Isolation of soil Streptomyces strains capable of degrading humic acids and analysis of their peroxidase activity

Abstract Fifteen Streptomyces strains capable of decolorizing humic acids in presence of glucose were isolated from soil samples using the dilute suspension technique and spread on agar plates. Six strains, displaying a significant and stable activity, were selected for further characterization. Some features of these isolates (carbon source utilization, enzyme production, antibiotic resistance) were compared with those of the reference strain Streptomyces viridosporus ATCC 39115. Degradation properties studied in batch cultures at pH 7.0 showed that the catabolic activity on humic acids was generally stimulated by incubation with 100% oxygen and was cell surface-associated. Peroxidase activity from cell-free extracts was analysed by using the oxidation of N,N,N′,N′-tetramethyl-phenylene-diamine. PAGE analysis revealed the existence of two major types of peroxidases (molecular mass: about 39.2 and 61.6 kDa), dividing the strains into two groups. The role of cell surface-associated peroxidase activity in the breakdown of humic acids is discussed.     

Low reaction temperature increases the selectivity in an enzymatic reaction due to substrate solvation effects

Immobilized a-chymotrypsin was used as catalyst for studying temperature effects on acyl transfer reactions (acyl-donor: Bz-TyrOEt) in a water-immiscible organic solvent. The solubility of the two nucleophiles, Phe-NH and water, decreased with decreasing temperature. The relative decrease for the amide was larger (2.4-fold) than for water. Therefore the thermodynamic activity (estimated by the relative saturation) increased more for this substrate and hence the selectivity in the reaction was increased.     

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

Role of pagL and lpxO in Bordetella bronchiseptica Lipid A Biosynthesis

PagL and LpxO are enzymes that modify lipid A. PagL is a 3-O deacylase that removes the primary acyl chain from the 3 position, and LpxO is an oxygenase that 2-hydroxylates specific acyl chains in the lipid A. pagL and lpxO homologues have been identified in the genome of Bordetella bronchiseptica, but in the current structure for B. bronchiseptica lipid A the 3 position is acylated and 2-OH acylation is not reported. We have investigated the role of B. bronchiseptica pagL and lpxO in lipid A biosynthesis. We report a different structure for wild-type (WT) B. bronchiseptica lipid A, including the presence of 2-OH-myristate, the presence of which is dependent on lpxO. We also demonstrate that the 3 position is not acylated in the major WT lipid A structures but that mutation of pagL results in the presence of 3-OH-decanoic acid at this position, suggesting that lipid A containing this acylation is synthesized but that PagL removes most of it from the mature lipid A. These data refine the structure of B. bronchiseptica lipid A and demonstrate that pagL and lpxO are involved in its biosynthesis.     

Genetic and Anatomical Basis of the Barrier Separating Wakefulness and Anesthetic-Induced Unresponsiveness

A robust, bistable switch regulates the fluctuations between wakefulness and natural sleep as well as those between wakefulness and anesthetic-induced unresponsiveness. We previously provided experimental evidence for the existence of a behavioral barrier to transitions between these states of arousal, which we call neural inertia. Here we show that neural inertia is controlled by processes that contribute to sleep homeostasis and requires four genes involved in electrical excitability: Sh, sss, na and unc79. Although loss of function mutations in these genes can increase or decrease sensitivity to anesthesia induction, surprisingly, they all collapse neural inertia. These effects are genetically selective: neural inertia is not perturbed by loss-of-function mutations in all genes required for the sleep/wake cycle. These effects are also anatomically selective: sss acts in different neurons to influence arousal-promoting and arousal-suppressing processes underlying neural inertia. Supporting the idea that anesthesia and sleep share some, but not all, genetic and anatomical arousal-regulating pathways, we demonstrate that increasing homeostatic sleep drive widens the neural inertial barrier. We propose that processes selectively contributing to sleep homeostasis and neural inertia may be impaired in pathophysiological conditions such as coma and persistent vegetative states.     

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.     

Ocean Acidification Accelerates Reef Bioerosion

In the recent discussion how biotic systems may react to ocean acidification caused by the rapid rise in carbon dioxide partial pressure (pCO₂) in the marine realm, substantial research is devoted to calcifiers such as stony corals. The antagonistic process – biologically induced carbonate dissolution via bioerosion – has largely been neglected. Unlike skeletal growth, we expect bioerosion by chemical means to be facilitated in a high-CO₂ world. This study focuses on one of the most detrimental bioeroders, the sponge Cliona orientalis, which attacks and kills live corals on Australia’s Great Barrier Reef. Experimental exposure to lowered and elevated levels of pCO₂ confirms a significant enforcement of the sponges’ bioerosion capacity with increasing pCO₂ under more acidic conditions. Considering the substantial contribution of sponges to carbonate bioerosion, this finding implies that tropical reef ecosystems are facing the combined effects of weakened coral calcification and accelerated bioerosion, resulting in critical pressure on the dynamic balance between biogenic carbonate build-up and degradation.