FEMS Microbiology Letters 244 (2005) 243–250 www.fems-microbiology.org Stress-related Pseudomonas genes involved in production of bacteriocin LlpA Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 Paulina Estrada de los Santos 1, Annabel H.A. Parret 1,2, Rene´ De Mot * Centre of Microbial and Plant Genetics, Faculty of Applied Bioscience and Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B 3001 Heverlee Leuven, Belgium Received 14 December 2004; received in revised form 27 January 2005; accepted 27 January 2005 First published online 4 February 2005 Edited by Y. Oken Abstract Pseudomonas sp. BW11M1 produces a novel type of bacteriocin that inhibits the growth of Pseudomonas putida GR12-2R3 and some phytopathogenic fluorescent Pseudomonas. A collection of mutants was screened for altered bacteriocin production pheno- types. Strongly reduced bacteriocin production was found to be caused by inactivation of the recA gene or the spoT gene. Con- versely, in a recJ mutant, the bacteriocin was constitutively overproduced. The same phenotype was observed for a mutant hit in a gene of unknown function. The predicted gene product belongs to a distinct subgroup of prokaryotic helicase-like proteins within the SWI/SNF family of regulatory proteins. One mutant that also exhibited a bacteriocin overproducer phenotype was defi- cient in the production of the peptidoglycan-associated lipoprotein OprL. This study shows that various environmental stress response pathways are involved in controlling expression of the Pseudomonas sp. BW11M1 bacteriocin. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Pseudomonas; Lectin-like bacteriocin; SOS response; Stringent response; Lipoprotein OprL; Stress regulation 1. Introduction are often larger proteins among which the colicins from Escherichia coli represent the best-known examples [4– Bacteriocins are proteins or ribosomally synthesized 7]. The structural diversity is also reflected in widely dif- peptides with antibacterial properties, in most instances ferent modes of killing including membrane disruption, only targeting related bacteria of the same species or non-specific degradation of nucleic acids, and inhibition genus [1]. Bacteriocins from Gram-positive bacteria of peptidoglycan synthesis. such as lactobacilli typically are small peptides [2,3]. As compared to the extensive work that has been Conversely, bacteriocins from Gram-negative bacteria devoted to elucidate the nature and activity of colic- ins, surprisingly little is known about bacteriocins pro- duced by Pseudomonas bacteria. Bacteriocin typing of clinical P. aeruginosa isolates substantiates the abun- * Corresponding author. Tel.: +32 16 329681; fax: +32 16 321963. dance of bacteriocins in this species but only few of E-mail address: [email protected] (R. De Mot). these have been characterized at the molecular level 1 Both authors contributed equally to this work. 2 Present address: European Molecular Biology Laboratory, Struc- [8]. The R/F-type pyocins are multi-subunit protein tural and Computational Biology Programme, Meyerhofstrasse 1, complexes thought to have evolved from phage tails 69117 Heidelberg, Germany. [9]. The S-type pyocins share several structural and 0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.01.049 244 P.E. de los Santos et al. / FEMS Microbiology Letters 244 (2005) 243–250 functional features with colicins of E. coli. To date, 2.2. DNA and cellular manipulations four S pyocins from different P. aeruginosa strains have been characterized. These bacteriocins all exhibit Restriction enzymes were used as specified by the DNase activity driving the killing process [8]. Colicins supplier (Roche Diagnostics, Vilvoorde, Belgium). and S pyocins also share a similar structural gene DNA fragments were recovered from agarose gels with organization with the cognate auto-immunity gene the QIAquick Gel Extraction kit (Qiagen Benelux, Ven- tightly linked to the gene encoding the killing protein. lo, The Netherlands). DNA ligations were performed Another common characteristic is the modular struc- using the Rapid DNA ligation kit (Roche Diagnostics). ture of the toxins with dedicated functions (receptor For electrotransformation of Pseudomonas, plasmids recognition, translocation and killing activity) being were first propagated in the non-methylating E. coli exerted by well-delineated domains. Furthermore, the strain GM2163 (Westburg, Leusden, The Netherlands). RecA-dependent SOS regulon that triggers enhanced Pseudomonas total DNA was isolated with the Pure- Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 expression of colicins is also involved in derepression Gene DNA purification kit (Gentra Systems, Minneap- of P. aeruginosa S pyocin synthesis [8]. olis, MN, USA). Plasmids were isolated using the Although, no pyocins have been functionally charac- QIAprep Spin Miniprep kit (Qiagen). Standard plate as- terized yet from Pseudomonas species other than P. aeru- says for detection of bacteriocin activity were performed ginosa, in silico analysis of genome sequence data as described previously [11]. revealed the presence of a variety of pyocin-like operons in P. fluorescens, P. putida, and P. syringae [10]. In the course of a study exploring the bacteriocinogenic poten- 2.3. Random transposon mutagenesis tial of fluorescent Pseudomonas isolates from the rhizo- sphere of different plants, we recently identified a new For the construction of a transposon mutant library type of bacteriocin in the banana rhizosphere isolate of strain BW11M1, the EZ::TN < KAN-2 > transpo- Pseudomonas sp. BW11M1, a strain of the P. putida some kit (Epicentre, Madison, WI, USA) was initially cluster [11]. This bacteriocin, LlpA (lectin-like putidacin used. Due to the low efficiency of this procedure, plaspo- A), shows remarkable similarity with a family of lectins son mutagenesis with pTnMod-OKm0 [15] was subse- mainly found in plants. Recently, we also identified and quently applied [11]. Transposase complexes and characterized two functional LlpA homologues in the plasmid pTnMod-OKm0 were introduced into Pseudo- biocontrol strain P. fluorescens Pf-5 [A. Parret, K. monas sp. BW11M1 by electroporation. Temmerman, R. De Mot, unpublished data]. The TnMod plasmid rescue procedure, used for mu- Here we report on the analysis of a mutant library of tants CMPG2064, CMPG2066 and CMPG2067, is de- the LlpA-producing strain BW11M1 that enabled iden- tailed in [11]. The transposome-mutated genomic tification of a number of genes involved in production of regions of CMPG2068, CMPG2069 and CMPG2070 this unusual bacteriocin. were isolated by inverse PCR. Total DNA was digested with restriction enzymes not cutting in the transposon, and, after ethanol purification, the digested DNA was divided in two batches. To one batch 1 U of T4 DNA 2. Materials and methods ligase and 5· ligase buffer were added. An equal amount of MilliQ water was added to the control batch. Purified 2.1. Bacterial strains, plasmids, media and growth ligation mixtures were used as template DNA in conditions PCR reactions with ThermalAce DNA polymerase (Invitrogen), using primers pseu-722 (50-ACCTA- Tryptic soy broth (TSB; Becton Dickinson, Erem- CAACAAAGCTCTCATCAACC-30) and pseu-723 bodegem, Belgium) was used for routine culturing of (50-GCAATGTAACATCAGAGATTTTGAG-30) com- Pseudomonas at 30 °C. E. coli was grown in Luria Broth plementary to the inserted cassette. PCR conditions (LB) at 37 °C. 5-bromo-4-chloro-3-indolyl-b-D-galacto- consisted of 30 cycles of annealing at 63 °C (30 s), pyranoside and isopropyl b-D-thiogalactoside polymerization at 74 °C (5 min), and denaturation at (40 lgmlÀ1) were added to detect the presence of insert 94 °C (30 s). Amplicons were cloned in pCRII-TOPO DNA cloned in pCRII-TOPO (Invitrogen, Merelbeke, and the nucleotide sequence of the transposon-flanking Belgium) in E. coli. Media were supplemented with DNA was sequenced with pseu-722 and pseu-723. ampicillin (100 lgmlÀ1), tetracycline (10 lgmlÀ1)or Nucleotide sequencing was performed using the ALFex- kanamycin (50 lgmlÀ1) when required. Plasmids were press2 automated sequencer (Amersham Biosciences introduced into E. coli by heat-shock transformation Benelux; Roosendaal, The Netherlands). Assembly [12] and into Pseudomonas by electroporation [11]. Pseu- and analysis of sequencing data were performed domonas sp. BW11M1 [13] and P. putida GR12-2R3 [14] with the Vector NTI Suite 8 software package were described previously. (Invitrogen). P.E. de los Santos et al. / FEMS Microbiology Letters 244 (2005) 243–250 245 2.4. Construction of plasmids for complementation of 2.5. Protein electrophoresis and immunodetection recA and spoT mutations For western blotting, total cellular proteins separated No CMPG2064 transformants could be obtained by SDS–PAGE were electroblotted onto polyvinylidene with E. coli Pseudomonas shuttle vectors carrying the difluoride (PVDF) membranes. Immunodetection was recA gene from P. fluorescens OE 28.3 [16]. Subse- performed according to Harlow and Lane [19] using quently, a construct was made that also contained the the OprL-specific monoclonal antibody MAI-6 (gift of downstream recX gene [17]. PCR primers were designed P. Cornelis). to anneal at positions À154 to À137 relative to the recA translational