ABSTRACT
Escherichia coli cells lacking the histone-like protein HU form filaments and have an abnormal number of anucleate cells. Furthermore, their phenotype resembles that of rfa mutants, the well-characterized deep-rough phenotype, as they show an enhanced permeability that renders them hypersensitive to chloramphenicol, novobiocin, and detergents. We show that, unlike rfa mutants, hupAB mutants do not have a truncated lipopolysaccharide but do have an abnormal abundance of OmpF porin in their outer membrane. While the complete absence of HU does not abolish the osmoregulation of OmpF protein synthesis, the steady-state level of micF RNA, the negative regulator of OmpF, decreases in bacteria lacking HU, increasing the basal level of this membrane protein. These findings demonstrate a novel link between a bacterial chromosomal protein and the outer membrane composition.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Escherichia coli/chemistry , Escherichia coli/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial , Lipopolysaccharides/chemistry , Lipopolysaccharides/metabolism , Osmolar Concentration , Water-Electrolyte Balance/geneticsABSTRACT
Mutations in a number of loci, including the lon gene, dramatically increase the production of colanic acid capsular polysaccharide and render Escherichia coli K-12 mucoid. The lon gene, which encodes an ATP-dependent protease, is localized at ten minutes on the E. coli map and is very closely linked to the hupB gene coding for one of the two subunits of the histone-like protein HU. Surprisingly the introduction of a multi-copy plasmid carrying either the hupB or hupA gene into a wild-type E. coli strain, results in the overproduction of one of the HU subunits and repression of the synthesis of the other without changing the overall concentration of HU, also renders the cells mucoid. As in a lon strain, the transcription of the cps genes, the structural genes for the synthesis of colanic acid, is induced dramatically. Protease Lon negatively regulates cps genes by destabilizing RcsA, a positive regulator of capsule synthesis. Regulation of HU synthesis does not affect the steady state level of Lon, as judged by Western blotting. The UV sensitivity of the hup transformed lon+ bacteria is identical to the lon+ parental strain, suggesting that Lon activity for the degradation of SulA in these cells is normal. Using lac operon fusions to cps gene promoters and to the rcsA promoter we show that the deregulation of HU synthesis does not by pass the positive regulatory action of RcsA and RcsB for the expression of cps genes but functions by stimulating RcsA synthesis.
Subject(s)
Bacterial Capsules/metabolism , Bacterial Proteins/physiology , DNA-Binding Proteins/physiology , Escherichia coli Proteins , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/metabolism , Polysaccharides, Bacterial/metabolism , Protease La , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Bacterial Proteins/genetics , DNA Repair , Escherichia coli/genetics , Genes, Lethal , Phenotype , Polysaccharides/metabolism , RNA, Bacterial/genetics , RNA, Messenger/metabolismABSTRACT
The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter. Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression. The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose. Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein. This band was shown by a zymogram to correspond to beta-glucosidase activity. The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene. Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined. It was shown that the enzyme is competitively inhibited by glucose. The effects of different metallic ions and other agents were studied. Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect. Ethanol did not show the stimulating effect observed with other beta-glucosidases. The mechanism of enzyme action was investigated. High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound. Results presented here indicate that this compound is cellotriose formed by transglycosylation.
