A CtrA homolog affects swarming motility and encystment in Rhodospirillum centenum.

The α-proteobacterium, Rhodospirillum centenum, has a complex life cycle that allows adaptation to different environments. Transitions between vegetative swim cell and swarmer cell types depend on whether the organism is growing in liquid surroundings or on a solid substrate. Moreover, starvation can induce vegetative cells to differentiate into quiescent cysts. This paper describes the results of our investigation into the role of a putative DNA-binding response regulator that is homologous to CtrA, the cell cycle regulator from Caulobacter crescentus. Deletion of ctrA from the R. centenum genome resulted in a viable strain with impaired swarming motility coupled with an increased tendency to form cysts. Conversely, overexpression of wild type CtrA or a phosphomimetic allele, CtrAD51E, suppressed cyst cell formation, whereas overexpression of a CtrAD51A allele failed to suppress encystment but did prevent swarming motility. Thus, we propose that CtrA participates within a two-component signal transduction pathway that promotes swarming motility while contributing to the suppression of cyst cell formation.

Two putative histidine kinases are required for cyst formation in Rhodospirillum Centenum.

The photosynthetic bacterium, Rhodospirillum centenum, has a flexible life cycle that permits it to survive starvation as dormant cyst cells. Previous studies have identified some of the key regulators for encystment and demonstrated that the control of development is intricate. This complexity may arise from the need to integrate several environmental signals to mediate a switch from one mode of energy metabolism to another and to ensure that a transition to dormancy is initiated only when necessary. We searched for additional regulators of development by screening for encystment deficient strains after subjecting wild type R. centenum to mini-Tn5 mutagenesis. Analysis of "hypo-cyst" strains led to the identification of two genes that encode putative hybrid histidine kinases (cyd1 and cyd2). Cells with deletions of either gene fail to form cysts under conditions that normally induce development. Furthermore, the deletion strains exhibit altered swarming behavior suggesting that Cyd1 and Cyd2 affect behaviors utilized when the organism is attached to a substrate.

Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation.

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

Protein kinase Cepsilon mediates PMA-induced growth inhibition of low population density NIH 3T3 fibroblasts.

Phorbol 12-myristate-13-acetate (PMA), a potent tumor promoter and activator of most protein kinase C (PKC) isotypes, was found to significantly inhibit the growth of low population density (1-5% confluency) NIH 3T3 cells. Higher cell population density (above 10% confluency) provided protection from this growth inhibitory effect of PMA. PMA-induced growth arrest is accompanied by an elevation in the level of p21(Cip1) protein, along with cell cycle arrest at the G1/S transition. Activation of PKC is required for this growth inhibitory response since the pan PKC inhibitor GF109203 blocked this effect of PMA. However, the classical PKC inhibitor Gö6976 had no effect, strongly suggesting the involvement of novel PKC isotypes (delta and/or epsilon). Overexpression of PKCepsilon, but not PKCdelta, was found to potentiate PMA-induced growth inhibition. Overexpression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) decreased the growth inhibitory effect of PMA and also blocked the PMA-induced increase in the level of p21(Cip1) protein. Taken together, these results indicate that PMA has a cell population density-dependent effect on the growth of NIH 3T3 cells and that the PMA growth inhibitory effect at low cell population density is mediated through activation of PKCepsilon.