The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.     

Mutants and Intersexual Heterokaryons of Blakeslea trispora for Production of β-Carotene and Lycopene

The industrial production of β-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of β-ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N′-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having β-carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of β-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of β-carotene per g (dry mass). β-Ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.     

Transketolase A, an enzyme in central metabolism, derepresses the marRAB multiple antibiotic resistance operon of Escherichia coli by interaction with MarR

The Escherichia coli marRAB operon specifies two regulatory proteins, MarR (which represses) and MarA (which activates expression of the operon). The latter controls expression of multiple other chromosomal genes implicated in cell physiology, multiple drug resistance and virulence. Using randomly cloned E. coli DNA fragments in the bacterial adenylate cyclase two-hybrid system, we found that transketolase A (TktA) interacts with MarR. Purified (6H)-TktA immobilized on NiNTA resin-bound MarR. Overexpression or deletion of tktA showed that TktA interfered with MarR repression of the marRAB operon. Deletion of tktA increased antibiotic and oxidative stress susceptibilities, while its overexpression decreased them. Hydrogen peroxide induced tktA at 1 h treatment, while an increase in marRAB expression occurred only after 3 h exposure. This increase was dependent on the presence of tktA. Two MarR mutations which eliminated MarR binding to the marRAB operator and one which decreased dimerization of MarR had no effect on MarR interaction with TktA in the two-hybrid system. However, the interaction was disrupted by one of the three tested superrepressor mutant MarR proteins known to increase MarR binding to DNA. TktA inhibition of repression by MarR demonstrates a previously unrecognized level of control of the expression of marRAB operon.     

Biogeography and phylogenetic relationships of Hyrcanian wild apple using cpDNA and ITS noncoding sequences

The Hyrcanian forest of northern Iran is considered one of the potential centres for the evolution and domestication of the genus Malus (Rosaceae). However, the biogeography, phylogenetic position, and taxonomic status of the Hyrcanian wild apples have never been evaluated. In our study, the nucleotide sequences of the internal transcribed spacer (ITS) and the trnH-psbA intergenic spacer region from 14 natural populations were analysed. Phylogenetic analysis based on the ITS and the Maximum-likelihood (ML) tree showed that all Hyrcanian samples were closely related to M. orientalis and M. asiatica and can be placed within section Malus and series Malus. Furthermore, based on a comparison of ITS2 secondary structures, the Hyrcanian samples were identical to M. orientalis and M. sieversii. Biogeographic scenarios constructed using Statistical Dispersal-Vicariance Analysis (S-DIVA) and the Bayesian Binary Method (BBM) indicated that the ancestor of Malus originated during the Eocene, ～53 million years ago (Ma), and that China played a vital role in the expansion of the range of the genus. The members of Malus colonized the Hyrcanian region from China during the Miocene, ～22-10?Ma.     

Characterization of the peridinin–chlorophyll a-protein complex in the dinoflagellate Symbiodinium

The water-soluble peridinin–chlorophyll a-proteins (PCPs) are one of the major light harvesting complexes in photosynthetic dinoflagellates. PCP contains the carotenoid peridinin as its primary pigment. In this study, we identified and characterized the PCP protein and the PCP gene organization in Symbiodinium sp. CS-156. The protein molecular mass is 32.7 kDa, revealing that the PCP is of the monomeric form. The intronless PCP genes are organized in tandem arrays. The PCP gene cassette is composed of 1095-bp coding regions and spacers in between. Despite the heterogeneity of PCP gene tandem repeats, we identified a single form of PCP, the sequence of which exactly matches the deduced sequence of PCP gene clone 7 (JQ395030) by LC–MS/MS analysis of tryptic digested PCP, revealing the mature PCP apoprotein is 312 amino acids in length. Pigment analysis showed a peridinin-to-Chl a ratio of 4. The peridinin-to-Chl a Q_y energy transfer efficiency is 95% in this complex.     

Genetic variation in histidine rich proteins among Indian Plasmodium falciparum population: possible cause of variable sensitivity malaria of rapid diagnostic tests

ABSTRACT: BACKGROUND: Rapid diagnostic tests (RDTs) have revolutionized the diagnosis of malaria. Among the various factors affecting RDTs sensitivity is genetic variation of the antigen used. The genetic variation in PfHRP2 and PfHRP3 proteins was studied among the Indian Plasmodium falciparum isolates. METHODS: One hundred and forty isolates of P. falciparum were collected from six geographical regions of India. Target genes encoding PfHRP2 and PfHRP3 antigens were sequenced to study genetic polymorphism. Minimum detection limit giving a positive rapid diagnostic test was also determined. RESULTS: Extensive variations were observed in amino acid repeat types of PfHRP2 and PfHRP3. PfHRP2 exhibited more polymorphism than PfHRP3. Significant relation was observed between type 2 and type 7 repeats and RDT detection rate as higher number of these repeats showed better sensitivity with RDTs. CONCLUSION: The results provide insights into the genetic diversity of Pfhrp2 and Pfhrp3 genes among Indian P. falciparum population and its relation to RDT sensitivity.     

Side-chain specific isotopic labeling of proteins for infrared structural biology: The case of ring-D(4)-tyrosine isotope labeling of photoactive yellow protein

An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D(4)-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D(4)-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515cm(-1) that is shifted to 1436cm(-1), consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling.     

Cytokine profiles, CTL response and T cell frequencies in the peripheral blood of acute patients and individuals recovered from hepatitis E infection

Background

Hepatitis E is a major public health problem in the developing countries. Pathogenesis of hepatitis E virus (HEV) infection is poorly understood.

Methods

This case-control study included 124 Hepatitis E patients (46 acute and 78 recovered), 9 with prior exposure to HEV and 71 anti-HEV negative healthy controls. HEV induced CTL response by Elispot, cytokines/chemokines quantitation by Milliplex assay and peripheral CD4+ & CD8+ T cell frequencies by flow cytometry were assessed.

Results

Among the patient categories, HEV specific IFN-γ responses as recorded by Elispot were comparable. Comparisons of cytokines/chemokines revealed significantly high levels of IL-1α and sIL-2Rα during acute phase. Circulating peripheral CD4/CD8+ T-cell subsets in acute and recovered individuals were comparable compared to controls, while among patient categories CD8+T cell subset was significantly higher in recovered individuals.

Conclusions

Our findings suggest that IL-1α and sIL-2Rα play a role in the pathogenesis of acute Hepatitis E infection. Lack of robust HEV ORF2-specific CTL response in the peripheral blood of HEV infected patients during the acute and recovered phases of the disease may be associated with involvement of innate immune cells/localization of the immune events at the site of infection.     

Nine-Year Study of the Occurrence of Culturable Viruses in Source Water for Two Drinking Water Treatment Plants and the Influent and Effluent of a Wastewater Treatment Plant in Milwaukee, Wisconsin (August 1994 through July 2003)

Reoviruses, enteroviruses, and adenoviruses were quantified by culture for various ambient waters in the Milwaukee area. From August 1994 through July 2003, the influent and effluent of a local wastewater treatment plant (WWTP) were tested monthly by a modified U.S. Environmental Protection Agency Information Collection Rule (ICR) organic flocculation cell culture procedure for the detection of culturable viruses. Modification of the ICR procedure included using Caco-2, RD, and HEp-2 cells in addition to BGM cells. Lake Michigan source water for two local drinking water treatment plants (DWTPs) was also tested monthly for culturable viruses by passing 200 liters of source water through a filter and culturing a concentrate representing 100 liters of source water. Reoviruses, enteroviruses, and adenoviruses were detected frequently (105 of 107 samples) and, at times, in high concentration in WWTP influent but were detected less frequently (32 of 107 samples) in plant effluent and at much lower concentrations. Eighteen of 204 samples (8.8%) of source waters for the two DWTPs were positive for virus and exclusively positive for reoviruses at relatively low titers. Both enteroviruses and reoviruses were detected in WWTP influent, most frequently during the second half of the year.