Differential translocation of green fluorescent protein fused to signal sequences of<i>Ruminococcus albus</i>cellulases by the Tat and Sec pathways of<i>Escherichia coli</i>

Esbelin, Julia; Martin, Christine; Forano, Evelyne; Mosoni, Pascale

doi:10.1111/j.1574-6968.2009.01576.x

Cited by 3 publications

(3 citation statements)

References 22 publications

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“…According to the presence of the seemingly canonical Sec-signal peptides on both cell-envelope-targeted proteins and anammoxosome-targeted proteins, it is likely that the Sec translocase has a dual localization on both the anammoxosomal and the periplasmic membranes. However, recent studies have also shown that under certain conditions Sec-system exported proteins could as well be translocated by the Tat-system [69,70], and therefore we cannot exclude the possibility that the Tat system translocates more than just the predicted twin-arginine-motif-carrying substrates.…”

Section: Discussionmentioning

confidence: 99%

A predicted physicochemically distinct sub-proteome associated with the intracellular organelle of the anammox bacterium Kuenenia stuttgartiensis

et al. 2010

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BackgroundAnaerobic ammonium-oxidizing (anammox) bacteria perform a key step in global nitrogen cycling. These bacteria make use of an organelle to oxidize ammonia anaerobically to nitrogen (N2) and so contribute ~50% of the nitrogen in the atmosphere. It is currently unknown which proteins constitute the organellar proteome and how anammox bacteria are able to specifically target organellar and cell-envelope proteins to their correct final destinations. Experimental approaches are complicated by the absence of pure cultures and genetic accessibility. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" has recently been sequenced. Here, we make use of these genome data to predict the organellar sub-proteome and address the molecular basis of protein sorting in anammox bacteria.ResultsTwo training sets representing organellar (30 proteins) and cell envelope (59 proteins) proteins were constructed based on previous experimental evidence and comparative genomics. Random forest (RF) classifiers trained on these two sets could differentiate between organellar and cell envelope proteins with ~89% accuracy using 400 features consisting of frequencies of two adjacent amino acid combinations. A physicochemically distinct organellar sub-proteome containing 562 proteins was predicted with the best RF classifier. This set included almost all catabolic and respiratory factors encoded in the genome. Apparently, the cytoplasmic membrane performs no catabolic functions. We predict that the Tat-translocation system is located exclusively in the organellar membrane, whereas the Sec-translocation system is located on both the organellar and cytoplasmic membranes. Canonical signal peptides were predicted and validated experimentally, but a specific (N- or C-terminal) signal that could be used for protein targeting to the organelle remained elusive.ConclusionsA physicochemically distinct organellar sub-proteome was predicted from the genome of the anammox bacterium K. stuttgartiensis. This result provides strong in silico support for the existing experimental evidence for the existence of an organelle in this bacterium, and is an important step forward in unravelling a geochemically relevant case of cytoplasmic differentiation in bacteria. The predicted dual location of the Sec-translocation system and the apparent absence of a specific N- or C-terminal signal in the organellar proteins suggests that additional chaperones may be necessary that act on an as-yet unknown property of the targeted proteins.

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Section: Discussionmentioning

confidence: 99%

A predicted physicochemically distinct sub-proteome associated with the intracellular organelle of the anammox bacterium Kuenenia stuttgartiensis

et al. 2010

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“…The addition of multiple TorA signal sequences can enhance the effect [145]. Examples for heterologous Tat-dependent signal sequences that can be used for recombinant protein expression are those of organophosphorus hydrolase (OPH) from Flavobacterium [150], Cel9B signal sequence from Ruminococcus albus major glycoside hydrolase [151], as well as DagA from Streptomyces coelicolor agarase [140]. Examples for heterologous Tat-dependent signal sequences that can be used for recombinant protein expression are those of organophosphorus hydrolase (OPH) from Flavobacterium [150], Cel9B signal sequence from Ruminococcus albus major glycoside hydrolase [151], as well as DagA from Streptomyces coelicolor agarase [140].…”

Section: Secretion Signalsmentioning

confidence: 99%

“…Other Tat signal sequences such as CueO [96], Pac [146,147], and SufI [96,148,149] are typically used to investigate the translocation of their natural proteins. Examples for heterologous Tat-dependent signal sequences that can be used for recombinant protein expression are those of organophosphorus hydrolase (OPH) from Flavobacterium [150], Cel9B signal sequence from Ruminococcus albus major glycoside hydrolase [151], as well as DagA from Streptomyces coelicolor agarase [140].…”

Section: Secretion Signalsmentioning

confidence: 99%

Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

Mirande¹,

Mosoni²,

Béra-Maillet³

et al. 2010

Appl Microbiol Biotechnol

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A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K (m) and V (max) of 1.6 mg ml(-1) and 118 micromol min(-1) mg(-1) on oat spelt xylan, and its optimal temperature and pH for activity were 37 degrees C and pH 6.0, respectively. Its catalytic properties (k (cat)/K (m) = 3,300 ml mg(-1) min(-1)) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.

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Differential translocation of green fluorescent protein fused to signal sequences ofRuminococcus albuscellulases by the Tat and Sec pathways ofEscherichia coli

Cited by 3 publications

References 22 publications

A predicted physicochemically distinct sub-proteome associated with the intracellular organelle of the anammox bacterium Kuenenia stuttgartiensis

A predicted physicochemically distinct sub-proteome associated with the intracellular organelle of the anammox bacterium Kuenenia stuttgartiensis

Secretion of recombinant proteins from E. coli

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

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