Genetic manipulation of candidate phyla radiation bacteria provides functional insights into microbial dark matter.

The study of bacteria has yielded fundamental insights into cellular biology and physiology, biotechnological advances and many therapeutics. Yet due to a lack of suitable tools, the significant portion of bacterial diversity held within the candidate phyla radiation (CPR) remains inaccessible to such pursuits. Here we show that CPR bacteria belonging to the phylum Saccharibacteria exhibit natural competence. We exploit this property to develop methods for their genetic manipulation, including the insertion of heterologous sequences and the construction of targeted gene deletions. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth and a transposon insertion sequencing genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their Actinobacteria hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii , as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

Interactions and quantification of multiple influencing factors on cadmium accumulation in soil-rice systems at a large region.

The accumulation of Cd in soil-rice systems at a large region is often extremely complicated due to environmental heterogeneity and the interactions of multiple influencing factors. However, the interactive effects and quantification of the contributions of influencing factors on Cd accumulation in large regions remain unclear. In this study, conditional inference trees and random forest analysis were used to identify the interactions of various factors (soil properties, topography and demographic-economic), and quantify their contributions to Cd accumulation in soil-rice systems of Sichuan-Chongqing region, China. The results showed that Cd content in the soil was the most significant influencing factor on Cd accumulation in soil-rice systems, especially bioavailable Cd in soil contributed to 35.73 % and 54.78 % for soil total Cd (Cdsoil) and brown rice Cd (Cdrice), respectively. Population density (PD) and elevation contributed 31.16 % and 27.40 % to Cdsoil content, respectively, and their interaction promoted the increase in Cdsoil content. Moreover, PD played a leading role in Cdsoil content when the elevation exceeded 324 m. The relative importances of slope and elevation for Cdrice content were 16.81 % and 8.49 %, respectively, and their interaction facilitated the increment of Cdrice content. As soil pH, gross domestic product (GDP) and slope decreased, the interaction of soil pH with GDP led to the increase of bioavailability factor (BAF), and that with slope enhanced the bioaccumulation factor (BCF). In addition, soil pH, PD and elevation were of considerable importance for the migration and transformation of Cd, with contributions of 22.11 %, 12.90 % and 12.52 % to BAF, and 5.05 %, 5.62 % and 5.50 % to BCF, respectively. This study is hopeful to provide a scientific insight into the prevention and control of Cd contamination in soil-rice systems at a large region.

Enzymatic thioamidation of peptide backbones.

Thioamides are found in a few natural products and two known protein assemblies: the Escherichia coli ribosome and methyl-coenzyme M reductase (MCR) from methane-metabolizing archaea. Compared to an amide, thioamides alter the physical and chemical properties of peptide backbones, including the conformation dynamics, proteolytic stability, hydrogen-bonding capabilities, and possibly reactivity of a protein when installed. Recently, there has been significant progress in elucidating enzymatic post-translational thioamide installation, with most work leveraging the archaeal MCR-modifying enzymes. This chapter describes the protocols used for the in vitro enzymatic thioamidation of MCR-derived peptides, including polypeptide overexpression, purification, reaction reconstitution, and mass spectrometry-based product analysis. In addition, we highlight the protocols used for the biochemical, kinetics, and binding studies using recombinant enzymes obtained heterologously from E. coli. We anticipate that these methods will serve to guide future studies on peptide post-translational thioamidation, as well as other peptide backbone modifications using a parallel workflow.

Functional elucidation of TfuA in peptide backbone thioamidation.

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.

Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans.

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

Variations Between Sources of Social Support and Cancer Screen Behaviors in U.S. Chinese Older Adults.

BACKGROUND: Social support is a key indicator of utilization of preventive health care among older adults, but we have limited knowledge on these associations in U.S. Chinese older adults. This study aims to examine the association between sources of social support and cancer screening behaviors among Chinese older adults in the greater Chicago area. METHODS: Data were drawn from the Population Study of Chinese Elderly in Chicago. Social supports were measured by asking the frequency of receipt of support from spouse, non-spouse family members, and friends. Use of cancer screenings were evaluated by asking the history of utilization of colon, breast, cervical, and prostate cancer screenings. RESULTS: After adjusting for covariates, results indicated significant association between higher social support and higher utilization of cancer screenings. Regarding to different sources of social support, higher levels of social supports from family members (odds ratio [OR], 1.15 [1.07, 1.25]) and friends (OR, 1.14 [1.06, 1.23]) were associated with higher utilization of breast cancer screening. However, higher levels of social support from family members (OR, 0.94 [0.88, 0.99]) and friends (OR, 0.94 [0.88, 1.00]) were associated with lower utilization of colon cancer screening. No associations were found between social support and prostate cancer screening. CONCLUSIONS: This study provides evidence that different types of social support were associated with variations in the utilization of cancer screenings. Future longitudinal studies are needed to explore the causal relationship between social support and cancer screening use.