A Defect in Lipoprotein Modification by Lgt Leads to Abnormal Morphology and Cell Death in Escherichia coli That Is Independent of Major Lipoprotein Lpp.

Lgt is an essential enzyme in proteobacteria and therefore a potential target for novel antibiotics. The effect of Lgt depletion on growth, morphology, and viability was studied in Escherichia coli to assess whether absence of Lgt leads to cell death. Two Lgt depletion strains were used in which lgt was under the control of an arabinose-inducible promoter that allowed regulation of Lgt protein levels. Reduced levels of Lgt led to severe growth and morphological defects that could be restored by expressing lgt in trans, demonstrating that only Lgt is responsible for the distorted phenotypes. In the absence of major lipoprotein Lpp, growth defects were partially restored when low levels of Lgt were still present; however, lgt could not be deleted in the absence of Lpp. Our results demonstrate that Lpp is not the main cause of cell death under conditions of Lgt depletion and that other lipoproteins are important in cell envelope biogenesis and cell viability. Specific inhibitors of Lgt are thus promising for the development of novel antibiotics. IMPORTANCE Incomplete maturation and envelope mislocalization of lipoproteins, through inhibition or mutations in lipoprotein modification enzymes or transport to the outer membrane, are lethal in proteobacteria. Resistance to small-molecule inhibition or the appearance of suppressor mutations is often directly correlated with the presence of abundant outer membrane lipoprotein Lpp. Our results show that Lgt, the first enzyme of the lipoprotein modification pathway, is still required for growth and viability in the absence of Lpp and thus is necessary for the function of other essential lipoproteins in the cell envelope. This adds credence to the hypothesis that Lgt is essential in proteobacteria and an attractive target for the development of novel antibiotics.

Timing of FtsZ assembly in Escherichia coli.

The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli.

The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the gamma form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.

The structure and function of Escherichia coli penicillin-binding protein 3.

Escherichia coli penicillin-binding protein PBP3 is a key element in cell septation. It is presumed to catalyse a transpeptidation reaction during biosynthesis of the septum peptidoglycan but, in vitro, its enzymatic activity has only been demonstrated with thiolester analogues of the natural peptide substrate. It has no detectable transglycosylase activity with lipid II as substrate. This tripartite protein is constructed of an N-terminal membrane anchor-containing module that is essential for cell septation, a non-penicillin-binding (n-PB) module of unknown function and a C-terminal penicillin-binding (PB) module exhibiting all the characteristic motifs of penicilloyl serine transferases. The n-PB module, which is required for the folding and stability of the PB module, may provide recognition sites for other cell division proteins. Initiation of septum formation is not PBP3-dependent but rests on the appearance of the FtsZ ring, and is thus penicillin-insensitive. The control of PBP3 activity during the cell cycle is briefly discussed.

Analysis of a novel DNA-binding protein kinase CKII-like enzyme of Chironomus cells.

We have previously described a Chironomus tentans nuclear 42 kDa phosphoprotein preferentially associated with transcriptionally active chromatin. In an attempt to purify and identify the kinase responsible for the phosphorylation of the 42 kDa protein, a casein-phosvitin affinity chromatography was used. Unexpectedly, in the eluted kinase fraction, a novel 42 kDa casein kinase, designated protein kinase CK42, with a kinase activity similar, but not identical, to protein kinase CKII, could be identified. In other studies, a nuclear protein that comigrates with protein kinase CK42 in electrophoresis and is capable to bind different gene promoters in single-stranded forms in a sequence-selective manner was found. The observations that both protein kinase and ssDNA-binding activities could be ascribed to a 42 kDa protein raised the possibility that the ssDNA-binding 42 kDa phosphoprotein is a protein kinase. By specific ssDNA-binding affinity chromatography, using a biotinylated oligodeoxyribonucleotide promoter probe and Streptavidine-agarose matrix, evidence that both activities arise from the same protein molecules was obtained. The similarity in the enzyme activities between protein kinase CK42 and CKII raised the question of whether the former was an alpha subunit of the latter. To provide an answer to this issue, CKII, isolated and purified from an epithelial cell line of C. tentans, was characterized and compared with protein kinase CK42 purified from the same cell system. Like other purified CKII preparations, CKII from Chironomus is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). However, the heparin and DRB sensitivities of protein kinase CKII were substantially higher than those of the protein kinase CK42. Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual alpha and beta subunit pools of CKII could be detected. More than 80% of the nuclear alpha subunit was insoluble in 0.35 M NaCl, while all individual beta subunit were solubilized under the same conditions suggesting that a major portion of the nuclear CKII alpha subunit does not form heterooligomeric structures with the beta subunit, but binds tightly to nuclear components, probably to chromatin. The biochemical and immunological data taken together strongly suggest that CK42 is a novel DNA-binding protein kinase that is not the alpha subunit of CKII.

A majority of casein kinase II alpha subunit is tightly bound to intranuclear components but not to the beta subunit.

Nuclear casein kinase II (CK II) was purified from an epithelial cell line of Chironomus tentans and characterized. The intracellular distribution of CK II and its two intracellular subunits (alpha and beta) was analysed by immunoblotting. The apparent molecular weights of the alpha and beta subunits were estimated to be 36 and 28 kDa, respectively. Like other purified CK II preparations, CK II from Chironomus tentans is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and by the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual alpha and beta subunit pools could be detected. More than 85% of the total immunostainable alpha subunit and essentially all immunoreactive individual beta subunit and heterooligomeric enzyme molecules were localised to the nucleus. Unexpectedly, more than 80% of this nuclear alpha subunit was insoluble in 0.35 M NaCl, while all individual beta subunit and heterooligomeric enzyme molecules were solubilized under the same conditions. Of the 0.35 M NaCl soluble kinase fractions, the active multisubunit form of CK II precipitated in 50% (NH4)2SO4 and could thus be separated from the free beta subunit, which precipitated at 60% and 80% (NH4)2SO4. These results suggest that a major portion of the nuclear CK II alpha subunit does not form heterooligomeric structures with the beta subunit, but binds tightly to nuclear components.

A novel nuclear 42-kDa casein kinase identified in Chironomus tentans.

We have purified and characterised an apparently novel nuclear 42-kDa casein kinase from epithelial cells of Chironomus tentans which comigrates with a phosphoprotein associated with transcriptionally active salivary gland genes. The protein kinase promotes phosphorylation of casein and phosvitin, using either ATP or GTP as phosphate donors, and undergoes autophosphorylation. The casein kinase activity of the 42-kDa protein is sensitive to heparin, 5,6-dichloro-1-beta-D-ribofuranosylbezimidazole (DRB), spermine and spermidine indicating that it is a novel enzyme with similar but not identical properties to casein kinase II or nuclear protein kinase NII.

A cell-free extract from yeast cells for studying mRNA turnover.

We have isolated a cell-free extract from yeast cells that reproduces the differences observed in vivo in the rate of turnover of individual yeast mRNAs. Detailed analysis of the degradation of yeast phosphoglycerate kinase (PGK) mRNA in this system demonstrated that both natural and synthetically prepared PGK transcripts are degraded by the same pathway previously established by us in vivo, consisting of endonucleolytic cleavage at a number of 5'-GGUG-3' sequence motifs within a short target region located close to the 3'-end of the coding sequence followed by 5'-3' exonucleolytic removal of the resulting fragments. The extract, therefore, is suitable for studying the mechanistic details of mRNA turnover in yeast. As a first application of this system we have performed a limited mutational analysis of two of the GGUG motifs within the endonucleolytic target region of the PGK transcript. The results show that sequence changes in either motif abolish cleavage at the mutated site only, indicating the involvement of the residues in question in selection of the cleavage positions.