Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King’s B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the “ring loop” covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent “locked on” and “locked off” allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.

Screening and Identification of a Novel Anti-tuberculosis Compound That Targets Deoxyuridine 5′-Triphosphate Nucleotidohydrolase

Tuberculosis (TB) is still a threat to humans worldwide. The rise of drug-resistant TB strains has escalated the need for developing effective anti-TB agents. Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is essential for thymidylate biosynthesis to maintain the DNA integrity. In Mycobacterium tuberculosis, dUTPase provides the sole source for thymidylate biosynthesis, which also has the specific five-residue loop and the binding pockets absent in human dUTPase. Therefore, dUTPase has been regarded as a promising anti-TB drug target. Herein, we used a luminescence-based dUTPase assay to search for the inhibitors target M. tuberculosis dUTPase (Mt-dUTPase) and identified compound F0414 as a potent Mt-dUTPase inhibitor with an IC50 of 0.80 ± 0.09 μM. F0414 exhibited anti-TB activity with low cytotoxicity. Molecular docking model and site-directed mutation experiments revealed that P79 was the key residue in the interaction of Mt-dUTPase and F0414. Moreover, F0414 was shown to have stronger binding with Mt-dUTPase than with Mt-P79A-dUTPase by surface plasmon resonance (SPR) detection. Interestingly, F0414 exhibited insensitivity and weak directly binding on human dUTPase compared with that on Mt-dUTPase. All the results highlight that F0414 is the first compound reported to have anti-TB activity by inhibiting Mt-dUTPase, which indicates the potential application in anti-TB therapy.

Modified Catalytic Performance of Lactobacillus Fermentum L-lactate Dehydrogenase by Rational Design

L-lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum. L-lactate dehydrogenase gene (LF-L-LDH0845) with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248. To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates, site-directed mutation of Tyr221 was performed by analyzing the amino acids in active center. Kinetic parameters show that the k cat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate, 4-methyl-2-oxopentanoate, and glyoxylate are 1.21 s -1 , 1.35 s -1 , and 0.72 s -1 , respectively, which are 420%, 150% and 130% of the wild-type LDH0845. This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845, making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.

A Comparison of Two Monoterpenoid Synthases Reveals Molecular Mechanisms Associated With the Difference of Bioactive Monoterpenoids Between Amomum villosum and Amomum longiligulare

The fruits of Amomum villosum and Amomum longiligulare are both used medicinally as Fructus Amomi the famous traditional Chinese medicine, however, the medicinal quality of A. villosum is better than that of A. longiligulare. Volatile terpenoids in the seeds, especially bornyl acetate and borneol, are the medicinal components of Fructus Amomi. The volatile terpenoids and transcriptome of developing seeds of A. villosum and A. longiligulare were compared in this study. The result revealed that the bornyl acetate and borneol contents were higher in A. villosum than in A. longiligulare. Additionally, six terpenoid synthase genes (AlTPS1–AlTPS6) were screened from the transcriptome of A. longiligulare, and AlTPS2 and AlTPS3 were found to share 98 and 83% identity with AvTPS2 and AvBPPS (bornyl diphosphate synthase) from A. villosum, respectively. BPPS is the key enzyme for the biosynthesis of borneol and bornyl acetate. Biochemical assays improved that AlTPS2 had an identical function to AvTPS2 as linalool synthase; however, AlTPS3 produced camphene as the major product and bornyl diphosphate (BPP) as the secondary product, whereas AvBPPS produced BPP as its major product. There was only one different amino acid between AlTPS3 (A496) and AvBPPS (G495) in their conserved motifs, and the site-directed mutation of A496G in DTE motif of AlTPS3 changed the major product from camphene to BPP. Molecular docking suggests that A496G mutation narrows the camphene-binding pocket and decreases the BPP-binding energy, thus increases the product BPP selectivity of enzyme. In addition, the expression level of AvBPPS was significantly higher than that of AlTPS3 in seeds, which was consistent with the related-metabolites contents. This study provides insight into the TPS-related molecular bases for the biosynthesis and accumulation differences of the bioactive terpenoids between A. villosum and A. longiligulare. BPPS, the key gene involved in the biosynthesis of the active compound, was identified as a target gene that could be applied for the quality-related identification and breeding of Fructus Amomi.