1 Wageningen Centre for Food Sciences, NIZO Food Research, Kernhemseweg 2, PO Box 20, 6710 BA Ede, The Netherlands
2 Unité de Génétique, Institut des Sciences de la Vie, Université catholique de Louvain, 5 Place Croix du Sud, Louvain-la-Neuve, 1348, Belgium
Correspondence
Michiel Kleerebezem
Michiel.Kleerebezem{at}nizo.nl
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ABSTRACT |
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INTRODUCTION |
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While evidence for the presence of an active Mn2+ uptake system in L. plantarum has been available for a long time (Archibald & Duong, 1984), the only uptake system described for this species to date is a Mn2+- and Cd2+-specific P-type ATPase (MntA) (Hao et al., 1999a
). Functionality of MntA from L. plantarum has been demonstrated in Escherichia coli where its expression conferred increased sensitivity and uptake of Cd2+, which is a known alternative substrate for Mn2+ (Hao et al., 1999a
). An mntA-mutant derivative of strain ATCC 14917 was unable to grow in medium containing less than 20 mM Mn2+.
Two additional families of Mn2+ transport systems have been identified in several other bacterial species. One of these Mn2+ transporters belongs to the ATP-binding cassette (ABC) family of transporters and was first described for Synechocystis sp. PCC6803 (Bartsevich & Pakrasi, 1995) and later also identified in several Gram-positive bacteria (reviewed by Claverys, 2001
). These transporters are encoded by an operon and contain a solute-binding extracytoplasmic protein, a cytoplasmic ATP-binding protein and an integral membrane protein. In Gram-positive bacteria, the cell-surface substrate-binding components of these Mn2+ transporters belong to the lipoprotein receptor antigen I (LraI) family and were initially identified as adhesins that play a role in virulence (Sampson et al., 1994
). However, subsequent research established their role as extracytoplasmic substrate recognition subunit of Mn2+-specific ABC transporters in Streptococcus pneumoniae (PsaA) and Streptococcus gordonii (ScaA) (Dintilhac et al., 1997
; Kolenbrander et al., 1998
). S. gordonii sca mutants displayed decreased Mn2+ uptake and impaired growth in media containing less than 0·5 µM Mn2+, and mutant cells were hypersensitive to oxygen in Mn2+-deficient medium (Jakubovics et al., 2002
). By analogy, a requirement for increased Mn2+ concentrations in the growth medium has been reported for an S. pneumoniae psaA mutant (Dintilhac et al., 1997
) and a Streptococcus pyogenes mtsA mutant (Janulczyk et al., 2003
). PsaA/ScaA homologues have been identified in at least nine additional species of Streptococcus and also in bacteria belonging to other genera (for a review see Claverys, 2001
). Mn2+ uptake by ABC transporters has been experimentally demonstrated in Salmonella enterica serovar Typhimurium (Kehres et al., 2002
; Kehres & Maguire, 2003
) and specificity for both iron and manganese was shown for Yersinia pestis (Bearden & Perry, 1999
), S. pyogenes (Janulczyk et al., 2003
) and Streptococcus mutans (Paik et al., 2003
).
A third type of transporter has been reported to be involved in Mn2+ uptake in bacteria. These bacterial transporters are homologues of the mammalian Nramp (natural resistance-associated macrophage protein) transporters for divalent metal ions, which act as regulators of host susceptibility to intracellular infections (reviewed by Forbes & Gros, 2001). Similar systems have been identified in Bacillus subtilis (Que & Helmann, 2000
), Salmonella typhimurium (Kehres et al., 2000
) and E. coli (Makui et al., 2000
), and confer high-affinity uptake of Mn2+. Molecular studies of the enterobacterial (Kehres et al., 2000
) and B. subtilis (Que & Helmann, 2000
) nramp genes demonstrated that they encode proton-stimulated, highly selective Mn2+ transporters that play a role in the bacterial response to oxidative stress (Kehres et al., 2000
).
Since Mn2+ serves an important function in oxygen tolerance of L. plantarum, one or more highly efficient systems to import this metal ion are expected to be encoded by this organism. Of the three known classes of bacterial Mn2+ transporters, to date only the P-type ATPase has been described for L. plantarum, while Nramp and ABC-transporters have been described in species that have low Mn2+ requirements and that have superoxide dismutase activity. In this paper we use protein sequences of established bacterial Mn2+ transport systems to identify candidate Mn2+ transport system encoding genes in the L. plantarum WCFS1 genome. In addition, we establish their induced expression by L. plantarum in response to Mn2+ limitation and, by mutation analysis, study their contribution to the extraordinary high Mn2+ levels accumulated by this bacterium.
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METHODS |
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Construction of mntA, mtsA and mntH2 mutants.
To inactivate the mtsA and mntH2 genes in L. plantarum WCFS1 by double cross-over recombination, we used the non-replicating vector pUC18ery (Van Kranenburg et al., 1997). For the introduction of a deletion in mtsA, two fragments flanking the region to be deleted were amplified by PCR. Using primers LPATPF3 and LPATPR3, a 860 bp fragment was obtained corresponding to the sequence downstream of the 3' end of the coding region of mtsA (fragment B). Primers LPATPF4 and LPATPR4 generated an 822 bp PCR fragment that contains 355 bp of the 5' end of mtsA and 467 bp of the 3' end of mtsB (fragment A). Both PCR products were cloned in pGEM-T (Promega) and the resulting plasmids were used for sequence verification of the fragments. Subsequently, the mtsA-flanking fragments were reisolated as a NcoISacI (fragment A) or NcoISalI fragments (fragment B) and cloned in SalI/SacI-digested pUC18ery, resulting in pNZ7257. This construct was introduced into L. plantarum by electroporation and primary integrants were selected on plates containing Em. Integration of pNZ7257 at the mts locus was confirmed by Southern blotting and one of the integrants was cultured without selection pressure to allow the second cross-over event, resulting in an EmS phenotype. After 100 generations, three candidate mutants (EmS) were obtained and Southern analysis confirmed that two strains contained the 575 bp deletion in mtsA (data not shown).
The mntH2 gene was inactivated by a 617 bp deletion at the 3' end of the gene using the double cross-over recombination strategy described above. A 649 bp fragment, located at the 5' end of mntH2, was amplified using primers LPMNTH2AF and LPMNTH2AR and cloned into pCRblunt (Promega), reisolated from the resulting plasmid as a SacIXbaI fragment and subcloned into SacI/XbaI-digested pUC18ery, yielding pNZ7258. A 714 bp fragment located downstream of the stop codon of mntH2 was amplified using primers LPMNTH2BF and LPMNTH2BR. The PCR product obtained was digested with SalI and XbaI and cloned in similarly digested pNZ7258, yielding the mntH2 knock-out plasmid pNZ7259. This plasmid was used to obtain the mntH2 mutant derivatives of both wild-type L. plantarum WCFS1 and its mtsA deletion derivative. Mutant strains were designated NZ7257, NZ7259 and NZ7260 for the mtsA, mntH2 and the mtsA-mntH2 mutants, respectively.
The mntA gene in L. plantarum WCFS1 was disrupted by single cross-over plasmid integration. To create the integration plasmid pNZ7256, an 1143 bp internal fragment of mntA was amplified by PCR using the primers LPATP3 and LPATPR3, and cloned into pUC18ery using the EcoRI and BamHI restriction sites introduced in the primer sequences. Plasmid pNZ7256 was transformed into L. plantarum by electroporation and candidate integrants were selected on MRS agar plates containing 10 µg Em ml1. Correct integration of pNZ7256 in the mntA locus was confirmed by PCR and Southern blotting, and a single mntA disruption mutant (NZ7256) was used in subsequent studies.
Intracellular manganese analysis.
The amount of Mn2+ accumulated in the wild-type and its mutant derivatives was determined. Overnight-grown cells in CDM (300 µM or 1·5 µM Mn2+) were diluted 1 : 100 in CDM (300 µM or 1·5 µM Mn2+) and cultured overnight. Cells were harvested by centrifugation and washed three times with CDM without manganese. The resulting pellet was suspended in Millipore water and cells were mechanically disrupted in the presence of zirconium beads in a FastPrep FP120 (Savant Instruments). Cell debris was removed by centrifugation and the remaining supernatant was diluted in MilliQ for determination of Mn and protein (Bradford, 1976) concentration. Mn2+ concentrations were determined by Inductivity-Coupled Plasma Atomic Emission Spectrometry (ICP-AES).
RNA isolation, Northern blotting and primer extension.
Overnight-grown cells of L. plantarum in CDM (containing 300 µM MnSO4) were washed in CDM without Mn2+ and diluted 1 : 100 in CDM with variable MnSO4 concentrations (1·5300 µM), and incubated overnight at 37 °C. One millilitre of each of these cultures was used to inoculate 50 ml CDM with the same MnSO4 concentration as used for the overnight culture. Cells were harvested at an OD600 of 0·40·5 and RNA was isolated by the Macaloid method described by Kuipers et al. (1993) with the adaptation that prior to disruption cells were incubated with lysozyme for 5 min on ice. RNA was separated on 1 % formaldehyde-MOPS agarose gels, blotted and hybridized as was described previously (Van Rooijen & de Vos, 1990
). Blots were probed with PCR-amplified fragments of mtsA (primers LPATPF6 and LPATPR6), mntH2 (LPMNTH2AF and LPMNTH2AR), mntH1 (LPMNTH1F and LPMNTH1R) and mntH3 (LPMNTH3F and LPMNTH3R). Probes were radiolabelled with [
-32P]dATP by nick translation. Quantification of the transcripts in Northern blotting was performed using the Dynamics Phosphor Imaging System (Molecular Dynamics). Signal intensities were corrected for background radiation and for the total amount of RNA loaded by correlation of the signal intensity to the amount of 16S rRNA as determined by hybridization with a 700 bp PCR-amplified probe (primers 16SP1 and 16SP2). The values presented for the mtsA and mntH2 mutant strains are means of two independent experiments and varied less than 12 % from the mean.
For primer extension, 20 ng oligonucleotides was annealed to 15 µg total RNA according to the method described by Kuipers et al. (1993). The oligonucleotides used were LPABCSEQ, LPH1SEQ and LPH2SEQ, which are complementary to the 5' sequence of mtsC, mntH1 and mntH2, respectively.
Real-time RT-PCR.
Total RNA of cells grown at either 1·5 or 300 µM Mn2+ was isolated as described above and mRNA levels for mntA, mntH2, mntH1 and mtsA were detected by reverse transcription (RT) followed by real-time PCR. Expression levels of the target genes were normalized using 16S rRNA levels. Prior to the RT step, RNA was treated with deoxyribonuclease I (Invitrogen Life Technologies) following the instructions of the manufacturer. RNA concentrations in the DNase-treated samples were determined using the RiboGreen RNA quantification reagent kit (Molecular Probes). Four (for samples) or 40 ng (standard curve) total RNA was reverse transcribed with Superscript II RNaseH reverse transcriptase (Invitrogen Life Technologies) using 2 pmol either QMNTAR3, QMNTH2R, QMTSAR, QMNTH2R or Q16SREV, according to the instructions of the manufacturer. Real-time PCR products were quantified using the SYBR Green PCR mastermix (Applied Biosystems). The dynamic range and the efficiency of both target and normalizer reactions were examined by running in triplicate dilutions of the cDNA pools (0·4 pg4 ng) using primers QMNTH2F/QMNTH2R (mntH2), QMNTAF3/QMNTAR3 (mntA), QMNTH1F/QMNTH1R (mntH1), QMTSAF/QMTSAR (mtsA) or Q16SFORW/Q16SREV (16S). Real-time PCR was performed on an ABI Prism 7700 sequence detector instrument (PE Applied Biosystems) in 50 µl reactions containing 25 µl SYBR Green mastermix, 2 µl forward primer (5 µM stock), 2 µl reverse primer (5 µM stock), 4 µl template (corresponding to 0·4 ng RNA per well) and 17 µl water. The following PCR conditions were used: 5 s at 50 °C and denaturation (95 °C, 10 min) followed by 40 cycles of 95 °C for 15 s followed by 54 °C for 1 min. Both target and normalizer reactions were run in triplicate and the relative error for the determination of the crossing point (CP) values was below 2 %. Relative expression levels and PCR efficiencies were calculated according to the method described by Pfaffl (2001) with the following modification: CP values of the individual samples were corrected for background values caused by small amounts of residual DNA in the RNA samples. This was done by running in triplicate real-time PCR reactions for the normalizer and for each target using RNA that was not reverse transcribed. The CP values of these samples were considered DNA-derived and the difference in CP value between the reverse transcribed and the non-transcribed sample was used for the calculation of relative expression levels of the various target genes. Statistical analysis was performed by using the REST software tool (Pfaffl et al., 2002
).
DNA and deduced protein analysis.
Computer analysis of DNA sequences and the deduced amino acid sequences was performed using the program Clone Manager 6.0 (Scientific and Educational Software, Durham, USA). For sequence similarity searches and genome searches, the BLAST facility of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the CLUSTALW facility of the European Bioinformatics Institute (http://www.ebi.ac.uk) were used.
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RESULTS |
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Using an mtsA-specific probe, a single transcript of 2·6 kb was detected in cells grown at 1·5 and 3 µM Mn2+, albeit the signal intensity was significantly lower in the latter sample (Fig. 1a). This transcript size corresponds to the length required to encompass the three mts genes. In cells grown at Mn2+ concentrations above 3 µM, no mtsA transcript could be detected. No increase of mtsCBA expression was observed upon aeration as compared to anaerobic growth at 1·5 µM Mn2+ (results not shown). Real-time RT-PCR was used to determine the relative quantity of mtsA-specific mRNA in the 1·5 µM Mn2+ sample compared to the 300 µM Mn2+ sample and revealed a 29-fold increase (P=0·001) in mtsCBA expression in the Mn2+-limited cells. The transcription initiation start site of the mtsCBA gene cluster was determined by primer extension using RNA isolated from cells grown at 1·5 µM Mn2+. Two transcription start sites, 35 and 40 nt upstream of the start codon of the mtsC gene were identified (Fig. 2a
). These results demonstrate that mtsC, B and A form an operon that is activated upon Mn2+ starvation, which strongly supports a role of the MtsCBA system in Mn2+ transport.
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No transcripts were detected using the mntH3- or the mntA-specific probes under the conditions tested. The presence of a putative ferrochelatase-encoding gene, upstream of mntH3, could suggest a role for MntH3 in iron rather than Mn2+ transport. Expression of mntA under Mn2+ starvation has been demonstrated in L. plantarum strain ATCC 14917 using reverse transcriptase PCR (Hao et al., 1999a), which is a more sensitive technique compared to Northern analysis. However, mntA expression in L. plantarum WCFS1 was barely detectable in our real-time RT-PCR experiments and did not appear to be significantly affected by the Mn2+ concentration in the medium. These data suggest that neither mntA nor mntH3 plays a prominent role in maintenance of Mn2+ homeostasis in L. plantarum WCFS1 under any of the conditions tested.
Analysis of mtsA, mntA and mntH2 mutant strains
The expression patterns of mtsA and mntH2 strongly support a role of these genes in Mn2+ transport in L. plantarum WCFS1 under Mn2+-limiting conditions. Therefore, these genes were selected as targets for mutation analysis in this strain. Despite the observation that strain WCFS1 did express mntA only at a very low level that was not influenced by Mn2+ limitation, this gene was also selected as a target for mutation analysis based on the reported role of this gene in Mn2+ transport in strain ATCC 14917 (Hao et al., 1999b).
The mtsA and mntH2 genes were functionally deleted via the double cross-over strategy described in Methods, while the mntA gene was functionally disrupted via a single cross-over plasmid integration. Unexpectedly, the growth rates of the mtsA, mntH2, mntA and the mtsA-mntH2 double mutants were not affected under Mn2+ limitation or excess as compared to those observed for the parent strain (Table 5). In addition, the final culture density after overnight growth of these mutants in media containing a range of Mn2+ concentrations was virtually identical to those observed for the wild-type strain (data not shown). Finally, the intracellular Mn2+ concentration in none of these mutant strains was significantly reduced compared to the parental strain under any of the conditions tested (data not shown). Notably, the mntA mutant strain appeared to form aggregates when grown in liquid medium under both aerobic and anaerobic conditions, independent of the Mn2+ concentration added to the medium. Complementation of the mntA mutant with a plasmid-encoded copy of mntA did not relieve aggregate formation (data not shown), suggesting that this phenotype results from proximal or downstream polar effects of the mutation. These growth characteristics of the L. plantarum WCFS1 mntA mutant are markedly different from those previously reported for the mntA mutant of strain ATCC 14917 which was unable to grow in liquid medium unless supplemented with at least 20 mM Mn2+ (Hao et al., 1999b
).
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DISCUSSION |
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The expression of mtsCBA is 29-fold increased (1·5 µM compared to 300 µM Mn2+) under manganese starvation in L. plantarum. The L. plantarum MtsCBA system shows a high degree of homology to systems in Lactobacillus casei (GenBank accession no. AJ276708) and in the Lactococcus lactis IL1403 genome sequence (Bolotin et al., 2001), indicating that this type of cation transporter represents a common system in lactic acid bacteria. The crystal structure of the MtsA homologue in S. pneumoniae (PsaA) revealed that the side chains of His67, His139, Glu205 and Asp280 form the metal-binding site (Lawrence et al., 1998
). These four residues are conserved in the MtsA protein of L. plantarum. Remarkably, the N-terminal prolipoprotein signal sequence common for Gram-positive bacteria (Sutcliffe & Russel, 1995
) is absent in MtsA of L. plantarum, but a type I signal peptidase cleavage site is predicted (Nielsen et al., 1997
). This suggests that in L. plantarum, MtsA is tethered to the cell surface via an alternative structure.
The two Nramp-type transporters that are expressed under Mn2+ starvation in L. plantarum, MntH1 and MntH2, are widespread in both Gram-positive and -negative bacteria, but are absent in S. pneumoniae and S. pyogenes genomes (Tettelin et al., 2001; Ferretti et al., 2001
). The increased expression levels of mntH1 (6·6-fold) and mntH2 (294-fold) under Mn2+ starvation strongly suggests that these genes encode Mn2+ transporters. By analogy, similar genes in B. subtilis (Que & Helmann, 2000
), E. coli and Salmonella typhimurium (Kehres et al., 2000
; Makui et al., 2000
) have been shown to be involved in Mn2+ transport. MntH1 and MntH2 of L. plantarum share a high level of identity with the HitA protein from a beer spoilage isolate of Lactobacillus brevis, which appeared to be expressed upon addition of bitter hop compounds (Hayashi et al., 2001
). Notably, one of these compounds is known to exchange extracellular protons for intracellular Mn2+ (Simpson, 1993
), which is likely to influence intracellular Mn2+ levels and could support a role for HitA in Mn2+ transport.
Overall the expression data as well as the functional analyses of homologues of the established Mn2+ transport systems support a role for MntH1 and MntH2 and MtsCBA in Mn2+ transport. However, the mtsA and mntH2 mutants and the mtsA-mntH2 double mutant derivative of L. plantarum exhibited no growth defects or decreased internal Mn2+ levels. This is in clear contrast to similar studies in a variety of other species (Dintilhac et al., 1997; Kolenbrander et al., 1998
; Que & Helmann, 2000
; Janulczyk et al., 2003
; Kehres et al., 2000
). This may suggest that L. plantarum can adapt effectively to inactivation of these genes. Indeed, inactivation of mtsA and mntH2 results in moderate upregulation of mntH2 and mtsCBA, respectively. The increased mtsCBA transcript levels in the mntH2 mutant, and vice versa, may suggest that cross-regulation occurs. Comparison of the promoter regions of mtsC and mntH2 revealed the presence of possible cis-acting elements that could act as metalloregulator target regions based on their similarity to the MntR-binding site described for B. subtilis (Que & Helmann, 2000
) and the ScaR-binding site described for S. gordonii (Jakubovics et al., 2000
) (Fig. 2
). In the cross-regulation scenario, it remains unknown which Mn2+ transporter is induced in the mntH2-mtsA double mutant. Obvious candidates would be mntH1 and mntA; however, expression of these two genes was not increased in the double mutant strain. Moreover, mntA disruption does not affect Mn2+ homeostasis in L. plantarum WCFS1 in contrast to the mntA mutant phenotype described for strain ATCC 14917 (Hao et al., 1999b
). Although strain-specific effects cannot be excluded, it seems likely that the ATCC 14917-derived mutant is not only affected in mntA but carries mutations in one or more genes involved in Mn2+ homeostasis.
An alternative explanation for the lack of phenotype of the mutants could be that one or more other proteins annotated as cation transporters accomplish Mn2+ uptake in L. plantarum. In L. plantarum, genes encoding 42 complete transport systems are present that have been annotated as cation transporters. For 13 of these transporters, the homology to transporters with experimentally verified specificity is not sufficient to predict the substrate of these systems. Moreover, annotation of the cation specificity of transporters is a prediction and requires experimental verification to substantiate the postulated role. For example, transporters belonging to the P-type ATPase family were reported to cluster in phylogenetic trees according to their substrate specificity (Axelsen & Palmgren, 1998). However, recent studies show that two members of the calcium cluster (PMR1 from yeast and ATP2A2 from humans) actually transport Mn2+ into the Golgi apparatus (Maeda et al., 2004
; Ton et al., 2002
). This illustrates that prediction of the cation specificity of transporters is difficult. Interestingly, the L. plantarum genome is predicted to encode nine P-type ATPases, three of which resemble the recently described P-type Ca2+/Mn2+ ATPase (PMR1) in Schizosaccharomyces pombe (Maeda et al., 2004
) (encoded by lp_0567, lp_3398 and lp_0124). None of the bacterial homologues of this eukaryote-derived transporter has been associated with Mn2+ transport to date. Moreover, several bacterial homologues of this eukaryotic protein are annotated as calcium transporters. Nonetheless, a role for these transport systems in Mn2+ transport in L. plantarum appears to be a realistic option and should be targeted by future functional studies in this species.
In conclusion, although transcription analyses support the postulated role of mntH2, mntH1 and mtsA in Mn2+ transport, mutation analysis has demonstrated that these genes are not essential for Mn2+ homeostasis in L. plantarum WCFS1. These findings exclude a central role for these proteins in a phenotypic characteristic that was described for L. plantarum several decades ago and that has been shown to fulfil highly relevant functions in this species. In addition, the results either suggest highly adaptive behaviour of L. plantarum in response to mutations affecting genes involved in Mn2+ homeostasis or could hint at the presence of alternative Mn2+ transporters that have not been identified in bacteria to date.
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Received 3 June 2004;
revised 16 November 2004;
accepted 14 January 2005.
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