Widespread Distribution and Adaptive Degradation of Microcystin Degrader (mlr-Genotype) in Lake Taihu, China

Microbial degradation is an important route for removing environmental microcystins (MCs). Here, we investigated the ecological distribution of microcystin degraders (mlr-genotype), and the relationship between the substrate specificity of the microcystin degrader and the profile of microcystin congener production in the habitat. We showed that microcystin degraders were widely distributed and closely associated with Microcystis abundance in Lake Taihu, China. We characterized an indigenous degrader, Sphingopyxis N5 in the northern Lake Taihu, and it metabolized six microcystin congeners in increasing order (RR > LR > YR > LA > LF and LW). Such a substrate-specificity pattern was congruent to the order of the dominance levels of these congeners in northern Lake Taihu. Furthermore, a meta-analysis on global microcystin degraders revealed that the substrate-specificity patterns varied geographically, but generally matched the profiles of microcystin congener production in the degrader habitats, and the indigenous degrader typically metabolized well the dominant MC congeners, but not the rare congeners in the habitat. This highlighted the phenotypic congruence between microcystin production and degradation in natural environments. We theorize that such congruence resulted from the metabolic adaptation of the indigenous degrader to the local microcystin congeners. Under the nutrient microcystin selection, the degraders might have evolved to better exploit the locally dominant congeners. This study provided the novel insight into the ecological distribution and adaptive degradation of microcystin degraders.

Feather mites of the new genus Bernierinyssus gen. n. (Acariformes: Pteronyssidae) from endemic Malagasy warblers (Passeriformes: Bernieridae)—a lineage showing symbiotic cospeciation with their avian hosts

A new feather mite genus Bernierinyssus gen. n. (Analgoidea: Pteronyssidae), associated with endemic Malagasy warblers (Passeriformes: Bernieridae), is proposed based on morphological evidence and DNA sequence data. Within this genus, we detected six mite species, including five new species described here: Bernierinyssus angulatus sp. n. from Crossleyia xanthophrys, B. bernieriae sp. n. from Bernieria madagascariensis, B. bifenestratus sp. n. from Hartertula flavoviridis, B. randiae sp. n. from Randia pseudozosterops, B. xanthomixis sp. n. from Xanthomixis zosterops (type host) and X. cinereiceps, and B. oxylabis (Mironov and Wauthy 2005) comb. n. (transferred from Pteronyssoides Hull). Phylogenetic relationships of these mites were nearly perfectly congruent with those of their hosts, indicating that ancestral Bernierinyssus probably co-dispersed to Madagascar on the common ancestor of Malagasy warblers and then cospeciated with their hosts. Species of Bernierinyssus are well-delimited based on several lines of evidence: morphology (clear among-specific differences in discrete characters), host associations (one mite species per one host species, except for B. xanthomixis), genetic distances (large COX1 barcoding gap between among- and within-species K2P distances: 8.22¨C12.38% vs 0¨C2.9%, respectively), and molecular phylogenetics (all species are well-supported, monophyletic clades). Our study suggests that species of the genus Bernierinyssus have evolved slower than their avian hosts or co-associated feather lice. Despite the discordance in the mitochondrial DNA evolutionary rates, speciation events in mites largely corresponded to bird species divergences, resulting in a nearly perfect correlation between mite and bird species richness (Eichler's Rule). The mite B. xanthomixis was associated with two avian species, but still formed two distinct shallow lineages (COX1 distance: 1.65%) separated by the host species. The nearly strict host-specificity pattern found in Bernierinyssus contrasts with that of continental feather mites, which tend to be less host-specific and have nearly equal proportions of single-host vs multi-host species.

Evaluation of various phenotypic methods with genotypic screening for detection of methicillin-resistant Staphylococcus aureus

BackgroundStaphylococcus aureus is one of the common opportunistic gram-positive pathogens which are often associated with nosocomial infections. Detection of methicillin-resistant S. aureus (MRSA) has become complicated due to the complex phenotypic and genomic pattern.ObjectiveTo evaluate the sensitivity and specificity pattern of various phenotypic methods used in screening mec genes harboring MRSA.MethodsClinical isolates of S. aureus were collected from diagnostic centers in Tamil Nadu. Phenotypic identification methods such as Minimal Inhibitory Concentration for oxacillin, oxacillin screen agar (OSA), oxacillin disk diffusion, and cefoxitin disk diffusion (CFD) tests were compared. The clinical isolates were classified into MRSA and methicillin-susceptible S. aureus (MSSA) based on the polymerase chain reaction (PCR) amplification of the mecA gene.ResultOut of 50 S. aureus, 21 were found to be MRSA based on the presence of the mecA gene. All 21 mecA-positive isolates were found to be resistant through minimum inhibitory concentration (MIC) and CFD test, having a sensitivity of 100% and specificity of 52% and 62%, respectively. OSA and oxacillin disk tests were found to have a sensitivity of 86% and specificity of 48% and 52%, respectively.ConclusionThe combination of two phenotypic methods, CFD and oxacillin MIC, can be used for the detection of MRSA in clinical laboratories.

Host relations and DNA reveal a cryptic gall crab species (Crustacea: Decapoda: Cryptochiridae) associated with mushroom corals (Scleractinia: Fungiidae)

Mushroom corals of the Indo-West Pacific Fungiidae (Scleractinia) provide habitats for a rich associated fauna, including three species of gall crabs (Cryptochiridae). During the course of the present study gall crabs were sampled from many different fungiid hosts. Based on this ‘reversed’ approach - by studying coral symbionts from a host perspective - a previously unnoticed host specificity pattern was detected. The sampling of gall crab fauna per host coral combined with molecular analyses of H3 nDNA, 16S and COI mtDNA revealed a cryptic gall crab species closely related to Fungicola fagei. This new species, described hereafter as Fungicola syzygia sp. nov., is predominantly associated with the mushroom coral genera Cycloseris and Pleuractis, whereas its sibling species F. fagei is only known to be associated with the host genera Podabacia and Sandalolitha. Based on morphology F. syzygia sp. nov. is difficult to distinguish from F. fagei, but there are subtle differences in carapace shape, the lateral carapace margins, the border between the orbital angles and the merus of the third maxilliped, as well as in the carapace length/width ratio. The type material of F. utinomi and F. fagei is figured for comparison.

Amino acid transamination in permeabilised cells of Lactobacillus helveticus, Lb. paracasei and Lb. danicus

Aminotransferase (AT) activity against 18 amino acids was studied in ten strains of three species of Lactobacillus. A method for permeabilisation of cells was developed using toluene and ethanol combined with mechanical treatment. It was found that the AT activities in the washed permeabilised cells (W-PC) corresponded well to that in cell-free extracts (CFE). The AT specificity pattern was species as well as strain dependant. Strains of Lb. helveticus had high specificity for aromatic amino acids (ArAA) and lower activity against branched-chain amino acids (BcAA) and Asp, while strains of Lb. paracasei subsp. paracasei degraded BcAA and Asp, but had a lower and variable specificity against ArAA. One of the Lb. paracasei strains was characterised by having very high AT activity against all three BcAA (Ile, Leu, Val) compared with any of the other Lb. paracasei strains tested. Strains of Lb. danicus, which is a newly discovered Lactobacillus species isolated from cheese, had up to about 20 times higher AT activity against Leu than Lb. paracasei and Lb. helveticus. The permeabilised cells of Lb. danicus had also considerably higher AT activity against ArAA than Lb. paracasei and Lb. helveticus strains, and also higher AT activity against Asp. All Lactobacillus strains tested had AT activity against Met, but at a much lower rate than against other amino acids. Results of this study also demonstrated a chemical reaction between α-ketoglutaric acid and Asp that was catalysed by pyridoxal-5-phosphate without any AT present.

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage.

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.

IDENTIFICATION OF beta-LIKE ADRENOCEPTORS ASSOCIATED WITH BIOLUMINESCENCE IN THE SEA PANSY RENILLA KOELLIKERI

Previous studies have reported pharmacological and biochemical evidence for the involvement of adrenergic substances in the regulation of neuroeffector activities in the bioluminescent cnidarian Renilla koellikeri (Cnidaria, Anthozoa). Therefore, direct radiobinding assays were developed to identify and characterize beta-adrenergic binding in membrane preparations from this species, using the two beta-antagonists [3H]dihydroalprenolol and [3H]CGP12177 as tracers. In addition, the effect of various beta-adrenergic agents on luminescence was examined. Binding of the radioligands at 25°C was rapid, reversible, saturable and specific. Saturation studies revealed the presence of two different and independent classes of binding site, site1 and site2, in the body of the colony (rachis). In contrast, homogeneous populations of binding sites corresponding to site1 were detected in autozooid polyps and to site2 in the peduncle. The pharmacological profile of beta-adrenergic binding in R. koellikeri membrane preparations displayed properties consistent with the presence of two sites and followed a pattern similar to beta2- and beta1-adrenergic receptor subtypes for site1 and site2, respectively. Bioluminescence in polyps was induced by beta-agonists as well as by one beta1-selective antagonist, atenolol, and was blocked by several beta-blockers including (+/−)CGP12177. The specificity pattern of the physiological effect of beta-adrenergic drugs on luminescence mirrors that of the radioligand interaction with site1. This suggests that radioligand binding to site1 represents binding to the receptor that mediates luminescence excitation in R. koellikeri. Blockade of the luminescent responses to site1 agonists by isotonic MgCl2 indicates that this beta-adrenergic mechanism must rely on interneuronal transmission. Collectively, these results suggest the evolutionary conservation of beta-adrenoceptors and of their dual character from coelenterates to higher vertebrates.

The Use of Monoclonal Antibodies for the Detection of Faecal Bacteria in Water

Monoclonal antibodies (MAbs) against heat-killed Escherichia coli and Klebsiella oxytoca originating from wastewater effluent were raised in BALB/C mice. The fusion was highly successful and three hybridomas cloned were selected to study the affinity and specificity pattern of the MAbs. The MAbs were found to react equally well with heat-killed and live bacteria when tested against their original immunogens. The MAbs showed lower specificity towards environmentally isolated faecal bacteria than a pure bacterial strain grown in the laboratory. A commercially obtained MAb showed the same specificity pattern.