Advanced analytical imaging techniques, including matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) imaging, can be used to visualize the distribution, localization, and dynamics of target compounds and their precursors with limited sample preparation. Herein we report an application of MALDI-HRMS imaging to map, in high spatial resolution, the accumulation of the medicinally important naphthodianthrone hypericin, its structural analogues and proposed precursors, and other crucial phytochemical constituents in the leaves of two hypericin-containing species, Hypericum perforatum and Hypericum olympicum. We also investigated Hypericum patulum, which does not contain hypericin or its protoforms. We focused on both the secretory (dark glands, translucent glands, secretory canals, laminar glands, and ventral glands) and the surrounding non-secretory tissues to clarify the site of biosynthesis and localization of hypericin, its possible precursors, and patterns of localization of other related compounds concomitant to the presence or absence of hypericin. Hypericin, pseudohypericin, and protohypericin accumulate in the dark glands. However, the precursor emodin not only accumulates in the dark glands but is also present outside the glands in both hypericin-containing species. In hypericin-lacking H. patulum, however, emodin typically accumulates only in the glands, thereby providing evidence that hypericin is possibly biosynthesized outside the dark glands and thereafter stored in them. The distribution and localization of related compounds were also evaluated and are discussed concomitant to the occurrence of hypericin. Our study provides the basis for further detailed investigation of hypericin biosynthesis by gene discovery and expression studies.

We examined fiber fermentation capacity of captive chimpanzee fecal microflora from animals (n = 2) eating low-fiber diets (LFDs; 14% neutral detergent fiber (NDF) and 5% of cellulose) and high-fiber diets (HFDs; 26% NDF and 15% of cellulose), using barley grain, meadow hay, wheat straw, and amorphous cellulose as substrates for in vitro gas production of feces. We also examined the effects of LFD or HFD on populations of eubacteria and archaea in chimpanzee feces. Fecal inoculum fermentation from the LFD animals resulted in a higher in vitro dry matter digestibility (IVDMD) and gas production than from the HFD animals. However, there was an interaction between different inocula and substrates on IVDMD, gas and methane production, and hydrogen recovery (P <0.001). On the other hand, HFD inoculum increased the production of total short-chain fatty acids (SCFAs), acetate, and propionate with all tested substrates. The effect of the interaction between the inoculum and substrate on total SCFAs was not observed. Changes in fermentation activities were associated with changes in bacterial populations. DGGE of bacterial DNA revealed shift in population of both archaeal and eubacterial communities. However, a much more complex eubacterial population structure represented by many bands was observed compared with the less variable archaeal population in both diets. Some archaeal bands were related to the uncultured archaea from gastrointestinal tracts of homeothermic animals. Genomic DNA in the dominant eubacterial band in the HFD inoculum was confirmed to be closely related to DNA from Eubacterium biforme. Interestingly, the predominant band in the LFD inoculum represented DNA of probably new or yet-to-be-sequenced species belonging to mycoplasms. Collectively, our results indicated that fecal microbial populations of the captive chimpanzees are not capable of extensive fiber fermentation; however, there was a positive effect of fiber content on SCFA production.

Aims:  Purification and partial characterization of an extracellular bacteriocin produced by the ruminal isolate Enterococcus faecalis II/1 and determine the frequency of occurrence of enterolysin A structural gene within the ruminal cocci. Methods and Results:  Bacteriocin produced by E. faecalis II/1 was purified to homogeneity. Purified bacteriocin exhibited a single band on sodium dodecylsulphate polyacrylamide gel electrophoresis with an apparent molecular weight of about 35 kDa. The amino acid sequence of the first 30 amino acids of purified bacteriocin was identical with the enterolysin A sequence. The DNA sequence of the nearly complete E. faecalis II/1 bacteriocin structural gene was identical to the enterolysin A gene sequence, confirming that this bacteriocin is identical to enterolysin A, a cell wall‐degrading bacteriocin from E. faecalis LMG 2333. Enterolysin A structural genes were detected in approximately one‐sixth of the Gram‐positive ruminal cocci examined by PCR using primers targeting the enterolysin A structural gene. Conclusions:  Bacteriocin produced by E. faecalis II/1 is identical to enterolysin A. Enterolysin A structural gene homologues are frequently encountered in rumen enterococcal and streptococcal bacterial strains. Significance and Impact of the study:  This is the first evidence of a large heat‐labile bacteriocin produced by rumen E. faecalis strain, enlarging the number and types of known anti‐bacterial proteins produced by rumen bacteria.