Effect of increased ppGpp concentration on DNA replication of different replicons in <i>Escherichia coli</i>

Herman, Anna; Wèrzyn, Grzegorz

doi:10.1002/jobm.3620350110

Cited by 23 publications

(10 citation statements)

References 24 publications

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“…This phenomenon can possibly be attributed to the occurrence of the uncharged tRNAs (23,44) that do interact with the plasmid replication control mechanism and additionally trigger the stringent response (45,46). Consequently, the high levels of ppGpp detected for the plasmid-based system may also contribute to the observed growth cessation (47). This assumption is also supported by the significant drop in PCN that we observed ( Fig.…”

Section: Discussionsupporting

confidence: 74%

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Mairhofer

Scharl

Marisch

et al. 2013

Appl Environ Microbiol

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Plasmid-based Escherichia coli BL21(DE3) expression systems are extensively used for the production of recombinant proteins. However, the combination of a high gene dosage with strong promoters exerts extremely stressful conditions on producing cells, resulting in a multitude of protective reactions and malfunctions in the host cell with a strong impact on yield and quality of the product. Here, we provide in-depth characterization of plasmid-based perturbations in recombinant protein production. A plasmid-free T7 system with a single copy of the gene of interest (GOI) integrated into the genome was used as a reference. Transcriptomics in combination with a variety of process analytics were used to characterize and compare a plasmid-free T7-based expression system to a conventional pET-plasmid-based expression system, with both expressing human superoxide dismutase in fed-batch cultivations. The plasmid-free system showed a moderate stress response on the transcriptional level, with only minor effects on cell growth. In contrast to this finding, comprehensive changes on the transcriptome level were observed in the plasmid-based expression system and cell growth was heavily impaired by recombinant gene expression. Additionally, we found that the T7 terminator is not a sufficient termination signal. Overall, this work reveals that the major metabolic burden in plasmid-based systems is caused at the level of transcription as a result of overtranscription of the multicopy product gene and transcriptional read-through of T7 RNA polymerase. We therefore conclude that the presence of high levels of extrinsic mRNAs, competing for the limited number of ribosomes, leads to the significantly reduced translation of intrinsic mRNAs. P lasmid-based expression systems have been used for the production of recombinant proteins for more than 4 decades (1, 2). They can be manipulated quickly and easily, and a variety of replicons for use in Escherichia coli have become available (3), allowing, e.g., different expression levels by using plasmids with different copy numbers (4, 5). Plasmids equipped with additional functions can be used to facilitate, for instance, coexpression of proteins assisting correct folding (6) or of tRNAs supporting transcription of rare codons (7). The dissemination of E. coli systems was further strongly supported by the availability of well-established, easy-to-use protocols from molecular manipulation to cell cultivation up to a large scale (8) and by the FDA-proven status of E. coli as a host for production of proteins for clinical use (9).However, E. coli-based production processes are still far from optimal for exploitation of the cellular system, as the expression of heterologous proteins is performed with excessive strength, leading to a rapid exhaustion of the host cell (10) and, hence, loss of yield. One prominent example is the T7 system, combining highcopy-number plasmids with an orthogonal transcription system in the form of the T7 phage RNA polymerase, an enzyme showing an average elongation ...

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Section: Discussionsupporting

confidence: 74%

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Mairhofer

Scharl

Marisch

et al. 2013

Appl Environ Microbiol

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“…In designing experiments to detect RelA, RTP‐dependent replication arrest sites, it was essential to establish conditions in which the production of ppGpp did not inhibit replication by a mechanism distinct from that correlated with LSTer sites. The replication of many replicons, such as colE1‐type plasmids, pSC101 and λ, is inhibited when the intracellular concentration of ppGpp is increased either by amino acid starvation or by overexpression of relA (Szalewska‐Palasz et al ., 1994; Herman and Wegrzyn, 1995; Wröbel and Wegrzyn, 1998). In contrast, replication of a plasmid carrying the ori region from RK2 is inhibited after amino‐acid starvation, but not when the intracellular level of ppGpp is increased without nutritional downshift.…”

Section: Resultsmentioning

confidence: 99%

The replication checkpoint control in Bacillus subtilis: identification of a novel RTP‐binding sequence essential for the replication fork arrest after induction of the stringent response

Autret

Levine

Vannier

et al. 1999

Molecular Microbiology

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SummaryWe have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA-DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (¹128 kb from oriC ). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17 bp sequence (BЈ-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This BЈ-1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.

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“…Our study used the P. aeruginosa relA gene, which may have altered ppGpp and pppGpp levels differently. We avoided the use of reporter fusions carried on plasmids, as the stringent response could affect the copy number of such plasmids, potentially interfering with the measurements (22)(23)(24). We measured ppGpp and pppGpp production by the relA strains created in these studies to ensure that the levels of these nucleotides were being affected (Fig.…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa relA Contributes to Virulence in Drosophila melanogaster

Erickson¹,

et al. 2004

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The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-L-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

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Effect of increased ppGpp concentration on DNA replication of different replicons in Escherichia coli

Cited by 23 publications

References 24 publications

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

The replication checkpoint control in Bacillus subtilis: identification of a novel RTP‐binding sequence essential for the replication fork arrest after induction of the stringent response

Pseudomonas aeruginosa relA Contributes to Virulence in Drosophila melanogaster

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