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The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.

6S RNA is a small, non-coding RNA that interacts directly with [[sigma].sup.70]-RNA polymerase and regulates transcription at many [[sigma].sup.70-]dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from [[sigma].sup.70]-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of Cs activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions. DOI 10.1099/mic.0.043992-0

6S RNA is a highly abundant small RNA that regulates transcription through direct interaction with RNA polymerase. Here we show that 6S RNA directly inhibits transcription of pspF, which subsequently leads to inhibition of pspABCDE and pspG expression. Cells without 6S RNA are able to survive at elevated pH better than wild-type cells due to loss of 6S RNA-regulation of pspF. This 6S RNA-dependent phenotype is eliminated in pspF-null cells, indicating that 6S RNA effects are conferred through PspF. Similar growth phenotypes are seen when PspF levels are increased in a 6S RNA-independent manner, signifying that changes to pspF expression are sufficient. Changes in survival at elevated pH most likely result from altered expression of pspABCDE and/or pspG, both of which require PspF for transcription and are indirectly regulated by 6S RNA. 6S RNA provides another layer of regulation in response to high pH during stationary phase. We propose that the normal role of 6S RNA at elevated pH is to limit the extent of the PW response under conditions of nutrient deprivation, perhaps facilitating appropriate allocation of diminishing resources.

The present knowledge of regulatory RNA function is discussed and how the RNAs act through base-pairing interactions in a variety of organisms is reviewed. Other regulatory RNAs act by modifying protein activity and in some cases by imitating the structures of other RNA or DNA molecules.

6S RNA was identified in Escherichia coli >30 years ago, but the physiological role of this RNA has remained elusive. Here, we demonstrate that 6S RNA-deficient cells are at a disadvantage for survival in stationary phase, a time when 6S RNA regulates transcription. Growth defects were most apparent as a decrease in the competitive fitness of cells lacking 6S RNA. To decipher the molecular mechanisms underlying the growth defects, we have expanded studies of 6S RNA effects on transcription. 6S RNA inhibition of [[sigma].sup.70]-dependent transcription was not ubiquitous, in spite of the fact that the vast majority of [[sigma].sup.70]-RNA polymerase is bound by 6S RNA during stationary phase. The [[sigma].sup.70]-dependent promoters inhibited by 6S RNA contain an extended -10 promoter element, suggesting that this feature may define a class of 6S RNA-regulated genes. We also discovered a secondary effect of 6S RNA in the activation of [[sigma].sup.S]-dependent transcription at several promoters. We conclude that 6S RNA regulation of both [[sigma].sup.70] and [[sigma].sup.S] activities contributes to increased cell persistence during nutrient deprivation.

When living cells encounter suboptimal chemical or physical conditions, global regulatory networks, at cellular level, are to be coordinated for optimization of available nutrients and cell survival. This review delineates the function of bacterial small, untranslated RNAs in such a regulatory role and their modulation of cellular metabolism in response to environmental cues.

Novel small RNAs (sRNAs) and their identification using comparative genomics and microarrays are discussed. High conservation of sRNAs among closely related bacterial species were used to predict presence of novel sRNA genes in the intergenic regions of the Esherichia coli genome. Analysis of transcripts detected by high-density oligonucleotide probe arrays was also used.

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity. [ABSTRACT FROM AUTHOR]

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system ( T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5′ untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion. [ABSTRACT FROM AUTHOR]

The bacterium X enorhabdus nematophila is a mutualist of entomopathogenic S teinernema carpocapsae nematodes and facilitates infection of insect hosts. X . nematophila colonizes the intestine of S . carpocapsae which carries it between insects. In the X . nematophila colonization-defective mutant nilD6:: Tn 5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X . nematophila strains from S . anatoliense and S . websteri nematodes. Only nilD from the S . carpocapsae strain of X . nematophila rescued the colonization defect of the nilD6:: Tn 5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I- E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression. [ABSTRACT FROM AUTHOR]

6S RNA is a small, non-coding RNA that interacts with σ70-RNA polymerase and downregulates transcription at many promoters during stationary phase. When bound to σ70-RNA polymerase, 6S RNA is engaged in the active site of σ70-RNA polymerase in a manner similar enough to promoter DNA that the RNA can serve as a template for RNA synthesis. It has been proposed that 6S RNA mimics the conformation of DNA during transcription initiation, suggesting contacts between RNA polymerase and 6S RNA or DNA may be similar. Here we demonstrate that region 4.2 of σ70 is critical for the interaction between 6S RNA and RNA polymerase. We define an expanded binding surface that encompasses positively charged residues throughout the recognition helix of the helix–turn–helix motif in region 4.2, in contrast to DNA binding that is largely focused on the N-terminal region of this helix. Furthermore, negatively charged residues in region 4.2 weaken binding to 6S RNA but do not similarly affect DNA binding. We propose that the binding sites for promoter DNA and 6S RNA on region 4.2 of σ70 are overlapping but distinct, raising interesting possibilities for how core promoter elements contribute to defining promoters that are sensitive to 6S RNA regulation. [ABSTRACT FROM AUTHOR]

6S RNA binds σ70-RNA polymerase and downregulates transcription at many σ70-dependent promoters, but others escape regulation even during stationary phase when the majority of the transcription machinery is bound by the RNA. We report that core promoter elements determine this promoter specificity; a weak −35 element allows a promoter to be 6S RNA sensitive, and an extended −10 element similarly determines 6S RNA inhibition except when a consensus −35 element is present. These two features together predicted that hundreds of mapped Escherichia coli promoters might be subject to 6S RNA dampening in stationary phase. Microarray analysis confirmed 6S RNA-dependent downregulation of expression from 68% of the predicted genes, which corresponds to 49% of the expressed genes containing mapped E. coli promoters and establishes 6S RNA as a global regulator in stationary phase. We also demonstrate a critical role for region 4.2 of σ70 in RNA polymerase interactions with 6S RNA. Region 4.2 binds the −35 element during transcription initiation; therefore we propose one mechanism for 6S RNA regulation of transcription is through competition for binding region 4.2 of σ70. [ABSTRACT FROM AUTHOR]

6S RNA binds σ70-RNA polymerase and downregulates transcription at many σ70-dependent promoters, but others escape regulation even during stationary phase when the majority of the transcription machinery is bound by the RNA. We report that core promoter elements determine this promoter specificity; a weak −35 element allows a promoter to be 6S RNA sensitive, and an extended −10 element similarly determines 6S RNA inhibition except when a consensus −35 element is present. These two features together predicted that hundreds of mapped Escherichia coli promoters might be subject to 6S RNA dampening in stationary phase. Microarray analysis confirmed 6S RNA-dependent downregulation of expression from 68% of the predicted genes, which corresponds to 49% of the expressed genes containing mapped E. coli promoters and establishes 6S RNA as a global regulator in stationary phase. We also demonstrate a critical role for region 4.2 of σ70 in RNA polymerase interactions with 6S RNA. Region 4.2 binds the −35 element during transcription initiation; therefore we propose one mechanism for 6S RNA regulation of transcription is through competition for binding region 4.2 of σ70. [ABSTRACT FROM AUTHOR]

6S RNA is a small RNA regulator of RNA polymerase (RNAP) that is present broadly throughout the bacterial kingdom. Initial functional studies in Escherichia coli revealed that 6S RNA forms a complex with RNAP resulting in regulation of transcription, and cells lacking 6S RNA have altered survival phenotypes. The last decade has focused on deepening the understanding of several aspects of 6S RNA activity, including (i) addressing questions of how broadly conserved 6S RNAs are in diverse organisms through continued identification and initial characterization of divergent 6S RNAs; (ii) the nature of the 6S RNA-RNAP interaction through examination of variant proteins and mutant RNAs, cross-linking approaches, and ultimately a cryo-electron microscopic structure; (iii) the physiological consequences of 6S RNA function through identification of the 6S RNA regulon and promoter features that determine 6S RNA sensitivity; and (iv) the mechanism and cellular impact of 6S RNA-directed synthesis of product RNAs (i.e., pRNA synthesis). Much has been learned about this unusual RNA, its mechanism of action, and how it is regulated; yet much still remains to be investigated, especially regarding potential differences in behavior of 6S RNAs in diverse bacteria.

RNA-protein interactions are critical in diverse aspects of gene expression and often serve to mediate regulatory events. Many procedures are available to gain information about RNA-protein interactions. They span from initial identification of an interaction, such as through co-immunoprecipitation studies, to highly detailed atomic resolution definition of the interaction gained from crystallographic and NMR studies. One of the most versatile techniques uses native gel electrophoresis to study RNA-protein complexes, which is often called band shift, gel retardation, or electrophoretic mobility shift assays. Gel shift assays have been used to study a plethora of RNA-protein interactions in all organisms, but here we will use the 6S RNA:RNA polymerase interaction from Escherichia coli as an example to direct discussion of questions that can be addressed, including the ability to follow the dynamics of complexes over time.

6S RNA is a small, non-coding RNA that interacts with sigma(70)-RNA polymerase and downregulates transcription at many promoters during stationary phase. When bound to sigma(70)-RNA polymerase, 6S RNA is engaged in the active site of sigma(70)-RNA polymerase in a manner similar enough to promoter DNA that the RNA can serve as a template for RNA synthesis. It has been proposed that 6S RNA mimics the conformation of DNA during transcription initiation, suggesting contacts between RNA polymerase and 6S RNA or DNA may be similar. Here we demonstrate that region 4.2 of sigma(70) is critical for the interaction between 6S RNA and RNA polymerase. We define an expanded binding surface that encompasses positively charged residues throughout the recognition helix of the helix-turn-helix motif in region 4.2, in contrast to DNA binding that is largely focused on the N-terminal region of this helix. Furthermore, negatively charged residues in region 4.2 weaken binding to 6S RNA but do not similarly affect DNA binding. We propose that the binding sites for promoter DNA and 6S RNA on region 4.2 of sigma(70) are overlapping but distinct, raising interesting possibilities for how core promoter elements contribute to defining promoters that are sensitive to 6S RNA regulation.

6S RNA binds sigma70-RNA polymerase and downregulates transcription at many sigma70-dependent promoters, but others escape regulation even during stationary phase when the majority of the transcription machinery is bound by the RNA. We report that core promoter elements determine this promoter specificity; a weak -35 element allows a promoter to be 6S RNA sensitive, and an extended -10 element similarly determines 6S RNA inhibition except when a consensus -35 element is present. These two features together predicted that hundreds of mapped Escherichia coli promoters might be subject to 6S RNA dampening in stationary phase. Microarray analysis confirmed 6S RNA-dependent downregulation of expression from 68% of the predicted genes, which corresponds to 49% of the expressed genes containing mapped E. coli promoters and establishes 6S RNA as a global regulator in stationary phase. We also demonstrate a critical role for region 4.2 of sigma70 in RNA polymerase interactions with 6S RNA. Region 4.2 binds the -35 element during transcription initiation; therefore we propose one mechanism for 6S RNA regulation of transcription is through competition for binding region 4.2 of sigma70.