Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.     

Pilus formation and protein secretion by the same machinery in Escherichia coli

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram-negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus-like bundles. Assembly of the 'secreton pilus' required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane-associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K-12 gene ppdD, led to secreton-dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus-like structures.     

Topology of microtubules and actin in the life cycle of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The morphology of budding and conjugating cells and associated changes in microtubules and actin distribution were studied in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by phase-contrast and fluorescence microscopy. The non-budding interphase cell showed a nucleus situated in the central position and bundles of cytoplasmic microtubules either stretching parallel to the longitudinal cell axis or randomly distributed in the cell; none of these, however, had a character of astral microtubules. During mitosis, the nucleus divided in the daughter cell, cytoplasmic microtubules disappeared and were replaced by a spindle. The cytoplasmic microtubules reappeared after mitosis had finished. Actin patches were present both in the bud and the mother cell. Cells were induced to mate by transfer to ribitol- containing medium without nitrogen. Partner cells fused by conjugation projections where actin patches had been accumulated. Cell fusion resulted in a zygote that produced a basidium with parallel bundles of microtubules extended along its axis and with actin patches concentrated at the apex. The fused nucleus moved towards the tip of the basidium. During this movement, nuclear division was taking place; the nuclei were eventually distributed to basidiospores. Mitochondria appeared as vesicles of various sizes; their large amounts were found, often lying adjacent to microtubules, in the subcortical cytoplasm of both vegetative cells and zygotes.     

Detection of intracellular phosphatidylserine in living cells

To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.     

Disulfide bond formation in secreton component PulK provides a possible explanation for the role of DsbA in pullulanase secretion

When expressed in Escherichia coli, the 15 Klebsiella oxytoca pul genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these pul genes, efficient pullulanase secretion also requires the host dsbA gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by a dsbA mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a dsbA mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.     

Development and use of chlorotetracycline fluorescence as a measurement assay of chloroplast envelope-bound mg

Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg²⁺ bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cell and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg²⁺ was altered by pretreatment with Mg²⁺ and divalent cation chelating agents and by additions of Mg²⁺ to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg²⁺ can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with ²⁸Mg²⁺ confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg²⁺. It was concluded that changes in Mg²⁺ associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg²⁺ may be exclusively due to increased envelope-associated Mg²⁺. The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg²⁺ inhibit photosynthetic capacity. This inhibition can be partially overcome by the presence of K⁺ in the photosynthetic reaction media.     

Structure and assembly of the pseudopilin PulG

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.     

Membrane Association and Multimerization of Secreton Component PulC

The PulC component of the Klebsiella oxytoca pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.     

Type IV-like pili formed by the type II secreton: specificity,composition, bundling,polar localization,and surface presentation of peptides

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).     

Chromosomes and their relationship to nuclear components during the cell cycle in Chinese hamster cells

Summary Chromosomes and their relationship to nuclear components during various phases of the cell cycle were studied with different fixation, embedding, and enzyme techniques. The results showed that interphase chromosomes may have oriented in such a way that a given locus became associated with the nuclear membrane. Some chromosomes also appeared to interact with the nucleolus. The nuclear matrix materials, however, were distributed between the chromosomes and formed a delineating boundary for the chromosomes. These matrix materials, furthermore, formed channel-like structures within the nucleus and towards the cytoplasm through their interaction with nuclear pore complexes. During mitosis, chromosomes were encapsulated with material that appeared to be derived from the matrix, disintegrated residues and fragments of the nuclear envelope, the lamina, and nucleolar material. These chromosome-associated materials seen in mitosis appeared to serve as foci for formation of new nuclear components in subsequent interphase.     

Distribution of protein A on the surface of Staphylococcus aureus

Surface proteins of Staphylococcus aureus fulfill many important roles during the pathogenesis of human infections and are anchored to the cell wall envelope by sortases. Although the chemical linkage of proteins to cell wall cross bridges is known, the mechanisms whereby polypeptides are distributed on the staphylococcal surface have not been revealed. We show here that protein A, the ligand of immunoglobulin, is unevenly distributed over the staphylococcal surface. Upon removal with trypsin, newly synthesized polypeptide is deposited at two to four discrete foci. During subsequent growth, protein A appears to be slowly distributed from these sites. When viewed through multiple focal planes by laser scanning microscopy, protein A foci are arranged in a circle surrounding the bacterial cell. This pattern of distribution requires the LPXTG sorting signal of protein A as well as sortase A, the transpeptidase that anchors polypeptides to cell wall cross bridges. A model is presented whereby protein A deposition at discrete sites coupled with cell wall synthesis enables distribution of protein A on the staphylococcal surface.     

Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa

In Gram-negative bacteria, most of the sec-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of 12 Xcp proteins (XcpA and XcpP to XcpZ) organized as a multiproteic complex within the envelope. In this study, by a co-purification approach using a His-tagged XcpZ as a bait, XcpY and XcpZ were found associated together to constitute the most stable functional unit so far isolated from the P. aeruginosa secreton. This subcomplex was also found to interact with XcpR and XcpS to form a XcpRSYZ complex which was isolated under native conditions. Another component, XcpP was not found to be associated to the complex but the results suggest that it can transiently interact with the XcpYZ subcomplex in vivo.     

Dynamic association of topoisomerase II to the mitotic chromosomes in live cells of Aspergillus nidulans

DNA topoisomerase II (Topo II) is an essential enzyme that catalyzes topological changes of DNA and consists of a major member of mitotic chromosomes. To investigate the dynamic localization of Topo II in nuclei, we engineered the strain of Aspergillus nidulans expressing Topo II fused with green fluorescent protein (GFP). Time-lapse microscopy revealed that the distribution of Topo II-GFP in nuclei varied depending on the cell cycle. In interphase, Topo II-GFP distributed evenly in the nucleoplasm and at the onset of G2 phase became concentrated into nucleolus. During mitosis, Topo II-GFP accumulated on chromosomes, when the chromosomes condensed. In the early mitosis, the Topo II also showed a single or two brighter spots among the fluorescence of clumped chromosomes. The spots once divided into several spots and then concentrated again into a spot per nucleus in the dividing nuclei of anaphase. Along with the subsequent decondensation of chromosomes, Topo II diffused back into nucleoplasm.     

The pseudorabies virus US3 protein is a component of primary and of mature virions

Herpesviruses acquire a primary envelope by budding of capsids at the inner leaflet of the nuclear membrane. They then traverse into the cytoplasm after fusion of the primary envelope with the outer leaflet of the nuclear membrane. In the alphaherpesvirus pseudorabies virus (PrV), the latter process is impaired when the US3 protein is absent. Acquisition of final tegument and envelope occurs in the cytoplasm. Besides the capsid components, only the UL31 and UL34 gene products of PrV have unequivocally been shown to be part of primary enveloped virions, whereas they lack several tegument proteins present in mature virions (reviewed by T. C. Mettenleiter, J. Virol. 76:1537-1547, 2002). Using immunoelectron microscopy, we show that the US3 protein is present in primary enveloped as well as in mature virions. It is also detectable in intracytoplasmic inclusions produced in the absence of other viral tegument components or envelope-associated glycoproteins. In particular, inclusions formed in the absence of the inner tegument protein UL37 contained the US3 protein. Thus, the US3 protein is a tegument component of both forms of enveloped alphaherpes virions. We hypothesize that US3 protein in primary virions modulates deenvelopment at the outer leaflet of the nuclear membrane and is either lost from primary virions during nuclear egress and subsequently reacquired early during tegumentation or is retained during transit of the nucleocapsid through the nuclear membrane.     

Cloning and mutagenetic modification of the firefly luciferase gene and its use for bioluminescence microscopy of engrailed expression during Drosophila metamorphosis

Bioluminescence microscopy is an area attracting considerable interest in the field of cell biology because it offers several advantages over fluorescence microscopy, including no requirement for excitation light and being phototoxicity free. This method requires brighter luciferase for imaging; however, suitable genetic resource material for this purpose is not available at present. To achieve brighter bioluminescence microscopy, we developed a new firefly luciferase. Using the brighter luciferase, a reporter strain of Drosophila Gal4-UAS (Upstream Activating Sequence) system was constructed. This system demonstrated the expression pattern of engrailed, which is a segment polarity gene, during Drosophila metamorphosis by bioluminescence microscopy, and revealed drastic spatiotemporal change in the engrailed expression pattern during head eversion in the early stage of pupation.     

Changes in the phosphorylation and distribution of vinculin during nerve growth factor induced neurite outgrowth

The mechanism of neurite initiation and elongation was studied using nerve growth factor (NGF) treatment of PC12 cells. The distribution of focal adhesion sites and of the cytoskeletal protein vinculin was determined in large, fused, multinucleated PC12 cells. In the absence of NGF, focal adhesion sites as seen by interference reflection microscopy were restricted to the cell periphery in a regular distribution. Vinculin assemblies (foci), observed by indirect immunofluorescence microscopy using affinity purified anti-vinculin antibodies, were restricted to the cell periphery at focal adhesion sites. Within 4 hr after NGF treatment of the cells, the distribution of both vinculin and focal adhesion sites began to change. Focal adhesion sites became restricted to discrete protruding portions of the cell periphery. Larger, brighter vinculin foci appeared at the tips of the cell margin extensions, concomitant with the loss of foci at locations between the protrusions. As neurites elongated focal adhesion sites and vinculin foci remained with the tips of the growth cone extensions. Both focal adhesion sites and vinculin foci were rarely seen in the perikarya of cells with elongating neurites, and these were always confined to extended portions of the cell body margin. Occasionally, vinculin foci could be seen at the proximal portion of the neurite, at bending elbows, and at discrete expansions along the length. By immunoprecipitation of vinculin from 32P-labeled cells, vinculin phosphorylation was found to be increased within 1 hr of NGF treatment. The role of vinculin phosphorylation and assembly in the formation and directional elongation of neuritic processes in response to NGF is discussed.     

IMMUNOLOCALIZATION OF CENTRIN DURING FERTILIZATION AND THE FIRST CELL CYCLE IN FUCUS DISTICHUS AND PELVETIA COMPRESSA (FUCALES,PHAEOPHYCEAE)1

Antibodies that recognize the centrosome-associated protein centrin were used to characterize centrosomal origin and positioning during fertilization and the first cell cycle in Fucus distichus subsp. evanescens (C. Agardh) Powell and Pelvetia compressa (J. Agardh) De Toni. Centrin was identified in sperm, eggs, and zygotes on protein blots, indicating the protein is present in both gametes. Using immunofluorescence microscopy, centrin was found in discrete foci in sperm. In contrast, eggs lack centrosomes and centrin was not detectable by immunofluorescence, indicating that centrin was probably dispersed in the cytoplasm. Two foci of centrin were present on the nuclear envelope of zygotes, but microtubules remained dispersed over the zygotic nucleus. Centrin foci separated over the nuclear envelope as the first cell cycle progressed. Microtubules became concentrated at the centrin foci to form centrosomes that gave rise to the spindle poles at mitosis.     

Localization of Parental Deoxyribonucleic Acid from Superinfecting T4 Bacteriophage in Escherichia coli

High-resolution autoradiography has been employed to localize the nonsolubilized but genetically excluded deoxyribonucleic acid (DNA) of T4 bacteriophage superinfecting endonuclease I-deficient Escherichia coli. This DNA was found to be associated with the cell envelope (this term is used here to include all cellular components peripheral to and including the cytoplasmic membrane); in contrast, T4 DNA in primary infected cells, like host DNA in uninfected E. coli, was found to be near the cell center. The envelope-associated DNA from super-infecting phage was not located on the outermost surface of the cell since it was insensitive to deoxyribonuclease added to the medium. These results suggest that DNA from superinfecting T-even phage is trapped within the cell envelope.     

Root plasticity of native and invasive Great Basin species in response to soil nitrogen heterogeneity

Soil nutrients are heterogeneously distributed in natural systems. While many species respond to this heterogeneity through root system plasticity, little is known about how the magnitude of these responses may vary between native and invasive species. We quantified root morphological and physiological plasticity of co-occurring native and invasive Great Basin species in response to soil nitrogen heterogeneity and determined if trade-offs exist between these foraging responses and species relative growth rate or root system biomass. The nine study species included three perennial bunchgrasses, three perennial forbs, and three invasive perennial forbs. The plants were grown in large pots outdoors. Once a week for 4 weeks equal amounts of ¹⁵NH₄ ¹⁵NO₃ were distributed in the soil either evenly through the soil profile, in four patches, or in two patches. All species acquired more N in patches compared to when N was applied evenly through the soil profile. None of the species increased root length density in enriched patches compared to control patches but all species increased root N uptake rate in enriched patches. There was a positive relationship between N uptake rate, relative growth rate, and root system biomass. Path analysis indicated that these positive interrelationships among traits could provide one explanation of how invasive forbs were able to capture 2 and 15-fold more N from enriched patches compared to the native grasses and forbs, respectively. Results from this pot study suggest that plant traits related to nutrient capture in heterogeneous soil environments may be positively correlated which could potentially promote size-asymmetric competition belowground and facilitate the spread of invasive species. However, field experiments with plants in different neighbor environments ultimately are needed to determine if these positive relationships among traits influence competitive ability and invader success.     

Flow cytometric measurements and electron microscopy of cell surface glycosaminoglycans using Acridine Orange

Summary Cell surface glycosaminoglycans (GAGs) were measured, after various treatments, by their binding to Acridine Organge using flow cytometry. Using a critical electrolyte concentration and combining it with specific degradation of individual GAG elements, it was found possible to differentiate between GAG components. The technique was adapted for electron microscopy level to reveal characteristics of membrane-associated GAG. By this means, the cell membrane of the human leukaemic cell line K562 was shown to contain a large amount of GAG; 75% of it was highly sulphated GAG, mostly heparan sulphate. This component was evenly distributed in the outer plasma membrane layer. In the presence of other GAGs, the appearance of complex proteoglycan granules was detected.