The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

Li, Shyr Jiann; Cronan, John E.

doi:10.1016/s0021-9258(18)41860-1

Cited by 132 publications

(14 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6C). The conjugated streptavidin probe was again employed to control for loading and lysis and likely bound the cytoplasmic, biotinylated protein AccB in E. coli (38). Significant levels of lysis were detected in E. coli supernatants but only in strains expressing TpeE (Fig.…”

Section: Resultsmentioning

confidence: 99%

Holin-Dependent Secretion of the Large Clostridial Toxin TpeL by Clostridium perfringens

Saadat

Melville

2021

J Bacteriol

View full text Add to dashboard Cite

Large clostridial toxins (LCTs) are secreted virulence factors found in several species, including Clostridioides difficile, Clostridium perfringens, Paeniclostridium sordellii, and Clostridium novyi. LCTs are large toxins that lack a secretion signal sequence and studies by others have shown the LCTs of C. difficile, TcdA and TcdB, require a holin-like protein, TcdE, for secretion. The TcdE gene is located on the PaLoc pathogenicity locus of C. difficile and holin-encoding genes are also present in the LCT-encoded PaLocs from P. sordellii and C. perfringens. However, the holin (TpeE) associated with the C. perfringens LCT, TpeL, has no homology and a different membrane topology than TcdE. In addition, TpeE has an identical membrane topology as the TatA protein, which is the core of the twin-arginine (Tat) secretion system. To determine if TpeE was necessary and sufficient to secrete TpeL, the genes from a type C strain of C. perfringens were expressed in a type A strain of C. perfringens, HN13, and secretion was measured using western blotting methods. We found that TpeE was required for TpeL secretion and secretion was not due to cell lysis. Mutant forms of TpeE lacking an amphipathic helix and a charged C-terminal domain failed to secrete TpeL and mutations that deleted conserved LCT domains in TpeL indicated only the full length protein could be secreted. In summary, we have identified a novel family of holin-like proteins that can function, in some cases, as a system of protein secretion for proteins that need to fold in the cytoplasm. Importance: Little is known about the mechanism by which LCTs are secreted. Since LCTs are major virulence factors in clostridial pathogens, we wanted to define the mechanism by which an LCT in C. perfringens, TpeL, is secreted by a protein (TpeE) lacking homology to previously described secretion-associated holins. We discovered TpeE is a member of widely dispersed class of holin proteins, and TpeE is necessary for secretion of TpeL. TpeE bears a high degree of similarity in membrane topology to TatA proteins, which form the pore through which Tat-secretion substrates pass through the cytoplasmic membrane. Thus, the TpeE-TpeL secretion system may be a model for understanding not only holin-dependent secretion, but also how TatA proteins function in the secretion process.

show abstract

Section: Resultsmentioning

confidence: 99%

Holin-Dependent Secretion of the Large Clostridial Toxin TpeL by Clostridium perfringens

Saadat

Melville

2021

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…The first 500 amino acid residues from the N-terminus (encoded by exons 1-10), known as the "transcarboxylation domain," are responsible for the ATP-dependent fixation of CO 2 into carboxyphosphate (Lim et al 1988;Toh et al 1993). Within this domain, there are a conserved GGGGRG (amino acids 198-203) motif that is known to bind ATP (Samols et al 1988), and an ERDCSIQRR (amino acids 261-270) motif that plays a role in CO 2 fixation (Li and Cronan 1992). Exon 13, which codes for the conserved motif WGGA-TATX 5 RXLRXLXXPW (amino acids 607-624) and has 32% and 34% identity to transcarboxylase and oxaloacetate decarboxylase, respectively, is the segment responsible for holding the substrate pyruvate in place for the carboxylation reaction (Samols et al 1988).…”

Section: Discussionmentioning

confidence: 99%

Amerindian Pyruvate Carboxylase Deficiency Is Associated with Two Distinct Missense Mutations

Carbone

MacKay

Ling

et al. 1998

The American Journal of Human Genetics

View full text Add to dashboard Cite

We characterized the pyruvate carboxylase (PC) gene by PCR amplification, subcloning, and sequencing. The coding region has 19 exons and 18 introns spanning approximately 16 kb of genomic DNA. Screening both the cDNA and the gene of individuals with the simple A form of PC deficiency revealed an 1828G-->A missense mutation in 11 Ojibwa and 2 Cree patients and a 2229G-->T transversion mutation in 2 brothers of Micmac origin. Carrier frequency may be as high as 1/10 in some groupings. The two point mutations are located in a region of homology conserved among yeast, rat, and human PC, in the vicinity of the carboxylation domain of the enzyme. These data provide the first characterization of the human PC gene structure, the identification of common pathogenic mutations, and the demonstration of a founder effect in the Ojibwa and Cree patients.

show abstract

“…The initiation of DNA replication relies on the initiator protein DnaA binding the origin of replication, oriC . − Therefore, we designed sgRNAs to target either the transcription of dnaA , abrogating the crucial function of replication initiation, or oriC , allowing dCas9 to physically occlude DnaA from binding to this region. Additionally, we also designed a sgRNA to target the essential gene accA , which encodes a subunit of the acetyl-CoA carboxylase in the fatty acid synthetic pathway . For dnaA and accA repression, we designed sgRNAs targeting the nontemplate strand, as this is typically more effective than targeting the template strand .…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, we also designed a sgRNA to target the essential gene accA, which encodes a subunit of the acetyl-CoA carboxylase in the fatty acid synthetic pathway. 34 For dnaA and accA repression, we designed sgRNAs targeting the nontemplate strand, as this is typically more effective than targeting the template strand. 20 For oriC, we designed sgRNAs that could bind to either strand, as the replication from oriC is bidirectional and this permits the testing of more potential sgRNA options.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Dynamic Cell Programming with Quorum Sensing-Controlled CRISPRi Circuit

et al. 2020

View full text Add to dashboard Cite

Synthetic biology is enabling rapid advances in the areas of biomanufacturing and live therapeutics. Dynamic circuits that can be used to regulate cellular resources and microbial community behavior represent a defining focus of synthetic biology, and have attracted tremendous interest. However, the existing dynamic circuits are mostly gene editing-dependent or cell lysis-based, which limits their broad and convenient application, and in some cases, such lysis-based circuits can suffer from genetic instability due to evolution. There is limited research in quorum sensing-assisted CRISPRi, which can function in a gene editing-independent manner. Here, we constructed a series of quorum sensing controlled CRISPRi systems (Q-CRISPRi), which can dynamically program bacteria by using customized sgRNA without introducing cell lysis. We successfully applied Q-CRISPRi circuits to dynamically program gene expression, population density, phenotype, physical property, and community composition of microbial consortia. The strategies reported here represent methods for dynamic cell programming and could be effective in programming industrially and medically important microorganisms to offer better control of their metabolism and behavior.

show abstract

The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

Cited by 132 publications

References 26 publications

Holin-Dependent Secretion of the Large Clostridial Toxin TpeL by Clostridium perfringens

Holin-Dependent Secretion of the Large Clostridial Toxin TpeL by Clostridium perfringens

Amerindian Pyruvate Carboxylase Deficiency Is Associated with Two Distinct Missense Mutations

Dynamic Cell Programming with Quorum Sensing-Controlled CRISPRi Circuit

Contact Info

Product

Resources

About