PubMed Health⌕ Search

Biomedical subjects

M Whiteley

Publications and source records attributed to M Whiteley.

At least 19 recordsLinked to original sources
PubMed2001

Gene expression in Pseudomonas aeruginosa biofilms.

Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms. Some of the regulated genes are known to affect antibiotic sensitivity of free-living P. aeruginosa. Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes. We propose that this response is critical for the development of biofilm resistance to tobramycin. Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences. Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics.

Animals↗
PubMed2001

QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa.

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR-LasI and RhlR-RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.

Amino Acid Sequence↗
PubMed2001

Biochemical confirmation and characterization of the family-57-like alpha-amylase of Methanococcus jannaschii.

The gene encoding a family-57-like alpha-amylase in the hyperthermophilic archaeon Methanococcus jannaschii, has been cloned into Escherichia coli. Extremely thermoactive alpha-amylase was confirmed in partially purified enzyme solution of the recombinant culture. This enzyme activity had a temperature optimum of 120 degrees C and a pH optimum 5.0-8.0. The amylase activity is extremely stable against denaturants. Hydrolysis of large sugar polymers with alpha-1-6 and alpha-1-4 linkages yields products including glucose polymers of 1-7 units. Highest activity is exhibited on amylose. The catalyst exhibited a half-life of 50 h at 100 degrees C, among the highest reported thermostabilities of natural amylases.

Amino Acid Sequence↗
PubMed2001

Novel, thermostable family-13-like glycoside hydrolase from Methanococcus jannaschii.

A novel glycoside hydrolase from the hyperthermophilic archaeon Methanococcus jannaschii has been cloned into Escherichia coli. Extremely thermoactive and thermostable amylolytic activity was confirmed in partially purified enzyme solution. This enzyme exhibited a temperature optimum of 100 degrees C and a pH optimum pH 5.0-8.0. Hydrolysis of large 1,6-alpha- and 1,4-alpha-linked polysaccharides yielded glucose polymers of 1-7 units. Incubation with amylose displayed the highest activity. The catalyst was activated and stabilized by Ca2+ and exhibited extreme thermostability at 100 degrees C with a half-life of 78 h.

Amino Acid Sequence↗
PubMed2001

Effects of community composition and growth rate on aquifer biofilm bacteria and their susceptibility to betadine disinfection.

Biofilm formation and function was studied in mixed culture using 20 bacterial strains isolated from a karst aquifer. When co-cultured in a glucose-limited chemostat, Vogesella indigofera and Pseudomonas putida were the dominant planktonic and biofilm organisms respectively. Biofilm formation and resistance to the iodine disinfectant betadine were then studied with monoculture and binary cultures of V. indigofera and P. putida and a 20-strain community. Biofilm population size [measured as colony-forming units (CFU) cm(-2)] increased with increasing species diversity. Significantly larger populations formed at dilution rates (DRs) of 0.0083 h(-1) than at 0.033 h(-1). P. putida populations were higher and V. indigofera lower in binary than in monoculture biofilms, suggesting that P. putida outcompeted V. indigofera. In binary biofilms, V. indigofera, a betadine-resistant organism, enhanced the survival of P. putida, a betadine-susceptible organism. In the 20-strain biofilms, this protective effect was not observed because of low concentrations of V. indigofera (< 1% of the total population), suggesting that resistant organisms contribute to overall biofilm disinfectant resistance. Growth at 0.033 h(-1) enhanced survival of V. indigofera biofilms against betadine. Although DR did influence survival of the other communities, its effects were neither consistent nor significant. All told, biofilm formation and betadine resistance are complex phenomena, influenced by community composition, growth rate and betadine concentration.

Bacteriological Techniques↗
PubMed2001

Promoter specificity elements in Pseudomonas aeruginosa quorum-sensing-controlled genes.

The LasR-dependent and RhlR-dependent quorum-sensing systems are global regulators of gene expression in Pseudomonas aeruginosa. Previous studies have demonstrated that promoter elements of the quorum-sensing-controlled genes lasB and hcnABC are important in density-dependent regulation. We have identified LasR- and RhlR-dependent determinants in promoters of quorum-sensing-controlled genes qsc102, qsc117 (acpP), and qsc131 (phzA to -G) by in silico, deletion, point-mutational, and primer extension analyses. Each of these genes (in addition to lasI and rsaL) is activated by LasR, and qsc117 and qsc131 also respond to RhlR. Point mutations in the promoters of the LasR-specific gene, qsc102, relax specificity so that this promoter can respond to RhlR in addition to LasR. Our findings indicate that quorum-sensing-controlled promoters in P. aeruginosa are either specific for LasR or respond to both LasR and RhlR and that critical bases in the promoter elements determine specificity.

Bacterial Proteins↗
PubMed2000

Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa.

The LasR-LasI and RhlR-RhlI quorum-sensing systems are global regulators of gene expression in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies suggest that the RhlR-RhlI system activates expression of rpoS. We constructed merodiploid strains of P. aeruginosa containing the native rpoS gene and an rpoS-lacZ fusion. Studies of lacZ transcription in these strains indicated that rpoS was not regulated by RhlR-RhlI. We also generated an rpoS null mutant. This rpoS mutant showed elevated levels of rhlI (but not rhlR) transcription, elevated levels of the RhlI-generated acylhomoserine lactone quorum-sensing signal, and elevated levels of RhlR-RhlI-regulated gene transcription. These findings indicate that there is a relationship between RpoS and quorum sensing, but rather than the RhlR-RhlI system influencing the expression of rpoS, it appears that RpoS regulates rhlI.

Bacterial Proteins↗
PubMed1999

Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa.

Bacteria communicate with each other to coordinate expression of specific genes in a cell density-dependent fashion, a phenomenon called quorum sensing and response. Although we know that quorum sensing via acyl-homoserine lactone (HSL) signals controls expression of several virulence genes in the human pathogen Pseudomonas aeruginosa, the number and types of genes controlled by quorum sensing have not been studied systematically. We have constructed a library of random insertions in the chromosome of a P. aeruginosa acyl-HSL synthesis mutant by using a transposon containing a promoterless lacZ. This library was screened for acyl-HSL induction of lacZ. Thirty-nine quorum sensing-regulated genes were identified. The genes were organized into classes depending on the pattern of regulation. About half of the genes appear to be in seven operons, some seem organized in large patches on the genome. Many of the quorum sensing-regulated genes code for putative virulence factors or production of secondary metabolites. Many of the genes identified showed a high level of induction by acyl-HSL signaling.

4-Butyrolactone↗
PubMed1998

The Drosophila Polycomb group gene pleiohomeotic encodes a DNA binding protein with homology to the transcription factor YY1.

Genes of the Polycomb group (PcG) of Drosophila encode proteins necessary for the maintenance of transcriptional repression of homeotic genes. PcG proteins are thought to act by binding as multiprotein complexes to DNA through Polycomb group response elements (PREs); however, specific DNA binding has not been demonstrated for any of the PcG proteins. We have identified a sequence-specific DNA binding protein that interacts with a PRE from the Drosophila engrailed gene. This protein (PHO) is a homolog of the ubiquitous mammalian transcription factor Yin Yang-1 and is encoded by pleiohomeotic, a known member of the PcG. We propose that PHO acts to anchor PcG protein complexes to DNA.

Amino Acid Sequence↗
PubMed1997

Evidence of autoinducer activity in naturally occurring biofilms.

N-Acyl homoserine lactone (AHL) molecules have been shown to act as mediators of population density-dependent (quorum-sensing) gene expression in numerous Gram-negative bacteria. Functions associated with AHL include light production in Vibrio fischeri, expression of virulence factors in Pseudomonas aeruginosa, and conjugation in Agrobacterium tumefaciens. In nature, bacteria often grow as surface-adherent biofilm communities. As biofilms typically contain high concentrations of cells, AHL activity and quorum-sensing gene expression have been proposed as essential components of biofilm physiology. However, proof of AHL production within biofilms has heretofore been lacking. In this study we have employed a cross-feeding assay, using A, tumefaciens A136 (traI::lacZ) as an AHL-responsive reporter strain, to show the presence of naturally occurring AHL production in aquatic biofilms growing on submerged stones. AHL was detected in living biofilms and biofilm extracts, but was not present in rocks lacking a biofilm. This represents the first report of AHL activity in naturally occurring biofilms.

4-Butyrolactone↗
PubMed1997

Expression pattern of an axolotl floor plate-specific fork head gene reflects early developmental differences between frogs and salamanders.

Gastrulation is one of the most important stages of animal development and, as such, tends to be remarkably conserved. Therefore it is interesting to see that the two amphibian species, Xenopus laevis (frog) and Ambystoma mexicanum (axolotl), are different in the arrangement of cell types just before and during gastrulation. In Xenopus, the cells that will form dorsal mesoderm are located deep in the dorsal marginal zone, while in the axolotl, these are on the surface of the embryo. In this study we investigated whether homologous genes known to be involved in the formation of dorsal structures show a different pattern of expression in these two species. For this purpose, we isolated a fork head gene (AxFKH 1) from the axolotl, which is likely to be the homologue of the Xenopus fork head gene, XFKH 1 (Pintallavis, XFD-1). We find that AxFKH 1 and XFH 1 have a similar pattern of expression, but there are some important differences. In early gastrulae, transcripts are detected in the organizer region of both species. In late gastrulae, the transcripts in Xenopus are located in both the superficial and deep layers, but they are only found in the superficial layer of axolotl embryos. During neurulation, XFKH 1 is expressed in notochord and neural floor plate, whereas AxFKH 1 is expressed in the neural floor plate only. We propose that the differences in expression pattern of these two genes are due to a difference in formation of dorsal structures between these two species. Furthermore, the expression pattern of these two genes early in gastrulation is consistent with the idea that at least some of the neural floor plate cells are already determined at this time.

Ambystoma mexicanum↗
PubMed1997

Rescue of Drosophila engrailed mutants with a highly divergent mosquito engrailed cDNA using a homing, enhancer-trapping transposon.

Specific fragments of Drosophila regulatory DNA can alter the insertional specificity of transposable elements causing them to 'home' to their parent gene. We used this property to insert a transposon-encoded functional coding region near a defective one and rescue a null mutation. This approach differs from homologous recombination in that the endogenous defective coding region is left in place and the genomic DNA is altered by the addition of the therapeutic transposon. We constructed a P-element-based transposon in which an engrailed cDNA from Anopheles gambiae (a mosquito) is expressed from a Drosophila engrailed minimal promoter. The promoter fragment used includes 2.6 kb of regulatory DNA that causes transposons to home to the endogenous Drosophila engrailed gene at high frequencies. We inserted this transposon onto a Drosophila chromosome that produces no functional engrailed proteins. When this transposon integrated near the engrailed promoter, adult viability was restored to engrailed mutant flies showing that the highly divergent mosquito engrailed protein can replace the Drosophila engrailed protein at all stages of development. Insertion of this transposon into the adjacent invected gene, which is transcribed in a pattern similar to engrailed, led to only embryonic rescue, suggesting an important difference in the regulation of these two genes.

Amino Acid Sequence↗