Laboratory for Microbial Gene Technology, Department of Biotechnological Sciences, Agricultural University of Norway, PO Box 5051,N-1432 s, Norway1
MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 s, Norway2
Author for correspondence: Dzung B. Diep. Tel: +47 64 94 85 44. Fax: +47 64 94 14 65. e-mail: dzung.diep{at}ikb.nlh.no
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ABSTRACT |
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Keywords: three-component regulatory systems, temperature-sensitive bacteriocin production, peptide pheromones
Abbreviations: BU, bacteriocin unit; IF, induction factor; IU, induction unit; HPK, histidine protein kinase; RR, response regulator
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INTRODUCTION |
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To exploit their potential in various applications, these compounds need to be studied in detail. We have recently shown that bacteriocin production in both Lactobacillus plantarum C11 and Lactobacillus sakei LTH673 is an inducible process, triggered by small induction factor (IF) peptides, plantaricin A and the mature product of orfY, respectively (the latter is hereafter referred to as Spp-Ph) (Diep et al., 1995 , 1996
; Eijsink et al., 1996
; Brurberg et al., 1997
). A similar regulatory mechanism has also been reported for nisin production, where nisin itself serves as the induction factor (Kuipers et al., 1995
). The genes (plnA and orfY) encoding plantaricin A and Spp-Ph are both followed by genes encoding histidine protein kinases (HPKs) and response regulators (RRs), components of so-called signal-transducing pathways which are widely used in bacterial response to environmental changes (Stock et al., 1989
). Such a three-component (IF, HPK and RR) regulatory unit has also been noticed in the gene cluster responsible for sakacin A production (Axelsson & Holck, 1995
; Nes et al., 1996
), i.e. orf4sapKR, but the role of orf4 and its gene product has not been investigated.
In this communication we show that sakacin A production is a temperature-sensitive process, being reduced or totally abolished at elevated temperatures, and that the putative mature product of orf4 (termed Sap-Ph) possesses a regulatory role, triggering bacteriocin production as well as its own synthesis.
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METHODS |
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Bacteriocin and induction assays.
Bacteriocin production was quantified as previously described (Nissen-Meyer et al., 1993 ). Sap-Ph (sakacin A induction factor) synthesis was assayed according to Diep et al. (1995)
, with the following modifications: (i) a fully grown overnight Bac- culture of LTH1174 (to be induced), was diluted 200-fold, and (ii) induction of bacteriocin production was tested by mixing 40 µl of cell-free culture supernatant from each microtitre plate culture with 200 µl of a diluted indicator culture (40-fold dilution of a fully grown overnight culture). Bacteriocin production was considered to have been induced when growth inhibition was at least 50%. One induction unit (IU) was defined as the minimal amount of Sap-Ph that induced bacteriocin production in a microtitre plate culture as described above. Induction on plates was performed according to Diep et al. (1995)
, with the following modifications: (i) the supporting layer consisted of 1215 ml and each of the three soft-agar layers was of 3 ml, and (ii) the second layer contained approximately 2050 c.f.u. Induction of the transformant L. sakei Lb790 containing the plasmids pSAK17B and pSAK20E in liquid cultures was performed as follows. Diluted cultures of the transformant were grown at 25 °C until the OD600 was approximately 0·1 before appropriate amounts of synthetic Sap-Ph were added to induce bacteriocin production. Samples were collected at the indicated time-points and bacteriocin production in culture supernatants was assayed as described previously (Nissen-Meyer et al., 1993
).
Mutagenesis of orf4.
A mutation in orf4 was introduced by modifying the plasmid pSAK20A, encompassing a functional orf4sapKRTE cassette (Axelsson & Holck, 1995 ) by the PCR overlap extension technique (Horton & Pease, 1991
). PCRs were done as described by Axelsson & Holck (1995)
. The derivative plasmid, pSAK20E, contained a frame-shift mutation in orf4 by insertion of a G in position 2578 and replacing A at position 2583 with C, together creating a new EcoRI site (numbering according to the published sap sequence, GenBank accession number z46867; Axelsson & Holck, 1995
). pSAK20E was introduced into L. sakei Lb790, already containing the plasmid pSAK17B, as described previously (Axelsson & Holck, 1995
). Sakacin A production (seen as inhibition zones on plates) was tested qualitatively by overlaying spot colonies on plates containing no Sap-Ph or 10 ng synthetic Sap-Ph ml-1 (see below) with an antibiotic-resistant derivative of the indicator strain Lb790 (Axelsson & Holck, 1995
), or monitored quantitatively in liquid cultures as described elsewhere.
RNA isolation and Northern analysis.
Bac- and Bac+ cultures of sakacin A producers were obtained as described above. Appropriate volumes of well-grown overnight cultures were inoculated into 100 ml volumes of a prewarmed growth medium and cells were grown at the indicated temperatures for 12 h (OD600 approximately 0·1) before adding synthetic Sap-Ph at final concentration of 40 ng ml-1 to induce bacteriocin production. After induction for 4·5 h and 6·5 h, samples were collected and RNA was isolated by the method of Igo & Losick (1986) . RNA concentration was determined spectrophotometrically at 260 nm and trace amounts of DNA in RNA samples were removed by DNase treatment. Three microlitres of RNA at a concentration of either 0·1 or 1 µg µl-1 was spotted directly on a cellulose membrane (Gene Screen Plus; Dupont), which was subsequently dried for 2 h at room temperature for fixation. Hybridization was performed as described by Lillehaug et al. (1991)
, using a DNA fragment as probe specific to the genes saiA and sapA (see Fig. 2
). The DNA fragment was obtained by PCR using primers SakAP1 and SakAP2 (see below) and the correct PCR product was purified by gel electrophoresis before random 32P labelling according to the manufacturers recommendations (Random Primed DNA Labelling Kit; Boehringer Mannheim). The primers SakAP1 and SakAP2 have coordinates 664682 and 10651047, respectively, with regard to the published sequence (accession number z46867).
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RESULTS |
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Sap-Ph induces bacteriocin production
The sap locus responsible for sakacin A production in Lb706 encompasses a three-component regulatory unit (orf4sapKR) (Axelsson & Holck, 1995 ) similar to the plantaricin A and sakacin P systems (Nes et al., 1996
). orf4 encodes a peptide containing a so-called double-glycine leader (Axelsson & Holck, 1995
) and could give rise to a mature 23-amino-acid peptide (hence referred to as Sap-Ph), analogous to plantaricin A and Spp-Ph (Nes et al., 1996
), while sapKR encode an HPK and an RR, respectively (see Fig. 2a
). The amino acid sequence of Sap-Ph is shown in Fig. 2(b)
. The instability of sakacin A production, observed at the elevated temperatures, was thought to be due to an intrinsic regulation involving the three-component regulatory unit. To test this hypothesis, synthetic Sap-Ph was obtained. Using the multilayer induction plate assay, we found that addition of Sap-Ph in the third layer (40 ng ml-1) induced bacteriocin production in both Lb706 (at 33·5 °C) (Fig. 1d
) and LTH1174 (at 30 °C) (data not shown). Synthetic plantaricin A and Spp-Ph, and protease-treated Sap-Ph, did not induce bacteriocin production (data not shown), demonstrating that the induction was Sap-Ph-specific.
Genetic evidence for the role of orf4 and Sap-Ph
The sap genes can be divided into two operons, i.e. sapAsaiA and orf4sapKRTE, respectively (see Fig. 2a). These two operons complement each other when present on two separate plasmids (pSAK20A containing orf4sapKRTE and pSAK17B containing sapAsaiA) in the same strain, resulting in sakacin A production and immunity (Axelsson & Holck, 1995
). A derivative of pSAK20A, pSAK20E, differing only in an introduced frame-shift mutation in orf4, was constructed. The resulting recombinant L. sakei Lb790 containing plasmids pSAK17B and pSAK20E was sakacin A negative in a standard agar-overlay assay at 25 °C (data not shown). However, adding 10 ng ml-1 of Sap-Ph in the plates resulted in sakacin A producing colonies (data not shown). orf4 was thus essential for sakacin A production and exogenously added Sap-Ph complemented a mutation in orf4.
Coordinated production of sakacin A and Sap-Ph
The synthesis of Sap-Ph in strain Lb706 was further examined with regard to its production in relation to sakacin A production. As shown in Table 1, only cell-free supernatants derived from Bac+-conditioned cultures (grown at 30 °C) were capable of inducing bacteriocin production; those derived from Bac--conditioned cultures (grown at 33·5 °C or higher) were not. The inducer was found to be heat-stable: its activity in culture supernatants resisted boiling for at least 15 min (data not shown). The induction efficiency was also examined at the elevated temperatures. Some bacteriocin activity was observed at the elevated temperatures when cells were grown in the presence of the inducer (40 ng ml-1); however, the production appeared to be less at the highest temperature (34·5 °C) than at the lower temperatures (3033·5 °C), see Table 1
. Furthermore, when using the recombinant strain L. sakei Lb790 that contained both the plasmids pSAK17B and pSAK20E, the amounts of sakacin A produced seemed to be somewhat correlated with that of Sap-Ph added to the cultures, e.g. higher bacteriocin production was found in cultures with high amounts of Sap-Ph added (Fig. 3
).
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DISCUSSION |
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The sakacin A producers L. sakei Lb706 and L. curvatus LTH1174 contain an almost identical genetic background for bacteriocin production and regulation (Tichaczek et al., 1992 ; Axelsson & Holck, 1995
). It is therefore not very surprising that their bacteriocin production was regulated in a somewhat similar manner, i.e. both being temperature-sensitive and regulated by the same pheromone peptide. However, they also displayed some small differences in their response to various cardinal temperatures. The molecular basis of these dissimilarities is still unknown. We have noted previously that the operator regions of both the sap operons have some features in common; each operon is preceded by a pair of conserved direct repeats with a defined spacer length resembling regulatory elements see Fig. 2(c)
(Axelsson & Holck, 1995
; Diep et al., 1996
). Such regulatory-like elements are also present in the operator regions associated with the pln and spp systems and we have recently demonstrated that these direct repeats are intrinsic DNA regulatory elements on which the cognate response regulators bind and regulate gene transcription (Risøen et al., 1998
). Thus it is reasonable to assume that the two sap operons are regulated in a similar manner, i.e. the product of sapR, which encodes a response regulator, binds on the promoter-proximal repeats and activates transcription of both the sap operons. This model is also supported by the fact that synthesis of Sap-Ph was found to be coordinated with that of the bacteriocin, i.e. the inducer was found only in the Bac+-conditioned culture supernatants but not in the Bac--conditioned culture supernatants (Table 1
).
The presence of a regulatory system for sakacin A production has been somewhat of a puzzle, since production always has appeared to be constitutive (Holck et al., 1992 ; Axelsson & Holck, 1995
). In this report, we have shown that the system is necessary for production and that Sap-Ph is needed for induction. Such a three-component regulatory system involving a pheromone peptide and a cognate signal-transducing network not only seems to be frequently used in the regulation of bacteriocin production from different species (Kleerebezem et al., 1997
), but has also been found to play key roles in other biological pathways. Examples are the com and agr systems that control competence development in streptococci and the expression of virulence proteins during stationary growth phase in Staphylococcus aureus, respectively (Novick et al., 1995
; Pestova et al., 1996
). For the sakacin A system, however, the question remains whether the temperature switch has some function in vivo, and/or if the system works as a quorum-sensing device as suggested for the nisin system (Kuipers et al., 1995
; Kleerebezem et al., 1997
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 December 1999;
revised 19 April 2000;
accepted 24 May 2000.