Jinsong Li

In order to study the relationship between the structure and function of AmpG, structure, site-specific mutation, and gene complementary experiments have been performed against the clinical isolates ofPseudomonas aeruginosa. We found that there are 51 nucleotide variations at 34 loci over theampGgenes from 24 of 35P. aeruginosastrains detected, of which 7 nucleotide variations resulted in amino acid change. TheampGvariants with the changed nucleotides (amino acids) could complement the function ofampGdeleted PA01 (PA01ΔG). The ampicillin minimum inhibitory concentration (MIC) of PA01ΔG complemented with 32ampGvariants was up to 512 μg/ml, similar to the original PA01 (P. aeruginosa PA01). Furthermore, site-directed mutation of two conservative amino acids (I53 and W90) showed that when I53 was mutated to 53S or 53T (I53S or I53T), the ampicillin MIC level dropped drastically, and the activity of AmpCβ-lactamase decreased as well. By contrast, the ampicillin MIC and the activity of AmpCβ-lactamase remained unchanged for W90R and W90S mutants. Our studies demonstrated that although nucleotide variations occurred in most of theampGgenes, the structure of AmpG protein in clinical isolates is stable, and conservative amino acid is necessary to maintain normal function of AmpG.

Ovarian cancer is the most lethal disease among the malignant tumors of female reproductive organs. Few successful therapeutic options exist for patients with ovarian cancer. The common therapeutic methods are surgical operation, chemotherapy, radiotherapy, and combination of these treatments. In recent years, studies have indicated that Pinellia pedatisecta Schott (PPS), a traditional Chinese medicine, could inhibit tumor growth. In this study, we demonstrated that PPS extract could induce apoptosis in SKOV3 cells in a dose- and time-dependent manner. We further conducted transcriptome sequencing on PPS extract-treated SKOV3 cells along with controls, and identified 1,754 transcripts whose expression differs at least 3-fold over the controls. These differentially expressed transcripts include the apoptosis-related genes such as the caspase family members, and were significantly enriched in steroid biosynthesis in the KEGG pathway database compared with the transcriptome background. Most of the differentially expressed transcripts from this pathway were upregulated in PPS extract-treated cell line, indicating that PPS extract-induced apoptosis was accompanied by increased steroid biosynthesis (e.g. zymosterol). These results suggest that PPS extract could be a new cytostatic therapeutic agent for ovarian cancer.

The aim of this study was to analyze the molecular epidemiologic characteristics of Acinetobacter baumannii. A total of 398 isolates were collected in 7 regions of South China from January to June of 2012. Drug sensitivity was tested toward 15 commonly used antibiotics; thus, 146 multi-drug-resistant strains (resistant to more than 7 drugs) were identified, representing 36.7% of all isolates. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for molecular subtyping. According to the PFGE results (with a cutoff of 70% similarity for the DNA electrophoretic bands), 146 strains were subdivided into 15 clusters, with cluster A being the largest (33.6%, distributed in all districts except Jiaxing). Cluster B was also widespread and included 14.4% of all strains. In addition, MLST results revealed 11 sequence types (ST), with ST208 being the most prevalent, followed by ST191 and ST729. Furthermore, 4 novel alleles and 6 novel STs were identified. Our results showed that multi-drug-resistant A. baumannii in South China shares the origin with other widespread strains in other countries. The nosocomial infections caused by A. baumannii have been severe in South China. Continuous monitoring and judicious antibiotic use are required.

In order to get insights into plasmid evolution and the dissemination of multidrug resistance, we performed extensive comparative genomics analyses of the Klebsiella pneumoniae plasmid pKF3-94 and some of its related plasmids. pKF3-94 is one of three plasmids isolated from the K. pneumoniae strain KF3. Of the 144 putative genes it harbors, 69 can be functionally assigned to be involved in transfer conjugation, transfer leading, antimicrobial resistance, transposon function and plasmid replication. Comparison of plasmid replicon sequence types revealed that pKF3-94 carries two replicons that are distinct from those carried on the two sibling K. pneumonia plasmids pKF3-70 and pKF3-140, thereby allowing pKF3-94 to coexist with these latter plasmids in the same host cell. Comparative genomics analyses further showed that pKF3-94 is more similar to plasmids pK1HV and pC15-k, which were isolated from different K. pneumonia strains, than to pKF3-70 and pKF3-140. Interestingly, pK1HV contains a unique 49 kb region rich in mobile genetic elements and drug resistance genes, while pKF3-94 and pC15-k share a 15 kb homology region partitioned into a region rich in drug resistance genes and one containing a replicon. It is conceivable, therefore, that pK1HV and pC15-k have both arisen from a common pKF3-94-like plasmid. The comparisons lend further support for the role horizontal gene transfer plays in genome evolution and in the dissemination of genetic elements including drug resistance genes.

The homocysteine methyltransferase encoded by mmuM is widely distributed among microbial organisms. It is the key enzyme that catalyzes the last step in methionine biosynthesis and plays an important role in the metabolism process. It also enables the microbial organisms to tolerate high concentrations of selenium in the environment. In this research, 533 mmuM gene sequences covering 70 genera of the bacteria were selected from GenBank database. The distribution frequency of mmuM is different in the investigated genera of bacteria. The mapping results of 160 mmuM reference sequences showed that the mmuM genes were found in 7 species of pathogen genomes sequenced in this work. The polymerase chain reaction products of one mmuM genotype (NC_013951 as the reference) were sequenced and the sequencing results confirmed the mapping results. Furthermore, 144 representative sequences were chosen for phylogenetic analysis and some mmuM genes from totally different genera (such as the genes between Escherichia and Klebsiella and between Enterobacter and Kosakonia) shared closer phylogenetic relationship than those from the same genus. Comparative genomic analysis of the mmuM encoding regions on plasmids and bacterial chromosomes showed that pKF3-140 and pIP1206 plasmids shared a 21 kb homology region and a 4.9 kb fragment in this region was in fact originated from the Escherichia coli chromosome. These results further suggested that mmuM gene did go through the gene horizontal transfer among different species or genera of bacteria. High-throughput sequencing combined with comparative genomics analysis would explore distribution and dissemination of the mmuM gene among bacteria and its evolution at a molecular level.

Streptococcus thermophilus, a gram-positive facultative anaerobe, is one of the most important lactic acid bacteria widely used in the dairy fermentation industry. In this study, we have analyzed the global transcriptional profiling of S. thermophilus upon temperature change. During a temperature shift from 42°C to 50°C, it is found that 196 (10.4%) genes show differential expression with 102 up-regulated and 94 down-regulated at 50°C. In particular, 1) Heat shock genes, such as DnaK, GroESL and clpL, are identified to be elevated at 50°C; 2) Transcriptional regulators, such as HrcA, CtsR, Fur, MarR and MerR family, are differentially expressed, indicating the complex molecular mechanisms of S. thermophilus adapting to heat shock; 3) Genes associated with signal transduction, cell wall genes, iron homeostasis, ABC transporters and restriction-modification system were induced; 4) A large number of the differentially expressed genes are hypothetical genes of unknown function, indicating that much remains to be investigated about the heat shock response of S. thermophilus. Experimental investigation of selected heat shock gene ClpL shows that it plays an important role in the physiology of S. thermophilus at high temperature and meanwhile we confirmed ClpL as a member of the CtsR regulon. Overall, this study has contributed to the underlying adaptive molecular mechanisms of S. thermophilus upon temperature change and provides a basis for future in-depth functional studies.