Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). In Pseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI and rhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development, lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI and rhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.     

Osmotic Stimulation of the Na+/H+ Exchanger NHE1: Relationship to the Activation of Three MAPK Pathways

The Na⁺/H⁺ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s) responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4, an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events. Received: 27 November 2000/Revised: 20 March 2001     

Dispersal and evolution of the Sinorhizobium meliloti group II RmInt1 intron in bacteria that interact with plants

Group II introns are both self-splicing RNAs and mobile retroelements found in bacterial and archaeal genomes and in organelles of eukaryotes. They are thought to be the ancestors of eukaryote spliceosomal introns and non-long terminal repeat retrotransposons. We show here that RmInt1, a bacterial group II intron first described in the nitrogen-fixing symbiont of alfalfa (Medicago sativa) Sinorhizobium meliloti, is also present in other Sinorhizobium and Rhizobium species. The intron-homing sites in these species are IS elements of the ISRm2011-2 group as in S. meliloti, but ectopic insertion is also observed. We present evidence that these related bacteria have acquired RmInt1 by vertical inheritance from a common ancestor and by independent horizontal transfer events. We also show that RmInt1 is mobile in related taxa of bacteria that interact with plants and tends to evolve toward an inactive form by fragmentation, with loss of the 3' terminus including the intron-encoded protein. Our results provide an overview of the evolution and dispersion of a bacterial group II intron.     

Glucose does not activate nonadrenergic, noncholinergic inhibitory neurons in the rat stomach

We reported previously that intravenously administered d-glucose acts in the central nervous system to inhibit gastric motility induced by hypoglycemia in anesthetized rats. The purpose of this study was to determine whether this effect is due to inhibition of dorsal motor nucleus of the vagus (DMV) cholinergic motoneurons, which synapse with postganglionic cholinergic neurons, or to excitation of DMV cholinergic neurons, which synapse with postganglionic nonadrenergic, noncholinergic (NANC) neurons, particularly nitrergic neurons. Three approaches were employed: 1) assessment of the efficacy of d-glucose-induced inhibition of gastric motility in hypoglycemic rats with and without inhibition of nitric oxide synthase [10 mg/kg iv nitro-l-arginine methyl ester (l-NAME)], 2) assessment of the efficacy of intravenous bethanechol (30 mug.kg(-1).min(-1)) to stimulate gastric motility in hypoglycemic rats during the time of d-glucose-induced inhibition of gastric motility, and 3) determination of c-Fos expression in DMV neurons after intravenous d-glucose was administered to normoglycemic rats. Results obtained demonstrated that l-NAME treatment had no effect on d-glucose-induced inhibition of gastric motility; there was no reduction in the efficacy of intravenous bethanechol to increase gastric motility, and c-Fos expression was not induced by d-glucose in DMV neurons that project to the stomach. These findings indicate that excitation of DMV cholinergic motoneurons that synapse with postganglionic NANC neurons is not a significant contributing component of d-glucose-induced inhibition of gastric motility.     

A novel role for the CBF3 kinetochore-scaffold complex in regulating septin dynamics and cytokinesis

In budding yeast, the kinetochore scaffold complex centromere binding factor 3 (CBF3) is required to form kinetochores on centromere DNA and to allow proper chromosome segregation. We have previously shown that SKP1 and SGT1 balance the assembly and turnover of CBF3 complexes, a cycle that we suggest is independent of its role in chromosome segregation (Rodrigo-Brenni, M.C., S. Thomas, D.C. Bouck, and K.B. Kaplan. 2004. Mol. Biol. Cell. 15:3366-3378). We provide evidence that this cycle contributes to a second, kinetochore-independent function of CBF3. In this study, we show that inhibiting the assembly of CBF3 causes disorganized septins and defects in cell polarity that give rise to cytokinesis failures. Specifically, we show that septin ring separation and disassembly is delayed in anaphase, suggesting that CBF3 regulates septin dynamics. Only mutations that affect the CBF3 cycle, and not mutants in outer kinetochore subunits, cause defects in septins. These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins. Consistent with this possibility, we find that CBF3 interacts with Bir1p, the homologue of the passenger protein Survivin. Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.     

Morphology and mechanics of myosepta in a swimming salamander (Siren lacertina)

In contrast to the complex, three-dimensional shape of myomeres in teleost fishes, the lateral hypaxial muscles of salamanders are nearly planar and their myosepta run in a roughly straight line from mid-lateral to mid-ventral. We used this relatively simple system as the basis for a mathematical model of segmented musculature. Model results highlight the importance of the mechanics of myosepta in determining the shortening characteristics of a muscle segment. We used sonomicrometry to measure the longitudinal deformation of myomeres and the dorsoventral deformation of myosepta in a swimming salamander (Siren lacertina). Sonomicrometry results show that the myosepta allow some dorsoventral lengthening, indicating an amplification of myomere shortening that is greater than that produced by muscle fiber angle alone (10% muscle fiber shortening produces 28.7% myomere shortening). Polarized light and DIC microscopy of isolated hypaxial myosepta revealed that the collagen fiber orientation in hypaxial myomeres is primarily mediolateral. The mediolateral collagen fiber orientation, combined with our finding that the hypaxial myosepta lengthen dorsoventrally during swimming, suggests that one possible function of hypaxial myosepta in S. lacertina is to increase the strain amplification of the muscle fibers by reducing the mediolateral bulging of the myomeres and redirecting the bulging toward the dorsoventral direction.     

Beating the cold: the functional evolution of troponin C in teleost fish

The sensitivity of the cardiac myocyte contractile element for Ca(2+) decreases with temperature. As myocyte contractility is regulated by changes in cytosolic [Ca(2+)], this desensitizing effect represents a challenge for temperate fish such as the rainbow trout, Oncorhynchus mykiss, living in environments where temperatures are low and variable. To allow cardiac function in a temperate environment it is thought that the comparatively high Ca(2+) sensitivity of trout cardiac myocytes compensates for the effects of low temperature on myocyte contractility. The high Ca(2+) sensitivity of the trout myocyte is due, at least in part, to changes in the amino acid sequence of the thin filament protein, cardiac troponin C (cTnC). cTnC is the Ca(2+)-activated switch that triggers myocyte contraction. The isoform of cTnC cloned from trout ventricle (ScTnC) is 92% identical to mammalian cTnC (McTnC) and is significantly more sensitive to Ca(2+). This result suggests that ScTnC has evolved in trout to allow cardiac function at low temperatures. cTnC also appears to play a role in maintaining cardiac function when temperatures change. Increasing myofibrillar pH according to alpha-stat regulation, as would occur when temperature decreases, increases Ca(2+) sensitivity. A similar increase in pH also sensitizes cTnC to Ca(2+). ScTnC therefore appears critical in maintaining cardiac function in trout at low temperatures as well as during changes in temperature.     

NIPBL mutational analysis in 120 individuals with Cornelia de Lange syndrome and evaluation of genotype-phenotype correlations

The Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder characterized by facial dysmorphia, upper-extremity malformations, hirsutism, cardiac defects, growth and cognitive retardation, and gastrointestinal abnormalities. Both missense and protein-truncating mutations in NIPBL, the human homolog of the Drosophila melanogaster Nipped-B gene, have recently been reported to cause CdLS. The function of NIPBL in mammals is unknown. The Drosophila Nipped-B protein facilitates long-range enhancer-promoter interactions and plays a role in Notch signaling and other developmental pathways, as well as being involved in mitotic sister-chromatid cohesion. We report the spectrum and distribution of NIPBL mutations in a large well-characterized cohort of individuals with CdLS. Mutations were found in 56 (47%) of 120 unrelated individuals with sporadic or familial CdLS. Statistically significant phenotypic differences between mutation-positive and mutation-negative individuals were identified. Analysis also suggested a trend toward a milder phenotype in individuals with missense mutations than in those with other types of mutations.     

Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal

Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0-0.25 m and 0.25-0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.     

Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes

We characterized the bacterioplankton community and its seasonal dynamics in two neighbouring hypertrophic lakes by denaturing gradient gel electrophoresis (DGGE) analysis of short (193 bp) 16S ribosomal DNA polymerase chain reaction (PCR) products obtained with primers specific for the domain Bacteria. Lake Blankaart is turbid and has a high phytoplankton biomass and episodic cyanobacterial blooms, whereas biomanipulated Lake Visvijver is characterized by clearwater conditions and the establishment of a dense charophyte vegetation. Both lakes were dominated by bacterial groups commonly found in freshwater habitats (e.g. ACK4 cluster of Actinomycetes; ACK stands for clones isolated from the Adirondack mountain lakes) . Yet, cluster analysis and principal components analysis (PCA) revealed that taxon composition of the bacterioplankton community of the two lakes differs substantially and consistently throughout the season. During the study year (1998), the bacterioplankton community of both lakes showed a distinct seasonal pattern. Lake Blankaart showed a clear differentiation between winter, spring, summer and autumn. In Lake Visvijver, summer samples differed greatly from spring, autumn and winter samples. We hypothesize that the contrasting bacterioplankton in the two neighbouring shallow lakes is determined largely by the presence or absence of macrophytes.