A bistable prokaryotic differentiation system underlying development of conjugative transfer competence

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7–4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.     

Building and degradation of secondary cell walls: are there common patterns of lamellar assembly of cellulose microfibrils and cell wall delamination?

During cell wall formation and degradation, it is possible to detect cellulose microfibrils assembled into thicker and thinner lamellar structures, respectively, following inverse parallel patterns. The aim of this study was to analyse such patterns of microfibril aggregation and cell wall delamination. The thickness of microfibrils and lamellae was measured on digital images of both growing and degrading cell walls viewed by means of transmission electron microscopy. To objectively detect, measure and classify microfibrils and lamellae into thickness classes, a method based on the application of computerized image analysis combined with graphical and statistical methods was developed. The method allowed common classes of microfibrils and lamellae in cell walls to be identified from different origins. During both the formation and degradation of cell walls, a preferential formation of structures with specific thickness was evidenced. The results obtained with the developed method allowed objective analysis of patterns of microfibril aggregation and evidenced a trend of doubling/halving lamellar structures, during cell wall formation/degradation in materials from different origin and which have undergone different treatments.     

Climate and sea‐level changes across a shallow marine Cretaceous–Palaeogene boundary succession in Patagonia,Argentina

Upper Maastrichtian to lower Paleocene, coarse‐grained deposits of the Lefipán Formation in Chubut Province, (Patagonia, Argentina) provide an opportunity to study environmental changes across the Cretaceous–Palaeogene (K–Pg) boundary in a shallow marine depositional environment. Marine palynological and organic geochemical analyses were performed on the K–Pg boundary interval of the Lefipán Formation at the San Ramón section. The palynological and organic geochemical records from the San Ramón K–Pg boundary section are characteristic of a highly dynamic, nearshore setting. High abundances of terrestrial palynomorphs, high BIT‐index values and the occasional presence of plant fossils are indicative of a large input of terrestrial organic material. The organic‐walled dinoflagellate cyst (dinocyst) assemblage is generally dominated by Senegalinium and other peridinioid dinocyst taxa, indicative of high‐nutrient conditions and decreased salinities, probably associated with a large fluvial input. The reconstructed sea surface temperatures range from 25°C to 27°C, in accordance with the tropical climate inferred by palynological and megafloral studies. As in the Bajada del Jagüel section, ~500 km north‐north‐east of San Ramón, peaks of Senegalinium spp. were recorded below and above the K–Pg boundary, possibly related to enhanced runoff resulting from more humid climatic conditions. The lithological, palynological and organic geochemical records suggest the occurrence of a sea‐level regression across the K–Pg boundary, resulting in a hiatus directly at the boundary in both sections, followed by a transgression in the Danian.     

Maximizing the value of systematic reviews in ecology when data or resources are limited

Systematic reviews provide a rigorous, repeatable and quantitative method for assessing and synthesizing all available empirical evidence to evaluate a specific research, management, or policy question. They are particularly well suited for evaluating the effectiveness of environmental management actions, and thus for underpinning evidence‐based adaptive natural resource management. However, their current utility may be limited in countries like Australia, where both the amount of research relative to land area and of well‐monitored, active land management for environmental purposes are relatively low. Based on our experience conducting two of the first ecological systematic reviews in Australia, we have developed a number of recommendations for conducting systematic reviews in situations where resources and/or primary research data are limited. We discuss potential modification or augmentation of most aspects of the systematic review process including selection of a review team, question formulation, search strategy, data analysis, and the communication of results, as well as the inherent tradeoffs between systematic thoroughness and available resources that are involved in these changes. We hope that our recommendations will encourage more ecologists to undertake systematic reviews even if primary research and resources to conduct the review appear to be limited, as even a modified systematic review can provide more defensible evidence‐based guidelines for management of natural resources.     

Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium

A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.     

An indispensable gene for NAD biosynthesis in Salmonella typhimurium.

We have located the nadD locus between lip and leuS at 14 min on the Salmonella typhimurium chromosome, and we have shown it to be the structural gene for nicotinic acid mononucleotide adenylyltransferase. This is the first indispensable gene of pyridine nucleotide metabolism that has been identified. Mutants altered at this locus, isolated by their 6-aminonicotinamide resistance phenotype, accumulate abnormally large pools of nicotinic acid mononucleotide in vivo; many exhibit a temperature-sensitive lethal phenotype. Enzyme assays reveal markedly lower transferase activity in mutant extracts than in nadD+ extracts. The partial dominance of nadD mutants when placed in a nadD+/nadD diploid suggests that nicotinic acid mononucleotide adenylyltransferase is a multimeric enzyme.     

The effect of physicochemical conditions and nutrient sources on maximizing the growth and lipid productivity of green microalgae

Biodiesel is a renewable fuel produced mostly from edible and non‐edible vegetables, by transesterification of neutral lipids (triacylglycerols). However, vegetable oil‐based biodiesel production competes with food crops for arable land, increasing food prices and leading to biodiversity loss. The production of biodiesel from oleaginous microorganisms – particularly microalgae – has attracted attention due to the higher lipid productivity of these organisms, when compared with vegetables. Several environmental factors – including light, temperature, pH and the presence of nutrients (particularly nitrogen, phosphorus and iron) – influence directly the ability of microalgae to produce and store triacylglycerols and other lipids, and also modulate microalgal growth. Although some environmental factors affect several species in a similar manner, differential responses between species are frequent, highlighting the importance of identifying optimal cultivation conditions for each species, to balance growth and lipid productivity for biodiesel production. Here, we reviewed the particular influence of the physicochemical and nutritional factors on the growth and lipid productivity of different green oleaginous microalgae species.     

Monitoring of aphid flight activities in?seed?potato?crops?in Serbia

Aphid flight activities in seed potato fields have been studied by the yellow water traps. It is a good method for monitoring aphids as vectors of viruses, but this study also showed it is a suitable method for insect-diversity research. During the four-year studies, over 11.500 specimens were collected and a total of 107 different taxa of aphids were identified. The most abundant species were polyphagous species, such as: Acyrthosiphon pisum (Haris), Aphis fabae Scopoli, Aphis gossypii Gloverand Brachycaudus helichrysi (Kaltenbach). The results of the studies show that diversity of aphids in different regions of Serbia is similar regardless of the altitude and the diversity of terrain. At most sites it ranged from 2 to 3. The highest value was recorded in Begeč, locality in northern part of Serbia, in year 2008, and it was 2.92. The maximum values of the Shannon-Weaver diversity index at all sites were recorded in the first weeks of the monitoring of aphid flight activities. Morisita-Horn similarity index shows no significant differences between sites regardless of altitudes. The sites are grouped by year, not by similarity of relief. In spite of these results, the Chi-square analysis showed highly significant difference in vector frequencies among seasons and sites with more pronounced differences for PVY. As a consequence of differences in vector frequencies, the vector pressure index in some regions was different also. The number of vectors and vector pressure index vary depending on the altitude of localities. At localities at altitudes under 1000 m, they were high. The highest index was at Kotraža, locality in central part of Serbia, in 2007, when PVY index exceeded the value of 180, while for PLRV it was 60. At high altitudes on mountain Golija, above 1100 m, the number of aphids was low, as well as the vector pressure index which indicates that these regions are suitable for producing virus-free seed potato.     

Human mucin MUC1 RNA undergoes different types of alternative splicing resulting in multiple isoforms

MUC1 is a transmembrane mucin with important functions in normal and transformed cells, carried out by the extracellular domain or the cytoplasmic tail. A characteristic feature of the MUC1 extracellular domain is the variable number of tandem repeats (VNTR) region. Alternative splicing may regulate MUC1 expression and possibly function. We developed an RT-PCR method for efficient isolation of MUC1 mRNA isoforms that allowed us to evaluate the extent of alternative splicing of MUC1 and elucidate some of the rules that govern this process. We cloned and analyzed 21, 24, and 36 isoforms from human tumor cell lines HeLa, MCF7, and Jurkat, respectively, and 16 from normal activated human T cells. Among the 78 MUC1 isoforms we isolated, 76 are new and different cells showed varied MUC1 expression patterns. The VNTR region of exon 2 was recognized as an intron with a fixed 5′ splice site but variable 3′ splice sites. We also report that the 3506 A/G SNP in exon 2 can regulate 3′ splice sites selection in intron 1 and produce different MUC1 short isoform proteins. Furthermore, the SNP A to G mutation was also observed in vivo, during de novo tumor formation in MUC1^+/?Kras^G12D/+Pten^loxP/loxP mice. No specific functions have been associated with previously reported short isoforms. We now report that one new G SNP-associated isoform MUC1/Y-LSP, but not the A SNP-associated isoform MUC1/Y, inhibits tumor growth in immunocompetent but not immunocompromised mice.     

Structural studies and biological evaluation of T30695 variants modified with single chiral glycerol-T reveal the importance of LEDGF/p75 for the aptamer anti-HIV-integrase activities

Some G-quadruplex (GQ) forming aptamers, such as T30695, exhibit particularly promising properties among the potential anti-HIV drugs. T30695?G-quadruplex binds to HIV-1 integrase (IN) and inhibits its activity during 3′-end processing at nanomolar concentrations. Herein we report a study concerning six T30695-GQ variants, in which the R or S chiral glycerol T, singly replaced the thymine residues at the T30695?G-quadruplex loops. CD melting, EMSA and HMRS experiments provided information about the thermal stability and the stoichiometry of T30695-GQ variants, whereas CD and ¹H NMR studies were performed to evaluate the effects of the modifications on T30695-GQ topology. Furthermore, LEDGF/p75 dependent and independent integration assays were carried out to evaluate how T loop modifications impact T30695-GQ biological activities. The obtained results showed that LEDGF/p75 adversely affects the potencies of T30695 and its variants. The IN inhibitory activities of the modified aptamers also depended on the position and on the chirality (R or S) of glycerol T loop in the GQ, mostly regardless of the G-quadruplex stabilities. In view of our and literature data, we suggest that the allosteric modulation of IN tetramer conformations by LEDGF/p75 alters the interactions between the aptamers and the enzyme. Therefore, the new T30695 variants could be suitable tools in studies aimed to clarify the HIV-1 IN tetramers allostery and its role in the integration activity.