The ALS (agglutinin-like sequence) gene family of Candida albicans encodes eight cell-surface glycoproteins, some of which are involved in adherence to host surfaces. A mutational analysis of each ALS gene is currently being performed to deduce the functions of the encoded proteins and to better understand the role of these proteins in C. albicans biology and pathogenesis. This paper describes construction of an als3/als3 mutant and comparison of its phenotype to an als1/als1 strain. Efforts to disrupt ALS3 indicated that the gene could be deleted in two transformation steps, suggesting that the gene is encoded by a single locus and that the ALS3-like locus, ALS8, does not exist. Strains lacking ALS3 or ALS1 did not exhibit a defect in germ tube formation when grown in RPMI 1640 medium, but the als1/als1 mutant formed significantly fewer germ tubes in Lee medium. Analysis of ALS3 and ALS1 promoter activity using green fluorescent protein (GFP) reporter strains and flow cytometry showed that when cells are placed into medium that promotes germ tube formation, ALS1 is transcribed prior to ALS3. Comparison of the mutant strains in adhesion assays showed that the als3/als3 strain was defective in adhesion to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC), but not to fibronectin-coated plastic plates. In contrast, the als1/als1 strain showed decreased adherence to HUVEC, but adherence to BEC and fibronectin were the same as wild-type controls. Inoculation of the buccal reconstituted human epithelium (RHE) model of oral candidiasis with the mutant strains showed nearly a total lack of adhesion and epithelial destruction by the als3/als3 mutant while the als1/als1 strain showed only a slightly reduced degree of epithelial destruction compared to the wild-type control. Adhesion data presented here suggest that, in the assays performed, loss of Als3p affects C. albicans adhesion more than loss of Als1p.Collectively, these results demonstrate functional similarities and differences between Als1p and Als3p, and suggest the potential for more complex interrelationships between the ALS genes and their encoded proteins.

Microarrays were used to identify changes in gene expression associated with Candida albicans biofilm development. Two biofilm substrates (denture and catheter), and two C. albicans strains for each substrate, were tested to remove model-and strain-dependent variability from the overall dataset. Three biofilm developmental phases were examined: early (6 h), intermediate (12 h), and mature (48 h). Planktonic specimens were collected at the same time points. Data analysis focused primarily on gene expression changes over the time-course of biofilm development. Glycolytic and non-glycolytic carbohydrate assimilation, amino acid metabolism, and intracellular transport mechanisms were important during the early phase of biofilm formation. These early events increase intracellular pools of pyruvate, pentoses and amino acids, which prepare the biofilm for the large biomass increase that begins around 12 h of development. This developmental stage demands energy and utilizes specific transporters for amino acids, sugars, ions, oligopeptides and lactate/pyruvate. At mature phase (48 h), few genes were differentially expressed compared with the 12 h time point, suggesting a relative lack of initiation of new metabolic activity. Data analysis to assess biofilm model-specific gene expression showed more dynamic changes in the denture model than in the catheter model. Data analysis to identify gene expression changes that are associated with each strain/substrate combination identified the same types of genes that were identified in the analysis of the entire dataset. Collectively, these data suggest that genes belonging to different, but interconnected, functional categories regulate the morphology and phenotype of C. albicans biofilm. INTRODUCTIONMicro-organisms attach to surfaces in many natural, industrial and medical environments and can develop into biofilms. Biofilms are a functional association of microbial cells that are enveloped within extracellular polymer matrices and associated with surfaces (Costerton et al., 1987;Donlan, 2002;Donlan & Costerton, 2002). Phenotypically, biofilm cells are different from planktonic (free-floating) cells (Costerton et al., 1995), with one of the most important manifestations of these differences being the significantly decreased susceptibility of microbial biofilms to antimicrobial agents (Rupp, 2005;Schwank et al., 1998;Stewart et al., 2004;Wilson, 1996). Although bacterial biofilms have been the focus of many studies, detailed investigations into the biology and pathogenesis of fungal biofilms have only recently been initiated (reviewed by Douglas, 2003;Ghannoum & O'Toole, 2004). Candida albicans is the most common fungal pathogen associated with colonization and biofilm formation on the surfaces of indwelling medical devices (IMDs) such as dentures and intravenous catheters (Kojic & Darouiche, 2004). Biofilm formation by C. albicans can promote superficial or systemic disease. For example, denture stomatitis, a superficial candidiasis, occurs in 65 % of denture-wearing Abbrevi...

Rice sheath blight (ShB), caused by the soilborne pathogen Rhizoctonia solani, annually causes severe losses in yield and quality in many rice production areas worldwide. Jasmine 85 is an indica cultivar that has proven to have a high level of resistance to this pathogen. The objective of this study was to determine the ability of controlled environment inoculation assays to detect ShB resistance quantitative trait loci (QTLs) in a cross derived from the susceptible cv. Lemont and the resistant cv. Jasmine 85. The disease reactions of 250 F(5) recombinant inbred lines (RILs) were measured on the seedlings inoculated using microchamber and mist-chamber assays under greenhouse conditions. In total, 10 ShB-QTLs were identified on chromosomes 1, 2, 3, 5, 6, and 9 using these two methods. The microchamber method identified four of five new ShB-QTLs, one on each of chromosomes 1, 3, 5, and 6. Both microchamber and mist-chamber methods identified two ShB-QTLs, qShB1 and qShB9-2. Four of the ShB-QTLs or ShB-QTL regions identified on chromosomes 2, 3, and 9 were previously reported in the literature. The major ShB-QTL qShB9-2, which cosegregated with simple sequence repeat (SSR) marker RM245 on chromosome 9, contributed to 24.3 and 27.2% of total phenotypic variation in ShB using microchamber and mistchamber assays, respectively. qShB9-2, a plant-stage-independent QTL, was also verified in nine haplotypes of 10 resistant Lemont/Jasmine 85 RILs using haplotype analysis. These results suggest that multiple ShB-QTLs are involved in ShB resistance and that microchamber and mist-chamber methods are effective for detecting plant-stage-independent QTLs. Furthermore, two SSR markers, RM215 and RM245, are robust markers and can be used in marker-assisted breeding programs to improve ShB resistance.

With the aim of identifying rice (Oryza spp.) germplasm having enhanced grain nutritional value, the mineral nutrient and trace element concentrations (or ionome) of whole (unmilled) grains from a set of 1763 rice accessions of diverse geographic and genetic origin were evaluated. Seed for analysis of p, Mg, K, S, Ca, As, Cd, Co, Cu, Fe, Mn, Mo, Ni, rb, Sr, and Zn concentrations by inductively coupled plasma mass spectrometry was produced over 2 yr in Beaumont, TX, under both flooded and unflooded watering regimes. The distributions of all element concentrations analyzed were skewed toward higher concentration. A significant portion of this ionomic variation has a genetic basis (broad sense heritabilities 0.14-0.75), indicating an ability to breed for improved grain concentration of all elements except possibly Ni. Variation in grain elemental concentrations was not strongly associated with plant height, heading time, or grain shape, suggesting these physiological factors are not of primary importance in controlling ionomic variation in rice grain. Accessions high in specific elements were sometimes found to have similar genetic or geographic origins, suggesting they share a heritable mechanism underlying their enhanced ionomes. For example, accessions with high Ca, Mg, or K were more common in the indica than in the japonica subgroup; low As was most common among temperate japonica accessions; and several lines high in Mo originated in Malaysia or adjacent Brunei.

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.The vaginal microbiota is important for maintaining vaginal health and preventing infections of the reproductive tract (10, 25, 34). However, studies using 16S rRNA gene clone libraries for identifying vaginal microbes have revealed considerably more diversity in the vaginal microbial communities of healthy premenopausal women than previously realized (14,17,31,33,35,36), thereby calling into question currently existing models for a healthy vaginal ecosystem and how it might be assessed. Although the vaginal microbiota in healthy individuals was traditionally thought to be dominated by Lactobacillus species, more recent studies have demonstrated that Lactobacillus is not the predominant bacterial genus within the vaginal tracts of a significant number of healthy women (1,17,31,35,36). Although 60 to 70% of the women had Lactobacillus-dominated vaginal microbiota, there were also individuals who lacked Lactobacillus altogether and instead had Gardnerella, Atopobium, Prevotella, Pseudomonas, or Streptococcus as the predominant bacteria in the vagina (1,17,31,35,36). The significant bacterial diversity observed among these individuals suggests that defining a healthy vaginal environment is more complex than originally thought.Although the most common method of collecting vaginal samples for clinical analysis is to swab the middle or deep vaginal canal (6,14,31,35), the extent to which this approach yields samples representative of the entire vaginal microbiota is unclear. Indeed, the size and anatomical complexity of the vaginal tract suggest the possibility that distinct microbial populations may reside at discrete sites (e.g., cervix, fornix, outer vaginal canal). However, within individuals, the presence or absence of vaginal microniches capable of supporting discrete microbial populations has not been evaluated. Differences in microbial population distributions within individuals may impede identification of the specific bac...