Identification and characterization of proteins involved in Myxococcus xanthus sporulation
Myxococcus xanthus is a Gram-negative, rod-shaped soil dweller that undergoes a complex developmental cycle in response to nutrient starvation. A complex multi-cellular structure, referred to as a fruiting body, is formed following the aggregation of approximately 10 5 cells under starvation conditions. The rod-shaped cells within this structure differentiate into stress-resistant, dormant spores that are relatively resistant to dessication, UV irradiation, toxic molecules, and extreme temperatures. Myxococcus xanthus ( M. xanthus ) spores are composed of a core, cortex, and coat, but the proteins associated with the layers of the spore are not well studied. Two approaches were taken to identify proteins involved in sporulation of M. xanthus. We looked for genes with sequence similarity to the well studied sporulation model, Bacillus subtilis. We also took a proteomic approach to identify spore related proteins in M. xanthus.
CbgA was first identified as a potential M. xanthus sporulation protein based upon its sequence similarity to the Bacillus subtilis sporulation protein SpoVR. SpoVR is known to play an important role in the formation the endospore cortex, a protective layer of peptidoglycan. Here we show that the cbgA mutant aggregates and forms fruiting bodies, although cbgA fruiting bodies have abnormal shapes. Transmission electron microscopy revealed that cbgA mutant spores lack cortexes or have relatively thin cortex layers. Given that cbgA spores are spherical like their wild-type counterparts, this finding suggests that the cortex is not essential for M. xanthus spores to maintain their characteristic shape. Heat sensitivity is a property associated with spore cortex defects, and we found that spores produced by the cbgA mutant are more sensitive to heat than wild-type spores. We also found that cbgA spores are more sensitive to sodium dodecyl sulfate (SDS) than wild-type spores. However, cbgA mutant spores show no loss in sonication, UV irradiation or lysozyme resistance. These results indicate that CbgA plays an important role in spore cortex formation and the acquisition of a subset of spore stress-resistance properties.
The proteome of liquid-grown vegetative cells was compared with the proteome of mature fruiting body spores to look for proteins involved in forming resistant fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores as has been previously reported. In addition, we identified three previously uncharacterized proteins differentially expressed in spores. The genes for the three novel m ajor s pore p roteins ( mspA, mspB, and mspC ), were inactivated by insertion mutagenesis, and the resulting mutant strains were characterized for development. All three mutants formed aggregates, but the aggregates of two of the strains the fruiting bodies remained as flattened mounds of cells instead of taking on the characteristic dome shape of wild-type fruiting bodies. The most pronounced structural defect of spores produced by all three of these mutants was an altered cortex layer. mspA and mspB mutant spores are more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores are more sensitive to all stress treatments tested. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive stress.