Masters Thesis Research

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

Myxococcus xanthus is a developmental bacterium (Kuner and Kaiser, 1982)

Two forms of gliding motility (Shimkets, 1986) ,[object Object],[object Object],[object Object],[object Object]

Type IV pili are required for S motility (Kaiser 1979) Bar = 0.5 µ m ,[object Object],[object Object],[object Object]

EPS is required for S motility (Merroun et al., 2003) ,[object Object],[object Object]

Proposed mechanism for S motility ,[object Object],[object Object],[object Object]

The dif locus in EPS regulation (Black and Yang, 2004) ,[object Object],[object Object]

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

sglK and stkA regulate EPS production ,[object Object],[object Object],[object Object],[object Object],[object Object]

Specific Aims ,[object Object],[object Object],[object Object]

[object Object],[object Object],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

EPS production of sglK locus mutants WT sglK fibR 6670 kinS grpS fibR grpS sglK 6670 kinS

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

difA , pilA and sglK are epistatic to kinS pilA kinS pilA difA kinS sglK kinS WT kinS sglK difA

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

Future directions of specific aim 1 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

[object Object],[object Object],Specific Aim 2. To determine which domains of SglK and StkA are important for their function .

SglK N terminal homology model ,[object Object],[object Object]

SglK C terminal domain homology model ,[object Object],[object Object],[object Object]

SglK C terminal domain – “Arch” ,[object Object],[object Object],[object Object],[object Object]

SglK C terminal domain - “Latch” ,[object Object],[object Object],Tyr 543 Lys 547 Arg 466 Asp 539 Asp 430

StkA N terminal domain ,[object Object],[object Object]

StkA C terminal domain ,[object Object],[object Object],[object Object],[object Object],[object Object]

Complementation of sglK WT sglK 1 2 3

Complementation of stkA WT stkA 1 2 3

Future directions of specific aim 2 ,[object Object]

[object Object],Specific Aim 3. To determine what SglK and StkA interact with .

DnaK Chaperone Machine (Mayer et al., 2000)

Yeast Mating ,[object Object],[object Object],MATa MAT α MATa MAT α MATa/MAT α shmoo

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

Identify “co-chaperones” of SglK and StkA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Masters Thesis Research

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