Promoter activity of a putative pollen monosaccharide transporter in Petunia hybrida and characterisation of a transposon insertion mutant

Summary. For the growth of the male reproductive cells of plants, the pollen, the presence of sufficient sucrose or monosaccharides is of vital importance. From Petunia hybrida a pollen-specific putative monosaccharide transporter designated PMT1 (for petunia monosaccharide transporter) has been identified previously. The present work provides an in-depth analysis and characterisation of PMT1 in the context of pollen development with the GUS reporter gene and an insertion mutant. The promoter of the pollen-specific putative PMT1 gene has been isolated by inverse PCR and sequenced. Analysis of plants transformed with the promoter-GUS fusion confirmed the specificity of this gene, belonging to the late pollen-specific expressed genes. GUS activity was detected even after 24 h of in vitro pollen germination, at the pollen tube tip. To elucidate the importance of PMT1 for gametophyte development and fertilisation, we isolated a mutant plant containing a transposon insertion in the PMT1 gene by the dTph1 transposon-tagging PCR-based assay. The PMT1 mutant contained a dTph1 insertion in position 1474 bp of the transcribing part of the gene, before the last two transmembrane-spanning domains. Analysis of the progeny of the heterozygous mutant after selfing revealed no alterations in pollen viability and fertility. Mature pollen grains of a plant homozygous for the transposon insertion were able to germinate in vitro in a medium containing sucrose, glucose, or fructose, which indicates that PMT1 is not essential for pollen survival. Several explanations for these results are discussed in the present work. Correspondence and reprints (present address): Department of Plant Biology, University of Granada. Fuentenueva s/n, 18001 Granada, Spain. Present address: Swammerdam Institute for Life Sciences, Amsterdam, the Netherlands.     

New insights into <Emphasis Type="Italic">SRY</Emphasis> regulation through identification of 5' conserved sequences

Background  

SRY is the pivotal gene initiating male sex determination in most mammals, but how its expression is regulated is still not understood. In this study we derived novel SRY 5' flanking genomic sequence data from bovine and caprine genomic BAC clones.     

A petunia MADS box gene involved in the transition from vegetative to reproductive development.

We have identified a novel petunia MADS box gene, PETUNIA FLOWERING GENE (PFG), which is involved in the transition from vegetative to reproductive development. PFG is expressed in the entire plant except stamens, roots and seedlings. Highest expression levels of PFG are found in vegetative and inflorescence meristems. Inhibition of PFG expression in transgenic plants, using a cosuppression strategy, resulted in a unique nonflowering phenotype. Homozygous pfg cosuppression plants are blocked in the formation of inflorescences and maintain vegetative growth. In these mutants, the expression of both PFG and the MADS box gene FLORAL BINDING PROTEIN26 (FBP26), the putative petunia homolog of SQUAMOSA from Antirrhinum, are down-regulated. In hemizygous pfg cosuppression plants initially a few flowers are formed, after which the meristem reverts to the vegetative phase. This reverted phenotype suggests that PFG, besides being required for floral transition, is also required to maintain the reproductive identity after this transition. The position of PFG in the hierarchy of genes controlling floral meristem development was investigated using a double mutant of the floral meristem identity mutant aberrant leaf and flower (alf) and the pfg cosuppression mutant. This analysis revealed that the pfg cosuppression phenotype is epistatic to the alf mutant phenotype, indicating that PFG acts early in the transition to flowering. These results suggest that the petunia MADS box gene, PFG, functions as an inflorescence meristem identity gene required for the transition of the vegetative shoot apex to the reproductive phase and the maintenance of reproductive identity.     

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.     

Voice Prosthetic Biofilm Formation and Candida Morphogenic Conversions in Absence and Presence of Different Bacterial Strains and Species on Silicone-Rubber

Morphogenic conversion of Candida from a yeast to hyphal morphology plays a pivotal role in the pathogenicity of Candida species. Both Candida albicans and Candida tropicalis, in combination with a variety of different bacterial strains and species, appear in biofilms on silicone-rubber voice prostheses used in laryngectomized patients. Here we study biofilm formation on silicone-rubber by C. albicans or C. tropicalis in combination with different commensal bacterial strains and lactobacillus strains. In addition, hyphal formation in C. albicans and C. tropicalis, as stimulated by Rothia dentocariosa and lactobacilli was evaluated, as clinical studies outlined that these bacterial strains have opposite results on the clinical life-time of silicone-rubber voice prostheses. Biofilms were grown during eight days in a silicone-rubber tube, while passing the biofilms through episodes of nutritional feast and famine. Biofilms consisting of combinations of C. albicans and a bacterial strain comprised significantly less viable organisms than combinations comprising C. tropicalis. High percentages of Candida were found in biofilms grown in combination with lactobacilli. Interestingly, L. casei, with demonstrated favorable effects on the clinical life-time of voice prostheses, reduced the percentage hyphal formation in Candida biofilms as compared with Candida biofilms grown in absence of bacteria or grown in combination with R. dentocariosa, a bacterial strain whose presence is associated with short clinical life-times of voice prostheses.     

Quantification and Qualification of Bacteria Trapped in Chewed Gum

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including caries and periodontal disease, are caused by bacteria. However, it is unknown whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the first method, known numbers of bacteria were finger-chewed into gum and chewed gums were molded to standard dimensions, sonicated and plated to determine numbers of colony-forming-units incorporated, yielding calibration curves of colony-forming-units retrieved versus finger-chewed in. In a second method, calibration curves were created by finger-chewing known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested to chew gum up to 10 min after which numbers of colony-forming-units and total numbers of bacteria trapped in chewed gum were determined using the above methods. The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a slow decrease over time up to 10 min was observed. Around 10⁸ bacteria were detected per gum piece depending on the method and gum considered. The number of species trapped in chewed gum increased with chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria from the oral cavity.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe ‘cytokinin-like’ phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Biosurfactants from thermophilic dairy streptococci and their potential role in the fouling control of heat exchanger plates

Recent work on biosurfactant release by thermophilic dairy streptococci is reviewed. There is a suggestion thatStreptococcus thermophilus isolates may release biosurfactants that stimulate detachment of already-adhering cells and leave an anti-adhesive coating on a substratum. A previously published rapid screening method is described for the identification of biosurfactant-releasing microorganisms, and growth medium supplements to enhance biosurfactant release by thermophilic dairy streptococci are reported. New experimental work described includes the isolation and purification of biosurfactants from dairy isolates by thin layer chromatography. Many compounds isolated were extremely surface-active and reduced the water surface tension to values around 30 mJ m^–2 at a concentration of 10 mg ml^–1. Most importantly, the thin layer chromatograms of various isolates resembled each other, and an adsorbed purified compound from one isolate retarded the deposition to glass of another isolate by a factor of two. Provided our findings implicate that these biosurfactants could also be adsorbed to heat exchanger plates in pasteurizers and thereby retard colonization by thermophilic streptococci, these compounds may have major economic implications. Further work is required, however.     

Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion

An experimental technique is described to determine contact angles on bacterial layers deposited on cellulose triacetate filters. Measurements with water, water-n-propanol mixtures, and alpha-bromonaphthalene were employed to calculate surface free energies of various oral bacteria. Differences of 30 to 40 erg cm-2 were obtained for four different bacterial species isolated from the human oral cavity, if the concept of dispersion and polar surface free energies is applied. The free energies obtained were used to calculate interfacial free energies of adhesion of these bacteria from saliva onto tooth surfaces. Bacterial adhesion is energetically unfavorable, if the enamel surface free energy is less than 50 erg cm-2.