Protein kinase A is involved in the control of morphology and branching during aerobic growth in Mucor circinelloides.

LÜBBEHÜSEN T (EQUAL CONTRIBUTION); GONZALEZ POLO, V. (EQUAL CONTRIBUTION); ROSSI S; NIELSEN J; MORENO S; MCINTYRE M; ARNAU J

MICROBIOLOGY-UK

Año: 2004 vol. 150 p. 143 - 150

1350-0872

The cAMP signal transduction pathway controls many processes in fungi. The Mucorcircinelloides pkaR and pkaC genes, encoding the regulatory (PKAR) and catalytic(PKAC) subunit of the cAMP-dependent protein kinase A (PKA), have been clonedrecently. Expression analysis during the dimorphic shift and colony morphologysuggested a role for PKAR in the control of morphology and branching. Herestrain KFA121, which overexpresses the M. circinelloides pkaR gene, was used toquantify growth and branching under different aerobic growth conditions in aflow-through cell by computerized image analysis. An inverse relationshipbetween the pkaR expression level in KFA121 and the hyphal growth unit lengthwas observed in KFA121, suggesting a central role for PKAR in branching. Abiochemical analysis of PKAR using antibodies and enzyme assay demonstrated thatthe level of PKAR is higher in KFA121 under inducing conditions, i.e. in thepresence of high glucose, than in the vector control strain KFA89. Measurementof cAMP binding demonstrated a significant increase (two- to threefold) in PKARlevel for KFA121 at the time of germ-tube emission in medium containing 10 gglucose l(-1). The level of PKA activity was determined using kemptide in thesame crude cell extracts used to determine cAMP binding. Strain KFA121 showed atwofold increase in PKA activity. An excess of free PKAR subunit over PKAholoenzyme was determined using sucrose gradient centrifugation of extracts fromKFA89 and KFA121. The data indicate that cAMP-dependent PKA in M. circinelloidesmight be down-regulated during hyphal-tube emergence and that an increase inPKAR levels results in increased branching.