Early Neonatal Meconium Does Not Have a Demonstrable Microbiota Determined through Use of Robust Negative Controls with cpn 60-Based Microbiome Profiling

Much like the recent placental microbiome controversy, studies of neonatal meconium reporting bacterial communities within the fetal and neonatal gut imply that microbial colonization begins prior to birth. However, recent work has shown that placental microbiomes almost exclusively represent contamination from lab reagents and the environment.

Comparison of the Moonlighting Actions of the Two Highly Homologous Chaperonin 60 Proteins of Mycobacterium tuberculosis

ABSTRACT Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophagelike cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophagelike cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4−/− mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages.

Effects of Transgenic Hybrid Aspen Overexpressing Polyphenol Oxidase on Rhizosphere Diversity

ABSTRACT This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.

Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES)

In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal ‘substrate’ for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.

A chaperonin from a thermophilic bacterium, Thermus thermophilus

Unlike Escherichia coli chaperonins, a chaperonin (cpn) from a therm ophilic bacterium , Thermus thermophilus , consisting of homologues to GroEL (cpn 60) and GroES (cpn 10) is co-purified as a large complex. Thermus chaperonin shows a bullet-like shape in the side view seen by electron microscopy, and antibody against cpn 10 binds only to the round side of the bullet. We conclude that a single cpn 60-heptam er ring with two stripes stacks into two layers and a cpn 10 oligomer binds to one side of the layers. The purified Thermus chaperonin contains endogenously bound ADP, and incubation with ATP causes a partial dissociation of chaperonin into cpn 60 monomers and a cpn 10 heptam er. The effect of Thermus chaperonin on protein refolding upon dilution from guanidine HCl is different at three temperature ranges. At high temperatures above 55°C, where the native proteins are stable but their spontaneous foldings fail, the chaperonin induces productive folding in an ATP-dependent manner. At middle temperatures (25-55°C) where spontaneous foldings of the enzymes occur, the chaperonin slows down the rate of folding without changing the final yield of productive folding. At lower temperatures below 25°C where spontaneous foldings also occur, the chaperonin arrests the folding even in the presence of ATP. When a solution of relatively heat labile protein is incubated at high temperatures, and then residual activity of the protein is measured at its optimal temperature after incubation with ATP, the temperature that causes irreversible heat denaturation of the protein is elevated about 10°C by inclusion of Thermus chaperonin in the solution. Furthermore, once the folding intermediate of a protein is captured by Thermus chaperonin, it retains the ability to resume productive folding even after exposure to the otherwise denaturing high temperature. These results indicate that during heat denaturation proteins assume the common structure which is recognizable by the chaperonin. Finally, a ‘folding intermediate reservoir’ model to explain the effect of chaperonin is proposed, and is compared with a ‘marsupium ’ model.