Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa.
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Alginates↗
Biomedical subjects
M Strathmann
Publications and source records attributed to M Strathmann.
Agarose hydrogels as EPS models.
For investigating the influence of extracellular polymeric substances (EPSs) on biofilm properties, artificial models of defined composition and structure can be very helpful. Different immobilised cell systems can be useful in the fitting of experimental results. Two different types of of artificial EPS matrix model were developed earlier. Homogeneous agarose beads (50-500 microns diameter) and porous beads (260 microns mean diameter) containing pores with diameters from 10 to 80 microns (28 microns on average), allowed the embedding of cells, particles and typical EPS matrix components such as proteins and polysaccharides. In this paper, some physico-chemical properties of the artificial EPS matrices were described and compared with results known from natural EPS. The stability of the artificial matrix polymer against solution in the aqueous medium was studied in batch experiments. The water binding and water retaining capabilities of the EPS models were investigated by drying and re-swelling experiments. The simulation of protective effects of the artificial EPS matrix against toxic substances like biocides in comparison to such known protective effects of the EPS of native biofilms were proved by the application of sodium hypochlorite (0.5 mg/l, 30 min) and subsequent microscopic investigation of the cell population after LIVE/DEAD staining (Molecular Probes).
Biofilms↗
Artificial biofilm model--a useful tool for biofilm research.
For biofilm studies, artificial models can be very helpful in studying processes in hydrogels of defined composition and structure. Two different types of artificial biofilm models were developed. Homogeneous agarose beads (50-500 microm diameter) and porous beads (260 microm mean diameter) containing pores with diameters from 10 to 80 microm (28 microm on average) allowed the embedding of cells, particles and typical biofilm matrix components such as proteins and polysaccharides. The characterisation of the matrix structures and of the distribution of microorganisms was performed by confocal laser scanning microscopy. The physiological condition of the embedded bacteria was examined by redox activity (CTC-assay) and membrane integrity (Molecular Probes LIVE/DEAD-Kit). Approximately 35% of the immobilised cells (Pseudomonas aeruginosa SG81) were damaged due to the elevated temperature required for the embedding process. It was shown that the surviving cells were able to multiply when provided with nutrients. In the case of homogeneous agarose beads, cell growth only occurred near the bead surface, while substrate limitation prevented growth of more deeply embedded cells. In the porous hydrogel, cell division was observed across the entire matrix due to better mass transport. It could be shown that embedding in the artificial gel matrix provided protection of immobilized cells against toxic substances such as sodium hypochlorite (0.5 mg/l, 30 min) in comparison to suspended cells, as observed in other immobilized systems. Thus, the model is suited to simulate important biofilm matrix properties.
Biofilms↗
Identification of Dictyostelium G alpha genes expressed during multicellular development.
Guanine nucleotide-binding protein (G protein)-mediated signal transduction constitutes a common mechanism by which cells receive and respond to a diverse set of environmental signals. Many of the signals involved in the developmental life cycle of the slime mold Dictyostelium have been postulated to be transduced by such pathways and, in some cases, these pathways have been demonstrated to be dependent on specific G proteins. Using the polymerase chain reaction, we have identified two additional Dictyostelium G alpha genes, G alpha 4 and G alpha 5, that are developmentally regulated. Transcripts from both of these genes are primarily expressed during the multicellular stages of development, suggesting possible roles in cell differentiation or morphogenesis. The entire G alpha 4 gene was sequenced and found to encode a protein consisting of 345 amino acids. The G alpha 4 subunit is homologous to other previously identified G alpha subunits, including the Dictyostelium G alpha 1 (43% identity) and G alpha 2 (41% identity) subunits. However, the G alpha 4 subunit contains some unusual sequence divergences in residues highly conserved among most eukaryotic G alpha subunits, suggesting that G alpha 4 may be a member of another class of G alpha subunits.
Amino Acid Sequence↗
Transposon-facilitated DNA sequencing.
We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating. A miniplasmid was designed to select against transposon insertions into the vector. Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates. A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced. Sequencing primers corresponding to the transposon ends allow sequencing in both directions. Thus, the entire sequence of both strands can be easily determined.
Base Sequence↗
Alternative splicing produces transcripts encoding two forms of the alpha subunit of GTP-binding protein Go.
The alpha subunit of the guanine nucleotide-binding protein Go ("o" for other) is believed to mediate signal transduction between a variety of receptors and effectors. cDNA clones encoding two forms of Go alpha subunit were isolated from a mouse brain library. These two forms, which we call GoA alpha and GoB alpha, appear to be the products of alternative splicing. GoA alpha differs from GoB alpha over the C-terminal third of the deduced protein sequence. Both forms are predicted to be substrates for ADP-ribosylation by pertussis toxin. GoA alpha transcripts are present in a variety of tissues but are most abundant in brain. The GoB alpha transcript is expressed at highest levels in brain and testis. It is possible that GoA alpha and GoB alpha have different functions.
Amino Acid Sequence↗
G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) are integral to the signal transduction pathways that mediate the cell's response to many hormones, neuromodulators, and a variety of other ligands. While many signaling processes are guanine nucleotide dependent, the precise coupling between a variety of receptors, G proteins, and effectors remains obscure. We found that the family of genes that encode the alpha subunits of heterotrimeric G proteins is much larger than had previously been supposed. These novel alpha subunits could account for some of the diverse activities attributed to G proteins. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha q and G alpha 11, that are 88% identical. They lack the site that is ordinarily modified by pertussis toxin and their sequences vary from the canonical Gly-Ala-Gly-Glu-Ser (GAGES) amino acid sequence found in most other G protein alpha subunits. Multiple mRNAs as large as 7.5 kilobases hybridize to G alpha q specific probes and are expressed at various levels in many different tissues. G alpha 11 is encoded by a single 4.0-kilobase message which is expressed ubiquitously. Amino acid sequence comparisons suggest that G alpha q and G alpha 11 represent a third class of alpha subunits. A member of this class was found in Drosophila melanogaster. This alpha subunit, DG alpha q, is 76% identical to G alpha q. The presence of the Gq class in both vertebrates and invertebrates points to a role that is central to signal transduction in multicellular organisms. We suggest that these alpha subunits may be involved in pertussis toxin-insensitive pathways coupled to phospholipase C.
Amino Acid Sequence↗
Diversity of the G-protein family: sequences from five additional alpha subunits in the mouse.
Biochemical analysis has revealed a number of guanine nucleotide-binding regulatory proteins (G proteins) that mediate signal transduction in mammalian systems. Characterization of their cDNAs uncovered a family of proteins with regions of highly conserved amino acid sequence. To examine the extent of diversity of the G protein family, we used the polymerase chain reaction to detect additional gene products in mouse brain and spermatid RNA that share these conserved regions. Sequences corresponding to six of the eight known G protein alpha subunits were obtained. In addition, we found sequences corresponding to five newly discovered alpha subunits. Our results suggest that the complexity of the G protein family is much greater than previously suspected.
Amino Acid Sequence↗