Osmotic regulation of PhoE porin synthesis in Escherichia coli.

In Escherichia coli, adaptation to hyperosmotic conditions alters the expression of the outer membrane porins OmpF and OmpC. The amount of PhoE porin, which is normally induced by phosphate deprivation, was greatly reduced in cells adapted to high-osmolarity conditions. Osmoregulation of PhoE operated independently of the activity of the PhoR phosphate sensor and did not involve cross-talk from the homologous osmosensor EnvZ. PhoE synthesis was partially restored by additional copies of the positive regulator phoB+ and by the osmoprotectant glycine betaine.

Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli.

The envZ11 missense mutation in the regulatory gene envZ pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon. Procaine treatment of wild-type cells resulted in the same phenotype through an envZ+-mediated mechanism. Here we show that envZ11-procaine act differently on the mal and pho regulons. In the mal system, the expression of the positive regulator gene malT, measured as beta-galactosidase activity of a malT-lac+ operon fusion, was drastically reduced by procaine treatment or by the envZ11 mutation. In contrast, expression of the positive regulator of the pho regulon phoB was not reduced by procaine treatment. The products of the regulatory genes phoM, phoR, and phoU were also not required for procaine action. Procaine and envZ11 inhibited expression of only two products of the pho regulon, alkaline phosphatase and the PhoE porin. The conclusion that envZ11-procaine act differently on the mal and the pho regulons is supported by our ability to isolate second-site mutations with a Mal+ PhoA- phenotype in an envZ11 strain.

Osmoregulation of alkaline phosphatase synthesis in Escherichia coli K-12.

Alkaline phosphatase, the phoA product, is synthesized constitutively in phoR mutants. This constitutive synthesis, which is independent of phosphate control, varies with changes in the osmolarity of the growth medium; phoA expression increases with increasing osmolarity. Maximum expression of the osmoregulated genes phoA, ompC, and ompF was achieved by osmotic manipulation of minimal medium; complex media repressed their expression.

Selective inhibition of carbohydrate transport by the local anesthetic procaine in Escherichia coli.

Maltose and lactose transport systems have been used to investigate the action of procaine on insertion and activity of membrane proteins and translocation of exported proteins in Escherichia coli. Procaine mildly inhibited growth on lactose. The level of inhibition was consistent with the small reduction observed in active and facilitated transport functions of the lac permease. However, procaine caused a severe reduction of growth rate on maltose, as well as an inhibition of induction of maltose regulon activities. In both constitutive and inducible strains, the synthesis of both maltose transport activity (malB operon) and amylomaltase activity (malA operon) was inhibited. Coordinate inhibition of soluble and membrane products was not observed with the lac operon. beta-Galactosidase synthesis proceeded normally during growth on procaine, whereas, the appearance of new transport activity was reduced. Regardless of carbon source, procaine specifically inhibited the appearance of ompF protein in the membrane fraction.

Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules.

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.

Directionality of brain microtubule assembly in vitro.

Chicks were injected intra-cerebrally with [(3)H]leucine, and (3)H-labeled microtubules were assembled in vitro in the brain supernatants. Pieces of these (3)H-labeled microtubules were then incubated with unlabeled brain tubulin subunits under conditions where the subunits assembled onto the labeled pieces. Electron microscopic autoradiography of the negatively-stained microtubules showed all of the radioactivity at one end of the tubules as they increased in length. This clearly demonstrated that the microtubules of brain in this in vitro system were being assembled unidirectionally.