Combined physical and genetic map of the Pseudomonas putida KT2440 chromosome

Abstract

A combined physical and genetic map of the Pseudomonas putida KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization. Circular genome size was estimated at 6.0 Mb by adding the sizes of 19 SwaI, 9 PmeI, 6 PacI, and 6 I-CeuI fragments. A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome with PmeI, SwaI, I-CeuI, and PacI as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I and Escherichia coli, as P. putida DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of the P. putida genome eluted from agarose gels. As an alternative, 10 random mini-Tn5-Km mutants of P. putida KT2440 were used as a source of DNA, and the band carrying the mini-Tn5 in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn5 as a probe. We established a circular genome map with an average resolution of 160 kb. Among the 63 genes located on the genetic map were key markers such as oriC, 6 rrn loci (rnnA to -F), recA, ftsZ, rpoS, rpoD, rpoN, and gyrB; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds. The genetic map of P. putida KT2440 was compared to those of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens SBW25. The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.

FIG. 1

CHEF electrophoresis of fragments of P. putida KT2440 genomic DNA predigested with different restriction enzymes. All gels were 1.2% (wt/vol) agarose and were run in 0.5× TBE buffer with the exception of gel A, which was a 0.8% (wt/vol) agarose gel run in 1× TBE buffer. (A) Running conditions: 50 V for 216 h; pulse times, 1,000 s for 24 h, followed by 1,000- to -4,000-s linearly ramped pulse for 192 h. Running conditions: (B and C) 150 V for 70 h; pulse times, 200 s for 24 h, 120 s for 24 h, and 80 s for 22 h. In gel C, partial I-CeuI fragments are indicated by arrows. (D and E) Running conditions: 145 V for 66 h; pulse times, 120 s for 24 h, 70 s for 22 h, and 60 s for 20 h. (F) Running conditions: 100 V for 72 h; 5- to 100-s linearly ramped pulse. (G) Running conditions: 450 V for 4 h, pulse times, 0.5 s. Lanes: S, SwaI; P, PmeI; Pa, PacI; C, I-CeuI; Sp, SpeI; 6, SwaI plus PmeI; 7, PacI plus PmeI; 8, SwaI plus PacI; 9, I-CeuI plus PmeI. DNA size markers were phage λ DNA concatemers (lane 1), intact phage λ DNA plus phage λ DNA digested with HindIII (lanes 2), and chromosomes of S. cerevisiae (lanes 3) and H. wingei (lanes 4). Lane 5 contains total P. putida DOT-OX3 DNA digested with SwaI. Sizes are indicated in kilobases.

FIG. 2

Identification of genomic fragments of P. putida KT2440 smaller than 15 kb after digestion with SwaI, PmeI, and SwaI plus PmeI and ³²P end labeling. Total DNA of P. putida KT2440 genomic DNA was digested with SwaI (lane S), PmeI (lane P), and SwaI plus PmeI (lane S/P) and then end labeled with ³²P as described in Materials and Methods. Fragments were separated in a conventional 0.8% (wt/vol) agarose gel run in 1× TAE buffer for 3 h at 5 V cm⁻¹. The gel was exposed to Kodak photographic film and developed. Size fragments are indicated in kilobases. Bands of interest and their sizes are indicated by arrows.

FIG. 3

Locations of SwaI restriction sites within PmeI fragments. (A) Separation of P. putida KT2440 PmeI fragments in the first electrophoresis; (B) separation of SwaI fragments generated upon digestion of different PmeI fragments. In gel A, two different lanes of total DNA digested with PmeI were run side by side. After ethidium bromide staining of one of them (A), the agarose plugs containing the PmeI fragment of interest were excised and digested with SwaI. Finally DNA fragments were separated in the second electrophoresis (B). Lanes: 1, phage lambda DNA undigested and digested with HindIII; 9, phage lambda DNA concatemers: S, P. putida KT2440 genomic DNA digested with SwaI. Lanes 2, 3, 4, 5, 6, 7, and 8 correspond to fragments PmeI-A, PmeI-B, PmeI-C, PmeI-D, PmeI-E, PmeI-F, and PmeI-G, respectively, digested with SwaI. Sizes are indicated in kilobases.

FIG. 4

Hybridization analysis with several gene probes. P. putida KT2440 DNA was digested with SwaI (lanes S), PmeI (lanes P), and SwaI plus PmeI (lanes 1), and DNA fragments were separated by CHEF electrophoresis. The three lanes on the left were separated from the two other lanes, and each set of DNA was transferred to a nylon membrane. In panel B, the three lanes on the left correspond to hybridization with the npt gene probe, while the other two lanes correspond to hybridization with the dnaJ-dapB-carAB set of genes. Sizes are indicated in kilobases.

FIG. 5

Hybridization analysis with small chromosomal fragments. Fragment SwaI-J was extracted from a gel and used as a probe against P. putida KT2440 DNA digested with SwaI (lane S), PmeI (lane P), or SwaI plus PmeI (lane 1).

FIG. 6

Hybridization analysis with the rrs gene as a probe. P. putida KT2440 genomic DNA was digested with SwaI (lanes S), PmeI (lanes P), I-CeuI (lanes C), SwaI plus PmeI (lanes 1), or I-CeuI plus PmeI (lanes 2). The sizes (in kilobases) indicated on the right correspond to S. cerevisiae chromosomes used as a size marker.

FIG. 7

Physical and genetic map of P. putida KT2440. The circular chromosome is represented as a series of overlapping fragments for the enzymes SwaI (Sw), PmeI (Pm), Pac (Pa), and I-CeuI (Ce). Genetic loci were assigned to restriction fragments by Southern hybridization analysis; groups of markers that hybridize at the same restriction fragments are underlined. The exact positions of the six rDNA operons are indicated.

FIG. 8

Comparison of the maps of P. fluorescens SBW25, P. putida KT2440, and P. aeruginosa PAO1. To facilitate comparisons, the circular maps were opened at an arbitrarily chosen point and positioned with respect to the origin of replication. Only the genes found in both P. putida and one of the other bacteria are shown, connected by a dashed line. The position of each gene is expressed as a ratio of the position of that gene relative to oriC.