MbnC Is Not Required for the Formation of the N-Terminal Oxazolone in the Methanobactin from Methylosinus trichosporium OB3b.

Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.

Identification of tylosin photoreaction products and comparison of ELISA and HPLC methods for their detection in water.

Tylosin is a widely used macrolide antibiotic for therapeutics and growth promotion in swine, beef cattle, and poultry production. Through various routes such as manure application, emission, inappropriate disposal, etc., tylosin enters the environment. The fate of tylosin in the environment is not yet fully understood. In this study, two photoreaction products of tylosin in water were identified as isotylosin A alcohol (E,Z) and isotylosin A aldol (E,Z). Tylosin A, B, C, D, isotylosin A alcohol, and isotylosin A aldol were purified, and immunological cross-reactivities of these tylosin-related compounds were tested with a specificity of 26% for tylosin B, 19% for tylosin C, 106% for tylosin D, 121% for isotylosin A alcohol, and 46% for isotylosin A aldol, compared to 100% for tylosin A. Competitive direct enzyme-linked immunosorbent assay (ELISA) for tylosin detection in water was compared with a high-performance liquid chromatography (HPLC) method by analyzing the same water samples from a study of tylosin dissipation in water. ELISA kits detect the other tylosin-related compounds besides tylosin A, which can result in differences in tylosin determination in water.