TnAraOut, A transposon-based approach to identify and characterize essential bacterial genes

Judson, Nicholas; Mekalanos, John J.

doi:10.1038/77305

Cited by 156 publications

(101 citation statements)

References 30 publications

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“…The introduction of TnAraOut into V. cholerae and the screen for insertion mutants that are arabinose-dependent for growth were done as described (8) with the following modification. NJ348 was isolated by mating Tn10cm (pBSL181; ref.…”

Section: Methodsmentioning

confidence: 99%

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae , localizes in codependent fashion with FtsL to the division site

Buddelmeijer

Judson

Boyd

et al. 2002

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

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108

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YgbQ is a cell division protein in Escherichia coli and Vibrio cholerae. In E. coli the ygbQ gene was discovered as a result of a computer search of the E. coli genome designed to find potential interacting partners for cell division protein FtsL. In V. cholerae, ygbQ was identified as an essential gene by using a transposon that fuses genes to an arabinose promoter. The role of YgbQ in cell division is supported by the following. Cells depleted of YgbQ in both organisms form long filaments, but DNA segregation is not affected. YgbQ localizes to the constriction site in wild-type E. coli cells. Localization of E. coli YgbQ to the constriction site depends on cell division proteins FtsQ and FtsL but not FtsW and FtsI, placing YgbQ in the sequential dependency order of proteins localizing to the division site. Localization of green fluorescent protein-FtsL also depends on YgbQ, indicating that FtsL and YgbQ colocalize to the division site in E. coli. Our results show colocalization of proteins to the bacterial midcell in E. coli and raise the possibility that these proteins interact in a coiled-coil structure.C ell division in bacteria takes place at the midcell and occurs after the DNA has been duplicated and segregated into two daughter nucleoids. In Escherichia coli, this process requires a set of at least nine proteins that localize to the constriction site or septum. These proteins coordinate invagination of the cell membrane, inward growth of the peptidoglycan layer, and, finally, separation of daughter cells. The nine proteins, FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN, have been identified largely through genetic approaches. Because no systematic genetic approach for identifying such proteins has been performed or devised, there could be a number of thus-farundiscovered proteins that play a role in cell division. Information is available regarding function for only a few of these proteins (reviewed in refs. 1 and 2).Studies from a number of laboratories have led to a sequential dependency model for the assembly of this group of proteins at the cell septum (3). FtsZ arrives first at the cell septum, providing a scaffold for the recruitment of subsequent proteins. FtsA and ZipA localize to the septum independently of each other, but each depends on the presence of FtsZ for localization. The remaining proteins assemble at midcell in a strictly sequential dependency order as follows: FtsK-FtsQ-FtsL-FtsW-FtsIFtsN. The mechanisms for this order of recruitment are not understood.The work reported here arose from projects with different goals in the Beckwith and Mekalanos laboratories. The Beckwith laboratory has studied cell division in E. coli by focusing attention on three proteins, FtsQ, FtsL, and FtsI. These proteins have similar membrane topologies, i.e., a short amino-terminal domain facing the cytoplasm, a single transmembrane segment, and a carboxyl-terminal domain located in the periplasm. FtsL has a leucine zipper-like motif in its periplasmic domain, indicating that it may form coiled-co...

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Section: Methodsmentioning

confidence: 99%

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae , localizes in codependent fashion with FtsL to the division site

Buddelmeijer

Judson

Boyd

et al. 2002

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

Self Cite

108

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“…The large chromosome encodes the majority of recognizable ''housekeeping'' gene products involved in transcription, translation, metabolism, and cell biology, whereas the small chromosome encodes many more hypothetical gene products. Recently, the V. cholerae genome was scanned genetically for genes encoding ''essential gene products'' defined as those required for optimal growth on rich laboratory media (9). This analysis revealed that the majority of essential genes reside on the large chromosome.…”

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confidence: 99%

Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

Dziejman

Mekalanos

2003

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

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218

177

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Vibrio cholerae is the etiologic bacterial agent of cholera, a severe diarrheal disease endemic in much of the developing world. The V. cholerae genome contains 3,890 genes distributed between a large and a small chromosome. Although the large chromosome encodes the majority of recognizable gene products and virulence determinants, the small chromosome carries a disproportionate number of hypothetical genes. Thus, little is known about the role of the small chromosome in the biology of this organism or other Vibrio species. We have used the rabbit ileal loop model of V. cholerae infection to obtain in vivo-grown cells under near midexponential conditions in the small-intestinal environment. We compared the global transcriptional pattern of these in vivo-grown cells to those grown to midexponential phase in rich medium under aerobic conditions. Under both conditions, the genes showing the highest levels of expression reside primarily on the large chromosome. However, a shift occurs in vivo that results in many more small chromosomal genes being expressed during growth in the intestine. Our analysis further suggests that nutrient limitation (particularly iron) and anaerobiosis are major stresses experienced by V. cholerae during growth in the rabbit upper intestine. Finally, relative to in vitro growth, the intestinal environment significantly enhanced expression of several virulence genes, including those involved in phenotypes such as motility, chemotaxis, intestinal colonization, and toxin production.

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“…Insertions can then be screened on medium that does not turn on the transposon promoter, resulting in loss of viability of those cells that depend on the conditional transposon promoter for growth. This results in the ability to positively identify essential genes and has been used for this purpose (Judson and Mekalanos 2000b). However, the technique is hindered by the fact that few of the transposon inserts within a population will be inserted into gene promoter regions.…”

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confidence: 99%

“…However, the experimentalist is still faced with the challenge of determining the role of various putative genes. Transposition-based strategies have been developed recently for identifying essential genes (for review, see Judson and Mekalanos 2000a;Hamer et al 2001;Gerdes et al 2002). Conceptually, these methods are based on the fact that transposon insertion into a gene causes loss of gene function (gene knockout), and insertion into an essential gene is lethal to the organism and cannot be observed (Akerley et al 1998;Hare et al 2001).…”

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confidence: 99%

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

Goryshin¹,

Naumann²,

Apodaca³

et al. 2003

Genome Res.

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In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes

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TnAraOut, A transposon-based approach to identify and characterize essential bacterial genes

Cited by 156 publications

References 30 publications

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae , localizes in codependent fashion with FtsL to the division site

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae , localizes in codependent fashion with FtsL to the division site

Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

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

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