The length of the 5' leader of Escherichia coli tRNA precursors influences bacterial growth

Based on a computational analysis of the 5' regions of tRNA-encoding genes, the average length of the 5' leaders in tRNA precursors in Escherichia coli appears to be 17-18 residues long. An in vivo assay based on tRNA nonsense suppression was developed and used to investigate the function of the 5' leader of the tRNA precursors on tRNA processing and bacterial growth. Our data indicate that the 5' leader influences bacterial growth but is surprisingly not absolutely necessary for growth. These findings are consistent with previous in vitro data where it was demonstrated that the 5' leader plays a role in the interaction with RNase P, the endoribonuclease responsible for removing the 5' leader in the cell. We discuss the plausible role of the 5' leader in processing and tRNA gene expression.     

Suppression of the Lethal Effect of Acidic-Phospholipid Deficiency in Escherichia coli by Bacillus subtilis Chromosomal Locus ypoP

An acidic-phospholipid deficiency caused by the pgsA3 allele that encodes a defective phosphatidylglycerophosphate synthase in Escherichia coli is lethal. The only known mutations that suppress this lethality fully have been related to the major outer-membrane lipoprotein. We isolated a Bacillus subtilis chromosomal locus that suppresses the lethality when harbored in a low copy-number plasmid, without restoring the synthase activity or phospholipid composition to normal. The locus was first recognized to suppress the conditional lethality of E. coli YA5512 (pgsA3) that harbored an unidentified mutation(s), allowing its growth in LB medium but not in media of lower osmolarities. The locus was then found to suppress the lethality of pgsA3 in wild-type E. coli W3110. This locus, named ypoP in the database, had 37% nucleotide identity with the E. coli mprA gene, but the amplification of mprA had no suppressive effect. Plasmid pPOP1 containing ypoP completely prevented the decrease in the amount of a porin protein, OmpF, in the outer membrane and also cell mucoidy caused by pgsA3. The mechanisms underlying these unusual effects are discussed in relation to a putative stress signal(s) generated by the acidic-phospholipid deficiency.     

Recognition of tRNA backbone for aminoacylation with cysteine: evolution from Escherichia coli to human

The underlying basis of the genetic code is specific aminoacylation of tRNAs by aminoacyl-tRNA synthetases. Although the code is conserved, bases in tRNA that establish aminoacylation are not necessarily conserved. Even when the bases are conserved, positions of backbone groups that contribute to aminoacylation may vary. We show here that, although the Escherichia coli and human cysteinyl-tRNA synthetases both recognize the same bases (U73 and the GCA anticodon) of tRNA for aminoacylation, they have different emphasis on the tRNA backbone. The E. coli enzyme recognizes two clusters of phosphate groups. One is at A36 in the anticodon and the other is in the core of the tRNA structure and includes phosphate groups at positions 9, 12, 14, and 60. Metal-ion rescue experiments show that those at positions 9, 12, and 60 are involved with binding divalent metal ions that are important for aminoacylation. The E. coli enzyme also recognizes 2'-hydroxyl groups within the same two clusters: at positions 33, 35, and 36 in the anticodon loop, and at positions 49, 55, and 61 in the core. The human enzyme, by contrast, recognizes few phosphate or 2'-hydroxy groups for aminoacylation. The evolution from the backbone-dependent recognition by the E. coli enzyme to the backbone-independent recognition by the human enzyme demonstrates a previously unrecognized shift that nonetheless has preserved the specificity for aminoacylation with cysteine.     

Escherichia coli tRNAs Are Resistant to the Hyperprocessing Reaction of Homologous E. coli Ribonuclease P Ribozyme

Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.     

改善大肠杆菌胞内氨基酰tRNA池提高外源基因表达水平

大肠杆菌是研究和高效表达异源蛋白的常用宿主细胞。由于大肠杆菌和真核生物及大部分古细菌在同义密码子上的使用有很大的差异,来源于真核或古细菌的外源基因在大肠杆菌中表达时,常常由于其带有稀有密码子而带来翻译方面的问题,如产生移码突变和表达量下降等,从而改变异源蛋白的表达质量和表达水平。在研究常见嗜热菌tRNA结构和组成的基础上,以古细菌α淀粉酶基因为例,采用增加有argU、ileY和leuWtRNA基因的大肠杆菌DE3为表达宿主,改善了胞内氨基酰tRNA池的大肠杆菌表达异源蛋白,表达量提高约9倍。     

The Yersinia high-pathogenicity island is highly predominant in virulence-associated phylogenetic groups of Escherichia coli

The high-pathogenicity island (HPI) present in pathogenic Yersinia and encoding the siderophore yersiniabactin, has recently been identified in the asnT tRNA region of various Escherichia coli pathotypes, especially those responsible for bacteremia and urosepsis. Most E. coli strains causing such extra-intestinal infections belong to phylogenetic groups B2 and D. In this study we investigated (i) the distribution and localization of HPI among the different E. coli phylogenetic groups, using the ECOR reference collection; and (ii) the prevalence of HPI among a set of 124 phylogenetically characterized E. coli strains responsible for neonatal meningitis. Ninety-three percent of the ECOR strains belonging to groups B2 and D harbored HPI. In contrast, the island was present in 32% and 25% of strains belonging to groups A and B1, respectively, which are considered to be non-pathogenic. HPI was found in 100% of the neonatal meningitis strains, 13 of which belonged to groups A and B1, suggesting that HPI might contain virulent factors required for the development of neonatal meningitis. Moreover, we observed for the first time that HPI can be inserted in a site different from the asnT tRNA region.     

大肠杆菌精氨酰tRNA合成酶基因（argS）的上游非编码区存在一个负调控区

含大肠杆菌精氨酰tRNA合成酶（ArgRS）基因(argS）的pUC18重组质粒,在大肠杆菌TG1转化子中能够高表达ArgRS近1000倍。为了研究大肠杆菌argS的表达调控,构建了一系列的缺失突变。分别缺失全部上游序列（argS△1）、Shine-Dalgarno（SD）区（argS△2）和缺失启动子－10区下游（相当于翻译起始位点－65nt,argS△3）前的上游序列后的变种argS都不能表达ArgRS。而缺失－122nt（距翻译起始位点－180nt,argS△8）、－70nt（距翻译起始位点－128nt,argS△7）、－52nt（距翻译起始位点－110nt,argS△6）、－35区9距翻译起始位点－94nt,argS△5）、启动子－10区（距翻译起始位点－71nt,argS△4）前的上游序列后,这些缺失突变基因的表达水平与野生型argS接近。但argS△4、argS△5、argS△6都会形成部分包涵体。通过RNA斑点杂交测定发现,argS△4、argS△5和argS△6的mRNA转录量为argS及argS△7的2到3倍。即－52nt和－70nt之间的19个碱基（AATAGTGAAAACGGCAATA)可能是大肠杆菌argS舞场康负调控区。该元件的缺失将使得ArgRS过快表达并导致部分蛋白质形成包涵体。凝胶阻滞分析也发现在细胞粗抽液中有一个因子可以专一地与这个负调控元件结合,它可能参与该基因的表达调控。精氨酸专一性地诱导argS的转录,其作用与上述转录负调控区相关。     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

Nucleotide sequence of the ethidium efflux gene from Escherichia coli

The nucleotide sequence of the gene specifying the ethidium efflux system of Escherichia coli has been determined. The translated open reading frame has identified a membrane-bound polypeptide of 110 amino acids (11,960 Da) which shares 42% identity with a staphylococcal protein specifying resistance to ethidium.     

An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin-binding protein 5 of Escherichia coli

A mutant of Escherichia coli has been described that produces an altered form of penicillin-binding protein 5 which still binds penicillin but is unable to catalyse the release of the bound penicilloyl moiety. We show that the mutation is caused by a single nucleotide transition that results in a change from glycine at residue 105 of the wild-type sequence of penicillin-binding protein 5 to aspartate in the mutant.     

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate‐based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase‐coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell‐free protein synthesis activity. The resulting tRNA‐depleted lysate is compatible with in vitro‐transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real‐time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401–1407, 2017     

Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E. coli strain from a diseased animal

Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E. coli fimbrial adhesins. This strain produces two fimbriae: F165(1) and F165(2). F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon. Data from this study suggest that these two operons are part of two PAIs. PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E. coli chromosome. PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes. PAI II(4787) is found between the proA and yagU at 6 min of the E. coli chromosome.     

多基因在大肠杆菌中的共表达策略

多基因共表达在多领域有重要的应用价值,大肠杆菌共表达系统包括多顺反子系统和双质粒系统,两者各有优缺点。多顺反子系统不需外界2种抗生素的同时存在,但操作较为复杂。通常认为,具有相同复制子的质粒是不相容的,但近来的实验表明,在双抗生素的选择压力下,不相容的双质粒系统也能稳定传代,且操作简单,周期较短。双质粒系统已经应用于生产、医学等各个领域。     

Nucleotide sequence of the sfaA gene coding for the S-fimbrial protein subunit of Escherichia coli

The sfaA gene of the uropathogenic Escherichia coli O6 strain 536, which is responsible for the determination of the S fimbrial protein subunit, was sequenced. The structural gene codes for a polypeptide of 180 amino acids including a 24-residue N-terminal signal sequence. A size of 15.95 kDa was calculated for the processed SfaA protein. The nucleotide and deduced amino acid sequences show significant homology to those of the F1C fimbria and, to a lesser extent, of the mannose-sensitive hemagglutinating fimbria (FimA, PilA). Only week homology to P fimbriae subunits (F7₂, Pap) was found.     

The immunological dissection of the Escherichia coli molecular chaperone DnaJ

In Escherichia coli, one of the main molecular chaperones is DnaJ (hsp40), which mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes. DnaJ is characterised by the presence of four distinct domains: the J-domain, glycine/phenylalanine-rich (G/F), cysteine-rich (Zn-finger) and C-terminal regions. Truncated DnaJ polypeptides (DnaJ 1-108, DnaJ Delta1-108, DnaJ Delta1-199) representing these domains were over-produced and used as a source of immunogens for the generation of sequence-specific polyclonal antibodies. Epitope mapping was achieved by Western blotting, which demonstrated the presence of antibodies directed against these domains. These characterised affinity-purified antibodies were then used to assess the role of DnaJ in the protection of firefly luciferase from irreversible heat-inactivation. In this study we have demonstrated the involvement of J-, G/F and Zn-finger domains in the protection of luciferase from heat-inactivation. The C-terminal region had only partial involvement in luciferase protection.     

Genome diversity at the serA-linked capsule locus in Escherichia coli

Individual isolates of Escherichia coli synthesize one of more than 70 chemically distinct polysaccharides which form the capsule. In this article we review the genetics of capsule production in E. coli and highlight what this is beginning to reveal in terms of the genetic basis of the structural diversity of polysaccharides. The serA-linked capsule locus can take three different allelic forms. Two of these are associated with capsule genes and are themselves internally variant, whilst the third form has not so far been implicated in capsule biogenesis. Thus the serA-linked region of the E. coli genome is strikingly polymorphic.