PHB Biosynthesis in Catabolite Repression Mutant of Burkholderia sacchari

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.     

The Effects of Combinatorial Treatments With Stress Inducing Molecules on Growth of <Emphasis Type="Italic">E. coli</Emphasis> Colonies

Stress inducing molecules affect both the mean behavior of bacterial growth and also variations in the growth. While the mechanisms that cause changes in the mean behavior are well understood, little is known about changes in the variation of the population. A true understanding of how organisms respond to stress must include an understanding of the mechanisms and purposes of changes in variation and the distribution not directly related to changes in the mean of the population. We have explored the results of combinatorial treatments using EDTA, copper sulfate, hydrogen peroxide, and hydrochloric acid as stress inducing molecules on bacterial colony formation and area on LB-agar plates. Three different combinations of X-gal and IPTG were used to create different background conditions. Some treatments alter the variation and/or the distribution of the area without having a significant effect on the mean, others affect the mean without altering the distribution, and yet others affect distribution and the mean.     

Simplified RNA secondary structure mapping by automation of SHAPE data analysis

SHAPE (Selective 2'-hydroxyl acylation analysed by primer extension) technology has emerged as one of the leading methods of determining RNA secondary structure at the nucleotide level. A significant bottleneck in using SHAPE is the complex and time-consuming data processing that is required. We present here a modified data collection method and a series of algorithms, embodied in a program entitled Fast Analysis of SHAPE traces (FAST), which significantly reduces processing time. We have used this method to resolve the secondary structure of the first ~900 nt of the hepatitis C virus (HCV) genome, including the entire core gene. We have also demonstrated the ability of SHAPE/FAST to detect the binding of a small molecule inhibitor to the HCV internal ribosomal entry site (IRES). In conclusion, FAST allows for high-throughput data processing to match the current high-throughput generation of data possible with SHAPE, reducing the barrier to determining the structure of RNAs of interest.     

Does aposymbiotically cultivated fungus Ramalina produce isolichenan?

The main α-glucan synthesized by lichens of the genera Ramalina in the symbiotic state is isolichenan. This polysaccharide was not found in the aposymbiotically cultivated symbionts. It is still unknown if this glucan is produced by the mycobiont only in the presence of a photobiont, in a lichen thallus, or if the isolichenan suppression is influenced by the composition of culture medium used in its aposymbiotic cultive. Consequently, the latter hypothesis is tested in this study. Cultures of the mycobiont Ramalina complanata were obtained from germinated ascospores and cultivated on 4% glucose Lilly and Barnett medium. Freeze-dried colonies were defatted and their carbohydrates extracted successively with hot water and aqueous 10% KOH, each at 100 °C. The polysaccharides nigeran, laminaran and galactomannan were liberated, along with a lentinan-type β-glucan and a heteropolysaccharide (Man : Gal : Glc, 21 : 28 : 51). Nevertheless, the α-glucan isolichenan was not found in the extracts. It follows that it was probably a symbiotic product, synthesized by the mycobiont only in this particular microenvironment, in the presence of the photobiont in the lichen thallus. A discussion about polysaccharides found in the symbiotic thallus as well as in other aposymbiotic cultivated Ramalina mycobionts is also included.     

SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem-loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.     

The Effect of Social Defeat on Tyrosine Hydroxylase Phosphorylation in the Rat Brain and Adrenal Gland

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated acutely by protein phosphorylation and chronically by protein synthesis. No studies have systematically investigated the phosphorylation of these sites in vivo in response to stressors. We specifically investigated the phosphorylation of TH occurring within the first 24 h in response to the social defeat stress in the rat adrenal, the locus coeruleus, substantia nigra and ventral tegmental area. Five groups were investigated; home cage control (HCC), two groups that underwent social defeat (SD+) which were sacrificed either 10 min or 24 h after the end of the protocol and two groups that were put into the cage without the resident being present (SD−) which were sacrificed at time points identical to the SD+. We found at 10 min there were significant increases in serine 40 and 31 phosphorylation levels in the locus coeruleus in SD+ compared to HCC and increases in serine 40 phosphorylation levels in the substantia nigra in SD+ compared to SD−. We found at 24 h there were significant increases in serine 19 phosphorylation levels in the ventral tegmental area in SD+ compared to HCC and decreases in serine 40 phosphorylation levels in the adrenal in SD+ compared to SD−. These findings suggest that the regulation of TH phosphorylation in different catecholamine-producing cells varies considerably and is dependent on both the nature of the stressor and the time at which the response is analysed.     

Heat stress screening of peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) seedlings for acquired thermotolerance

Heat can be one of the major abiotic stresses that adversely affect crop production worldwide at different stages of development. As field screening for heat tolerance can be inconsistent and seasonally-limited, it is important to develop a reliable protocol under controlled conditions that allows simultaneous screening of multiple genotypes. The objective of this research was to develop a straightforward laboratory protocol using acquired thermotolerance (ATT) in peanut seedlings as a measure of one mechanism of heat stress tolerance. Sixteen genotypes, including selected accessions of the US peanut minicore collection along with standard checks, were evaluated for acquired themotolerance in two independent experiments. A change in the temperature sensitivity of chlorophyll accumulation was used as an indicator of acquired thermotolerance. Pre-incubation at 38°C for 4 h before the 30-min 50°C challenge triggered the acquired thermotolerance system of the leaf disks, resulting in chlorophyll accumulation upon exposure to light. There was considerable variation among genotypes for ATT in both experiments. Genotypic ranking for mean ATT values were highly correlated (0.949) in both experiments. The effect of seed weight on ATT was not significant. This method is relatively simple and inexpensive and can be used to screen a large number of genotypes.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Pityopsis ruthii</Emphasis>

Pityopsis ruthii is an endangered herbaceous perennial species from the United States. In vitro multiplication of this species can be valuable for germplasm conservation. Flower receptacles of P. ruthii were cultured on Murashige and Skoog medium (MS) supplemented with 11.4 μM indole-3-acetic acid (IAA) in combination with 2.2, 4.4 or 8.8 μM 6-benzyladenine (BA). Shoots were visible within 14–28 days and three plants were successfully rooted on MS medium supplemented with 5.7 μM IAA. A two tailed t-test for paired-variates revealed that shoot regeneration on MS medium amended with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than on other treatments. Leaf explants were also cultured on MS not supplemented with growth regulators or supplemented with 11.4 μM IAA in combination with 0, 2.2, 4.4 or 8.8 μM BA. Shoots were visible within 21–35 days and one plant was successfully rooted on MS medium supplemented with 5.4 μM NAA. Shoot regeneration on MS medium augmented with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than the other treatments according to analysis of variance (ANOVA) with a rank transformation. Hyperhydricity and rooting of shoots was problematic for explants derived from flower receptacles and leaf tissue, but viable plants were regenerated using both explants sources indicating the potential role for micropropagation in the ex situ conservation of the species.     

Tropospheric 14CO2 at Wellington, New Zealand: the world??s longest record

Measurements of near-sea-level tropospheric ??¹⁴CO₂ have been made at Wellington, New Zealand since December 1954; these measurements comprise the longest such record available. The ??¹⁴C rose from ?10?? in 1955 peaking at 695?? in 1965 as a result of ??bomb ¹⁴C?? production, before falling thereafter to the present day (2005) value of 73??. The ??¹⁴C peak occurred about 1?year later in the southern hemisphere than in the northern hemisphere. The post-1965 fall is due to the transfer of ¹⁴C-enriched CO₂ to the biospheric and oceanic pools together with ongoing release of ¹⁴C-free CO₂ from fossil fuel combustion, during an era without major atmospheric nuclear-weapon tests. Time series analysis of the data using Loess decomposition and filtering indicates an approximately exponential decline in excess ??¹⁴CO₂ over 1967?C2005 with an e-folding time of 18?years. The seasonal cycle from 1954 until 1980 had a maximum in the late (austral) summer, a minimum in winter, with peak-to-trough amplitude that peaked at 20?? in 1966. For the period 1980?C1989, a new seasonal cycle emerged, with a maximum in winter and a minimum in late summer/early autumn and peak-to-trough amplitude of 3.5??, transitioning to a new seasonal structure after about 1990.     

Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in west Alabama (USA) in 2009

Three isolates (AL09-71, AL09-72, and AL09-73) of Aeromonas hydrophila were cultured from infected channel catfish Ictalurus punctatus during a disease outbreak in west Alabama, USA, in August 2009. Sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR), cpn60, gyrB, and rpoD genes of the 3 strains revealed that the 3 strains were closely related to each other, sharing 97 to 99% nucleotide sequence similarities. However, ISR sequences of the 3 isolates from 2009 shared only 64% nucleotide sequences with AL98-C1B, a 1998 isolate of A. hydrophila cultured from diseased fish in Alabama. Sequences of cpn60, gyrB, and rpoD from the 3 isolates from 2009 shared 91 to 95% homologies with AL98-C1B. Based on both LD50 and LD95 values of intraperitoneal injection assays, the virulences of the 3 isolates from 2009 were not significantly different from each other, but were at least 200-fold more virulent than AL98-C1B, indicating that the 3 west Alabama isolates of A. hydrophila from 2009 were highly virulent to channel catfish.