Laura Schaefer

Maturation of the 5' end of Bacillus subtilis 16s rRNA by the essential ribonuclease YkqC/RNase J1

Functional ribosomal RNAs are generated from longer precursor species in every organism known. Maturation of the 5′ side of 16S rRNA in Escherichia coli is catalysed in a two‐step process by the cooperative action of RNase E and RNase G. However, many bacteria lack RNase E and RNase G orthologues, raising the question as to how 16S rRNA processing occurs in these organisms. Here we show that the maturation of Bacillus subtilis 16S rRNA is also a two‐step process and that the enzyme responsible for the generation of the mature 5′ end is the widely distributed essential ribonuclease YkqC/RNase J1. Depletion of B. subtilis of RNase J1 results in an accumulation of 16S rRNA precursors in vivo. The precursor species are found in polysomes suggesting that they can function in translation. Mutation of the predicted catalytic site of RNase J1 abolishes both 16S rRNA processing and cell viability. Finally, purified RNase J1 can correctly mature precursor 16S rRNA assembled in 70S ribosomes, showing that its role is direct.

Probiotic L. reuteri Treatment Prevents Bone Loss in a Menopausal Ovariectomized Mouse Model

Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post‐menopausal bone loss. Recent studies suggest an important role for gut‐bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri‐treated mice. Consistent with this, L. reuteri suppressed Ovx‐induced increases in bone marrow CD4+ T‐lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost‐effective approach to reduce post‐menopausal bone loss. J. Cell. Physiol. 229: 1822–1830, 2014.

The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups.

Reuterin is an antimicrobial compound produced by Lactobacillus reuteri, and has been proposed to mediate, in part, the probiotic health benefits ascribed to this micro-organism. Despite 20 years of investigation, the mechanism of action by which reuterin exerts its antimicrobial effects has remained elusive. Here we provide evidence that reuterin induces oxidative stress in cells, most likely by modifying thiol groups in proteins and small molecules. Escherichia coli cells subjected to sublethal levels of reuterin expressed a set of genes that overlapped with the set of genes composing the OxyR regulon, which senses and responds to various forms of oxidative stress. E. coli cells mutated for oxyR were more sensitive to reuterin compared with wild-type cells, further supporting a role for reuterin in exerting oxidative stress. The addition of cysteine to E. coli or Clostridium difficile growth media prior to exposure to reuterin suppressed the antimicrobial effect of reuterin on these bacteria. Interestingly, interaction with E. coli stimulated reuterin production or secretion by L. reuteri, indicating that contact with other microbes in the gut increases reuterin output. Thus, reuterin inhibits bacterial growth by modifying thiol groups, which indicates that reuterin negatively affects a large number of cellular targets.

The essential GTPase RbgA (YlqF) is required for 50S ribosome assembly in Bacillus subtilis

In this paper the essential GTPase YlqF is shown to participate in the biogenesis of the 50S ribosomal subunit in Bacillus subtilis. Cells depleted of YlqF displayed gene expression profiles and nucleoid morphologies that were consistent with a function for YlqF in translation. In addition, YlqF is evolutionarily linked to two eukaryotic GTPases, Nog2p and Nug1p, that are involved in the biogenesis and the nuclear export of the 60S ribosomal subunit. Analysis of ribosomes from cells depleted of YlqF demonstrated that the formation of 70S ribosomes was greatly reduced and the large subunit sedimented at 45S. Cells grown with varying depleted levels of YlqF, yielding doubling times ranging from 38 min to 150 min, all displayed the 45S intermediate. Purified YlqF‐His6 protein associates with the 45S intermediate, but not the mature 50S subunit in vitro. Analysis of proteins from the 45S intermediate indicated that ribosomal protein L16, which is added late during in vitro Escherichia coli 50S ribosome biogenesis, was missing from the 45S intermediate. These results support a model in which YlqF participates in the formation of active 70S ribosomes in the cell by functioning in a late step of 50S subunit biogenesis. Based on these results we propose to rename the ylqF gene rbgA (ribosome biogenesis GTPase A).

Multiple GTPases Participate in the Assembly of the Large Ribosomal Subunit in Bacillus subtilis

GTPases have been demonstrated to be necessary for the proper assembly of the ribosome in bacteria and eukaryotes. Here, we show that the essential GTPases YphC and YsxC are required for large ribosomal subunit biogenesis in Bacillus subtilis. Sucrose density gradient centrifugation of large ribosomal subunits isolated from YphC-depleted cells and YsxC-depleted cells indicates that they are similar to the 45S intermediate previously identified in RbgA-depleted cells. The sedimentation of the large-subunit intermediate isolated from YphC-depleted cells was identical to the intermediate found in RbgA-depleted cells, while the intermediate isolated from YsxC-depleted cells sedimented slightly slower than 45S, suggesting that it is a novel intermediate. Analysis of the protein composition of the large-subunit intermediates isolated from either YphC-depleted cells or YsxC-depleted cells indicated that L16 and L36 are missing. Purified YphC and YsxC are able to interact with the ribosome in vitro, supporting a direct role for these two proteins in the assembly of the 50S subunit. Our results indicate that, as has been demonstrated for Saccharomyces cerevisiae ribosome biogenesis, bacterial 50S ribosome assembly requires the function of multiple essential GTPases.

The Essential GTPase YqeH Is Required for Proper Ribosome Assembly in Bacillus subtilis

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in Bacillus subtilis.

Evaluation of Reuterin Production in Urogenital Probiotic Lactobacillus reuteri RC-14

ABSTRACT Classified as a distinct species in 1980, Lactobacillus reuteri strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of L. reuteri SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic Lactobacillus reuteri RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous Lactobacillus species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (gldC), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin. L. reuteri RC-14 was determined to be negative for gldC sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of L. reuteri RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production.