Essentiality of ribosomal and transcription antitermination proteins analyzed by systematic gene replacement in Escherichia coli - PubMed (original) (raw)

Essentiality of ribosomal and transcription antitermination proteins analyzed by systematic gene replacement in Escherichia coli

Mikhail Bubunenko et al. J Bacteriol. 2007 Apr.

Abstract

We describe here details of the method we used to identify and distinguish essential from nonessential genes on the bacterial Escherichia coli chromosome. Three key features characterize our method: high-efficiency recombination, precise replacement of just the open reading frame of a chromosomal gene, and the presence of naturally occurring duplications within the bacterial genome. We targeted genes encoding functions critical for processes of transcription and translation. Proteins from three complexes were evaluated to determine if they were essential to the cell by deleting their individual genes. The transcription elongation Nus proteins and termination factor Rho, which are involved in rRNA antitermination, the ribosomal proteins of the small 30S ribosome subunit, and minor ribosome-associated proteins were analyzed. It was concluded that four of the five bacterial transcription antitermination proteins are essential, while all four of the minor ribosome-associated proteins examined (RMF, SRA, YfiA, and YhbH), unlike most ribosomal proteins, are dispensable. Interestingly, although most 30S ribosomal proteins were essential, the knockouts of six ribosomal protein genes, rpsF (S6), rpsI (S9), rpsM (S13), rpsO (S15), rpsQ (S17), and rpsT (S20), were viable.

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Figures

FIG. 1.

Gene disruption recombineering in E. coli. Replacement of an entire chromosomal gene ORF with an antibiotic resistance cassette orf by recombineering is shown in the upper row. If the gene is dispensable (A), the antibiotic resistance orf simply replaces the gene ORF without affecting cell survival. In case of essential gene replacement, the cells with a replaced gene can only survive if there is an additional wild-type copy of the gene, a chromosomal duplication (B) or a complementing plasmid expressing this gene in trans (C).

FIG. 2.

Flow chart of the procedure for analysis of gene essentiality. A gene is disrupted with recombineering by exactly replacing the gene ORF with an antibiotic resistance cassette orf. A group of genes can be analyzed by choosing different antibiotic resistance cassettes for disruption (see “Construction of multiple knockouts for minor ribosome-associated proteins” in the text). A COG-based prediction procedure may aid in selecting the genes for analysis. Essential versus dispensable genes are determined by their different recombination frequencies and detection of a partial duplication in the chromosomal region for a targeted gene if the gene is essential. If needed, the gene essentiality can be further tested by gene disruption in the presence of a complementing plasmid carrying the wild-type allele of this gene.

FIG. 3.

Three patterns of gene knockouts observed during analysis of gene essentiality by recombineering. A. Nonessential gene pattern characterized by a standard high recombination frequency on LB plates (left panel) and single configuration of a replaced gene as analyzed by agarose gel electrophoresis (right panel). The agarose gel shows a gene replaced with an antibiotic resistance cassette (lane 1) versus the original wild-type gene (lane 2). B. Essential gene pattern characterized by a low recombination frequency of colonies and duplicated configuration of the disrupted gene (lane 1) versus its wild-type allele (lane 2) by gel analysis. The duplication includes the gene replaced with an antibiotic resistance cassette (lane 1, upper band) and the wild-type gene (lane 1, lower band). C. Growth-impaired pattern characterized by a mixed pattern of two colony sizes with different recombination frequencies. The large colonies appear first with a low recombination frequency and a duplicated configuration (lane 1) of the essential gene. The small colonies have a high recombination frequency and a single configuration of the replaced gene (lane 2) versus its wild-type allele (lane 3).

FIG. 4.

Characterization of gene duplications with essential (nusG) and impaired (nusB) gene knockout patterns. A. Genetic mapping of nusG and nusB duplications with a set of Tn_10_ (Tcr) auxotrophic markers was done by plating the cells on LB-Tc and M63 minimal agar to select for Tcr and prototrophy, respectively. The gene markers and their position (min) in the E. coli chromosome are indicated. Positions of nusG and nusB are shown in bold. The dotted line defines the inverted chromosomal region found in W3110 (25). The duplicated regions are shown in bold. The bold dotted line indicates that duplication was not precisely mapped and may extend to the flanking area. B. Stability of nusG and nusB diploids. Every 12 h the diploid cultures were passed through LB medium. Every 24 h the number of diploids in cultures was estimated as a ratio of Cmr cells to the total number of cells in the cultures by plating them on LB-Cm and on plain LB plates, respectively. Note that because of the very different stabilities of diploids, the days of incubation of nusG and nusB diploids are plotted against the percentage or the actual number of Cmr cells in the cell cultures and are shown as a linear or a log plot, respectively.

FIG. 5.

Survival of rmf<>bla and quadruple ribosome-associated protein mutants in stationary phase. Stationary-phase cultures of rmf and quadruple knockouts, as well as of W3110, were grown at 37°C for 24 h. The mixed cultures were obtained by combining 1 ml of each of the 24-h culture knockouts together with 1 ml of W3110 and grown at 37°C for 10 days (bottom panel) along with an equal volume (2 ml) of the original monoculture (top panel). Samples were taken as indicated, and the number of Apr survivors in the total cell population was counted by plating them on LB-Ap and plain LB plates, respectively.

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Essentiality of ribosomal and transcription antitermination proteins analyzed by systematic gene replacement in Escherichia coli - PubMed (original) (raw)