The Activity of the Lactose Transporter from <i>Streptococcus thermophilus</i> Is Increased by Phosphorylated IIA and the Action of β-Galactosidase

Geertsma, Eric R.; Duurkens, Ria H.; Poolman, Berend

doi:10.1021/bi051638w

Cited by 20 publications

(16 citation statements)

References 39 publications

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“…All vectors used in this work were derivatives of pBADMycHisB (Invitrogen). The construction of pBADLacSC320A⌬IIA, pBADLacSC320A⌬IIA-GFP, and pBADcLIC has been described (16,17). Plasmid pBADcLIC-GFP was constructed from a 4,045-bp SwaI-XbaI fragment of pBADcLIC and an XbaI-digested PCR product (784 bp) holding an NcoI-free sequence coding for EGFP.…”

Section: Methodsmentioning

confidence: 99%

“…17. LacS⌬IIA is a truncation mutant of LacS devoid of the regulatory soluble IIA domain (16); for convenience, in the main text and figures we refer to LaSDIIA as LacS. The construction of derivatives of pBADcLIC and pBADcLIC-GFP, containing gltP, dctA, ydjN, and sstT, will be described elsewhere.…”

Section: Methodsmentioning

confidence: 99%

“…Lactose counterflow transport was assayed in E. coli MC1061/pBADLacSC320A⌬IIA as described in ref. 16. LacY.…”

Section: Methodsmentioning

confidence: 99%

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Quality control of overexpressed membrane proteins

Geertsma

Groeneveld²,

Slotboom³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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169

189

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Overexpression of membrane proteins in Escherichia coli frequently leads to the formation of aggregates or inclusion bodies, which is undesirable for most studies. Ideally, one would like to optimize the expression conditions by monitoring simultaneously and rapidly both the amounts of properly folded and aggregated membrane protein, a requirement not met by any of the currently available methods. Here, we describe a simple gel-based approach with green fluorescent protein as folding indicator to detect well folded and aggregated proteins simultaneously. The method allows for rapid screening and, importantly, pinpointing the most likely bottlenecks in protein production.folding indicator ͉ in gel GFP fluorescence ͉ optimization of overexpression ͉ inclusion bodies

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Quality control of overexpressed membrane proteins

Geertsma

Groeneveld²,

Slotboom³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

169

189

View full text Add to dashboard Cite

show abstract

“…By carrying out in vitro and in vivo complementation studies with truncated LacS lacking the EIIA Glc -like domain, it could be established that the unphosphorylated EIIA Glc -like domain does not bind to truncated LacS. In contrast, the phosphorylated EIIA Glclike domain interacted with truncated LacS and stimulated its lactose/galactose counterflow activity (63).…”

Section: Proteins Containing a Pts Component Fused To The N Or C Termmentioning

confidence: 99%

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Deutscher

Aké

Derkaoui

et al. 2014

Microbiol Mol Biol Rev

354

374

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SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

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“…3A). Such kinetics are indicative of so-called counterflow transport and suggest that BcaP is able to facilitate bidirectional transport and most likely constitutes a secondary transporter (14,63).…”

Section: Characterization Of the L Lactis Mg1363 Bcap (Ctra) Locusmentioning

confidence: 99%

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

2006

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Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

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The Activity of the Lactose Transporter from Streptococcus thermophilus Is Increased by Phosphorylated IIA and the Action of β-Galactosidase

Cited by 20 publications

References 39 publications

Quality control of overexpressed membrane proteins

Quality control of overexpressed membrane proteins

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

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