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Abstracts 1992-199956. Kimsey HH; Kaiser D. (1992) The orotidine-5'-monophosphate decarboxylase gene of Myxococcus xanthus. Comparison to the OMP decarboxylase gene family. J Biol Chem 267:819-24. (UI: 92112814) The nucleotide sequence of the Myxococcus xanthus orotidine-5'-monophosphate decarboxylase (OMP DCase) gene was determined. The derived protein sequence is not closely related to other prokaryotic OMP DCase sequences; nor is it closely related to any eukaryotic OMP DCase sequences. Progressive multiple alignment of the M. xanthus OMP DCase protein sequence with 19 other OMP DCase sequences revealed four conserved regions present in all 20 sequences. Ten entirely conserved residues were found in these four regions and one region contains a tight cluster of 5 conserved residues, certain of which may be catalytically active residues. A second open reading frame was found upstream of uraA and oriented in the same direction as uraA. A stretch of 21 consecutive pyrimidine (C or T) residues were found in the intercistronic region between the potential ribosome-binding site of uraA and the UGA stop codon of the upstream open reading frame. RNA directly upstream of the pyrimidine run, including the UGA stop codon of the upstream open reading frame, could be folded into a stable hairpin structure resembling Rho-independent terminators of Escherichia coli. Expression of the uraA gene may be regulated by an intercistronic transcription termination mechanism. 57. Plamann L; Kuspa A; Kaiser D. (1992) Proteins that rescue A-signal-defective mutants of Myxococcus xanthus. J Bacteriol 174:3311-8. (UI: 92250427) The asg mutants of Myxococcus xanthus are defective in the production of an extracellular substance, called A-factor, that is required for expression of a set of fruiting body-specific genes. A-factor is released by wild-type cells (asg+) after 1 to 2 h of development. When A-factor is added to asg mutant cells, it restores expression of their A-factor-dependent genes. Rescue of beta-galactosidase production in an asg mutant carrying the A-factor-dependent lacZ transcriptional fusion (omega 4521) was used to assay A-factor activity. According to this assay, two types of substances with A-factor activity are present in conditioned medium. One type is heat stable and of low molecular weight; the other is heat labile and of high molecular weight. An approximately 27-kDa protein with heat-labile A-factor activity was purified from conditioned medium. The purified protein has proteolytic activity as well as A-factor activity. The substrate specificity of the 27-kDa protease resembles that of trypsin. A smaller protein with both heat-labile A-factor activity and proteolytic activity was identified. Its substrate specificity differs from that of the 27-kDa protein. In addition, trypsin and other proteases were found to have heat-labile A-factor activity. Trypsin inhibitory protein from soybeans neutralizes the A-factor activity of trypsin in parallel with its neutralization of protease activity, showing that the proteolytic activity of trypsin is necessary for its A-factor activity. The 27-kDa protein rescues the aggregation and sporulation defects of an asgB mutant in submerged culture as well as its ability to express beta-galactosidase from an asg-dependent lac fusion. 58. Kuspa A; Plamann L; Kaiser D. (1992) Identification of heat-stable A-factor from Myxococcus xanthus. J Bacteriol 174:3319-26. (UI: 92250428) The asg mutants of Myxococcus xanthus fail to produce a set of related substances called A-factor. A-factor is released into the medium and is required early in fruiting body development. Lacking A-factor, the asg mutants are defective in aggregation, sporulation, and expression of most genes whose products appear later than 1 h after development is induced by starvation. Previous work has shown that these defects are reversed when A-factor, released by developing wild-type cells, is added to asg mutant cells. Part of the material in conditioned medium with A-factor activity is heat stable and dialyzable. This low-molecular-weight A-factor consists of a mixture of amino acids and peptides. Fifteen single amino acids have A-factor activity, and 11 of these are found in conditioned medium. Mixtures of amino acids have a total activity approximately equal to the sum of the activities of their constituents. Conditioned medium also contains peptides with A-factor activity. Pure peptides have A-factor activity, and their specific activities are equal to or less than the sum of the activities of their constituent amino acids. There is no evidence for a specialized A-factor peptide in conditioned medium, one with a specific activity greater than the sum of its constituent amino acids. About half of the heat-stable A-factor activity in conditioned medium can be accounted for by free amino acids, and the remaining half can be accounted for by peptides. It is argued that heat-stable A-factor induces A-dependent gene expression not by the nutritional action of amino acids but through a chemosensory circuit. 59. Kim SK; Kaiser D; Kuspa A. (1992) Control of cell density and pattern by intercellular signaling in Myxococcus development. Ann Rev Microbiol 46:117-39. (UI: 93073707) Myxococcus xanthus cells feed, move, and develop cooperatively. Genetic, biochemical, and cell mosaic studies demonstrate that cells coordinate their multicellular behavior by transmission of intercellular signals. Starvation for amino acids at sufficiently high density on a solid surface initiates a series of events culminating in the formation of a multicellular structure called a fruiting body filled with dormant, environmentally resistant spores. This review discusses how myxobacteria use extracellular signals to sequentially check the density and arrangement of cells at different stages during development. For at least one early and one late developmental signal, cell density determines the efficiency of intercellular signaling. In turn, proper signaling insures that the appropriate cell density exists, thus controlling the progress of multicellular development in M. xanthus. 60. Kuspa A; Plamann L; Kaiser D. (1992) A-signalling and the cell density requirement for Myxococcus xanthus development. J Bacteriol 174:7360-9. (UI: 93054352) Mutations in any of three asg (A-signalling) loci cause fruiting body development of Myxococcus xanthus to arrest at about the 2-h stage. Development can be restored to asg mutants by the addition of conditioned buffer in which wild-type cells have been developing or of A-factor purified from the conditioned buffer. Two forms of A-factor have been identified: heat-stable A-factor, which is composed of amino acids and peptides, and heat-labile A-factor, which consists of at least two proteases. A-factor is found in conditioned buffer in rough proportion to the cell density. As decreasing amounts of either form of A-factor are added, the developmental response of asg cells decreases until a threshold concentration is reached, below which no response is detected. In addition, wild-type cells fail to develop when their density is decreased below the point at which the level of A-factor is predicted to fall short of this threshold. The development of low-density asg+ cells can, however, be restored by the addition of either form of A-factor. These experiments show that A-factor is important for the development of wild-type cells. Moreover, the development of an asgB mutant that produces 5 to 10% the wild-type level of A-factor can be restored when the cell density is increased 10-fold above the standard density. We propose that the A-signal is used by M. xanthus to specify the minimum cell density required for the initiation of development. Differences in the response to A-factor between different asg mutants suggest that the different asg loci govern A-factor production in diverse ways.
61. Sager B; Kaiser D. (1993) Two cell-density domains within the Myxococcus xanthus fruiting body. Proc Natl Acad Sci (USA) 90:3690-4. (UI: 93234563) 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. 62. Russo-Marie F; Roederer M; Sager B; Herzenberg LA; Kaiser D. (1993) Beta-galactosidase activity in single differentiating bacterial cells. Proc Natl Acad Sci (USA) 90:8194-8. (UI: 93376770) Myxococcus xanthus strains containing transcriptional fusions to lacZ were analyzed and fractionated by differences in their levels of beta-galactosidase expression. The fluorogenic substrate for beta-galactosidase, fluorescein di-beta-galactopyranoside, was introduced into M. xanthus cells during a rapid decrease in osmolarity of the medium followed by a return to isoosmolarity. Fluorescein, the product of hydrolysis, was retained within the cells and their viability was preserved. Fluorescence increased linearly with time and was proportional to beta-galactosidase activity. beta-Galactosidase expression in most fusion strains, though beginning at different phases of growth or development, was distributed unimodally amongst cells. However, fusion strain Tn5 lac omega 4473 was shown to be heterogeneous at 9 hr of development. It was possible to separate physically cells that expressed beta-galactosidase at a high level from other, still viable, cells with no expression. The approach described here could be adapted to study differentiation in plants and animals as well, where transcriptional fusions and fluorogenic substrates for enzyme probes of gene expression also can be used. 63. Sager B; Kaiser D. (1993) Spatial restriction of cellular differentiation. Genes and Devel 7:1645-53. (UI: 93380641) Myxococcus xanthus cells differentiate into myxospores within a fruiting body, an aggregate of approximately 10(5) cells. Previous work had discerned an inner and outer domain within the fruiting body differentiated by cell density and cell alignment. To test whether the two domains might play different roles in spore differentiation, developmentally regulated gene fusions were screened for expression restricted to one domain or the other. Transcriptional lacZ fusions to 80 developmentally regulated genes were examined and eight fusions were found that restricted expression to the inner domain, while one fusion, omega 7621, showed initial expression in the outer domain. Initial omega 7621 expression coincided with patches of spore precursors evident in bright-field microscopy. Later in development, both omega 7621 expression and the patches expanded inward, eventually filling both the inner and outer domains. Previous work had also shown that high cell density and cell alignment are required for transmission of the C-signal, which is needed to initiate spore differentiation. Evidence is presented for a novel morphogenetic mechanism in which C-signaling in the outer (high density) domain initiates spore differentiation. It is proposed that spore precursors are passively transported from the outer to the inner domain by the movements of undifferentiated rod cells. Reconstruction experiments showed that developing rod cells move with sufficient force to displace spores. Spore precursors thus accumulate in the inner domain where they express spore-specific genes at high levels and account for inner domain specific expression. 64. Kaiser D. (1993) Roland Thaxter's legacy and the origins of multicellular development. Genetics 135:249-54. (UI: 94063466) 65. Thony-Meyer L; Kaiser D. (1993) devRS, an autoregulated and essential genetic locus for fruiting body development in Myxococcus xanthus. J Bacteriol 175:7450-62. (UI: 94042921) Two Tn5 lac insertions into the Myxococcus genome at sites omega 4414 and omega 4473, which are separated by 550 nucleotides, inactivate fruiting body development. Sporulation is decreased 100- to 10,000-fold. At least two genes, devR and devS, are transcribed in this region, probably as an operon. Expression of devR begins by 6 h after starvation has initiated development. On the basis of their nucleotide sequences, devR and devS are expected to encode proteins of 302 and 214 amino acids, respectively. Dev+ function can be restored by a segment of 7.8 kb cloned from the devRS region of wild-type cells. Two experiments show that devR expression is under strong negative autoregulation. beta-Galactosidase is expressed at a higher level from a transcriptional devR::lacZ fusion when the fused operon is in a dev strain than when it is in the dev/dev+ genetic background of a partial diploid. There is more mRNA accumulation from the devRS region in the dev strain than in a rescued dev/dev+ tandem duplication strain. Sporulation rescue is correlated with some degree of negative autoregulation, even though sporulation is not inversely proportional to beta-galactosidase expression from omega 4414. A second level of regulation is suggested by complementation of dev by dev+ in duplication strains. The expression of devRS, measured by sporulation levels, differs 1,000-fold when devRS+ is moved from a distance of 20 kb to 3 Mb from the mutant devRS locus. Expression of devR is also dependent on the cell density at which development is initiated, a third level of regulation. Multiple levels of regulation suggest that devRS is a switch required to activate completion of aggregation and sporulation.
66. Cheng YL; Kalman LV; Kaiser D. (1994) The dsg gene of Myxococcus xanthus encodes a protein similar to translation initiation factor IF3. J Bacteriol 176:1427-33. (UI: 94156849) The dsg mutants of Myxococcus xanthus are defective in fruiting body development and sporulation, yet they grow normally. The deduced amino acid sequence of the dsg gene product is 50 and 51% identical to the amino acid sequence of translation initiation factor IF3 of both Escherichia coli and Bacillus stearothermophilus, respectively. However, the Dsg protein has a carboxy-terminal extension of 66 amino acids, which are absent from its E. coli and B. stearothermophilus homologs. The Shine-Dalgarno sequence GGAGG and 5 bases further upstream are identical in M. xanthus and several enteric bacteria, despite the wide phylogenetic gap between these species. The infC gene, which encodes IF3 in enteric bacteria, starts with the atypical translation initiation codon AUU, which is known to be important for regulating the cellular level of IF3 in E. coli. Translation of the Dsg protein overexpressed from the M. xanthus dsg gene in E. coli cells initiates at an AUC codon, an atypical initiation codon in the AUU class. The dsg mutants DK429 and DK439 carry the same missense mutation that changes Gly-134 to Glu in a region of amino acid identity. 67. Kalman LV; Cheng YL; Kaiser D. (1994) The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo. J Bacteriol 176:1434-42. (UI: 94156850) The amino acid sequence of the Dsg protein is 50% identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene. Anti-E. coli IF3 antibodies cross-react with the Dsg protein. Tn5 insertion mutations in dsg are lethal. When ample nutrients are available, however, certain dsg point mutant strains grow at the same rate as wild-type cells. Under the starvation conditions that induce fruiting body development, these dsg mutants begin to aggregate but fail to develop further. The level of Dsg antigen, as a fraction of total cell protein, does not change detectably during growth and development, as expected for a factor essential for protein synthesis. The amount of IF3 protein in E. coli is known to be autoregulated at the translational level. This autoregulation is lost in an E. coli infC362 missense mutant. The dsg+ gene from Myxococcus xanthus restores normal autoregulation to the infC362 mutant strain. Dsg is distinguished from IF3 of E. coli, other enteric bacteria, and Bacillus stearothermophilus by having a C-terminal tail of 66 amino acids. Partial and complete deletion of this tail showed that it is needed for certain vegetative and developmental functions but not for viability. 68. Sager B; Kaiser D. (1994) Intercellular C-signaling and the traveling waves of Myxococcus. Genes and Development 8:2793-804. (UI: 95087881) Early in their development into fruiting bodies, Myxococcus xanthus cells organize themselves into dense bands that move as trains of traveling waves. C-factor, a 20-kD cell-surface bound protein, is a short-range developmental signal molecule required for these waves. What is the role of C-factor in the wave pattern? It is proposed that oriented collisions between cells initiate C-signaling, which, in turn, causes individual cells to reverse their direction of gliding. Cells would move about one wavelength and then reverse. Several lines of experimental evidence support these proposals: (1) Cells that suffered a mutation in the signal transduction pathway that controls the spontaneous reversal frequency lost the ability to form waves; (2) presentation of developing cells with detergent-solubilized C-factor increased the mean frequency of single cell reversal by three-fold; and (3) fluorescently labeled cells in the waves were tracked, and it was found that they moved and reversed on linear paths along the axis of wave propagation. Similar numbers of cells were found moving in the direction of ripple propagation, and in the reverse direction, as expected. (4) Dilution of C-signaling-competent cells with C-factor-deficient cells increased the wavelength as the probability of productive collision decreased. The waves exemplify a way that a multicellular pattern of stripes can be produced de novo, one that maintains a uniform 50-microns separation between stripes over a distance as large as 1 cm.
69. Singer M; Kaiser D. (1995) Ectopic production of guanosine penta- and tetraphosphate can initiate early developmental gene expression in Myxococcus xanthus. Genes and Devel 9:1633-44. (UI: 95354935) Amino acid or carbon limitation is sufficient to initiate fruiting body development in Myxococcus xanthus. In both Escherichia coli and M. xanthus the levels of guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp] rise transiently when cells are starved for amino acids or carbon. Ectopic increase in the intracellular concentration of (p)ppGpp was achieved in M. xanthus by introducing a copy of the E. coli relA gene, whose product catalyzes pyrophosphate transfer from ATP- to GTP-forming pppGpp. The E. coli RelA protein was detected in these M. xanthus strains, and a rise in (p)ppGpp was observed chromatographically. This increase in the intracellular (p)ppGpp levels was sufficient to activate developmentally specific gene expression. Although (p)ppGpp is made from GTP, the intracellular GTP pool from these strains was not significantly decreased. Moreover, when the GTP pool was lowered by either of two specific inhibitors of GTP synthesis, mycophenolic acid or decoyinine, development was not induced. These results suggest that M. xanthus cells can assess their nutritional status by monitoring the internal availability of amino acids through (p)ppGpp levels. 70. Keseler IM; Kaiser D. (1995) An early A-signal-dependent gene in Myxococcus xanthus has a sigma 54-like promoter. J Bacteriol 177:4638-44. (UI: 95370140) A-signaling plays an essential role in the early stages of Myxococcus xanthus fruiting body development. Expression of the 452I gene, which is regulated at the level of RNA accumulation, depends on starvation and on A-signaling. To identify the cis-acting regulatory elements which allow gene 4521 to respond to the nutritional and A-factor signals, the 4521 transcription start site was mapped. The region just upstream of the start site showed sequence similarity to the sigma 54 family of promoters and to the developmentally regulated mbhA promoter of M. xanthus. A mutational analysis of this region established that the bases which were conserved between the sigma 54 consensus, mbhA, and 4521 promoters were also important for 4521 promoter activity. Changes which altered the spacing between two conserved regions centered around positions -14 and -24 abolished promoter activity. In contrast, mutations in a putative -10 region for a sigma 70-like promoter had little effect on expression of 4521. Despite their similar promoter regions, the regulation of the 4521 and mbhA genes was shown to differ with respect to timing of expression and requirement for a solid surface and extracellular signals. This suggests a model in which different activator proteins may be responsible for regulating expression of these two genes. 71. Wu SS; Kaiser D. (1995) Genetic and functional evidence that type IV pili are required for social gliding motility in Myxococcus xanthus. Mol Microbiol 18:547-58. (UI: 96342383) The social gliding behavior of Myxococcus xanthus has previously been associated with the presence of polar pili. A Tn5 transposon insertion was isolated which introduces a defect in social gliding and is genetically linked to a known sgl locus; this insertion was found also to cause a piliation defect. 2.7 kb of DNA were isolated from either side of this transposon and sequenced, revealing three genes which encode amino acid sequences with substantial similarity to components of the Type IV pilus biogenesis pathway in Pseudomonas aeruginosa. The myxococcal pilA gene encodes a putative pilin precursor with a short signal sequence and processing site similar to those of other Type IV pilins. Myxococcal pilS and pilR encode amino acid sequences with similarity to PilS and PilR of P. aeruginosa, as well as to other members of the NtrB/C family of two component regulators. Mutations within pilR and pilA that have no polar effect were demonstrated to be responsible for pilus and social motility defects. These results indicate that the pili of M. xanthus belong to the Type IV family of pili, and demonstrate that these pili are actually required for social motility. 72. Spormann AM; Kaiser AD. (1995) Gliding movements in Myxococcus xanthus. J Bacteriol 177:5846-52. (UI: 96011371) Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.
73. Sogaard-Andersen L; Slack FJ; Kimsey H; Kaiser D. (1996) Intercellular C-signaling in Myxococcus xanthus involves a branched signal transduction pathway. Genes and Devel 10:740-54. (UI: 96178541) C-factor, the product of the csgA gene, is a cell-surface associated short-range intercellular signaling protein in Myxococcus xanthus. C-factor is required for at least four responses during starvation-induced fruiting body morphogenesis: rippling, aggregation, sporulation, and full expression of the csgA gene, all of which fail in a csgA mutant. To analyze the C-factor signaling pathway, eight Tn5 lac insertion mutants that began but failed to complete fruiting body aggregation were characterized. Seven of the insertions identified genes whose products function in the csgA signaling pathway. The seven mutants were differentially deficient in the C-factor responses, and could be divided into two classes on the basis of those differences. On one hand, the four mutants in class I were deficient in rippling and aggregation, but sporulated and produced C-factor at wild-type levels. The Tn5 lac insertions in the class I mutants mapped to the frz locus, which encodes a signal transduction system that controls the frequency of single cell reversals. On the other hand, mutants carrying any of the three closely linked class II Tn5 lac insertions had deficiencies in all four C-factor responses. Because the sporulation defect in the class 11 mutants is cell autonomous, the data suggest that the primary defect in these mutants is an inability to respond to the C-factor signal. All the data can be explained by a model in which the first part of the C-factor signaling pathway is common to all four C-factor-dependent responses. The genes identified by the class 11 insertions would function in the common part. Downstream of class II, the pathway branches. One branch includes the frz genes and leads to aggregation and rippling; the second branch leads to sporulation and controls the level of csgA gene expression. This model was confirmed in epistasis tests with characterized frz mutations, a csgA null mutation, and a class II mutation. 74. Sogaard-Andersen L; Kaiser D. (1996) C factor, a cell-surface-associated intercellular signaling protein, stimulates the cytoplasmic Frz signal transduction system in Myxococcus xanthus. Proc Natl Acad Sci (USA) 93:2675-9. (UI: 96181465) 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. 75. Kaiser D. (1996) Bacteria Also Vote. Science 272:1598-9. (UI: 96247310) 76. Wu SS; Kaiser D. (1996) Markerless deletions of pil genes in Myxococcus xanthus generated by counterselection with the Bacillus subtilis sacB gene. J Bacteriol 178:5817-21. (UI: 96422018) In-frame deletions of pilA and pilS were constructed in Myxococcus xanthus, using a plasmid integration/excision strategy facilitated by sacB. sacB conferred sucrose-sensitivity upon its M. xanthus host only when it lay in the same orientation as adjacent M. xanthus genes. Gene orientation also affected the efficiency of sucrose counterselection in the sucrose-sensitive strains. The pilA deletion mutant lacked pili and social motility, while the pilS deletion mutant showed no defect in either phenotype.
77. Wu SS; Wu J; Kaiser D. (1997) The Myxococcus xanthus pilT locus is required for social gliding motility although pili are still produced. Mol Microbiol 23:109-21. Social gliding motility in Myxococcus xanthus depends on the presence of Type IV pili. To begin to dissect out the role of pili in social motility, 17 mutants were identified which had lost social motility, but still expressed pili. Four of these mutants carry point mutations which mapped to a locus upstream of the recently identified pilS, pilR, and pilA genes. Sequencing of this locus revealed a gene with homology to pilT from Pseudomonas aeruginosa. Sequencing of the four point mutations revealed that they occurred within the M. xanthus pilT locus. A markerless deletion within M. xanthus pilT, like the four point mutations, disrupted social gliding behavior but did not interfere with pilus formation or pilus-dependent cell-cell agglutination. Using time-lapse videomicroscopy, residual social motility was observed in dsp- strains (known to be deficient in fibril but not pilus production); this was not observed in a pilT deletion dsp- double mutant. Two genes flanking pilT were also sequenced, and found to have homology to pilB and pilC from P. aeruginosa. Markerless deletions within these genes caused both pilus and social motility defects. These results indicate that M. xanthus pilB and pilC are required for pilus biogenesis, while pilT is required for assembled pili to play their role in social motility. Thus, pilB, pilT, pilC, pilS, pilR and pilA form a contiguous cluster of pil genes required for social motility. 78. Losick R; Kaiser D. (1997) Why and how bacteria communicate. Scientific American 276: 68-73. (UI: 97153832) 79. Keseler IM; Kaiser D. (1997) Sigma-54, a vital protein for Myxococcus xanthus. Proc Natl Acad Sci (USA) 94:1979-84. (UI: 97203172) The rpoN gene encoding the transcription factor sigma-54 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 sigma-54 family of factors. Low- as well as high-stringency hybridizations detected only a single rpoN gene in the M. xanthus chromosome. In other bacteria, sigma-54 is an alternative sigma factor, 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, sigma-54, therefore appears to be essential for growth in M. xanthus. 80. Rodriguez-Soto JP; Kaiser D. The tgl gene: social motility and stimulation in Myxococcus xanthus. J Bacteriol 179:4361-71. (UI: 97352696) Mutations in the tgl locus inactivate social gliding motility in Myxococcus xanthus and block production of pili. The tgl locus is distinctive among the genes for social motility because social gliding and pili can be restored transiently to tgl mutant cells by mixing them with tgl+ cells, a process known as stimulation. The tgl locus was cloned with a linked insertion of transposon Tn5 by using the kanamycin resistance encoded by that transposon. A 16-kb segment of chromosomal DNA complemented the social motility defect when introduced into tgl mutant cells to form a tandem duplication tgl+/tgl heterozygote. To delimit the autonomous tgl transcription unit, subfragments of this 16-kb piece were integrated at the ectopic Mx8 prophage attachment site. A 1.7-kb DNA fragment was identified which, when integrated at the Mx8 site, simultaneously rescued social motility and pilus production. The ability to stimulate tgl mutants was also rescued by the 1.7-kb fragment. Because rescue of stimulation from an mgl-deficient donor strain which cannot swarm was observed, this demonstrates that a stimulation donor requires a tgl+ allele but does not require the capacity to swarm actively. The nucleotide sequence of the 1.7-kb fragment revealed two protein coding regions, open reading frame A and open reading frame B (ORFB). ORFB is the tgl gene, because a 613-bp DNA fragment which includes 75% of ORFB rescues tgl-1, -2, and -3 mutants and because disruption of ORFB by deletion or insertion of transposon Tn5lac constitutes a tgl mutation. 81. Rodriguez-Soto JP; Kaiser D. (1997) Identification and localization of the Tgl protein, which is required for Myxococcus xanthus social motility. J Bacteriol 179:4372-81. (UI: 97352697) Tgl protein is required for the production of the type IV pili found at a pole of the Myxococcus xanthus cell. These pili are essential for social motility. Evidence is presented that Tgl is a membrane protein, based on experiments with polyclonal antibody specific for Tgl that was raised against the fusion proteins beta-galactosidase-Tgl and TrpE-Tgl. Immunoaffiity-purified antibody reacted with a protein in M. xanthus having an apparent molecular mass of 27.5 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sequence of the tgl gene translates into a polypeptide of 27 kDa. Although these numbers are close, it is likely that the primary tgl translation product is processed and modified in M. xanthus. The N terminus has a signal peptidase II recognition sequence, cleavage of which is expected to remove 19 amino acid residues. When the tgl gene is expressed in Escherichia coli, the protein product consistently migrates faster in the gel than mature Tgl expressed in M. xanthus, suggesting a second modification by addition which slows migration of the protein from M. xanthus. Tgl, as detected by its specific antibody, sediments with the membrane fraction of cells. It can be extracted with detergents but not with salt or by the addition of chelators for divalent cations. In an equilibrium gradient, Tgl bands at the buoyant density of membranes and with the NADH-oxidase activity. Intact cells failed to bind anti-Tgl antibody, and less than 2% of the total Tgl is released in soluble form from the periplasm. Yet, cells that had been osmotically shocked and treated with paraformaldehyde were able to react with the specific antibody--a reaction absent from cells with a deletion of the tgl transcription unit. Assuming that osmotic shock disrupts the outer membrane, the fractionation and localization data imply that Tgl is attached to the inner or outer membranes, from which it is exposed to the intermembranous space. Tgl is necessary for synthesis of pili in M. xanthus and is the only pilus protein that can be donated by other cells (stimulation). Tgl contains six tandem copies of the tetratrico peptide repeat structural motif. Its membrane localization, capacity for stimulation, and content of tetratrico structural repeats together suggest that Tgl may be necessary for the assembly of pilin subunits into filaments. 82. Wu SS; Kaiser D. (1997) Regulation of expression of the pilA gene in Myxococcus xanthus. J Bacteriol 179:7748-58 (UI: 98062206)Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA , consistent with the homology of PilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.
83. Wall D; Wu SS; Kaiser D. (1998) Contact stimulation of Tgl and type IV pili in Myxococcus xanthus. J Bacteriol 180:759-61. (UI: 98117065)Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation. 84. Wall D; Kaiser D. (1998) Alignment enhances the cell-to-cell transfer of pilus phenotype. Proc Natl Acad Sci (USA) 95:3054-58. (UI: 98169495)Social gliding motility of Myxococcus xanthus requires polar type IV pili. Tgl mutants lack pili and lack social motility. However, both defects can be rescued phenotypically, but not genotypically, when tgl+ donor and tgl- recipient cells make physical contact with each other. What is the cellular and molecular basis of this transfer of phenotype, which is called stimulation? Stimulation does not occur in liquid nor in soft (0.5%) agar; however, on a more firm surface (1.0% agar) cells stimulate each other efficiently. Microscopy revealed that cells placed on 1.0% agar readily became aligned whereas they remained poorly aligned on 0.5% agar. It was observed, moreover, that stimulation is greatly reduced when donor and recipient cells lack the ability to move because of mutations in any of several different motility genes. Microscopy showed that motile cells became highly aligned, and can even align nonmotile cells. Using a stimulation assay based on the assembly of pili, a strong correlation was found between conditions that promote cell alignment and the speed or extent of stimullation. Because pili are assembled only at the end(s) of a cell, and a parallel alignement of cells in contact brings them end-to-end, we would suggest that end-to-end contacts are important for stimulation of pilus assembly. 85. Wu SS; Wu J; Cheng YC; Kaiser D. (1998) The pilH gene encodes an ABC transporter required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus. Mol Microbiol 29:1249-61. Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus. We report the discovery of four additional pil genes: pilD, a homolog of type IV prepilin leader peptidases, and pilG, pilH and pilI, which have no known homologs in other Type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homolog, the first such homolog to be required for biogenesis of any bacterial pilus type. pilG and pilI are cotranscribed with pilH and appear to be functionally related to pilH. Null mutants of pilG, pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies, and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in supernatant after cells are sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, where PilI is an integral membrane protein with membrane spanning domains and PilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.
86. Gorski, L.; Kaiser, D. (1998) Targeted Mutagenesis of Sigma-54 Activator Proteins in Myxococcus xanthus. J. Bacteriol.180:5896-5905. Myxococcus xanthus DNA segments related to the highly conserved central sequence of sigma54 activator proteins have been investigated. A genetic technique designed to inactivate a gene that encodes such an activator by inserting a plasmid-borne internal fragment of the putative gene has been tested. When the internal fragment inserted by homologous recombination into the corresponding chromosomal locus, the expected duplication of the gene was observed by Southern hybridization. The single restriction fragment characteristic of each segment was replaced in the insertion strains by two hybridizing fragments, and one of these fragments hybridized with the kanamycin resistance gene of the plasmid vector. The combined molecular weights of the two fragments from the insertion strains were equal to the molecular weight of the original fragment plus the expected molecular weight contributed by the vector. In the duplication, one copy is expected to have an N-terminal deletion and the other copy is expected to have a C-terminal deletion. In most cases, the net result should be loss of activator function. If an activator is essential for vegetative growth, then it should not be possible to obtain the insertion strain by plasmid integration. Indeed, integrants for three of the segments were not obtained in repeated trials; however, a plausible explanation for these results other than lethality can be offered. Of the seven insertions validated by Southern hybridization, four strains exhibited defects in the development of fruiting bodies. One of these failed to develop in submerged culture, though it developed normally on agar. The other three showed arrested development of fruiting bodies, each at a morphologically different stage of aggregation. One of the mutants may be defective in the reception pathway of A-signal. 87. Wall, D., Kolenbrander, P.E. & Kaiser, AD. (1999) Myxococcus xanthus pilQ (sglA) gene encodes a seretin homolog required for type IV biogenesis, S motility and development. J. Bacteriol. 1999 181(1): 24-33. The Myxococcus xanthus sglA1 spontaneous mutation was originally isolated because it allowed dispersed cell growth in liquid yet retained the ability to form fruiting bodies. Consequently, most of today's laboratory strains either contain the sglA1 mutation or were derived from strains that carry it. Subsequent work showed that sglA was a gene for social gliding motility, a process which is mediated by type IV pili. Here sglA is shown to map to the major pil cluster and to encode a 901-amino-acid open reading frame (ORF) that is homologous to the secretin superfamily of proteins. Secretins form a channel in the outer membrane for the transport of macromolecules. The closest homologs found were PilQ proteins from Pseudomonas aeruginosa and Neisseria gonorrhoeae, which are required for type IV pili biogenesis and twitching motility. To signify these molecular and functional similarities, we have changed the name of sglA to pilQ. The hypomorphic pilQ1 (sglA1) allele was sequenced and found to contain two missense mutations at residues 741 (G-->S) and 762 (N-->G). In addition, 19 independent social (S)-motility mutations are shown to map to the pilQ locus. In-frame deletions of pilQ and its downstream gene, orfL, were constructed. pilQ is shown to be essential for pilus biogenesis, S-motility, rippling, and fruiting body formation, while orfL is dispensable for these processes. The pilQ1 allele, but not the DeltapilQ allele, was found to render cells hypersensitive to vancomycin, suggesting that PilQ1 alters the permeability properties of the outer membrane. Many differences between pilQ1 and pilQ+ strains have been noted in the literature. We discuss some of these observations and how they may be rationalized in the context of our molecular and functional findings 88. Spormann, A. M.&Kaiser, D.(1999) Gliding mutants of Myxococcus xanthus with high reversal frequencies and small displacements. J. Bacteriol.181(8):2593-2601. Myxococcus xanthus cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the mgl and frz genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective in mglAB or in cglB, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min-1 for ÆmglAB mutants and 2.7 min-1 for cglB mutants, compared to 0.17 min-1 for wild-type cells). The average gliding speed of ÆmglAB mutant cells was 40% of that of wild-type cells (on average 1.9 micrometers/min for ÆmglAB mutants, compared to 4.4 micrometers/min for wild-type cells). The mglA-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein: mglB mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min-1 and an average speed of 2.6 micrometers/min. These values range between those exhibited by wild-type cells and by ÆmglAB mutant cells. Epistasis analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation, but not a frzE mutation, partially suppressed the mglA phenotype. In contrast to mgl mutants, cglB mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of mglAB cells was only partially reduced by a pilR mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S motility. 89. Wall D & Kaiser, AD. (1999) Type IV pili and cell motility.(MicroReview) Molecular Microbiogy. 1999 32(1), 1-10. |