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PubMed2003

Identification of an activator protein required for the induction of fruA, a gene essential for fruiting body development in Myxococcus xanthus.

Myxococcus xanthus exhibits social behavior and multicellular development. FruA is an essential transcription factor for fruiting body development in M. xanthus. In the present study, the upstream promoter region was found to be necessary for the induction of fruA expression during development. A cis-acting element required for the induction was identified and was located between nucleotides -154 and -107 with respect to the transcription initiation site. In addition, it was found that two binding sites exist within this element of the fruA promoter. By using DNA affinity column chromatography containing the cis-acting element, a fruA promoter-binding protein was purified. The purified protein was shown by N-terminal sequence analysis to be identical to MrpC, a protein identified previously by transposon insertion mutagenesis as an essential locus for fruiting body development [Sun, H. & Shi, W. (2001) J. Bacteriol. 183, 4786-4795]. Furthermore, fruA mRNA was not detectable in the mrpC::km strain, demonstrating that MrpC is essential for fruA expression. Moreover, mutational analysis of the binding sites for MrpC in the fruA promoter indicates that binding of MrpC activates transcription of fruA in vivo. This report provides evidence for a direct molecular interaction involved in temporally regulated gene expression in M. xanthus.

Amino Acid Sequence↗
PubMed2000

Myxococcus xanthus fibril appendages are essential for excitation by a phospholipid attractant.

Isolated (A-motile) Myxococcus xanthus cells glide over solid surfaces and display excitation, a suppression of direction reversals, when presented with phosphatidylethanolamine (PE) purified from its own membranes or synthetic dilauroyl PE and dioleoyl PE. Although the mechanism of PE signal transduction is unknown, we hypothesized that M. xanthus might use surface-associated factors to detect exogenous PE to prevent endogenous lipids from self-stimulating the sensory system. Peritrichous protein and polysaccharide appendages called fibrils were correlated with dilauroyl PE excitation. Wild-type cells treated with Congo red, an inhibitor of fibril assembly, and mutants defective in fibril biosynthesis showed an elevated reversal period, which suggested that fibrils regulate the gliding motor. Furthermore, the loss of fibrils resulted in loss of excitation to dilauroyl PE but not dioleoyl PE. Restoration of fibril production to these mutants restored the dilauroyl PE response. In addition, the dif cytoplasmic signal transduction system and starvation conditions were required for dilauroyl PE excitation. The chemically specific nature of the response and the dependence on the dif system suggests that fibrils define a novel sensory organelle whose evolution may have been necessary to prevent autostimulation by endogenous membrane lipids. Because the hydrophobic nature of dilauroyl PE would be inaccessible to periplasmic chemosensors, we suggest that fibrils act as extracellular signal transducers to probe surfaces for insoluble chemical signals.

Cell Membrane↗
PubMed2001

Identification of a developmental chemoattractant in Myxococcus xanthus through metabolic engineering.

Fruiting body formation of Myxococcus xanthus requires the ordered migration of tens of thousands of cells by using a form of surface motility known as gliding and chemical signal(s) that have yet to be elucidated. Directed movement is regulated by phosphatidylethanolamine (PE) purified from M. xanthus cell membranes. Because the purified PE preparation contains a remarkably diverse mixture of fatty acids, metabolic engineering was used to elucidate the biologically active fatty acid component. The mutational block in an esg mutant, which renders it defective in producing primers for branched-chain fatty acid biosynthesis, was bypassed with one of a series of primers that enriches for a particular family of branched-chain fatty acids. Each PE enrichment was observed for chemotactic activity by using an excitation assay and for fatty acid content. The excitation activity of a PE preparation was generally proportional with the concentration of the fatty acid 16:1 omega 5c. 1,2-O-Bis[11-(Z)-hexadecenoyl]-sn-glycero-3-phosphoethanolamine (PE-16:1 omega 5c/16:1 omega 5c) was synthesized and elicited an excitation peak at 2 ng. This peak activity occurred at a 1,000-fold lower concentration than dilauroyl PE (PE-12:0/12:0) and the peak magnitude was 2-fold higher. PE containing 16:1 omega 5c is likely to play a role in development because it is active at physiological concentrations and only under developmental conditions.

Chemotactic Factors↗
PubMed1993

The two motility systems of Myxococcus xanthus show different selective advantages on various surfaces.

Myxococcus xanthus, a bacterium that forms fruiting bodies, moves by gliding motility utilizing dual motility systems that differ both genetically and morphologically [system A, having at least 21 genetic loci and moving mainly single cells, and system S, having at least 10 genetic loci and moving groups (rafts) of cells] [Hodgkin, J. & Kaiser, D. (1979) Mol. Gen. Genet. 172, 177-191]. In this study, we found that A- and S-gliding-motility systems have different selective advantages on surfaces containing different concentrations of agar. We observed that colonies of A+S- cells (A-motile cells) swarmed better than A-S+ cells (S-motile cells) on relatively firm and dry surfaces (e.g., 1.5% agar). In contrast, colonies of A-S+ cells swarmed much better than A+S- cells on soft and wet surfaces (e.g., 0.3% agar). Individual A-motile cells moved at a rate of 2-4 microns/min on 1.5% agar but they barely moved on 0.3% agar (< 0.5 microns/min); in contrast S-motile cells moved 3-5 times faster on 0.3% agar than on 1.5% agar. Wild-type cells with both A- and S-motility systems were able to move well over a wide range of surfaces. These results suggest that dual motility systems enable the myxobacteria to adapt to a variety of physiological and ecological environments and show similarities in function to the dual motility systems of flagellated bacteria such as Vibrio spp.

Agar↗
PubMed1993

Two cell-density domains within the Myxococcus xanthus fruiting body.

Myxococcus xanthus, one of the simplest of multicellular organisms, develops into an organized, multicellular aggregate, called a fruiting body. Examination of the internal structure of the nascent fruiting body showed it to consist of a hemispherical outer domain of densely packed and ordered cells. Inside this dense shell is an inner domain of less ordered cells at 3-fold lower cell density. Single cells move in a bidirectional stream in the outer domain, orbiting the fruiting body throughout development, whereas in the inner domain, cell movement ceases as the fruiting body matures. The fruiting body thus consists of two domains, distinguished from each other by differential cell density, cell arrangements, and cell movements.

Cell Movement↗
PubMed1994

A physical map of the Myxococcus xanthus chromosome.

A physical map of the 9.2-Mbp Myxococcus xanthus DK1622 chromosome at a resolution of 25 kbp was constructed by using a strategy that is applicable to virtually all microorganisms. Segments of the chromosome were used as hybridization probes to subdivide a yeast artificial chromosome (YAC) library into groups of linked clones. The clones were aligned by comparing their EcoRI restriction patterns. The groups of YAC clones ("contigs") were oriented and aligned with the genomic restriction map by means of common genetic and physical markers such as rare restriction sites and transposon insertions. Over 95% of the genome is represented by cloned DNA. Sixty genetic loci including > 100 genes, many of which play a role in fruiting body development, have been mapped in this way. Additional genes can now be located on the chromosome map by hybridization of their sequences to the ordered set of YAC chromosomes. The mapped genetic loci account for approximately 2% of the genome.

Base Sequence↗
PubMed1996

C factor, a cell-surface-associated intercellular signaling protein, stimulates the cytoplasmic Frz signal transduction system in Myxococcus xanthus.

C factor, an intercellular signaling protein, is required for aggregation and sporulation of the social bacterium, Myxococcus xanthus. We report that C factor, which normally is associated with the cell surface, provides input to the Frz signal transduction cascade. Elements of this cascade have sequence homology to bacterial chemotaxis systems and are known to control the frequency of gliding reversal. Exposure of developing cells of a C-factor-less mutant (csgA) to purified C factor increases the ratio of methylated to nonmethylated FrzCD protein, the Frz homolog of the methyl-accepting chemotaxis proteins. Methylation depends on the cognate methyltransferase FrzF, and its extent increases with the concentration of C factor. C-factor-induced methylation also depends on the product of a gene, called class II, which is necessary in vivo for all known responses to C factor. A model for aggregation is proposed in which C factor stimulates the Frz cascade and thereby decreases cell reversals in a way that preferentially leads cells into an aggregate.

Bacterial Proteins↗
PubMed1996

Cell density regulates cellular reversal frequency in Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium that aggregates to form fruiting bodies when nutrients are limiting. Previous studies showed that the frz mutants that are defective in chemotaxis exhibited irregular and infrequent patterns of cellular reversal. In contrast, wild-type cells, when examined individually, reverse relatively frequently, about once every 6 min. It is not known how the change of reversal frequency effects cellular aggregation during fruiting body formation in M. xanthus. In this study, we stained cells with a tetrazolium dye so that we could track the reversal frequencies of single cells and cells in groups. We found that developmental cells in large groups reverse much less than cells in small groups or as single cells. This reduced cellular reversal frequency is related to the frz signal transduction system and correlated with the methylation of FrzCD (a methyl-accepting chemotaxis protein). Cells containing a mutation in the frz genes or in the genes required for social motility do not respond in this way. The reduction in cellular reversals as developmental cells accumulate in groups suggests a simple hypothesis for the aggregation of cells into discrete mounds during fruiting body formation. We also found that M. xanthus cells glide with equal frequency in the forward or reverse directions, indicating that cells do not contain a "head" or "tail."

Bacterial Proteins↗
PubMed1997

sigma54, a vital protein for Myxococcus xanthus.

The rpoN gene encoding the transcription factor sigma54 in Myxococcus xanthus has been cloned using a heterologous rpoN probe. The sequence of the cross-hybridizing DNA confirmed the existence of an ORF 1518 bp long that encodes a well conserved member of the sigma54 family of sigma factors. Low- as well as high-stringency hybridizations detected only a single rpoN gene in the M. xanthus chromosome. In other bacteria, sigma54 is an alternative sigma, and null mutants are viable. However, all attempts to construct a strain containing a null mutation in the M. xanthus rpoN have been unsuccessful. Partial diploids of rpoN+/rpoN null are viable. Recombination experiments with such partial diploids showed the impossibility of constructing, either by segregation or by transduction, a viable null haploid under any of a wide range of growth conditions. The product of the rpoN gene, sigma54, therefore appears to be essential for growth in M. xanthus.

Amino Acid Sequence↗
PubMed1999

Myxococcus cells respond to elastic forces in their substrate.

Elasticotaxis describes the ability of Myxococcus xanthus cells to sense and to respond to elastic forces within an agar gel on which they rest. Within 5 min of the application of stress, each cell begins to reorient its long axis perpendicular to the stress force. The cells then glide in that direction, and the swarm becomes asymmetric. A quantifiable assay for the strength of elasticotaxis is based on the change in swarm shape from circular to elliptic. By using a collection of isogenic motility mutants, it has been found that the ability to respond to stress in agar depends totally on adventurous (A) motility, but not at all on social (S) motility or on the frz genes. In fact, S- mutants (which are moving only by means of A motility) respond to the applied stress more strongly than does the wild type, despite the fact that their spreading rates are slower than that of the wt strain. Based on the swarming and elasticotactic phenotypes of isogenic frizzy strains that were also defective either in A or S motility, frz behaves as if part of the S motility system.

Agar↗
PubMed1999

Effect of cellular filamentation on adventurous and social gliding motility of Myxococcus xanthus.

Filamentous bacterial cells often provide biological information that is not readily evident in normal-size cells. In this study, the effect of cellular filamentation on gliding motility of Myxococcus xanthus, a Gram-negative social bacterium, was investigated. Elongation of the cell body had different effects on adventurous and social motility of M. xanthus. The rate of A-motility was insensitive to cell-body elongation whereas the rate of S-motility was reduced dramatically as the cell body got longer, indicating that these two motility systems work in different ways. The study also showed that filamentous wild-type cells glide smoothly with relatively straight, long cell bodies. However, filamentous cells of certain social motility mutants showed zigzag, tangled cell bodies on a solid surface, apparently a result of a lack of coordination between different fragments within the filaments. Further genetic and biochemical analyses indicated that the uncoordinated movements of these mutant filaments were correlated with the absence of cell surface fibril materials, indicating a possible new function for fibrils.

Cell Wall↗
PubMed1999

The cell surface-associated intercellular C-signal induces behavioral changes in individual Myxococcus xanthus cells during fruiting body morphogenesis.

Fruiting body formation in Myxococcus xanthus depends on ordered changes in cell movements from swarming to aggregation in response to starvation. We show that appropriately starved individual cells change behavior during fruiting body formation. Specifically, from the time of initiation of aggregation, individual wild-type cells began to move with increased gliding speeds, the duration of the mean gliding interval increased, and the stop frequency decreased whereas the duration of the mean stop interval and the reversal frequency remained unchanged. Mutants lacking the cell surface-associated, intercellular C-signal (csgA mutants) failed to aggregate. Likewise, appropriately starved individual csgA cells did not change their behavior during development. In the absence of other cell-cell interactions, the motility defect of individual csgA cells was corrected in a time- and concentration-dependent manner after C-signaling was reestablished by exogenous MalE-CsgA protein. The C-signal-induced stimulation of motility depended on the cytoplasmic Frz signal transduction system. We propose that C-signal instructs cells to move with high speed and low stop and reversal frequencies into aggregation centers during development.

Bacterial Proteins↗
PubMed2000

Spatial control of cell differentiation in Myxococcus xanthus.

Myxococcus xanthus develops species-specific multicellular fruiting bodies. Starting from a uniform mat of cells, some cells enter into nascent fruiting body aggregates, whereas other cells remain outside. The cells within the fruiting body differentiate from rods into spherical, heat-resistant spores, whereas the cells outside the aggregates, called peripheral cells, remain rod-shaped. Early developmentally regulated genes are expressed in peripheral cells as well as by cells in the fruiting bodies. By contrast, late developmental genes are only expressed by cells within the nascent fruiting bodies. The data show that peripheral cells begin to develop, but are unable to express genes that are switched on later than about 6 h after the start of development. All of the genes whose expression is limited to the fruiting body are dependent on C-signaling either directly or indirectly, whereas the genes that are equally expressed in peripheral rods and in fruiting body cells are not. One of the C-signal-dependent and spatially patterned operons is called dev, and the dev operon has been implicated in the process of sporulation. It is proposed that expression of certain genes, including those of the dev operon, is limited to the nascent fruiting body because fruiting body cells engage in a high level of C-signaling. Peripheral cells do less C-signaling than fruiting body cells, because they have a different spatial arrangement and are at lower density. As a consequence, peripheral cells fail to express the late genes necessary for spore differentiation.

Artificial Gene Fusion↗
PubMed1996

Photolyase of Myxococcus xanthus, a Gram-negative eubacterium, is more similar to photolyases found in Archaea and "higher" eukaryotes than to photolyases of other eubacteria.

We report the identification of the gene encoding a DNA photolyase (phrA) from the Gram-negative eubacterium Myxococcus xanthus. The deduced amino acid sequence of M. xanthus photolyase indicates that the protein contains 401 amino acids (Mr 45,071). By comparison of the amino acid and DNA sequences with those of other known photolyases, it has been found that it is more similar to the deduced amino acid sequences of the photolyases of "higher" eukaryotes than to the photolyases of other eubacteria. Recombinant plasmids carrying M. xanthus phrA rescue the photoreactivation activity of an irradiated strain of Escherichia coli with a deletion in phrA. This rescue is light-dependent.

Amino Acid Sequence↗
PubMed1996

Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme.

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.

Amino Acid Sequence↗
PubMed1998

Identification of an FAD superfamily containing protoporphyrinogen oxidases, monoamine oxidases, and phytoene desaturase. Expression and characterization of phytoene desaturase of Myxococcus xanthus.

A large number of FAD-containing proteins have previously been shown to contain a signature sequence that is referred to as the dinucleotide binding motif. Protoporphyrinogen oxidase (PPO), the penultimate enzyme of the heme biosynthetic pathway, is an FAD-containing protein that catalyzes the six electron oxidation of protoporphyrinogen IX. Sequence analysis demonstrates the presence of the dinucleotide binding motif at the amino-terminal end of the protein. Analysis of the current data base reveals that PPO has significant sequence similarities to mammalian monoamine oxidases (MAO) A and B, as well as to bacterial and plant phytoene desaturases (PHD). Previously MAOs have been shown to contain FAD, but there are no publications demonstrating the presence of FAD in purified PHDs. We have carried out the expression and purification of PHD from the bacterium Myxococcus xanthus and demonstrate the presence of noncovalently bound FAD. Sequence analysis demonstrate that PPO is closely related to bacterial PHDs and more distantly to plant PHDs and animal MAOs. Interestingly bacterial MAOs are no more closely related to PPOs, PHDs, and animal MAO's than they are to the unrelated Pseudomonas phenyl hydroxylase. All of the related sequences contain not only the basic putative dinucleotide binding motif that is found frequently for FAD-binding proteins, but they also have high similarity in an approximately 60-residue long region that extends beyond the dinucleotide motif. This region is not found among any other proteins in the current data base and, therefore, we propose that this region is a signature motif for a superfamily of FAD-containing enzymes that is comprised of PPOs, animal MAOs, and PHDs.

Amino Acid Sequence↗
PubMed2001

Transcriptional activation of a heat-shock gene, lonD, of Myxococcus xanthus by a two component histidine-aspartate phosphorelay system.

In vitro transcription of lonD, a heat-shock gene from Myxococcus xanthus, was stimulated in the presence of extract from heat-shocked cells. For this stimulation the upstream promoter region of lonD was found to be essential. Activation of lonD transcription was also observed when extract from non-heat-shocked cells was heat treated in vitro at 42 degrees C for 10 min. A DNA binding assay and footprinting analysis revealed that a factor(s) binds to the upstream region from -122 to -107 with respect to the transcription initiation site. This region was required for heat-shock induction of lonD expression both in vitro and in vivo. The lonD promoter-binding protein named HsfA was purified, and its gene was cloned. Analysis of the DNA sequence reveals that HsfA is a response regulator of the two-component system and shows high sequence similarity to the NtrC family or the enhancer-binding proteins. Upstream of hsfA, a gene encoding a histidine kinase was identified and named hsfB. HsfB was found to be autophosphorylated and able to phosphorylate HsfA. HsfA with HsfB activated in vitro transcription of lonD in a manner dependent on RNA polymerase containing SigA, the housekeeping sigma factor of M. xanthus.

Base Sequence↗
PubMed2001

A repressor-antirepressor pair links two loci controlling light-induced carotenogenesis in Myxococcus xanthus.

The light-inducible carB operon encodes all but one of the structural genes for carotenogenesis in Myxococcus xanthus. It is transcriptionally controlled by two proteins expressed from two unlinked genetic loci: CarS from the light-inducible carQRS operon, and CarA from the light-independent carA operon. CarA represses transcription from the carB promoter (P(B)) in the dark, and CarS counteracts this on illumination. The CarA sequence revealed a helix-turn-helix DNA-binding motif of the type found in bacterial MerR transcriptional factors, whereas CarS contains no known DNA-binding motif. Here, we examine the molecular interplay between CarA and CarS. We demonstrate the following. (i) Whereas CarS exhibits no DNA binding in vitro, CarA binds specifically to a region encompassing P(B) to form at least two distinct complexes. (ii) A palindrome located between positions -46 and -63 relative to the transcription start point is essential but not sufficient for the formation of the two CarA-DNA complexes observed. (iii) CarS abrogates the specific DNA binding of CarA. CarA is therefore a repressor and CarS an antirepressor. (iv) CarS physically interacts with CarA; thus, the functional interaction between them is mediated by protein-protein interactions.

Bacterial Proteins↗