FEMS Microbiology Letters 244 (2005) 243–250 www.fems-microbiology.org

Stress-related 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. 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 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 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 lgml1) 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 lgml1), tetracycline (10 lgml1)or Nucleotide sequencing was performed using the ALFex- kanamycin (50 lgml1) 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 154 to 137 relative to the recA translational start (pseu-719, 50-TCGACAAACGG- TAAAGGC-30) and at positions 4 to +13 relative to the predicted stop codon of recX (primer pseu-676, 50- 3. Results Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 CCAGCCTGAAGACTTAATCG-30). PCR reactions were performed on strain OE 28.3 total DNA using 3.1. Identification of Pseudomonas sp. BW11M1 mutants Platinum Pfx polymerase (Invitrogen). The 1702-bp with altered bacteriocin production fragments were cloned in pGEM-T Easy (Promega Ben- elux, Leiden, The Netherlands) for sequence verifica- The transposon mutant library of Pseudomonas sp. tion. The amplicon was subsequently cloned as a BW11M1 (some 10,500 strains) that was used previously SacI-SphI fragment in pJB3Tc20 [18], giving to isolate the structural gene of bacteriocin LlpA [11], pCMPG6028. was further screened with P. putida GR12-2R3 as an The primers pseu-774 (50-TAGAGCTCAAAAC- indicator strain in order to identify other genetic deter- AGCGCTTCCCTATTGGC-30 with underlined SacI minants of bacteriocin production. In addition, a small site) and pseu-775 (50-TATCTAGACGACATGTAC- independent library of about 250 transposome-gener- ACGGTGTTGCC-30 with underlined XbaI site) en- ated Pseudomonas sp. BW11M1 mutants was screened abled amplification of the spoT region from P. putida for strains with altered bacteriocin activity. Mutants KT2440 as a 2662-bp fragment (position 6,048,707 to with three different bacteriocin production phenotypes, 6,051,354 of the genome; GenBank Accession No. as judged from halo formation in bacteriocin plates, NC_002947). Sequence-verified amplicons were digested were identified: (i) diminished for mutants CMPG2064 with SacIandXbaI, and ligated into pJB3Tc20, yielding and CMPG2067; (ii) increased for mutants CMPG2068, plasmid pCMPG6063. CMPG2069 and CMPG2070; or (iii) abolished for

Fig. 1. Bacteriocin phenotype of Pseudomonas sp. BW11M1 wild-type and mutants with altered activity against P. putida GR12-2R3. 2-ll drops of producer cell cultures at OD600 = 0.3 were allowed to grow for 5 h at 30 C on TSB agar before application of the indicator overlay. 246 P.E. de los Santos et al. / FEMS Microbiology Letters 244 (2005) 243–250 mutant CMPG2066, similar to the previously character- Complementation of the recA mutation in ized LlpA-minus mutant CMPG2065 (Fig. 1). CMPG2064 was first attempted with recA of P. fluores- In order to identify the genes interrupted by insertion cens OE 28.3 [16] but no transformants could be ob- of the transposons, the respective flanking DNA regions tained. In P. aeruginosa, the downstream region of the were isolated and sequenced. Blast similarity searches recA gene, carrying the transcriptionally linked recX enabled the identification of the gene disrupted in each gene, is required for viability of cells when RecA is of the mutant strains. highly expressed in a recA background [22]. Recently, it was shown that RecX is a potent negative modulator 3.2. Strongly reduced bacteriocin production in recA and of RecA activity [23]. Therefore, complementation was spoT mutants also attempted with plasmid pCMPG6028, carrying the contiguous recA–recX genes of P. fluorescens OE Mutant CMPG2064 displayed a strongly reduced 28.3 [17]. Introduction of recA, along with the cognate Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 bacteriocin activity compared to wild type BW11M1 recX, restored wild-type bacteriocin production in mu- (Fig. 1). Apparently, this resulted from inactivation of tant CMPG2064 (Fig. 3). the DNA recombinase gene, recA, as sequence analysis Sequence analysis mapped the insertion in mutant of the affected BW11M1 genomic region revealed CMPG2067 to an ORF encoding the SpoT homologue 100% sequence identity of the deduced amino acid of strain BW11M1 (92% amino acid identity with P. put- sequence (partial ORF covering 268 amino acids) ida KT2440 SpoT in a 490-amino acid overlap). SpoT is with P. putida PpG2 RecA [20] and 97% identity with a key enzyme in the metabolism of the stringent P. fluorescens OE 28.3 RecA [16]. This was confirmed response alarmone ppGpp [24]. E. coli spoT mutants by phenotypic characterization showing that growth of strain CMPG2064 was strongly affected in the presence of the DNA-crosslinking agent mitomycin C (Fig. 2). This feature stems from the impaired postreplication recombinational DNA repair capacity of a recA mutant [21]. Bacteriocin production by CMPG2064, unlike the wild type [11], could not be enhanced by UV exposure of cells (data not shown).

Fig. 3. Complementation of mutations in CMPG2064, CMPG2067, Fig. 2. Sensitivity of Pseudomonas sp. BW11M1, recA mutant and CMPG2070 with plasmids carrying P. fluorescens OE 28.3 recA, P. CMPG2064, and recJ mutant CMPG2068 to mitomycin C. TSB agar putida KT2440 spoT, and P. putida KT2440 oprL, respectively. plates with mitomycin C (0.5 lg/ml) were spotted with 10-fold Bacteriocin phenotype towards P. putida GR12-2R3 was assayed with dilutions of each culture harvested at OD600 = 0.6. Plates were 2-ll drops of cell cultures at OD600 = 1.5 spotted on TSB agar and photographed after incubation for 24 h at 30 C. grown for 24 h before applying the indicator overlay. P.E. de los Santos et al. / FEMS Microbiology Letters 244 (2005) 243–250 247 display elevated ppGpp basal levels in balanced growth and slower growth rates [25]. A similar growth retarda- tion effect was observed for CMPG2067 in rich medium, which was much more pronounced than the effect seen with the recA mutant CMPG2064 (Fig. 4). Given the similarity with P. putida KT2440 SpoT, the correspond- ing gene was used to complement the deficiency of CMPG2067. Upon introduction of pCMPG6063, carry- ing spoT, bacteriocin production by the mutant was in- creased. The level attained was higher than that of the wild-type, which may be due to overexpression of plas- Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 mid-borne spoT (Fig. 3). Fig. 5. Immunodetection of OprL with monoclonal antibody MA1-6 through western blot of total cellular proteins from Pseudomonas sp. 3.3. Increased bacteriocin activity of an OprL-lacking BW11M1 (lane 1), mutant CMPG2070 (lane 2), and CMPG2070/ mutant pJB3Tc200D (lane 3). Two positive controls were included, namely P. putida KT2440 (lane 4) and P. aeruginosa PAO1 (lane 5). The positions of the marker proteins are indicated. In mutant CMPG2070, the transposon insertion was located in the oprL homologue of Pseudomonas sp. BW11M1 (identification based on 98% identity with P. CMPG2070 bacteriocin production was reduced to the putida KT2440 OprL in a 155-amino-acid overlap for level observed for wild-type cells (Fig. 3). the deduced partial amino acid). Sequence analysis of the flanking region further indicated a gene organization 3.4. Enhanced bacteriocin production by mutants with a (tolB–oprL–orf2) similar to the one previously reported deficient recJ gene or putative helicase-like regulatory for P. putida [26] and P. aeruginosa [27]. Western blot gene using a monoclonal antibody directed against P. aeru- ginosa PAO1 OprL [27] confirmed the absence of OprL In strain CMPG2068 an ORF with strong similarity in mutant strain CMPG2070 (Fig. 5). The growth rate to recJ homologues in several Pseudomonas genomes of strain CMPG2070 was comparable to the parent was hit. For the available deduced BW11M1 sequence, strain but the final cell density of the culture was some- identity (covering 176 amino acids) ranged from 92% what lower (Fig. 4). for P. putida KT2440 to 81% for P. aeruginosa. The recJ As shown in Fig. 5, immunodetection demonstrated gene encodes a 50-to-30 single strand-specific exonucle- successful expression of P. putida KT2440 oprL in ase, that participates in the RecF pathway for repair CMPG2070 transformed with pJB3Tc200D [gift of of DNA lesions and in mismatch DNA repair [29].An J.L. Ramos] carrying oprL and orf2, which constitute E. coli recJ null mutant is only slightly affected by UV an operon in P. putida [28]. In OprL-complemented irradiation due to the presence of other exonucleases with functionally redundant roles in DNA repair [30]. Compared with the strong growth-inhibitory effect of 2.0 mitomycin C on the recA mutant of Pseudomonas sp. BW11M1, this compound had much less effect on the

) 1.5 growth of CMPG2068 (Fig. 2), which suggests func- 600 tional redundancy in Pseudomonas as well. Unlike the other genes identified in this screen, the 1.0 putative gene affected in strain CMPG2069, tentatively named helX, does not have a homologue in other Pseu-

Cell density (OD 0.5 domonas strains. Based on the partial sequence available for the predicted gene product (300 deduced amino acids) it can be assigned to a distinct group of hypothet- 0.0 ical proteins with a patchy phylogenetic distribution. 0 1020304050Members are found in the genomes of only two other Time (h) strains of c-, the uropathogen E. coli Fig. 4. Growth curves of Pseudomonas sp. BW11M1 (d), and mutants CFT073 [31] and Pasteurella multocida subsp. multocida CMPG2064 (recA, h), CMPG2066 (gpsA, ¤), CMPG2067 (spoT, j), Pm70, with the E. coli sequence (C5378) giving the clos- n m s CMPG2068 (recJ, ), CMPG2069 ( helX, ), CMPG2070 (oprL, ) est match (74% identity over 301 amino acids). Other in TSB medium at 30 C. Cells from an overnight culture at representatives (with >40% amino acid identity OD600 = 1.5 were diluted 5000-fold in TSB medium. Growth was monitored in a Bioscreen C apparatus (Labsystems). Data represent to C5378) occur in specific strains of Bordetella means of 5 replicates from 2 independent experiments. bronchiseptica (b-proteobacteria), Mesorhizobium sp. 248 P.E. de los Santos et al. / FEMS Microbiology Letters 244 (2005) 243–250

(a-proteobacteria), Fusobacterium nucleatum (Fusobac- cluded that the bacteriocin phenotype of strain teria), Chlorobium tepidum (Chlorobia), the Firmicutes CMPG2069 would be the result of a polar effect on (a) Listeria monocytogenes, L. innocua and Clostridium ther- downstream gene(s). Our sequencing data indicate that mocellum, and in a Magnetococcus sp. The function of the gene organization gpsA–PA1615–PA1616 of P. aeru- none of these putative helicase-like proteins, that all ginosa PAO1, which is conserved among Pseudomonas emerged from genome sequencing, has been investi- species, is also present in strain BW11M1. The function gated. They share the Pfam domains PF00271 (Helicase of PA1615 and PA1616 is unknown. PA1615 shows C-terminal domain) and PF00176 (SNF2 family N-ter- weak similarity to GDSL-like lipases and other acyl minal domain). The latter domain occurs in eukaryotic hydrolases (Pfam accession number PF00657), whereas proteins involved in a variety of processes including PA1616 displays a phosphoglycerate mutase family sig- transcription regulation (e.g. SNF2), chromatin remod- nature (Pfam accession number: PF00300). Clearly, fur- elling, DNA repair and DNA recombination. The E. ther work is required to elucidate the possible role of the Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 coli bacterial SNF2-like protein, RapA [32], equally pos- Pseudomonas sp. BW11M1 homologues in LlpA sesses these two domains but lacks significant similarity production. to the E. coli CFT073 protein C5378 in the remainder of the sequence. In the latter strain both rapA and c5378 are present, indicating that the c5378 gene is not merely 4. Discussion the functional counterpart of rapA. Inactivation of recJ (mutant CMPG2068) or helX Given the many similarities between colicins and S (mutant CMPG2069) did not have a significant effect pyocins with respect to gene organization, domain archi- on the growth rate or final cell density of Pseudomonas tecture, amino acid sequence similarity and mode of sp. BW11M1 in rich medium (Fig. 4). action, it is not surprising that their expression is con- trolled by a common basic regulatory mechanism, the 3.5. A bacteriocin non-producing mutant not affected in RecA-dependent SOS response [8,34]. This alarm system the bacteriocin structural gene helps the cell to deal with potentially lethal stresses and is primarily activated upon exposure to DNA-damaging Unexpectedly, mutant CMPG2066, which had lost agents such as UV irradiation and DNA-modifying the ability to inhibit the growth of P. putida GR12- chemicals [35]. Our finding that LlpA production is 2R3, was not affected in the llpA structural gene, as is reduced to a basal level in a recA mutant of Pseudomonas the case for the previously described mutant sp. BW11M1 indicates that this regulatory system also CMPG2065 [11], but contained an insertion in the puta- plays a major role in controlling expression of a bacterio- tive gpsA homologue of Pseudomonas sp. BW11M1. cin that is unrelated to S pyocins or colicins. This gene assignment is based on the pronounced simi- The opposite phenotype, enhanced bacteriocin pro- larity (>80% identity) of the available deduced amino duction, was observed for a recJ mutant of strain acid sequence (154 amino acids) to GpsA proteins of BW11M1. The single strand-specific exonuclease RecJ several Pseudomonas species, including P. aeruginosa operates in the mismatch DNA repair pathway and is PAO1 GpsA (PA1614). The E. coli gpsA gene product, a component of the RecF pathway that allows cells to the ÔbiosyntheticÕ glycerol-3-phosphate dehydrogenase recover from DNA lesions blocking replication, which GpsA, generates glycerol-3-phosphate from dihydroxy- also involves RecA [29]. The different phenotypes engen- acetone phosphate for bacterial phospholipid biosynthe- dered by inactivation of recA and recJ suggest that two sis [33].InE. coli, GpsA is the sole enzyme that different, but not necessarily independent, stress-respon- produces glycerol-3-phosphate for phospholipid synthe- sive regulatory systems are involved in bacteriocin sis in cells grown on carbon sources other than glycerol expression. and, consequently, a gpsA knock-out mutation results A bacteriocin phenotype similar to that of the recJ in glycerol auxotrophy. However, the growth of mutant was also found for a mutant with another type CMPG2066 was not impaired on minimal medium sup- of DNA-interacting protein inactivated. This putative plemented with a carbon source other than glycerol, helicase-like enzyme belongs to a group of as yet such as glucose (data not shown). Also, growth kinetics uncharacterized bacterial members of the SWI/SNF of this mutant in rich medium was indistinguishable family. Although lacking substantial sequence similar- from the wild type (Fig. 4). ity, it shares some features with E. coli RapA that acts It should be noted that transposon insertion in gpsA as a transcriptional activator by stimulating RNA poly- occurred near the 30-end of the gene and may still allow merase recycling and is important for transcription un- synthesis of a functional enzyme. Since we were not yet der the stress conditions induced by high salt able to obtain the Pseudomonas BW11M1 gpsA clone concentration [36]. Recently, three helicase genes of P. for complementation analysis (which is also the case fluorescens SBW25 were shown to be upregulated upon for recJ and the helicase-like gene), it can not be ex- colonization of plant surfaces [37]. Together with our re- P.E. de los Santos et al. / FEMS Microbiology Letters 244 (2005) 243–250 249 sults on bacteriocin production, this points to a role for [2] Ennahar, S., Sashihara, T., Sonomoto, K. and Ishizaki, A. (2000) helicases in the ecology of Pseudomonas. Class IIa bacteriocins: biosynthesis, structure and activity. FEMS From the low level of bacteriocin expression detected Microbiol. Rev. 24, 85–106. [3] McAuliffe, O., Ross, R.P. and Hill, C. (2001) Lantibiotics: in a spoT mutant, it appears that production is also structure, biosynthesis and mode of action. FEMS Microbiol. influenced by another global stress response system. Rev. 25, 285–308. SpoT is a bifunctional enzyme primarily responsible [4] Braun, V., Patzer, S.I. and Hantke, K. (2002) Ton-dependent for the degradation of the alarmone ppGpp, although colicins and microcins: modular design and evolution. Biochimie it also has synthase activity under certain conditions 84, 365–380. [5] James, R., Penfold, C.N., Moore, G.R. and Kleanthous, C. (2002) [24]. The ppGpp-dependent stringent response reduces Killing of E. coli cells by E group nuclease colicins. Biochimie 84, the cellular protein synthesis capacity and controls fur- 381–389. ther global responses upon nutritional downshift. Our [6] Lazzaroni, J.-C., Dubuisson, J.-F. and Vianney, A. (2002) The data suggest that nutrient-related growth-unfavourable Tol proteins of Escherichia coli and their involvement in the Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 conditions, linked with enhanced ppGpp levels, affect translocation of group A colicins. Biochimie 84, 391–397. [7] Masaki, H. and Ogawa, T. (2002) The modes of action of colicins LlpA production. E5 and D, and related cytotoxic tRNAses. Biochimie 84, 433–438. A mutant strain lacking the peptidoglycan-associated [8] Michel-Briand, Y. and Baysse, C. (2002) The pyocins of Pseudo- lipoprotein OprL produced strongly increased levels of monas aeruginosa. Biochimie 84, 99–510. LlpA. OprL is involved in maintaining cell envelope [9] Nakayama, K., Takashima, K., Ishihara, H., Shinomiya, T., integrity in P. putida [38]. In the absence of OprL, the Kageyama, M., Kanaya, S., Ohnishi, M., Murata, T., Mori, H. and Hayashi, T. (2000) The R-type pyocin of Pseudomonas cell envelope structure is disturbed and therefore the aeruginosa is related to P2 phage, and the F-type is related to overproducer phenotype may be explained as a non-spe- lambda phage. Mol. Microbiol. 38, 213–231. cific effect due to leakiness of the cells. However, such [10] Parret, A.H.A. and De Mot, R. (2002) Bacteria killing their own overproduction may also be mediated through a consti- kind: novel bacteriocins of Pseudomonas and other c-proteobac- tutive stress response initiated as a consequence of the teria. Trends Microbiol. 10, 107–112. [11] Parret, A.H.A., Schoofs, G., Proost, P. and De Mot, R. (2003) crippled cell envelope. It was recently shown that the Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseu- RecA-dependent SOS response can be activated in E. domonas isolate. J. Bacteriol. 185, 897–908. coli cells with defective cell wall synthesis [39]. This alter- [12] Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: a native explanation is in line with the observed involve- Laboratory Manual, third ed. Cold Spring Harbor Laboratory, ment of RecA in derepression of LlpA expression Cold Spring Harbor, NY. [13] Vlassak, K., Van Holm, L., Duchateau, L., Vanderleyden, J. and following DNA damage. De Mot, R. (1992) Isolation and characterization of fluorescent Collectively, the results of this study on mutant bac- Pseudomonas associated with the roots of rice and banana grown teriocin producers point to the involvement of various in Sri Lanka. Plant Soil 145, 51–63. environmental stress regulons in controlling expression [14] Lifshitz, R., Kloepper, J.W., Kozlowski, M., Simonson, C., of the lectin-like bacteriocin LlpA by Pseudomonas. This Carlson, J., Tipping, E.M. and Zaleska, I. (1987) Growth promotion of canola (rapeseed) seedlings by a strain of Pseudo- is consistent with the idea that bacteriocins help bacte- monas putida under gnotobiotic conditions. Can. J. Microbiol. 33, riocinogenic strains to defend their environmental niche 390–395. when challenged by related competitors [40,41]. [15] Dennis, J.J. and Zylstra, G.J. (1998) Plasposons: modular self- cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl. Environ. Microbiol. 64, 2710–2715. Acknowledgments [16] De Mot, R., Laeremans, T., Schoofs, G. and Vanderleyden, J. (1993) Characterization of the recA gene from Pseudomonas P.E.d.l.S. (F/04/026) and A.P. (PDM/02/143-PDM fluorescens OE 28.3 and construction of a recA mutant. J. Gen. 03/197) acknowledge the Research Council K.U.Leuven Microbiol. 139, 49–57. for granting a postdoctoral fellowship. Part of this re- [17] De Mot, R., Schoofs, G. and Vanderleyden, J. (1994) A putative regulatory gene downstream of recA is conserved in gram- search was supported by a Grant (Onderzoeksproject negative and gram-positive bacteria. Nucleic Acids Res. 22, 1313– G.0303.04 to R.D.M.) from the Fund for Scientific Re- 1314. search (FWO-Vlaanderen). The authors thank P. Corn- [18] Blatny, J.M., Brautaset, T., Winther-Larsen, H.C., Haugan, K. elis (Flanders Interuniversity Institute of Biotechnology, and Valla, S. (1997) Construction and use of a versatile set of Belgium) for supplying OprL-specific antibodies and broad-host-range cloning and expression vectors based on the RK2 replicon. Appl. Environ. Microbiol. 63, 370–379. J.L. Ramos (CSIC, Granada, Spain) for plasmids [19] Harlow, E. and Lane, D. (1988) Antibodies: a Laboratory pJB3Tc20 and pJB3Tc200D. Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [20] Luo, J., Burns, G. and Sokatch, J.R. (1993) Construction of chromosomal recA mutants of Pseudomonas putida PpG2. Gene References 136, 263–266. [21] Courcelle, J. and Hanawalt, P.C. (2003) RecA-dependent recov- [1] Riley, M.A. and Wertz, J.E. (2002) Bacteriocin diversity: ecolog- ery of arrested DNA replication forks. Annu. Rev. Genet. 37, ical and evolutionary perspectives. Biochimie 84, 355–604. 611–646. 250 P.E. de los Santos et al. / FEMS Microbiology Letters 244 (2005) 243–250

[22] Sano, Y. (1993) Role of the recA-related gene adjacent to the recA and Blattner, F.R. (2002) Extensive mosaic structure revealed by gene in . J. Bacteriol. 175, 2451–2454. the complete genome sequence of uropathogenic Escherichia coli. [23] Drees, J.C., Lusetti, S.L. and Cox, M.M. (2004) Inhibition of Proc. Natl. Acad. Sci. USA 99, 17020–17024. RecA protein by the Escherichia coli RecX protein: modulation by [32] Sukhodolets, M.V. and Jin, D.J. (2001) RapA, a novel RNA the RecA C-terminus and filament functional state. J. Biol. Chem. polymerase-associated protein, is a bacterial homolog of SWI2/ 279, 52991–52997. SNF2. J. Biol. Chem. 273, 7018–7023. [24] Chatterji, D. and Ojha, A.K. (2001) Revisiting the stringent [33] Cronan, J.E. (2003) Bacterial membrane lipids: where do we response, ppGpp and starvation signaling. Curr. Opin. Microbiol. stand? Annu. Rev. Microbiol. 57, 203–224. 4, 160–165. [34] Riley, M.A. (1998) Molecular mechanisms of bacteriocin evolu- [25] Cashel, M., Gentry, D.R., Hernandez, V.J. and Vinella, D. (1996) tion. Annu. Rev. Genet. 32, 255–278. The stringent response In: Escherichia Coli and Salmonella. [35] Walker, G.C. (1996) The SOS response of Escherichia coli In: Cellular and Molecular Biology (Neidhardt, F.C., Ed.), pp. 1458– Escherichia Coli and Salmonella. Cellular and Molecular Biology 1496. ASM Press, Washington, DC. (Neidhardt, F.C., Ed.), pp. 1400–1416. ASM Press, Washington,

[26] Rodrı´guez-Herva, J.J. and Ramos, J.L. (1996) Characterization of DC. Downloaded from https://academic.oup.com/femsle/article/244/2/243/470295 by guest on 30 September 2021 an OprL null mutant of Pseudomonas putida. J. Bacteriol. 178, [36] Sukhodolets, M.V., Cabrera, J.E., Zhi, H. and Jin, D.J. (2001) 5836–5840. RapA, a bacterial homolog of SWI2/SNF2, stimulates RNA [27] Lim Jr., A, De Vos, D., Brauns, M., Mossialos, D., Gaballa, A., polymerase recycling in transcription. Genes Dev. 15, 3330–3341. Qing, D. and Cornelis, P. (1997) Molecular and immunological [37] Zhang, X.-X., Lilley, A.K., Bailey, M.J. and Rainey, P.B. (2004) characterization of OprL, the 18 kDa outer-membrane peptido- The indigenous Pseudomonas plasmid pQBR103 encodes plant- glycan-associated lipoprotein (PAL) of Pseudomonas aeruginosa. inducible genes, including three putative helicases. FEMS Micro- Microbiology 143, 1709–1716. biol. Ecol. 51, 9–17. [28] Llamas, M.A., Ramos, J.L. and Rodrı´guez-Herva, J.J. (2003) [38] Rodrı´guez-Herva, J.J., Ramos, M.-I. and Ramos, J.L. (1996) The Transcriptional organization of the Pseudomonas putida tol–oprL Pseudomonas putida peptidoglycan-associated outer membrane genes. J. Bacteriol. 185, 184–195. lipoprotein is involved in maintenance of the integrity of the cell [29] Yamagata, A., Kakuta, Y., Masui, R. and Fukuyama, K. (2002) envelope. J. Bacteriol. 178, 1699–1706. The crystal structure of exonuclease RecJ bound to Mn2+ ion [39] Miller, C., Thomsen, L.E., Gaggero, C., Mosseri, R., Ingmer, H. and suggests how its characteristic motifs are involved in exonuclease Cohen, S.N. (2004) SOS response induction by b-lactams and activity. Proc. Natl. Acad. Sci. USA 99, 5908–5912. bacterial defense against antibiotic lethality. Science 305, 1629–1631. [30] Viswanathan, M. and Lovett, S.T. (1998) Single-strand DNA- [40] Kirkup, B.C. and Riley, M.A. (2004) Antibiotic-mediated antag- specific exonucleases in Escherichia coli: roles in repair and onism leads to a bacterial game of rock-paper-scissors in vivo. mutation avoidance. Genetics 149, 7–16. Nature 428, 412–414. [31] Welch, R.A., Burland, V., Plunkett III, G.D., Redford, P., [41] Validova, S., Mavrodi, O., De La Fuente, L., Boronin, A., Weller, Roesch, P., Rasko, D.A., Buckles, E.L., Liou, S.-R., Boutin, A., D., Thomashow, L. and Mavrodi, D. (2005) Antagonistic activity Hackett, J., Stroud, D., Mayhew, G.F., Rose, D.J., Zhou, S., among 2,4-diacetylphloroglucinol-producing fluorescent Pseudo- Schwartz, D.C., Perna, N.T., Mobley, H.L.T., Donnenberg, M.S. monas spp. FEMS Microbiol. Lett. 242, 249–256.