Multiple defects in Escherichia coli mutants lacking HU protein.

The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, alpha and beta, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a perturbation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize, or to carry out transposition.     

Introduction of Escherichia coli cells and spheroplasts into Vinca protoplasts

In the presence of 10% polyvinyl alcohol (PVA), Escherichia coli cells or spheroplasts can be easily introduced into Vinca protoplasts by endocytosis. Uptake proceeded quite rapidly; bacterial cells or spheroplasts were found within the cytoplasm of Vinca protoplasts after 10 min of incubation with PVA.     

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.     

Cell surface growth in Escherichia coli: distribution of matrix protein.

Autoradiography of cell envelope "ghosts" from Escherichia coli was used to demonstrate that newly synthesized molecules of "matrix" protein are inserted at random locations over the entire surface of the outer membrane and that, once inserted, these molecules are not thereafter conserved in any fixed spatial location.     

Topographical distribution of penicillin-binding proteins in the Escherichia coli membrane.

The penicillin-binding proteins (PBPs) found in the membranes of Escherichia coli X925 minicells (primarily cell ends or septa) were compared with those found in rod-shaped cells (primarily sidewalls) in an effort to determine whether certain PBPs are unevenly distributed over the bacterial cell membrane. The seven major PBPs of E. coli were all present in minicell membranes. PBP 1B was altered in minicells, however, appearing as two bands on sodium dodecyl sulfate-polyacrylamide gels rather than the usual three. PBP 2, which is needed for longitudinal growth of the cell but not for septum formation, was significantly reduced in minicell membranes. This observation is consistent with the fact that minicells contain very little sidewall material and raises the possibility that the specialized function of PBP 2 may be determined or regulated by its uneven topographical distribution in the membrane. None of the PBPs appeared to be selectively enriched in minicell membranes.     

Delivery of antibody-captured proteins into living cells using PTD-fused protein A

Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.     

Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes.

When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, alpha-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells' native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, alpha-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throughout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC6[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.     

Serratia marcescens contains a heterodimeric HU protein like Escherichia coli and Salmonella typhimurium.

Homologs of the dimeric HU protein of Escherichia coli can be found in every prokaryotic organism that has been analyzed. In this work, we demonstrate that Serratia marcescens synthesizes two distinct HU subunits, like E. coli and Salmonella typhimurium, suggesting that the heterodimeric HU protein could be a common feature of enteric bacteria. A phylogenetic analysis of the HU-type proteins (HU and IHF) is presented, and a scheme for the origin of the hup genes and the onset of HU heterodimericity is suggested.     

Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF.

Opening of the three tandem repeats of a 13-mer in the replication origin (oriC) of Escherichia coli is a prime event in the replication in vitro of minichromosomes (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). DnaA, the initiator protein, requires protein HU or IHF, along with a millimolar level of ATP and negative superhelical density in the plasmid to open this region. The extent of opening, as judged by cleavage by a single-strand-specific endonuclease (i.e. P1 nuclease), correlated closely with replication of the oriC plasmid. In an initial complex, preceding opening of the 13-mers, the footprint of DnaA protein bound by ATP covered its four 9-mer recognition sequences. The footprint of the nucleotide-free form of the protein, by contrast, was more extensive and thus, less specific.     

Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU

The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes. A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype. The motility of hupA or hupB single mutants was similar to that of wild-type cells. SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain, suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella.     

ADP-ribosylation of proteins in non-infected Escherichia coli cells

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-(3)H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radioactive group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them.