Masters Thesis Research

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Understanding the roles of the sglK and stkA genes in the regulation of EPS production in Myxococcus xanthus

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Masters Thesis Research

  1. 1. KRISTEN HUNTINGTON YANG LAB MEETING SEPTEMBER 11, 2008 Understanding the roles of the sglK and stkA genes in the regulation of EPS production in Myxococcus xanthus
  2. 2. Myxococcus xanthus is a developmental bacterium (Kuner and Kaiser, 1982)
  3. 3. Two forms of gliding motility (Shimkets, 1986) <ul><li>Adventurous motility (A) </li></ul><ul><ul><li>Movement of single cells </li></ul></ul><ul><li>Social Motility (S) </li></ul><ul><ul><li>Movement of cells in a group </li></ul></ul>
  4. 4. Type IV pili are required for S motility (Kaiser 1979) Bar = 0.5 µ m <ul><li>Polarly located </li></ul><ul><li>Polymeric protein filament </li></ul><ul><li>pil locus </li></ul>
  5. 5. EPS is required for S motility (Merroun et al., 2003) <ul><li>EPS = Extracellular polysaccharide </li></ul><ul><li>Peritrichous </li></ul>
  6. 6. Proposed mechanism for S motility <ul><li>Pili is the motor for S motility </li></ul><ul><li>EPS serves as an anchor to which TFP attach to </li></ul><ul><li>Contact with EPS stimulates TFP retraction </li></ul>
  7. 7. The dif locus in EPS regulation (Black and Yang, 2004) <ul><li>Do not produce EPS: difA, difC, difE </li></ul><ul><li>Overproduce EPS: difD, difG </li></ul>
  8. 8. The EPS regulatory pathway EPS Production Machinery cell membrane DifA ~ MCP DifC ~ CheW DifE ~ CheA EPS DifD ~ CheY DifG ~ CheC DifX’s DifA DifA DifG DifD + - ? DifE DifE C
  9. 9. sglK and stkA regulate EPS production <ul><li>Role in EPS production </li></ul><ul><ul><li>sglK mutants don’t produce EPS </li></ul></ul><ul><ul><li>stkA mutants overproduce EPS </li></ul></ul><ul><li>SglK and StkA are DnaK homologs </li></ul><ul><ul><li>Likely regulatory rather than biosynthetic </li></ul></ul>
  10. 10. Specific Aims <ul><li>1. To establish epistatic relationships among genes at the sglK , stkA , dif and pil loci. </li></ul><ul><li>2. To determine which domains of SglK and StkA are important for their function. </li></ul><ul><li>3. To determine what SglK and StkA interact with using yeast two-hybrid analysis. </li></ul>
  11. 11. <ul><ul><li>Construction of mutations at the sglK locus </li></ul></ul><ul><ul><li>Examination of genetic epistasis </li></ul></ul>Specific Aim 1. To establish epistatic relationships among genes at sglK, stkA, dif , and pil loci . The EPS regulatory pathway DifX’s EPS Production Machinery cell membrane DifA DifA DifG DifD DifA ~ MCP DifC ~ CheW DifE ~ CheA + - ? DifE DifE C EPS DifD ~ CheY DifG ~ CheC
  12. 12. EPS production of sglK locus mutants WT sglK fibR 6670 kinS grpS fibR grpS sglK 6670 kinS
  13. 13. DifD ~ CheY DifG ~ CheC SglK genetic epistasis results DifX’s EPS Production Machinery cell membrane DifA DifA DifG DifD DifA ~ MCP DifC ~ CheW DifE ~ CheA + - ? DifE DifE C EPS StkA SglK SglK ~ DnaK StkA ~ DnaK
  14. 14. difA , pilA and sglK are epistatic to kinS pilA kinS pilA difA kinS sglK kinS WT kinS sglK difA
  15. 15. DifD ~ CheY DifG ~ CheC KinS? KinS genetic epistasis results DifX’s EPS Production Machinery cell membrane DifA DifA DifG DifD DifA ~ MCP DifC ~ CheW DifE ~ CheA + - ? DifE DifE C EPS StkA SglK SglK ~ DnaK StkA ~ DnaK
  16. 16. Future directions of specific aim 1 <ul><li>Characterization of the role that KinS plays in EPS regulation. </li></ul><ul><ul><li>Triple mutants </li></ul></ul><ul><ul><ul><li>difD pilA kinS </li></ul></ul></ul><ul><ul><ul><ul><li>addition kinS insertion has no effect on phenotype </li></ul></ul></ul></ul><ul><ul><ul><li>difG pilA kinS </li></ul></ul></ul><ul><ul><ul><li>kapB pilA kinS </li></ul></ul></ul>
  17. 17. <ul><ul><li>Homology modeling </li></ul></ul><ul><ul><li>Site-directed mutagenesis of SglK and StkA </li></ul></ul>Specific Aim 2. To determine which domains of SglK and StkA are important for their function .
  18. 18. Homology to DnaK
  19. 19. SglK N terminal homology model <ul><li>Human Hsp70 ATPase domain </li></ul><ul><li>52 % sequence identity </li></ul>
  20. 20. SglK C terminal domain homology model <ul><li>E. coli DnaK substrate binding domain </li></ul><ul><li>56% sequence identity </li></ul><ul><li>Shown with heptapeptide in peptide binding pocket </li></ul>
  21. 21. SglK C terminal domain – “Arch” <ul><li>Arch necessary to enclose peptide backbone </li></ul><ul><li>DnaK vs SglK </li></ul><ul><li>Methionine 404  Alanine 403 </li></ul><ul><li>Alanine 429  Alanine 428 </li></ul>
  22. 22. SglK C terminal domain - “Latch” <ul><li>DnaK vs. SglK </li></ul><ul><li>Histidine 544  Tyrosine 543 </li></ul>Tyr 543 Lys 547 Arg 466 Asp 539 Asp 430
  23. 23. StkA N terminal domain <ul><li>E. coli DnaK </li></ul><ul><li>49% sequence identity </li></ul>
  24. 24. StkA C terminal domain <ul><li>E. coli HscA </li></ul><ul><li>25% sequence identity </li></ul><ul><li>Another E. coli Hsp70 </li></ul><ul><li>Biosynthesis of Fe-S clusters </li></ul><ul><li>Distinct substrate & co-chaperone specificity </li></ul>
  25. 25. Complementation of sglK WT sglK 1 2 3
  26. 26. Complementation of stkA WT stkA 1 2 3
  27. 27. Future directions of specific aim 2 <ul><li>Identification of key residues important for the function of both SglK and StkA </li></ul>
  28. 28. <ul><ul><li>To identify “co-chaperones” of SglK and StkA </li></ul></ul>Specific Aim 3. To determine what SglK and StkA interact with .
  29. 29. DnaK Chaperone Machine (Mayer et al., 2000)
  30. 30. Yeast Mating <ul><li>Budding yeast can exist stably as haploid or diploid cells </li></ul><ul><li>Haploid cells exist in 2 forms: MATa and MAT α </li></ul>MATa MAT α MATa MAT α MATa/MAT α shmoo
  31. 31. Yeast Matrix Experiment DnaK’s 1-10 in pGADT7 transformed into AH109 (MATa) PREY 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H
  32. 32. Identify “co-chaperones” of SglK and StkA <ul><li>Yeast matrix experiment </li></ul><ul><ul><li>Clontech Matchmaker TM two-hybrid system 3 </li></ul></ul><ul><ul><ul><li>Perfect Mating Pair AH109 (MATa) and Y187 (MAT α ) </li></ul></ul></ul><ul><ul><li>Primer design completed for amplification of 10 DnaK, 11 DnaJ and 2 GrpE homologs in M. xanthus genome. </li></ul></ul><ul><ul><li>Amplified inserts, prepared vectors </li></ul></ul><ul><ul><li>Problem with NdeI cutting inserts </li></ul></ul>

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