The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

The specificity and processivity of DNA methyltransferases have important implications regarding their biological functions. We have investigated the sequence specificity of CcrM and show here that the enzyme has a high specificity for GANTC sites, with only minor preferences at the central position. It slightly prefers hemimethylated DNA, which represents the physiological substrate. In a previous work, CcrM was reported to be highly processive [Berdis et al. (1998) Proc. Natl Acad. Sci. USA 95: 2874-2879]. However upon review of this work, we identified a technical error in the setup of a crucial experiment in this publication, which prohibits making any statement about the processivity of CcrM. In this study, we performed a series of in vitro experiments to study CcrM processivity. We show that it distributively methylates six target sites on the pUC19 plasmid as well as two target sites located on a 129-mer DNA fragment both in unmethylated and hemimethylated state. Reaction quenching experiments confirmed the lack of processivity. We conclude that the original statement that CcrM is processive is no longer valid.     

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42–114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal–host interactions and adaptation to this ecological niche.     

Patch occupancy and abundance of local populations in landscapes differing in degree of habitat fragmentation: a case study of the colonial black‐headed gull,Chroicocephalus ridibundus

Aim This study investigated whether habitat fragmentation at the landscape level influences patch occupancy and abundance of the black‐headed gull, Chroicocephalus ridibundus, and whether the response of the species to environmental factors is consistent across replicated landscape plots. Location Water bodies (habitat patches) in southern Poland. Methods Surveys were conducted in two landscape types (four plots in each): (1) more‐fragmented landscape, in which habitat patches were small (mean size 2.2–6.2 ha) and far apart (mean distance 2.5–3.1 km); and (2) less‐fragmented landscape, in which habitat patches were large (mean size 9.2–16.5 ha) and separated by short distances (mean 0.9–1.4 km). Observations were performed twice in 284 potential habitat patches during the 2007 breeding season. Results Colonies were significantly more frequent and larger in the less‐fragmented landscapes than in the more‐fragmented ones. Probability of patch occupancy and number of breeding birds were positively related with patch size and these relationships were especially strong in the more‐fragmented landscapes. In the less‐fragmented landscapes, the occurrence of black‐headed gulls was negatively related to the distance to the nearest local population, but in the more‐fragmented landscapes such a relationship was not detected. As distance to the nearest habitat patch increased, the probability of the patch occupancy decreased in the more‐fragmented landscapes. Moreover, abundance was negatively influenced by distance to the nearest habitat patch, especially strongly in more‐fragmented landscapes. Proximity of corridors (rivers) positively influenced the occupation of patches regardless of landscape type. The number of islets positively influenced occupancy and abundance of local populations, and this relationship was stronger in the more‐fragmented landscapes. Main conclusions Our results are in agreement with predictions from metapopulation theory and are the first evidence that populations of black‐headed gulls may have a metapopulation structure. However, patch occupancy and abundance were differentially affected by explanatory variables in the more‐fragmented landscapes than in the less‐fragmented ones. This implies that it is impossible to derive, a priori, predictions about presence/abundance patterns based on only a single landscape.     

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.     

NETosis occurs independently of neutrophil serine proteases

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.     

Conversion of oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F₁ progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5–0.9 mm, 1.0–1.4 mm, and ≥1.5 mm), and transferred into 190–2 regeneration medium with different growth regulators: 0.5 mg L^?1 kinetin (KIN) and 0.5 mg L^?1 1-naphthaleneacetic acid (NAA); 1 mg L^?1 zeatin (ZEA) and 0.5 mg L^?1 NAA; or 1 mg L^?1 dicamba (DIC), 1 mg L^?1 picloram (PIC), and 0.5 mg L^?1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0–1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L^?1 NAA and 0.5 mg L^?1 KIN, while the fewest germinated on medium with 1 mg L^?1 DIC, 1 mg L^?1 PIC, and 0.5 mg L^?1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.