Natural Selection Shapes Maintenance of Orthologous sRNAs in Divergent Host-Restricted Bacterial Genomes

Historically it has been difficult to study the evolution of bacterial small RNAs (sRNAs) across distantly related species. For example, identifying homologs of sRNAs is often difficult in genomes that have undergone multiple structural rearrangements. Also, some types of regulatory sRNAs evolve at rapid rates. The high degree of genomic synteny among divergent host-restricted bacterial lineages, including intracellular symbionts, is conducive to sRNA maintenance and homolog identification. In turn, symbiont genomes can provide us with novel insights into sRNA evolution. Here, we examine the sRNA expression profile of the obligate symbiont of psyllids, Carsonella ruddii, which has one of the smallest cellular genomes described. Using RNA-seq, we identified 36 and 32 antisense sRNAs (asRNAs) expressed by Carsonella from the psyllids Bactericera cockerelli (Carsonella-BC) and Diaphorina citri (Carsonella-DC), respectively. The majority of these asRNAs were associated with genes that are involved in essential amino acid biosynthetic pathways. Eleven of the asRNAs were conserved in both Carsonella lineages and the majority were maintained by selection. Notably, five of the corresponding coding sequences are also the targets of conserved asRNAs in a distantly related insect symbiont, Buchnera. We detected differential expression of two asRNAs for genes involved in arginine and leucine biosynthesis occurring between two distinct Carsonella-BC life stages. Using asRNAs identified in Carsonella, Buchnera, and Profftella which are all endosymbionts, and Escherichia coli, we determined that regions upstream of these asRNAs encode unique conserved patterns of AT/GC richness, GC skew, and sequence motifs which may be involved in asRNA regulation.     

Identification of bacterial small non-coding RNAs: experimental approaches

Almost 140 bacterial small RNAs (sRNAs; sometimes referred to as non-coding RNAs) have been discovered in the past six years. The majority of these sRNAs were discovered in Escherichia coli, and a smaller subset was characterized in other bacteria, many of which were pathogenic. Many of these genes were identified as a result of systematic screens using computational prediction of sRNAs and experimental-based approaches, including microarray and shotgun cloning. A smaller number of sRNAs were discovered by direct labeling or by functional genetic screens. Many of the discovered genes, ranging in size from 50 to 500 nucleotides, are conserved and located in intergenic regions, in-between open reading frames. The expression of many of these genes is growth phase dependent or stress related. As each search employed specific parameters, this led to the identification of genes with distinct characteristics. Consequently, unique sRNAs such as those that are species-specific, sRNA genes that are transcribed under unique conditions or genes located on the antisense strand of protein-encoding genes, were probably missed.     

链球菌非编码小RNA研究进展

非编码小RNA(Small non-coding RNA,sRNA)是一种存在于原核和真核生物中的新型调控RNA,长度约为40～500个核苷酸。作为一类关键的调控因子,sRNA通过与靶mRNA或蛋白质结合来调控细胞内的基因表达。大部分细菌sRNA在大肠杆菌等革兰氏阴性菌中被发现并研究,但近十年来越来越多的sRNA在革兰氏阳性菌中被逐步发现。作为一类革兰氏阳性菌,链球菌属中sRNA目前研究主要集中在毒力调节,鲜有其他调控的报道。本文总结了链球菌中sRNA的最新进展,并介绍其主要功能和机理,以期为细菌sRNA研究提供借鉴。     

Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence

The emergence of pathogenic strains of enteric bacteria and their adaptation to unique niches are associated with the acquisition of foreign DNA segments termed ‘genetic islands’. We explored these islands for the occurrence of small RNA (sRNA) encoding genes. Previous systematic screens for enteric bacteria sRNAs were mainly carried out using the laboratory strain Escherichia coli K12, leading to the discovery of ~80 new sRNA genes. These searches were based on conservation within closely related members of enteric bacteria and thus, sRNAs, unique to pathogenic strains were excluded. Here we describe the identification and characterization of 19 novel unique sRNA genes encoded within the ‘genetic islands’ of the virulent strain Salmonella typhimurium. We show that the expression of many of the island-encoded genes is associated with stress conditions and stationary phase. Several of these sRNA genes are induced when Salmonella resides within macrophages. One sRNA, IsrJ, was further examined and found to affect the translocation efficiency of virulence-associated effector proteins into nonphagocytic cells. In addition, we report that unlike the majority of the E. coli sRNAs that are trans regulators, many of the island-encoded sRNAs affect the expression of cis-encoded genes. Our study suggests that the island encoded sRNA genes play an important role within the network that regulates bacterial adaptation to environmental changes and stress conditions and thus controls virulence.     

Translational control and target recognition by Escherichia coli small RNAs in vivo

Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.     

病原菌非编码sRNA的功能及研究新方法

细菌中的非编码小RNA（small RNA, sRNA）作为一种靶向调控分子在细胞生理代谢过程中具有重要作用。sRNA作用于特定靶标,调控基因的表达。大肠杆菌大约有100种sRNA,其中1/3 sRNA需要伴侣蛋白Hfq的介导。病原细菌中sRNA分子如何调控致病基因的表达,目前研究仍处于初级阶段。本文将从生物膜形成、细菌耐药性以及对宿主的影响等方面,结合新颖的sRNA的研究方法,综述sRNA在调控代谢网络及控制病原菌致病性方面的作用。     

A screening system for artificial small RNAs that inhibit the growth of Escherichia coli

We have developed a screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli. In this system, we used a plasmid library to express artificial sRNAs (approximately 200 bases long) containing 60 bases of random nucleotide sequence. The induced expression of the known rydB sRNA in the system reduced the amount of its possible target mRNA, rpoS, supporting the reliability of the method. To isolate clones of sRNAs that inhibited the growth of E. coli, we used two successive screening steps: (i) colony size selection on plates and (ii) monitoring E. coli growth in a 96-well plate format. As a result, 83 artificial sRNAs were identified that showed a range of inhibitory effects on bacterial growth. We also introduced nucleotide replacements into one of the highly inhibitory sRNA clones, H12, which partially abolished the inhibition of bacterial growth, suggesting that bacterial growth was inhibited in a sequence-specific manner.     

Regulatory RNAs in Haloferax volcanii

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.     

Competition among Hfq-binding small RNAs in Escherichia coli

A major class of small bacterial RNAs (sRNAs) regulate translation and mRNA stability by pairing with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq, related to the Lsm/Sm families of splicing proteins, binds the sRNAs and stabilizes them in vivo and stimulates pairing with mRNAs in vitro. Although Hfq is abundant, the sRNAs, when induced, are similarly abundant. Therefore, Hfq may be limiting for sRNA function. We find that, when overexpressed, a number of sRNAs competed with endogenous sRNAs for binding to Hfq. This correlated with lower accumulation of the sRNAs (presumably a reflection of the loss of Hfq binding), and lower activity of the sRNAs in regulating gene expression. Hfq was limiting for both positive and negative regulation by the sRNAs. In addition, deletion of the gene for an expressed and particularly effective competitor sRNA improved the regulation of genes by other sRNAs, suggesting that Hfq is limiting during normal growth conditions. These results support the existence of a hierarchy of sRNA competition for Hfq, modulating the function of some sRNAs.     

sRNAbase: A Web Resource for Small RNAs in Enterobacteriaceae\m{1}

Bacterial small RNAs (sRNAs) have gained considerable attention due to their multivalent roles in the survival and pathogenesis of bacteria and mostly identified through bio-computational methods. A manually curated web-resource, sRNAbase has been constructed to give comprehensive and exhaustive information on non-coding small RNAs excluding tRNAs and rRNAs in Enterobacteriaceae family. The sRNA entries curated in sRNAbase contain experimentally verified small RNAs available in the literature and their partial/non-homologs reported within the related genomes from our earlier studies. The sRNAbase aims to facilitate the scientific community by providing information on the physical genomic location of the non-coding small RNAs, its alias names, sequences, strand orientation, gene identification numbers of the conserved genes that sandwiches the particular sRNA with possible functional role and a link to the PubMed literatures. Currently, sRNAbase holding information on 1986 entries belongs to 80 sRNA families spread over 45 Enterobacteriaceae genomes. The sRNAbase is accessible on the web at http://bicmku.in:8081/srnabase/.