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The next International Meeting on The Biology of the Myxobacteria (which will be our SilverAnniversary Meeting!) will be held in Delphi , Greece.
25th International Conference on the Biology of Myxobacteria
Arahova (Greece), June 21-24, 1998
For more detailed information, click on Myxococcus labs and then select Yannis Karamanos' home page.
Alternatively, contact:
Secretariat :
Yannis Karamanos
Laboratoire de Biochimie Moleculaire et Cellulaire
Universite d'Artois- Faculte des Sciences
Rue Souvraz - SP18 - 62307 Lens Cedex
fax + 33 3 21 79 17 55
E-mail : karamanos@univ-artois.fr
The following has been submitted by Phil Youderian for your consideration:
Nov. 14, 1997
Myxococcus xanthus Has Multiple Systems for Hexose Transport
Phil Youderian, Department of Microbiology, Molecular Biology and
Biochemistry, University of Idaho, Moscow, Idaho 83844-3052
The vegetative growth of Myxococcus xanthus is inhibited by low
concentrations of the glucose (Glc) analog, 2-deoxyglucose (2dGlc). This
inhibition can be supressed phenotypically by the addition of the hexoses
glucose (Glc) and N-acetylglucosamine (GlcNAc), but not by the addition of
fructose (Fru) or mannitol (Mtl). Mutants of M. xanthus which can grow in
the presence of 5 mM 2dGlc (tdg, for two-deoxyglucose-resistant) arise at
a low spontaneous frequency and fall into two distinct phenotypic classes.
One class of resistant mutants, like its wild-type parent, cannot form
fruiting bodies or heat-resistant spores at 2dGlc concentrations
permissive for the multicellular development of the other class of
resistant mutants. Ectopic expression of E. coli glucokinase in thse
resistant mutants restores sensitivity to 2-deoxyglucose. Furthermore,
these resistant mutants regain sensitivity to 2dGlc when grown in the
presence of low concentrations of GlcNAc. These results suggest that
Myxococcus xanthus can uptake hexoses by at least three different
mechanisms, a group translocation (phosphotransferase, or PTS) system
specific for Glc, an additional Glc transport system, and an inducible PTS
system specific for GlcNAc. The PTS systems provide M. xanthus with
activities that carry out the initial and terminal steps in glycolysis.
Presently we are confirming this hypothesis, in collaboration with
Milt Saier at UCSD, by assaying M. xanthus grown in Glc, GlcNAc, Fru, and
Mtl for PTS activities, and by performing 14C-Glc and 14C-2dGlc uptake
experiments with both wild-type and 2dGlc-resistant mutants of M. xanthus.
Dr. Linda Plamann has has departed Texas A&M and has begun an exciting new position at the University of Missouri, Kansas City, where she will continue her work on A signaling in Myxococcus xanthus. Her new address is as follows:
Dr. Lynda S. Plamann
Associate Professor
School of Biological Sciences
Division of Cell Biology and Biophysics
University of Missouri-Kansas City
2411 Holmes, Room M3-403
Kansas City, Missouri 64108-2792
The following pertains to the recent 24th Myxo meeting held on June 22-25, 1997 at the John Newcombe Tennis Ranch in New Braunfels, Texas. The information consists of the program, a list of abstracts and the names and addresses of attendees.
MYXOBACTERIAL MEETING PROGRAM
Sunday, June 22
2:00 - 6:00 PM Registration and Check-In - Main Lodge
6:30 - 7:30 PM Dinner - Deck and Pool Area
7:30 PM Meeting begins - Conference Center
7:30 - 7:35 Announcements: Heidi Kaplan
7:35 - 7:40 Welcome: Laura Wilson, Meetings Coordinator,
John Newcombe Tennis Ranch
7:40 - 10:00 PM SESSION 1
Transcriptional Regulators and Genetic Tools
Chair: Lynda Plamann
7:40 - 8:05 Characterization of sigma factors of Myxococcus xanthus
Toshiyuki Ueki* and Sumiko Inouye p. 1
8:05 - 8:30 CarQ is a sigma factor, CarR is an inner membrane protein of Myxococcus xanthus and CarS appears not to be a DNA binding protein
Douglas F. Browning, Nazrin Khoshkhoo and David A. Hodgson* p. 2
8:30 - 8:55 Detection of protein-protein interactions in the Yeast two-hybrid system using the Myxococcal response regulator FrzZ as 'bait'
Mandy Ward* and David Zusman p. 3
8:55 - 9:10 BREAK
9:10 - 9:35 The role of sigma-54 in the development of Myxococcus xanthus:: mutational analysis of sigma-54 activator proteins
Lisa Gorski* and Dale Kaiser p. 4
9:35 - 10:00 PCR-based gene disruption in M. xanthus and M. virescens
Don Walthers*, David Chapman, Vincent Magrini, David White, Patricia L. Hartzell and Philip Youderian p. 5
10:00 - midnight Myxo Mixer - Billabong Bar (Main Lodge)
Monday, June 23
7:30 - 8:30 AM Breakfast - Waltzing Matilda Dinning Room
8:30 - 12:10 AM Session 2
Motility and Directed Cell Movement
Chair: Mitch Singer
8:30 - 8:55 CglB and single cell gliding in Myxococcus xanthus
Ana M. Rodriguez* and Alfred Spormann p. 6
8:55 - 9:20 A mutation in the gidA operon of Myxococcus xanthus affects adventurous motility and a late stage of spore maturation
David White* and Patricia Hartzell p. 7
9:20 - 9:45 Cell movements, alignments and PilQ (SglA) are involved in Tgl contact stimulation in Myxococcus xanthus
Daniel Wall* and Dale Kaiser p. 8
9:45 - 10:10 Alterations in the Myxococcus xanthus O-antigen result in defective motility and A signal-independent 4521 developmental gene expression
M. Gabriela Bowden* and Heidi B. Kaplan p. 9
10:10 - 10:30 BREAK
10:30 - 10:55 Identification and characterization of a second chemosensory system of Myxococcus xanthus involved in cellular aggregation and development gene expression
Zhaomin Yang*, Dick Xu, Yongzhi Geng, Heidi B. Kaplan, and Wenyuan Shi p. 10
10:55 - 11:20 Directed cell movements of developmental mutants of Myxococcus xanthus p. 11
Yongzhi Geng, Zhaomin Yang, Yiping Han, and Wenyuan Shi*
11:20 - 11:45 Chemotaxis in Myxococcus xanthus
Daniel B. Kearns* and Lawrence J. Shimkets p. 12
11:45 - 12:10 Genetic analysis of Cytophaga johnsonae gliding motility
Mark J. McBride* p. 13
12:10 - 1:00 PM Lunch - Waltzing Matilda Dinning Room
FREE AFTERNOON
5:45 - 6:30 PM POSTERS - A/V ROOM (adjacent to meeting area)
6:30 - 7:30 PM Dinner - Waltzing Matilda Dinning Room
7:30 - 9:50 PM SESSION 3
Starvation and Bsg Control of Development
Chair: Lee Kroos
7:30 - 7:55 Organization and expression of the sdeII locus which mediates fruiting body formation in Myxococcus xanthus p. 14
Brandon Greenberg*, Anthony Garza, and Mitch Singer
7:55 - 8:20 Characterization of the sde locus of Myxococcus xanthus
Jeffrey Pollack* and Mitchell Singer p. 15
8:20 - 8:45 The guanosine nucleotide (p)ppGpp and RelA are necessary and sufficient to initiate Myxococcus xanthus development
Baruch Z. Harris* and Mitchell Singer p. 16
8:45- 9:00 BREAK
9:00 - 9:25 Genetic identification of a negative regulator of developmental gene transcription in the social prokaryote Myxococcus xanthus
Elizabeth Hager* and Ron Gill p. 17
9:25 - 9:50 The spdRS operon
Hubert M. Tse* and Ron Gill p. 18
9:50 - midnight Myxo Mixer - Billabong Bar (Main Lodge)
Tuesday, June 24
7:30 - 8:30 AM Breakfast - Waltzing Matilda Dinning Room
8:30 - 11:45 AM SESSION 4
M. xanthus A signaling and Stigmatella development
Chair: Lotte Søgaard-Andersen
8:30 - 9:55 Cloning of ?4530, a gene that requires functional asg genes, but not A-signal, for proper developmental expression in Myxococcus
John M. Davis* and Lynda Plamann p. 19
8:55 - 9:20 Genetic suppression analysis of an asgA missense mutant
Valerie Kessler*, Cathy Eylem, Andrea Tews, and
Lynda Plamann p. 20
9:20 - 9:45 A histidine protein kinase and a response regulator control early Myxococcus xanthus developmental gene expression
Chun Yang* and Heidi B. Kaplan p 21
9:45 - 10:10 Molecular and genetic analysis of the Myxococcus xanthus sasB5 gene
Di Xu*, Chun Yang and Heidi B. Kaplan p. 22
10:10 - 10:30 BREAK
10:30 - 10:55 Cloning and analysis of LPS O-antigen suppressors
Dongchuan Guo*, Yun Wu and Heidi B. Kaplan p. 23
10:55 - 11:20 Genes involved in fruiting body formation of Stigmatella aurantiaca
Barbara Silakowski*, Heidi Ehret and Hans Ulrich Schairer p. 24
11:20 - 11:45 Intercellular signalling in Stigmaella aurantiaca: Proof, purification and structure of a myxobacterial pheromone p. 25
William Hull, Albrecht Berkessel, Irmela Stamm and Wulf Plaga*
11:45 - 12:30 PM SEQUENCING THE MYXO GENOME
OPEN DISCUSSION
Chair: Alfred Spormann
12:30 - 1:30 PM Lunch - Waltzing Matilda Dinning Room
FREE AFTERNOON
6:30 - 7:30 PM Dinner - Waltzing Matilda Dinning Room
7:30 - 10:15 PM SESSION 5
C-signaling
Chair: Yannis Karamanos
7:30 - 7:55 The ClassII protein, a key-protein in the C-factor signal transduction pathway, is a response regulator with a C-terminal DNA binding
domain and may act in a C-factor dependent phosphorelay system p. 26
Eva Ellehauge*, Sune Lobedanz and Lotte Søgaard-Andersen
7:55 - 8:20 fruA, an essential locus in the C-factor signaling pathway, may serve to coordinate early developmental signals and C-factor signaling during fruiting body morphogenesis in M. xanthus p. 27
Lotte Søgaard-Andersen*, Peter Søholt, Eva Ellehauge and Anders Boysen
8:20 - 8:45 Analysis of the devTRS operon from M. xanthus
Bryan Julien* and Dale Kaiser p. 28
8:45 - 9:00 BREAK
9:00 - 9:25 Characterization of the regulatory regions of C-signal-dependent genes of Myxococcus xanthus
Lee Kroos*, Janine Brandner, Dvora Biran, Makda Fisseha, and Tong Hao p. 29
9:25 - 9:50 A developmental checkpoint for C-signaling
Eugene W. Crawford Jr.* and Lawrence J. Shimkets p. 30
9:50 - 10:15 Rescue of aggregation and sporulation in Bsg, Csg, and Dsg mutants
Kathleen A. O'Connor* and David R. Zusman p. 31
10:15 - midnight Myxo Mixer - Billabong Bar (Main Lodge)
Wednesday, June 25
7:30 - 8:30 AM Breakfast - Waltzing Matilda Dinning Room
8:30 - 11:15 AM Session 6
Phage, Metabolism, Stress Responses and Evolution
Chair: Wenyuan Shi
8:30 - 8:55 Genetic analysis of temperate Myxococcus xanthus phage Mx8 int and xis: evidence for a prokaryotic origin of the protamines
Vincent Magrini*, Daniel Salmi, and Philip Youderian p. 32
8:55 - 9:20 Genetic analysis of temperate Myxococcus xanthus phage Mx8 immunity: the primary repressor gene, imm, encodes a histone H1-like product
Daniel Salmi*, Vincent Magrini, and Philip Youderian p. 33
9:20 - 9:45 Cloning and characterization of the genes flanking the glyoxylate bypass regulon in Myxococcus xanthus p. 34
David R. Chapman*, Daniel Salmi and Philip Youderian
9:45 - 10:00 BREAK
10:00 - 10:25 Further studies on endo-N-acetyl-ß-D-glucosaminidase from Myxococcus xanthus
Yannis Karamanos* p. 35
10:25 - 10:50 The CspA (cold-shock protein) family in M. xanthus p. 36
Kunitoshi Yamanaka*, Masayori Inouye, and Sumiko Inouye
10:50 - 11:15 Loss of social behaviors by Myxococcus xanthus during evolution in an unstructured habitat
Gregory Velicer*, Lee Kroos, and Richard E. Lenski p. 37
11:15 - 12:00 BUSINESS MEETING
12:00 - 1:00 LUNCH - Waltzing Matilda Dinning Room
CHECK OUT BY 2:00 PM
BACK TO THE LAB
Abstracts from the 24th Annual Meeting on the Biology of the Myxobacteria
- Abstracts are listed in their presentation order (see the included presentation schedule).
- Abstracts # 1, 3, 10, 11, 14, 15, 16, 17, 25, 35, and 36 are not included because we did not receive electronic versions.
- Abstracts # 38 42 were poster presentations.
Abstract #2
CarQ is a sigma factor, CarR is an inner membrane protein of Myxococcus xanthus and CarS appears not to be a DNA binding protein
Douglas F. Browning, Nazrin Khoshkhoo & David A. Hodgson.
Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, England
We will report on our recent attempts to confirm our model of mechanism of light-regulation of carotenoid biosynthesis in Myxococcus xanthus using more biochemical approaches. Central to our model is that CarQ is an RNA polymerase sigma factor and CarR is an inner membrane bound anti-sigma factor that binds and inactivates CarQ in the dark and releases it in active form in the light.
The only evidence that we had that CarQ was a sigma factor was the observation of homology to other proteins of the ECF (extra-chromosomal function) sigma factor family, other members of which had been shown to have sigma factor activity in vitro. The carQ open reading frame was tagged with a 6-His peptide and over-expressed in E. coli. All the protein formed inclusion bodies and all attempts at resolubilisation failed. At the same time we constructed the same 6-His-tagged gene in M. xanthus. This construct failed to complement a carQ3 mutant and, when introduced as a extra copy in a wild-type strain, failed to generate a CarC phenotype. We concluded that the tag had destroyed all activity of the protein. Over-expression of wild-type CarQ in E. coli also led to inclusion body formation but these inclusion-bodies could be solubilised and a protein isolated that had specific sigma factor activity on the carQRS promoter DNA in vitro in the presence of E. coli core RNA polymerase.
CarR fused with protein A has been shown to be located in the inner membrane when expressed in E. coli. A 6-His tagged truncation of CarR was over-expressed in E. coli which proved to be soluble and so was purified and used to raise antibodies. These anti-CarR antisera were used to probe M. xanthus cells constitutively expressing CarR in the dark. A protein of the expected size was found. Fractionation of the myxobacterial cells using sucrose gradients failed to yield pure inner and outer membrane fractions. However, detergent fractionation did yield separate fractions enriched in succinate dehydrogenase (inner membrane) and LPS (outer membrane). The CarR protein present in CarC cells all proved to be present in the M. xanthus inner membrane, by western blotting. Cross linking experiments failed to demonstrate CarQ:CarR interaction in the membrane.
CarS was also 6His-tagged and over-expressed in E. coli. The protein was soluble and used to raise antibodies. Construction of strains that carried the 6-His-tagged CarS protein in M. xanthus showed the 6-His-tagged protein was not active.
Abstract # 4
The role of s54 in the development of Myxococcus xanthus: mutational analysis of s54 activator proteins.
Lisa Gorski and Dale Kaiser
Department of Biochemistry, Stanford University School of Medicine
Beckman Center, Room B400, Stanford, CA 94305-5307
Analysis of developmentally regulated genes in Myxococcus xanthus has revealed that at least two are transcribed by s54 type promoters. This type of promoter requires a specific activator protein to bind upstream in order to initiate an open complex to achieve transcription. Understanding the role s54 promoters play during development has been hampered by the finding that the gene transcribing the s54ubunit, rpoN, is essential for the viability of M. xanthus (Keseler and Kaiser, 1997); therefore, rpoN cannot be deleted. To better understand the role of s54 in regulating development, we have been creating null mutations in s 54 activator proteins using a collection of 500 bp fragments (made in collaboration with the Tracy Nixon lab) generated for the internal, heavily conserved ATP binding region common to these activator proteins (Kaufman and Nixon, 1996).
Among mutants in 13 genes, five have strong developmental defects. Two are inhibited due to a defect in social motility; the remaining three have normal motility. The three with normal motility arrest development at different stages. One arrests before aggregation, while the other two arrest at different stages during aggregation. None of the three can sporulate. Neither can they sporulate in co-culture with the wild-type strain. Thus all three are cell autonomous. All three make the A and C signals since mutants in those signal classes can be rescued for sporulation when co-cultured with these activator mutants.
It is possible that these three activators play a role in signal transduction pathways that respond to the A and C signals. The early mutant might be involved with A signalling since, in addition to its sporulation defects, it fails to transcribe the A-signal dependent gene W4521 while it does transcribe other non-A dependent fusions such as W4469, W4455, and W4411. The two later mutants may be involved with C signalling. Efforts are underway to clone and sequence the two activators possibly involved in the C signal receptor pathway and to locate the mutant arrest points within the response pathway for C-signal reception (Søgaard-Andersen et al., 1996).
Kaufman, R. I. & Nixon, B. T. 1996. Use of PCR to isolate genes encoding s54-dependent activators from diverse bacteria. J. Bacteriol. 178:3967-3970.
Keseler, I. M. & Kaiser, D. 1997. s 54, a vital protein for Myxococcus xanthus. Proc. Natl. Acad. Sci. USA 94: 1979- 1984.
Sogaard, Andersen L., Slack, F. J., Kimsey, H., & Kaiser, D. 1996. Intercellular C-signaling in Myxococcus xanthus involves a branched signal transduction pathway. Genes Dev. 10: 740-754.
Abstract #5
PCR-based gene disruption in M. xanthus and M. virescens
Don Walthers, David Chapman, Vincent Magrini, David White, Patricia L. Hartzell
and Philip Youderian
Department of Microbiology, Molecular Biology and Biochemistry
University of Idaho, Moscow, ID 83844-3052
A combination of the PCR-mediated amplification of dominant resistance markers with the electroporation of linear DNA has been used as a rapid method for targeted gene disruption in yeast (Wach et al. (1994) Yeast 10: 1793-1808). We have tested this method in both Myxococcus xanthus and Myxococcus virescens. Disruption cassettes were constructed by amplification of resistance determinants with primers (ca. 70 bp) having 5' homology to flanking chromosomal regions of interest (ca. 50 bp) and 3' homology to the resistance genes (ca. 20 bp).
Plasmids containing kanamycin-, tetracycline-, and sulfisoxazole- resistance genes were used as templates for PCR. Whereas electroporation of M. virescens with a linear Kmr disruption cassette targeted for replacement of the mglBA region results in non-motile kanamycin-resistant transformants, as expected, electroporation of M. xanthus strain DK1622 gives rise to both a majority of non-motile and a minority of motile recombinants. This result suggests that, in some cases, either targeted replacement can give rise to homeologous recombinants, or linear PCR fragments may circularize prior to integration into the genome.
We have observed at least two additional limitations of this method. Only about 10% of Kmr or Tcr cassettes used for disruption give rise to recombinants with wild-type strain DK1622. Nonetheless, when successful, the method appears to yield a majority of products with the expected phenotype. For example, targeted disruption of the sglK gene in wild-type M. xanthus strain DK1622 results in kanamycin-resistant recombinants that have lost social gliding motility. Whereas the Tn5 aph and Tn10 tetA genes can be used for targeted replacement, the sul2 gene does not prove to be a suitable marker for replacement. When flanked with homologies internal to Tn5-lac, this gene yields sulfisoxazole-resistant recombinants with DK1622.
Previously, we have found that M. xanthus strain DK476 (asg) is a more efficient recipient of circular DNA after electroporation than is wild-type strain DK1622. DK476 is also a much better host for targeted disruption, and almost all cassettes we have tested give rise to replacements in this host, which can be moved into the wild-type genetic background by generalized transduction with phage Mx4.
Abstract #6
CglB and single cell gliding in Myxococcus xanthus
Ana M. Rodriguez and Alfred M. Spormann
Environmental Engineering and Science
Stanford University
Stanford, CA 94305-4020
Single cell gliding in Myxococcus xanthus is controlled by genes of the A-motility system. Genes of the A-system can be divided into two classes: cgl and agl genes. Mutants of the cgl class have the quality that the motility defect can be extracellularly rescued by mixing the mutant cells with wild type or mutant cells that are defective in a different motility gene. These stimulated cgl mutant cells transiently regain the ability to glide as individual cells.
Previously, we have reported the cloning and sequencing of cglB. CglB is a 416 aa protein and contains a signal sequence that is typical of lipoproteins.
Currently, we are interested in the localization and function of CglB. Western blot analysis showed that CglB was localized to the membrane fraction of M. xanthus. In experiments using a truncated promoter region of cglB, we observed that the intracellular level of CglB was lower than in wild type and that swarming of these mutant cells was significally reduced. Using a recombinant cglB gene that contains a His tag fusion to the C-terminus (cglB.His), we were able to complement the A-motility defect of cglB null mutants. However, M. xanthus strains carrying cglB.His as the only cglB allele were unable to extracellularly rescue single cell gliding of cglB null mutants. The function of CglB in single cell gliding will be discussed.
Abstract #7
A mutation in the gidA operon of Myxococcus xanthus affects Adventruous motility and a late stage of spore maturation.
David White and Patricia Hartzell
Department of Microbiology, Molecular Biology and Biochemistry,
University of Idaho, Moscow, ID 83844-3052
A final step in the developmental program of M. xanthus is the darkening of the fruiting body concurrent with the appearance of refractile, heat-resistant spores. MxH1273 is a mutant carrying an insertion of Tn5-lac that affects adventurous gliding. Because the mutant retains social gliding, it is able to form fruiting bodies when starved for nutrients. However, the mutant fails to produce mature spores. During development, cells of MxH1273 differentiate into spores that do not become refractile, fail to germinate after heat treatment and cannot be rescued by the additiong of wild-type cells during development. This mutant defines a new class of genes involved in spore maturation and motility.
Sequence analysis of the region disrupted by the Tn5-lac insertion reveals an open reading frame that is 43% identical to GidA of Bacillus subtilis. Northern analysis suggests that the gidA gene is in an operon with other genes including the gene disrupted by the Tn5-lac insertion. In E. coli and B. subtilis, gidA is near the origin of replication and is thought to be involved in controlling the initiation of replication. Upon vegetative passage of MxH1273 cells, a rearrangement occurs in which a portion of the gidA gene and the 5' region of Tn5-lac is lost. The resulting double mutant exhibits the same vegetative phenotype as the MxH1273 parent, but can no longer initiate development. Experiments are underway to determine if a gidA mutation results in the same phenotype as the double mutant.
Abstract #8
Cell movements, alignments and PilQ (SglA) are involved in Tgl contact stimulation in Myxococcus xanthus
Daniel Wall and Dale Kaiser
Departments of Biochemistry and Developmental Biology
Stanford University, Stanford, CA 94305.
Social gliding motility involves group movements of cells on a solid surface and is required for rippling, fruiting body formation and development in M. xanthus. Genetic studies have demonstrated that polar type IV pili are required for S-motility. A similar type of motility has been shown to involve type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae, establishing type IV pili as common component in some forms of gliding motility. In M. xanthus, tgl- cells lack S-motility and pili, but can be transiently stimulated for S-motility and pili production by contact with a tgl+ cell. The tgl gene encodes a lipoprotein which contains six tandem tetratricopeptide repeats (TPR), suggesting Tgl is involved in protein-protein interactions. Evidence is provided that Tgl contact stimulation is greatly enhanced by cell movements, cell alignment, and particularly by end-to-end contacts between cells. The pilQ (sglA) gene product, which belongs to the pIV/PulD outer membrane gated channel family, is characterized and shown to be required for Tgl contact stimulation. This result suggests that Tgl is associated with or can be transported through the putative PilQ gated channel.
Abstract #9
Alterations in the Myxococcus xanthus O-antigen
result in defective motility and A signal-independent 4521 developmental
gene expression
M. Gabriela Bowden* and Heidi B. Kaplan
Department of Microbiology and Molecular Genetics
The University of Texas Medical School, Houston, Texas 77030
Myxococcus xanthus is a gliding bacterium that aggregates to form spore-filled fruiting bodies when nutrients are limiting. We have isolated wzm wzt wbgA (formerly rfbABC) mutants that are defective in developmental aggregation and sporulate at 0.02% of the wild-type level.
The wzm wzt wbgA genes are required for the biosynthesis of LPS O-antigen. The wzm wzt wbgA mutants form glossy colonies that are about half the diameter of wild-type colonies when grown on nutrient agar. Genetic analysis indicates that the wzm wzt wbgA deletion mutant exhibits wild-type A motility. Initial transductants exhibit poor S motility that increases after subsequent plate transfer. Individual cell motility of wzm wzt wbgA deletion mutant cells spotted on nutrient agar is different than the motility of wild-type cells. The wzm wzt wbgA mutant exhibits a wild-type pattern of cellular reversals, at about one reversal every 8 inutes. However, cellular translocation is decreased to about 25% of the wild-type velocity. Wild-type cells glide on average at 1.5 µm/min, whereas the mutant cells glide on average at 0.4 µm/min. The O-antigen defective cells appear to deposit significantly less slime on the agar surface than the wild-type cells. It is possible that released wild-type polysaccharides create a hydrated film that facilitates translocation.
The wzm wzt wbgA mutations confer A signal-independent expression of the 4521 reporter gene. All of the O-antigen mutants tested have this same effect on 4521 expression. In addition, all of the O-antigen mutants overexpress 4521 in wild-type and other backgrounds. To
determine if adding M. xanthus polysaccharides extracellularly could reduce 4521 overexpression to its parental level, polysaccharides extracted from vegetative and developing cells were added to starving wzm wzt wbgA asgB480 cells. Addition of polysaccharides purified from developing cells caused 4521 expression to be reduced to the asgB480 parental level. In contrast, addition of polysaccharides purified from growing cells did not affect 4521 gene expression. The mechanism of this extracellular polysaccharide control of 4521 gene expression is unclear, but it is likely to function through the SasS sensor kinase (formerly
HpkA), which is epistatic to wzm wzt wbgA.
Abstract #12
Chemotaxis in Myxococcus xanthus
Daniel B. Kearns and Lawrence J. Shimkets
Department of Microbiology
University of Georgia, Athens, 30601
It was originally noted that when starving M. xanthus cells were separated from a field of preformed fruiting bodies by a semi-permeable membrane, the nascent and preformed fruiting bodies would become superimposed suggesting that the preformed structures secreted a morphogenic determinant with chemotactic properties1. The role of chemotaxis in fruiting body development was later challenged when an exhaustive survey of biological compounds failed to identify any positive chemoattractants2. Furthermore, the diffusion rate of small, soluble molecules is high enough that the use of such compounds as attractants would be of limited usefulness to these slow moving bacteria.
We have isolated a cell-associated compound which has a dramatic effect on M. xanthus motility. When extracts containing this compound were added to a filter disk atop a lawn of wild type M. xanthus DK1622 cells, thick radial ridges containing cells that seemed to migrate towards the disk were observed. The bioactive fraction was purified from a M. xanthus DK1622 cell pellet via sequential chloroform:methanol extraction 3, silica gel G adsorption chromatography4, and thin layer chromatography (TLC)5. The compound was identified as phosphatidylethanolamine (PE), the predominant phospholipid produced by M. xanthus6, based upon its polarity during adsorption chromatography, its Rf value during TLC, and its reactivity with ninhydrin.
Phospholipid gradients were demonstrated on agar surfaces using fluorescently labeled phosphatidylethanolamine (Molecular Probes). The phospholipid diffused at a rate of 11 mm/min, a rate comparable to that of M. xanthus motility. Cells in the presence of PE gradients purified from vegetative M. xanthus cells preferentially migrated up the phospholipid gradient and PE purified from developing cells induced a similar motility bias but at a 10-fold lower concentration.
Cellular reversal periods were determined using time lapse video microscopy in the presence of PE. M. xanthus cells normally reverse direction once every 6.8 minutes but reversal period increased with increasing amounts of vegetative PE, reached a peak activity, and dropped at higher concentrations. A similar dose response curve was observed with developmental PE but the peak activity occurred at a 10-fold lower PE concentration. Chemokinesis could not account for directed movement because individual M. xanthus cells moved 25% slower in the presence of PE. Adaptation was not observed to PE as cells maintained the same reversal period over each quarter of the 45 minute time lapse video.
We analyzed M. xanthus reversal periods in response to six different synthetic phosphatidylethanolamine species: dilauroyl PE (diC12:0), dimyristoyl PE (diC14:0), dipalmitoyl PE (diC16:0), deheptadecanoyl PE (diC17:0), distearoyl PE (diC18:0), and dioleoyl PE (diC18:1 w9c). Only dilauroyl and dioleoyl PE increased M. xanthus reversal period and induced directed movement up a concentration gradient.
Fatty acid analysis of PE purified from vegetative and developmental cells suggested that neither dilauroyl nor dioleoyl PE was responsible for the activity in the purified fractions: in both cases, dilauroyl PE was absent andthe dioleoyl PE concentration was not sufficiently high to account for the magnitude of the effect. An as yet unidentified PE species is presumed to be responsible for the activity found in the extracts.
PE purified from E. coli was also capable of directing M. xanthus motility and suppressing direction reversals.
1. Lev, M. Nature 173, 501 (1954).
2. Dworkin, M., & Eide, D. J. Bact. 154, 437-442 (1983).
3. Bligh, E. G., & Dyer, W. J. Can. J. Biochem. Physiol. 37, 911-917 (1959).
4. Hamilton, J. G., & Comai, K. Lipids 23, 1146-1149 (1988).
5. Yamanaka, S., Fudo, R., Kawaguchi, A., & Komogata, K. J. Gen. Appl. Microbiol. 34, 57-66 (1988).
6. Orndorff, P., & Dworkin, M. J. Bact. 141, 914-927 (1980).
Abstract #13
Genetic analysis of Cytophaga johnsonae gliding motility
Mark J. McBride
Department of Biological Sciences
The University of Wisconsin-Milwaukee.
Gliding motility is a trait shared by numerous bacteria belonging to different branches of the eubacterial phylogenetic tree. The mechanism(s) responsible for gliding movement are not known. Cytophaga johnsonae is a member of the Cytophaga-Flavobacterium-Bacteroides branch of the eubacterial phylogenetic tree that exhibits rapid gliding motility. Techniques to genetically manipulate C. johnsonae have recently been developed. Here we report the use of these techniques to clone and analyze genes that are required for C. johnsonae gliding motility.
We have taken two independent approaches to identify genes that are required for gliding motility. In the first approach we attempted to identify gliding motility genes by complementation of nonmotile mutants that were previously isolated by Pate and colleagues. We prepared cosmid libraries of wild-type C. johnsonae DNA in the shuttle cosmids pCP17 and pCP26 and identified several cosmids that complemented some of the completely nonmotile mutants. pCP100 complemented 4 of 61 mutants, and pCP200 complemented 4 different mutants. Subcloning of pCP100 identified one gene (gldA) that was required for complementation. Disruption of the chromosomal copy of gldA in wild-type cells eliminated gliding motility. GldA is similar to ATP binding cassette (ABC) transport proteins. Subcloning of pCP200 identified one gene that is required for gliding motility (gldB) and one gene that appears to be involved in gliding motility, but is not absolutely required (gldC). GldB is not similar to sequences in the databases, but may be a membrane protein. GldC exhibits some similarity to fungal lectins. The second approach to identify gliding motility genes involved the isolation of Tn4351 induced completely nonmotile mutants. We have isolated a number of mutants, and have cloned the disrupted genes from nine of these. Some of the insertions fall within gldA or gldB, while others identify new genes that we are currently analyzing.
Abstract # 18
The spdRS operon
Hubert M. Tse* and Ron Gill
Department of Microbiology, University of Colorado Health Sciences Center
Denver, CO
The Gill lab has been characterizing the substrate(s) of the BsgA intracellular protease by identifying genetic suppressor mutants of the protease defect. Earlier work demonstrated that the bsgA gene product is required for early developmental gene transcription and its DNA sequencedisplayed homology to the Lon protease of E. coli. Mutants of the BsgA protease are affected quite early in the developmental pathway and are unable to produce fruiting bodies or sporulate under normal starvation conditions.
We initially hypothesized that the BsgA protease may be involved in degrading a transcriptional repressor and thereby allow expression of early developmental genes necessary for entry into the developmental pathway. One may assume that during vegetative growth, the repressor is bound to developmental genes and transcription is inhibited. Once development has initiated, the BsgA protease may degrade the repressor and allow for early developmental gene transcription.
In order to identify this substrate/repressor, we have identified suppressor mutants of a bsgA- strain by performing UV and Tn5 mutagenesis. Many suppressor mutants were identified by their ability to restore the developmental phenotype after selecting on starvation media. One particular mutant (M575) was further characterized and shown to contain a mutation in a locus (spdR - Suppressor of Protease Defect) having DNA sequence similarity to the ntrC family of two component system response regulators.
Mutations in spdR bypassed the developmental defect of a bsgA mutation, suggesting that spdR acts negatively with respect to development either as a transcriptional repressor or as an activator of a transcriptional repressor. The possibility that SpdR is a substrate of the BsgA protease was explored by performing Western blot analysis with SpdR polyclonal serum directed against various M. xanthus vegetative and developmental cell extracts (WT, bsgA-, bsgA-, spdR-). Based on equivalent SpdR protein levels in the WT and bsgA- backgrounds during both vegetative and developmental growth, it does not appear that SpdR is a substrate of the BsgA protease.
The potential cognate sensor kinase (spdS) of SpdR has been identified by DNA sequence analysis and displays high homology to various two component sensor kinases from an assortment of bacteria. The DNA sequence also suggests that spdS and spdR are transcribed as an operon and translationally coupled. An in-frame knockout of spdS is in the process of being constructed and will be discussed at a later date.
The prototypical two component system response regulator is usually involved in transcriptional activation. We have identified a novel response regulator that appears to function by transcriptional repression. Mutants of spdR display an increase in developmental gene transcription when grown in rich media, suggesting that SpdR functions as a transcriptional repressor either directly or indirectly. In order to identify gene(s) that the SpdS,R two component system regulates, we have constructed a M. xanthus strain that is able to induce expression of spdR. By using this strain in conjunction with a promoter probe fusion such as Tn5 - lacZ, we can identify other genes involved in the bsgA and spdS,R pathway. In the near future, we will fully characterize the relationship between the SpdS,R two component regulatory system and the BsgA protease with respect to their roles during early transcriptional events in the M. xanthus developmental lifecycle.
Abstract #19
Cloning of W530, a gene that requires functional asg genes, but not
A-signal, for proper developmental expression in Myxococcus xanthus.
John M. Davis and Lynda Plamann. Department of Biology, Texas A&M University
College Station, TX 77843
Early development in Myxococcus requires the production of extracellular A-signal, which has been identified as a combination of amino acids and small peptides. The A-signal is thought to act as a cell density signal during development. The three asg (A-signal generating) mutants fail to produce A-signal and are blocked early in development. AsgA is a histidine protein kinase, AsgB is a putative DNA-binding protein, and AsgC is the major sigma factor of M. xanthus. Because all three asg genes encode regulatory proteins, we hypothesize that the asg gene products act together in a signal transduction pathway that leads to A-signal production. Tn5-lac promoter probes have previously
identified developmentally regulated genes. Some of these gene fusions were found to require functional asg genes for expression. Bowden and Kaplan (J. Bacteriol., 178: 6628-6631) proposed that the asg-dependent gene fusions can be subdivided into two groups: 1) those that are part of the A-signal sensing pathway, and 2) those that are part of the A-signal generating pathway and are most likely targets for regulation by the Asg proteins. W4530 is a Tn5 lacZ fusion that is expressed at 1-2 hours after initiation of starvation and requires wild-type asg genes for expression but does not require extracellular A-signal.
Therefore, W4530 may be a component of the A-signal generating pathway. We will show the expression of W4530 in the different asg mutant backgrounds, as well as the cloning of this gene. Besides starvation-induced expression, W4530 was previously found to be expressed during glycerol sporulation. We have examined expression of W4530 during glycerol sporulation in wild-type and the three asg mutant backgrounds. W4530 may be a potential target of regulation by the asg gene products and characterization of this insertion may provide a useful tool for understanding regulation by the Asg proteins.
Abstract #20
Genetic suppression analysis of an asgA missense mutant
Valerie Kessler, Cathy Eylem, Andrea Tews, and Lynda Plamann
Department of Biology, Texas A&M University, College Station, TX 77843
Developing Myxococcus xanthus cells undergo a complex series of events involving intercellular signaling that culminates in the production of a spore-filled fruiting body. A-signal, necessary for early development, has been identified as a combination of extracellular amino acids and short peptides. Three genes have been identified that are thought to encode regulatory components in the A-signal production pathway. Mutations in any of these three genes caused dramatically reduced production of extracellular A-signal. One of the three genes, asgA, encodes a protein with similarity to both histidine kinases and response regulators found in two-component signal transduction systems. To identify other components in the A-signal production pathway, we have UV-mutagenized an asgA missense mutant and selected for pseudorevertants capable of producing heat and sonication-resistant myxospores. Forty-two independent pseudorevertants have been isolated. Presumably, mutations could be of three classes: bypass, allele-specific interaction, or intragenic reversion. To determine which pseudorevertants contain bypass mutations, we replaced asgA473 with a "knockout" asgA containing the gene for kanamycin resistance inserted in the middle of the sequence by electroporating linear DNA into each pseudorevertant. Strains containing a suppressing mutation that bypasses the need for AsgA should still be able to develop after such a replacement, whereas strains containing either a reversion or an allele-specific interaction suppressing mutation would not be able to develop. To distinguish between interaction suppressing mutations and reversions, we are checking linkage of the suppressing mutation to asgA473 using Mx8 phage grown on a strain containing Tn5 that is 80% linked to asgA473. When an intragenic revertant is transduced with this phage, 80% of the kanamycin-resistant transductants will fail to develop because they have regained the asgA473 mutation. A pseudorevertant containing an interaction suppressing mutation will give rise only to fruiting competent kanamycin-resistant transductants. Of the 25 pseudorevertants that have been tested, 20 appear to contain bypass mutations, and 5 appear to contain allele-specific interaction suppressing mutations. Assays to determine if pseudorevertants produce extracellular A-signal are currently in progress, as are direct and viable spore counts of the pseudorevertants.
Abstract #21
A histidine protein kinase and a response regulator control early
Myxococcus xanthus developmental gene expression
Chun Yang* and Heidi Kaplan
Department of Microbiology and Molecular Genetics
UT- Medical School, Houston, TX 77030
Progression through Myxococcus xanthus early development requires starvation and A signal (1). A suppressor mutation in the M. xanthus sasB locus that permits expression of the 4521 gene independent of the starvation and A signal requirement (2) has been characterized. This point
mutation maps to the sasS (formerly hpkA) gene and has been determined to be a gain-of-function mutation. The deduced amino acid sequence reveals that SasS is a member of the histidine protein kinase family that is predicted to be located in the cytoplasmic membrane. A sasS null mutation in an otherwise wild-type background results in very low 4521 expression
during growth and development. The sasS null mutant is defective in
fruiting body formation and sporulation. SasS has been overexpressed and
purified as a MalE-SasS fusion protein from E. coli. A NtrC-like response
regulator, sasR (formerly mrrA), mapping 2.2-kb downstream of sasS in the
sasB locus has also been identified. A sasR null mutation in an otherwise
wild-type background results in very low 4521 expression during growth and
development. Since the 4521 promoter is a member of the sigma-54 family
(3), it is possible that SasS and SasR form a two-component signal
transduction system that regulates 4521 expression. Epistasis experiments
support this idea. SasR has been overexpressed and purified as a MalE-SasR
fusion protein from E. coli. We are currently testing if SasR directly
regulates 4521 expression.
References:
1. Kuspa, A., L. Plamann, and D. Kaiser. 1992. A-signaling and the
cell-density requirement for Myxococcus xanthus development. J. Bacteriol.
174: 7360-7369.
2. Kaplan, H. B., A. Kuspa, and D. Kaiser. 1991. Suppressors that permit
A-signal-independent developmental gene expression in Myxococcus xanthus.
J. Bacteriol. 173: 1460-1470.
3. Keseler, I. M. and D. Kaiser. 1995. An early A-signal-dependent gene in
Myxococcus xanthus has a s54-like promoter. J. Bacteriol. 177: 4638-4644.
Abstract #22
Molecular and genetic analysis of the Myxococcus xanthus sasB5 gene
Di Xu*, Chun Yang and Heidi B. Kaplan
Department of Microbiology and Molecular Genetics
University of Texas Medical School at Houston, Houston, TX 77030
Initiation of Myxococcus xanthus multicellular development requires
integration of information concerning the cell's nutrient status and cell
density. Five clustered suppressor mutations (sasB5, 14-17) have been
isolated that bypass both the starvation and high cell density requirements
for expression of the 4521 reporter gene. A 22 kb region of the M. xanthus
chromosome containing the sasB5 locus was cloned using a marker linked to
these mutations. Subclones of this region were used to map the mutations
to a 2 kb region. This region was sequenced and is predicted to contain one
open reading frame encoding a protein of 363 amino acids. This putative
protein has a strong transmembrane domain and a leucine-zipper motif in the
N-terminus, and a proline-rich region at the C-terminus (21 prolines among
129 amino acids). Comparison of the sequence with the available databases
does not reveal any homologues. The five mutant alleles were cloned and
sequenced. Four of them (sasB5, 14,15 17) code for an identical threonine
to proline substitution at residue 280; one (sasB16) codes for a nonsense
codon at residue 47. A sasB5 null strain has been constructed in a
wild-type background by replacing an internal fragment of sasB5 with a
kanamycin resistant gene. The expression of 4521 in this strain is very
high during growth and development, suggesting that SasB5 functions as a
negative regulator of 4521 expression. Epistasis experiments indicate that
SasB5 functions upstream of a histidine protein kinase (SasS, formerly
HpkA) in the A signaling pathway. The null mutation of sasB5 in a
wild-type background causes abnormal fruiting body morphology and a
sporulation deficiency (10% of the wild-type level). The function of SasB5 in the A signal transduction pathway is under investigation.
Abstract #23
Cloning and analysis of LPS O-antigen suppressors
Dongchuan Guo*, Yun Wu and Heidi B. Kaplan
Department of Microbiology and Molecular Genetics
University of Texas Medical School, Houston, Texas 77030
Mutations that block Myxococcus xanthus LPS O-antigen biosynthesis cause A
signal-independent 4521 developmental expression. To study the connection
between 4521 expression and O-antigen biosynthesis, LPS O-antigen suppressors were obtained by transposon mutagenesis. Eleven mutants were isolated that abolish or reduce 4521 developmental expression in a DK6621 (wztA1 asgB480 sglA1 ¦4521 Tn5 lac[Tcr]) background. The effect of these
mutations on 4521 expression suggests that the wild-type genes identified
by these mutations encode proteins that promote early developmental gene
expression. All of the TnV insertions have been located on the M. xanthus
chromosomal physical map. Eight of these mutation map to the sasB locus.
The los1 TnV insertion mutation maps to the sasS (formerly hpkA) gene of
the sasB locus. The los2, los3 and los4 TnV insertion mutations map to the
sasR (formerly mrrA) gene of the sasB locus located 2.2 kb downstream of
sasS. The sasS and sasR genes appear to belong to a two component signal
transduction system that positively regulates 4521 expression. The los5,
los6, los7 and los8 TnV insertions all map to one open reading frame (ORF)
directly downstream of the negative regulator of 4521 expression, sasB5,
which itself maps directly downstream of sasR. This ORF appears to encode
a positive regulator of 4521 expression because los5, los6, los7 and los8
abolish 4521 expression in an otherwise wild-type background.
Interestingly, these los5, los6, los7 and los8 mutants are not
distinguishable from wild type in their growth and development. The los9
TnV insertion mutation maps to a putative ORF about 15 kb away from the
4521 gene. The insertion in this ORF abolishes 4521 expression both in
wild type and sasB7 (a gain-of-function sasS point mutation) backgrounds
suggesting that this protein functions downstream of sasS. The los9
mutation in an otherwise wild-type background confers defective early
developmental aggregation and fruiting body formation. Sporulation of this
mutant is 1% of the wild-type level. This los9 mutant grew more slowly
than wild type in nutrient medium. The predicted amino acid sequence of
this ORF does not show homology to any known protein sequence in the
databases. The los10 TnV inserted in an ORF that is 53% identical to
Arabidopsis thaliana dTDP-glucose 4,6-dehydratase. The Salmonella
typhimirium equivalent of this enzyme is required for LPS biosynthesis.
Immuno-slot analysis showed that the los10 mutant does not react to an
anti-LPS-core monoclonal antibody. The los10 mutation in an otherwise
wild-type background confers defective early developmental aggregation and
fruiting body formation. In this los10 mutant sporulation is greatly
reduced (less than 0.1% of the wild-type level). The los11 TnV insertion
mutation maps to an ORF that is 41% identical to a Rhodobacter capsulatus
periplasmic thioredoxin-like protein, HelX, proposed to maintain heme in
the reduced state required for cytochrome c biogenesis. The los11 mutant
grows more slowly than wild type and sporulates poorly (less than 0.1% of
the wild-type level). We anticipate that future studies of these mutants
will help to clarify the LPS-dependent regulation of 4521 gene expression.
Abstract #24
Genes involved in fruiting body formation of Stigmatella aurantiaca
Barbara Silakowski, Heidi Ehret and Hans Ulrich Schairer
ZMBH, Zentrum fur Molekulare Biologie der Universitat Heidelberg
Im Neuenheimer Feld 282, D-69120 Heidelberg
For studying fruiting body formation in Stigmatella aurantiaca, mutants impaired in fruiting have been induced. One of the genes, fbfA, whose inactivation leads to a defect in fruiting body formation, has been described recently (Silakowski et al., 1996, J. Bacteriol. 178, 6706-6713). It encodes a putative polypeptide with homology to chitin synthases such as NodC from Rhizobia. Fruiting body formation of fbfA mutants is partially restored by mixing the cells of this mutants with cells of some other mutants unable to form fruiting bodies before starvation. 152 bp upstream of the coding region of fbfA an open reading frame, fbfB, on the other DNA strand encoding a putative polypeptide with a homology to the galactose oxidase from the fungus Dactylium dendroides was detected. Inactivation of fbfB leads to a defect in fruiting which is also partially suppressed by phenotypic complementation. FbfB is expressed about 14 hours after the beginning of starvation as shown by Northern analysis and b-galactosidase activity determination using a merodiploid strain with an indicator gene.
Sequencing of the region around the above genes resulted in the detection of three further open reading frames: hesA, pksA and sspA, which encode putative polypeptides with similarity to small peptides involved in secretion of E. coli, Anabaena and Bacillus sp., polyketide synthases of different organisms, and synapsin of the human brain, respectively. The analyse of these open reading frames and the determination of their function in development is in progress.
Abstract #26
The ClassII protein, a key-protein in the C-factor signal transduction pathway, is a response regulator with a C-terminal DNA binding domain and may act in a C-factor dependent phosphorelay system.
Eva Ellehauge, Sune Lobedanz & Lotte Søgaard-Andersen,
Dept. of Molecular Biology, Univ. Odense, Campusvej 55, 5230 Odense, Denmark.
C-factor, a 17 kD cell-surface-associated intercellular signaling protein, is required for aggregation, rippling, sporulation and developmentally regulated gene expression during fruiting body formation in M. xanthus. The Class II protein has a central role in the C-factor signal transduction pathway and is required for multiple events including transduction of the C-factor signaling event on the cell surface to the cytoplasmic Frz proteins to alter the motility pattern of starved cells and C-factor dependent expression of devRS which is essential for sporulation.
Here, we have cloned the classII gene. The ClassII protein is identical to the FruA protein (Ogawa et al., Mol. Microbiol. 22, 757-767) and is from now on referred to as FruA. Homology analyses show that FruA is a member of the DegU/NarL/UhpA family of DNA binding response regulators. FruA contains an N-terminal receiver domain and a C-terminal DNA binding domain. In the receiver domain FruA contains a putative phosphorylation site, Asp59. Interestingly, immediately downstream from Asp59 eight additional amino acid residues are present which are not found in other
receiver domains. To test whether phosphorylation of Asp59 is required for FruA activity, Asp59 was changed to Ala and Glu by site directed mutagenesis. The fruAAsp59Ala allele did not complement a fruA knock-out mutant whereas the fruAAsp59Glu allele could complement the fruA knock-out mutant. These data suggest that phosphorylation at Asp59 is important for FruA activity. We propose that FruA acts in a C-factor dependent phosphorelay signal transduction system to control C-factor dependent events during development.
Abstract #27
fruA, an essential locus in the C-factor signaling pathway, may serve to coordinate early developmental signals and C-factor signaling during fruiting body morphogenesis in M. xanthus.
Lotte Søgaard-Andersen, Peter Søholt, Eva Ellehauge & Anders Boysen.
Dept. of Molecular Biology, Univ. Odense, Campusvej 55, 5230 Odense, Denmark.
In the model for C-factor signal transduction pathway the signaling event is the interaction between C-factor on one cell with a C-factor sensor on a different cell. This interaction results in the activation of fruA (formerly classII) which is required for all C-factor dependent responses. Downstream of fruA, the pathway branches: one branch leads to the motility response to C-factor; the Frz proteins are components in this pathway. The second branch leads to the sporulation response and C-factor dependent gene expression. Here, the essential sporulation locus devRS has been identified as coding for components in the sporulation/gene expression pathway. Aggregation and rippling by the devRS mutant is similar to that of wildtype. However, the sporulation efficiency of the devRS mutant is strongly reduced. Epistasis experiments indicate that devRS is positioned downstream of fruA in the C-signaling pathway. It has previously been shown that expression of devRS is C-factor dependent. Using a devRS-lacZ fusion (devR::Tn5 lacW4414) to monitor devRS expression, we find that FruA is required for C-factor dependent activation of devRS.
To analyse how C-factor interacts with fruA, expression of fruA was measured using a transcriptional fruA-lacZ fusion. Expression of the fusion initiated after approximately 6 hrs. of starvation and was independent of C-factor. Ogawa et al. (Mol. Microbiol. 22, 757-767) have previouly shown that expression of a fruA-lacZ fusion depends on asgA, asgB and esg. fruA is positioned downstream of C-factor in the C-factor signal transduction pathway and FruA is, most likely, a response regulator. We suggest that there are two levels of control of fruA: the early acting A- and E- signals are required for transcriptional activation of fruA after 6 hrs. of starvation and the late acting C-signal acts post-translationally to activate FruA protein. We suggest that the C-factor dependent modification of FruA is phosphorylation of D59 in the receiver domain in FruA. Models will be discussed to explain how these two levels of control may contribute to coordinate early and late developmental events.
Abstract #28
Analysis of the devTRS operon from M. xanthus
Bryan Julien* and Dale Kaiser
Department of Biochemistry, Stanford University, Stanford, CA. 94305-5307
The devR locus was identified by two Tn5 lac insertions, W4414 and W4473. These insertions decrease sporulation efficiencies by a factor of 100 to 1000. Analysis of the regulation of the devTRS operon demonstrated that expression begins at approximately 4 hours after the onset of starvation and that it is highly regulated (1). Transcription of this operon requires the A and C signals. It is negatively autoregulated and expression is affected by cell density. Also, during development, the operon is only expressed in a subpopulation of cells (2). Preliminary evidence indicates that devTRS is expressed only in cells that have entered into an aggregation center. Thus, it appears that this operon is regulated spatially as well as temporally.
To further characterize this operon, we have worked on completing its sequence. To date, 6611 bp have been sequenced and a total of five open reading frames (orfs) have been identified (orf-1, orf-2, devT, devR, and devS). Analysis of these open reading frames reveals little homology
to known proteins.
We have currently constructed in-frame deletions in devT, devR, and devS and have analyzed their effects on development. The devT mutation causes a delay in aggregation and sporulation by at least 24 hours. After three days of development, no viable spores are produced, but after five days, spores are produced at 2% wild type levels. The devR mutant aggregates normally, but the fruiting bodies do not completely darken. However, there is only a 50% reduction in the number of spores produced after three and five days. The devS mutant also aggregates normally but the fruiting bodies do not darken. The percentage of viable spores after three and five days is about 4% of wild type.
We have also measured the amount of devTRS RNA produced in these mutant strains in order to determine which gene product(s) are responsible for the negative autoregulation. Preliminary evidence indicates that DevS is negatively regulating the operon.
We are currently trying to define the promoter region and have determined that sequences further upstream are required for expression of this operon. Sequencing of this region is underway.
1. Thony-Meyer, L. and D. Kaiser. 1993. J. Bacteriol. 175:7450-7462.
2. Russo-Marie et al. 1993. Proc. Natl. Acad. Sci. USA 90:8194-8198.
Abstract #29
Characterization of the regulatory regions of C-signal-dependent genes of Myxococcus xanthus
Lee Kroos, Janine Brandner, Dvora Biran, Makda Fisseha, and Tong Hao
Department of Biochemistry, Michigan State University, East Lansing, MI
To determine how C-signaling regulates gene expression during M. xanthus development, we have been characterizing the regulatory regions of several C-signal-dependent genes that were originally identified by insertions of Tn5 lac. Fisseha et al. (1996) [J. Bacteriol. 178:2539-2550] characterized the 4403 promoter region. DNA between -80 and -72 was essential for promoter activity, suggesting that a transcriptional activator might bind to this region. Using DNA spanning from -105 to +53 as a probe in mobility shift assays, a complex was observed by 4 h with extracts of developing cells, but not with extracts from growing cells. However, the binding was not specific for the -80 to -72 region and it did not depend on C-signaling. Moreover, the promoter regions of several other genes, including some expressed during growth, were shifed by extracts from developing cells, but not by extracts from growing cells. We conclude that assays demanding greater specificity of binding are needed to identify the relevant transcription factors using this approach.
We have characterized the promoter region of a second C-signal-dependent gene, 4400, and it shows some striking similarities to the 4403 promoter region. DNA between -101 and -76 is essential for promoter activity. However, inspection of this region reveals no obvious sequence similarity to the -80 to -72 region of the 4403 promoter. On the other hand, the two promoters share the sequence CATCCCT centered at -49 and the sequence TACA-C in the -10 regions. Thus, the two promoters may be recognized by some of the same transcription factors that bind proximally, but may require specific regulators that bind farther upstream. A csgA mutation reduces expression of Tn5 lac 4400 or the deletion to -101 about twofold. Expression of Tn5 lac 4400, as well as expression of Tn5 lacs 4403, 4499 (see below), and 4459, was unaffected by mutations in sigB or sigC, which encode development-specific sigma factors [Apelian and Inouye (1990) Genes Dev. 4:1396-1403; Apelian and Inouye (1993) J. Bacteriol. 175:3335-3342].
The promoter region of a third C-signal-dependent gene, 4499, is somewhat different. DNA between -218 and -49 is required for full developmental promoter activity, but the deletion to -49 retains about 30% as much activity as observed for Tn5 lac 4499, and the timing of expression during development is similar. In addition, expression from the -49 deletion is C-signal-dependent since expression is abolished in csgA mutant cells but restored upon co-development with wild-type cells. Interestingly, two sequences in the 4499 promoter region resemble the sequence centered at -49 in the 4403 and 4400 promoters. One, CATTCCT, is centered at -33 and the other, CATTCCCT, is centered at -54.5. The importance of this sequence for regulation of C-signal-dependent genes can be tested by mutational analyses.
Abstract #30
A developmental checkpoint for C-Signaling
Eugene W. Crawford Jr.* and Lawrence J. Shimkets
Department of Microbiology, University of Georgia, Athens GA 30602-2605
In developmental systems checkpoints exist to prevent premature and/or inappropriate transitions between the growth, division or differentiation phases of a cell's life cycle. The concept of developmental checkpoints is well established in eukaryotic systems where mechanisms exist to prevent entry into mitosis before the completion of DNA replication (Murray, 1992). Evidence will be presented for a new developmental checkpoint mediated by socE which acts in concert with C-signaling.
Myxococcus xanthus development is coordinated by a series of extracellular signals. At least two of these signals, the A-signal and the C-signal, act as checkpoints for the continuation of the developmental process. A-signaling is initiated by starvation and acts as a quorum sensing system very early in development (Kuspa et al., 1992). Specifically, if cell density is not high enough to support fruiting body formation, A-signal production will not reach the critical threshold required to initiate development. The C-signal is a cell-associated signal which is used to determine cell density through a tactile mechanism (reviewed recently in Dworkin, 1996). If cell density and cell alignment are too low to support efficient C-signaling, aggregation and fruiting body formation do not occur.
To elucidate the C-signaling pathway and its regulation, a series of mutants isolated by Rhie & Shimkets (1989) which bypass the developmental requirement of a functional csgA gene. One locus isolated by Rhie & Shimkets (1989) as a Tn5 insertion (socE-537) has been cloned and sequenced. Disruption of the socE gene in a csgA background does not restore rippling or the ability to extracellularly complement other csgA mutants, but does restore wild-type levels of fruiting body formation and sporulation. This mutant phenotype argues against a model for aggregation in which CsgA is both the C-signal and the sole means of directed movement during fruiting body development (Sogaard-Andersen and Kaiser, 1996). SocE encodes a putative 53.6 kDa protein that has no significant homologies to any protein currently in Genbank. socE is expressed at a constant level during vegetative growth and expression decreases early in development. This decrease in socE expression does not depend on a functional socE or csgA gene.
Elimination of the socE is lethal in a csgA+ background. When socE is expressed ectopically under the control of the light-inducible promoter, pcarQRS, in liquid (CYE) culture, growth stops approximately 2 generations (12 hours) after expression ceases (i.e. the light is turned off). This growth inhibition is accompanied by a concomitant halt in DNA and stable RNA synthesis. Seven days after ectopic socE expression is eliminated >99% of all cells in the culture become spherical, light-refractile spores which are ultrustructurally indistinguishable from fruiting body spores though
unable to germinate. Sporulation is not accelerated by conditioned media, implying that there is not a slow build-up of an extracellular signal. Restoration of socE expression, prior to sporulation, results in a resumption of vegetative growth.
Our tentative model for socE function has two feedback loops working in opposition to each other. One loop involves a putative starvation signal which stimulates C-signaling and responds to C-signaling by inducing fruiting body morphogenesis. This loop also inhibits growth by stimulating production of the starvation signal. The second feedback loop consists of socE attenuating the starvation signal in an attempt to maintain vegetative growth.
Dworkin, M. 1996. Microbiol. Rev. 60: 70-102.
Kuspa, A., Plamann, L. and D. Kaiser. 1992. J. Bacteriol. 174: 7360-7369.
Murray, A.W. 1992. Nature. 359:599-604.
Rhie, H.G. and L.J. Shimkets. 1989. J Bacteriol. 171: 3268-3276.
Søgaard-Andersen, L., and D. Kaiser. 1996. PNAS. 93: 2675-2679.
Abstract #31
Rescue of aggregation and sporulation in Bsg, Csg, and Dsg mutants
Kathleen A. O'Connor and David R. Zusman
Department of Molecular and Cell Biology
University of California
Berkeley, CA 94720-3204
Shimkets and Kaiser reported that aggregation and sporulation of CsgA mutants could be rescued by the addition of a mixture of peptidoglycan components to developmental medium. Last year, we reported that the CsgA mutant could also be rescued by the addition of Ampicillin to developmental medium. Rescue of CsgA by peptidoglycan components and by Ampicillin, which disrupts assembly of peptidoglycan, may occur by a single mechanism. The details of this mechanism are not yet known.
Can Ampicillin rescue the development of other mutants or is the rescue specific to strains with defects in the csgA gene? Development in the presence of Ampicillin of strains carrying mutations in genes other than csgA was examined. We observed that BsgA and Dsg mutants aggregated and sporulated in the presence of Ampicillin under the appropriate conditions (the strains did not aggregate or sporulate on control medium lacking Ampicillin). The development of AsgA, AsgB, or AsgC mutants was not fully rescued in the presence of Ampicillin. Frz mutants were not rescued by Ampicillin.
These results show that rescue of development by Ampicillin is not restricted to CsgA mutants: BsgA and Dsg mutants can also be rescued. It is likely that BsgA, CsgA, and Dsg mutants are all rescued by the same mechanism. It is possible that the disruption of peptidoglycan by Ampicillin mimics a natural step in development which occurs after the points at which BsgA, CsgA, and Dsg mutants are blocked. If these functions are needed only at the initial stages of development, then mimicking a later step in development may allow the mutants to continue development from the later step.
Abstract #32
Genetic analysis of temperate Myxococcus xanthus phage Mx8 int and xis: evidence for a prokaryotic origin of the protamines
Vincent Magrini, Daniel Salmi, and Philip Youderian
Department of Microbiology, Molecular Biology and Biochemistry
University of Idaho, Moscow, ID 83844-3052
Temperate phage Mx8 of Myxoccocus xanthus encodes both trans-acting and cis-acting elements required for site-specific integration into the M. xanthus chromosome. Sequence analysis shows six potential int translational starts, the first of which, GTG-5085, is used for translation initiation. Whereas a change of GTG-5085 to TAG (amber) abolishes int function, a change of the next potential start codon, GTG-5208, to GCG (Val42 to Ala) reduces int function less than two-fold. The predicted sequence of the N-terminus of Int is extremely arginine-rich, and is similar to that of sperm protamines. Surprisingly, many M. xanthus phage- and host-encoded DNA-binding proteins also appear to have "basic tail" motifs similar in sequence to protamines or histones.
Overlapping and immediately upstream of int is a second open reading frame with 12 potential start codons. The predicted product of one of these, designated xis, shows similarity with the Salmonella phage P22 xis and Methylobacterium dcmR (dichloromethane-responsive) repressor genes. The predicted sequence of Mx8 Xis begins with a striking helix-turn-helix motif. Many elements of this motif are conserved among both phage and plasmid excisionases. Indeed, the secondary structural analysis of the N-terminus of phage lambda Xis suggests that this excisionase also has an N-terminal helix-loop-helix DNA-binding motif.
We have located the int promoter within a 200 bp region upstream of GTG-5085, and are using b-galactosidase fusions to assign unambiguous translational starts to the int and xis genes by an independent, biochemical approach, using Western blot analysis of Int/LacZ and Xis/LacZ fusion products expressed in M. xanthus.
Abstract # 33
Genetic analysis of temperate Myxococcus xanthus phage Mx8 immunity: the primary repressor gene, imm, encodes a histone H1-like product.
Daniel Salmi, Vincent Magrini, and Philip Youderian
Department of Microbiology, Molecular Biology and Biochemistry
University of Idaho, Moscow, ID 83844-3052
The temperate bacteriophage Mx8 imm gene encodes a product necessary for superinfection immunity; the predicted sequence of this product is similar to that of sea urchin histone H1. The level of superinfection immunity conferred by the Imm repressor is dependent upon the concentration of Imm within the cell. Thus, lysogens with tandem prophages are more resistant to superinfection than are lysogens with a single integrated prophage. When the imm gene is fused to the mglBA or aph promoters, and introduced into DZ1, these operon fusions confer different levels of superinfection immunity. When imm is placed under the control of the glycerol-inducible fumerate hydratase (fhy) promoter, superinfection immunity becomes dependent on the addition of glycerol to induce the fhy promoter.
Expression of imm from the wild-type Mx8 genome requires additional genetic elements located in a region 450 bp immediately upstream of imm. Presently, we are identifying the features of this element critical for imm expression by (1) sequencing mutations of Mx8 that map to this region and which prevent the establishment of lysogeny, and (2) determining the phenotypes of mutations introduced into this region by site-directed mutagenesis of plasmids.
Abstract #34
Cloning and characterization of the genes flanking the glyoxylate bypass regulon in Myxococcus xanthus
David R. Chapman, Daniel Salmi and Philip Youderian
Department of Microbiology, Molecular Biology and Biochemistry
University of Idaho, Moscow, ID 83844-3052
Previously, we have shown that the genes encoding the enzymes of the glyoxylate bypass, malate synthase and isocitrate lyase, are critical for both fruiting body morphogenesis and sporogenesis during the multicellular developmental cycle of Myxococcus xanthus. An 8 kb region upstream of the glyoxylate genes was cloned and sequenced. Immediately upstream of the glyoxylate genes is a small open reading frame of unknown function (ufo) and the gene encoding fumarate hydratase (fhy), transcribed divergently from the glyoxylate genes. Immediately downstream of fhy is the clpP gene, which encodes the major subunit of the proteosome. Downstream of fhy, ufo and clpP, and convergently transcribed, is the aco gene, which has a predicted product similar in sequence to eukaryotic peroxisomal acyl-coenzymeA oxidases.
We are characterizing the roles that each of these genes plays in the multicellular development of M. xanthus, and their regulation during both vegetative growth and development.
Abstract #37
Loss of social behaviors by Myxococcus xanthus during evolution in an unstructured habitat
Gregory Velicer, Lee Kroos, and Richard E. Lenski
Department of Biochemistry, Michigan State University
East Lansing, MI 48824
Twelve independent replicate populations of Myxococcus xanthus underwent 1000 generations of evolution in a nutrient-rich, unstructured habitat. The twelve populations were descended from a single common ancestor and underwent daily serial dilutions into fresh liquid medium. Derived lineages were compared with their ancestor with respect to maximum exponential growth rate, motility rates on hard and soft agar, fruiting body formation ability and sporulation frequencies during starvation. Motility phenotypes and transduction analysis showed that most of the derived lines were overtaken by mutants that are defective in S (social) motility, while all derived lines maintained functional A (adventurous) motility. Only three of the derived lines were able to develop into fruiting bodies, and sporulation frequencies among the derived lines ranged from zero to near wild-type levels. Sporulation ability, however, was not always associated with fruiting ability. One lineage developed into large fruiting bodies but produced no detectable spores, while two lines showed relatively high sporulation levels without forming fruiting bodies. The rapid loss of social motility, developmental ability, and sporulation ability under a liquid culture selective regime suggests that M. xanthus social behaviors require strong and sustained selective pressures to be maintained over time in natural populations.
Abstract #38
Characterization of two suppressor mutations of the BsgA protease during development of Myxococcus xanthus
John Cusick and Ron Gill
Department of Microbiology, University of Colorado Health Sciences Center
Denver, CO
I am currently trying to clone and characterize a pair of bypass suppressors of the BsgA protease requirement for development. The bypass suppressor strains M951 and M955 were created by transposon mutagenesis of strain M853 which is defective in BsgA proteolysis. The two strains were shown to be able to develop despite the absence of the protease when plated on starvation agar. The locations of the transposon insertions in strains M951 and M955 were shown to be in different cosmids with respect to each other. Additionally, the DNA adjacent to the transposon insertions for both mutant strains did not cross-hybridize with the cosmid clone containing the newly discovered two component system being characterized by Elizabeth Hager and Hubert Tse. The phenotypes of the mutant strains were shown to be due to the transposon insertions by complementing the strains with wild-type DNA from a cosmid library. Only when the strains were complemented with wild-type DNA adjacent to the transposon insertion was the inability to develop without the protease restored.
I am currently characterizing the phenotypes of the mutant strains by measuring their effect on a collection of developmental inducible lac fusions. I have cloned and sequenced approximately 2 kb of DNA surrounding the transposon insertions from both strains. Although obvious homologies have not yet been found, there are several stretches of DNA that are good candidates for being legitimate open reading frames. I am currently attempting to identify the smallest segments of DNA that are required for complementing the mutant phenotype in the hopes of identifying an open reading frame(s) that would be expected to act as an inhibitor of development. Further down the line, I would like to test the expression of such an open reading frame(s) and purify the corresponding protein(s) in an attempt to characterize the function of the gene product.
Abstract #39
Use of the Yeast two-hybrid system to detect possible interactions between the Asg proteins.
Catherine Eylem and Lynda Plamann
Department of Biology, Texas A&M University, College Station, TX 77843
Three genes, asgA, asgB, and asgC are required for production of extracellular A-signal, a necessary component for starting fruiting body development in M. xanthus. These genes encode regulatory proteins that are thought to act together in a signal transduction pathway that leads to A-signal production. Therefore, it is reasonable to speculate that the Asg proteins directly interact with each other. The yeast two-hybrid system was used to identify possible interactions between these proteins. The asgA, asgB, and asgC genes were cloned into plasmids pACT2 and pAS2-1 to produce hybrid genes that encode Asg sequences fused to either the GAL4 binding domain (BD) or the GAL4 activation domain (AD). Yeast strains containing a GAL4-dependent lacZ reporter gene were transformed with BD and AD pairs of plasmids. If the Asg sequences present in the two fusion proteins interact, they will tether the GAL4 DNA binding domain with the transcriptional activation
domain and allow transcription of lacZ and therefore detection of b-galactosidase activity. Ten plasmids were constructed and tested separately and together in three different yeast strains, Y187, Y190, and CG1945 (Y187 produces the highest levels of ss-galactosidase activity). The ten plasmids consisted of the asgA and asgB genes fused to both the BD and AD vectors, asgC with the AD vector, and the histidine protein kinase (HPK) and response regulator (RR) regions of asgA to both vectors. Two different lengths of the asgA HPK were inserted into the BD vector. Low-level b-galactosidase activity was sometimes detected in strains carrying asgB-BD fusion plasmids. The
detected interactions were not consistently reproducible, and occurred only in the Y187 yeast strain. It therefore seems that the asgB gene alone can produce a positive signal. No reproducible interactions were detected between the Asg proteins. Later experiments will focus on identifying interactions between the Asg proteins and other unknown M. xanthus proteins.
Abstract #40
Analysis of eight Tn5 lac insertion mutants perturbed in C-factor dependent activities during fruiting body morphogenesis in M. xanthus.
Lars Jelsbak 1, Ellen Licking2, & Lotte Søgaard-Andersen1
1Dept. of Molecular Biology, University of Odense, Campusvej 55, 5230 Odense
M, Denmark
2Dept. of Biochemistry, Stanford University, Stanford, CA. 94305-5307.
C-factor, the product of the csgA gene, is a cell surface-associated, extracellular signal protein that is required for the sequential developmental steps of rippling, aggregation and sporulation. Moreover, C-factor is required for expression of genes, including the csgA gene, that are normally expressed after 6 hrs. of starvation. We have previously suggested a model for the C-factor signal transduction pathway in which the signaling event is the interaction between C-factor on the end of one cell with a C-factor sensor on the end of a different cell. This interaction results in the activation of the FruA protein (formerly the ClassII protein). Downstream of FruA, the pathway branches: one branch leads to the motility response to C-factor and one branch leads to the sporulation and gene expression response.
To identify additional components of this pathway, we screened a collection of Tn5lac insertion mutants for those that were perturbed in aggregation, while still maintaining normal A and S motility. Subsequently, these mutants were analysed for their ability to perform other C-factor dependent activities. In the initial screening eight Tn5lac mutants were identified. A detailed analysis of these mutants showed that two of them, DK7703 and DK7528, fulfilled the screening criteria.
DK7703 forms large, branched aggregates during development and is unable to ripple. It is unable to sporulate and is cell autonomous with respect to this defect. Moreover, C-factor production is strongly reduced. Southern blot analysis with a fruA specific probe identified the location of the insertion within the fruA gene. Thus, Tn5lac W7703 is the fourth identified Tn5lac in the fruA gene.
DK7528 exhibits an unusual developmental phenotype. Development on TPM agar results in the formation of abnormally sized and shaped fruiting bodies that are organized in a spiraling pattern. Moreover, ripples are still observed after 72 hrs of starvation on TPM agar. DK7528 is severly reduced in sporulation and this defect is cell autonomous. Finally, DK7528 produce reduced levels of C-factor. The expression pattern of the gene tagged by Tn5lac W7528 was monitored by following specific activity of b-galactosidase. The Tn5lac W7528 fusion appears to be expressed constitutively during growth and development. We are currently in the process of characterizing the gene affected by the insertion in more details. Hypotheses to explain the developmental defect of DK7528 will be
presented.
fruA, an essential locus in the C-factor signaling pathway, may serve to coordinate early developmental signals and C-factor signaling during fruiting body morphogenesis in M. xanthus
Abstract #41
Searching for additional regulators within the Myxococcus xanthus sasB locus
Jose J. Rivera* and Heidi B. Kaplan
Department of Microbiology and Molecular Genetics
The University of Texas Medical School, Houston TX 77030
The Myxococcus xanthus sasB locus encodes genes necessary for multicellular
fruiting body development of this gram-negative soil bacterium. The sasB
locus was first identified by suppressor mutations that allow expression of
a development-specific gene, 4521, independent of starvation and
extracellular A signal. The sasB locus has been cloned and most of it has
been sequenced. Six open reading frames have been found clustered within
the sasB locus region and mutagenesis of these open reading frames indicate
they encode positive and negative regulators of 4521 expression. A
previously uncharacterized 2 kb fragment within this locus was subcloned
and sequenced to identify any potential open reading frames involved in M.
xanthus development. The DNA sequence of the 2 kb fragment was analyzed by
sequence analysis software and was compared to other sequences in the
databases. No homologues were identified from the databases. Analysis
using a third-codon bias algorithm based on the high GC content of the M.
xanthus chromosome does not predict an open reading frame. Mutagenesis of
this region will be performed to determine if it encodes any regulators of
4521 gene expression or multicellular development.
Abstract #42
An indirect role for CarA in the regulation CarQ dependent promoters
David E. Whitworth and David A. Hodgson. Department of Biological Sciences,
University of Warwick, Coventry, CV4 7AL, England
Myxococcus xanthus produces carotenoids as a protective response against illumination with blue light. The regulation of this response requires an alternative sigma-factor, CarQ, which is released from a membrane bound anti-sigma factor, CarR. CarQ mediates transcription from specific promoters within the carotenoid regulon, causing expression of the car structural genes and consequent production of carotenoids. The photosensitisers responsible for the release of free CarQ from the membrane complex are quenched by the carotenoids and the result is a down-regulation of
carotenogenesis.
The negative feedback effect due to carotenoid production, leads to a reduction in the activity of the promoters transcribed by CarQ. This phenomenon is especially important for mutants which produce carotenoids constitutively. Prevention of carotenoid synthesis, either through chemical inhibition of the synthetic enzymes or by genetic modification, causes an enhancement of promoter activity for the CarQ-dependent promoters. Removal of the influence of carotenoids allows the accurate analysis of promoter activities in strains constitutive for car gene expression, where low promoter activities may be an artefact due to carotenoid production. This is shown to be the case for carA mutants, which exhibit promoter activities identical to the wild-type, when the production of carotenoids is prevented. Mutants of carA therefore exhibit reduced promoter activities due to their constitutive production of carotenoids, rather than through a loss of direct positive regulation by CarA.
The following individuals attended the 24th Myxo meeting on June 22-25,1997:
Name
Department
Institution
City, State, Zip, COUNTRY
Phone (area code) country code
FAX (area code) [country code]
Lotte Sogaard-Andersen
Department of Molecular Biology
University of Odense
5230 Odense M, DENMARK
[45] 65572372
[45] 65 932781
sogaard@molbiol.ou.dk
Gabriela Bowden
Microbiology & Molecular Genetics
Univ Texas Medical School-Houston
Houston, TX 77030
(713) 500-5449
(713) 500-5499
gbowden@utmmg.med.uth.tmc.edu
David Chapman
Dept. of Microbiology, Molecular Biology and Biochemistry
University of Idaho
Moscow, ID 83844-3052
(208) 885-7161
(208) 885-6518
chapman@vetmed.wsu.edu
Kyungyun Cho
Dept. of Molecular & Cell Biology
University of California-Berkeley
Berkeley, CA 94720
(510) 643-5457
kc28@uclink4.berkeley.edu
Gene Crawford
Department of Microbiology
University of Georgia
Athens, Georgia 30602
(706) 542-2682
(706) 542-2674
crawford@bscr.uga.edu
John Cusick
Department of Microbiology
University of Colorado
Denver, CO 80262
(303) 315-7735
(303) 315-6785
cusick_j@defiance.uchsc.edu
John Davis
Department of Biology
Texas A & M University
College Station, TX 77843-3258
(409) 847-9239
jdavis@bio.tamu.edu
John Downard
Department Botany & Microbiology
University of Oklahoma
Norman, OK 73019
(405) 325-6302
(405) 325-7619
jdownard@ou.edu
Marty Dworkin
Department of Microbiology
Minneapolis, MN 55455-0312
martin@lenti.med.umn.edu
Eva Ellehauge
Department of Molecular Biology
University of Odense
5230 Odense M, DENMARK
[45] 65572426
[45] 65 932781
ellehauge@molbiol.ou.dk
Anthony Garza
Section of Microbiology
Division of Biological Sciences
University of California-Davis
Davis, CA 95616
(916) 752-2093
(916) 752-9014
aggarza@ucdavis.edu
Ron Gill
Department of Microbiology
University of Colorado
Denver, CO 80262
(303) 315-7832
(303) 315-6785
ron.gill@uchsc.edu
Lisa Gorksi
Department of Biochemistry
Stanford University
Stanford, CA 94305-5307
(415) 723-5347
(415) 725-7739
lgorski@cmgm.stanford.edu
Brandon Greenberg
Section of Microbiology
Division of Biological Sciences
University of California-Davis
Davis, CA 95616
Thomas Gronewold
Department of Microbiology
Stanford University
Stanford, CA 94305-5307
(415) 723-5347
gronewol@cmgm.stanford.edu
Dongchuan Guo
Microbiology & Molecular Genetics
Univ Texas Medical School-Houston
Houston, TX 77030
(713) 500-5449
(713) 500-5499
dguo@utmmg.med.tmc.edu
Elizabeth Hager
Department of Microbiology
University of Colorado
Denver, CO 80262
(303) 315-7474
(303) 315-6785
Baruch Harris
Section of Microbiology
Division of Biological Sciences
University of California-Davis
Davis, CA 95616
(916) 752-2093
(916) 752-9014
bzharris@ucdavis.edu
David Hodgson
Department of Biological Sciences
University of Warwick
Coventry, England CV4 7AL
UNITED KINGDOM
[44]-1203-523559
[44]-1203-523701
dm@dna.bio.warwick.ac.uk
Lars Jelsbak
Department of Molecular Biology
University of Odense
5230 Odense M, DENMARK
jelsbak@molbiol.ou.dk
Bryan Julien
Department of Biochemistry
Stanford University
Stanford, CA 94305-5307
(415) 723-5347
(415) 725-7739
bjulien@cmgm.stanford.edu
Dale Kaiser
Dept. Of Developmental Biology
Stanford University
Stanford, CA 94305-5307
(415) 723-6165
(415) 725-7739
luttman@cmgm.stanford.edu
Heidi Kaplan
Microbiology & Molecular Genetics
Univ. Texas Medical School-Houston
Houston, TX 77030
(713) 500-5448
(713) 500-5499
hkaplan@utmmg.med.tmc.edu
Yannis Karamanos
Laboratoire de Biochimie
Univ. d=Artois - Faculte des Sciences
Rue Souvraz - SP18
62307 Lens Cedex, FRANCE
[33] 21 79 17 14
[33] 3 21 79 17 55
karamanos@univ-artois.fr
Daniel Kearns
Department of Microbiology
University of Georgia
Athens, Georgia 30602
(706) 542-2682
(706) 542-2674
dkearns@uga.cc.uga.edu
Valerie Kessler
Department of Biology
Texas A & M University
College Station, TX 77843-3258
(409) 847-9237
(409) 845-2891
vkessler@bio.tamu.edu
Paul Kolenbrander
Department of Biochemistry
Stanford University
Stanford, CA 94305-5307
(415) 723-5685
(415) 725-7739
kolenb@cmgm.stanford.edu
Lee Kroos
Department Of Biochemistry
Michigan State University
East Lansing, MI 48824
(517) 355-9726
(517) 353-9334
kroos@pilot.msu.edu
Vince Magrini
Department of Microbiology, Molecular Biology and Biochemistry
University of Idaho
Moscow, ID 83844-3052
(208) 885-7161
(208) 885-6518
magri892@uidaho.edu
Mark McBride
Department of Biological Sciences
University of Wisconsin-Milwaukee
Milwaukee, WI 53201
(414) 229-5844
(414) 229-3926
mcbride@csd.uwm.edu
Wulf Plaga
Zentrum fur Molekulare Biologie (ZMBH)
Universitat Heidelberg
Im Neuenheimer Feld 282
D-69120 Heidelberg, GERMANY
[49] 6221-546882
[49] 6221-545893
wplaga@sun0.urz.uni-heidelberg.de
Lynda Plamann
Department of Biology
Texas A & M University
College Station, TX 77843-3258
(409) 847-9237
(409) 845-2891
lplamann@bio.tamu.edu
Jeff Pollack
Section of Microbiology
Division of Biological Sciences
University of California-Davis
Davis, CA 95616
(916) 752-2093
jspollack@ucdavis.edu
Kathleen O=Connor
Department of Molecular & Cell Biology
University of California-Berkeley
Berkeley, CA 94720
(510) 643-5457
(510) 643-5035
kathyo@uclink2.berkeley.edu
Jose Rivera
Microbiology & Molecular Genetics
Univ Texas Medical School-Houston
Houston, TX 77030
(713) 500-5449
(713) 500-5499
jrivera@utmmg.med.tmc.uth.edu
Ana Rodriguez
Civil Engineering
Stanford University
Stanford, CA 94305-4020
(415) 723-0315
(415) 725-3162
rodriguez@ce.stanford.edu
N. Jamie Ryding
Department of Microbiology
University of Georgia
Athens, Georgia 30602
ryding@bscr.uga.edu
Dan Salmi
Dept. of Microbiology, Molecular Biology and Biochemistry
University of Idaho
Moscow, ID 83844-3052
((208) 885-7161
(208) 885-6518
salm9457@uidaho.edu
Hans U. Schairer
Zentrum fur Molekulare Biologie (ZMBH)
Universitat Heidelberg
Im Neuenheimer Feld 282
D-69120 Heidelberg, GERMANY
[49]-6221-56-6880/6881/6830
(49)-6221-56-5893
hus@sun0.urz.uni-heidelberg.de
Wenyuan Shi
School of Dentistry and Molecular Biology Institute
University of California-Los Angeles
Los Angeles, CA 90095-1668
(310) 825-8356
(310) 206-5539
wenyang@ucla.edu
Larry Shimkets
Department of Microbiology
University of Georgia
Athens, Georgia 30602-2605
(706) 542-2681
(706) 542-2674
shimkets@bscr.uga.edu
Barbara Silakowski
Zentrum fur Molekulare Biologie (ZMBH)
Universitat Heidelberg
Im Neuenheimer Feld 282
D-69120 Heidelberg, GERMANY
[49]-6221-56-6881
[49]-6221-56-5893
silakowski@sun0.urz.uni-heidelbeg.de
Mitchell Singer
Section of Microbiology
Division of Biological Sciences
University of California-Davis
Davis, CA 95616
(916) 752-9005
(916) 752-9014
mhsinger@ucdavis.edu
Alfred Spormann
Civil Engineering
Stanford University
Stanford, CA 94305-4020
(415) 723-3668
(415) 725-3164
spormann@stanford.edu
Hubert Tse
Department of Microbiology
University of Colorado
Denver, CO 80262
(303) 315-7735
hubert.tse@uchsc.edu
Toshiyuki Ueki
Department of Biochemistry
Univ. of Medicine & Dentistry-NJ
Piscataway, NJ 08854
(908) 235-4116
(908) 235-4559
Gregory Velicer
Department of Biochemistry
Michigan State University
East Lansing, MI 48824
(517) 353-0809
(517) 353-2917
velicerg@pilot.msu.edu
Dan Wall
Department of Biochemistry
Stanford University
Stanford, CA 94305-5307
(415) 723-5437
(415) 725-7739
dwall@cmgm.stanford.edu
Don Walthers
Dept. of Microbiology, Molecular Biology and Biochemistry
Moscow, ID 83844-3052
(208) 885-7161
walt9693@uidaho.edu
Mandy Ward
Department of Molecular & Cell Biology
University of California-Berkeley
Berkeley, CA 94720
(510) 643-5457
(510) 642-7000
mjward@socrates.berkeley.edu
David White
Dept. of Microbiology, Molecular Biology and Biochemistry
University of Idaho
Moscow, ID 83844-3052
(208) 885-5914
(208) 885-6518
whit9441@uidaho.edu
David White
Department of Biology
Indiana University
Bloomington, IN 47405
white@indiana.edu
David Whitworth
Department of Biological Sciences
University of Warwick
Coventry, England CV4 7AL
UNITED KINGDOM
[44] 1203 522572
mhvi.@dna.bio.warwick.ac.uk
Kunitoshi Yamanaka
Department of Biochemistry
Univ. Of Medicine & Dentistry-NJ
Piscataway, NJ 08854
(908) 235-4116
(908) 235-4559
Chun Yang
Microbiology & Molecular Genetics
Univ Texas Medical School-Houston
Houston, TX 77030
(713) 500-5449
(713) 500-5499
cyang@utmmg.med.uth.tmc.edu
Zhaomin Yang
School of Dentistry and Molecular Biology Institute
University of California-Los Angeles
Los Angeles, CA 90095-1668
(310) 825-3856
(310) 206-5539
zhaomin@ucla.edu
Phil Youderian
Department of Microbiology, Molecular Biology and Biochemistry
University of Idaho
Moscow, ID 83844-3052
(208) 885-0571
(208) 885-6518
pay@uidaho.edu
David Zusman
Dept. of Molecular & Cell Biology
University of California-Berkeley
Berkeley, CA 94720
(510) 642-2293
(510) 642 7000
zusman@mendel.berkeley.edu
Dick Xu
Microbiology & Molecular Genetics
Univ Texas Medical School-Houston
Houston, TX 77030
(713) 500-5449
(713) 500-5499
dixu@utmmg.med.uth.tmc.edu