Effects of FIS Overexpression on Cell Growth, rRNA Synthesis, and Ribosome Content in <i>Escherichia</i><i>coli</i>

Richins, Richard D.; Chen, Wilfred

doi:10.1021/bp000170f

Cited by 10 publications

(8 citation statements)

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“…It can be speculated that the cell is diverting its resources to ext-a1 expression perturbed rRNA and ribosomal protein synthesis. A correlation between an increase in rRNA synthesis and a decrease in cellular growth has been previously reported in the literature (10). In this work, ext-a1 expression is observed to result in reduced final OD and cell viability.…”

Section: Vol 193 2011supporting

confidence: 82%

“…Wild-type E. coli W3110 cells transformed with plasmids pklr1 and pKQV4 (designated W3110 pklr1 cells) were used for this study. Plasmid pklr1 derives its identity from pKFis4 and consists of RhsA components ext-a1 and a partial sequence of dsORF-a1, under the control of an IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible tac promoter, and a kanamycin (Km) resistance gene (10). This plasmid contains the entire sequence of ext-a1 and the first 341 nucleotides of dsORF-a1, corresponding to nucleotides 4611 to 5446 in GenBank accession no.…”

mentioning

confidence: 99%

“…The dsORF-a1 sequence is followed by the constitutive transcription terminators present in plasmid pklr1. Plasmid pKQV4 carries lacI q and encodes ampicillin (Amp) resistance (10). Plasmids pklr1 and pKQV4 were obtained from Wilfred Chen (University of California, Riverside, CA).…”

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confidence: 99%

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Overexpression of Cloned RhsA Sequences Perturbs the Cellular Translational Machinery in Escherichia coli

Aggarwal

Lee

2011

J Bacteriol

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RhsA is a member of the multigene Rhs family and consists of a complex genetic sequence. This sequence consists of several distinct components, including a GC-rich core (core open reading frame [ORF]), an AT-rich extension (ext-a1) of the core ORF and an AT-rich region following the core extension (dsORF-a1). The functions of RhsA and the different distinct components, which can include open reading frames, are not well understood. Here, we study the effect of overexpression of the ext-a1 sequence and the ext-a1 3 region, which includes a partial sequence of dsORF-a1, on Escherichia coli cells. Cells expressing these sequences show reduced cell growth and cell viability. The expression of these sequences dramatically affects different components of the transcription and translation machinery. Transcriptomic analysis reveals an increase in the expression of genes involved in transcription, RNA processing, and nucleotide biosynthesis and metabolism and a decrease in the expression of amino acid biosynthesis genes and transfer RNAs. Further, expression of the above-mentioned RhsA components increases ribosomal gene expression, as well as rRNA and ribosome abundance. Proteomic analysis reveals an overall reduction of protein expression at the genome-wide level in cells expressing the above-mentioned RhsA components. Based on these observations, we suspect a translation product of ext-a1 affects different regulatory mechanisms that control rRNA synthesis.The Rhs family in Escherichia coli consists of five genetic elements. These elements are found in many, but not all, natural E. coli strains (4, 7, 11, 16). The Rhs (or rearrangement hot spot) elements were originally detected as components of recAdependent intrachromosomal recombination (8). Subsequent studies have defined the Rhs loci on the chromosome and elucidated the evolutionary history of these elements (4,6,7,19). Members of the Rhs family share one or more discrete homologous structures, one of which, a 3.7-kb GC-rich core, is common to all the elements. The Rhs family also contains unique sequences specific to each element, such as dissimilar sequences that flank the core of Rhs elements. Certain components of the Rhs elements also have a distinct GϩC content (approximately 40% or 60%) compared to the rest of the E. coli genome (approximately 50%), suggesting their origin is outside the E. coli species (4). While the function of Rhs elements is not fully understood, the amino acid sequence suggests that the elements encode hydrophilic proteins with repetitive sequence elements and divergent C termini (7).RhsA, a member of the Rhs family, is 8.2 kb long and consists of some distinct sequence features. The RhsA sequence can include five different open reading frames (ORFs). The 3.7-kb GC-rich core includes a long core ORF that extends 417 bases into a 1.3-kb AT-rich region. This extension is designated exta1. The 1.3-kb AT-rich region also contains dsORF-a1, an 840-bp ORF. Both ext-a1 and dsORF-a1 contain a high number of infrequently or rarely used codons; ...

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Section: Vol 193 2011supporting

confidence: 82%

mentioning

confidence: 99%

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Overexpression of Cloned RhsA Sequences Perturbs the Cellular Translational Machinery in Escherichia coli

Aggarwal

Lee

2011

J Bacteriol

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“…Plasmid pklr1 was derived from plasmid pkFis4 [20] by substitution of the fis gene with rhsA elements, ORF-ex and dsORF-a1, and is under the control of an isopropyl-b-D-thiogalactopyranoside (IPTG) inducible tac promoter, and contains the gene for kanamycin (Km) resistance. Plasmid pKQV4 encodes lacI q and ampicillin (Amp) resistance.…”

Section: Strains and Vectorsmentioning

confidence: 99%

A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging inEscherichia coli cells

Choe¹,

Aggarwal²,

Franck³

et al. 2005

Electrophoresis

109

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A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cellsAn important consideration in the measurement of quantitative changes in protein expression is the consistency of the observations for a given technique as well as the reproducibility of the experiment. A quantitative assessment of the technical and biological variability is crucial to avoid erroneous inferences and conclusions. Two methods for measuring quantitative changes in protein expression are two-dimensional electrophoresis (2-DE) and shotgun proteomics of isobaric-tagged samples using iTRAQ  reagents. An assessment of changes in Escherichia coli protein expression in response to rhsA induction demonstrates that half of the quantified protein expression ratios have a coefficent of variation (CV) less than 0.31 using 2-DE and less than 0.24 using isobaric tags; whereas 95% of the quantified protein expression ratios have a CV less than 0.81 using 2-DE and less than 0.53 using isobaric tags. The selective removal of outlier data points from the shotgun method using Grubb's and Rosner's statistical outlier tests improves the consistency of the quantitation data obtained.

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“…The rrnB P1P2 promoter is sensitive to the concentrations of two small molecules, the initiating NTP (iNTP) and guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) . FIS is a small nucleoid‐associated protein involved in many cellular processes, including the activation of rRNA synthesis, and the expression of FIS has certain physiological effects on E. coli , suggesting that FIS plays a global role in the adaption of E. coli to changing environmental and nutritional conditions . For rrnB P1 promoter most of the activation by FIS is due to the three FIS‐binding sites located upstream and FIS acts as a transcription factor that trans activates the rrnB promoter (Figure ).…”

Section: Introductionmentioning

confidence: 99%

Design of growth‐dependent biosensors based on destabilized GFP for the detection of physiological behavior of Escherichia coli in heterogeneous bioreactors

Han

Delvigne

Brognaux

et al. 2013

Biotechnology Progress

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In this work, we present the design and characterization of Green Fluorescent Protein (GFP)-based reporter systems designed to describe cellular activity in "complex," heterogeneous bioreactors. The reporter systems consist of Escherichia coli strains carrying growth dependent promoters fused to genes expressing stable and unstable variants of GFP, respectively. The response of Escherichia coli cells to transient exposure to glucose was studied in a two-compartment scale down bioreactor (SDR) consisting of a well-stirred tank reactor (STR) connected to a plug-flow reactor (PFR). Such a SDR system is employed to mimic the situation of high glucose concentration and oxygen limitation that often encountered in large-scale, fed-batch bioreactors and the response of E. coli was simulated by continuously pumping microbial cells from STR to the PFR. We found that repeated addition of concentrated glucose pulses with varied frequency at the entrance of the PFR had consequences on strain physiological behavior. The GFP expressions were significantly marked after 10 h of cultivation in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat with programmed glucose perturbation suggested that the activities of the promoters were linked with the substrate limitation signal. Taken together with immunoblot analysis, we suppose protein leakage is responsible for the overexpression of fis and the related promoters, such as rrnB in this case study, but additional works are required in order to confirm this relationship. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large-scale bioreactors.

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Effects of FIS Overexpression on Cell Growth, rRNA Synthesis, and Ribosome Content in Escherichiacoli

Cited by 10 publications

References 32 publications

Overexpression of Cloned RhsA Sequences Perturbs the Cellular Translational Machinery in Escherichia coli

Overexpression of Cloned RhsA Sequences Perturbs the Cellular Translational Machinery in Escherichia coli

A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging inEscherichia coli cells

Design of growth‐dependent biosensors based on destabilized GFP for the detection of physiological behavior of Escherichia coli in heterogeneous bioreactors

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