Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome.

Backman, Keith; Chen, Yumei; Magasanik, Boris

doi:10.1073/pnas.78.6.3743

Cited by 205 publications

(109 citation statements)

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“…We determined that the ΔglnA mutant did not respond to magnesium because it is a glutamine auxotroph (10). Glutamine is the scarcest amino acid in tryptone, at 0.1% (8); supplementation of TB7 with glutamine permitted the ΔglnA mutant to respond to magnesium.…”

Section: Discussionmentioning

confidence: 99%

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Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Christensen

Orr

Rao

et al. 2017

Appl Environ Microbiol

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Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth.IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.KEYWORDS acetylation, complex media, improved cell growth, magnesium, tryptone B acteria are routinely grown in complex media containing a rich assortment of nutrients. For example, lysogeny broth (LB) contains tryptone and yeast exact. Together, these components provide a rich mixture of nutrients that supports growth of numerous bacteria. However, the complexity of such media obscures factors governing cell behavior, especially those that limit cell growth.We routinely grow Escherichia coli aerobically in buffered tryptone broth (TB7) supplemented with 4 g/liter glucose (TB7-glucose) when studying protein acetylation.

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Section: Discussionmentioning

confidence: 99%

“…Of the mutants tested, only a ΔglnA mutant lost the magnesium-dependent growth yield increase ( Table 3). The glnA gene encodes glutamine synthetase, and thus the ΔglnA mutant is a glutamine auxotroph (10). We hypothesized that these cells could not respond to magnesium because glutamine now was limiting.…”

Section: Magnesium Limitation In Peptide-based Mediamentioning

confidence: 99%

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Christensen

Orr

Rao

et al. 2017

Appl Environ Microbiol

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“…The glutamine synthetase from B. fragilis BF-1, GlnN, was expressed constitutively from a pEcoR1-derived low copy number plasmid, pJS139, in an auxotrophic E. coli strain, YMC11 (glnA À , ntrB À , ntrC À , ApS) [18] as described previously [3] but without the low-nitrogen induction and CTAB steps. Following 16 h growth in Luria broth [19] containing ampicillin (100 lg/ml) at 37°C with aeration, cells were collected by centrifugation for 15 min at 7500 rpm at 4°C and resuspended in 1/50th of the original culture volume of extraction buffer (50 mM imidazole, 50 mM KCl, pH 7.1).…”

Section: Expression and Cell Lysismentioning

confidence: 99%

Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol

Rooyen¹,

Abratt²,

Belrhali³

et al. 2010

Protein Expression and Purification

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a b s t r a c tGlutamine synthetase enzymes (GSs) are large oligomeric enzymes that play a critical role in nitrogen metabolism in all forms of life. To date, no crystal structures exist for the family of large ($1 MDa) type III GS enzymes, which only share 9% sequence identity with the well characterized GSI and GSII enzymes. Here we present a novel protocol for the isolation of untagged Bacteroides fragilis GlnN expressed in an auxotrophic Escherichia coli strain. The rapid and scalable two-step protocol utilized differential precipitation by divalent cations followed by affinity chromatography to produce suitable quantities of homogenous material for structural characterization. Subsequent optimizations to the sample stability and solubility led to the discovery of conditions for the production of the first diffraction quality crystals of a type III GS enzyme.Ó 2010 Elsevier Inc. All rights reserved. IntroductionGlutamine synthetases (GSs) 1 are large oligomeric enzymes that play a central role in nitrogen assimilation, catalysing the condensation of ammonium and glutamate to form glutamine, a precursor for the synthesis of many critical bio-molecules. The ancient [1] and ubiquitous [2] GS superfamily is, therefore, evolutionarily diverse and can be divided into three main groupings, namely GSI, GSII and GSIII, on the basis of amino-acid divergence [3].GSI and GSII enzymes have been purified from numerous bacterial and eukaryotic sources, respectively, over the past five decades of biochemical and structural investigations into their functioning and regulation [4]. The first crystal structure of a GSI enzyme [5] was solved using material prepared from an adenylylation deficient Salmonella typhimurium strain using a combination of simple differential precipitation techniques and nucleotide-analogue affinity chromatography [6]. Since then, advances in recombinant DNA technology and the advent of affinity purification technologies have greatly facilitated the isolation of GS material suitable for crystallization. The first crystal structure of a GSII enzyme was of the Zea mays GS1a protein [7], which was heterologously expressed in Escherichia coli and purified using a combination of anion exchange and affinity chromatography steps [8]. More recently, several structures of GSII enzymes purified with affinity tags [9,10] have been reported.In comparison, GSIII enzymes have only been isolated and characterized from a small number of sources [11][12][13] despite the widespread occurrence of homologues in a number of evolutionarily divergent organisms [14][15][16], including, the anaerobic pathogenic protozoan Trichomonas vaginalis [17]. To date, the only structural information describing the large GSIII enzymes ($1 MDa), which share only $9% sequence identity with the GSI enzymes, is from a low-resolution cryo-EM reconstruction of the GlnN enzyme from the opportunistic human pathogen Bacteroides fragilis [3]. A detailed understanding of the functioning and evolution of these divergent enzymes in light of the known ...

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“…This was accomplished using single-stranded template obtained from a plasmid constructed by subcloning the EcoRI-Hind111 fragment from the previously described pgln6 (Backman et al, 1981) into pTZ18 obtained from Amersham. Template was isolated from E. coli strain RZ1032 harboring this construction and coinfected with M13K07 helper phage.…”

Section: Site-directed Mutagenesis and Cell Growthmentioning

confidence: 99%

“…W 158s was also expressed in pgln35 after construction by analogous methods. All mutant plasmids were transformed into the E. coli strain YMCl 1 , which contains a deletion in chromosomal glutamine synthetase (Backman et al, 1981). Wild-type protein was obtained from an overexpression system consisting of the strain YMC10 harboring the plasmid pgln35.…”

Section: Site-directed Mutagenesis and Cell Growthmentioning

confidence: 99%

Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes

Atkins

Villafranca

1992

Protein Science

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Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry30,3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the yglutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.Keywords: Escherichia coli; fluorescence studies; glutamine synthetase; transition-state complexes; tryptophan mutants Glutamine synthetase (GS) plays a central role in the nitrogen metabolism of both prokaryotes and eukaryotes, catalyzing the ATP-dependent condensation of glutamate with ammonia to form glutamine (Ginsburg, 1972;Stadtman & Ginsburg, 1974). The bacterial enzymes consist of a highly symmetrical dodecamer formed by two face-toReprint requests to: Joseph J. Villafranca, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.Abbreviations: W57L, the site-directed mutant that has Trp-57 replaced by leucine; W158S or WlSSF, the site-directed mutants that have Trp-158 replaced by serine or phenylalanine; glutamylphosphate-ADP-GS, the ternary complex formed after the addition of ATP and glutamate to glutamine synthetase.

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Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome.

Cited by 205 publications

References 22 publications

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol

Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes

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