Genome scanning in Haemophilus influenzae for identification of essential genes - PubMed (original) (raw)

Genome scanning in Haemophilus influenzae for identification of essential genes

K A Reich et al. J Bacteriol. 1999 Aug.

Abstract

We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.

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Figures

FIG. 1

FIG. 1

Features and partial restriction maps of in vitro transposition cassettes. Relevant restriction sites, positions of start and stop codons, and positions of ORFs coding for antibiotic resistance determinants are indicated. Solid bars indicate the positions of U3 termini recognized by Ty-1 transposase. (Top diagram) AT-2. (Bottom diagram) AT-Cm. The position of the AT-Cm-specific insert-anchored primer is indicated by the half arrow. DHFR, dihydrofolate reductase.

FIG. 2

FIG. 2

Southern analysis of antibiotic-resistant H. influenzae isolates. (A) Genomic Southern blot of trimethoprim-resistant colonies. (B) Genomic Southern blot of chloramphenicol-resistant colonies. Lanes: 1 to 24, 1 colony/lane; 25 to 30, three colonies/lane. (A) Lanes 1 to 31, _Eco_RI digest; 31 to 36, _Eco_RI-_Bam_HI double digest. (B) Lanes 1 to 31, _Eco_RI digest; +, positive controls for Southern hybridization with AT-2 and AT-Cm, respectively.

FIG. 3

FIG. 3

Detection of metE insert mutant by PCR and Southern analysis. A Southern blot of dilutions of metE mutant DNA with genomic DNA from a small insert library is shown. The positions of known metE insert and library mutants are shown. Genome equivalents indicate the calculated copies of PCR template in the reactions. Lanes: 1, 50 ng of metE mutant DNA and 50 ng of insert library DNA; 2, 5 ng of metE DNA and 50 ng of library DNA; 3, 0.5 ng of metE DNA and 50 ng of library DNA; 4, 50 pg of metE DNA and 50 ng of library DNA; 5, 5 pg of metE DNA and 50 ng of library DNA; 6, 0.5 pg of metE DNA and 50 ng of library DNA; 7, 50 fg of metE DNA and 50 ng of library DNA; 8, 5 fg of metE DNA and 50 ng of library DNA; 9, 0.5 fg of metE DNA and 50 ng of library DNA; 10, 50 ng of insert library DNA. The schematic shows the positions of the PCR primers relative to the metE coding region and AT-Cm insert.

FIG. 4

FIG. 4

Zero time analysis of metE insertion loss. Aliquots from growing cultures were removed at the indicated times and processed for PCR and Southern analysis (see text). Results are from minimal media with (upper panel) and without (lower panel) methionine. The optical densities at 660 nm (OD660) of bacterial cultures (right panel) for minimal media with (solid line) and without (dashed line) methionine are shown. The schematic illustrates the positions of PCR primers used in the analysis.

FIG. 5

FIG. 5

Zero time analysis of focused mutant library. (Top left panel) Ethidium-stained agarose gel of insert-anchored PCRs with primers specific for HI 992 to 994. (Top right panel) Southern analysis of gel probed with [33P]dCTP random-primed region probe. (Bottom) ORF map of chromosomal region. Arrows indicate the direction of transcription and relative sizes of ORFs. The deduced locations of inserts are indicated by the vertical bars above the ORF map for the individual time points. Size standards (1-kb ladder) are indicated.

FIG. 6

FIG. 6

Mutation exclusion analysis of HI 991 to 999. Ethidium-stained agarose gel and Southern analysis of insert-anchored PCRs with primers specific for HI 991 to 999 are shown (see text for details), as is an ORF map of the chromosomal region. Arrows indicate the direction of transcription and relative sizes of the ORFs. (A) Ethidium-stained agarose gel. (B) Southern analysis of gel probed with [33P]dCTP random-primed region probe. The positions and orientations of ORF-specific primers are shown by half arrows. The deduced locations of inserts are indicated by the vertical bars above the ORF map.

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