Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs

The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Qbeta replicase. More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation. Despite their stability under certain conditions, E. coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures. We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5'-untranslated region of E. coli ompA mRNA as well as with that in the sRNA, DsrA. Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E. These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq(-) strain. It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets. This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack.     

Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur.     

Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.     

Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens.     

Negative effect of glucose on ompA mRNA stability: a potential role of cyclic AMP in the repression of hfq in Escherichia coli

Glucose is a carbon source that is capable of modulating the level of cyclic AMP (cAMP)-regulated genes. In the present study, we found that the stability of ompA mRNA was reduced in Escherichia coli when glucose (40 mM) was present in Luria-Bertani (LB) medium. This effect was associated with a low level of cAMP induced by the glucose. The results were confirmed with an adenylyl cyclase mutant with low levels of cAMP that are not modulated by glucose. Northern blot and Western blot analyses revealed that the host factor I (Hfq) (both mRNA and protein) levels were downregulated in the presence of cAMP. Furthermore, we showed that a complex of cAMP receptor protein (CRP) and cAMP binds to a specific P3(hfq) promoter region of hfq and regulates hfq expression. The regulation of the hfq gene was confirmed in vivo using an hfq-deficient mutant transformed with an exogenous hfq gene containing the promoter. These results demonstrated that expression of hfq was repressed by the CRP-cAMP complex. The presence of glucose resulted in increased Hfq protein levels, which decreased ompA mRNA stability. An additional experiment showed that cAMP also increased the stability of fur mRNA. Taken together, these results suggested that the repression of Hfq by cAMP may contribute to the stability of other mRNA in E. coli.     

肺炎克雷伯菌Hfq蛋白特性及对耐药的影响

Hfq(host factor for RNA phage QB replicase)蛋白是一个全局性调节因子,广泛参与细菌生长、趋化、毒力、耐药及应对外界选择压力等方面的调节,但在肺炎克雷伯菌(Klebsiella pneumoniae,KP)中的功能尚不清楚。本研究从临床病例中分离到59株KP,将其hfq基因与11例常见临床感染菌株hfq基因〔从美国国立生物技术信息中心(National Center for Biotechnology Information, NCBI)数据库下载〕进行了比较。所有hfq基因经EMBOSS Transeq翻译成氨基酸序列,用MAFFT软件进行多序列比对,并通过NCBI数据库中的保守结构域预测Hfq蛋白结构域。分别采用ESPript3.0、Phyre2分析Hfq蛋白的二、三级结构。59株KP中仅3株hfq基因的5个密码子位点存在差异,而其蛋白质氨基酸序列完全一致。KP与大肠埃希菌、阴沟肠杆菌、痢疾志贺菌之间,Hfq蛋白的氨基酸序列相似度较高,主要区别在C末端上;与金黄色葡萄球菌、产单核细胞李斯特菌相比,KP Hfq蛋白在N末端和C末端上差别较大;所有菌株C末端均呈酸性。三级结构预测提示68(66.67%)个氨基酸与模板序列一致, 较为保守的功能结构为54-VYKHAI-59序列。采用CRISPR/Cas9同源重组技术敲除KP的hfq基因,并对其进行药物敏感性测试,结果显示,基因敲除菌株对抗生素的耐药性较野生株有显著下降(P<0.05),差异有统计学意义,提示KP的Hfq蛋白氨基酸序列非常保守,可能参与了KP的耐药调节。     

RNase ES of Streptomyces coelicolor A3(2) can complement the rne and rng mutations in Escherichia coli

The Streptomyces coelicolor gene SCC88.10c encodes a protein (RNase ES) which is homologous to endoribonucleases in the RNase E/G family. We expressed S. coelicolor RNase ES as a 6 x His-tagged protein in an Escherichia coli mutant carrying a rng (which encodes RNase G) or a rne (which encodes RNase E) mutation to study whether S. coelicolor RNase ES is able to complement these mutations in host E. coli cells. The results clearly indicated that the S. coelicolor RNase ES can partially abrogate either the rng::cat or rne-1 mutation, as measured by the ability to suppress the several aberrant phenotypes resulting from the rng or rne mutation. Thus, S. coelicolor RNase ES appears to have the dual ability to supplant the functions of both RNase G and RNase E in E. coli.