Chromosomal Deletion Formation System Based on Tn<i>5</i> Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis

Goryshin, Igor Y.; Naumann, Todd A.; Apodaca, Jennifer; Reznikoff, William S.

doi:10.1101/gr.611403

Cited by 58 publications

(45 citation statements)

References 29 publications

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“…Further studies using a YmgF-GFP fusion revealed that YmgF is a membranebound protein, although it did not localize to the E. coli septum site, at least when cells were grown in rich medium (N. Buddelmeijer, personal communication). Interestingly, ymgF is located near the minCDE region within the genome of E. coli K-12, yet ymgF seems to be nonessential, as the corresponding chromosomal DNA region can be deleted without affecting cell viability (22). At present, the role of YmgF in cell division remains elusive, but as this polypeptide interacted specifically with several Fts proteins, one may speculate that it may be involved in the fine tuning of E. coli cell division.…”

Section: Resultsmentioning

confidence: 99%

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

2005

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Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.In Escherichia coli, the cell division process, also referred to as cytokinesis, constriction, or septation, is one of the most central yet poorly understood aspects of the bacterial physiology (for reviews, see references 5, 33, and 45). The event takes place at the midcell and starts after the bacterial chromosomal DNA has been duplicated and segregated into two daughter nucleoids. Cell division genes, named fts, have been identified mainly through conditional mutants that form long filamentous cells at nonpermissive temperatures (4, 5). At present, at least fourteen proteins are known to be specifically required for the E. coli cell septation (for reviews, see references 1, 5, 33, 42, and 45). The majority of the Fts proteins are anchored to the cell membrane, and most of them appear to localize to the bacterial septum in a sequential order (for reviews, see references 5, 33, 35, and 40). Fluorescence microscopy studies using immunofluorescence or the green fluorescent protein (GFP) fused to the Fts proteins have revealed that assembly of the septum starts with the positioning of an FtsZ ring in the cell center. The FtsZ ring is stabilized by FtsA and ZipA, which localize to the septum independently of each other but only in the presence of the FtsZ protein. FtsQ follows FtsK, whose localization requires both FtsA and ZipA proteins, in this hierarchical assembly. Then FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC are successively recruited to the FtsZ ring (for reviews, see refere...

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Section: Resultsmentioning

confidence: 99%

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

2005

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“…In addition, when overproduced, YmgF could partially overcome the thermosensitivity of the ftsQ1(Ts) mutant and restore the ftsQ1 viability under lowosmolarity growth conditions. Taken together, these data suggest that YmgF, although previously shown to be not essential for cell viability (6,23,31), might be a novel component of the E. coli cell division machinery.…”

mentioning

confidence: 59%

“…We attempted to further characterize this putative inner membrane-associated protein and, particularly, to examine its potential association with E. coli cell division proteins, although prior reports indicated that the ymgF gene is not essential and could be inactivated without affecting the cell viability (6,23,31). YmgF association with the E. coli cell division machinery.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

2009

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Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.Cell division is a fundamental process in all organisms. In Escherichia coli, it requires the coordinated constriction of the three layers of the gram-negative cell envelope: the invagination of the inner membrane and the biosynthesis of septal peptidoglycan, accompanied by the invagination of the outer membrane. The inner membrane invagination is mediated by the divisome, an assembly of proteins that forms a ring-like structure at midcell. In E. coli, the divisome includes at least 10 essential proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, and FtsN) and a set of nonessential proteins (ZapA, ZapB, FtsE, FtsX, FtsP [SufI], AmiC, and EnvC) (19,34,45,55,56). The cell divisome formation starts with the polymerization of the FtsZ protein at midcell to form the so-called Z ring. This structure marks the future division site and provides a scaffold for the recruitment of the other cell division proteins (3,7,43,52,53). Numerous studies showed that the divisome proteins are sequentially recruited to the septum in E. coli. First, two FtsZ binding proteins, FtsA and ZipA, almost simultaneously localize at midcell independently of each other, presumably to stabilize the Z-ring structure. The nonessential accessory proteins ZapA and ZapB that are able to directly associate with FtsZ in vivo (27, 28, 32) may also participate in stabilizing the Z ring (51). Then, a series of integral membrane proteins are recruited to the septum in a sequential manner in the following order: FtsK, FtsQ, FtsL/ FtsB, FtsW, FtsI, and FtsN (for reviews, see references 34, 45, 54, and 56). The assembly of these proteins with the divisome is essential for the progression and completion of cytokinesis. Several nonessential prot...

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“…For Escherichia coli, the development of genomic engineering techniques utilizing bacteriophage recombinases, homologous recombination, or transposable elements has been reported recently (6,10,12,20,39). By use of such techniques, the construction of a deletion mutant whose genome size was reduced by 1.38 Mb was reported (12).…”

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confidence: 99%

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Suzuki

Nonaka

Tsuge

et al. 2005

Appl Environ Microbiol

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Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.The whole-genome sequences of more than 200 organisms have been deciphered since the first genome sequence was determined in 1995. These genome sequences have become an important resource for a more comprehensive understanding of cellular life. The availability of whole-genome sequences allows us to decipher the roles of thousands of genes. Corynebacterium glutamicum is a well-known industrial strain widely used for the production of amino acids, nucleic acids, and organic acids (18,23). It has had two strains sequenced: R (3,314,179 bp [our unpublished data]) and ATCC 13032 (3,309,401 bp [14] or 3,282,708 bp [17]). Based on whole-genome sequences, strain reconstruction studies for improved industrial applications have been initiated (28). In the field of bacterial genomics and metabolic engineering in the postgenome era, the concept of minimum genome factories (MGFs) has been proposed (16). These can be defined as recombinant strains whose metabolism has been streamlined to the optimal minimal subset in order to maximize product formation for targeted applications. C. glutamicum is one of the most widely used bacteria for bioindustry, and the improvement of its genome is important for enhanced production of biochemicals.To implement the concept of MGFs by the rearrangement of bacterial genomes, molecular biology tools that make multiple excisions and insertions possible are a prerequisite. For Escherichia coli, the development of genomic engineering techniques utilizing bacteriophage recombinases, homologous recombination, or transposable elements has been reported recently (6,10,12,20,39). By use of such techniques, the construction of a deletion mutant whose genome size was reduced by 1.38 Mb was reported (12). However, despite its industrial usefulness, similarly us...

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Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis

Cited by 58 publications

References 29 publications

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

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