Polynucleotide phosphorylase-deficient mutants of Pseudomonas putida

Abstract

In bacteria, polynucleotide phosphorylase (PNPase) is one of the main exonucleolytic activities involved in RNA turnover and is widely conserved. In spite of this, PNPase does not seem to be essential for growth if the organisms are not subjected to special conditions, such as low temperature. We identified the PNPase-encoding gene (pnp) of Pseudomonas putida and constructed deletion mutants that did not exhibit cold sensitivity. In addition, we found that the transcription pattern of pnp upon cold shock in P. putida was markedly different from that in Escherichia coli. It thus appears that pnp expression control and the physiological roles in the cold may be different in different bacterial species.

FIG. 1.

(A) Map of P. putida TMB rpsO-pnp region. The E. coli K-12 region is included at the top for comparison. Relevant restriction sites and cloned regions are shown; the corresponding plasmids (designations on the right) are described in Table 1. Promoters and intrinsic terminators are indicated by bent arrows and lollipops, respectively (dashed lines indicate that such features are putative). The terminator downstream of pnp is outside the sequence established in this work and was inferred from the complete genomic sequence of P. putida KT2442 (accession number NC_002947). Scissors indicate the RNase III cut site in the E. coli mRNA. Primers (Table 2) are indicated by arrowheads flanked by the oligonucleotide numbers and delimit the regions amplified by PCR and cloned. The triangle in pMA9 and pKRF7A indicates the Ω-Km cassette cloned into the HincII site. The fragment cloned in pURF10 was obtained by PCR of P. putida TMB genomic DNA with degenerate primers FG143 and FG144 (Table 2) designed with two amino acid motifs (PK/GRREIGHG and KAA/PVAGIAMG) conserved in the PNPases of E. coli (SwissProt accession number P05055), Photorhabdus luminescens (P41121), Haemophilus influenzae (P44584), B. subtilis (P50849), and Streptomyces antibioticus (Q53597). (B) Mutagenesis of P. putida pnp gene. An internal fragment of pnp (regions 2 and 3) interrupted by a kanamycin resistance cassette and cloned in the suicide plasmid pKNG101 (giving pKRF7A) recombined with homologous region 3 of the chromosomal gene, producing a merodiploid with two mutant pnp alleles (pnp-1 mutation in PPM101 and PPM103).

FIG. 2.

(A) Western blot analysis of PNPase in wild-type and mutant P. putida strains. Cultures of E. coli and P. putida strains KT2440 (KT), PPM103 (103), TMB, and PPM101 (101) were grown in LD broth at 30°C (P. putida strains) or 37°C (E. coli) to an optical density at 600 nm of 0.4 and analyzed by Western blotting by using polyclonal antibodies raised against E. coli PNPase (a generous gift from A. J. Carpousis). Equal portions (1.5 μg) of proteins were loaded in all lanes. Immunoreactive bands were revealed by using the Amersham ECL Western blotting reagent. (B) Expression of pnp in different culture growth phases at 30°C. pnp mRNA and PNPase from samples of P. putida TMB and KT2440 cultures in different growth phases were analyzed by Northern and Western blotting, respectively, as described previously (43). The size of the mRNA is about 2.2 kb. (C) Growth curves. Solid symbols indicate the times when the samples analyzed in panel B were taken. OD₆₀₀, optical density at 600 nm.

FIG. 3.

Growth of P. putida pnp mutants at low temperatures. (A) Overnight cultures (grown in LD broth at 30°C with aeration) of the wild-type and pnp mutant strains indicated on the left were serially diluted (10⁰ to 10⁻⁴) in Bertani tray wells, replica plated onto five LD agar plates, and incubated at different temperatures and for different times, as indicated below the images, until visible colonies were observed. (B) The same strains were grown in LD broth at 30°C to an optical density at 600 nm (OD₆₀₀) of about 0.5 and then shifted to 9°C (time zero). The optical density at 600 nm is plotted versus time in hours before the temperature downshift (left graph) or in days after the temperature downshift (right graph). Symbols: □, TMB; ▪, PPM101; ▵, KT2440; ▴, PPM103.

FIG. 4.

Expression profile of pnp and rpsO upon cold shock. Cultures of KT2440 (pnp⁺), PPM103 (pnp), and PPM103 containing pJB3Pnp2 (a plasmid expressing P. putida pnp from the plac promoter) were grown in LD broth at 30°C with aeration to an optical density at 600 nm of 0.4, quickly cooled to 9°C in an ice-water bath, and transferred to 9°C in a refrigerated water bath with aeration. Aliquots were obtained immediately before the temperature downshift (zero time) and at different times after the temperature downshift (indicated in hours above the lanes); RNA was extracted as described previously (12) and processed as follows. (A) Northern blot analysis of pnp transcripts. Fifteen micrograms of RNA was resolved by 1.5% agarose gel electrophoresis and analyzed by Northern blot hybridization with the radiolabeled riboprobe pnp transcribed with T7 polymerase from pGERF1, as previously described (7, 12). The approximate sizes of the RNAs (in kilobases) are indicated on the left. (B) Expression of PNPase upon cold shock. Western blotting with PNPase polyclonal antibodies was performed by using 1-μg portions of total P. putida protein extracts. Lane 103 contained a sample of PPM103 (pnp) extract. (C) Primer extension of pnp transcripts. Oligonucleotide FG243 labeled at the 5′ end with ³²P was hybridized to 10 μg of total RNA, extended with reverse transcriptase, and electrophoresed in a 6% polyacrylamide sequencing gel. The sequencing reactions (lanes A, C, G, and T) were performed by using the FG243 primer and plasmid pRF17 as a template. The coordinates of the 5′ end signals are indicated on the right. (D) Northern blot analysis of rpsO transcripts. RNA was resolved by 5% acrylamide denaturing gel electrophoresis and analyzed by Northern blot hybridization with radiolabeled oligonucleotide FG432.