Accepted adjunct faculty position at University of Houston-Downtown after receiving MS
Myxococcus xanthus is a Gram-negative soil bacterium that goes through a multicellular developmental program upon starvation at high cell density. The 4445 gene is expressed early in development and requires for expression conditions of starvation, high density, aeration, and at least 8 mM Mg++. The expression of 4445 is also dependent on the asg regulators that control secretion in response to starvation, and a low molecular weight, heat-stable activity that is present in high density starving wild-type cell supernatants. Using random mutagenesis with the Himar-tet transposon and subsequent genetic experiments, the regulators of 4445 have been identified to be a positive regulator, EcfA, and two negative regulators, ReaA and ReaB. This M. xanthus EcfA/ReaA/ReaB signal transduction system is encoded by one operon and is analogous to the Escherichia coli sigmaE/RseA/RseB signal transduction system that is activated by certain envelope stresses.
To begin to test the hypothesis that ReaA and ReaB are functional homologues of E. coli RseA and RseB, respectively, their topologies were studied by constructing alkaline phosphatase (PhoA) translational fusions to ReaA and ReaB and testing their activities in E. coli. The results support the computer predictions that ReaA has a central transmembrane domain, a cytoplasmic N-terminus and a periplasmic C-terminus, and ReaB has a signal peptide sequence and is secreted to the periplasm. The topologies and localizations of ReaA and ReaB are similar to the E. coli RseA and RseB, suggesting that they are functional homologues.
Mutants that lack either of the negative regulators express 4445 at levels more than two orders of magnitude greater than wild-type cells during growth and development. Western immunoblot analysis in which anti-EcfA antiserum was reacted with crude extracts of wild-type cells and a mutant containing an in-frame mutation in reaA revealed that the ecfA operon is auto-regulated because in the absence of the negative regulator ReaA, EcfA is greatly overproduced during growth and development.
The expression of 4445 can be induced by the addition of cell-envelope damaging agents to growing and starving cells. The addition of 25 mM ammonium metavanadate or 0.5 M glycerol increased 4445 expression in growing cells by about 4 fold. The addition of 0.3 mg/ml lysozyme to starving low-density cells increased 4445 expression 2 fold. As some of these cell-wall damaging agents directly affect the integrity of the cell-wall peptidoglycan (PGN), which is a dynamic component of all Gram-negative and Gram-positive bacterial cell walls and is critical for cell growth and survival, it was hypothesized that a component of PGN is the activating signal of 4445 expression present in high cell density starving cell supernatants. PGN was isolated from growing wild-type M. xanthus cells and added to low-density starving cells containing the 4445-lacZ fusion. The addition of crude hydrolyzed PGN (50 to 300 ug/ml) increased 4445 expression to 75 to 100% of the level measured during normal development of these cells at high density. These data support the hypothesis that a component present in the crude hydrolyzed PGN extracts functions in a dual role to reflect both cell density and membrane integrity during early M. xanthus development.
Characterization of the Extracytoplasmic Function (ECF) Sigma Factor Signal Transduction System that Senses Envelope Integrity During Early Myxococcus xanthus Multicellular Development