Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina1
Department of Microbiology, Miami University, Oxford, OH 45056, USA2
Instituto de Investigaciones Bioquímicas "Fundación Campomar", Buenos Aires, Argentina3
Institute of Molecular Biology and Nutrition, Department of Biological Science, California State University Fullerton, Fullerton, CA 92834-6850, USA4
Author for correspondence: Luis A. Actis. Tel: +1 513 529 5424. Fax: +1 513 529 2431. e-mail: actisla{at}muohio.edu
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ABSTRACT |
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Keywords: adhC copies, formaldehyde metabolism, iron regulation, gene duplication
Abbreviations: EDDHA, ethylenediamine-di(o-hydroxyphenylacetic) acid; FURTA, Fur titration assay; GSH-FDH, glutathione-dependent formaldehyde dehydrogenase
This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague.
The GenBank accession number for the sequence reported in this paper is AF130307.
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INTRODUCTION |
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It was initially proposed that the primary function of bacterial glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) is the metabolism of formaldehyde formed endogenously by oxidative demethylation of compounds such as monomethyl-L-amino acids (Ling & Tung, 1948 ). However, it was found later that this enzyme is essential in the oxidation of methanol and other methylated compounds when they are the sole carbon source in methylotrophic bacteria such as Paracoccus denitrificans (Ras et al., 1995
) and the facultative phototroph Rhodobacter sphaeroides (Barber et al., 1996
; Barber & Donohue, 1998a
). It was also shown that GSH-FDH activity is involved in microbial resistance to exogenous formaldehyde (Kaulfers & Wollman, 1988
; Sasnauskas et al., 1992
). In the case of Escherichia coli, this resistance activity is due to the GSH-FDH-mediated degradation of formaldehyde, which is encoded by a gene located within a large self-transmissible plasmid (Kummerle et al., 1996
).
In some bacterial species, the production of GSH-FDH is induced when the cells are growing in culture media containing C1 substrates that are sources of formaldehyde (Barber et al., 1996 ; Hanson & Hanson, 1996
). Furthermore, examination of GSH-FDH activity in E. coli and Haemophilus influenzae showed that this enzymic activity is induced by formaldehyde at concentrations between 7·9 µM and 264 µM, whereas higher concentrations of this disinfectant were toxic to the bacterial cells (Gutheil et al., 1997
). The expression of GSH-FDH activity in these two bacteria is controlled at the transcriptional level. In addition, it was recently shown that the transcription of the adhI gene in R. sphaeroides is upregulated by exogenous formaldehyde and S-(hydroxymethyl)glutathione (Barber & Donohue, 1998b
). The redox state of the cell is also an important signal controlling the expression of adhI in R. sphaeroides through the global regulator PrrA (Barber & Donohue, 1998b
). This positive regulator controls the expression of bacterial photosynthetic genes in response to changes in oxygen tension, and it is required to fully stimulate the expression of this gene. However, this is not the only factor controlling adhI expression because its transcription is also increased by the trans-acting mutation spd-7 (Barber & Donohue, 1998b
). These data show that the expression of the gene encoding this activity in bacteria is indeed controlled by a complex regulatory circuitry in which some of the components remain to be identified and characterized.
The genus Acinetobacter includes bacterial species that are widely distributed (Baumann, 1968 ). A. baumannii, previously known as A. calcoaceticus subsp. anitratus, is the most prevalent genospecies associated with human infections, mainly in hospitalized and ambulatory compromised patients (Bergogne-Berenzin & Towner, 1996
). Although numerous reports describing different aspects related to this pathogen and the infections it causes in compromised patients have been published (Bergogne-Berenzin et al., 1996
; Bergogne-Berenzin & Towner, 1996
), not much is known about the basic metabolic processes and the regulation of gene expression.
In this work, we provide evidence that all A. baumannii clinical isolates examined produce a 46·5 kDa protein immunologically related to GSH-FDH, which is encoded by a constitutively expressed gene named adhC2. However, some of these strains harbour an additional copy, identified as adhC1, that encodes a slightly smaller GSH-FDH whose expression is controlled by the iron concentration of the culture media. In addition, our data indicate that the presence and expression of two adhC copies does not confer resistance to exogenous formaldehyde, nor does it enable these strains to utilize some methylated compounds as sole carbon sources.
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METHODS |
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Recombinant DNA techniques.
Standard DNA recombinant methods were done as described by Sambrook et al. (1989) . DNA restriction fragments used as probes were either isolated from agarose gels with the GeneClean kit (Bio101) or amplified by PCR using the conditions described previously (Barancin et al., 1998
). The probes were labelled with [
-32P]dATP or [
-32P]dCTP using the oligolabelling method (Feinberg & Vogelstein, 1983
). The adhC1.5' (5'-CTTGCTTAGACGTATGTGAC-3') and adhC1.3' (5'-CCTACTCCAGTATGGGTAATC-3') primers were used to amplify the 5' region of adhC1. DNA nucleotide sequences were determined manually (Sanger et al., 1979
) or by automated DNA sequencing with the BigDye terminator cycle sequencing kit (PE-Applied Biosystems). DNA and protein sequences were analysed using the BLAST network service at NCBI, the GCG University of Wisconsin Genetics Computer software package, and ProfileScan, which is available at http://www.ch.embnet.org/software/PFSCAN.
Construction of a genomic library and transposition mutagenesis.
The library was constructed using pVK102 and 8399 chromosomal DNA partially digested with HindIII as described previously (Genti-Raimondi et al., 1991 ). The Tn3-HoHo1 transposon (Stachel et al., 1985
) was used as described previously (Tolmasky et al., 1988
) to generate insertional derivatives of recombinant cosmids harbouring A. baumannii 8399 DNA. Recombinant cosmids and Tn3-HoHo1 insertional derivatives were introduced into E. coli DH5
by electroporation. The A. baumannii 8399M6 adhC1::Tn3-HoHo1 isogenic derivative was obtained using pM6 and the allelic-exchange method described previously (Tolmasky et al., 1993
). The cosmid pM6 was conjugated into the 8399 wild-type strain and then cured by incompatibility with pPH1JI. The 8399M6 transconjugant was selected on L agar containing 10 µg chloramphenicol ml-1, 500 µg ampicillin ml-1 and 50 µg gentamicin ml-1. Generation of the appropriate derivative was confirmed by Southern hybridization and nucleotide sequencing after PCR amplification and cloning into pCR2.1. DNA hybridization experiments were carried out under high- and low-stringency conditions as previously described (Graber et al., 1998
).
Cloning of promoter elements, RNA analysis and S1 mapping.
The 500 bp ClaIEcoRI fragment, containing the left end of Tn3-HoHo1 and the predicted adhC1 promoter region, was cloned into pSK+ to generate pPE9. This derivative was used to create the reporter construct pPE9-lacZ by cloning at the EcoRI site the DNA cassette from pAB2001 (Becker et al., 1995 ), which harbours a promoterless lacZ gene and encodes gentamicin resistance. Total RNA from A. baumannii 8399 cells, grown to mid-exponential phase at 37 °C in Tris/M9 containing either 50 µM EDDHA or 300 µM FeCl3, was isolated using hot phenol (Barancin et al., 1998
). Northern blots were prepared and probed with the 32P-labelled ClaI-EcoRI fragment using standard techniques (Sambrook et al., 1989
). The adhC1 transcription initiation site(s) was determined by S1 mapping as described previously (Bidinost et al., 1999
; Wu & Janssen, 1997
). Primer #188 (5'-CGATACTACTCAAGCTCCAAATGGC-3') was end-labelled and used together with unlabelled primer #189 (5'-CCTGGAGCAAAGGCGACAGCTGCACG-3') to PCR amplify the adhC1 promoter region. The purified amplicon and total RNA were coprecipitated, dissolved in hybridization buffer and allowed to anneal at 53 °C. E. coli tRNA was used as negative control. The samples were digested with nuclease S1 and analysed by electrophoretic comparison under denaturing conditions to a ladder obtained by dideoxynucleotide sequencing using the primer #189 and the plasmid pMU6 as a template.
Overexpression of GSH-FDH.
E. coli BL21(DE3) cells harbouring pMU6, a pBCSK+ derivative with adhC1 cloned downstream of the T7 promoter, were grown in L broth at 37 °C until the OD600 reached a value of 0·50·6. Gene expression was then induced by the addition of IPTG as previously described (Studier et al., 1990 ). After washing with 1 vol. TBS, whole-cell lysates were prepared and analysed by SDS-PAGE (Actis et al., 1985
; Smoot et al., 1998
).
Protein electrophoresis and immunoblot analysis.
Samples containing either 25 µg or 50 µg proteins were loaded onto mini (5 cm longx8 cm wide separating gel) and large (16 cm longx15 cm wide separating gel) polyacrylamide gels, respectively. GSH-FDH and the Fur repressor were detected by immunoblotting using standard protocols (Towbin et al., 1979 ). The antiserum against the E. coli Fur was raised and absorbed as described previously (Tolmasky et al., 1994
). The anti-GSH-FDH serum was prepared using the protein isolated by SDS-PAGE and electroelution, and immunopurified with nitrocellulose strips containing the cognate antigen band as described previously (Olmsted, 1981
). The immunocomplexes were detected using horseradish-peroxidase-labelled protein A and the SuperSignal CL-HRP substrate (Pierce Chemical).
Cloning of fur and detection of Fur boxes.
The A. baumannii 8399 fur gene was PCR amplified using the primers #265 (5'-CCAAAGATTGAACAACAAGCTAGG-3') and #267 (5'-CGATTATTTCTTGCGCAATGCTTCC-3'), which were designed using the nucleotide sequence reported by Daniel et al. (1999) . The reaction and cycling conditions were as described previously (Barancin et al., 1998
) with the exception that 55 °C was used as the annealing temperature. The amplicon was cloned with the Invitrogen TA system using E. coli Top10F' competent cells. The presence of the A. baumannii fur homologue and the expression of Fur activity by recombinant clones were confirmed by determining the nucleotide sequence of the cloned amplicon and testing it using the E. coli RRJC1 reporter strain (Wertheimer et al., 1994
), respectively. The BamHIXbaI insert of one of these clones, named pMU45, was ligated into pACYC184 to create the plasmid pMU45.184 that was used to examine the iron-regulated expression of cloned adhC1. The presence of Fur (iron) boxes was assessed using the Fur titration assay (FURTA) (Stojiljkovic et al., 1994
). The FURTA phenotype was confirmed by determining the ß-galactosidase activity (Miller, 1972
) of cells cultured in liquid medium supplemented with 100 µM FeCl3 at least twice. Each time, the assays were done in duplicate and the results are reported as the mean value±1SD of a representative experiment.
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RESULTS AND DISCUSSION |
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The predicted amino acid sequence of the A. baumannii GSH-FDH is highly similar to bacterial enzymes such as those found in H. influenzae Rd (83·7% identity, accession no. P44557), P. denitrificans (62·9% identity, accession no. L36327), R. sphaeroides (61·7% identity, accession no. L47326), Methylobacter marinus (28·0% identity, accession no. L33464), E. coli K-12 strain MG1655 (85·6% identity, accession no. AAC73459·1) and E. coli VU3695 (85·9% identity, accession no. X73835). The latter is a plasmid-encoded gene product that confers formaldehyde resistance (Kummerle et al., 1996 ). The A. baumannii 8399 GSH-FDH also showed significant similarity to the glutathione-independent formaldehyde dehydrogenase produced by Pseudomonas putida (30·7% identity, accession no. D21201). The alignment of these predicted amino acid sequences showed that the A. baumannii GSH-FDH enzyme contains several conserved amino acids required for the structure and enzymic activity of this protein, such as the C-40, H-62 and C-169 ligands to the active Zn, the cysteine residues at positions 92, 95, 98 and 106 that bind to the second Zn atom, and the typical NAD+-binding site 193-GLGGIG-200 (Ras et al., 1995
; Whener et al., 1993
).
Generation of an adhC isogenic mutant
One of the metabolic pathways that detoxifies formaldehyde by using it as a carbon source and generates essential reducing power in the form of NADH includes the enzymic action of GSH-FDH (Hanson & Hanson, 1996 ; Large, 1983
). To test whether this enzyme activity plays a similar role in A. baumannii, the pM6 cosmid harbouring the adhC::Tn3-HoHo1 copy was used to create the 8399M6 (adhC::Tn3-HoHo1) isogenic mutant by marker exchange (Tolmasky et al., 1993
). Southern blot analysis showed that the adhC probe hybridized with a 6·4 kb HindIII fragment present in the chromosome of the 8399 wild-type strain and the pPA6 cosmid clone (Fig. 1
, lanes 1 and 2). As predicted, the pM6 HindIII fragment harbouring adhC increased its size to about 20 kb, a 14 kb increase due to the insertion of Tn3-HoHo1 within this gene (Fig. 1
, lane 3). A HindIII fragment with a size similar to the latter was detected in the genome of the A. baumannii 8399M6 strain, with the concomitant disappearance of the 6·4 kb HindIII fragment detected in the wild-type strain (Fig. 1
, lane 4). The Tn3-HoHo1 insertion site in the chromosome of A. baumannii 8399M6 was confirmed by Southern blot hybridization and by sequencing a PCR fragment encompassing the putative adhC promoter region and the 5' end of the leaderless ß-galactosidase gene present at one of the ends of Tn3-HoHo1. This approach showed that the transposon inserted between the T116 and the A117 of the predicted adhC ORF.
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Growth studies in L broth revealed that strain 8399M6 (adhC1::Tn3-HoHo1) was viable and showed a growth rate slightly higher than the 8399 wild-type strain during the exponential phase (data not shown). However, both cultures reached similar cell density during stationary phase. Both strains also showed similar growth rate and final cell density when cultured in Tris/M9 minimal medium, although the final cell mass was not as high as that observed with L broth.
Analysis of the expression of adhC
GSH-FDH assays showed that the 8399 parental strain and the 8399M6 isogenic derivative produced 160·9±6·9 and 59·7±2·61 µmol NADH min-1 (mg protein)-1, respectively. These data demonstrate that the insertion of Tn3-HoHo1 in adhC1 results in a 2·7-fold reduction but not complete elimination of enzyme activity, providing further support for the presence of adhC isogenes in the genome of A. baumannii 8399. It is noteworthy that no formaldehyde dehydrogenase activity was detected in either of these A. baumannii strains when glutathione was not added to the reaction mixture, suggesting that the 8399 strain does not express glutathione-independent formaldehyde dehydrogenase activity.
Enzyme levels similar to those detected in the 8399 parental strain were also measured in E. coli DH5 cells harbouring pPA6, in which adhC1 was cloned into the cosmid pVK102, whilst the activity was increased almost 10-fold when the cells contained this gene cloned in the higher-copy-number recombinant plasmid pMU6. In contrast, DH5
cells that harboured the cloning vectors pVK102 or pBCSK+ produced 106·8±29·2 and 116·0±34·5 µmol NADH min-1 (mg protein)-1, respectively. These lower levels represent the enzyme activity encoded by the chromosome of this bacterium (Gutheil et al., 1997
). Comparable lower enzyme levels were also detected in E. coli DH5
cells containing pM6, the pPA6 derivative that has Tn3-HoHo1 inserted within the adhC1 gene.
The adhC1 gene product was characterized by PAGE under denaturing and non-denaturing conditions using cells that overexpressed this gene. For this purpose, we used the T7 promoter-RNA polymerase system (Tabor & Richardson, 1985 ) and exploited the fact that the adhC1 cloned in pMU6 was inserted downstream and transcribed in the same direction as the T7 promoter present in the pBCSK+ vector. SDS-PAGE analysis showed the presence of a 45 kDa protein in the extract of E. coli BL21(DE3) cells harbouring pMU6 when induced with IPTG (Fig. 2a
, lane 4). This protein was also detected, although at a lower amount, in the extract of non-induced cells (lane 3). In contrast, no significant changes of protein bands within this molecular size range were detected in the extracts of non-induced and induced E. coli BL21(DE3) cells harbouring either pBCSK+ (lanes 1 and 2) or pMU6.
N (lanes 5 and 6). Both these constructs were used as negative controls, with the latter being a derivative of pMU6 in which a 941 bp NsiI fragment that contains most of adhC1 was deleted. When the samples obtained from IPTG-induced cells (Fig. 2a
, lanes 2, 4 and 6) were analysed by PAGE under non-denaturing conditions and stained to detect GSH-FDH, the extract of cells containing pMU6 (Fig. 2b
, lane 2) was the only sample that showed a protein band with enzymic activity. A faint band with similar mobility representing the GSH-FDH produced by the E. coli host strain was detected in the other two samples only after prolonged staining (data not shown).
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These data, taken together with the results obtained using enzymic assays, confirm that A. baumannii 8399 carries and expresses two adhC isogenes, which encode proteins with molecular sizes similar to those detected in other bacteria that produce GSH-FDH activity. Cloning and sequencing experiments have shown so far that, generally, bacteria carry a single adhC gene, with the apparent exception of E. coli VU3695. This clinical strain contains a large self-conjugative plasmid that harbours an adhC copy encoding resistance to exogenous formaldehyde (Kummerle et al., 1996 ). This observation, together with the fact that E. coli DH5
(Gutheil et al., 1997
) and MG1655 (Blattner et al., 1997
) have an adhC chromosomal copy, suggest that E. coli VU3695 indeed carries chromosomal and plasmid adhC copies. Whether either A. baumannii 8399 adhC isogene is located on a plasmid element remains to be determined.
Regulation of the expression of adhC
It has been established that C1 compounds such as formaldehyde control the production of GSH-FDH in bacteria like E. coli and H. influenzae (Gutheil et al., 1997 ). Similarly, the enzyme activity produced by the A. baumannii 8399 wild-type strain and the 8399M6 adhC1::Tn3-HoHo1 isogenic derivative was increased between five- and tenfold when the cells were cultured in the presence of sub-inhibitory concentrations of formaldehyde. However, our initial observation that an adhC1lacZ transcriptional fusion expressed iron-regulated ß-galactosidase appears to be novel, and it prompted us to examine the effect of this metal on the expression of GSH-FDH in more detail. Spectrophotometric assays showed that GSH-FDH activity in E. coli DH5
harbouring pPA6 was about twofold higher in cells cultured under iron limitation than in cells cultured under iron-rich conditions (see Table 2
). A similar response to changes in iron concentration was observed in E. coli DH5
cells harbouring pMU6, although the enzymic activity detected in iron-starved cells was much higher due to the higher copy number of this clone. This table also shows that the enzymic activity of cells harbouring either the vector pVK102 or pM6 (adhC1::Tn3-HoHo1) was comparable under iron-limiting or iron-rich conditions. The effect of iron on the expression of GSH-FDH was also tested in the A. baumannii 8399 wild-type strain. Table 2
shows that the GSH-FDH activity increased significantly as the minimal medium was supplemented with increasing concentrations of the iron chelator EDDHA, with an almost tenfold difference between the enzymic activity detected in 8399 cells cultured under iron-rich and iron-limiting conditions. The enzymic activity of A. baumannii 8399M6 (adhC1::Tn3-HoHo1) cells cultured in the presence of 300 µM FeCl3 was similar to that of the parental strain grown under the same conditions and was increased only about 1·5-fold when the cells were cultured under iron-limiting conditions. The iron regulation of adhC1 transcription in the A. baumannii 8399 wild-type strain was further examined by Northern blot analysis. Fig. 3
shows the presence of an RNA band of 1400 nucleotides which coincides with the predicted size of the transcription product of adhC1. Scanning densitometry of the autoradiogram showed that the amount of adhC1 transcripts in the total RNA sample obtained from iron-starved cells (lane 2) was six- to eightfold higher than the amount present in total RNA isolated from iron-replete cells (lane 1). Taken together, the data indicate that although the expression of both A. baumannii 8399 adhC copies are regulated by formaldehyde, only the transcription of adhC1 is affected by the iron concentration of the media.
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Concluding remarks
The data presented in this report show that some strains of A. baumannii contain two adhC isogenes, one of which is expressed in a Fur- and Fe-dependent manner. However, the presence of two adhC copies and the differential expression of one of them does not affect, at least under the conditions used in this study, the growth rate of the cells, the ability to resist exogenous formaldehyde, or the use of different methylated compounds as sole carbon sources. This gene duplication may represent simply an intermediate step or an incomplete gene transfer process that left two adhC isogenes in some strains of A. baumannii. However, the fact that only adhC1 is iron regulated may suggest that the product of this gene plays a role additional to its well-established function in the detoxification of intracellular formaldehyde. It was recently reported that organic acids such as formate, which is produced by the successive enzymic action of GSH-FDH and S-formylglutathione hydrolase on formaldehyde, protect stationary-phase E. coli and Salmonella cells from killing by cationic peptides (Barker et al., 2000 ). Thus, a potential protective function of increased expression of GSH-FDH, which would result in higher intracellular levels of formate, is in agreement with the fact that pathogens such as A. baumannii encounter antimicrobial peptides produced by the host. Furthermore, the Fur-regulated expression of adhC1 is consistent with the well-known fact that iron-restricted conditions of the host control the expression of a large number of bacterial genes. Also, it has recently been proposed that Fur works more like a global regulator that controls different cell functions in addition to those involved in iron acquisition (Escolar et al., 1999
). Another aspect that should be considered is that other metals, besides iron, could be involved in the Fur-mediated regulation of the adhC1 expression in A. baumannii 8399. Bacillus subtilis contains the yqkL, yqfV and ygaG fur homologues in its genome (Cummings & Connerton, 1997
; Mizuno et al., 1996
), which encode distantly related regulatory proteins that control different regulons. The first two homologues encode a Fur iron repressor, which controls the transcription of siderophore biosynthetic and ferri-siderophore transport genes, while the PerR regulator controls the response of this bacterium to peroxide stress (Bsat et al., 1998
). The ygaG gene encodes the B. subtilis Zur homologue that controls the expression of two operons involved in zinc transport. The potential presence of such a homologue, which would control the biosynthesis of the GSH-FDH zinc metalloenzyme, seems quite reasonable. However, the expression of more than one fur homologue in A. baumannii 8399 that can control the expression of unrelated regulons remains to be seen.
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ACKNOWLEDGEMENTS |
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Received 19 March 2001;
revised 12 June 2001;
accepted 18 June 2001.