Signal peptide mutants ofEscherichia coli

Numerous secretory proteins of the Gram-negative bacteriaE. coli are synthesized as precursor proteins which require an amino terminal extension known as the signal peptide for translocation across the cytoplasmic membrane. Following translocation, the signal peptide is proteolytically cleaved from the precursor to produce the mature exported protein. Signal peptides do not exhibit sequence homology, but invariably share common structural features: (1) The basic amino acid residues positioned at the amino terminus of the signal peptide are probably involved in precursor protein binding to the cytoplasmic membrane surface. (2) A stretch of 10 to 15 nonpolar amino acid residues form a hydrophobic core in the signal peptide which can insert into the lipid bilayer. (3) Small residues capable of -turn formation are located at the cleavage site in the carboxyl terminus of the signal peptide. (4) Charge characteristics of the amino terminal region of the mature protein can also influence precursor protein export. A variety of mutations in each of the structurally distinct regions of the signal peptide have been constructedvia site-directed mutagenesis or isolated through genetic selection. These mutants have shed considerable light on the structure and function of the signal peptide and are reviewed here.     

Unusual genetic phenomena associated with Tn5 mutagenesis in Alcaligenes eutrophus strain H1

Tn5 was introduced into Alcaligenes eutrophus strain H1 by a suicide vector pSUP1011. Physical characterization of mutants obtained after Tn5 mutagenesis revealed a relatively high frequency of plasmid curing, or deletion of a 50 kb plasmid DNA segment. Results of Southern hybridization and chromosomal walking indicate that the same continuous stretch of plasmid DNA (designated as D region of plasmid) is deleted in four independent isolates. Moreover, the same deletion of plasmid DNA is also observed in a mitomycin C-generated mutant strain H1-4.Journal Paper No. J-12095 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2607, supported in part by a grant from the Iowa High Technology Council     

利用Tn5转座子介导突变提高大肠杆菌丁醇生产水平

目前,对于构建高产丁醇大肠杆菌工程菌株的工作,主要是对丁醇通路和相关途径的基因进行理性改造。为进一步提升菌株的丁醇生产能力,需要发掘基因组上可影响丁醇生产能力的基因,但这很难通过已有认识或计算机模型进行预测。本工作以一株实验室前期构建的产丁醇大肠杆菌工程菌株为研究对象,利用Tn5转座子构建了一个含有1 196个菌株的突变文库。丙酮酸是丁醇的前体,并且在发酵终产物中,副产物丙酮酸的含量与丁醇的含量呈反相关,因此,可以利用丙酮酸的含量来间接反映丁醇的含量,而丙酮酸可用二硝基苯肼显色法进行快速测定,基于此,建立了96孔板——酶标仪快速筛选方法。利用该方法成功筛选到了比对照菌株丁醇产量提高了29%、49%、56%的3个突变体菌株。利用反向PCR及测序的方法,确定了其转座子插入位置分别为:pyk A、tdk、cad C基因。这些基因可以作为进一步提高菌株丁醇产量的靶点,同时这种利用Tn5转座子筛选基因靶标的策略也为构建其他微生物细胞工厂提供了新思路。     

Genetic and physical analysis of plasmid genes expressing inducible resistance of tellurite in Escherichia coli

Summary A large (>250 kb) conjugative plasmid, pMER610, specifying resistance to tellurium and mercury was isolated from an Alcaligenes strain and transferred by conjugation to Escherichia coli AB1157. The acquisition of pMER610 by AB1157 increased the resistance to both tellurite and tellurate by 100-fold. Expression of tellurite resistance by pMER610 and the cloned Te^r determinant was inducible by prior exposure to tellurite at levels sub-toxic to the sesitive AB1157. Physical analysis of the cloned Te^r fragment located the resistance determinant to a 3.55 kb region. Insertion of Tn 1000 () into this region produced two classes of sensitive mutations, fully sensitive and intermediate or hyposensitive, which map in adjacent regions and form two complementation groups. Maxicell analysis identified four polypeptides (15.5, 22, 23 and 41 kDa) expressed by the Te^r clone. The 23 kDa polypeptide may not be required for resistance since tellurium-sensitive insertion mutations were not detected in the 23 kDa coding region.     

Voltage-sensitive ion channel ofEscherichia coli

Summary A voltage-sensitive, cation-selective ion channel ofEscherichia coli has been reconstituted into liposomes and studied with the patch-clamp method. The single channel conductance was 91 pS in symmetric solutions of 150mm KCl. Many channels were open most of the time, with frequent brief transitions to closed levels. Multiple conducting units could close and reopen simultaneously, and this apparent cooperativity in gating was increases with depolarizing voltages. Above a voltage threshold, the channels closed irreversibly, often in groups.     

Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli

In the context of recombinant DNA technology, the development of feasible and high-yielding plasmid DNA production processes has regained attention as more evidence for its efficacy as vectors for gene therapy and DNA vaccination arise. When producing plasmid DNA in Escherichia coli, a number of biological restraints, triggered by plasmid maintenance and replication as well as culture conditions are responsible for limiting final biomass and product yields. This termed "metabolic burden" can also cause detrimental effects on plasmid stability and quality, since the cell machinery is no longer capable of maintaining an active metabolism towards plasmid synthesis and the stress responses elicited by plasmid maintenance can also cause increased plasmid instability. The optimization of plasmid DNA production bioprocesses is still hindered by the lack of information on the host metabolic responses as well as information on plasmid instability. Therefore, systematic and on-line approaches are required not only to characterise this "metabolic burden" and plasmid stability but also for the design of appropriate metabolic engineering and culture strategies. The monitoring tools described to date rapidly evolve from laborious, off-line and at-line monitoring to online monitoring, at a time-scale that enables researchers to solve these bioprocessing problems as they occur. This review highlights major E. coli biological alterations caused by plasmid maintenance and replication, possible causes for plasmid instability and discusses the ability of currently employed bioprocess monitoring techniques to provide information in order to circumvent metabolic burden and plasmid instability, pointing out the possible evolution of these methods towards online bioprocess monitoring.     

Genetics of pentose-phosphate pathway enzymes ofEscherichia coli K-12

The pentose-phosphate pathway ofEscherichia coli K-12, in addition to its role as a route for the breakdown of sugars such as glucose or pentoses, provides the cell with intermediates for the anabolism of amino acids, vitamins, nucleotides, and cell wall constituents. Through its oxidative branch, it is a major source of NADPH. The expression of the gene for NADP-dependent 6-phospho-gluconate dehydrogenase (gnd) is regulated by the growth rate inE. coli. The recently identified gene for ribulose-5-phosphate 3-epimerase (rpe) is part of a large operon that comprises among others genes for the biosynthesis of aromatic amino acids. In recent years, genes for all enzymes of the pathway have been cloned and sequenced. Isoenzymes have been found for transketolase (genestktA andtktB), ribose-5-phosphate isomerase (rpiA andrpiB) and transaldolase (talA andtalB).     

The hydrogenases and formate dehydrogenases ofEscherichia coli

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.     

Host controlled plasmid replication: Escherichia coli minichromosomes

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.     

Adenosine monophosphate-induced amplification of ColE1 plasmid DNA in Escherichia coli

ColE1 plasmid copy number was analyzed in relaxed (relA) and stringent (relA(+)) Escherichia coli cells after supplementation of culture media with adenosine monophosphate (AMP). When a relaxed E. coli strain bearing ColE1 plasmid was cultured in LB medium for 18 h and induced with AMP for 4h, the plasmid DNA yield was significantly increased, from 2.6 to 16.4 mgl(-1). However no AMP-induced amplification of ColE1 plasmid DNA was observed in the stringent host. Some plasmid amplification was observed in relA mutant cultures in the presence of adenosine, while adenine, ADP, ATP, ribose, potassium pyrophosphate and sodium phosphate caused a minor, if any, increase in ColE1 copy number. A mechanism for amplification of ColE1 plasmid DNA with AMP in relA mutant bacteria is suggested, in which AMP interferes with the aminoacylation of tRNAs, increases the abundance of uncharged tRNAs, and uncharged tRNAs promote plasmid DNA replication. According to this proposal, in relA(+) cells, the AMP induction could not increase ColE1 plasmid copy number because of lower abundance of uncharged tRNAs. Our results suggest that the induction with AMP can be used as an effective method of amplification of ColE1 plasmid DNA in relaxed strains of E. coli.     

Comparative genomics and phylogeny of the IncI1 plasmids: a common plasmid type among porcine enterotoxigenic Escherichia coli

Increasing reports of multidrug resistance conferred by conjugative plasmids of Enterobacteriaceae necessitate a better understanding of their evolution. One such group is the narrow-host-range IncI1 plasmid type, known for their ability to carry genes encoding resistance to extended-spectrum beta lactamases. The focus of this study was to perform comparative sequencing of IncI1 plasmids from porcine enterotoxigenic Escherichia coli (ETEC), isolated irrespective of antimicrobial susceptibility phenotype. Five IncI1 plasmids of porcine ETEC origin and one IncI1 plasmid from a Salmonella enterica serovar Kentucky isolate from a healthy broiler chicken were sequenced and compared to existing IncI1 plasmid sequences in an effort to better understand the overall genetic composition of the IncI1 plasmid lineages. Overall, the sequenced porcine ETEC IncI1 plasmids were divergent from other sequenced IncI1 plasmids based upon multiple means of inferred phylogeny. High occurrences of IncI1 and IncA/C plasmid-associated genes and the bla_TEM and bla_CMY-2 beta lactamase genes were observed among porcine ETEC. However, the presence of bla_TEM and bla_CMY-2 did not strongly correlate with IncI1 plasmid possession, suggesting that these plasmids in porcine ETEC are not primarily associated with the carriage of such resistance genes. Overall, this work suggests a conservation of the IncI1 plasmid backbone among sequenced plasmids with a single locus for the acquisition of accessory genes, such as those associated with antimicrobial resistance. Furthermore, the high occurrence of IncI1 and IncA/C plasmids among clinical E. coli from commercial swine facilities is indicative of extensive horizontal gene transfer among porcine ETEC.     

Requirement of the Escherichia coli dnaA gene function for integrative suppression of dnaA mutations by plasmid R100-1

Summary The phenotype of Escherichia coli dnaA missense and nonsense mutations was integratively suppressed by plasmid R100-1. The suppressed strains, however, could not survive when the dnaA function was totally inactivated. This was demonstrated by the inability of replacing the dnaA allele in the suppressed strain by a dnaA::Tn10 insertion using phage P1-mediated transduction. When the intact dnaA ⁺ allele was additionally supplied by a specialized transducing phage, imm ²¹ dnaA ⁺, which integrated at the att site on the E. coli chromosome, then the dnaA::Tn10 insertion, together with a oriC deletion, were able to be introduced into the suppressed strain. Thus, the mechanisms of dnaA function for oriC and for the replication origin of R100-1 may not be quite the same.     

A temperature-sensitive mutant ofEscherichia coli with an alteration in ribosomal protein L22

A temperature-sensitive, protein synthesis-defective mutant ofEscherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to therplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.     

The close proximity ofEscherichia coli genes: Consequences for stop codon and synonymous codon use

It is shown that synonymous codon usage is less biased in favor of those codons preferred by highly expressed genes at the end ofEscherichia coli genes than in the middle. This appears to be due to the close proximity of manyE. coli genes. It is shown that a substantial number of genes overlap either the Shine-Dalgarno sequence or the coding sequence of the next gene on the chromosome and that the codons that overlap have lower synonymous codon bias than those which do not. It is also shown that there is an increase in the frequency of A-ending codons, and a decrease in the frequency of G-ending codons at the end ofE. coli genes that lie close to another gene. It is suggested that these trends in composition could be associated with selection against the formation of mRNA secondary structure near the start of the next gene on the chromosome. Stop codon use is also affected by the close proximity of genes; many genes are forced to use TGA and TAG stop codons because they terminate either within the Shine-Dalgarno or coding sequence of the next gene on the chromosome. The implications these results have for the evolution of synonymous codon use are discussed.     

Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide

Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

Summary The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.     

A single amino acid substitution alters conductance and gating of OmpC porin ofEscherichia coli

Summary We have reconstituted into liposomes outer-membrane fractions fromEscherichia coil strains which express OmpC porins with altered pore properties. Single-channel experiments were performed with the patch-clamp technique on blisters generated from the reconstituted liposomes. Our goal was to identify positively the activity pattern of OmpC in our reconstituted system. The properties of the parent strain were compared to those of a strain whose OmpC porin has a single amino acid substitution in a postulated transmembrane segment. The parent and the mutant strain each exhibit a cation-selective channel of high open probability and gating to closed levels of various amplitudes. However, the mutant channel appeared to be 9 to 30% larger in unit conductance. It tended to close and reopen most often in groups of three units, as opposed to two units in the parent channel. The results are discussed in terms of the observed phenotype and of their implication as to the structure-function relationship of the porin channels.     

Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5

Drug-resistance element Tn5 coding for kanamycin resistance was used for mutagenesis of Alcaligenes eutrophus strain H16. The vehicle for introducing Tn5 into A. eutrophus was plasmid pJB4JI harboured by Escherichia coli. Kanamycin-resistant transconjugants occurred at a frequency of approximately 5×10^-8. One third of the transconjugants exhibited other plasmid-coded resistances such as gentamycin and spectinomycin. However, the latter markers were not stably maintained in the new host. Among the kanamycin-resistant transconjugants three classes of mutants were found: (i) Auxotrophic mutants occurred at a frequency of 0.8% and showed requirements for histidine, methionine, aspartate orisoleucine. Out of eleven auxotrophic mutants examined eight reverted to prototrophy. However, none of the revertants was kanamycin-sensitive. (ii) Mutants unable to grow with fructose as the carbon source occurred at a frequency of almost 10%. (iii) Mutants which had lost the ability to grow autotrophically with hydrogen and carbon dioxide were found at a frequency of 1%. Further analyses revealed that this class of mutants was either defective in carbon dioxide fixation or impaired in hydrogen metabolism.     

转座子Tn5-Mob对菜豆根瘤菌共生基因的诱变、转移和初步定位

转座子Tn5-Mob在质粒RP4-4配合下能诱动(Mobilization)菜豆根瘤菌RCR3622内源质粒的诱动转移。在种间根瘤菌杂交过程中,二个巨型质粒的转移频率均大于10~(-3);分子量约为285kb的psym3622是带有结瘤(nod)和产黑素(mel)基因的共生质粒(Symbiotic plasmid);这二个基因的最大距离不超过70kb左右。另一个分子量约为135kb的质粒在试验中为不具结瘤功能的隐蔽质粒。将psym3622共生质粒导入不结瘤(Nod-)的豌豆根瘤菌菌株B151,能够使后者在菜豆植物上表达结瘤的特性,形成无效根瘤。将psym3622共生质粒导入不结瘤的菜豆根瘤菌菌株JI8400,能够在菜豆植物上形成正常发育的有效根瘤。