Colicin V-treated Escherichia coli does not generate membrane potential.

Colicin V-treated Escherichia coli was inhibited in its capacity to carry out active transport of proline and was unable to generate a membrane potential. Colicin V also prevented membrane potential formation by isolated cytoplasmic membrane vesicles. We conclude that a primary effect of this colicin involves the cytoplasmic membrane as a target.     

The biology of colicin M

Abstract This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma , has been sequenced and the protein isolated and purified. With a deduced molecular size of 29 453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13 890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

The biology of colicin M

This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma, has been sequenced and the protein isolated and purified. With a deduced molecular size of 29,453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13,890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

Biosynthesis and export of colicin A in Citrobacter freundii CA31

Synthesis of colicin A after induction with mitomycin C was studied. Specific inhibition of chromosomal protein synthesis occurred very shortly after mitomycin addition. There was no coordinate synthesis of colicin A (61000 Mr) and low-molecular-weight protein. Free and membrane-bound polysome fractions were isolated from cells induced with mitomycin C. Colicin A is synthesized in vitro in the free polysomes and not in the membrane-bound polysomes. Conditions are described which allow a practically specific labelling of colicin A in vivo. By using this system it was possible to demonstrate that colicin A is not transferred cotranslationally across the cytoplasmic membrane. In contrast, this protein leaves the cell where it was made long after synthesis. Preliminary evidence, suggesting that pauses occur during synthesis of colicin A, is presented.     

Leucine induces initial lowering of cytoplasmic Ca2+ in pancreatic beta-cells without concomitant inhibition of insulin release

The early effects of glucose and leucine on cytoplasmic Ca2+ and insulin release were compared in suspensions of cells prepared by dispersal of the beta-cell-rich pancreatic islets of ob/ob-mice. Adequate temporal resolution was achieved by continuously recording the 340/380 nm fluorescence excitation ratio from cells loaded with the Ca2+ indicator fura-2 and measuring insulin in the perifusate from cells mixed with polyacrylamide beads. Raising the glucose concentration from 3 to 20 mM resulted in concomitant reductions of cytoplasmic Ca2+ and insulin release during the first minute. Whereas 10 mM leucine was as efficient as glucose in inducing temporary lowering of cytoplasmic Ca2+, this amino acid did not depress insulin release. It is concluded that the initial decrease of cytoplasmic Ca2+ is a phenomenon coupled to stimulation of the metabolism. The leucine-induced lowering of Ca2+ may essentially reflect changes in cytoplasmic pools other than in a peripheral one regulating insulin release.     

Nucleocytoplasmic translocation of ribosomal RNA in the rabbit blastocyst: participation of sulfhydryl groups

The release of ribosomes from the nucleus in the rabbit blastocyst was investigated by pulse-labeling embryos to within 5 min of the earliest appearance of radiolabeled ribosomal RNA (rRNA) in the cytoplasmic fraction. The accumulation of radiolabeled 4.7 and 1.9 kilobase mature rRNA species in the cytoplasm was then followed during a 2 hour chase period, using polyacrylamide gel electrophoresis to identify the rRNAs. Colchicine, cytochalasin B, KCN, and EDTA were found to have no effect on the release of radiolabeled rRNA from the blastocyst nucleus during the 2 hour chase. Oligomycin, a known inhibitor of the nuclear envelope nucleoside triphosphatase, and the protein synthesis inhibitors puromycin and cycloheximide blocked rRNA release after a short delay. In contrast, actinomycin D and the sulfhydryl-reactive agents N-ethylmaleimide and diamide produced an abrupt and complete block to further rRNA release. The results indicate that ribosomes leave the nuclear compartment by an energy-dependent process. They further underscore the importance of reduced sulfhydryl groups in a rapidly growing blastocyst with a high level of oxidative metabolism.     

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.     

Studies on colicin B translocation: FepA is gated by TonB

Colicin B is a 55 kDa dumbbell-shaped protein toxin that uses the TonB system (outer membrane transporter, FepA, and three cytoplasmic membrane proteins TonB/ExbB/ExbD) to enter and kill Escherichia coli. FepA is a 22-stranded beta-barrel with its lumen filled by an amino-terminal globular domain containing an N-terminal semiconserved region, known as the TonB box, to which TonB binds. To investigate the mechanism of colicin B translocation across the outer membrane, we engineered cysteine (Cys) substitutions in the globular domain of FepA. Colicin B caused increased exposure to biotin maleimide labelling of all Cys substitutions, but to different degrees, with TonB as well as the FepA TonB box required for all increases. Because of the large increases in exposure for Cys residues from T13 to T51, we conclude that colicin B is translocated through the lumen of FepA, rather than along the lipid-barrel interface or through another protein. Part of the FepA globular domain (residues V91-V142) proved relatively refractory to labelling, indicating either that the relevant Cys residues were sequestered by an unknown protein or that a significant portion of the FepA globular domain remained inside the barrel, requiring concomitant conformational rearrangement of colicin B during its translocation. Unexpectedly, TonB was also required for colicin-induced exposure of the FepA TonB box, suggesting that TonB binds FepA at a different site prior to interaction with the TonB box.     

Colicins and bacterial membranes: structures and functions. I. Effects of colicins on the protein composition of the membranes of sensitive and tolerant Escherichia coli.

Treatment of Escherichia coli K12 C600 with colicin K or E1, but not E3, caused changes in the protein composition of the bacterial cytoplasmic membrane and an impairment of the membrane-associated ATP-linked transhydrogenase activity. The major compositional changes were loss and/or reduction in the levels of protein bands 4, 8, 9, 10, 13, and 18 with approximate molecular weights of 122,000, 81,000, 75,000, 73,000, 62,000, and 44,000, respectively. Colicin K or E1 treatment had no significant effect on the protein composition or the ATP-linked transhydrogenase activity of the cytoplasmic membranes of the isogenic tolerant strain E. coli K12 C600 TolII (A592). The cytoplasmic membranes of the untreated tolerant mutant were characteristically devoid of protein bands 4 and 13. It is proposed that protein bands 4 and/or 13 participate in colicin action by acting as receptors for colicins at the cytoplasmic membrane level. Some observations on the structural and functional heterogeneity of the cytoplasmic membrane preparations were made.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

A morphometric study of 24-hour variations in subcellular structures of the rat pancreatic acinar cell

Summary Subcellular structures of pancreatic acinar cells were examined at six evenly spaced time points in the 24-h period (light cycle: 06.00 h–18.00 h) in four Wistar male rats at each time point. At each sampling point, the area and circumference of acinar cell bodies and the area, number and circumference of their cytoplasmic organelles were measured using a semiautomatic computer system for morphometry and a point-counting method.The area, number and circumference-area ratio of the cytoplasmic organelles were subject to strong circadian variations, and the cellular area and circumference exhibited weak circadian variations. Variation pattern of the cytoplasmic organelles suggested an intracellular route for secretory proteins during a 24-h span. From the results it was possible to divide the 24-h period into three stages. 1. The resting or protein synthetic stage (00.00 h to 08.00h): the area of the rough surfaced endoplasmic reticulum (rER) was strongly increased, and that of zymogen granules was clearly decreased. 2. The granule accumulation stage (08.00h to 16.00h): the area of the rER was markedly decreased; that of zymogen granules was increased. 3. The secretion stage (16.00 h to 00.00): as a result of the release of zymogen granules from the acinar cell, the area of zymogen granules decreased, and that of the rER increased. The relationship between the area of the rER and zymogen granules varied in a reciprocal manner. Other cytoplasmic organelles, namely the Golgi complex, condensing vacuoles, mitochondria and lysosomes also varied prominently during the 24-h span, corresponding to variations in the rER and zymogen granules.     

Genetic Basis of Colicin E Susceptibility in Escherichia coli I. Isolation and Properties of Refractory Mutants and the Preliminary Mapping of Their Mutations

Colicin E-resistant mutants were isolated in Escherichia coli K-12 which, although still apparently possessing the E receptor and adsorbing colicin, were nevertheless insensitive (refractory) to its effect. Eight phenotypic groups were obtained, but some mutants from three of these groups were all shown to map at gal, whereas a second refractory locus, giving resistance to E1 alone, mapped close to thy. It is suggested that the successful fixation of any of the three distinct colicins of group E may involve a dual role for the cell surface "receptor," the first for the binding of the protein and the second for the correct orientation of the bound molecule relative to the cytoplasmic membrane. The majority of the refractory mutants isolated may derive from changes in components concerned with the second of these receptor functions. Two groups of mutants, however, refractory to only E1 or E2, probably reflect changes in the intracellular transmission systems which specifically mediate the effects of these two colicins, the changes not allowing transmission through the cytoplasmic membrane to the respective targets of the colicins. The E1 adsorption site was shown to be distinct from that for E2 and E3, indicating an early separation of the colicin E transmission systems.     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Binding of the immunity protein inactivates colicin M

Colicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma-producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N-terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.     

Structural and functional properties of colicin M.

Colicin M of Escherichia coli Cl139 was isolated in pure form. It consisted of a single polypeptide with a molecular weight of 27,000 +/- 2,000. Colicin M lysed sensitive cells of E. coli but had to act continuously up to the point when lysis commenced (after 20 min). Colicin M was largely resistant to hydrolysis by trypsin except when adsorbed to cells. Within 4 to 5 min after addition of colicin M, cells could be rescued by trypsin or sodium dodecyl sulfate. Later, colicin M was apparently inaccessible to these inactivating agents. Killing of cells by colicin M required Ca2+ ions. Cells could be rescued with ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) immediately before the onset of lysis. Under these conditions, colicin M remained bound to the cells, and it became again sensitive to trypsin. We conclude that under the influence of EGTA colicin M is removed from its site of action and becomes again accessible to trypsin at the cell surface.     

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3.

Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, ColE8-J was shown to be situated between ColE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids.     

去极化时Ca~(2+) 内流引起蛋白激酶C_α浆膜转位

 为观察胞外Ca2 + 内流和肌浆网Ca2 + 释放两种来源的Ca2 + 对cPKCα转位激活的影响 ,揭示PKC在去极化 nAChR转录偶联中的作用 ,构建了pPKCα EGFP N1融合蛋白真核基因表达载体 .转染C2C1 2肌细胞后 ,采用激光共聚焦显微镜记录了KC1或咖啡因处理所引起的细胞Ca2 + 波变化及PKCα GFP融合蛋白在细胞内的分布 .结果提示 ,只有用KC1处理引起细胞膜去极化时 ,伴随Ca2 +内流 ,才能观察到PKCα GFP绿色荧光在细胞内发生的细胞浆至细胞膜分布变化 .然而 ,采用肌浆网Ca2 + 通道激动剂咖啡因刺激肌细胞 ,使肌浆网中Ca2 + 释放 ,未见PKCα GFP绿色荧光在浆、膜分布发生任何变化 .结果提示 ,去极化时外Ca2 + 内流可引起PKCα转位激活 ,肌浆网Ca2 + 释放对PKCα的转位激活没有影响 .     

Immunity proteins to pore-forming colicins: structure-function relationships

Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be Involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins.     

Amount of Colicin Release in Escherichia coli Is Regulated by Lysis Gene Expression of the Colicin E2 Operon

The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of these dynamic processes. Here, we quantitatively study expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. DNA damage, triggering SOS response leads to the heterogeneous expression of this operon including the cea gene encoding the toxin, Colicin E2, and the cel gene coding for the induction of cell lysis and subsequent colicin release. Advancing previous whole population investigations, our time-lapse experiments reveal that at low exogenous stress levels all cells eventually respond after a given time (heterogeneous timing). This heterogeneous timing is lost at high stress levels, at which a synchronized stress response of all cells 60 min after induction via stress can be observed. We further demonstrate, that the amount of colicin released is dependent on cel (lysis) gene expression, independent of the applied exogenous stress level. A heterogeneous response in combination with heterogeneous timing can be biologically significant. It might enable a bacterial population to endure low stress levels, while at high stress levels an immediate and synchronized population wide response can give single surviving cells of the own species the chance to take over the bacterial community after the stress has ceased.