Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum / 生物工程学报

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

Diverse aminoglycoside phosphotransferase types conferring aminoglycoside resistance in Enterobacteriaceae: A single-centre study from Northeast India

The present study investigates the molecular basis of aph-mediated aminoglycoside resistance and their transmission dynamics in a tertiary care hospital of Northeast India. Two hundred forty one isolates (230 Escherichia coli and 11 Klebsiella pneumoniae) were collected and screened for aminoglycoside resistance genes. Various aph types were amplified using polymerase chain reaction (PCR) assay. Plasmid incompatibilty, horizontal transferability and ERIC-PCR based typing were carried out for all the positive isolates. Among them, 67 isolates showed the presence of aph gene. Aph (3“)-IIIa and aph (3')-Via were predominant and horizontally transferable. All the plasmids were of incompatibility I1 group. Twenty-eight different haplotypes of E. coli were found harbouring aph gene types. This study was able to identify diverse aph types in a single centre and their corresponding phenotypic trait.

HCMV UL97 phosphotransferase gene mutations may be associated with antiviral resistance in immunocompromised patients in Belém, PA, Northern Brazil

Abstract INTRODUCTION: Human cytomegalovirus is one of the causes of opportunist infections in immunocompromised patients, and is triggered by factors such as state of viral latency, weakened immune responses, and development of antiviral resistance to ganciclovir, the only drug offered by the public health system in Brazil to treat the infection. The goal of this study was to identify mutations that may be associated with antiviral resistance in immunocompromised patients. METHODS: Molecular analysis was performed in 82 blood samples and subjected to genomic DNA extraction by a silica-based method. Three sequences of the HCMV UL97 gene, which encodes a phosphotransferase protein required for activation of ganciclovir, were amplified by polymerase chain reaction. Pyrosequencing methods were applied to one external 2096-bp segment DNA and two internal sequences between nucleotides 1087 to 1828 to detect mutations in this gene. RESULTS: Approximately 10% of sequences contained mutations between nucleotides 377 and 594, in conserved regions of the UL97 gene, leading to amino acid changes. Eleven coding mutations were identified, including changes leading to amino acid substitutions, E596K and S604F, which were observed in 100% of samples and are described for the first time in Brazil. In addition, one mutation (A594V) that is associated with ganciclovir resistance was detected in a kidney transplant patient. CONCLUSIONS: Further studies to detect mutations associated with HCMV resistance to antiviral drugs are required to demonstrate the need to increase the variety and availability of drugs used to treat viral infections in the public health care system in Brazil.

Effect of PTS modifications on L-tryptophan production in Escherichia coli / 生物工程学报

L-tryptophan, one of the aromatic amino acids, is widely used in the fields of medicine, food and feed additives. The phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) plays an important role in glucose transport and phosphorylation in Escherichia coli. PTS-mediated regulation dominates the carbohydrates' uptake and metabolism in E. coli. We constructed L-tryptophan-producing bacteria containing two typical PTS mutations (ptsHIcrrglf⁻ glk⁺ and ptsG⁻) by Red homologous recombination system, and studied in 50 L jar fermenter using fed-batch fermentation. Both PTS system mutants had a great impact on the biomass (increasing 47.0% and 17.6%, respectively), L-tryptophan production (increasing 25.9% and 9.4%, respectively), glucose conversion rate (increasing 26.5% and 17.4%, respectively) and byproduct acetic acid generation (slightly increased and decreased，respectively).

Weakening resistance marker for establishing a process of screening high-producing CHO cell lines / 中国药科大学学报

To optimize Chinese hamster ovary (CHO)expression system and establish a process of screening CHO cell lines with high productivity;neomycin-phosphotransferase (NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G.After selection by culturing with G418;the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bear-ing wide type NPT.An enhanced green fluorescent protein (EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene;which verified that the resistance of mutant-NPT to G418 was weakened.By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks;mutant-selected pools expressed more exogenous protein than the WT-selected pools.Therefore;the ratio of high producers in a transfected cell population greatly increased.

Xylitol Sensitivity among Oral Streptococci

Xylitol is a five-carbon sugar alcohol that inhibits the growth of oral streptococci, including Streptococcus mutans. In this study, we tested xylitol sensitivity among the oral streptococci. We also compared nucleotide homology of putative fructose phosphotransferase system (PTS) and xylitol sensitivity, since xylitol is transported via the fructose PTS. Among the tested Streptococci, S. pneumonia showed the highest resistance to xylitol while S. gordonii and S. sanguinis showed the most sensitive growth inhibition. These streptococci could be grouped according to their xylitol sensitivity. S. mutans and S. salivarius showed similar bacterial growth inhibition by xylitol. S. mitis, S. oralis, S. pneumonia, S. intermedius and S. anginosus showed relatively low sensitivity to xylitol. When the genetic homologies of five fructose PTSs were compared among the tested streptococci, closely related streptococci showed similar sensitivity to xylitol. Taken together, fructose PTSs may mediate the sensitivity to xylitol in oral streptococci.

Streptococcus mutans gcp gene knockout strains expression profile gene chip / 中国组织工程研究

BACKGROUND:Previous studies have confirmed the presence of bis-(3'-5')-cyclic dimeric guanosine monophosphate signaling pathway in Streptococcus mutans, which construct the streptococcus mutans gcp gene knockout strains. OBJECTIVE:To compare the gene expression differences between Streptococcus mutans wild strains and gcp mutant strains, and to screen the biofilm-related genes from them for the fol ow-up study. METHODS:The total RNA of two kinds of strains were extracted and stained with cy3 and cy5 respectively after reverse transcription. The gene chip was scanned after hybridization and the differential gene were obtained through the data analysis. The different expression genes were verified by real-time PCR. RESULTS AND CONCLUSION:Differential genes were mainly relative about glucose metabolism and biofilm formation. We selected two genes for real-time PCR verification. The PCR results were consistent with the microarray results. After Streptococcus mutans gcp gene knockout, the gene expressions of gcp mutant strains were upregulated and the gene expressions of phosphotransferase system were downregulated, this result suggested that two different genes were related with the c-di-GMP signal pathway downstream.

Cloning, over-expression and in vitro activity of aminoglycoside phosphotransferase from clinical MRSA isolates / 中国药科大学学报

Aim: To clone and over-express the gene encoding aminoglycoside(AG)phosphotransferase(APH)from clinical MRSA isolates in E.coli and to develop an assay method for the recombinant APH.Methods: The susceptibility of clinical MRSA isolates to AGs was tested by disk diffusion.A nucleic acid sequence encoding APH was amplified from the genomic DNA of an isolate and ligated to expression vector pET-28a,and then trans-formed into E.coli BL21(DE3).After purification of the recombinant protein by affinity chromatography,the phosphorylation activity of the enzyme was determined by ESI-MS and disk diffusion.Flesults: All 6 clinical MRSA isolates were unsusceptible to AGs.After cloning and expression,the recombinant APH was purified to90%.The in vitro activity assay indicated that the recombinant protein could inactivate kanamycin B in the assay mixture within 2 h.Conclusion: The recombinant APH showed excellent enzymatic activity.The assay method was simple and convenient,which may provide the basis of developing a screening model for APH inhibitors.