Concentration of a major outer membrane protein at the cell poles in Escherichia coli

Autoradiography of cell envelope ghosts obtained from a strain of Escherichia coli which lacks two major outer membrane proteins has been used to demonstrate the polar concentration of another major outer membrane protein, ompA protein. The beta-lactam antibiotic cephalexin prevents the insertion of newly synthesized ompA protein into the poles but removal of the antibiotic allows the randomly dispersed protein to migrate to the polar and possibly the septal areas of the cell. Labelling of whole cells with bacteriophage K3 has confirmed a polar concentration of ompA protein.     

Isolation and identification of new inner membrane-associated proteins that localize to cell poles in Escherichia coli

Several bacterial structures, processes and proteins are localized primarily to the poles of rod-shaped cells. To better understand this cellular organization, we devised a new method for identifying proteins that localize to the poles of Escherichia coli. Pole-derived membrane fragments were isolated by affinity capture of vesicles containing the chemotaxis protein, Tar; and for comparison, vesicles representing all parts of the cytoplasmic membrane were captured by expressing a Tar variant that was no longer pole-specific. A combination of one-dimensional SDS-PAGE and semi-quantitative mass spectrometry identified 31 proteins that were highly enriched in polar vesicles. Five were chemotaxis proteins known to be pole-specific and another, Aer, was an aerotaxis protein that had not yet been localized to the pole. The behaviour of these internal controls validated the overall approach. GFP-fused derivatives of four candidates (Aer, YqjD, TnaA and GroES) formed polar foci that were distinct from inclusion bodies. TnaA-GFP and GroES-GFP were functional, formed a single focus per cell, and competed for polar localization with the wild-type versions of these proteins. Polar localization of TnaA, GroES and YqjD was disrupted in cells lacking the MinCDE proteins, suggesting that this system may help localize proteins not involved in cell division.     

Transposable elements can be used to study cell lineages in transgenic plants.

The beta-glucuronidase reporter gene has been used to develop a sensitive assay for the excision of transposable elements introduced into transgenic plants. The reporter gene, inactivated by the insertion of the maize transposable element Activator (Ac) into the 5'-untranslated leader, was introduced into the genome of tobacco by Agrobacterium-mediated transformation. Reactivation of the beta-glucuronidase gene was detected in transgenic plants using a fluorometric or histochemical assay. Reactivation of the reporter gene was dependent on the presence of the transposase of Ac, and resulted from the excision of the Ac element. This assay, together with the improved methods for visualization, will provide a valuable and rapid method for studying the basic mechanism of transposition in plants and for developing modified transposable element systems suitable for gene tagging in transgenic plants.     

Cardiolipin synthase A colocalizes with cardiolipin and osmosensing transporter ProP at the poles of Escherichia coli cells

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)‐dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two‐Hybrid System (BACTH). The BACTH‐tagged variants T18‐ProP and T25‐ProP retained ProP function and localization. Their interaction confirmed the ProP homo‐dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL‐dependent. ClsA was shown to be N‐terminally processed and membrane‐anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.     

3-Azi-1-methoxybutyl D-maltooligosaccharides specifically bind to the maltose/maltooligosaccharide-binding protein of Escherichia coli and can be used as photoaffinity labels

Maltooligosaccharides with two to six (alpha 1-4)-linked glucose residues, carrying at their reducing end a 3-azi-1-methoxybutyl group in either alpha or in beta glycosidic linkage, were synthesized. These maltooligosaccharide analogues inhibit maltose uptake via the maltose-binding-protein-dependent transport system in Escherichia coli. The concentration of half-maximal inhibition of maltose transport, at 15 nM concentration, decreases with increasing chain length of the analogue, levelling off at 40 microM after a chain length of four glucose residues in the alpha series and at 350 microM after a chain length of three glucose residues in the beta series. The inhibition of maltose transport occurs at the level of the periplasmic maltose-binding protein. 3-Azi-1-methoxybutyl alpha-D-[3H]maltotrioside was bound by the maltose-binding protein with a Kd of 0.18 mM. Irradiation at 350 nm of purified maltose-binding protein in the presence of 4 microM of this substrate labeled the protein covalently; labeling was prevented by 1 mM maltose. Using a crude preparation of periplasmic proteins two proteins were labeled, the maltose-binding protein and alpha-amylase. Thus, 3-azi-1-methoxybutyl alpha-D-maltooligosaccharides are potent photoaffinity labels for proteins with maltooligosaccharides-binding sites.     

Embryo production through somatic embryogenesis can be used to study cell differentiation in plants

Somatic embryogenesis is the process by which somatic cells, under induction conditions, generate embryogenic cells, which go through a series of morphological and biochemical changes that result in the formation of a somatic embryo. Somatic embryogenesis differs from zygotic embryogenesis in that it is observable, its various culture conditions can be controlled, and a lack of material is not a limiting factor for experimentation. These characteristics have converted somatic embryogenesis into a model system for the study of morphological, physiological, molecular and biochemical events occurring during the onset and development of embryogenesis in higher plants; it also has potential biotechnological applications. The focus of this review is on embryo development through somatic embryogenesis and especially the factors affecting cell and embryo differentiation.     

Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted host-toxic single-chain antibody fragment in Escherichia coli

In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding. High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important. We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible Pm/xylS promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx). Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences. Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields. By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of Escherichia coli.     

A stochastic process determines the time at which cell division begins in Escherichia coli

The theoretical distributions of cell masses in exponential cultures of bacteria were derived for both total cells and cells having formed a constriction in preparation for division. The parameters used for this derivation include the mass doubling time, tau, the T-period, and 3 statistical parameters (h, sigma 1, sigma 2) which describe the variability of the cell cycle. The theoretical distributions were compared with observed distributions from E. coli B/rA growing in glucose minimal medium (45 min doubling time) to determine whether a stochastic process in the division pathway affects the time of initiation of constriction or the duration of the constriction process. The results indicate that the stochastic process determines the onset rather than the completion of constriction and that the timing of this process is coupled (6% variability, = sigma 1) to a given cell mass. The values obtained for the duration of the T-period, T = 9.3 min, and for a half-life parameter associated with the stochastic process, h = 4.3 min, agree with previously reported data.     

Autoencoder based local T cell repertoire density can be used to classify samples and T cell receptors

Recent advances in T cell repertoire (TCR) sequencing allow for the characterization of repertoire properties, as well as the frequency and sharing of specific TCR. However, there is no efficient measure for the local density of a given TCR. TCRs are often described either through their Complementary Determining region 3 (CDR3) sequences, or theirV/J usage, or their clone size. We here show that the local repertoire density can be estimated using a combined representation of these components through distance conserving autoencoders and Kernel Density Estimates (KDE). We present ELATE–an Encoder-based LocAl Tcr dEnsity and show that the resulting density of a sample can be used as a novel measure to study repertoire properties. The cross-density between two samples can be used as a similarity matrix to fully characterize samples from the same host. Finally, the same projection in combination with machine learning algorithms can be used to predict TCR-peptide binding through the local density of known TCRs binding a specific target.     

Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome

Microcell-mediated chromosome transfer is a useful technique for the study of gene function, gene regulation, gene mapping, and functional cloning in mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have been developed. These donor cell lines contain a single human chromosome marked with a dominant selectable gene in a rodent cell background. However, a similar panel does not exist for murine chromosomes. To produce mouse monochromosomal donor hybrids, we have utilized embryonic stem (ES) cells with targeted gene disruptions of known chromosomal location as starting material. ES cells with mutations in aprt, fyn, and myc were utilized to generate monochromosomal hybrids with neomycin phosphotransferase-marked murine Chr 8, 10, or 15 respectively in a hamster or rat background. This same methodology can be used to generate a complete panel of marked mouse chromosomes for somatic cell genetic experimentaion. Received: 28 July 1998 / Accepted: 15 December 1998     

Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.     

Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli.

Glutathione S-transferase (GST) from Schistosoma japonicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time. For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cystatin, whose binding properties for papain were to be studied. The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin. The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST. Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction. Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose. Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C terminus. The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Furthermore, strong binding activity for papain was detected in an ELISA. The signal with the cystatin-GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST. The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size. Thus, engineered GST is suitable as a moderately sized, secretion-competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity.     

Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis-elements unrelated to canonical translation initiation signals

In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5' neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis-elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome.     

Asymmetric disposal of individual protein aggregates in Escherichia coli, one aggregate at a time

Escherichia coli cells employ an asymmetric strategy at division, segregating unwanted substances to older poles, which has been associated with aging in these organisms. The kinetics of this process is still poorly understood. Using the MS2 coat protein fused to green fluorescent protein (GFP) and a reporter construct with multiple MS2 binding sites, we tracked individual RNA-MS2-GFP complexes in E. coli cells from the time when they were produced. Analyses of the kinetics and brightness of the spots showed that these spots appear in the midcell region, are composed of a single RNA-MS2-GFP complex, and reach a pole before another target RNA is formed, typically remaining there thereafter. The choice of pole is probabilistic and heavily biased toward one pole, similar to what was observed by previous studies regarding protein aggregates. Additionally, this mechanism was found to act independently on each disposed molecule. Finally, while the RNA-MS2-GFP complexes were disposed of, the MS2-GFP tagging molecules alone were not. We conclude that this asymmetric mechanism to segregate damage at the expense of aging individuals acts probabilistically on individual molecules and is capable of the accurate classification of molecules for disposal.     

Expression of the Escherichia coli pmi gene, encoding phosphomannose-isomerase in Zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker.

Wild-type Zymomonas mobilis can utilize only three substrates (sucrose, glucose, and fructose) as sole carbon sources, which are largely converted into ethanol and carbon dioxide. Here, we show that although D-mannose is not used as a growth substrate, it is taken up via the glucose uniport system (glucose facilitator protein) with a Vmax similar to that of glucose. Moreover, D-mannose was phosphorylated by a side activity of the resident fructokinase to mannose-6-phosphate. Fructokinase was purified to homogeneity from an frk-recombinant Z. mobilis strain showing a specific activity of 205 +/- 25 U of protein mg-1 with fructose (K(m), 0.75 +/- 0.06 mM) and 17 +/- 2 U mg-1 (relative activity, 8.5%) with mannose (K(m), 0.65 +/- 0.08 mM). However, no phosphomannoseisomerase activity could be detected for Z. mobilis, and this appeared to be the reason for the lack of growth on mannose. Therefore, we introduced the Escherichia coli gene pmi (manA) in Z. mobilis under the control of a lacIq-Ptac system on a broad-host-range plasmid (pZY507; Cmr). Subsequently, in pmi-recombinant cells of Z. mobilis, phosphomannoseisomerase was expressed in a range of from 3 U (without isopropyl-beta-D-thiogalactopyranoside [IPTG]) to 20 U mg-1 of protein in crude extracts (after IPTG induction). Recombinant cells of different Z. mobilis strains utilized mannose (4%) as the sole carbon source with a growth rate of 0.07 h-1, provided that they contained fructokinase activity. When the frk gene was additionally expressed from the same vector, fructokinase activities of as much as 9.7 U mg-1 and growth rates of as much as 0.25 h-1 were detected, compared with 0.34 h-1 on fructose for wild-type Z. mobilis. Selection for growth on mannose was used to monitor plasmid transfer of pZY507pmi from E. coli to Z. mobilis strains and could replace the previous selection for antibiotic resistance.     

The activities of the Escherichia coli MalK protein in maltose transport, regulation, and inducer exclusion can be separated by mutations

The maltose regulon consists of several genes encoding proteins involved in the uptake and utilization of maltose and maltodextrins. Five proteins make up a periplasmic binding-protein-dependent active transport system. One of these proteins, MalK, contains an ATP-binding site and is thought to couple the hydrolysis of ATP to the accumulation of substrate. Beside its function in transport, MalK has two additional roles: (i) it negatively regulates mal regulon expression and (ii) it serves as the target for regulation of transport activity by enzyme IIIGlc of the phosphotransferase system. To determine whether the three functions of MalK are separable, we have isolated and characterized three classes of malK mutations. The first type (class I) exhibited constitutive mal gene expression but still allowed normal transport of maltose; the second type (class II) lacked the ability to transport maltose but retained the ability to repress the mal genes. Class I mutations were localized in the last third of the gene, at amino acids 267 (Trp to Gly) and 346 (Gly to Ser). Mutations of class II were found at the positions 137 (Gly to Ala), 140 (delta Gln Arg), and 158 (Asp to Asn). These mutations are near or within the region of MalK that exhibits extensive homology to the B site of an ATP-binding fold. In addition, site-directed mutagenesis was used to add or remove one amino acid in the A site of the ATP-binding fold. Plasmids carrying these mutations also behaved as class II mutants. The third class of malK mutations resulted in resistance to the enzyme IIIGlc-mediated inhibitory effects of alpha-methylglucoside. These mutations did not interfere with the regulatory function of MalK. One of these mutations (exchanging a serine at position 282 for leucine) is located in a short stretch of amino acids that exhibits homology to a sequence in the Escherichia coli Lac permease in which alpha-methylglucoside-resistant mutations have been found.     

A novel high-copy plasmid, pEC, compatible with commonly used Escherichia coli cloning and expression vectors

A 66164-bp cryptic plasmid, pEIB1, was isolated from strain Vibrio anguillarum MVM425 and sequenced. A plasmid carrying a 1089-bp fragment, containing the minimal replication region of pEIB1, a kanamycin-resistance marker and an l-arabinose promoter, designated pEC, was maintained as a high copy plasmid in E. coli and stably inherited in the absence of antibiotic selection. Significantly, pEC was compatible with the widely used ColE1, pSC101 and p15A replicons making it a useful tool for a dual-plasmid expression system.