The individual contributions of blaB, blaGOB and blaCME on MICs of ß-lactams in Elizabethkingia anophelis.

BACKGROUND: The blaB, blaGOB and blaCME genes are thought to confer ß-lactam resistance to Elizabethkingia anophelis, based on experiments conducted primarily on Escherichia coli. OBJECTIVES: To determine the individual contributions of ß-lactamase genes to increased MICs in E. anophelis and to assess their impact on the in vivo efficacy of carbapenem therapy. METHODS: Scarless gene deletion of one or more ß-lactamase gene(s) was performed in three clinical E. anophelis isolates. MICs were determined by broth microdilution. Hydrolytic activity and expressions of ß-lactamase genes were measured by an enzymatic assay and quantitative RT-PCR, respectively. In vivo efficacy was determined using Galleria mellonella and murine thigh infection models. RESULTS: The presence of blaB resulted in >16-fold increases, while blaGOB caused 4-16-fold increases of carbapenem MICs. Hydrolysis of carbapenems was highest in lysates of blaB-positive strains, possibly due to the constitutionally higher expression of blaB. Imipenem was ineffective against blaB-positive isolates in vivo in terms of improvement of the survival of wax moth larvae and reduction of murine bacterial load. The deletion of blaB restored the efficacy of imipenem. The blaB gene was also responsible for a >4-fold increase of ampicillin/sulbactam and piperacillin/tazobactam MICs. The presence of blaCME, but not blaB or blaGOB, increased the MICs of ceftazidime and cefepime by 8-16- and 4-8-fold, respectively. CONCLUSIONS: The constitutionally and highly expressed blaB gene in E. anophelis was responsible for increased MICs of carbapenems and led to their poor in vivo efficacy. blaCME increased the MICs of ceftazidime and cefepime.

Closer vein spacing by ectopic expression of nucleotide-binding and leucine-rich repeat proteins in rice leaves.

KEY MESSAGE: Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a "Kranz-like" anatomy in leaves that includes decreased interveinal spacing close to that in C4 plant species. As C4 photosynthesis has evolved from C3 photosynthesis independently in multiple lineages, the genes required to facilitate C4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty.

Rice Big Grain 1 promotes cell division to enhance organ development, stress tolerance and grain yield.

Grain/seed yield and plant stress tolerance are two major traits that determine the yield potential of many crops. In cereals, grain size is one of the key factors affecting grain yield. Here, we identify and characterize a newly discovered gene Rice Big Grain 1 (RBG1) that regulates grain and organ development, as well as abiotic stress tolerance. Ectopic expression of RBG1 leads to significant increases in the size of not only grains but also other major organs such as roots, shoots and panicles. Increased grain size is primarily due to elevated cell numbers rather than cell enlargement. RBG1 is preferentially expressed in meristematic and proliferating tissues. Ectopic expression of RBG1 promotes cell division, and RBG1 co-localizes with microtubules known to be involved in cell division, which may account for the increase in organ size. Ectopic expression of RBG1 also increases auxin accumulation and sensitivity, which facilitates root development, particularly crown roots. Moreover, overexpression of RBG1 up-regulated a large number of heat-shock proteins, leading to enhanced tolerance to heat, osmotic and salt stresses, as well as rapid recovery from water-deficit stress. Ectopic expression of RBG1 regulated by a specific constitutive promoter, GOS2, enhanced harvest index and grain yield in rice. Taken together, we have discovered that RBG1 regulates two distinct and important traits in rice, namely grain yield and stress tolerance, via its effects on cell division, auxin and stress protein induction.

Emission Spectrometry for the Detection of Methane Based on Gas Ionization Discharge Microplasma at Room Temperature.

A microplasma-generating device was developed by using needle-plate electrode discharge with the incorporation a Pt/carbon nanotube (CNT) nanocomposite-decorated FTO electrode. When an alternating current voltage of 1.32 kV and a low power consumption of 13 W in nitrogen (N2) carrier gas are applied, the system can be applied to detect methane at room temperature. The main characteristic lines were assigned to CH, C2 and Hα during the discharge process of CH4 at room temperature.The emission intensity of C2 at 516 nm is linear with the concentration of CH4 from 0.5% to 4.0% (φ), and the detection limit (S/N=3) is 0.19% (φ). The emission intensity of Hα at 656 nm is linear with the concentration of CH4 from 0.1% to 3.0%(φ)with the detection limit (S/N=3) is 0.03% (φ). The relative standard deviation (RSD) is less than 2% from 11 repetitive analyses using 3.2% CH4. The Pt/CNT nanocomposite-modified FTO electrode exhibited enhanced sensing performance with precise, repeatability and linear correlation compared with that of the pure MWNT/FTO electrode and bare FTO electrode. When CH4 were discharged in air, the emission spectra of CH4 was different from that in N2. It was found that C2 peak was disappeared and the Hα intensity was also liner to the concentration of CH4 in the range of 0.5%～4%. The established system exhibited advantages with small size, simple fabrication and operation at room temperature compared to other detection system.

The analytical determination and electrochemiluminescence behavior of amoxicillin.

A novel electrochemiluminescence (ECL) luminophor of amoxicillin was studied and found to generate ECL following the oxidation or reduction of amoxicillin. The amoxicillin oxidation state was also found to eliminate the reduction state, generating ECL. When solutions of amoxicillin were scanned between +1.5 V and -1.0 V with a graphite electrode in the presence of cetyltrimethyl ammonium bromide using KC1 as the supporting electrolyte, ECL emissions were observed at potentials of -0.7 V and +0.5 V. The ECL intensity at -0.7 V was enhanced by H2O2. Based on these findings, an ECL method for the determination of the amoxicillin concentration is proposed. The ECL intensities were linear with amoxicillin concentrations in the range of 1.8 × 10-8 g/mL to 2.5 × 10-7 g/mL, and the limit of detection (signal/noise = 3) was 5 × 10-9 g/mL. The florescence of amoxicillin had the greatest emission intensity in a neutral medium, with the emission wavelength dependent on the excitation wavelength. The experiments on the ECL mechanism for amoxicillin found that the electrochemical oxidation products of dissolved oxygen and active oxygen species contributed to the ECL process. The data also suggest that the hydroxyl group of amoxicillin contributed to its ECL emission.

Influence of signal-peptide truncations on the functional expression of Escherichia coli gamma -glutamyltranspeptidase.

The full-length Escherichia coli gamma -glutamyltranspeptidase (EcGGT) gene and five truncations lacking 33, 51, 54, 60, and 78 bp respectively at the 5' end were prepared by polymerase chain reaction and cloned into the expression vector pQE-30. Isopropyl-beta -D-thiogalactopyranoside induction of E. coli M15 cells bearing the recombinant plasmids resulted in the intracellular production of the expressed proteins, EcGGT, EcGGT/DeltaN11, EcGGT/DeltaN17, EcGGT/DeltaN18, EcGGT/DeltaN20, and EcGGT/DeltaN26. The overexpressed enzymes were purified to near homogeneity by Ni(2+)-NTA resin. The specific activity for EcGGT, EcGGT/DeltaN11 and EcGGT/DeltaN17 was 5.3, 4.9, and 4.8 U/mg protein respectively, whereas the rest three enzymes had shown no GGT activity under the enzyme assay conditions. More than 94% of the activity was found in the cytoplasmic fraction of E. coli M15 cells harboring pQE-EcGGT, pQE-EcGGT/DeltaN11 or pQE-EcGGT/DeltaN17. Western blot analysis confirmed that the majority of N-terminally truncated enzymes were present in the cytoplasm.

Site-directed mutagenesis of conserved Thr407, Asp433 and Met464 residues in small subunit of Escherichia coli gamma-glutamyltranspeptidase.

Sequence comparison showed that residues Thr407, Asp433, and Met464 in the small subunit of Escherichia coli gamma-glutamyltranspeptidase (EcGGT) were conserved in the aligned enzymes. In this study, we further investigated the functional significance of these conserved residues by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 cells and purified to near homogeneity by Ni2+-NTA resin. Except M464L, other mutants had shown no GGT activity under enzyme assay conditions and activity staining. Furthermore, mutations on these residues impaired the capability of autocatalytic processing of the enzyme. Based on these observations, it is concluded that these residues play an important role in the enzyme maturation.

Identification of glutamate residues important for catalytic activity or thermostability of a truncated Bacillus sp. strain TS-23 alpha-amylase by site-directed mutagenesis.

The importance of 17 glutamate residues of a truncated Bacillus sp. strain TS-23 alpha-amylase (BACdeltaNC) was investigated by site-directed mutagenesis. The Ala- and Asp-substituted variants were overexpressed in the recombinant E. coli cells and the 54-kDa proteins were purified to nearly homologous by nickel-chelate chromatography. Glu-295, which locates in the conserved region III of amylolytic enzymes, mutations resulted in a complete loss of enzyme activity. The specific activity for E151A was decreased by more than 30%, while other variants showed activity comparable to that of BACdeltaNC. A decreased half-life at 70 degrees C was observed for Glu-219 variants with respective to the wild-type enzyme, suggesting that replacement of Glu-219 by either Ala or Asp might have a significant destabilizing effect on the protein structure.