Culture of Desulfovibrio vulgaris in a medium supplemented with 5-aminolevulinic acid and L-methioninemethyl-d 3 resulted in the formation of porphyrins (sirohydrochlorin, coproporphyrin III, and protoporphyrin IX) in which the methyl groups at the C-2 and C-7 positions were deuterated. A previously unknown hexacarboxylic acid was also isolated, and its structure was determined to be 12,18-didecarboxysirohydrochlorin by mass spectrometry and 1 H NMR. These results indicate a primitive pathway of heme biosynthesis in D. vulgaris consisting of the following enzymatic steps: (i) methylation of the C-2 and C-7 positions of uroporphyrinogen III to form precorrin-2 (dihydrosirohydrochlorin); (ii) decarboxylation of acetate groups at the C-12 and C-18 positions of precorrin-2 to form 12,18-didecarboxyprecorrin-2; (iii) elimination of acetate groups of the C-2 and C-7 positions of 12,18-didecarboxyprecorrin-2 to form coproporphyrinogen III; and (iv) conversion of coproporphyrinogen III to protoporphyrin IX via protoporphyrinogen IX. We isolated the following three enzymatic activities involved in steps i-iii from the soluble fraction of the cells by anionexchange chromatography: S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase, precorrin-2 12,18-acetate decarboxylase, and 12,18-didecarboxyprecorrin-2 2,7-decarboxymethylase; all enzymic products were converted into autooxidized methyl esters and analyzed by thin-layer chromatography, UV-visible (UV-VIS) absorption, and mass spectrometry. The enzymatic reactions in D. vulgaris shed new light on porphyrin biosynthesis at an early stage in the evolution of prokaryotes.

Biofilm formation, one of the most important virulence factors of pathogenic bacteria, protects bacteria against desiccation, antibiotics, phages and host immune responses. However, phage-derived depolymerases show antibiofilm activity and demonstrate great potential to treat infections caused by biofilm-forming bacteria. In this study, the Escherichia coli phage vB_EcoM_ECOO78 was isolated and characterised, and we observed its ability to lyse five out of 34 tested E. coli clinical isolates. The highest phage titre was observed at a multiplicity of infection of 10-5 and a burst size of approximately 74 plaque forming units (PFU)/infection. Electron micrographs indicated that vB_EcoM_ECOO78 belongs to the family Myoviridae. The presence of increasing halos surrounding the lysis plaques formed by vB_EcoM_ECOO78 indicated that this phage may encode a depolymerase. Based on a sequencing analysis, the complete genome of vB_EcoM_ECOO78 was found to be 41,289 bp in size, with a GC content of 53.07%. Additionally, vB_EcoM_ECOO78 has 56 predicted open reading frames, 51 (91.07%) of which are assumed to be functional. A BLAST analysis indicated that ORF42 of vB_EcoM_ECOO78 (Dpo42) has low identity with other reported phage-associated depolymerases. Dpo42 was expressed and purified as a soluble protein using E. coli BL21. The biofilm formation ability of E. coli isolates and the antibiofilm activity of Dpo42 were tested by performing spot assays and using a 96-well micro-titre plate method. Dpo42 degraded the capsular polysaccharides surrounding E. coli and exhibited dose-dependent biofilm-formation prevention activity. Based on these results, Dpo42 appears to be a novel phage-derived depolymerase that represents a new potential strategy for preventing E. coli biofilm formation.

Background: Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy. Methods: A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results. Results: The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls. Conclusions: This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.