Complete genome sequence of Acinetobacter baumannii MDR-TJ and insights into its mechanism of antibiotic resistance.

OBJECTIVES: To determine the genome sequence of Acinetobacter baumannii strain MDR-TJ and characterize the mechanisms of multidrug resistance in this strain. METHODS: The whole-genome sequence was determined using Roche 454 GS FLX Titanium. Subsequently, the gaps were closed by sequencing PCR products. The genome of strain MDR-TJ was annotated using IMG ER, the RAST annotation server and the BASys bacterial annotation system. The comM gene of MDR-TJ was examined to identify a possible antibiotic resistance island. Based on the results of multilocus sequence typing, we investigated seven multidrug-resistant A. baumannii strains belonging to global clone 2 (GC2) isolated from Asia, Australia and Europe to determine the backbone shared by resistance islands of GC2 isolates. RESULTS: The A. baumannii strain MDR-TJ genome consists of a circular chromosome and a plasmid, pABTJ1. Strain MDR-TJ was assigned to sequence type ST2. Strain MDR-TJ harbours a 41.6 kb resistance island designated RI(MDR-TJ), which can be derived from the backbone of Tn6167 through the insertion of a Tn6022 into the 3'-end of the tetA(B) gene. Comparative analysis showed that transposon Tn6022 and its truncated forms prevailed in the antibiotic resistance islands of GC2 isolates. The carbapenem resistance gene bla(OXA-23) carried by transposon Tn2009 is located on a putatively conjugative plasmid, pABTJ1. CONCLUSIONS: A. baumannii strain MDR-TJ belongs to GC2 and is resistant to multiple antibiotics. A. baumannii MDR-TJ harbours a genomic resistance island that interrupts the comM gene. The carbapenem resistance of MDR-TJ is mediated by a putatively conjugative plasmid, pABTJ1.

Genome sequence of Acinetobacter baumannii MDR-TJ.

Acinetobacter baumannii is a pathogenic species of bacteria, identified as an aerobic gram-negative bacterium, that is resistant to most antibiotics. In this study, the MDR-TJ strain was isolated at the Second Hospital of Tianjin Medical University, China, and was found to be resistant to penicillin, cephalosporins, aminoglycosides, quinolones, and also imipenem. The genome sequence of Acinetobacter baumannii strain MDR-TJ was determined by using a combination of 454 pyrosequencing and paired-end sequencing performed with the Roche Genome Sequencer FLX system to generate a scaffolded assembly.

Genetic modification of critical enzymes and involved genes in butanol biosynthesis from biomass.

Interest in biobutanol, a sustainable vehicle fuel, is increasing due to rising oil prices and concerns of surrounding climate change and the energy crisis. However, the costs of biobutanol with conventional ABE fermentation by Clostridium are higher than the cost of butanol from today's petrochemical processes. Two major problems in the economic production of biobutanol are difficulty controlling the induction of a metabolic shift from acidogenesis to solventogenesis and limitations imposed by severe product inhibition. With developments in biotechnology, and the completion of genome sequencing of Clostridium, genetic modification is a viable method to improve the solvent yield and the butanol production ratio. The present article aims to highlight the latest research progress on overexpressing, inserting, knocking out, and knocking down genes of the key enzymes in the ABE fermentation pathway and other relative genes (such as genes coding for heat-shock proteins, operon, transcription, etc). Recombinant manipulations of these genes in Escherichiacoli and yeast have also been reported recently, although their butanol yields are lower than in Clostridium. Butanol production with solventogenic clostridia from various feedstocks is also evaluated in this review.

Synthesis and activity of an octapeptide inhibitor designed for SARS coronavirus main proteinase.

The outbreak of SARS, a life-threatening disease, has spread over many countries around the world. So far there is no effective drug for the treatment of SARS. Stimulated by the binding mechanism of SARS-CoV Mpro with the octapeptide AVLQSGFR reported recently as well as the "Chou's distorted key" theory, we synthesized the octapeptide AVLQSGFR for conducting various biochemical experiments to investigate the antiviral potential of the octapeptide against SARS coronavirus (BJ-01). The results demonstrate that, compared with other compounds reported so far, AVLQSGFR is the most active in inhibiting replication of the SARS coronavirus, and that no detectable toxicity is observed on Vero cells under the condition of experimental concentration.

Determination of residual clenbuterol in pork meat and liver by HPLC with electrochemical detection.

AIM: To detect the residual clenbuterol in pork meat and liver using HPLC with Coulometric electrode array system. METHODS: Homogenized meat or liver sample was treated with 1 mol x L(-1) hydrochloric acid and centrifuged, the fat existing in meat or liver tissue was removed by diethyl ether. The pH of the remaining aqueous layer was adjusted to 10.8 +/- 0.2 or 11.6 +/- 0.2 for meat or liver and liquid-liquid extraction with diethyl ether was followed. The ether extract was evaporated to dryness, the residue was dissolved in the mobile phase. The mobile phase A consisted of 50 mmol x L(-1) phosphoric acid-30 mmol x L(-1) triethylamine and was adjusted to pH 4.0 with 2 mol x L(-1) sodium hydroxide solution. The mobile phase B consisted of methanol-acetonitrile (30:45). A mixture of mobile phase A and B (80:20) was used in the method. A four electrode array module was selected for quantitation, the electrode potentials were set at 450, 600, 650 and 680 mV respectively. RESULTS: The two calibration curves for meat and liver showed good linearity between 1.88 - 60.16 ng x g(-1), the detection limit of clenbuterol was 1.2 ng x g(-1). CONCLUSION: This method using HPLC-electrochemical detection is reproducible, and the sensitivity is good enough for the determination of clenbuterol in meat and liver.

Prokaryotic expression of Chinese bovine enterokinase catalytic subunit / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.</p><p><b>METHODS</b>Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from duodenal mucosa of a bovine obtained at wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His.Tag affinity chromatography, and it bioactivity was analyzed.</p><p><b>RESULTS</b>Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21 degrees C overnight and shown to have a high autocatalytic cleavage activity.</p><p><b>CONCLUSIONS</b>The EKL gene from Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.</p>

[Cloning and fusion expression of bovine enterokinase light chain gene in Escherichia coli].

The objective of the study was to obtain the gene of bovine enterokinase light chain, which would be used in the cleavage and purification of fusion proteins. The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from a sold bovine's duodenal mucosa, then cloned into the pUCmT cloning vector and sequenced. Compared with the sequence deposited in GenBank,the cloned gene sequence is correct. Then the interested gene fragment was inserted into the pET39b expression plasmid. The recombinant vector pET39b-EKL was transformed into E.coli BL21(DE3) and induced by IPTG. It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5'terminal multi-cloning site and recombinant fragment after the analysis of the nucleotide sequence. SDS-PAGE analysis indicated that target product was about 65 kDa which occupied 28% of the total protein. A pure fusion protein was obtained by nickel chelating chromatogram using His*Binding Resin. After desalting and changing buffer, the crude kinase was incubated at 21 degrees overnight and demonstrated a high autocatalytic cleavage activity. This investigation would be able to lay a foundation for enterokinase activity research and farther application of expression products on a large scale.