peptide fragments with homology to methyltransferases. These methyltransferase modules are assumed to be necessary for the formation of the proposed methoxy- and beta-methoxy-acrylate intermediates of myxothiazol biosynthesis. The last gene of the cluster, mtaG, again resembles a NRPS and provides insight into the mechanism of the formation of the terminal amide of myxothiazol. The carbon backbone of an amino acid added to the myxothiazol-acid is assumed to be removed via an unprecedented module with homology to monooxygenases within MtaG.

Glucocorticoids regulate the expression of the human osteoblastic endothelin A receptor gene.

The endothelial cell-derived peptide endothelin 1 (ET1) stimulates cell proliferation and differentiated functions of human osteoblastic cells (HOC), and HOC constitutively express the endothelin A receptor (ETRA). Therefore, ET1 may play an important role in the regulation of bone cell metabolism. As glucocorticoids (GC) exert a profound influence on bone metabolism and increase the effects of ET1 on bone cell metabolism in vitro, the effects of GC on ETRA expression in HOC were investigated. Dexamethasone (DEX) increased ETRA mRNA levels in a dose- and time-dependent fashion. The effects of dexamethasone, prednisolone, and deflazacort on the increase of ETRA mRNA levels correlate positively with their binding affinity to the GC receptor. Scatchard analysis of ET1 binding data to HOC revealed that DEX increased the binding capacity for ET1 from 25,300 to 62,800 binding sites per osteoblastic cell, leading to an enhanced mitogenic effect of ET1 on HOC after preincubation with DEX. Transiently transfected primary HOC with a reporter gene construct, containing the 5'-flanking region of the ETRA gene fused to luciferase gene, showed a promoter-dependent expression of the reporter gene and the induction of reporter gene expression by DEX treatment. Total RNA extracts of femoral head biopsies with osteonecrotic lesions from GC-treated patients showed threefold higher ETRA mRNA levels compared with extracts of bone biopsies from patients with traumatically induced osteonecrosis and coxarthrosis. Furthermore, GC treatment increased plasma ET1 levels by 50% compared with pretreatment values. These findings suggest that GC induced upregulation of ETRA, and ET1 plasma levels enhance ET1's anabolic action on bone cell metabolism. Increased ET1 concentrations may also impair bone perfusion by vasoconstriction in a metabolically activated skeletal region.

Intercellular signaling in Stigmatella aurantiaca: purification and characterization of stigmolone, a myxobacterial pheromone.

The myxobacterium Stigmatella aurantiaca passes through a life cycle that involves formation of a multicellular fruiting body as the most complex stage. An early step in this differentiation process depends on a signal factor secreted by the cells when nutrients become limited. The formation of a fruiting body from a small cell population can be accelerated by addition of this secreted material. The bioactive compound was found to be steam volatile. It was purified to homogeneity by steam distillation followed by reversed-phase and normal-phase HPLC. The pheromone was named stigmolone, in accordance with the structure 2,5, 8-trimethyl-8-hydroxy-nonan-4-one, as determined by NMR and mass spectrometry. Stigmolone represents a structurally unique and highly bioactive prokaryotic pheromone that is effective in the bioassay at 1 nM concentration.

Bacillus popilliae cry18Aa operon is transcribed by sigmaE and sigmaK forms of RNA polymerase from a single initiation site.

Bacillus popilliae is an obligate pathogen for larvae of the insect family Scarabaeidae (Coleoptera). It forms parasporal crystals upon sporulation. The gene cry18Aa coding for the parasporal crystal protein and an upstream open reading frame, orf1, were previously isolated from B.popilliae. Here we report an analysis of cry18Aa transcription in Bacillus thuringiensis. The only transcriptional start site of cry18Aa was found 29 bp upstream of the open reading frame orf1, suggesting that orf1 and cry18Aa are transcribed as an operon. lacZ fusion to the cry18Aa promoter was used to follow the time-course of cry18Aa transcription in wild type B.thuringiensis and in various B.thuringiensis sporulation-deficient mutants (spo0A, sigE or sigK). In wild type B.thuringiensis, the cry18Aa promoter was activated 2 h after the end of exponential growth and the expression lasted to the late sporulation phase. The results of promoter activity in Spo+or Spo-backgrounds together with the results of primer extension experiments suggest that the transcription from this promoter can be driven by both sigmaE and sigmaK types of RNA polymerase at a single start site. The promoter region of cry18Aa operon fits the consensus sequences of both sigmaE and sigmaK dependent promoters of Bacillus.

fbfB, a gene encoding a putative galactose oxidase, is involved in Stigmatella aurantiaca fruiting body formation.

Stigmatella aurantiaca is a gram-negative bacterium which forms, under conditions of starvation in a multicellular process, characteristic three-dimensional structures: the fruiting bodies. For studying this complex process, mutants impaired in fruiting body formation have been induced by transposon insertion with a Tn5-derived transposon. The gene affected (fbfB) in one of the mutants (AP182) was studied further. Inactivation of fbfB results in mutants which form only clumps during starvation instead of wild-type fruiting bodies. This mutant phenotype can be partially rescued, if cells of mutants impaired in fbfB function are mixed with those of some independent mutants defective in fruiting before starvation. The fbfB gene is expressed about 14 h after induction of fruiting body formation as determined by measuring beta-galactosidase activity in a merodiploid strain harboring the wild-type gene and an fbfB-delta trp-lacZ fusion gene or by Northern (RNA) analysis with the Rhodobacter capsulatus pufBA fragment fused to fbfB as an indicator. The predicted polypeptide FbfB has a molecular mass of 57.8 kDa and shows a significant homology to the galactose oxidase (GaoA) of the fungus Dactylium dendroides. Galactose oxidase catalyzes the oxidation of galactose and primary alcohols to the corresponding aldehydes.

Cloning and analysis of the first cry gene from Bacillus popilliae.

An 80-kDa parasporal crystal protein was detected in protein extracts of sporangia of Bacillus popilliae isolated from a diseased larva of the common cockchafer (Melolontha melolontha L.). Amino acid analysis of tryptic peptides revealed significant homology to the Cry2Aa endotoxins of Bacillus thuringiensis. The gene cryBP1 (cry18Aa1), which codes for the parasporal crystal protein, was found in a putative cry operon on the bacterial chromosome, which contains at least one further (smaller) open reading frame, orf1. The 706-amino-acid-long CryBP1 (Cry18Aa1) protein has a predicted molecular mass of 79 kDa and shows about 40% sequence identity to the Cry2 polypeptides of B. thuringiensis. In the light of published observations which suggest that the parasporal crystal proteins of B. popilliae are slightly toxic to their grub hosts, we propose the following survival strategy of B. popilliae. As an obligate pathogen of grubs, B. popilliae germinates in the gut of a grub and the parasporal crystal proteins are released and activated. The activated protein does not cause colloid osmotic lysis but instead damages the gut wall somehow to allow the vegetative cells to enter the hemolymph more easily. By becoming a parasite, B. popilliae can continue to proliferate efficiently while the living grub provides a food supply. This process is in contrast to that of B. thuringiensis, which rapidly kills the insect and is then limited to growth on the larval carcass.

Endothelin-1 is a potent regulator of human bone cell metabolism in vitro.

Endothelial cell products may affect bone cell function, since trabecular and cortical bone are in close proximity to vascular endothelial cells. Incubation of cultured human osteoblastic cells with the endothelial cell polypeptide endothelin-1 (ET-1) resulted in a time- and dose-dependent stimulation of cell proliferation. Furthermore, markers of differentiated osteoblastic function, i.e., alkaline phosphatase and type-I collagen, were dose-dependently increased in response to ET-1. The effects of ET-1 on cell growth and function reached a maximum at higher ET-1 concentrations, and osteoblastic cells bound ET-1 specifically with a KD of 35 pM, corresponding to the biologic effects of ET-1 on bone cells. Under baseline conditions osteoblastic cells expressed 16,800 binding sites per cell. The effect of ET-1 was dependent on its binding to the endothelin-1 receptor A (ETRA), since an inhibitor of ET-1 binding blocked the biologic effects of ET-1. Northern blot analyses revealed that cultured human osteoblastic cells possess the transcript for the ETRA. Expression of ETRA mRNA was under control of 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3]. Incubation of osteoblastic cells with 1,25(OH)2D3 increased ETRA mRNA levels, corresponding to an increased effect of ET-1 on osteoblastic proliferation and function. Thus, a concerted action of the endothelial cell polypeptide ET-1 and 1,25(OH)2D3 may mediate an osteoanabolic effect of the vascular and endocrine vitamin D system.

Stigmatella aurantiaca fruiting body formation is dependent on the fbfA gene encoding a polypeptide homologous to chitin synthases.

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. For analyzing this process, mutants defective in fruiting body formation have been induced by transposon mutagenesis using a Tn5-derived transposon. About 800 bp upstream of the transposon insertion of mutant AP182 which inactivates a gene (fbfB) involved in fruiting, a further gene (fbfA) needed for fruiting body formation was detected. Inactivation of fbfA leads to mutants which form only non-structured clumps instead of the wild-type fruiting body. The mutant phenotype of fbfA mutants can be partially suppressed by mixing the mutant cells with cells of some independent mutants defective in fruiting body formation. The fbfA gene is transcribed after 8 h of development as determined by measuring the induction of beta-galactosidase activity of a fbfA-delta(trp)-lacZ fusion gene and by Northern (RNA) analysis using an insertion encoding a stable mRNA. The predicted polypeptide FbfA shows a homology of about 30% to NodC of rhizobia, an N-acetylglucosamine-transferase which is involved in the synthesis of the sugar backbone of lipo-oligosaccharides. These induce the formation of the root nodules in the Papilionaceae. Besides the predicted molecular mass of 45.5 kDa, the hydropathy profile reveals a structural relationship to the NodC polypeptide.

Localization of the stress protein SP21 in indole-induced spores, fruiting bodies, and heat-shocked cells of Stigmatella aurantiaca.

The localization and distribution of the stress protein SP21 in indole-induced vegetative cells, fruiting bodies, and heat shocked cells of Stigmatella aurantiaca were determined by immunoelectron microscopy. SP21 was found at the cell periphery in heat-shocked cells and either at the cell periphery or within the cytoplasm in indole-induced cells, often concentrated in clusters. In fruiting-body-derived spores, SP21 was located mainly at the cell wall, preferentially at the outer periphery. Furthermore, SP21 antigen was associated with cellular remnants within the stalk and within the peripheral horizon next to the fruiting body.

Cloning and characterization of the gene encoding the major sigma factor of Stigmatella aurantiaca.

The gene (sigA) encoding the major sigma factor of the myxobacterium, Stigmatella aurantiaca, was cloned and sequenced. The deduced polypeptide contains 706 amino acids (aa) and has a deduced M(r) of 79,910. It exhibits four different aa sequence motifs which correlate with the conserved domains of the major sigma factors of Myxococcus xanthus (sigma 80), Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The sigma factor (sigma A) was detected in crude lysates of vegetative cells and in cells of different developmental stages from S. aurantiaca with an antiserum to M. xanthus sigma 80 by Western blot analysis. The SigA polypeptide copurified with RNA polymerase from vegetative S. aurantiaca cells. The aa sequence of its N terminus matches a sequence located 25 codons downstream from the proposed start codon. The sigA gene was expressed in E. coli and the corresponding gene product cross-reacted with the SigA antiserum as a polypeptide of 100 kDa, which is identical in size to the sigma A detected in vegetative cells of S. aurantiaca.

A physical and genetic map of the Stigmatella aurantiaca DW4/3.1 chromosome.

A physical map of the myxobacterium Stigmatella aurantiaca DW4/3.1 chromosome was constructed by pulsed-field gel (PFG) long-range mapping. One-and two-dimensional pulsed-field gel analyses were used together with reciprocal double-restriction, cross-hybridization and hybridization fingerprint analysis. These PFG results were confirmed by Smith-Birnstiel analysis, by Southern hybridization using linking clones and clones of a lambda genomic library for the determination of adjacent restriction fragments and by transposon insertion mapping using defined genomic sequences for hybridization. It was thus possible to construct a circular restriction map of the single 9.35 Mbp chromosome of S. aurantiaca based on the endonucleases Asel and Spel. Genetic loci as well as the replication origin were located on the physical map by Southern hybridization using heterologous (derived from Myxococcus xanthus, Escherichia coli and Streptomyces lividans) and homologous probes that are mainly involved in development and cell motility.

Heat shock and development induce synthesis of a low-molecular-weight stress-responsive protein in the myxobacterium Stigmatella aurantiaca.

In the fruiting body-forming myxobacterium Stigmatella aurantiaca a 21,000-M(r) protein, SP21, is synthesized during fruiting, heat shock, and stress induced by oxygen limitation. The corresponding gene was isolated from a gene expression library in lambda gt11 with an antiserum to the purified protein. The DNA sequence of the gene reveals that SP21 is a member of the alpha-crystallin family of low-molecular-weight heat shock proteins.

Purification and characterization of SP21, a development-specific protein of the myxobacterium Stigmatella aurantiaca.

Stigmatella aurantiaca is a gram-negative bacterium with a complex life cycle, including cellular aggregation resulting in the formation of a characteristic three-dimensional structure, the so-called fruiting body. During fruiting and upon chemical induction of sporulation, a major development-specific protein, SP21, is synthesized. SP21 was purified to homogeneity from the membranous fraction of chemically induced spores. Expression of SP21 was studied with an antiserum raised against the purified protein.

Size and stability of the genomes of the myxobacteria Stigmatella aurantiaca and Stigmatella erecta.

Genomic DNA of Stigmatella aurantiaca DW 4/3.1 was restricted with the rare-cutting endonucleases AseI and SpeI. The restriction pattern derived is composed of 33 AseI and 25 SpeI fragments, whose total size amounts to approximately 9,350 kbp. Genomic fingerprint analysis of chromosomal DNA from several S. aurantiaca isolates further revealed five completely different SpeI and AseI fingerprints and one distinct fingerprint for Stigmatella erecta. In addition, minor variations between the genome sizes of these isolates were observed.

Purification of the DNA-dependent RNA polymerase from the myxobacterium Stigmatella aurantiaca.

The DNA-dependent RNA polymerase (EC 2.7.7.6) of the myxobacterium Stigmatella aurantiaca has been purified. It shows three main polypeptide bands with apparent molecular weights of 146,000, 105,000, and 40,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. beta and beta' subunits of the S. aurantiaca polymerase were shown to migrate in the 146,000-molecular-weight polypeptide band and the main sigma factor was shown to migrate in the 105,000-molecular-weight band by using heterologous antisera.

Transfer of IncP plasmids into Stigmatella aurantiaca leading to insertional mutants affected in spore development.

Derivatives of the broad-host-range plasmid RP4, containing the wild-type or modified transposon Tn5 were transferred by conjugation to various Stigmatella aurantiaca isolates. The transposons and in some cases fragments of the plasmid as well were integrated into the chromosome. Thus, insertional mutants have been obtained affected in spore formation in liquid culture.

Platelet-derived growth factor. Phorbol ester induces the expression of the B-chain but not of the A-chain in HEL cells.

It was shown previously [(1984) EMBO J. 3, 453-459] that after treatment of the human erythroleukemia cell line HEL with phorbol ester and dimethyl sulfoxide there was a marked increase in the amounts of megakaryocytic markers, especially of platelet alpha-granule proteins and platelet glycoproteins. In order to investigate this differentiation process further we have studied the expression of the mRNA encoding PDGF-A and PDGF-B (c-sis). Upon addition of the phorbol ester to the culture medium the expression of the c-sis transcript was enhanced about 7-fold over a period of 4 days. With dimethyl sulfoxide there was no significant stimulation of the expression. Addition of cycloheximide to HEL cells treated for a short period with phorbol ester superinduced the expression of the c-sis gene. The HEL cells did not express the A-chain mRNA even in the presence of phorbol ester or dimethyl sulfoxide. This leads us to propose that synthesis of the PDGF-A chain and PDGF-B chain is differentially regulated in the megakaryocytic-like HEL cell line.