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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Purification and expression of a novel bacteriocin, JUQZ-1, against Pseudomonas syringae pv. Actinidiae (PSA), secreted by Brevibacillus laterosporus Wq-1, isolated from the rhizosphere soil of healthy kiwifruit

Kiwifruit canker, caused by Pseudomonas syringae pv. actinidiae (PSA), has led to significant losses in the kiwifruit industry each year. Due to the drug resistance feature of PSA, biological control is currently the most promising method. Developing biocontrol bacteria against PSA could help solve the issue of drug resistance generated during the chemical control of PSA to a certain extent. In this research, a Wq-1 strain that demonstrated excellent inhibitory activity against PSA was isolated from the rhizosphere soil of healthy kiwifruit. Based on the morphological characteristics and phylogenetic analysis of the 16S rRNA gene sequence, the isolated strain was identified as Brevibacillus laterosporus Wq-1. Bacteriostatic proteins were isolated from the cell-free culture filtrate of strain Wq-1 and were found to have a molecular weight of approximately 12 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection revealed that there were several peptides in the target band that were consistent with protein 01021 in the genome. The gene of the 01021 protein was cloned into the plasmid pPICZa, and the recombinant bacteriocin was successfully expressed using the Pichia pastoris X33 expression system. The recombinant protein 01021 effectively inhibited the growth of PSA. This is the first report of the protein’s antimicrobial activity, distinguishing it from previously identified bacteriocins. Therefore, we named this bacteriocin JUQZ-1. In addition, our results showed that the protein JUQZ-1 not only exhibited a broad bacteriostatic spectrum but also high thermal and pH stability suitable for harsh environmental conditions., JUQZ-1, a protein with antimicrobial properties and strong environmental tolerance, may serve as a promising alternative to antibiotics.

Shuai, Yang↗
DOE OSTI2024

Characterization of a widespread sugar phosphate-processing bacterial microcompartment

Many prokaryotes form Bacterial Microcompartments (BMCs) that encapsulate segments of specialized metabolic pathways to enhance catalysis. The various functions of metabolosomes, catabolic BMCs, are dictated by the signature enzyme that processes initial substrates of the confined pathway. The components and native functions of several metabolosomes have been experimentally characterized; however one of the most prevalent across all bacteria has yet to be studied. Sugar Phosphate Utilizing (SPU) BMC loci encode enzymes predicted to be involved in sugar phosphate metabolism. The SPU genetic loci are found in organisms occupying habitats ranging from soils to hot springs, highlighting the ubiquity of the SPU BMC. We bioinformatically characterized seven SPU subtypes, all which contain an enzyme unique to SPU BMCs, a deoxyribose 5-phosphate aldolase (DERA). Here, we define the fundamental characteristics of SPU BMCs and have expressed, purified, and characterized a set of SPU core enzymes. These include a protein-protein complex formed between a SPU BMC DERA and a predicted ribose 5-phosphate isomerase. Further, we show that the SPU BMC DERA is catalytically active and propose that it acts as the universal signature enzyme for the SPU BMC, with implications for fundamental understanding and biotechnological applications of SPU BMCs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum

The heliobacterial photochemical reaction center (HbRC) from the chlorophototrophic Firmicutes bacterium Heliobacterium modesticaldum is the only homodimeric type I RC whose structure is known. Using genetic techniques recently established in our lab, we have developed a rapid heterologous expression system for the HbRC core polypeptide PshA. Our system relies on rescue of the non-chlorophototrophic ΔpshA::cbp2p - aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. Furthermore, we constructed two tagged variants of PshA, one with an N-terminal octahistidine tag and one with an internal hexahistidine tag, which facilitate rapid purification of pure, active HbRC cores in milligram quantities. We constructed a suite of shuttle vectors bearing untagged or tagged versions of pshA driven by various promoters. Surprisingly, we found that the eno and gapDH_2 promoters from Clostridium thermocellum drive better expression of pshA than fragments of DNA derived from the region upstream of the pshA locus on the Hbt. modesticaldum genome. This “ pshA rescue” strategy additionally provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural Characterization of the Human Cytosolic Malate Dehydrogenase I

The first crystal structure of the human cytosolic malate dehydrogenase I (MDH1) is described. Structure determination at a high resolution (1.65 Å) followed production, isolation, and purification of human MDH1 using a bacterial expression system. The structure is a binary complex of MDH1 with only a bound malonate molecule in the substrate binding site. Comparisons of this structure with malate dehydrogenase enzymes from other species confirm that the human enzyme adopts similar secondary, tertiary, and quaternary structures and that the enzyme retains a similar conformation even when nicotinamide adenine dinucleotide (NAD + ) is not bound. A comparison to the highly homologous porcine (sus scrofa) MDH1 ternary structures leads to the conclusion that only small conformational differences are needed to accommodate binding by NAD + or other NAD + mimetics. Conformational differences observed in the second subunit show that the NAD + binding elements are nevertheless quite flexible. Comparison of hMDH1 to the human mitochondrial malate dehydrogenase (hMDH2) reveals some key differences in the α7–α8 loop, which lies directly beneath the substrate binding pocket. These differences might be exploited in the structure-assisted design of selective small molecule inhibitors of hMDH1, an emerging target for the development of anticancer therapeutics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗