Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Carrageenans with complex substitution patterns from red algae of the genus Erythroclonium

Extracellular polysaccharides from three Erythroclonium spp. were shown, by a combination of compositional, linkage analyses, and Fourier transform infrared and C-13-nuclear magnetic resonance spectroscopy, to be highly substituted carrageenans with at least five types of repeating disaccharide units. These are the carrabiose 2,4'-disulfate of iota-carrageenan, carrabiose 2-sulfate of alpha-carrageenan, the 6'-O-methylated counterparts of each of these repeating units, and 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate. The polysaccharides also contain significant amounts of unsubstituted, 4-linked galactopyranose and small amounts of 4-linked 3-O-methylgalactopyranose and terminal glycosyl residues. The carrageenan preparations of the three species are similar, differing only in the proportions of some components. (C) 1998 Elsevier Science Ltd.

Carrageenans from Australian representatives of the family Cystocloniaceae (Gigartinales, Rhodophyta), with description of Calliblepharis celatospora sp. nov., and transfer of Austroclonium to the family Areschougiaceae

Ten Australian representatives from seven of the 10 genera presently constituting the family Cystolcloniaceae have been analyzed for their cell-wall galactans. Included in our survey are the monotypic Australian-endemic genera Austroclonium, Gloiophyllis, Erythronaema, and Stictosporum, one species of Craspedocarpus, three species of Rhodophyllis, and two species of Calliblepharis. As one of the species of the latter genus is endemic to Western Australia and presently undescribed, we illustrate its habit and anatomical features in formally proposing to name it Calliblepharis celatospora Kraft, sp. nov. All the species surveyed essentially produce typical iota (iota)-carrageenans, with the exception of Austroclonium. The sulfated galactans from Austroclonium predominantly contain the repeating units of iota-, alpha (alpha)-, and 6'-O-methylated iota- and alpha-carrageenans; whether these exist as discrete polysaccharides or a complex hybrid structure was not resolved. Thus, Austroclonium carrageenans resemble the polysaccharides from Rhabdonia, Areschougia, and Erythroclonium. Although these latter three genera are currently included in the large gigartinalean family Solieriaceae, all produce significantly different carrageenans from Solieria itself and related genera such as Eucheuma, Kappaphycus, Betaphycus, Sarcodiotheca, Agardhiella, Sarconema, and Callophycus. In consideration of these findings, as well as of significant anatomical similarities, we provisionally recommend reestablishment of the family Rhabdoniaceae Kylin (as the family Areschougiaceae J. Agardh) for Rhabdonia, Areschougia, Erythroclonium, and Austroclonium.

Membrane-bounded nucleoids in microbial symbionts of marine sponges

In thin sections of resin-embedded samples of glutaraldehyde- and osmium tetroxide-fixed tissue from five genera of marine sponges, Stromatospongia, Astrosclera, Jaspis, Pseudoceratina and Axinyssa, cells of a bacteria-like symbiont microorganism which exhibit a membrane-bounded nuclear region encompassing the fibrillar nucleoid have been observed within the sponge mesohyl. The nuclear region in these cells is bounded by a single bilayer membrane, so that the cell cytoplasm is divided into two distinct regions. The cell wall consists of subunits analogous to those in walls of some Archaea. Cells of the sponge symbionts observed here are similar to those of the archaeal sponge symbiont Cenarchaeum symbiosum. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Novel nano-organisms from Australian sandstones

We report the detection of living colonies of nano-organisms (nanobes) on Triassic and Jurassic sandstones and other substrates. Nanobes have cellular structures that are strikingly similar in morphology to Actinomycetes and fungi (spores, filaments, and fruiting bodies) with the exception that they are up to 10 times smaller in diameter (20 nm to 1.0 mu m). Nanobes are noncrystalline structures that are composed of C, O, and N. Ultra thin sections of nanobes show the existence of an outer layer or membrane that may represent a cell wall. This outer layer surrounds an electron dense region interpreted to be the cytoplasm and a less electron dense central region that may represent a nuclear area. Nanobes show a positive reaction to three DNA stains, [4',6-diamidino-2 phenylindole (DAPI), Acridine Orange, and Feulgen], which strongly suggests that nanobes contain DNA. Nanobes are communicable and grow in aerobic conditions at atmospheric pressure and ambient temperatures. While morphologically distinct, nanobes are in the same size range as the controversial fossil nannobacteria described by others in various rock types and in the Martian meteorite ALH84001.

Pyruvated carrageenans from Solieria robusta and its adelphoparasite Tikvahiella candida

Solieria, the type genus of the commercially important red algal family Solieriaceae (Gigartinales), contains seven or eight species, three of which are represented in Australia. The cell-wall galactans of the most common Australian Solieria species, S. robusta (Greville) Kylin, were analysed by a combination of compositional assays, linkage analysis, and Fourier transform infrared (FTIR) and C-13 nuclear magnetic resonance (NMR) spectroscopy. They are shown to be composed predominantly of carrabiose 2,4'-disulphate units (the repeating unit of iota-carrageenan) and a significant proportion of 4',6'-pyruvated carrabiose 2-sulphate units. The constituent sugars, pyruvate content, FTIR spectrum, and linkage and substitution patterns of the galactans from Tikvahiella candida Kraft et Gabrielson, an adelphoparasite of Solieria robusta, closely resemble those of its host and furnish evidence in support of a close phylogenetic relationship between the two species.

Friedmanniella spumicola sp nov and Friedmanniella capsulata sp nov from activated sludge foam: Gram-positive cocci that grow in aggregates of repeating groups of cocci

Two Gram-positive, non-motile, non-spore-forming, strictly aerobic, pigmented cocci, strains Ben 107(T) and Ben 108(T), growing in aggregates were isolated from activated sludge samples by micromanipulation. Both possessed the rare type A3 gamma' peptidoglycan. Major menaquinones of strain Ben 107(T) were MK-9(H-4) and MK-7(H-2), and the main cellular fatty acid was 12-methyltetradecanoic acid (ai-C-15:0). In strain Ben 108(T), MK-9(H-4), MK-9(H-2) and MK-7(H-4) were the menaquinones and again the main fatty acid was 12-methyltetradecanoic acid (ai-C-15:0). Polar lipids in both strains consisted of phosphatidyl inositol, phosphatidyl glycerol and diphosphatidyl glycerol with two other unidentified glycolipids and phospholipids also present in both. These data, together with the 16S rDNA sequence data, suggest that strain Ben 107(T) belongs to the genus Friedmanniella which presently includes a single recently described species, Friedmanniella antarctica. Although the taxonomic status of strain Ben 108(T) is far less certain, on the basis of its 16S rRNA sequence it is also adjudged to be best placed in the genus Friedmanniella, The chemotaxonomic characteristics and DNA-DNA hybridization data support the view that Ben 107(T) and Ben 108(T) are novel species of the genus Friedmanniella. Hence, it is proposed that strain Ben 107(T) (=ACM 5121(T)) is named as Friedmanniella spumicola sp. nov. and strain Ben 108(T) (=ACM 5120(T)) as Friedmanniella capsulata sp. nov.

The mechanical properties of Saccharomyces cerevisiae

Cell-wall mechanical properties play an integral part in the growth and form of Saccharomyces cerevisiae, In contrast to the tremendous knowledge on the genetics of S. cerevisiae, almost nothing is known about its mechanical properties. We have developed a micromanipulation technique to measure the force required to burst single cells and have recently established a mathematical model to extract the mechanical properties of the cell wall from such data, Here we determine the average surface modulus of the S, cerevisiae cell wall to be 11.1 +/- 0.6 N/m and 12.9 +/- 0.7 N/m in exponential and stationary phases, respectively, giving corresponding Young's moduli of 112 +/- 6 MPa and 107 +/- 6 MPa, This result demonstrates that yeast cell populations strengthen as they enter stationary phase by increasing wall thickness and hence the surface modulus, without altering the average elastic properties of the cell-wall material. We also determined the average breaking strain of the cell wall to be 82% +/- 3% in exponential phase and 80% +/- 3% in stationary phase, This finding provides a failure criterion that can be used to predict when applied stresses (e,g,, because of fluid flow) will lead to wall rupture, This work analyzes yeast compression experiments in different growth phases by using engineering methodology.

Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 mum ceramic membrane successfully recovered the granulocyte macrophagecolony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by infermenter Benzonase(R) digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and re-suspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations. (C) 2004 Wiley Periodicals Inc.

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24 h elicited a marked increase in mRNA expression for IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway.. although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Sulfated galactans from Australian specimens of the red alga Phacelocarpus peperocarpos (Gigartinales, Rhodophyta)

Polysaccharides from the red alga Phacelocarpos peperocarpos were extracted with hot water, clarified, and precipitated with 2-propanol. The native preparation was highly sulfated (36.2% w/w). Alkali modification decreased the sulfate content by 2.0% w/w. The alkali-modified polysaccharide is composed mostly of galactose (Gal, 51 mol%) and 3,6-anhydrogalactose (AnGal, 41 mol%), with minor amounts of a mono-O-methylgalactose (MeGal, 1 mol%), xylose (Xyl, 6 mol%), and glucose (Glc, 1 mol%). The FTIR spectrum of the alkali-modified polysaccharide resembled K-carrageenan with absorption at 930 cm(-1) (indicative of AnGal) and 850 cm(-1) (Gal ii-sulfate). However, an additional, major band of absorption occurred at 820 cm(-1) indicating the presence of equatorial sulfate ester substitution at O-6 of Gal residues, A combination of linkage and C-13 NMR spectroscopic analyses showed that the polysaccharide was composed predominantly of a novel repeating-unit, O-beta-D-galactopyranosyl 4,6-disulfate)-(1 --> 4)-3,6-anhydro-alpha-D-galactopyranose. Minor structural variations also occurred, including alternative patterns of sulfation and the presence of terminal Xylp, The location of the terminal Xylp residues was not certain but evidence supported their attachment at O-3 of some 4-linked Galp residues. The cell-wall galactans remain unchanged during the life cycle of the alga. (C) 1996 Elsevier Science Ltd.

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae, The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented, A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria, Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group, Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species, A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.

Chemical treatment of Escherichia coli .1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetetraacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.

Chemistry, properties, and phylogenetic implications of the methylated carrageenans from red algae of the genus Areschougia (Areschougiaceae, Gigartinales, Rhodophyta)

The three Australian-endemic species comprising the genus Aresehougia have been examined to determine the structure of their nonfibrillar wall components. The polysaccharide extracted from the most widely distributed species, A. congesta (Turner) J. Agardh, was shown by compositional analyses, Fourier transform infrared (FTIR) spectroscopy, linkage analysis, and C-13-NMR spectroscopy to be a carrageenan composed predominantly of the repeating disaccharides 6'-O-methylcarrabiose 2,4'-disulfate, carrabiose 2,4-disulfate (the repeating unit of L-carrageenan), 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate, and 6'-O-methylcarrabiose 2-sulfate. The carrageenan also contained small amounts of 4-linked Galp residues, some bearing methyl ether substitution at O-3 and some possibly bearing sulfate ester and/or glycosyl substitutions at O-3. The A. congesta carrageenan had unique rheological properties, its gels having some similarities to those of commercial iota -carrageenan but with the viscosity of commercial lambda -carrageenan. Polysaccharides from A. ligulata Harvey ex J. Agardh and A. stuartii Harvey were shown by constituent sugar and FTIR analyses to be sulfated galactans rich in mono-O-methylgalactose. The carrageenan structures of Areschougia spp. were consistent with those of the genera Rhabdonia, Erythroclonium, and Austroclonium, the other genera constituting the family Areschougiaceae.