Widespread Occurrence of Secondary Lipid Biosynthesis Potential in Microbial Lineages

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as “Pfa synthases”. In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of “secondary lipids” to describe these biosynthetic pathways and products, a proposition consistent with the “secondary metabolite” vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages

The use of second derivative plots for the determination of mol% guanine plus cytosine of DNA by the thermal denaturation method

Procedures for the estimation of thermal denaturation temperatures for the determination of the mol% G+C base composition of bacterial DNA have been examined with a view to simplifying the procedure and taking advantage of developments in microprocessor controlled spectrophotometers. Thermal denaturation profiles of DNA from 40 strains with base compositions ranging from 35 to 70 mol% G+C were studied. Thermal denaturation temperatures were determined by the conventional Tm method and by using second derivative plots to determine the temperature of inflection (Ti). The results showed that there is a close linear correlation between mol% G+C values calculated using Tm or Ti as the thermal denaturation temperature, provided the thermal denaturation temperature of the internal reference DNA is estimated in the same way. Actual Ti values were approximately 0.3°C higher than corresponding Tm values but the increase was consistent across the range of DNA samples studied. The results also showed that the error introduced by disregarding correction for the effect of thermal expansion was not consistent across the range of DNA samples studied. However, the error was small and in practical terms could be neglected

Isolation and synthesis of falcitidin, a novel myxobacterial-derived acyltetrapeptide with activity against the malaria target falcipain-2

A 384-well microtitre plate fluorescence cleavage assay was developed to identify inhibitors of the cysteine protease falcipain-2, an important antimalarial drug target. Bioassay-guided isolation of a MeOH extract from a myxobacterium Chitinophaga sp. Y23 isolated from soil collected in Singapore, led to the identification of a new acyltetrapeptide, falcitidin (1), which displayed an IC 50 value of 6 μM against falcipain-2. The planar structure of 1 was secured by NMR and MS/MS analysis. Attempts to isolate further material for biological testing were hampered by inconsistent production and by a low yield