Genomic characteristics of a Klebsiella pneumoniae strain coproducing carbapenemases KPC-2 and NDM-5 / 中华微生物学和免疫学杂志

Objective:To analyze the characteristics of drug resistance genes in a Klebsiella pneumoniae strain coproducing carbapenemases KPC-2 and NDM-5. Methods:Klebsiella pneumoniae KPN-hnqyy was separated from the stool specimen of a patient in the Hematology Department of Affiliated Cancer Hospital of Zhengzhou University. The strain was identified with a BD Phenix-M50 automated microbiology system and the minimum inhibitory concentration against the strain was measured as well. The genotypes of the carbapenemases were tested by enzyme immunochromatographic assay and PCR method. The transferability of related plasmids was analyzed by conjugation test. Whole-genome sequencing of the strain was conducted using PacBio and Illumina platforms. The MLST type, resistance gene and plasmid type of the strain were retrieved in BacWGSTdb. The genome and open reading frame sequence of the strain were compared using Easyfig_2.2.3. Visual cycle graphs were generated using BRIG v0.95. Results:Klebsiella pneumoniae KPN-hnqyy was resistant to carbapenem antibiotics. It belonged to ST11 and carried two carbapenemase genes of blaKPC-2 and blaNDM-5. The conjugant only harbored the blaKPC-2 gene. Whole-genome sequencing revealed that the strain contained one chromosome and three plasmids. Its chromosome genome shared more than 99.9% similarity with that of Klebsiella pneumonia KP69 and KP19-2029. Moreover, a similar IncR and IncFⅠ resistance gene fusion region was contained in different types of plasmids carried by them: the blaKPC-2 gene was located in a structure—which evolved from the Tn3-△Tn4401-Tn1721/Tn1722 sequence—inside this fusion region with its ends inserted into the transposase IS26 gene; the blaNDM-5 gene was located on a transposon containing the special plasmids of the insertion fragment in phages, with its ends inserted into the transposase IS26 gene too. Conclusions:The IncR and IncFⅡ resistance gene fusion region of blaKPC-2 carried by Klebsiella pneumoniae ST11 might be widely coexistent with the chromosomal genome. The blaNDM-5 gene carried by special plasmids might be accidentally obtained through gene recombination mediated by transposable element IS26. The wide transmission of Klebsiella pneumoniae ST11 carrying the blaKPC-2 gene in China and its ability to obtain other carbapenemase genes through transposable element IS26 were well worth attention.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Cloning, High Expression of Single-Stranded DNA-Binding Protein and Its Interaction with ssDNA / 中国生物工程杂志

E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.

Telomere and Telomerase:to Decode 2009 Nobel Prize in Physiology or Medicine / 生物化学与生物物理进展

Nobel Prize 2009 in Physiology or Medicine is awarded to three American scientists, Elizabeth H.Blackburn, Carol W.Greider and Jack W.Szostak, for the discovery of"how the chromosomes are protected by telomeres and the enzyme telomerase".Telomere is the specific structure at the ends of the chromosomes and protects it from fusion and degradation.Telomerase synthesizes telomere DNA to maintain the telomere length.Studies suggest that telomere length and telomerase activity is directly associated with cell life and the genesis of many diseases.With the progress of study, how to control the telomere length and telomerase activity is helpful to shed light on the studies in"cancer, inherited diseases and senescence", and will stimulate the development of potential new therapies.

Observation of The Interaction Between MutS and MutL Mismatch Repair Proteins by Fusion Protein Systems / 生物化学与生物物理进展

MutL and MutS or their homologues are two crucial proteins of DNA mismatch repair (MMR) system. A new method was described for observation of the interaction between MutS and MutL which is based on the fusion gene/fusion protein technique. Three fusion proteins, MutL-GFP fusion (Trx-His6-GFP-(Ser-Gly)6-MutL), MutL-Strep tag Ⅱ fusion (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) and MutS fusion (Trx-His6-(Ser-Gly)6-MutS), were constructed and expressed in E. coli AD494 (DE3). Interaction assay between MutS and MutL was performed in a 96-well microtiter plate.MutS fusion protein was immobilized on the wells and provided a surface for the interaction between MutS and MutL.Results showed that only after binding of MutS to the mismatched DNA, there was an interaction between MutS and MutL.The binding events could be indicated by GFP signal or the signal generated from alkaline phosphatase and its substrate. In addition, the method based on fusion molecular system also serve as a model for studies on the interactions among other proteins or biomolecules.