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Keywords:

  • Alteromonas;
  • [NiFe] hydrogenase;
  • conjugation;
  • physiology of H2 metabolism;
  • Urania basin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Alteromonas macleodii Deep ecotype is a marine, heterotrophic, gammaproteobacterium isolated in the Mediterranean Sea between depths of 1000 and 3500 m. The sequenced strain was previously reported to contain a [NiFe] hydrogenase. We verified the presence of this hydrogenase in other strains of A. macleodii Deep ecotype that were previously isolated from several bathypelagic microenvironments. We developed a system for the genetic manipulation of A. macleodii Deep ecotype using conjugation and used this system to create mutant strains that lack the [NiFe] hydrogenase structural genes (hynSL). The mutants did not possess hydrogenase activity, and complementation of the mutant strain with the hynSL genes successfully restored hydrogenase activity. Both the mutant and the wild-type strains grew at the same rate in a variety of media and under different environmental conditions, indicating little effect of the hydrogenase mutation under the conditions tested.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bathypelagic environments exist well below the photic zone at depths between 1000 and 4000 m. At such depths, pressure increases to 10–40 MPa and temperatures decline; however, Mediterranean basins maintain warmer temperatures throughout the water column because they are sheltered from cold polar currents (Martín-Cuadrado et al., 2007). The Urania basin in the eastern Mediterranean is characterized by hypersaline, anoxic waters (Borin et al., 2009). A steep chemocline of 5 m separates the oxic seawater above from the anoxic brine layer below that contains 16% salinity and high concentrations of sulfide (10–16 mM), methane (5.5 mM), sulfate (85 mM), phosphate (41 μM), and manganese II (3.47 μmol kg−1) (Sass et al., 2001; Borin et al., 2009). The warmer waters and extreme geochemistry of Urania basin make for an unusual microbial ecosystem that is largely separated from surface inputs and that has only recently been characterized (Sass et al., 2001; Borin et al., 2009).

Several recent studies have profiled the microbial consortium inhabiting this deep water environment (Sass et al., 2001; Lopez-Lopez et al., 2005; Yakimov et al., 2007; Borin et al., 2009). One frequently isolated bacterium is Alteromonas macleodiii, a marine, heterotrophic, gammaproteobacterium. Alteromonas macleodii is globally distributed, but sequence analysis of ribosomal and housekeeping genes indicates that isolates obtained from the Mediterranean Sea at depths between 1000 and 3500 m are genetically distinct from their surface-isolated counterparts (Lopez-Lopez et al., 2005; Ivars-Martinez et al., 2008a, b). When the sequenced genomes of representative Deep ecotype (AltDE) and surface ecotype (ATCC 27126) strains were compared, many differences were identified, including the presence of a [NiFe] hydrogenase in AltDE, but not in ATCC 27126 (Ivars-Martinez et al., 2008b). The [NiFe] hydrogenase gene locus is present in a 95-kb gene island and includes hynS and hynL encoding the hydrogenase small and large subunits, respectively, and the genes predicted to encode the accessory proteins that are responsible for maturation of the hydrogenase. An environmental Alteromonas hydrogenase showing 99% identity to the AltDE hydrogenase was heterologously expressed in Thiocapsa roseopersicina and was confirmed to be active (Maroti et al., 2009). Later, the AltDE hydrogenase was characterized and was found to be active (Vargas et al., 2011). The presence of this hydrogenase in AltDE was suggested to help the organism survive in a nutritionally restricted environment (Ivars-Martinez et al., 2008b), but the physiological role of the hydrogenase in this species is unknown.

Genetic tools may supplement metagenomic approaches to study the microbial biochemistry of bathypelagic environments (Martín-Cuadrado et al., 2007; Borin et al., 2009). Transformation systems for other Alteromonas species have been described (Kato et al., 1998), but no genetic tools have been described as yet for the A. macleodii Deep ecotype. In this paper, we report a survey of hydrogenases in various A. macleodii Deep ecotype strains, the development of a conjugation system for the A. macleodii Deep ecotype, and the effect of hydrogenase mutations on the growth of A. macleodii Deep ecotype under various conditions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Strains and growth conditions

Unless noted otherwise, all Escherichia coli strains were grown at 37 °C in Luria–Bertani (LB) broth or LB agar plates and A. macleodii strains were grown at 28 °C in marine broth (MB, Difco) or MB agar plates. Antibiotic concentrations used for the growth of E. coli cultures were ampicillin (50 μg mL−1), tetracycline (12.5 μg mL−1), kanamycin (50 μg mL−1), spectinomycin (50 μg mL−1), and chloramphenicol (25 μg mL−1). Antibiotic concentrations used for the growth of Alteromonas cultures were kanamycin (100 μg mL−1), spectinomycin (50 μg mL−1), and chloramphenicol (25 μg mL−1). Minimal synthetic seawater, essentially marine broth without peptone or yeast extract, was prepared as described previously (Coolen & Overmann, 2000).

The sequenced strain of A. macleodii Deep ecotype (DSMZ 17117) was isolated from the Adriatic Sea at a depth of 1000 m (Lopez-Lopez et al., 2005; Ivars-Martinez et al., 2008a). Other strains of A. macleodii Deep ecotype were isolated from the Urania Basin in the Eastern Mediterranean Sea above the chemocline (3455 m, Um4b), at the chemocline (3475 m, Um7, Um8, U4), or from the brine below the chemocline (3500 m, U7, U8, U10, U12) (Sass et al., 2001, Table 1). To distinguish them, the sequenced strain was referred to as the strain AltDE, while the other isolates were referred to by their strain designation (i.e. U7, etc.).

Table 1.   Bacterial strains and plasmids used in this study
Bacterial strain or plasmidGenotype or featuresReference or source
Alteromonas macleodii‘Deep ecotype’, strain AltDE, DSMZ 17117Wild-type A. macleodii‘Deep ecotype’ isolated from the Adriatic SeaLopez-Lopez et al. (2005)
Alteromonas macleodii strains U4, U7, U8, U10, U12, Um4b, Um7, Um8Wild-type A. macleodii isolated from Urania Basin in the Eastern Mediterranean.Sass et al. (2001)
Escherichia coli
 DH10BF- mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZ ΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL nupGInvitrogen
 HB101F- recA13 mcrB mrrBoyer & Roulland-Dussoix (1969)
Plasmids:
 pRC41AltDE hydrogenase gene cluster Orf1/cyt/orf2/hynD/hupH/hynS/hynL/hypC/hypA/hypB/hypD/hypF/hypE in pTRC-NSI, SpRWeyman et al. (2011)
 pRL448AmpR, KmRElhai & Wolk (1988a)
 pRL2948aCmR, EmR, sacB, oriTC.P. Wolk, unpublished data
 pRL528Helper plasmid, M. AvaI, M. Eco47II, CmRElhai et al. (1997)
 pRL443Conjugal plasmid based on RP4, ApR, TcRElhai & Wolk (1988b)
 pPW418pRC41 Δorf2, hynD, hupH, hynS, hynL, hypC, hypA, SpR, KmRThis work
 pPW427Modified hydrogenase cluster from pPW418 ligated into ScaI site of pRL2948a, KmRThis work
 pPW437CmR, EmR, sacB, oriT from pRL2948a ligated into pUC19, ApRThis work
 pPW438Hydrogenase cluster from pRC41 ligated into pPW437, ApR, CmRThis work
 pPW440Plasmid to create targeted deletion of hynSL, KmR, ApR, CmRThis work

Construction of E. coli strains

All molecular biology techniques were performed according to Sambrook & Russell (2001). A previously described plasmid, pRC41, carries a c. 13-kb fragment containing the entire hydrogenase gene cluster from AltDE (Weyman et al., 2011). To knock out the hydrogenase region, a plasmid containing a deletion in a large portion of the hydrogenase gene cluster in AltDE was created based on pRC41. This plasmid, pPW418, was constructed by digesting pRC41 with AvrII and EcoNI and replacing with the kanamycin resistance gene C.K3 (KmR) digested from pRL448 with SmaI. Plasmid pPW418 contains a modified hydrogenase cluster with partial or complete deletions of the following genes: orf2, hynD, hupH, hynS, hynL, hypC, and hypA. The modified cluster was digested from pPW418 with SacI, blunted, and ligated into the ScaI site of pRL2948a that contains the origin of transfer (OriT) for conjugation and the sacB gene conferring sensitivity to sucrose. The resulting plasmid was confirmed by restriction digest and named pPW427.

A second plasmid, pPW440, was designed to specifically knock out only hynSL, the genes encoding the hydrogenase small and large subunits, by replacing most of the genes with the KmR antibiotic resistance cassette. To generate pPW440, we first created a plasmid capable of being conjugated (pPW437). A 5-kb fragment [containing genes with resistance to erythromycin (EmR) and chloramphenicol (CmR), the transfer origin oriT, and the gene sacB] from pRL2948a was digested using SpeI, blunted, and ligated to pUC19 that had been digested with HincII, resulting in pPW437. The pPW440 plasmid that contained about 1 kb of sequence upstream and downstream of hynSL, respectively, was constructed by four-piece ligation using the following fragments: (1) a 1-kb piece fragment containing kanamycin resistance gene C.K3 (KmR) generated by PCR with primers KmR-BamHI and KmR-XhoI and subsequent digestion with BamHI and XhoI, (2) a 1.8-kb fragment from AvrII- and BamHI-digested pRC41, (3) a 1.6-kb fragment from XhoI- and XbaI-digested pRC41, and (4) an XbaI-digested pPW437. The resulting plasmid, pPW440, was verified by restriction digest and sequencing.

To construct a plasmid that can complement the mutant, pRC41 was digested with SacI to release a 13.4-kb fragment containing the whole AltDE hydrogenase gene cluster. This fragment was ligated to a SacI-digested pPW437, creating plasmid pPW438.

Conjugation of the A. macleodii Deep ecotype

Plasmids to be conjugated were first electroporated into E. coli strain HB101 that contains plasmid pRL528 encoding AvaI and AvaII methyltransferases. Escherichia coli and A. macleodii cells in the log phase were washed twice with LB or marine broth and resuspended in 500 μL appropriate growth medium. For the conjugation of plasmids into A. macleodii, 100 μL each of the washed donor E. coli cells (HB101 containing pRL528 and plasmids pPW440 or pPW438), the helper E. coli cells (HB101 containing pRL443), and the A. macleodii recipient cells were mixed together, spread on a nitrocellulose filter (Protran BA85, Whatman) laid on top of a marine broth agar plate, and incubated overnight at 28 °C. The following day, the cells were washed from the filter and plated on marine broth agar plates containing the appropriate antibiotics. AltDE has a natural resistance to spectinomycin at 50 μg mL−1, and this resistance was exploited to eliminate the E. coli strains used in conjugation that were sensitive to the antibiotic. Colonies that were confirmed to contain the antibiotic cassette by PCR were further screened to select for fully segregated, double recombinants that lack the hydrogenase region by plating cultures on marine broth agar containing appropriate antibiotics and 5% sucrose. Plates were incubated overnight at 28 °C and colonies were selected for further testing by PCR and Southern blot to confirm that the sacB gene and the hydrogenase gene region had been eliminated by DNA homologous recombination.

PCR analysis and Southern blot

Southern blots were performed as described in Sambrook & Russell (2001). Probes for the Southern blots were constructed by incorporating digoxigenin-labeled nucleotides into a PCR product as described previously (Maroti et al., 2009). The primers used to construct the probes were KmF-BamHI (5′-GTAGGATCCGTTGACACGGGCGTATAAGACAT) and KmR-XhoI (5′-AGTTCCTCGAGGTGGGCGAAGAACTCCAGC) for the KmR probe and AmF2 (5′-CGTCTTTTGGCGGGATCCC) and AmR2 (5′-GTAAAATCAGTTCAATTCCC) for the hynSL probe.

In vitro hydrogen evolution assays

In vitro hydrogen evolution using methyl viologen as an electron donor to hydrogenase was performed as described in Maroti et al. (2009). Cultures were grown overnight in marine broth supplemented with 100 μM NiCl2 before being spun down for sonication and the assay.

Physiological tests

Growth curves were performed in 96-well plates with 2-mL wells covered with Airpore tape sheets (Qiagen). Starter cultures were grown aerobically overnight in marine broth, washed three times in minimal seawater, and diluted 100-fold in 800 μL per well containing the growth medium to be tested. The plates were shaken at room temperature in air or in an anaerobic chamber (3% H2/97% N2). Complete (marine broth) or minimal (synthetic seawater) media were used with KNO3 or MgSO4 added at a final concentration of 40 and 60 mM, respectively.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Presence of hydrogenase in A. macleodii Deep ecotype strains

The sequenced strain of A. macleodii Deep ecotype (AltDE) contains one hydrogenase (HynSL) and was isolated from the Adriatic Sea at a depth of 1000 m (Lopez-Lopez et al., 2005). Other A. macleodii Deep ecotype strains were found to be genetically related to AltDE and were isolated from the Urania basin in the Eastern Mediterranean at a depth of c. 3500 m (Sass et al., 2001). It was unknown whether the strains isolated from the Urania Basin also contained a hydrogenase. To test for the presence of the hydrogenase HynSL in these strains, we isolated genomic DNA from each strain and performed Southern blots using a probe that could detect the AltDE hydrogenase gene sequence. HindIII-digested DNA from all Urania Basin strains contained a hynSL-hybridizing restriction fragment that was the same size as AltDE, indicating that the hydrogenase genes, hynSL, are present in all A. macleodii Deep Ecotype strains isolated from the Urania basin (Fig. 1a). To determine whether the hydrogenase HynSL was expressed and functional, in vitro hydrogen evolution assays were performed. All strains expressed an active hydrogenase when grown under aerobic conditions, although the activities differed approximately fourfold among the different strains (Fig. 1b). Thus, not only do all the environmental strains possess an active hydrogenase, they also express it under aerobic conditions, although at different levels.

image

Figure 1.  Presence of the hydrogenase HynSL in Alteromonas macleodii isolates from the Urania Basin (Sass et al., 2001). (a) Southern blot of HindIII-digested genomic DNA from A. macleodii Deep ecotype isolates. Blots were probed with a labeled hynS PCR product. (b) Detection of hydrogenase activity in A. macleodii strains. In vitro H2 evolution assays were performed on crude cell extracts from cultures grown in marine broth supplemented with 100 μM NiCl2. Error bars indicate 1 SD of the mean from three replicate cultures.

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Mutation and complementation of hydrogenase in A. macleodii

To begin to explore the physiology of hydrogen metabolism in AltDE, we designed a vector to replace the hydrogenase genes with an antibiotic cassette using a conjugation-based approach. The first plasmid we constructed, pPW427, was designed to delete several genes including the hydrogenase structural genes, hynSL, and several adjacent hydrogenase accessory genes, orf2, hynD, hupH, hynS, hynL, hypC, and hypA (Fig 2a). The resistance cassette was flanked by 2.7 and 5.0-kb homology regions at the 5′ end and 3′ end, respectively, in which homologous recombination may occur with the A. macleodii chromosome (Fig. 2a). Plasmid pPW427 was conjugated from E. coli into AltDE, and colonies were selected on marine broth plates supplemented with kanamycin. The number of colonies obtained on the selective medium was 0.1% of the total number of colonies obtained on nonselective medium, indicating a conjugation efficiency of 1 × 10−3. When the selected colonies were examined by PCR, they were found to have both the KmR cassette and the hydrogenase genes, hynSL (Fig. 3a), indicating that insertion had not proceeded by double recombination and replacement of hynSL. To select for clones in which a double recombination event had occurred, we used the dominant negative selection marker, SacB, encoded by the sacB gene located in the plasmid pPW27 (Ried & Collmer, 1987). After selection on sucrose, colonies were picked and tested by PCR. These colonies lacked hynSL and the adjacent accessory genes, but contained the gene for the kanamycin resistance gene (Fig. 3a), suggesting that the double recombination event occurred.

image

Figure 2.  Construction of hynSL mutant strains PW427 (a) and PW440 (b) Arrows indicate ORFs drawn to scale. The positions of restriction enzymes used in cloning and probes used in Southern blotting experiments are indicated.

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image

Figure 3.  Verification of the AltDE hydrogenase mutants and hydrogenase-complemented mutant strain. (a) PCR amplification of the hynSL and KmR genes from AltDE PW427 mutants. Template PW427-pre is an aliquot of PW427-1 taken before the sucrose selection for the elimination of sacB. NTC, pPW427, and AltDE gDNA refer to a no-template control, plasmid pPW427, and AltDE wild-type genomic DNA, respectively. (b) PCR amplification of hynSL and KmR genes from the AltDE PW440 mutant and its complemented strain PW438/PW440. NTC, pPW440, pPW438, and AltDE gDNA refer to a no-template control, plasmids pPW440 or pPW438, and AltDE wild-type genomic DNA, respectively. (c) Southern blot probed with a labeled hynSL PCR product. Each lane contains 1 μg HindIII-digested genomic DNA isolated from the indicated strains. Plasmid pPW440 was digested with BamHI and XhoI, and plasmid pRC41 was digested with HindIII. (d) Southern blot probed with a labeled KmR PCR product. Samples were prepared and loaded as in (c). (e) Examination of hydrogenase activity in wild type, mutant, and complemented AltDE strains. In vitro H2 evolution assays were performed on crude cell extracts from cultures grown in marine broth supplemented with 100 μM NiCl2. Error bars indicate 1 SD of the mean from three replicate cultures.

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Once the methodology of conjugation into A. macleodii was established, we constructed the second plasmid that was designed to delete only the hydrogenase structural genes, hynSL. This plasmid, pPW440, was introduced into AltDE, selected on sucrose-containing medium, and screened by PCR as above. The PW440 mutant was confirmed to lack the hynSL genes while possessing the KmR cassette (Fig. 3b), suggesting that hynSL was deleted through a double recombination event.

Southern blot was performed to confirm that the PW427 and PW440 mutants lacked the hynSL genes. No bands were observed when the blots were hybridized with a probe corresponding to hynSL, confirming that the genes encoding the hydrogenase were deleted (Fig. 3c). When the blots were rehybridized with a probe to the kanamycin resistance gene, bands of the expected size were identified, confirming that double recombination had occurred and that the KmR cassette had replaced the hydrogenase genes (Fig. 3d). Mutants confirmed by Southern blot analysis were also found to lack hydrogenase activity in an in vitro hydrogen evolution assay (Fig. 3e).

To complement the PW440 hydrogenase mutant, a plasmid, pPW438, containing the wild-type AltDE hydrogenase gene cluster was conjugated into the mutant and a single recombination event in the mutant was selected by resistance to the antibiotics chloramphenicol and kanamycin. PCR amplification of hynSL confirmed that the complemented strain contained a copy of the wild-type hydrogenase genes (Fig. 3b). Hydrogenase activity measured by in vitro hydrogen evolution assay with cell extracts indicated that the complemented strain, PW438/PW440, regained hydrogenase activity to almost wild-type levels (Fig. 3e).

Examination of the AltDE mutant under different growth conditions

To learn more about the physiology of A. macleodii, we investigated the ability of AltDE to grow under various conditions. While AltDE grew well in the complete medium (marine broth) under aerobic conditions, growth under anaerobic conditions was inhibited unless nitrate was added to the medium as an electron acceptor (Fig. 4a). No growth was observed when sulfate was provided as an electron acceptor (data not shown). When grown in a complete medium with nitrate under anaerobic conditions containing 3% H2, no differences in the growth rate were observed between the wild type and the hydrogenase mutant strain, PW440 (Fig. 4a).

image

Figure 4.  Growth of wild-type and mutant AltDE strains under aerobic and anaerobic conditions. For all graphs, error bars indicate 1 SD of the mean from three replicate cultures. (a) Comparison of anaerobic growth in the marine broth supplemented with or without nitrate. Wild-type AltDE (diamond) or hydrogenase mutant PW440 (square) strains were grown under anaerobic conditions with (solid symbols) or without (open symbols) nitrate. (b, c). Comparison of aerobic or anaerobic growth in media supplemented with different nutrients or salts. Wild-type AltDE or hydrogenase mutant PW440 were grown under aerobic (b) or anaerobic (c) conditions in marine broth, marine broth supplemented with 2 M NaCl, or minimal seawater media. All cultures were supplemented with 20 mM KNO3 as an electron acceptor. The y-axis has been plotted on a logarithmic scale. Specific conditions are as follows: wild-type AltDE (diamond) or hydrogenase mutant PW440 (square) grown in marine broth, AltDE (triangle) or PW440 (X) grown in marine broth supplemented with 2 M NaCl, and AltDE (+) or PW440 (circle) grown in minimal seawater.

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Strains U7, U8, U10, and U12 were isolated by Sass et al. (2001) in the Urania Basin at a depth of 3500 m, where the chloride concentration was measured to be between 2.5 and 3.0 M. Thus, we tested the ability of A. macleodii to grow in the presence of additional salt. When the complete marine medium was supplemented with an additional 2 M NaCl, growth was slowed, but still detectable (Fig. 4b and c). This slower growth in the high-salt medium was detected when grown either aerobically or anaerobically (nitrate was supplied as the electron acceptor) (Fig. 4b and c). As expected, no growth occurred in minimal seawater media (Fig. 4b and c). No significant differences in the growth rates could be observed between the wild type and the hydrogenase mutant strains of AltDE under all growth conditions tested (Fig. 4b and c). Thus, the presence of the hydrogenase does not appear to affect growth rate in the presence of 3% H2 in a complete medium. The fact that no growth was detected in minimal seawater in the presence of 3% H2 is consistent with the designation of A. macleodii as a chemoheterotroph that requires fixed carbon sources. It remains an open question as to whether the hydrogenase plays a role in metabolism under some combination of environmental conditions not tested in this paper.

Recent studies have reported that sulfate is the main terminal electron acceptor in the Urania basin brine (Borin et al., 2009). Nitrate, oxygen, and manganese may be important electron acceptors in the upper parts of the interphase between the brine from which several cultures of A. macleodii were isolated, and that may support much higher levels of microbial life (Borin et al., 2009). AltDE was previously reported to possess nitrate reductase activity, but no growth assays were conducted (Ivars-Martinez et al., 2008b). In our growth assays, combined nitrogen was not a limiting factor due to the presence of peptone in the marine broth at a concentration of 5 g L−1. Thus, the inhibitory effect on growth by withholding nitrate was likely due to respiratory requirements.

Deep sea basins are some of the most remote and extreme environments on earth and little is known about their physiology. The existence of a mud volcano at the bottom of the Urania basin may indicate that hydrogen from geological sources is also present (Yakimov et al., 2007). More studies are needed to determine whether the hydrogenase present in all Deep ecotypes contributes to environmental adaptation. While metagenomic data have led to many new hypotheses about the microbial ecology in benthic environments, the development of genetic tools in A. macleodii Deep ecotype will facilitate the elucidation of the genetic basis for survival in these extreme deep sea environments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

This work was supported by the US Department of Energy Hydrogen, Fuel Cells, and Infrastructure Technology Program (DE-FG36-05GO15027). We thank Dr Francisco Rodriguez-Valera for kindly providing us with the A. macleodii Deep ecotype strains.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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