Role of sigmaD in regulating genes and signals during Myxococcus xanthus development

Abstract

Starvation-induced development of Myxococcus xanthus is an excellent model for biofilm formation because it involves cell-cell signaling to coordinate formation of multicellular mounds, gene expression, and cellular differentiation into spores. The role of sigma(D), an alternative sigma factor important for viability in stationary phase and for stress responses, was investigated during development by measuring signal production, gene expression, and sporulation of a sigD null mutant alone and upon codevelopment with wild-type cells or signaling mutants. The sigD mutant responded to starvation by inducing (p)ppGpp synthesis normally but was impaired for production of A-signal, an early cell density signal, and for production of the morphogenetic C-signal. Induction of early developmental genes was greatly reduced, and expression of those that depend on A-signal was not restored by codevelopment with wild-type cells, indicating that sigma(D) is needed for cellular responses to A-signal. Despite these early developmental defects, the sigD mutant responded to C-signal supplied by codeveloping wild-type cells by inducing a subset of late developmental genes. sigma(D) RNA polymerase is dispensable for transcription of this subset, but a distinct regulatory class, which includes genes essential for sporulation, requires sigma(D) RNA polymerase or a gene under its control, cell autonomously. The level of sigD transcript in a relA mutant during growth is much lower than in wild-type cells, suggesting that (p)ppGpp positively regulates sigD transcription in growing cells. The sigD transcript level drops in wild-type cells after 20 min of starvation and remains low after 40 min but rises in a relA mutant after 40 min, suggesting that (p)ppGpp negatively regulates sigD transcription early in development. We conclude that sigma(D) synthesized during growth occupies a position near the top of a regulatory hierarchy governing M. xanthus development, analogous to sigma factors that control biofilm formation of other bacteria.

FIG. 1.

Timeline of events during M. xanthus development. Signal inputs are below the line and genes induced are above.

FIG. 2.

Expression of A-signal-independent genes in a sigD mutant. β-Galactosidase specific activity during development was measured for lacZ fusions to csgA (A) and sdeK (B) in wild type (▪) or in a sigD mutant alone (•) or upon codevelopment of the fusion-containing sigD mutant with an equal number of fusionless wild-type cells (○).

FIG. 3.

ppGpp and GTP levels during nutritional downshift of wild type and a sigD mutant. Guanosine nucleotides were measured in extracts of wild-type cells (squares) or a sigD mutant (circles) as described in Materials and Methods during growth (time zero) or after resuspension in starvation buffer. ppGpp (closed symbols) and GTP (open symbols) levels are expressed relative to the time zero level in a representative experiment.

FIG. 4.

Expression of A-signal-dependent genes in a sigD mutant. β-Galactosidase specific activity during development was measured for lacZ fusions to Ω4521 (A), fruA (B), and Ω4514 (C) in wild type (▪) or in a sigD mutant alone (•) or upon codevelopment of the fusion-containing sigD mutant with an equal number of fusionless wild-type cells (○).

FIG. 5.

Expression of C-signal-dependent genes in a sigD mutant. β-Galactosidase specific activity during development was measured for lacZ fusions to dev (A), Ω7536 (B), Ω4400 (C), Ω4403 (D), and Ω4499 (E) in wild type (▪) or in a sigD mutant alone (•) or upon codevelopment of the fusion-containing sigD mutant with an equal number of fusionless wild-type (○) or csgA mutant (▵) cells.

FIG. 6.

Rescue of Ω4400, Ω4403, and Ω4499 expression in the sigD mutant depends on fruA. β-Galactosidase specific activity during development was measured for lacZ fusions to Ω4400 (A), Ω4403 (B), and Ω4499 (C) in the sigD fruA double mutant alone (•) or upon codevelopment of the fusion-containing sigD fruA mutant with an equal number of fusionless wild-type cells (○).

FIG. 7.

Rescue of dev but not Ω4403 expression in a csgA mutant upon codevelopment with the sigD mutant. β-Galactosidase specific activity during development was measured for lacZ fusions to dev (A) and Ω4403 (B) in a csgA mutant upon codevelopment with an equal number of fusionless wild-type (○), sigD mutant (□), or csgA mutant (▵) cells.

FIG. 8.

Model of regulatory events governing M. xanthus development. Arrows indicate positive regulation, which may be direct or indirect. The dashed arrows indicate regulatory inputs that this study suggests might be different for dev than for Ω4400, Ω4403, and Ω4499.