Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015, Lausanne, Switzerland1
Author for correspondence: Dieter Haas. Tel: +41 21 692 56 31. Fax: +41 21 692 56 35. e-mail: Dieter.Haas{at}lbm.unil.ch
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ABSTRACT |
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Keywords: Pseudomonas fluorescens, hydrogen cyanide, ANR, GacA, iron regulation
a Present address: Institut für Mikrobiologie und Weinforschung, Universität Mainz, D-55099 Mainz, Germany.
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INTRODUCTION |
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HCN synthase of P. fluorescens CHA0 is encoded by the hcnABC gene cluster (Laville et al., 1998 ). Two regulatory proteins have been demonstrated to control cyanogenesis in this bacterium: the global activator GacA and the anaerobic regulator ANR (Laville et al., 1992
, 1998
). The same regulatory elements are also involved in HCN formation in P. aeruginosa (Reimmann et al., 1997
; Zimmermann et al., 1991
). GacA, the response regulator of the two-component regulatory system GacS (formerly LemA)/GacA, is required not only for the synthesis of HCN, but also for that of other secondary metabolites and exoenzymes in strain CHA0 (Laville et al., 1992
; Sacherer et al., 1994
). GacA activates a regulatory network in which the RNA-binding protein RsmA is one of the downstream elements. The region of translation initiation of the hcnA gene is important for regulation by GacA and RsmA (Blumer et al., 1999
; Blumer & Haas, 2000
). The GacS/GacA system is conserved in many Gram-negative bacteria. In plant-beneficial strains of P. fluorescens and Pseudomonas aureofaciens, this system regulates biocontrol activity (Laville et al., 1992
; Gaffney et al., 1994
; Chancey et al., 1999
). In plant- and animal-pathogenic species of Pseudomonas, Erwinia, Vibrio and Salmonella, GacA function is required for virulence (Rich et al., 1994
; Ericksson et al., 1998
; Wong et al., 1998
; Rakeman & Miller, 1999
; Kinscherf & Willis, 1999
). The signals interacting with the GacS sensor are unknown.
Mutants of both P. fluorescens and P. aeruginosa lacking the anaerobic regulator ANR, an FNR homologue, are defective in HCN biosynthesis (Laville et al., 1998 ; Zimmermann et al., 1991
). FNR of Escherichia coli is a dimeric transcriptional activator which, by virtue of two bound [4Fe4S]2+ clusters, senses intracellular oxygen concentrations (Kiley & Beinert, 1999
). Below a threshold concentration (about 10 µM) of dissolved oxygen, FNR can activate or repress transcription of target genes by binding to a conserved sequence known as the FNR box (Spiro, 1994
; Rhodius & Busby, 1998
). The ANR protein of P. fluorescens CHA0 is structurally and functionally very similar to FNR (Laville et al., 1998
; Højberg et al., 1999
). The transcription start site of the hcnABC gene cluster of P. fluorescens has been mapped (Fig. 1
). The hcn promoter contains a typical FNR/ANR box in the -40 region (Laville et al., 1998
). Moreover, a translational hcnA''lacZ fusion is strongly induced under oxygen-limited conditions in the wild-type, whereas little expression occurs in an anr mutant (Laville et al., 1998
).
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METHODS |
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DNA manipulation.
Small-scale preparations of plasmid DNA were carried out by the CTAB (cetyltrimethylammonium bromide) method (Del Sal et al., 1988 ) for E. coli and by the alkaline lysis method (Sambrook et al., 1989
) for P. fluorescens. Large-scale preparation of plasmid DNA was performed using Qiagen-tip 100 columns. Transformation of E. coli and P. fluorescens strains was done by the standard CaCl2 procedure (Sambrook et al., 1989
) or by electroporation (Farinha & Kropinski, 1990
). DNA cloning experiments were carried out according to standard methods (Sambrook et al., 1989
). DNA fragments were generated by PCR as described previously (Laville et al., 1998
). All PCR and linker constructs were verified by nucleotide sequencing using the chain termination method with the Sequenase 2.0 kit (US Biochemical) and T7 polymerase (Pharmacia) following the manufacturers instructions.
Construction of plasmids.
Recombinant plasmids were constructed in the broad-host-range vector pME6010 (Heeb et al., 2000 ). The translational hcnA''lacZ fusion in pME3219 has been described previously (Laville et al., 1998
). Plasmids pME3223 and pME3225 were obtained by PCR-amplifying different parts of the hcn promoter region, using internal EcoRI-tagged primers which anneal to different sequences in the 5' region of the hcn promoter and an external primer (primer 3) which anneals within the lacZ sequence (Table 1
). PCR products were generated from pME3219 as the template, restricted with EcoRI and PstI and cloned into pME3219 previously cut with the same enzymes. The two-base substitution in the -10 region of the hcn promoter in plasmid pME3226 (Fig. 2
) was generated by the overlap extension method (Mikaelian & Sergeant, 1996
), using as flanking oligonucleotides primers 1, 2 (Laville et al., 1998
) and 3, together with the mutagenic primer 9 (Table 1
). Plasmid pME3224 is identical to pME3219 except for a 17 bp AvaII deletion in the 5' untranslated region of the hcn genes (Fig. 3
). For the construction of plasmid pME3238, a DNA linker with a 5' EcoRI and a 3' MaeI site carrying the ANR box (underlined) and a random downstream sequence (bold face) not homologous to the native hcn promoter sequence (5'-AATTCTTGGCCCAGATCAACGCTTACACTGGTAGCGTTAGC-3') was synthesized. This linker was fused to a MaeI/PstI fragment containing the authentic -10 region and the beginning of the hcnA gene (Laville et al., 1998
) and cloned into pME3219 cut with EcoRI and PstI. The resulting construct, pME3238, was thus equivalent to pME3225, except for an altered sequence of 21 bp between the FNR/ANR box and the -10 region. In the hcnlacZ transcriptional fusion of pME6520, the lacZ gene with its own ribosome-binding site (from pME3535; Højberg et al., 1999
) was joined to a 380 bp hcn promoter fragment which had been amplified by primers 1 and 2 (Table 1
) in vector pME6010. The fusion was performed between an artificial HindIII site created previously close to the hcnA translation start (Laville et al., 1998
) and a PstI site at the 5' end of the lacZ fragment.
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RESULTS AND DISCUSSION |
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The -10 region (TAGATT) was changed to a consensus 70 -10 sequence (TATAAT) in the construct pME3226, which was otherwise identical to pME3219 (Fig. 2
). This change of the -10 promoter element led to elevated aerobic expression, whereas anaerobic expression was reduced more than twofold. Thus, plasmid pME3226 showed a reduced ANR-dependent induction factor and higher expression levels in the anr mutant CHA21 (Fig. 2
). In conclusion, the expression of the native hcn promoter strongly depends on ANR and the ANR box.
The FNR consensus box at -40 (TTGAT . . . . ATCAA) combined with the consensus 70 -10 sequence (TATAAT) has previously been tested for induction by oxygen limitation in strain CHA0. When this artificial promoter was linked to the lacZ gene and integrated into the P. fluorescens chromosome, an anaerobic induction factor of about 25 was observed under batch conditions in flask cultures (Højberg et al., 1999
). When the same promoter construct was carried by the vector pME6010, which has about six copies per chromosome (Heeb et al., 2000
), the induction factor was the same (data not shown). Thus, the pME6010-based reporter plasmids used here are adequate to monitor ANR-dependent regulation.
The hcn promoter is not subject to GacA control
Although previous work (Blumer et al., 1999 ) suggested that GacA control has a major impact on hcn gene expression at a post-transcriptional level, additional effects of GacA on the hcn promoter and a synergy with ANR were not excluded. Therefore, the same hcnA''lacZ constructs were also tested in the gacA mutant CHA89 under aerobic and anaerobic conditions. All constructs that were anaerobically inducible in the wild-type strain CHA0 showed the same induction pattern in strain CHA89, but in general expression levels were about 20 times lower in the gacA mutant than in the wild-type (Fig. 2
). All constructs used in Fig. 2
also gave strongly GacA-dependent expression in mildly oxygen-limited cultures grown in NYB to an OD600 of 2·02·5 (data not shown). Thus, it is unlikely that GacA, or a regulator controlled by GacA, could activate the hcn promoter upstream of the ANR box or at the -10 hexamer. There was still the somewhat remote possibility that the hcn promoter sequence lying between the ANR box and -10 hexamer might have a role in GacA control. Therefore, a derivative of pME3225 (Fig. 2
), pME3238, was constructed in which the hcn sequence (TATCCGTGTCCGACAAACCGA) was replaced by a random sequence (CGCTTACACTGGTAGCGTTAG). However, this construct exhibited an unchanged dependence on GacA for expression (data not shown). Taken together, these results indicate that no element of the hcn promoter is involved in control by GacA. It appears that all effects of the GacS/GacA system on hcnA expression in P. fluorescens CHA0 occur post-transcriptionally, in agreement with earlier findings (Blumer et al., 1999
).
Iron regulation of hcn expression depends on ANR rather than on GacA
Under iron-limiting conditions, HCN production of P. fluorescens CHA0 is abolished (Voisard et al., 1989 ). Using again the translational hcnA''lacZ fusion constructs, we examined the question of how iron limitation affects hcn expression in P. fluorescens. Strain CHA0 was grown in a minimal medium (MMC) with or without addition of 20 µM FeCl3. ß-Galactosidase expression was determined when cells reached an OD600 of about 1·0. At this cell density, cultures grown without iron supplementation were in stationary phase and were green-fluorescent, which indicates siderophore (pyoverdin) production as a consequence of iron depletion, whereas cells grown in the presence of iron were in late-exponential-growth phase under mild oxygen limitation and did not produce siderophores. Iron depletion reduced the expression of the lacZ reporter fusion about 50-fold in strains CHA0/pME3219 and CHA0/pME3225 (Fig. 3a
).
Many iron-regulated genes in bacteria are controlled by Fur (ferric uptake regulator), an aporepressor, which is converted to the active repressor by binding Fe2+. Recent studies indicate that Fur could also act as an activator (Crosa, 1997 ). Therefore, as no fur mutant is available in P. fluorescens CHA0, we tested the expression of pME3219 in the fur mutant PAO1A4 of P. aeruginosa (Barton et al., 1996
) and compared it with the expression in the wild-type strain PAO1 in NYB. No difference in expression was observed in both strains (data not shown), suggesting that iron regulation of the P. fluorescens hcn genes may be independent of Fur.
In eukaryotes, the expression of the iron storage protein ferritin is upregulated by an increase in the intracellular iron concentration. A stemloop structure, the iron-response element, in the 5' leader of ferritin mRNA is involved in the regulation by iron (Addess et al., 1997 ). The untranslated 5' leader of the hcn mRNA contains a predicted stemloop structure (Laville et al., 1998
). By deleting a 17 bp AvaII fragment from the centre of the corresponding inverted repeat, plasmid pME3224 was created, the hcn mRNA of which could not form this specific secondary structure. The ß-galactosidase activity of strain CHA0/pME3224, assayed under high and low iron conditions, was reduced, but still regulated by iron limitation (Fig. 3a
), implying that the stemloop structure may stabilize the transcript rather than affect the response to iron.
In the wild-type strain CHA0 carrying plasmid pME3223, which is deleted for the ANR box, ß-galactosidase expression was no longer iron-dependent and in strain CHA0 harbouring pME3226, a construct which has lost most of its ANR-dependence because of an improved -10 consensus sequence (CTATAAT), ß-galactosidase expression was constitutively high, in the presence and in the absence of iron (Fig. 3a). These results raised the possibility that the effects of iron limitation depend on ANR. Indeed, in the anr mutant CHA21 the expression of all constructs was no longer stimulated upon addition of iron (Fig. 3a
).
In E. coli, iron-dependent transcriptional control can be mediated by FNR (Cotter et al., 1992 ; Niehaus et al., 1991
). The transcriptionally active form of the FNR protein is a dimer containing two [4Fe4S]2+ clusters. Oxygen inactivates FNR by converting the FeS cluster to a [2Fe2S]2+ form (Kiley & Beinert, 1999
). Iron depletion may limit the availability of FeS clusters to FNR and other Fe proteins (Cotter et al., 1992
). Since the cellular concentration of apo-FNR varies little under different growth conditions (Kiley & Beinert, 1999
), FNR itself may act as an iron sensor, in addition to being an oxygen sensor (Rouault & Klausner, 1996
). Considering the fact that FNR and ANR of P. fluorescens show a high degree of structural and functional conservation (Laville et al., 1998
; Højberg et al., 1999
), we suggest that P. fluorescens ANR could also sense iron availability and that iron depletion could convert ANR to an inactive state even under anaerobic conditions.
In the anr mutant CHA21, iron limitation and/or stationary-phase conditions caused somewhat elevated ß-galactosidase expression levels of all reporter constructs (Fig. 3a), but in particular with pME3226. This effect may be explained by assuming that the housekeeping
70 factor can recognize the hcn promoter, especially that of pME3226, even without ANR. Moreover, in iron-deprived stationary-phase cells, the hcn promoter might be co-transcribed by RNA polymerase associated with the stress sigma factor
38 (
S), which also recognizes the -10 sequence CTATAAT carried by pME3226 and which has no apparent need for a conserved -35 element, at least in E. coli (Wise et al., 1996
; Wösten, 1998
). It has been shown previously that the
70/
38 balance can affect the expression of secondary metabolism in P. fluorescens (Sarniguet et al., 1995
; Schnider et al., 1995
). Thus, in strain CHA0/pME3226, weak induction by ANR on the one hand and weak induction by iron depletion in stationary phase on the other hand might cancel each other out.
A transcriptional hcnlacZ fusion in pME6520 escaped GacA control (Fig. 3b), as predicted, and was positively controlled by iron in anr+ strains, whereas the same fusion was upregulated slightly by iron depletion in the anr mutant CHA21 (Fig. 3b
). These data reflect the fact that induction of hcn gene expression by high iron concentrations is essentially transcriptional and ANR-dependent. No evidence was obtained for iron effects at the post-transcriptional level, although the possibility that GacA might modulate siderophore synthesis and thereby indirectly influence the organisms response to iron availability cannot be excluded. Such an interpretation might explain why induction by iron sufficiency was less pronounced in a gacA background than in the wild-type (Fig. 3
).
Conclusions
The hcn promoter of P. fluorescens CHA0 is anaerobically activated by ANR and this transcriptional activation by ANR appears to be mediated exclusively via the ANR box in the hcn promoter (Fig. 2). Moreover, hcn expression is strongly dependent on iron availability. Iron limitation has the same negative effect on hcn expression as has good aeration and this type of iron regulation is not observed in an anr-negative background (Figs 2
and 3
). These results lead to the conclusion that ANR acts as an iron sensor, perhaps because iron limitation may restrict the assembly of the FeS clusters on the ANR apo-protein, a situation which would interfere with activation of ANR. As shown elsewhere (Blumer et al., 1999
), a region at or near the ribosome-binding site of the hcnA gene acts as a post-transcriptional target of the GacS/GacA regulatory network. The data obtained in this work suggest that ANR and the GacS/GacA system act sequentially, rather than synergistically, on cyanogenesis. Oxygen and iron availability determine the activity of ANR, whereas cell density appears to influence the activity of the GacS/GacA system, via unknown signals.
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ACKNOWLEDGEMENTS |
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Received 5 April 2000;
accepted 21 June 2000.