A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

Abstract

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.

Figure 1

Method of construction of the single-gene deletion mutants by creation of a spliced PCR integration cassette. P1–P6 are used for integration cassette construction and P7, P8, S1 and S2 for verifications. The kan^R integration cassette is obtained by PCR amplification using P1 and P2 primers on pEVL186 DNA template. The flanking regions, specific for the target gene, are amplified on wild-type DNA template by P3/P4 primers (R1 region) and P5/P6 primers (R2 region). Designations followed by a prime (′) represent reverse complement sequences. The primers P7 and P8 are used for external PCR verification of the correct replacement of the targeted gene by the integrative cassette. The primers S1 and S2 located within the kan^R cassette are used to sequence junctions of the cassette on the P7/P8 PCR product.

Figure 2

Flow diagram of the ADP1 mutant collection.

Figure 3

Chromosomal representation of the ADP1 mutant collection. Circles display (from the outside): (1) GC percent deviation (GC window—mean GC) in a 1000-bp window. (2) CDSs in blue are classified into three biosynthesis TIGR role categories: (i) amino acid, (ii) cofactors, prosthetic groups, and carriers, and (iii) purines, pyrimidines, nucleosides, and nucleotides. (3) CDSs in red corresponds to ADP1 essential genes. (4) ADP1 CDSs in purple are sharing orthologs with E. coli K12 genes, described as essential (Keio collection, in green) or conditionally essential (mutants growing very poorly on glucose minimal media (OD<0.1)). (5) GC skew (G+C/G−C) in a 1000-bp window.

Figure 4

Analysis of orthologous gene essentiality data between ADP1 and three other bacteria (E. coli, P. aeruginosa and B. subtilis). The number of orthologous genes for each comparison and the number of genes that are essential in the four organisms, are reported as Ess(A: ADP1, E: E. coli, P: P. aeruginosa, B: B. subtilis).

Figure 5

Comparison of ADP1, E. coli and P. aeruginosa essentiality data according to TIGR role classification on (A) the medium used to obtain the mutants, (B) on minimal medium. White and blue areas correspond to nonessential and essential genes, respectively. The red area concerns the main categories notably impacted by the medium used for the mutant construction.

Figure 6

Comparison of mutants corresponding to deletion of genes responsible for the formation of the rod shape or polar differentiation and wild-type strain phenotypes observed by microscopy (× 1000). The mreBCD, pbpA and rodA genes are involved in cell-shape determination in rod-shaped bacteria, whereas the minCDE genes are involved in cellular division. The cells were grown overnight in liquid media (minimal medium complemented with succinate as carbon source).

Figure 7

Methionine biosynthesis. The first steps of methionine biosynthesis in ADP1 and E. coli. Essential genes are shown in red, whereas nonessential genes are in green.

Figure 8

Acinetobacter genomic organization of the evp homolgous gene cluster involved in a type VI protein secretion system in E. tarda. Genes in green are those homologous to E. tarda genes; genes in orange are those with a conserved domain (COG or PUF domain) with E. tarda. Genes in gray are genes with no known function annotated as conserved hypothetical proteins in Acinetobacter species.