Both temperature and medium composition regulate RNase E processing efficiency of the rpsO mRNA coding for ribosomal protein S15 of Escherichia coli

Cleavage by RNase E is believed to be the rate-limiting step in the degradation of many RNAs. These cleavages are modulated by 5' end-phosphorylation, folding and translation of the mRNA in question. Here, we present data suggesting that these cleavages are also regulated by environmental conditions. We report that rpsO mRNA, 15 minutes after a shift to 44 degrees C, is stabilized in cells grown in minimal medium. This stabilization is correlated with a reduction in the efficiency of the RNase E cleavage which initiates its decay. We also observe the appearance of RNA fragments previously detected following RNase E inactivation and a defect in the adaptation of RNase E concentration. These observations, coupled to the fact that RNase E overproduction slightly reduces the accumulation of the rpsO mRNA, suggest that this stabilization is caused in part by a limitation in RNase E concentration. An increase in the steady-state level of rpsT mRNA is also observed following a shift to 44 degrees C in minimal medium; however, processing of the 9 S rRNA precursor is not affected under these conditions. We thus propose that RNase E concentration changes in the cell in response to environmental conditions and that these changes can selectively affect the processing and the stability of individual mRNAs. Our data also indicate that the efficiency of cleavage of the rpsO mRNA by RNase E is modified by other factor(s) which remain to be identified.     

抑制E.coli的SOS应答对恩诺沙星抗药性的影响

通过敲除SOS应答启动蛋白基因rec A,探讨SOS应答对E.coli恩诺沙星抗药性的影响,并体外评价Rec A抑制剂和恩诺沙星联用对细菌协同抑制作用的影响.利用Red重组系统,构建E.coli ATCC 25922的rec A缺失菌株E.coli ATCC 25922/?rec A;在恩诺沙星压力下,利用荧光定量PCR测定SOS应答系统相关基因rec A和umu C表达量的变化.用微量肉汤稀释法测定恩诺沙星等常用抗生素对两个菌株的MIC变化;利用梯度平板法测定恩诺沙星对两个菌株抗药性变异的影响;合成Rec A抑制剂,并评估其与恩诺沙星联合抑制E.coli生长及其抗药性的作用.结果表明,E.coli ATCC 25922/?rec A菌株对恩诺沙星的最低抑菌浓度值降低至原始菌株的1/8;经药物处理后,在梯度平板上,rec A缺失菌株较野生型不易产生抗药性;荧光定量PCR表明,rec A缺失菌株或在Rec A抑制剂作用下,SOS应答系统受到一定的抑制.敲除rec A,使菌株对恩诺沙星的抗药性和抗药率均明显降低;Rec A抑制剂在一定程度上能抑制SOS应答,起到协同抑菌作用.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Feedback control of ribosome function in Escherichia coli

We have previously proposed that the rate of ribosome function during balanced growth in E. coli, expressed as the rate of peptide chain elongation, is adjusted by a feedback mechanism: whenever that rate is submaximal (i.e. below 22 amino acid residues polymerized per active ribosome at 37 degrees C), the feedback signal ppGpp is generated by an activation of the ppGpp synthetase expressed from the spoT gene. The accumulation of ppGpp reduces the synthesis of additional ribosomes and thereby reduces the consumption of amino acids which, in turn, allows the remaining ribosomes to function at a higher rate. Here we have described with supporting evidence the proposed feedback loop in greater detail and provided a mathematical analysis which predicts that the SpoT ppGpp synthetase activity should be highest when the ribosomes function at their half-maximal rate. This prediction is consistent with reported observations and is independent of the particular (unknown) mechanism by which the rate of translation controls the ppGpp synthetase activity of SpoT.     

Macromolecular crowding in the Escherichia coli periplasm maintains alpha-synuclein disorder

The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. Most studies of this protein are performed in dilute solution, but its biologically relevant role is performed in the crowded environment inside cells. We addressed the effects of macromolecular crowding on alpha-synuclein by combining NMR data acquired in living Escherichia coli with in vitro NMR data. The crowded environment in the E.coli periplasm prevents a conformational change that is detected at 35 degrees C in dilute solution. This change is associated with an increase in hydrodynamic radius and the formation of secondary structure in the N-terminal 100 amino acid residues. By preventing this temperature-induced conformational change, crowding in the E.coli periplasm stabilizes the disordered monomer. We obtain the same stabilization in vitro upon crowding alpha-synuclein with 300 g/l of bovine serum albumin, indicating that crowding alone is sufficient to stabilize the disordered, monomeric protein. Two disease-associated variants (A30P and A53T) behave in the same way in both dilute solution and in the E.coli periplasm. These data reveal the importance of approaching the effects of macromolecular crowding on a case-by-case basis. Additionally, our work shows that discrete structured protein conformations may not be achieved by alpha-synuclein inside cells, implicating the commonly overlooked aspect of macromolecular crowding as a possible factor in the etiology of Parkinson's disease.     

A new cell division operon in Escherichia coli

Summary At 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.     

Structural characterization of Escherichia coli sialic acid synthase

Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.     

Role of the RuvAB protein in avoiding spontaneous formation of deletion mutations in the Escherichia coli K-12 endogenous tonB gene

The endogenous tonB gene of Escherichia coli was used as a target for spontaneous deletion mutations which were isolated from ruvAB-, recG-, and ruvC- cells. The rates of tonB mutation were essentially the same in ruv+, ruvAB-, recG-, and ruvC- cells. We analyzed tonB mutants by sequencing. In the ruv+, recG-, and ruvC- strains, the spectra were different from those obtained from the ruvAB- cells, where deletions dominated followed by IS insertions, base substitutions, and frameshifts, in that order. We then analyzed the tonB-trp large deletion, due to simultaneous mutations of the trp operon, and found that the frequency in ruvAB- was higher than those in ruv+, recG-, and ruvC- cells. To characterize deletion formation further, we analyzed all the tonB mutants from one colicin plate. Seven deletions were identified at five sites from the 45 tonB mutants of ruv+ cells and 24 deletions at 11 sites from the 43 tonB mutants of ruvAB- cells. Thus, the ruvAB- strain is a deletion mutator. We discuss the role of RuvAB in avoiding deletions.     

Characterization of the cheY genes from Leptospira interrogans and their effects on the behavior of Escherichia coli

The motility and chemotaxis system are critical for the virulence of pathogenic leptospire, which enable them to penetrate host tissue barriers during infection. The completed genome sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroups Icterohaemorrhagiae (L. interrogans strain Lai) suggested that there were multiple copies of putative chemotaxis homologues located at its large chromosome. In order to verify the function of these proteins, the putative cheY genes were cloned into pQE31 vector and then expressed, respectively, in wild-type Escherichia coli strain RP437 and cheY defective strain RP5232. The results showed that all the five cheYs could restore the swarming of RP5232 strain to some extend. Overexpression of CheYs in RP437 showed inhibited swarming of RP437. To investigate the mechanism of chemotaxis signaling in L. interrogans strain Lai, certain aspartates (Asp-53, Asp-61, Asp-70, Asp-62, and Asp-66 for L. interrogans strain Lai CheY1, CheY2, CheY3, CheY4, and CheY5, respectively) were mutated. Expression of these mutated cheYs manifested neither restoration of the swarming ability of RP5232 nor inhibition on swarming ability of RP437. Multiple amino acid sequence alignment predicted ternary structures and the result of mutation experiment suggested that these conserved aspartate residues of L. interrogans were analogous to that in E. coli CheY in function and structure. So, L. interrogans and E. coli may have similar mechanisms of activation of the chemotaxis phosphorelay pathway, but there are differences in their control by signal terminator.     

Phenotypic plasticity of Escherichia coli at initial stage of symbiosis with Dictyostelium discoideum

We observed the change in the physiological state of Escherichia coli cells at the initial stage for establishing a new symbiotic relationship with Dictyostelium discoideum cells. For the physiological state, we monitored green fluorescence intensity due to a green fluorescent protein (GFP) gene integrated into the chromosome by flow cytometry (FCM). On co-cultivation of the two species, a new population of E. coli cells with increased GFP concentration appeared, and when the formation of mucoidal colonies housing the coexisting two species began, most E. coli cells were from the new population. Further experiments suggest that the physiological change is induced by interaction with D. discoideum cells and is reversible, although the processes of the changes in both directions seem to proceed gradually. The observed phenotypic plasticity, together with natural selection under a co-cultivation environment, may be important for leading to the evolution of a new symbiotic system.     

The relation between translation and mRNA degradation in the lacZ gene

The technique of gene fusion, in which the gene of interest, severed from its 3' end, is in-phase fused to a reporter gene--usually lacZ--is widely used to study translational regulation in Escherichia coli. Implicit in these approaches is the assumption that the activity of the ribosome binding site (RBS) fused in-phase with lacZ, does not per se modify the steady-state level of the lacZ mRNA. Herein, we have tested this hypothesis, using a model system in which the RBS of the lamB gene is fused to lacZ. Several point mutations affecting translation initiation have been formerly characterized in this RBS, and we used Northern blots to study their effect upon the lacZ mRNA pattern. Two series of constructs were assayed: in the first one, a 51-bp fragment centered around the lamB initiator codon, was inserted in front of lacZ within the natural lactose operon, whereas in the second the lacZ gene was fused to the genuine malK-lamB operon just downstream from the lamB RBS. We observed that in the first series, the concentration and average molecular weight of the lacZ mRNA dropped sharply as the efficiency of the RBS decreased. This apparently arose from a decreased stability of the message, since the mRNA patterns are equalized when the endonuclease RNase E is inactivated. We suggest that in this case the rate limiting step in the decay process is an RNase E cleavage that is outcompeted by translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient disinfection of Escherichia coli in water by silver loaded alumina

The catalytic inactivation of Escherichia coli (E. coli) in water by silver loaded alumina as catalyst was investigated. Ag/Al₂O₃ and AgCl/Al₂O₃ catalysts exhibited high bactericidal activity at room temperature in water with no need for any light or electrical power input. Dissolved oxygen which can be catalyzed to reactive oxygen species (ROS) was found to be essential for the strong bactericidal activities of the catalysts. Decomposition of the cell wall leading to leakage of the intracellular component and the complete lysis of the whole cell were directly observed by transmission electron microscopy (TEM). The resultant change in cell permeability was confirmed by potassium ion leakage. The different morphological changes between E. coli cells treated with the catalysts and Ag⁺ were also observed. The formation of ROS involved in the bactericidal process by AgCl/Al₂O₃ was confirmed by addition of catalase and OH scavenger. Higher temperature and pH value were found to have positive effect on the bactericidal activity of AgCl/Al₂O₃. All these results indicated that the bactericidal effect of the catalyst was a synergic action of ROS and Ag⁺, not an additive one. A possible mechanism is proposed.     

The role of UbiX in Escherichia coli coenzyme Q biosynthesis

The reversible redox chemistry of coenzyme Q serves a crucial function in respiratory electron transport. Biosynthesis of Q in Escherichia coli depends on the ubi genes. However, very little is known about UbiX, an enzyme thought to be involved in the decarboxylation step in Q biosynthesis in E. coli and Salmonella enterica. Here we characterize an E. coli ubiX gene deletion strain, LL1, to further elucidate E. coli ubiX function in Q biosynthesis. LLI produces very low levels of Q, grows slowly on succinate as the sole carbon source, accumulates 4-hydroxy-3-octaprenyl-benzoate, and has reduced UbiG O-methyltransferase activity. Expression of either E. coli ubiX or the Saccharomyces cerevisiae ortholog PAD1, rescues the deficient phenotypes of LL1, identifying PAD1 as an ortholog of ubiX. Our results suggest that both UbiX and UbiD are required for the decarboxylation of 4-hydroxy-3-octaprenyl-benzoate in E. coli coenzyme Q biosynthesis, especially during logarithmic growth.