Roles of DJ41_1407 and DJ41_1408 in Acinetobacter baumannii ATCC19606 Virulence and Antibiotic Response.

Acinetobacter baumannii is a major cause of nosocomial infections, and its highly adaptive nature and broad range of antibiotic resistance enable it to persist in hospital environments. A. baumannii often employs two-component systems (TCSs) to regulate adaptive responses and virulence-related traits. This study describes a previously uncharacterized TCS in the A. baumannii ATCC19606 strain, consisting of a transcriptional sensor, DJ41_1407, and its regulator, DJ41_1408, located adjacent to GacA of the GacSA TCS. Markerless mutagenesis was performed to construct DJ41_1407 and DJ41_1408 single and double mutants. DJ41_1408 was found to upregulate 49 genes and downregulate 43 genes, most of which were associated with carbon metabolism and other metabolic pathways, such as benzoate degradation. MEME analysis revealed a putative binding box for DJ41_1408, 5'TGTAAATRATTAYCAWTWAT3'. Colony size, motility, biofilm-forming ability, virulence, and antibiotic resistance of DJ41_1407 and DJ41_1408 single and double mutant strains were assessed against wild type. DJ41_1407 was found to enhance motility, while DJ41_1408 was found to upregulate biofilm-forming ability, and may also modulate antibiotic response. Both DJ41_1407 and DJ41_1408 suppressed virulence, based on results from a G. mellonella infection assay. These results showcase a novel A. baumannii TCS involved in metabolism, with effects on motility, biofilm-forming ability, virulence, and antibiotic response.

Reactivity of human antisera to codon optimized SARS-CoV2 viral proteins expressed in Escherichia coli.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV2 virus continues to pose a serious threat to public health worldwide. The development of rapid diagnostic kits can assist the Tzu Chi Foundation in supporting global volunteers working to provide relief during the current pandemic. MATERIALS AND METHODS: In this study, nucleotide sequences derived from publicly available viral genome data for several domains of the SARS-CoV2 spike and nucleocapsid (N) proteins were chemically synthesized, with codon optimization for Escherichia coli protein expression. No actual viral particles were involved in these experiments. The synthesized sequences were cloned into an E. coli expression system based on pQE80L, and expressed viral proteins were subsequently purified using Ni-affinity chromatography. Western blotting was conducted using human antiviral sera to assess the response of codon-modified viral proteins to COVID-19 patient sera. RESULTS: N protein was expressed in amounts large enough to support large-scale production. The N-terminal domain, receptor-binding domain (RBD), Region 3, and the S2 domain were expressed in small but sufficient amounts for experiments. Immunoblotting results showed that anti-N IgG and anti-N IgM antibodies were detected in most patient sera, but only 60% of samples reacted with the recombinant RBD and S2 domain expressed by E. coli. CONCLUSION: The results indicated that codon-optimized SARS-CoV2 viral proteins can be expressed in E. coli and purified for rapid antibody detection kit preparation, with the codon-optimized N protein, RBD, and S2 protein demonstrating the most potential.

Production of optically pure (-)-borneol by Pseudomonas monteilii TCU-CK1 and characterization of borneol dehydrogenase involved.

(-)-Borneol is a bicyclic plant secondary metabolite. Optically pure (-)-borneol can only be obtained from plants, and demand exceeds supply in China. In contrast, chemically synthesized borneol contains four different stereoisomers. A strain of Pseudomonas monteilii TCU-CK1, isolated in Hualien, Taiwan, can accumulate (-)-borneol in growth culture and selectively degrades the other three isomers when chemically synthesized borneol is used as sole carbon source. This (-)-borneol production method can be scaled-up for production of large quantities in the future. More importantly, laborious plant cultivation and harvest is no longer required. The main enzyme that appears in this degradation pathway, borneol dehydrogenase (BDH), and the genome sequence of TCU-CK1 are reported. The kcat/Km values of TCU-CK1 BDH on (+)- and (-)-borneol are 538.4 ± 38.4 and 17.7 ± 1.1 (s-1 mM-1), respectively. About ∼30 fold difference in the kcat/Km value between (+)-borneol and (-)-borneol was observed, in good agreement with the fact that TCU-CK1 prefers to degrade (+)-borneol, rather than (-)-borneol. A BDH isozyme was identified in a strain in which the primary BDH gene had been knocked out. (-)-Camphor can work as an inhibitor of BDH with a Ki of 1.03 ± 0.11 mM at pH 7.0, leading to the accumulation of (-)-borneol in culture. (Patent pending).