Recent studies have uncovered dozens of regulatory small RNAs in bacteria. A large number of these small RNAs act by pairingto their target mRNAs. The outcome of pairing can be either stimulation or inhibition of translation. Pairing in vivo frequentlydepends on the RNA-binding protein Hfq. Synthesis of these small RNAs is tightly regulated at the level oftranscription; many of the well-studied stress response regulons have now been found to include a regulatory RNA.Expression of the small RNA can help the cell cope with environmental stress by redirecting cellular metabolism, exemplifiedby RyhB, a small RNA expressed upon iron starvation. Although small RNAs found in Escherichia coli can usually beidentified by sequence comparison to closely related enterobacteria, other approaches are necessary to find the equivalentRNAs in other bacterial species. Nonetheless, it is becoming increasingly clear that many if not all bacteria encode significantnumbers of these important regulators. Tracing their evolution through bacterial genomes remains a challenge.

RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli . Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB , negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA , encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σ ^E . The sequence upstream of the +1 of rybB contains a consensus σ ^E promoter. The activity of rybB - lacZ was increased in cells lacking the RseA anti-sigma factor and when σ ^E was overproduced from a heterologous promoter. The activity of rybB - lacZ and the detection of RybB were totally abolished in an rpoE -null strain. In vitro, σ ^E efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB - lacZ and rpoE - lacZ fusions, consistent with negative regulation of the σ ^E response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate σ ^E -dependent promoters in an RseA-independent fashion.

Appreciable evidence suggest that dysbiosis in microbiota, reflected in gut microbial imbalance plays a key role in the pathogenesis of neuropsychiatric disorders including depression and inflammatory diseases. Recently, the antidepressant properties of ketamine have gained prominence due to its fast and long lasting effects. Additional uses for ketamine in inflammatory disorders such as irritable bowel syndrome have been suggested. However, ketamine’s exact mechanism of action and potential effects on microbiome is not known. Here, we examined the effects of low dose ketamine, known to induce antidepressant effects, on stool microbiome profile in adult male Wistar rats. Animals (5/group) were injected intraperitoneally with ketamine (2.5 mg/kg) or saline, daily for 7 days and sacrificed on day 8 when intestinal stools were collected and stored at − 80 °C. DNA was extracted from the samples and the 16 S rRNA gene-based microbiota analysis was performed using 16S Metagenomics application. At genus–level, ketamine strikingly amplified Lactobacillus, Turicibacter and Sarcina by 3.3, 26 and 42 fold, respectively. Conversely, opportunistic pathogens Mucispirillum and Ruminococcus were reduced by approximately 2.6 and 26 fold, respectively, in ketamine group. Low levels of Lactobacillus and Turicibacter are associated with various disorders including depression and administration of certain species of Lactobacillus ameliorates depressive-like behavior in animal models. Hence, some of the antidepressant effects of ketamine might be mediated through its interaction with these gut bacteria. Additionally, high level of Ruminococcus is positively associated with the severity of irritable bowel syndrome (IBS), and some species of Mucispirillum have been associated with intestinal inflammation. Indirect evidence of anti-inflammatory role of Sarcina has been documented. Hence, some of the anti-inflammatory effects of ketamine and its usefulness in specific inflammatory diseases including IBS may be mediated through its interaction with these latter bacteria. Our data suggest that at least some of the antidepressant and anti-inflammatory effects of daily ketamine treatment for 7 days may be mediated via its interaction with specific gut bacteria. These findings further validate the usefulness of microbiome as a target for therapeutic intervention and call for more detailed investigation of microbiome interaction with central mediators of mood and/or inflammatory disorders.

Binary fission has been well studied in rod-shaped bacteria, but the mechanisms underlying cell division in spherical bacteria are poorly understood. Rod-shaped bacteria harbor regulatory proteins that place and remodel the division machinery during cytokinesis. In the spherical human pathogen Staphylococcus aureus, we found that the essential protein GpsB localizes to mid-cell during cell division and co-constricts with the division machinery. Depletion of GpsB arrested cell division and led to cell lysis, whereas overproduction of GpsB inhibited cell division and led to the formation of enlarged cells. We report that S. aureus GpsB, unlike other Firmicutes GpsB orthologs, directly interacts with the core divisome component FtsZ. GpsB bundles and organizes FtsZ filaments and also stimulates the GTPase activity of FtsZ. We propose that GpsB orchestrates the initial stabilization of the Z-ring at the onset of cell division and participates in the subsequent remodeling of the divisome during cytokinesis.

The stationary phase/general stress response sigma factor RpoS (σ ^S ) is necessary for adaptation and restoration of homeostasis in stationary phase. As a physiological consequence, its levels are tightly regulated at least at two levels. Multiple small regulatory RNA molecules modulate its translation, in a manner that is dependent on the RNA chaperone Hfq and the rpoS 5′ untranslated region. ClpXP and the RssB adaptor protein degrade RpoS, unless it is protected by an anti-adaptor. We here find that, in addition to these posttranscriptional levels of regulation, tRNA modification also affects the steady-state levels of RpoS. We screened mutants of several RNA modification enzymes for an effect on RpoS expression and identified the miaA gene, encoding a tRNA isopentenyltransferase, as necessary for full expression of both an rpoS750-lacZ translational fusion and the RpoS protein. This effect is independent of rpoS , the regulatory RNAs, and RpoS degradation. RpoD steady-state levels were not significantly different in the absence of MiaA, suggesting that this is an RpoS-specific effect. The rpoS coding sequence is significantly enriched for leu codons that use MiaA-modified tRNAs, compared to rpoD and many other genes. Dependence on MiaA may therefore provide yet another way for RpoS levels to respond to growth conditions.

The translation of rpoS (σ^S), the general stress/stationary phase sigma factor, is tightly regulated at the post-transcriptional level by several factors via mechanisms that are not clearly defined. One of these factors is MiaA, the enzyme necessary for the first step in the N⁶-isopentyl-2-thiomethyladenosinemethyladenosine 37 (ms²i⁶A37) tRNA modification. We tested the hypothesis that an elevated UUX-Leucine/total leucine codon ratio can be used to identify transcripts whose translation would be sensitive to loss of the MiaA-dependent modification. We identified iraP as another candidate MiaA-sensitive gene, based on the UUX-Leucine/total leucine codon ratio. An iraP-lacZ fusion was significantly decreased in the absence of MiaA, consistent with our predictive model. To determine the role of MiaA in UUX-Leucine decoding in rpoS and iraP, we measured β-galactosidase-specific activity of miaA⁻rpoS and iraP translational fusions upon overexpression of leucine tRNAs. We observed suppression of the MiaA effect on rpoS, and not iraP, via overexpression of tRNA^LeuX but not tRNA^LeuZ. We also tested the hypothesis that the MiaA requirement for rpoS and iraP translation is due to decoding of UUX-Leucine codons within the rpoS and iraP transcripts, respectively. We observed a partial suppression of the MiaA requirement for rpoS and iraP translational fusions containing one or both UUX-Leucine codons removed. Taken together, this suggests that MiaA is necessary for rpoS and iraP translation through proper decoding of UUX-Leucine codons and that rpoS and iraP mRNAs are both modification tunable transcripts (MoTTs) via the presence of the modification.