First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.     

Geography best explains global patterns of genetic diversity and postglacial co‐expansion in marine turtles

For many species, climate oscillations drove cycles of population contraction during cool glacial periods followed by expansion during interglacials. Some groups, however, show evidence of uniform and synchronous expansion, while others display differences in the timing and extent of demographic change. We compared demographic histories inferred from genetic data across marine turtle species to identify responses to postglacial warming shared across taxa and to examine drivers of past demographic change at the global scale. Using coalescent simulations and approximate Bayesian computation (ABC), we estimated demographic parameters, including the likelihood of past population expansion, from a mitochondrial data set encompassing 23 previously identified lineages from all seven marine turtle species. For lineages with a high posterior probability of expansion, we conducted a hierarchical ABC analysis to estimate the proportion of lineages expanding synchronously and the timing of synchronous expansion. We used Bayesian model averaging to identify variables associated with expansion and genetic diversity. Approximately 60% of extant marine turtle lineages showed evidence of expansion, with the rest mainly exhibiting patterns of genetic diversity most consistent with population stability. For lineages showing expansion, there was a strong signal of synchronous expansion after the Last Glacial Maximum. Expansion and genetic diversity were best explained by ocean basin and the degree of endemism for a given lineage. Geographic differences in sensitivity to climate change have implications for prioritizing conservation actions in marine turtles as well as for identifying areas of past demographic stability and potential resilience to future climate change for broadly distributed taxa.     

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens

Applied Microbiology and Biotechnology - Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes....     

Characterization and use of a bacterial lignin peroxidase with an improved manganese-oxidative activity

Peroxidases are well-known biocatalysts produced by all organisms, especially microorganisms, and used in a number of biotechnological applications. The enzyme DypB from the lignin-degrading bacterium Rhodococcus jostii was recently shown to degrade solvent-obtained fractions of a Kraft lignin. In order to promote the practical use, the N246A variant of DypB, named Rh_DypB, was overexpressed in E. coli using a designed synthetic gene: by employing optimized conditions, the enzyme was fully produced as folded holoenzyme, thus avoiding the need for a further time-consuming and expensive reconstitution step. By a single chromatographic purification step, > 100 mg enzyme/L fermentation broth with a > 90% purity was produced. Rh_DypB shows a classical peroxidase activity which is significantly increased by adding Mn²⁺ ions: kinetic parameters for H₂O₂, Mn²⁺, ABTS, and 2,6-DMP were determined. The recombinant enzyme shows a good thermostability (melting temperature of 63–65 °C), is stable at pH 6–7, and maintains a large part of the starting activity following incubation for 24 h at 25–37 °C. Rh_DypB activity is not affected by 1 M NaCl, 10% DMSO, and 5% Tween-80, i.e., compounds used for dye decolorization or lignin-solubilization processes. The enzyme shows broad dye-decolorization activity, especially in the presence of Mn²⁺, oxidizes various aromatic monomers from lignin, and cleaves the guaiacylglycerol-β-guaiacyl ether (GGE), i.e., the Cα-Cβ bond of the dimeric lignin model molecule of β-O-4 linkages. Under optimized conditions, 2 mM GGE was fully cleaved by recombinant Rh_DypB, generating guaiacol in only 10 min, at a rate of 12.5 μmol/min mg enzyme.

     

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> on <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick larvae

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT₅₀ values obtained were 134.6, 27.2 and 24.8 days for concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT₅₀ values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 10⁶, 10⁷ and 10⁸ conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.     

Relationships between reversion of hydroxyurea resistance in hamster cells and the co-amplification of ribonucleotide reductase M2 component, ornithine decarboxylase and P5-8 genes

Hydroxyurea is a specific inhibitor of ribonucleotide reductase, which is a rate-limiting enzyme activity in DNA synthesis. Cells selected for resistance to hydroxyurea contain alterations in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of hamster cells has been used to isolate a stable drug resistant cell line, and two stable revertant lines with different sensitivities to hydroxyurea cytotoxicity and different ribonucleotide reductase activity levels. We show for the first time that a decrease in hydroxyurea resistance is accompanied by a parallel decline in gene copies for the M2 component of ribonucleotide reductase, ornithine decarboxylase and a gene of unknown function called p5-8, indicating that the co-amplification of the three genes is associated with drug resistance, and supporting the concept that M2, ornithine decarboxylase and p5-8 are closely linked, and form part of a single amplicon in hamster cells.     

Promotion of Lipid and Protein Oxidative Damage in Rat Brain by Ethylmalonic Acid

High concentrations of ethylmalonic acid are found in tissues and biological fluids of patients affected by ethylmalonic encephalopathy, deficiency of short-chain acyl-CoA dehydrogenase activity and other illnesses characterized by developmental delay and neuromuscular symptoms. The pathophysiological mechanisms responsible for the brain damage in these patients are virtually unknown. Therefore, in the present work we investigated the in vitro effect of EMA on oxidative stress parameters in rat cerebral cortex. EMA significantly increased chemiluminescence and thiobarbituric acid-reactive species levels (lipoperoxidation), as well as carbonyl content and oxidation of sulfhydryl groups (protein oxidative damage) and DCFH. EMA also significantly decreased the levels of reduced glutathione (non-enzymatic antioxidant defenses). In contrast, nitrate and nitrite levels were not affected by this short organic acid. It is therefore presumed that oxidative stress may represent a pathomechanism involved in the pathophysiology of the neurologic symptoms manifested by patients affected by disorders in which EMA accumulates.     

Effect of nitrogen nutrition on amino acid composition of xylem sap and stem wood in Alnus glutinosa

Citrulline was the major amino acid in root pressure sap, stem sap and stem wood from Alnus glutinosa L. Gaertn. plants relying on fixed nitrogen or, partly or wholly, on mineral nitrogen for growth. Glutamine increased in prominence in plants assimilating mineral nitrogen but asparagine remained a relatively insignificant component. Differences in the relative amounts of the free amino compounds of stem sap from nitrogen-fixing and mineral nitrogen-fed plants were usually small compared to differences between plants fed different sources of mineral nitrogen. In contrast, relatively high values for the ratios of citrulline/total free amino nitrogen compounds and particularly of citrulline/amides in root pressure sap distinguished nitrogen-fixing plants from those receiving mineral nitrogen. Although the amino acid ratios of stem wood extracts showed closer similarity to those for root pressure sap than stem sap, the seasonal accumulation of citrulline, possibly as a storage amino acid, in stem wood from field-grown plants negated the possibility of utilising stem wood analyses as an indicator of the form of nitrogen assimilation. Comparative data on the levels of citrulline or other free amino acids in Alnus glutinosa are unlikely to be useful as an index of nitrogen fixation, under most experimental conditions.