PubMed Health⌕ Search

Biomedical subjects

S Molin

Publications and source records attributed to S Molin.

At least 73 records · Page 4Linked to original sources
PubMed1996

Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling.

Acylated homoserine lactones (AHLs) play a widespread role in intercellular communication among bacteria. The Australian macroalga Delisea pulchra produces secondary metabolites which have structural similarities to AHL molecules. We report here that these metabolites inhibited AHL-controlled processes in prokaryotes. Our results suggest that the interaction between higher organisms and their surface-associated bacteria may be mediated by interference with bacterial regulatory systems.

4-Butyrolactone↗
PubMed1995

Induction of phospholipase- and flagellar synthesis in Serratia liquefaciens is controlled by expression of the flagellar master operon flhD.

When a liquid culture of Serratia spp. reaches the last part of the logarithmic phase of growth it induces the synthesis of several extracellular hydrolytic enzymes. In this communication we show that synthesis and secretion of the extracellular phospholipase is coupled to expression of flagella. Expression of flagella is demonstrated to follow a growth-phase-dependent pattern. Cloning, complementation studies and DNA-sequencing analysis has identified a genetic region in Serratia liquefaciens which exhibits extensive homology to the Escherichia coli flhD flagellar master operon. Interruption of the chromosomal flhD operon in S. liquefaciens results in non-flagellated and phospholipase-negative cells, but the synthesis of other exoenzymes is not affected. By placing the flhD operon under the control of a foreign inducible promoter we have shown that increased transcription through the flhD operon leads to induction of flagellar synthesis and phospholipase expression.

Amino Acid Sequence↗
PubMed1995

Survival of, and induced stress resistance in, carbon-starved Pseudomonas fluorescens cells residing in soil.

We investigated the survival, cell length, and development of general stress resistance in populations of Pseudomonas fluorescens R2f and its rifampin-resistant mutant, R2f Rpr, following exposure to carbon starvation conditions in liquid cultures and residence in two different soils, Flevo silt loam (FSL) and Ede loamy sand (ELS). In much the same way as was recently shown for P. putida KT2442, carbon-starved P. fluorescens R2f populations revealed enhanced resistance to otherwise lethal treatments, such as exposure to ethanol, high temperature, osmotic tension, and oxidative stress. A large population of nonculturable P. fluorescens R2f Rpr cells arose shortly after their introduction into ELS soil, whereas the formation of nonculturable cells was not observed in FSL soil. Also, the inoculant cell (based on immunofluorescence) and CFU counts decreased faster in ELS soil than in FSL soil. Introduction of carbon-starved instead of exponential-growth-phase R2f Rpr cells into ELS soil did not affect bacterial survival. The inoculant cell length decreased in soil, and no large differences in cell length in the two soil types were observed. Addition of glucose to ELS soil resulted in a stable cell length of R2f Rpr cells, whereas carbon-starved cells introduced into ELS soil remained small. Exponentially growing R2f Rpr cells developed enhanced resistance to ethanol, high temperature, osmotic tension, and oxidative stress within 1 day in both soils, whereas cells introduced into ELS soil amended with glucose showed decreased resistance. Cells that were carbon starved prior to introduction into ELS soil showed unchanged stress resistance levels upon residence in soil.

Carbon↗
PubMed1995

A stochastic killing system for biological containment of Escherichia coli.

Bacteria with a stochastic conditional lethal containment system have been constructed. The invertible switch promoter located upstream of the fimA gene from Escherichia coli was inserted as expression cassette in front of the lethal gef gene deleted of its own natural promoter. The resulting fusion was placed on a plasmid and transformed to E. coli. The phenotype connected with the presence of such a plasmid was to reduce the population growth rate with increasing significance as the cell growth rate was reduced. In very fast growing cells, there was no measurable effect on growth rate. When a culture of E. coli harboring the plasmid comprising the containment system is left as stationary cells in suspension without nutrients, viability drops exponentially over a period of several days, in contrast to the control cells, which maintain viability nearly unaffected during the same period of time. Similar results were obtained with a strain in which the killing cassette was inserted in the chromosome. In competition with noncontained cells during growth, the contained cells are always outcompeted. Stochastic killing obtained by the fim-gef fusion is at present relevant only as a containment approach for E. coli, but the model may be mimicked in other organisms by using species-specific stochastic expression systems.

Cloning, Molecular↗
PubMed1995

Bacterial growth on surfaces: automated image analysis for quantification of growth rate-related parameters.

A fast routine method for estimating bacterial cell growth rates by using the metachromatic dye acridine orange is described. The method allows simultaneous estimates of cellular RNA and DNA contents of single cells. Acridine orange staining can be used as a nonspecific supplement to quantitative species-specific hybridizations with fluorescence-labelled ribosomal probes to estimate the single-cell concentration of RNA. By automated analysis of digitized images of stained cells, we determined four independent growth rate-related parameters: cellular RNA and DNA contents, cell volume, and the frequency of dividing cells in a cell population. These parameters were used to compare physiological states of liquid-suspended and surface-growing Pseudomonas putida KT2442 in chemostat cultures. The major finding is that the correlation between substrate availability and cellular growth rate found for the free-living cells was not observed for the surface-bound cells; in contrast, the data indicate an almost constant growth rate for attached cells which was independent of the dilution rate in the chemostat.

Journal Article↗
PubMed1995

Application of a strain-specific rRNA oligonucleotide probe targeting Pseudomonas fluorescens Ag1 in a mesocosm study of bacterial release into the environment.

Sequence analysis of domains 3 and 4 of 23S rRNA from Pseudomonas fluorescens Ag1 was carried out to allow the design of a strain-specific rRNA oligonucleotide probe targeting this strain. The specificity of the probe, Ps-Ag1, was assessed by dot blot analysis and whole-cell hybridization, and it was found to be specific for P. fluorescens Ag1. The correlation between the ribosomal content of P. fluorescens Ag1 and growth rate was determined during balanced growth conditions with generation times ranging from 1.2 to 31.8 h. Hybridization of the rRNA-targeting probes combined with charged coupled device-enhanced microscopy was used to determine the rRNA content. The total RNA content per cell was determined by staining with acridine orange and charged coupled device-enhanced microscopy. After 2 h under carbon starvation conditions, the rRNA content per cell decreased to 45% of the content of an exponentially growing cell. After 1 day of carbon starvation, the rRNA content had decreased to 20%. When cells were grown at different temperatures, it was found that the rRNA content per cell was only dependent on the substrate in the temperature range from 5 to 30 degrees C. P. fluorescens Ag1 was used in a mesocosm release experiment. The strain could be detected by use of the oligonucleotide probe targeting rRNA for 8 days in the water column and for 10 days on solid surfaces. The standard curve correlating growth rate with rRNA content was used to estimate the physiological activity of P. fluorescens Ag1 in the mesocosm experiment.

Base Sequence↗
PubMed1995

Construction and behavior of biologically contained bacteria for environmental applications in bioremediation.

The survival of microorganisms can be predicted through the use of active biological containment systems. We have constructed contained Pseudomonas putida strains that degrade alkylbenzoates. The modified strain carries a fusion of the Plac promoter to the gef gene, which encodes a killing protein. Expression from Plac is controlled through a regulatory cascade, so that Plac is switched on or off by the absence or presence of alkylbenzoates, respectively. Similar uncontained strains were also constructed and tested as a control. Contained and uncontained strains were genetically stable, and their survival and functionality in soil microcosms were as expected. Both contained and uncontained strains survived well in soils supplemented with alkylaromatics, whereas survival of the contained strain in soil microcosms without methylbenzoates was markedly reduced, in contrast to the control strain, which survived in these soils in the absence of alkylbenzoates. The TOL plasmid was transferred in soils between Pseudomonas strains but was not able to mobilize the elements of the containment system.

Bacteria↗
PubMed1995

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene. The high frequencies of marker excision obtained with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event. Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida. This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.

Bacterial Proteins↗
PubMed1995

Physiological state of Escherichia coli BJ4 growing in the large intestines of streptomycin-treated mice.

Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static.

Animals↗
PubMed1994

The behavior of bacteria designed for biodegradation.

Mineralization of organic molecules by microbes is essential for the carbon cycle to operate. The massive mobilization of compounds stored in natural resources, or the introduction of xenobiotics into the biosphere, leads to unidirectional fluxes, which result in the persistance of a number of chemicals in the biosphere, and thus constitute a source of pollution. Molecular biology offers the tools to optimize the biodegradative capacities of microorganisms, accelerate the evolution of "new" activities, and construct totally "new" pathways through the assemblage of catabolic segments from different microbes. Although the number of genetically engineered microbes (GEMs) for potential use in biodegradation is not large, these recombinant microbes function in microcosms according to their design. The survival and fate of recombinant microbes in different ecological niches under laboratory conditions is similar to what has been observed for the unmodified parental strains. rDNA, both on plasmids and on the host chromosome, is usually stably inherited by GEMs. The potential lateral transfer of rDNA from the GEMs to other microbes is significantly diminished, though not totally inhibited, when rDNA is incorporated on the host chromosome. The behavior and fate of GEMs can be predicted more accurately through the coupling of regulatory circuits that control the expression of catabolic pathways to killing genes, so that the GEMs survive in polluted environments, but die when the target chemical is eliminated.

Bacteria↗
PubMed1994

Spatial distribution of Escherichia coli in the mouse large intestine inferred from rRNA in situ hybridization.

Fluorescent oligonucleotide probes targeting rRNA were used to develop an in situ hybridization technique by which the spatial distribution of Escherichia coli in the large intestines of streptomycin-treated mice was determined. Single E. coli cells were identified in thin frozen sections from the large intestines by the use of a probe specific for E. coli 23S rRNA. Furthermore, the total bacterial population was visualized with an rRNA probe targeting the domain Bacteria. By this technique, all E. coli cells were seen embedded in the mucosal material overlying the epithelial cells of the large intestine, and no direct attachment to the epithelium was observed.

Animals↗
PubMed1994

Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content.

The physiology of Pseudomonas putida KT2442 with respect to growth and carbon starvation was studied. During the transition from growth to nongrowth, the cell shape changes from cylindrical to spheric, a change which is accompanied by reductions in cell size, DNA and ribosome content, and the rate of total protein synthesis. In addition, a pattern of general cross-protection develops, which enables the cells to survive environmental stresses such as high and low temperatures, elevated osmolarity, solvents, and oxidative agents. Cultures are almost fully viable during 1 month of carbon, nitrogen, and multiple-nutrient starvation and are considered to be in an active nondormant state. In contrast, strain KT2442 does not survive well under conditions of sulfate and phosphate starvation.

Adaptation, Physiological↗
PubMed1994

Responses to nutrient starvation in Pseudomonas putida KT2442: two-dimensional electrophoretic analysis of starvation- and stress-induced proteins.

The responses of Pseudomonas putida KT2442 to various forms of nutrient starvation and stress conditions were examined by two-dimensional polyacrylamide electrophoresis. Carbon deprivation resulted in a temporal expression of two classes of starvation-induced proteins: one class was transiently expressed during the initial phase of starvation, and the second class was expressed throughout the entire starvation period. Proteins of the second class could be further subdivided into proteins induced specifically under conditions of carbon starvation, proteins also induced by conditions of stress created by elevated temperature and osmolarity, and finally proteins that were also induced by conditions of nitrogen as well as phosphate starvation. Addition of glucose to a carbon-starved culture led to initiation of a recovery phase. During this phase, repression of starvation-induced proteins as well as induction of a new class of transiently expressed proteins, referred to as maturation proteins, took place.

Bacterial Proteins↗
PubMed1993

Environmental potential of suicide genes.

Environmental applications of genetically engineered microorganisms have caused concern among scientists, the authorities and the general public. Despite the many potential benefits offered by gene technology to agriculture, environment protection and the medical sector, only a few cases of released engineered bacteria have been permitted. In this review, the design of safer organisms for release purposes is discussed with specific emphasis on the use of suicide systems to limit the survival of bacteria in the environment.

Animals↗
PubMed1993

Secretion of Serratia liquefaciens phospholipase from Escherichia coli.

The Serratia liquefaciens phospholipase (PhlA) is secreted to the medium from its natural host. Here we present results which indicate that, when cloned and expressed in Escherichia coli, secretion can be mediated by a putative host-encoded pathway, expression of which is controlled by FlhD (formerly FlbB), the master regulator of the flagellar/chemotaxis regulon. In the absence of this secretion pathway, the synthesized phospholipase accumulates inside the host cell where it forms a complex with the PhlB protein. PhlB, which is encoded from the promoter distal gene of the phospholipase operon, inhibits the phospholipase activity of PhlA. Formation of this enzymatically inactive PhlA/PhlB complex is required for maintenance of cell viability.

Amino Acid Sequence↗
PubMed1993

A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene.

A model substrate-dependent suicide system to biologically contain Pseudomonas putida KT2440 is reported. The system consists of two elements. One element carries a fusion between a synthetic lac promoter (PA1-04/03) and the gef gene, which encodes a killing function. This element is contained within a transposaseless mini-Tn5 transposon so that it can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to control the first and bears a fusion between the promoter of the P. putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene, encoding the Lac repressor, plus xylS2, coding for a positive regulator of Pm. In liquid culture under optimal growth conditions and in sterile and nonsterile soil microcosms, P. putida KT2440 (pWWO) bearing the containment system behaves as designed. In the presence of a XylS effector, such as m-methylbenzoate, the LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the PA1-04/03::gef cassette is no longer prevented and a high rate of cell killing is observed. Fluctuation test analyses revealed that mutants resistant to cell killing arise at a frequency of around 10(-5) to 10(-6) per cell per generation. Mutations are linked to the killing element rather than to the regulatory one. In bacteria bearing two copies of the killing cassette, the rate of appearance of mutants resistant to killing decreased to as low as 10(-8) per cell per generation.

Benzoates↗
PubMed1993

Identification of coccoid Escherichia coli BJ4 cells in the large intestine of streptomycin-treated mice.

Escherichia coli BJ4, a rat isolate, was used to examine the growth and differentiation of the microorganism in its natural habitat, the intestine. Growth of E. coli BJ4 in the large intestine of streptomycin-treated mice was compared with its growth in laboratory media. By a number of methods, it was shown that E. coli BJ4 differentiates, during growth in the intestine, into two distinct populations, one that has the characteristics of the laboratory-grown strain and one that appears as a coccoid cell. Furthermore, it was shown that there is a natural selection for the coccoid-type cell in the intestine, while in laboratory media growth of rod-shaped E. coli BJ4 is enhanced.

Animals↗