Insights into 6S RNA in lactic acid bacteria (LAB)

Background 6S RNA is a regulator of cellular transcription that tunes the metabolism of cells. This small non-coding RNA is found in nearly all bacteria and among the most abundant transcripts. Lactic acid bacteria (LAB) constitute a group of microorganisms with strong biotechnological relevance, often exploited as starter cultures for industrial products through fermentation. Some strains are used as probiotics while others represent potential pathogens. Occasional reports of 6S RNA within this group already indicate striking metabolic implications. A conceivable idea is that LAB with 6S RNA defects may metabolize nutrients faster, as inferred from studies of Echerichia coli. This may accelerate fermentation processes with the potential to reduce production costs. Similarly, elevated levels of secondary metabolites might be produced. Evidence for this possibility comes from preliminary findings regarding the production of surfactin in Bacillus subtilis, which has functions similar to those of bacteriocins. The prerequisite for its potential biotechnological utility is a general characterization of 6S RNA in LAB. Results We provide a genomic annotation of 6S RNA throughout the Lactobacillales order. It laid the foundation for a bioinformatic characterization of common 6S RNA features. This covers secondary structures, synteny, phylogeny, and product RNA start sites. The canonical 6S RNA structure is formed by a central bulge flanked by helical arms and a template site for product RNA synthesis. 6S RNA exhibits strong syntenic conservation. It is usually flanked by the replication-associated recombination protein A and the universal stress protein A. A catabolite responsive element was identified in over a third of all 6S RNA genes. It is known to modulate gene expression based on the available carbon sources. The presence of antisense transcripts could not be verified as a general trait of LAB 6S RNAs. Conclusions Despite a large number of species and the heterogeneity of LAB, the stress regulator 6S RNA is well-conserved both from a structural as well as a syntenic perspective. This is the first approach to describe 6S RNAs and short 6S RNA-derived transcripts beyond a single species, spanning a large taxonomic group covering multiple families. It yields universal insights into this regulator and complements the findings derived from other bacterial model organisms.

6S-1 RNA Contributes to Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

6S RNA is a kind of high-abundance non-coding RNA that globally regulates bacterial transcription by interacting with RNA polymerase holoenzyme. Through bioinformatics analysis, we found that there are two tandem 6S RNA-encoding genes in the genomes of Bacillus cereus group bacteria. Using Bacillus thuringiensis BMB171 as the starting strain, we have explored the physiological functions of 6S RNAs, and found that the genes ssrSA and ssrSB encoding 6S-1 and 6S-2 RNAs were located in the same operon and are co-transcribed as a precursor that might be processed by specific ribonucleases to form mature 6S-1 and 6S-2 RNAs. We also constructed two single-gene deletion mutant strains ΔssrSA and ΔssrSB and a double-gene deletion mutant strain ΔssrSAB by means of the markerless gene knockout method. Our data show that deletion of 6S-1 RNA inhibited the growth of B. thuringiensis in the stationary phase, leading to lysis of some bacterial cells. Furthermore, deletion of 6S-1 RNA also significantly reduced the spore number and parasporal crystal content. Our work reveals that B. thuringiensis 6S RNA played an important regulatory role in ensuring the sporulation and parasporal crystal formation.

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells

Background Pathogenic Rickettsia species belonging to the spotted fever group are arthropod-borne, obligate intracellular bacteria which exhibit preferential tropism for host microvascular endothelium in the mammalian hosts, resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. Although rickettsiae are known to undergo evolution through genomic reduction, the mechanisms by which these pathogens regulate their transcriptome to ensure survival in tick vectors and maintenance by transovarial/transstadial transmission, in contrast to their ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells. Results We performed deep sequencing on total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conorii. Strand-specific RNA sequencing of R. conorii transcripts revealed the expression 32 small RNAs (Rc_sR’s), which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were differentially upregulated (> 2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5′-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 181 primary TSSs in R. conorii during the infection to tick and human host cells, respectively. Seventy-five coding genes exhibited different TSSs depending on the host environment. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche. Conclusions In sum, the findings of this study authenticate the presence of novel Rc_sR’s in R. conorii, reveal the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

Transcriptomic responses of the marine cyanobacteriumProchlorococcusto viral lysis products

Marine phytoplankton contributes to about one half of global primary production, and a significant proportion of their photosynthetically fixed organic carbon is released after viral infection as dissolved organic matter (DOM). This DOM pool is known to be consumed by heterotrophic microorganisms; however, its impact on the uninfected co-occurring phytoplankton remains largely unknown. Here, we conducted transcriptomic analyses to study the effects of viral lysis products on the unicellular cyanobacteriumProchlorococcus, which is the most abundant photosynthetic organism on Earth. WhileProchlorococcusgrowth was not affected by viral lysis products, many tRNAs increased in abundance, which was also seen after amino acid addition, suggesting that amino acids are one of the compounds in viral lysis products that affected the expression of tRNA genes. The decreased transcript abundances of N metabolism genes also suggested thatProchlorococcusresponded to organic N compounds, consistent with abundant amino acids in viral lysis products. The addition of viral lysis products toProchlorococcusreduced the maximum photochemical efficiency of photosystem II and CO2fixation while increased its respiration rate, consistent with differentially expressed genes related to photosynthesis and respiration. One of the highest positive fold-changes was observed for the 6S RNA, a non-coding RNA functioning as a global transcriptional regulator in bacteria. The high level of 6S RNA might be responsible for some of the observed transcriptional responses. Taken together, our results revealed the transcriptional regulation ofProchlorococcusin response to viral lysis products and suggested its metabolic potential to utilize organic N compounds.ImportancePhotosynthetic microorganisms called phytoplankton are abundant in the oceans and contribute to about one half of global CO2fixation. Phytoplankton are frequently infected by viruses and after infection their organic carbon is released into the ocean as dissolved organic matter (DOM). Marine DOM is important for the marine food web because it supports the growth of heterotrophic microorganisms. However, the impact of viral DOM on the uninfected phytoplankton is largely unknown. In this study, we conducted transcriptomic analyses and identified many differentially expressed genes when viral DOM was added to the marine cyanobacteriumProchlorococcus. One effect of viral DOM is that the carbon fixation ofProchlorococcuswas reduced by ~16%, which might affect carbon cycling in the world’s oceans sinceProchlorococcusis the most abundant photosynthetic organism on Earth.