Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

SSR based genetic diversity of pigmented and aromatic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes of the western Himalayan region of India

A set of 24 of SSR markers were used to estimate the genetic diversity in 16 rice genotypes found in Western Himalayas of Kashmir and Himachal Pradesh, India. The level of polymorphism among the genotypes of rice was evaluated from the number of alleles and PIC value for each of the 24 SSR loci. A total of 68 alleles were detected across the 16 genotypes through the use of these 24 SSR markers The number of alleles per locus generated varied from 2 (RM 338, RM 452, RM 171) to 6 (RM 585, RM 249, RM 481, RM 162). The PIC values varied from 0.36 (RM 1) to 0.86 (RM 249) with an average of 0.62 per locus. Based on information generated, the genotypes got separated in six different clusters. Cluster 1 comprised of 4 genotypes viz; Zag 1, Zag 13, Pusa sugandh 3, and Zag 14, separated from each other at a similarity value of 0.40. Cluster second comprised of 3 landraces viz; Zag 2. Zag 4 and Zag10 separated from each other at a similarity value of 0.45. Cluster third comprised of 3 genotypes viz; Grey rice, Mushk budji and Kamad separated from each other at a similarity value of 0.46. Cluster fourth had 2 landraces viz; Kawa kreed and Loual anzul, and was not sub clustered. Fifth cluster had 3 genotypes viz; Zag 12, Purple rice and Jhelum separated from each other at a similarity value of 0.28. Cluster 6 comprised of a single popular variety i.e. Shalimar rice 1 with independent lineage.     

Slipped-strand mispairing: a major mechanism for DNA sequence evolution

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing- over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.      

Complete sequences of the rRNA genes of Drosophila melanogaster

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.      

Ratio of free to bound polyamines during maturation in mung-bean hypocotyl cells

Free- and bound-polyamine levels were estimated in successive segments of the mung-bean hypocotyl. Three aliphatic polyamines (putrescine, spermidine and spermine) were found in proportions which depended on the state of maturation. In young cells, most of the polyamines were located in the protoplasm whereas in older cells they were mostly bound to the cell walls. Spermidine was always the main bound polyamine, and putrescine, the main free polyamine.Abbreviation EDTA ethylenediaminetetraacetic acid     

An overview of models of stomatal conductance at the leaf level

Stomata play a key role in plant adaptation to changing environmental conditions as they control both water losses and CO₂ uptake. Particularly, in the context of global change, simulations of the consequences of drought on crop plants are needed to design more efficient and water‐saving cropping systems. However, most of the models of stomatal conductance (g_s) developed at the leaf level link g_s to environmental factors or net photosynthesis (A_net), but do not include satisfactorily the effects of drought, impairing our capacity to simulate plant functioning in conditions of limited water supply. The objective of this review was to draw an up‐to‐date picture of the g_s models, from the empirical to the process‐based ones, along with their mechanistic or deterministic bases. It focuses on models capable to account for multiple environmental influences with emphasis on drought conditions. We examine how models that have been proposed for well‐watered conditions can be combined with those specifically designed to deal with drought conditions. Ideas for future improvements of g_s models are discussed: the issue of co‐regulation of g_s and A_net; the roles of CO₂, absissic acid and H₂O₂; and finally, how to better address the new challenges arising from the issue of global change.     

Evolution of xyloglucan-related genes in green plants

Background  

The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.