Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs.

Prokaryotic and eukaryotic RNA polymerases can use 2- to ∼4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.

RNase E Promotes Expression of Type III Secretion System Genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen that employs a type III secretion system (T3SS) to inject effector proteins into host cells. Using a protein depletion system, we show that the endoribonuclease RNase E positively regulates expression of the T3SS genes. We also present evidence that RNase E antagonizes the expression of genes of the type VI secretion system and limits biofilm production in P. aeruginosa Thus, RNase E, which is thought to be the principal endoribonuclease involved in the initiation of RNA degradation in P. aeruginosa, plays a key role in controlling the production of factors involved in both acute and chronic stages of infection. Although the posttranscriptional regulator RsmA is also known to positively regulate expression of the T3SS genes, we find that RNase E does not appreciably influence the abundance of RsmA in P. aeruginosa Moreover, we show that RNase E still exerts its effects on T3SS gene expression in cells lacking all four of the key small regulatory RNAs that function by sequestering RsmA.IMPORTANCE The type III secretion system (T3SS) is a protein complex produced by many Gram-negative pathogens. It is capable of injecting effector proteins into host cells that can manipulate cell metabolism and have toxic effects. Understanding how the T3SS is regulated is important in understanding the pathogenesis of bacteria with such systems. Here, we show that RNase E, which is typically thought of as a global regulator of RNA stability, plays a role in regulating the T3SS in Pseudomonas aeruginosa Depleting RNase E results in the loss of T3SS gene expression as well as a concomitant increase in biofilm formation. These observations are reminiscent of the phenotypes associated with the loss of activity of the posttranscriptional regulator RsmA. However, RNase E-mediated regulation of these systems does not involve changes in the abundance of RsmA and is independent of the known small regulatory RNAs that modulate RsmA activity.

NanoRNAs prime transcription initiation in vivo.

It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of oligoribonucleotides 2 to ∼4 nt in length, "nanoRNAs," which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. Our results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using only NTPs. Our findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism.

Bacillus subtilis RNase J1 endonuclease and 5' exonuclease activities in the turnover of DeltaermC mRNA.

RNase J1, a ribonuclease with 5' exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5' end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3' exonucleases; the downstream fragment is degraded by RNase J1 5' exonuclease activity. Previously, DeltaermC mRNA was used to show 5'-end dependence of mRNA turnover. Here we used DeltaermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. DeltaermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem-loop structure at +65 resulted in increased stability. Weakening this stem-loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3' end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5' ends of these fragments mapped to the upstream side of predicted stem-loop structures, consistent with an impediment to RNase J1 5' exonuclease processivity. A DeltaermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3' end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5' end.

Rsd family proteins make simultaneous interactions with regions 2 and 4 of the primary sigma factor.

Bacterial anti-sigma factors typically regulate sigma factor function by restricting the access of their cognate sigma factors to the RNA polymerase (RNAP) core enzyme. The Escherichia coli Rsd protein forms a complex with the primary sigma factor, sigma(70), inhibits sigma(70)-dependent transcription in vitro, and has been proposed to function as a sigma(70)-specific anti-sigma factor, thereby facilitating the utilization of alternative sigma factors. In prior work, Rsd has been shown to interact with conserved region 4 of sigma(70), but it is not known whether this interaction suffices to account for the regulatory functions of Rsd. Here we show that Rsd and the Rsd orthologue AlgQ, a global regulator of gene expression in Pseudomonas aeruginosa, interact with conserved region 2 of sigma(70). We show further that Rsd and AlgQ can interact simultaneously with regions 2 and 4 of sigma(70). Our findings establish that the abilities of Rsd and AlgQ to interact with sigma(70) region 2 are important determinants of their in vitro and in vivo activities.

Modeling Pollen-Mediated Virus Spread in Bee Colonies as a Classroom Activity.

Using a hands-on approach, this activity introduces students to the concept of viral spread and honey bee pathogenesis by illustrating pathogen transmission throughout the hive. This viral transmission activity, designed for introductory biology, virology, or microbiology classes, can be used in laboratory or lecture settings. Students are provided with information on viral transmission and hive structure. Students then retrieve "pollen" and distribute it to the colony. A UV light passed across students' hands determines which hive was infected, indicating the viral transmission pathways among bees. Students then discuss how viruses impact bees, how long it would take an infected hive to succumb to the pathogen, and what can be done to prevent viral spread.

Local and global regulators linking anaerobiosis to cupA fimbrial gene expression in Pseudomonas aeruginosa.

The cupA gene cluster of Pseudomonas aeruginosa encodes components and assembly factors of a putative fimbrial structure that enable this opportunistic pathogen to form biofilms on abiotic surfaces. In P. aeruginosa the control of cupA gene expression is complex, with the H-NS-like MvaT protein functioning to repress phase-variable (on/off) expression of the operon. Here we identify four positive regulators of cupA gene expression, including three unusual regulators encoded by the cgrABC genes and Anr, a global regulator of anaerobic gene expression. We show that the cupA genes are expressed in a phase-variable manner under anaerobic conditions and that the cgr genes are essential for this expression. We show further that cgr gene expression is negatively controlled by MvaT and positively controlled by Anr and anaerobiosis. Expression of the cupA genes therefore appears to involve a regulatory cascade in which anaerobiosis, signaled through Anr, stimulates expression of the cgr genes, resulting in a concomitant increase in cupA gene expression. Our findings thus provide mechanistic insight into the regulation of cupA gene expression and identify anaerobiosis as an inducer of phase-variable cupA gene expression, raising the possibility that phase-variable expression of fimbrial genes important for biofilm formation may occur in P. aeruginosa persisting in the largely anaerobic environment of the cystic fibrosis host lung.

Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens.

The CrbS/R two-component signal transduction system is a conserved regulatory mechanism through which specific Gram-negative bacteria control acetate flux into primary metabolic pathways. CrbS/R governs expression of acetyl-CoA synthase (acsA), an enzyme that converts acetate to acetyl-CoA, a metabolite at the nexus of the cell's most important energy-harvesting and biosynthetic reactions. During infection, bacteria can utilize this system to hijack host acetate metabolism and alter the course of colonization and pathogenesis. In toxigenic strains of Vibrio cholerae, CrbS/R-dependent expression of acsA is required for virulence in an arthropod model. Here, we investigate the function of the CrbS/R system in Pseudomonas aeruginosa, Pseudomonas entomophila, and non-toxigenic V. cholerae strains. We demonstrate that its role in acetate metabolism is conserved; this system regulates expression of the acsA gene and is required for growth on acetate as a sole carbon source. As a first step towards describing the mechanism of signaling through this pathway, we identify residues and domains that may be critical for phosphotransfer. We further demonstrate that although CrbS, the putative hybrid sensor kinase, carries both a histidine kinase domain and a receiver domain, the latter is not required for acsA transcription. In order to determine whether our findings are relevant to pathogenesis, we tested our strains in a Drosophila model of oral infection previously employed for the study of acetate-dependent virulence by V. cholerae. We show that non-toxigenic V. cholerae strains lacking CrbS or CrbR are significantly less virulent than are wild-type strains, while P. aeruginosa and P. entomophila lacking CrbS or CrbR are fully pathogenic. Together, the data suggest that the CrbS/R system plays a central role in acetate metabolism in V. cholerae, P. aeruginosa, and P. entomophila. However, each microbe's unique environmental adaptations and pathogenesis strategies may dictate conditions under which CrbS/R-mediated acs expression is most critical.

Cellular Light Scattering for the Identification of Bacteria and Its Application to the Identification of Staphylococcus.

Rapid identification of bacteria is critical in clinical and food safety applications. This paper describes a novel instrument and data analysis method for identifying bacteria based on the measurement of laser light scattering as the beam interacts with bacterial cells suspended in water. A description of the technology is followed by an identification performance study for a set of strains from the genus Staphylococcus (the inclusive target organisms) and a set of non-Staphylococcus strains (the exclusive organisms). Staphylococcus and non-Staphylococcus cells were grown on sheep blood agar (SBA), tryptic soy agar, brain heart infusion (BHI) agar, or Luria-Bertani (LB) agar and identified based on how cells scattered light. Bacteria from the genus Staphylococcus grown on solid media were correctly identified more than 92% of the time. To determine whether the system could also identify bacteria grown in liquid culture, six different Staphylococcus strains and six different non-Staphylococcus strains were grown in tryptic soy broth, BHI broth, or LB broth. This system accurately identified all targeted Staphylococcus samples tested, and no misidentifications occurred. A single-blind identification experiment was also performed on human clinical isolates obtained from the Upper Peninsula Health System. Ninety blind-coded clinical bacterial isolates on SBA were tested to determine whether they were from the genus Staphylococcus. All Staphylococcus were accurately identified, and no misidentifications occurred. This study demonstrated the proof of concept of a novel system that can rapidly and accurately identify bacteria from pure culture based on cellular light-scattering properties.

Effect of 5'-proximal elements on decay of a model mRNA in Bacillus subtilis.

Previous work showed that a 42-nucleotide sequence from an SP82 bacteriophage early RNA functions as a 5' mRNA stabilizer in Bacillus subtilis. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of decay of a model mRNA with alterations at the 5'-end was used to elucidate the mechanism of SP82-mediated stability. A predicted 5'-terminal stem-loop structure was essential for stabilization. Increasing the strength of the 5'-terminal structure above a minimum level did not result in increased stability. A thorough analysis of the context in which the stabilizing structure occurred included the effects of distance from 5'-end, translation of downstream coding sequence, and distance between the secondary structure and the ribosome binding site. Our data are consistent with the dominant mRNA decay pathway in B. subtilis being 5'-end dependent.

Effect of translational signals on mRNA decay in Bacillus subtilis.

A 254-nucleotide model mRNA, designated deltaermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis. DeltaermC mRNA features a strong ribosome-binding site (RBS) and a 62-amino-acid-encoding open reading frame, followed by a transcription terminator structure. Inactivation of the RBS or the start codon resulted in a fourfold decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability. Data for the decay of deltaermC mRNAs with stop codons at positions increasingly proximal to the translational start site showed that actual translation--even the formation of the first peptide bond--was not important for stability. The half-life of an untranslated 3.2-kb deltaermC-lacZ fusion RNA was similar to that of a translated deltaermC-lacZ mRNA, indicating that the translation of even a longer RNA was not required for wild-type stability. The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with a 5'-end-dependent activity, possibly a 5'-binding endonuclease, which is required for the initiation of mRNA decay. This model is supported by the finding that increasing the distance from the 5' end to the start codon resulted in a 2.5-fold decrease in the mRNA half-life. These results underscore the importance of the 5' end to mRNA stability in B. subtilis.

Endonuclease cleavage of messenger RNA in Bacillus subtilis.

A deletion derivative of the ermC gene was constructed that expresses a 254-nucleotide mRNA. The small size of this mRNA facilitated the detection of processing products that did not differ greatly in size from the full-length transcript. In the presence of erythromycin, which induces ribosome stalling near the 5' end of ermC mRNA, the 254-nucleotide mRNA was cleaved endonucleolytically at the site of ribosome stalling. Only the downstream product of this cleavage was detectable; the upstream product was apparently too unstable to be detected. The downstream cleavage product accumulated at times after rifampicin addition, suggesting that the stalled ribosome at the 5' end conferred stability to this RNA fragment. Neither Bs-RNase III nor RNase M5, the two known narrow-specificity endoribonucleases of Bacillus subtilis, was responsible for this cleavage. These results indicate the presence in B. subtilis of another specific endoribonuclease, which may be ribosome associated.