Stoichiometric modeling of oxidation of reduced inorganic sulfur compounds (Riscs) in Acidithiobacillus thiooxidans

The prokaryotic oxidation of reduced inorganic sulfur compounds (RISCs) is a topic of utmost importance from a biogeochemical and industrial perspective. Despite sulfur oxidizing bacterial activity is largely known, no quantitative approaches to biological RISCs oxidation have been made, gathering all the complex abiotic and enzymatic stoichiometry involved. Even though in the case of neutrophilic bacteria such as Paracoccus and Beggiatoa species the RISCs oxidation systems are well described, there is a lack of knowledge for acidophilic microorganisms. Here, we present the first experimentally validated stoichiometric model able to assess RISCs oxidation quantitatively in Acidithiobacillus thiooxidans (strain DSM 17318), the archetype of the sulfur oxidizing acidophilic chemolithoautotrophs. This model was built based on literature and genomic analysis, considering a widespread mix of formerly proposed RISCs oxidation models combined and evaluated experimentally. Thiosulfate partial oxidation by the Sox system (SoxABXYZ) was placed as central step of sulfur oxidation model, along with abiotic reactions. This model was coupled with a detailed stoichiometry of biomass production, providing accurate bacterial growth predictions. In silico deletion/inactivation highlights the role of sulfur dioxygenase as the main catalyzer and a moderate function of tetrathionate hydrolase in elemental sulfur catabolism, demonstrating that this model constitutes an advanced instrument for the optimization of At. thiooxidans biomass production with potential use in biohydrometallurgical and environmental applications. Biotechnol. Bioeng. 2013; 110: 2242–2251. © 2013 Wiley Periodicals, Inc.     

Developmental exposure to noninherited maternal antigens induces CD4+ T regulatory cells: relevance to mechanism of heart allograft tolerance

We hypothesize that developmental exposure to noninherited maternal Ags (NIMA) results in alloantigen-specific natural and adaptive T regulatory (T(R)) cells. We compared offspring exposed to maternal H-2(d) (NIMA(d)) with nonexposed controls. In vitro assays did not reveal any differences in T cell responses pretransplant. Adoptive transfer assays revealed lower lymphoproliferation and greater cell surface TGF-beta expression on CD4(+) T cells of NIMA(d)-exposed vs control splenocytes. NIMA(d)-exposed splenocytes exhibited bystander suppression of tetanus-specific delayed-type hypersensitivity responses, which was reversed with Abs to TGF-beta and IL-10. Allospecific T effector cells were induced in all mice upon i.v. challenge with B6D2F1 splenocytes or a DBA/2 heart transplant, but were controlled in NIMA(d)-exposed mice by T(R) cells to varying degrees. Some (40%) NIMA(d)-exposed mice accepted a DBA/2 allograft while others (60%) rejected in delayed fashion. Rejector and acceptor NIMA(d)-exposed mice had reduced T effector responses and increased Foxp3(+) T(R) cells (CD4(+)CD25(+)Foxp3(+) T(R)) in spleen and lymph nodes compared with controls. The key features distinguishing NIMA(d)-exposed acceptors from all other mice were: 1) higher frequency of IL-10- and TGF-beta-producing cells primarily in the CD4(+)CD25(+) T cell subset within lymph nodes and allografts, 2) a suppressed delayed-type hypersensitivity response to B6D2F1 Ags, and 3) allografts enriched in LAP(+), Foxp3(+), and CD4(+) T cells, with few CD8(+) T cells. We conclude that the beneficial NIMA effect is due to induction of NIMA-specific T(R) cells during ontogeny. Their persistence in the adult, and the ability of the host to mobilize them to the graft, may determine whether NIMA-specific tolerance is achieved.     

First Record of Acrocercops serrigera serrigera Meyrick (Lepidoptera: Gracillariidae) from Chile

Acrocercops Wallengren (Gracillariidae) is recorded for the first time from Chile. The little known Acrocercops serrigera serrigera Meyrick is reported from the Azapa Valley, Arica Province, northern Chilean coastal desert. Specimens were reared from Malva nicaeensis and Waltheria ovata (both Malvaceae). Specimen identification is based on comparisons of the male genitalia with that of the lectotype of the species originated from Peru. Sequences of a fragment of the mitochondrial gene cytochrome oxidase subunit I of specimens reared on both plants indicate that there is only one Acrocercops species involved as only one substitution site was found over the 524-bp sequenced.     

Virulence traits in Cronobacter species isolated from different sources

Cronobacter spp. ( Enterobacter sakazakii ) includes gram-negative opportunistic foodborne pathogens known as rare but important causes of life-threatening neonatal infections. However, the pathogenic mechanism is not yet clear. In this study, 43 isolates of Cronobacter, from human and nonhuman sources, were analyzed. A total of four clusters were identified and 32 DNA pulsotypes were observed by pulsed-field gel electrophoresis. In addition, 86% of the Cronobacter isolates were able to adhere to HEp-2 cells and 35% were invasive, Cronobacter sakazakii isolates being the most efficient. Twenty-six percent of Cronobacter isolates were able to form biofilms, mainly those from nonhuman sources, such as Cronobacter dublinensis and Cronobacter malonaticus . Three putative virulence genes (siderophore-interacting protein (sip), type III hemolysin (hly), and plasminogen activator (cpa)) were identified by bioinformatic analysis and then detected by PCR. The sip gene was the most frequently detected (60%; 26/43), followed by the hly gene (37%; 16/43) and the cpa gene (28%; 12/43). The three genes were identified primarily in C. sakazakii. Our data show that Cronobacter species harbor different virulence traits.     

Acidithiobacillus thiooxidans secretome containing a newly described lipoprotein Licanantase enhances chalcopyrite bioleaching rate

The nature of the mineral–bacteria interphase where electron and mass transfer processes occur is a key element of the bioleaching processes of sulfide minerals. This interphase is composed of proteins, metabolites, and other compounds embedded in extracellular polymeric substances mainly consisting of sugars and lipids (Gehrke et al., Appl Environ Microbiol 64(7):2743–2747, 1998). On this respect, despite Acidithiobacilli—a ubiquitous bacterial genera in bioleaching processes (Rawlings, Microb Cell Fact 4(1):13, 2005)—has long been recognized as secreting bacteria (Jones and Starkey, J Bacteriol 82:788–789, 1961; Schaeffer and Umbreit, J Bacteriol 85:492–493, 1963), few studies have been carried out in order to clarify the nature and the role of the secreted protein component: the secretome. This work characterizes for the first time the sulfur (meta)secretome of Acidithiobacillus thiooxidans strain DSM 17318 in pure and mixed cultures with Acidithiobacillus ferrooxidans DSM 16786, identifying the major component of these secreted fractions as a single lipoprotein named here as Licanantase. Bioleaching assays with the addition of Licanantase-enriched concentrated secretome fractions show that this newly found lipoprotein as an active protein additive exerts an increasing effect on chalcopyrite bioleaching rate.     

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.     

Expression and immunolocalization of ERG1 potassium channels in the rat kidney

Potassium (K⁺) channels participate in K⁺ secretion, K⁺ recycling, and cell volume regulation and help to maintain the resting potential in mammalian kidneys. Previously, we identified a set of voltage-gated K⁺ channels (Kv1) in the inner medullary collecting duct of the rat kidney. In the present work, we identified the voltage-gated K⁺ channel ether-à-go-go-related gene (ERG) in the rat kidney. mRNAs of ERG1a and its N-terminal splice-variant ERG1b were detected. Immunoblots of the cortex and medulla revealed two molecular mass proteins of 135 and 80 kDa, consistent in size with the nonglycosylated ERG1a and ERG1b isoforms, respectively. However, bands of 155 and 95 kDa, corresponding to mature glycosylated ERG1a and ERG1b, respectively, were also observed. In our immunohistochemical experiments, we could not differentiate the ERG1 isoforms because we used an antibody against a carboxy-terminal epitope. ERG1 was differentially localized in specific nephron segments: its localization was intracellular in the proximal tubule and medullary collecting ducts and in the apical membranes in the distal convoluted and connecting tubules. ERG1 was also abundant in glomerular arterioles and renal vessels. In summary, ERG1 displays a heterogeneous distribution in the rat kidney.     

Comparison of clinical pathology parameters with two different blood sampling techniques in rats: retrobulbar plexus versus sublingual vein

Blood samples were taken from the retrobulbar venous plexus or the sublingual vein of male HamIbm:Wist rats to compare clinical pathology parameters between the two sampling techniques. By analogy with a pharmacokinetic study, blood was sampled six times during one day from unfasted animals. After 3 weeks of recovery, blood was taken from fasted animals on a single occasion. In addition, prolactin and corticosterone levels were determined to compare stress-related effects between the two sampling methods. Body weight development and food consumption were similar after single as well as after repeated blood sampling for the two blood sampling techniques. Haemotological evaluation showed a gradual decrease in erythrocyte count, haemoglobin concentration and haematocrit after repeated blood sampling. Repeated withdrawal of blood samples over 24 h corresponding to approximately 22% of the total blood volume resulted in a decrease in red blood cell parameters by up to 30%. The withdrawal of approximately 10% of the total blood volume was associated with a decrease in these parameters by up to 10% and should not be exceeded for animal welfare reasons and to allow a reliable evaluation of data in a study. Repeated blood sampling was associated with an initial decrease in the number of white blood cells, mainly due to a reduction in lymphocytes; white blood cell counts were slightly increased one day after. The decrease in lymphocytes and the increase in neutrophils after repeated sampling were generally slightly more pronounced in the blood from the retrobulbar plexus than from the sublingual vein. Comparison of serum clinical chemistry data showed significantly higher activities of creatine kinase and aspartate aminotransferase in samples from the retrobulbar plexus. These findings suggest a higher degree of tissue damage with blood sampling from the retrobulbar plexus than from the sublingual vein. Despite a large inter-individual variability, higher mean values of prolactin on each occasion and corticosterone after a single sample in fasted animals indicate a higher stress associated with blood sampling from the retrobulbar plexus.