1. health and fitness
2. disease

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Pyruvic Oxime Nitrification and Copper and Nickel Resistance
3
by a Cupriavidus pauculus, an Active Heterotrophic Nitrifier-Denitrifier
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5
Miguel Ramirez, Jennifer Obrzydowski, Mary Ayers, Sonia Virparia, Meijing Wang, Kurtis
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Stefan, Richard Linchangco and Domenic Castignetti, Department of Biology, Loyola
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University of Chicago, 1032 West Sheridan Road, Chicago, IL, 60626, U.S.A.
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Corresponding Author: Domenic Castignetti, Telephone 773-508-3638, Fax 773-508-3646, email: [email protected]
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E-mail addresses- Miguel Ramirez ([email protected]), Jennifer Obrzydowski
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([email protected]), Mary Ayers ([email protected]), Sonia Virparia
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([email protected]), Meijing Wang ([email protected]), Kurtis Stefan
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([email protected]), Richard Linchangco ([email protected])
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Abstract
Heterotrophic nitrifiers synthesize nitrogenous gasses when nitrifying ammonium ion.
3
A Cupriavidus pauculus previously thought an Alcaligenes sp. and noted as an active
4
heterotrophic nitrifier-denitrifier, was examined for its ability to produce nitrogen gas (N2) and
5
nitrous oxide (N2O) while heterotrophically nitrifying the organic substrate pyruvic oxime
6
[CH3-C(NOH)-COOH]. Neither N2 nor N2O were produced. Nucleotide and phylogenetic
7
analyses indicated that the organism is a member of a genus (Cupriavidus) known for its
8
resistance to metals and its metabolism of xenobiotics. The microbe (a Cupriavidus pauculus
9
designated as C. pauculus strain UM1) was examined for its ability to perform heterotrophic
2+
2+
10
nitrification in the presence of Cu
and Ni and to metabolize the xenobiotic phenol. The
11
bacterium heterotrophically nitrified well when either 1mM Cu
12
in either enriched, or minimal, medium. The organism also used phenol as a sole carbon
13
source either in the presence or absence of 1mM Cu
14
isolate to perform a number of different metabolisms, its noteworthy resistance to copper and
15
nickel, and its potential use as a bioremediation agent are discussed.
2+
2+
or 0.5 mM Ni
2+
was present
2+
or 0.5 mM Ni . The ability of this
16
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Key Words: Cupriavidus pauculus, heterotrophic nitrification, copper and nickel tolerance,
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pyruvic oxime
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2
1
2
Introduction
Heterotrophic nitrification is the oxidation of nitrogenous compounds, both organic and
3
inorganic, to more oxidized products. Organic nitrogenous compounds, for example, may be
4
oxidized to organic nitro-containing compounds or can be converted to NO2- or NO3- [1,2].
5
Denitrification, the reduction of the nitrogen oxides NO3- and NO2- to N2O or N2 [3], is
6
performed by a diverse group of prokaryotes, including members of the Archaea and the
7
Bacteria [4,5]. Castignetti and Hollocher [6] described a bacterium, provisionally identified as
8
an Alcaligenes sp., which was capable of the two processes, that is, both vigorous heterotrophic
9
nitrification and denitrification. They also noted that a number of different denitrifiers,
10
representing some of the more commonly encountered soil denitrifying microbes [4,7],
11
heterotrophically nitrify [1].
12
Heterotrophic nitrification is performed by chemoheterotrophs [2,7]. The process has
13
the potential to result in nitrogen oxidation [2,8] and to effect bioremediation, especially with
14
respect to the removal of excess nitrogen [2,9]. Jetten et al. [2] noted that Thiosphera
15
pantotropha (now Paracoccus pantotrophus) and other heterotrophic nitrifiers, could
16
simultaneously oxidize and reduce N compounds with gaseous N products being formed.
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Similarly, heterotrophic nitrifiers have been noted to aerobically denitrify [10]. The
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simultaneous processes of heterotrophic nitrification and denitrification were postulated to
19
have resulted in the under estimation of the significance of heterotrophic nitrification [2,11].
20
Rather than inorganic N sources, organic N sources were the substrates for heterotrophic
21
nitrification, which, in some environments, was more significant than autotrophic nitrification
3
1
2
[11,12].
The role of heterotrophic nitrifiers is the subject of current research [13,14,15,16,17] as
3
such microbes may be of consequence to both natural (pastures, grasslands) [8,9,11,12] and
4
artificial (wastewater treatment) [2,10,13,18] environments. Some of these organisms have
5
significant capacities to both heterotrophically nitrify and denitrify [2,10,19] and some
6
synthesize N2O or N2 during the course of heterotrophic nitrification [17,20] or heterotrophic
7
nitrification-aerobic denitrification metabolism [10,14,15,16,18,21]. To date, studies have
8
focused on inorganic N compounds as the sources of nitrogenous gas production, with either
9
NH4+, NH2OH, or NO2- serving as the substrate for either N2 or N2O production
10
[10,14,16,17,18,19]. Whether organic-N substrates, such as pyruvic oxime (PO; H3C-
11
C(NOH)-COOH), serve as sources of N2 or N2O synthesis during heterotrophic nitrification or
12
heterotrophic nitrification coupled to aerobic denitrification has received little, if any, attention.
13
The current study was undertaken to examine whether the heterotrophic nitrification of
14
PO by the organism previously known as an Alcaligenes sp.: 1- resulted in the gaseous
15
nitrogen products N2 or N2O; 2-supported heterotrophic nitrification-aerobic denitrification
16
such that either N2 or N2O was produced; and 3-occurred when the organism was cultured in
17
the presence of the heavy metals Cu and Ni. In addition, 1-whether a representative
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xenobiotic, phenol, was used as a carbon source; 2-an analysis of the bacterium’s phylogenetic
19
identity; and 3-the identity of the crucial gene necessary for denitrification (nitrite reductase)
20
were conducted. At the time of its original characterization, the Alcaligenes sp.’s taxonomic
21
classification was based on phenotypic traits and its G+C content [66.1 mol%-D. Castignetti,
4
1
D. (1980) Characterization of a soil heterotrophic nitrifier and its synergistic interactions with
2
Nitrobacter agilis. Dissertation, University of Massachusetts, Amherst, MA]. In the current
3
study, the bacterium was characterized with respect to its 16S rRNA gene sequence and nitrite
4
reductase (nir) gene via cloning and nucleotide sequencing. Identification of the bacterium as
5
a Cupriavidus pauculus spurred an examination of its Cu
6
ability to heterotrophically nitrify PO in the presence of these metals and its use of the
7
xenobiotic phenol in the absence or presence of Cu
8
tolerance of metals and use of xenobiotics. (To avoid confusion, the provisionally identified
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Alcaligenes sp. will be referred to as C. pauculus UM1 in the remainder of this report.)
2+
2+
and Ni
2+
and Ni
2+
resistance capacities, its
as this genus is known for its
10
11
Materials and Methods
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Resting cell experiments To examine the ability of C. pauculus UM1 to produce nitrogenous
13
gasses while heterotrophically nitrifying PO, the bacterium was grown to mid- to late
14
logarithmic phase in the PO mineral salts medium described below, harvested by centrifugation
15
(10,000 x g for 10 min) and washed twice in 50 mM pH 7.0 phosphate buffer to make resting
16
cells. Cells were divided into control (boiled for 5 minutes) and experimental groups and 5 ml
17
were placed into a 33 ml vial along with a stirring bar. Vials were then sealed with a rubber
18
septum and flushed, for 5 min, with the appropriate gas (see below) to remove the air. At t = 0,
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1 ml of 60 mM PO in the phosphate buffer was added via syringe and needle to yield a final
20
concentration of 10 mM PO. Gasses used to flush the vials were either 100% helium (He) or
21
100% O2. Three sets of conditions were examined: no O2 (anaerobic), low O2 (3.6% O2) and
5
1
100% O2. Helium was used to flush the vials when either an anaerobic environment was to be
2
tested or when 1 ml of pure O2 was added, via syringe and needle, to yield an approximately
3
3.6% O2 atmosphere in the vials. During PO metabolism by the organism, the oxidation of
4
PO-nitrogen results in its conversion to NO2- [25]. NO2- synthesis from PO was therefore used
5
to monitor PO use throughout these experiments. At t = 0, approximately 3 and 22 hours, 0.11
6
ml of cells were removed from each vial (via a syringe and needle) and used to measure the
7
consequent production of NO2- from PO. 40 μl of gaseous headspace was simultaneously
8
removed to measure for the synthesis of N2, NO, N2O and CO2 by the cells. As C. pauculus
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UM1 is a respiratory microbe, it was not expected that cells without O2 (He only) would
10
nitrify. Hence, to vials that received only He, 10 mM NaNO2 was added to ensure the presence
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of an oxidant. This condition allowed for assessment of heterotrophic nitrification of the PO at
12
the expense of NO2- reduction, in which case the NO2- would be the oxidant and substitute for
13
O2 gas.
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Growth and metal tolerance To determine the bacterium’s tolerance of either Cu
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growth was performed using mid- to late log cultures as the inoculum [0.5% (v/v)] of the
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bacterium grown in nutrient broth (NB-Sigma Chemical Co., St. Louis, MO) without any metal
17
added. To perform a growth curve with Cu and Ni present, the medium used was NB to which
18
the metals were added from 2 M stocks of either CuCl2-2H2O or NiCl2-6H2O to result in the
19
desired final concentrations [22]. Controls consisted of NB devoid of metal additions.
20
Minimum inhibitory concentrations (MIC) experiments were conducted as described by Chess
21
[23] using NB supplemented with Cu
2+
or Ni
2+
2+
2+
or Ni ,
rather than antibiotics. For both growth and
6
1
MIC experiments, samples were run in triplicate and each experiment was performed at least
2
twice. For all growth experiments, cultures were incubated at 28-30 oC and shaken at 300
3
r.p.m.
4
To determine C. pauculus UM1’s ability to heterotrophically nitrify PO when either
2+
2+
5
Cu
6
first, the bacterium was cultured in NB with 10-12 mM PO with either 1 mM Cu
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Ni
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consisted of identical cultures but without PO. To determine if the organism could
9
heterotrophically nitrify PO without supplemental carbon nutrients (i.e., NB), a second
2+
or Ni
was present, three different types of growth experiments were conducted. For the
2+
or 0.5 mM
and PO use was monitored as described above (via conversion to NO2-). Controls
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experiment type was used where the C. pauculus UM1 was grown in a mineral salts medium,
11
previously described [24], containing either 1 mM Cu
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PO served as the sole C source and where the uninoculated medium was the control. The
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minerals salts medium was made and autoclaved without 0.5g l-1 MgSO4-7H2O, 0.5 g l-1
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NH4Cl and the 10-12 mM PO, which were filter sterilized and added to the cooled, sterile
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medium. To determine the effect of Cu
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ability to grow and nitrify PO, the third experiment type was conducted in which C. pauculus
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UM1 was grown in the above mineral salts medium without metals (control) or with either 1
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mM Cu
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source of carbon, the organism was grown to mid-log in NB and used at a 1:100 dilution to
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inoculate the above mineral salts medium containing filter sterilized 0.5g l-1 MgSO4-7H2O, 0.5
2+
2+
and Ni
2+
2+
2+
or 0.5 mM Ni , in which 10-12 mM
on the relative rate of C. pauculus UM1’s
2+
or 0.5 mM Ni . To determine if C. pauculus UM1 could use phenol as a sole
7
1
g l-1 NH4Cl and phenol (2 mM) as the sole C source. Culturing of C. pauculus UM1 into this
2
medium lacking phenol showed that “carry-over” growth of the bacterium due to residual NB
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did not occur as growth measurements noted a decline of culture turbidity with time as opposed
4
to an increase if phenol was present.
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To determine if C. pauculus UM1 could use phenol as a sole C source when cultured in
6
the presence of either 1 mM Cu2+ or 0.5 mM Ni2+, the bacterium was grown in ~2 mM phenol-
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mineral salts medium and used at a 1:100 dilution to inoculate the same medium containing
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either no metals, 1 mM Cu2+ or 0.5 mM Ni2+. As the medium with 1 mM Cu2+ had an initial
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absorbance above 0 and growth with phenol was fairly sparse, growth was spurred in the
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cultures by adding an aliquot of filter sterilized 200 mM phenol solution after 7 days, thereby
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reestablishing the cultures to a phenol concentration of ~ 2 mM.
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Nucleotide cloning, sequencing and gene identifications The subject of investigation is a
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bacterium originally isolated from an agricultural soil [24,25]. The 16S rRNA gene sequence
14
of the bacterium was analyzed by conducting PCR using the Escherichia coli equivalents from
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base pair 8 to base pair 926 and then from base pair 906 to1512. A total of 1501 nucleotides of
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the bacterium’s 16S rRNA gene were cloned and sequenced.
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Genomic DNA from the bacterium, for both 16S rRNA gene and nirS and nirK gene
18
analyses (see below) was isolated using the Wizard Genomic DNA Purification kit (Promega,
19
Madison, WI) and used as template DNA. The first primer pair employed was 8F and 926R,
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the 8F primer sequence being 5' AGAGTTTGATCCTGGCTCAG 3' [26]. The complement to
21
the 8F primer is the 926R primer which has the sequence of 5' CCGTCAATTCCTTTRAGTTT
8
1
3' [27]. The second primer pair consists of 906F and 1512R. The 1512R sequence was 5'
2
ACGGYTACCTTGTTACGACTT 3' [28] while the 906F sequence is the 5' to 3' reverse
3
complement of the 926R primer (16S rDNA). PCR reactions were performed using either a
4
Hybaid PCR Sprint Thermocycler (Cole-Parmer, Vernon Hills, IL) or a Techne-412 (Scie-Plas
5
Ltd., Cambridge, UK) thermocycler. PCR Master Mix (Promega, Madison, WI) was used, as
6
described by the manufacturer to make 50 µl samples. PCR conditions were one cycle of 94
7
o
8
min followed by one cycle of 72 oC for 7 min. Samples were then kept at either 4 or 10 oC until
9
removed for analysis. Gel analysis was routinely performed using a sodium borate system
10
[29]. Negative controls, with sterile water substituted for template DNA (water controls),
11
were performed to ensure that the DNA copied was from the bacterium being analyzed.
12
Genomic DNA of stock bacteria (e.g., Pseudomonas spp., E. coli) was used as a positive
13
control to insure that amplicons of the proper size were made.
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C for 4 min followed by 33 cycles of 94 oC for 0.75 min, 56.4 oC for 1 min and 72 oC for 1.5
Amplicons of interest were cloned as described in the instructions for the TOPO TA
15
Cloning Kit for Sequencing system (Invitrogen, Carlsbad CA). The resulting pCR4 plasmids,
16
containing the amplicons of interest, were isolated using the Wizard Plus SV Minipreps DNA
17
Purification System (Promega, Madison, WI), verified that they contained the clone of interest
18
by using the plasmid to produce the appropriate PCR product, and then sequenced by the
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University of Chicago Cancer Research Center DNA Sequencing Facility (Chicago IL) using
20
either an Applied Biosystems 3730XL 96-capillary or a 3130 16-capillary automated DNA
21
sequencer. Sequences were analyzed using Chromas Lite
9
1
(www.technelysium.com.au/chromas_lite.html Technelysium Pty Ltd), to make an initial
2
decision as to the quality and acceptability of the sequence, followed by a more thorough
3
analysis to align and append sequences using Lasergene (DNASTAR, Inc. Madison, WI)
4
software. Analysis of the 16S rRNA gene nucleotide sequence allowed for the construction of
5
appended nucleotide sequences of the E. coli equivalent of base pairs 8-1512. Sequences were
6
aligned, appended and analyzed via BLAST-N (GenBank-National Center for Biotechnology
7
Information - http://www.ncbi.nlm.nih.gov/) to search for matches and to identify the closest
8
sequenced relatives. The data generating the best matches were used to characterize the
9
bacterium. In addition, the validity and the quality of the GenBank 16S rRNA gene sequences
10
were investigated via submitting the sequences to Greengenes [30]. Sequence alignments and
11
phylogenetic analysis of the bacterium were performed via the use of the SeaView
12
(http://pbil.univ-lyon1.fr/software/seaview.html) software [31]. Phylogenetic tree construction
13
was done via the SeaView program using the parameters of 100 bootstrap replicates and the NJ
14
algorithm. The dissimilarity bar and accession numbers of the sequences used for comparison
15
are included in the figure.
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Denitrifying bacteria contain the crucial, key enzyme (nitrite reductase), which is
17
necessary to reduce nitrite to nitric oxide [3,32,33]. A denitrifying bacterium contains nitrite
18
reductase as either the cytochrome cd1 (NirS) or the copper-containing (NirK) enzyme and an
19
individual organism contains a single type of the nitrite reductase gene, either nirS or nirK
20
[3,32,33]. As C. pauculus UM1 had previously been identified as an active denitrifier [6], we
21
examined it for the presence of either the nirS or nirK gene by using the primer pairs and
10
1
amplification conditions described by either Braker et al. [32] or Throback et al. [33]. The
2
only modification was that the annealing temperature of the Throback et al. [33] procedure was
3
adjusted to 57 oC. For the examination of the presence of the nirS gene, the primer sets were
4
nirS1F and nirS6R [32] or cd3aF and Rc3d [33]. The former yields a theoretical 873 bp
5
amplified product while the latter yields a theoretical amplified product of 425 bp. Primer sets
6
for the examination of the presence of the nirK gene were nirK1F and nirK5R [32] or FlaCu
7
and R3Cu [33]. The former yields a theoretical 514 bp amplified product while the latter
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yields a theoretical amplified product of 475 bp. DNA from Pseudomonas stutzeri (ATCC
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11607) served as a positive control for the amplification of the nirS amplicon while DNA from
10
Achromobacter xylosoxidans (ATCC 15173) served as a positive control for the amplification
11
of the nirK amplicon. Once the amplicon of the correct size for nirS was identified (the
12
bacterium contains only the nirS gene-see below), it was cloned using the TOPO TA Cloning
13
Kit for Sequencing system noted above. The resultant plasmid was isolated, verified via PCR
14
as containing a nirS amplicon and sequenced. Aligned and appended sequences were analyzed
15
by BLAST-N to identify the closest matches.
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Nucleotide sequences The partial 16S rRNA sequence of the bacterium, as well as the nirS
17
gene sequences, have been deposited in GenBank and are listed below.
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Chemicals and analytic methods All chemicals used to make the above media were reagent
19
grade or better. The sodium salt of PO was synthesized via the method of Quastel et al. [34].
20
NO2- synthesis was measured to monitor PO usage and was determined by the sulfanilamide-
21
N-(1-napthyl)ethylenediamine method [35]. Molecular sieve (Linde-Union Carbide, South
11
1
Plainfield NJ) and Poropak Q (Waters, Milford MA) columns, in a Shimadzu GC-8A (Kyoto,
2
Japan) gas chromatograph, were used to determine CO2, N2, NO and N2O as described [36].
3
Growth was measured via absorbance at 600 nm and phenol was quantified via the method of
4
Slinkard and Singleton [37], adjusted to accommodate 1 ml samples.
5
Statistical analyses Comparison of mean values (Tables 1 and 2, Figure 3) was performed via
6
T-testing using SYSTAT 13 or Excel. Comparison of the curves and of the individual points
7
(means of three replicates) for a given time in Figures 2 and 3 were done using the ANOVA
8
and Tukey’s honestly-significantly-different tests in SYSTAT 13.
9
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Results and Discussion
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To determine if the bacterium produces either N2 or N2O while heterotrophically
12
nitrifying, we attempted to grow the bacterium in sealed test tubes from which gasses could be
13
sampled. Due to insufficient growth of the bacterium, these experiments were discontinued.
14
C. pauculus UM1 was thus grown in the mineral salts-PO medium aerobically at 30 oC,
15
harvested at mid- to late-logarithmic growth and resting cells were prepared. Under either 0%
16
(He), 3.6% or 100% O2 atmospheres, resting cells of the bacterium produced neither N2, NO
17
nor N2O. Although control (boiled) vials failed to produce either NO2- or CO2, experimental
18
vials clearly produced NO2- by the 3 hours and CO2 was noted by the 22 hour time point.
19
A number of organisms produce either N2 or N2O while heterotrophically nitrifying or
20
during heterotrophic nitrification-aerobic denitrification [2,14,16,17,20]. When PO was
21
present under anaerobic conditions, with NO2- present to serve as an oxidant, neither N2 nor
12
1
N2O was synthesized. With PO as a nitrogen source and under full or limited O2 conditions, C.
2
pauculus UM1 produced neither N2 nor N2O but did make NO2-, indicating that while
3
heterotrophic nitrification had occurred, aerobic, or anaerobic, denitrification had not. While
4
previously shown to be clearly capable of heterotrophic nitrification ([24, 25] and this study)
5
and denitrification [6], the current experiments indicate that C. pauculus UM1 produced
6
neither N2 nor N2O via heterotrophic nitrification or via heterotrophic nitrification-aerobic
7
denitrification.
8
9
NH3 or NH4+ are the likely sources of N2 and N2O produced via heterotrophic
nitrification [2,16,17,20]. The failure of C. pauculus UM1 to make either of these gasses likely
10
reflects that PO oxidation to NO2- probably occurs via an organic route, that is, via a carbon
11
intermediate and not via NH2OH [38,34,39]. While C. pauculus UM1 vigorously denitrifies
12
[6] and avidly oxidizes both the organic-N source PO and the inorganic N-source
13
hydroxylamine [24,25], the inability of C. pauculus UM1 to synthesize either N2 or N2O during
14
the oxidation of PO limits its ability to remove N as a gas from aerobic environments.
15
As the bacterium was originally described prior to today’s nucleotide sequencing
16
techniques, the phylogenetic identities of the bacterium and the nitrite reductase gene of the
17
organism were of interest. PCR primers [40] specifically designed to amplify 16S rRNA genes
18
from Ralstonia (i.e., Ralstonia and Cupriavidus–see below) resulted in an amplicon of the
19
proper size from genomic DNA of the bacterium but not from genomic DNA from a non-
20
Cupriavidus (Pseudomonas) bacterium. The cloning, sequencing and BLAST-N analysis of
21
1501 nucleotides of C. pauculus UMI’s 16S rRNA gene resulted in the greatest similarity
13
1
(98.0% identity) match to Cupriavidus pauculus strain KPS201 and Ralstonia sp. HB1 and
2
HB2. A similar match (97.9%) to C. pauculus strain KPS201 was also obtained from
3
Greengenes. A multiple alignment of similar 16S ribosomal DNA sequences indicates that the
4
bacterium is most closely related to C. pauculus KPS201 and other Cupriavidus species
5
(Figure 1). The nucleotide sequence of the 1501 nucleotides of C. pauculus UM1’s 16S rRNA
6
gene has been deposited in GenBank under the accession number GQ504718. Of the first 24
7
GenBank matches, including 20 of which were noted as being members of the genus
8
Cupriavidus, Ralstonia or the family Burkholderiaceae, [of which Cupriavidus (Ralstonia) are
9
included genera-the other matches were to unidentified isolates], the sequence is a perfect
10
match between nucleotides 906-926, strongly indicating that the primers used for this region
11
did not result in errant sequence data.
12
A 98% identity match to a species’ 16S rRNA nucleotide sequence is considered
13
species identification [41,42]. Analyses of the 16S rRNA gene sequence of C. pauculus UM1
14
revealed that its greatest homology was to the microbes C. pauculus strain KPS201 and
15
Ralstonia sp. HB1 or HB2, all of which had a GenBank 98.0% similarity match and E values
16
of 0.0. Nucleotide analysis by Greengenes [the more reliable database as it screens for
17
chimeras, performs standard alignments and taxonomic classifications based on multiple
18
published taxonomies [30], indicated a 97.9% match to Cupriavidus pauculus strain KPS201,
19
with the second closest match being to Ralstonia eutropha strain H16 (97.4%).
20
21
We note that Alcaligenes eutrophus has been reclassified as Ralstonia eutrophus, then
Wautersia eutropha and finally as Cupriavidus necator, the type species [43]. Cupriavidus
14
1
pauculus is now the accepted nomenclature for the microbes previously known as Ralstonia
2
paucula and Wautersia paucula [43]. As the Ralstonia spp. bear no species identification, and
3
is thus of less consequence than the bacterium (C. pauculus KPS201) identified as being the
4
closest match in Greengenes, we propose that the identity of our isolate is a C. pauculus strain
5
(C. pauculus UM1). Phylogenetic analysis (Figure 1) suggests a close association with C.
6
pauculus KPS201 and with organisms originally classified as either Ralstonia or Cupriavidus.
7
The claim is supported by the bacterium’s phenotypic traits [24] and its G+C content (66.1
8
mol%) as the genus has a G+C content ranging from 63-69 mol% [43].
9
Both of the primer sets used to generate nitrite reductase amplicons yielded highly
10
similar matches to sequences of the nirS gene, that is, the cytochrome cd1 (nirS) nitrite
11
reductase gene (data not shown). nirS gene sequences GQ504717, generated from the Braker et
12
al. [32] amplicon, and GQ504716, from the Throback et al. [33] amplicon, have been
13
deposited in GenBank.
14
The ability to denitrify is widespread and precludes identifying denitrifiers by using
15
16S rRNA gene analyses [3,44,45]. That the genus Cupriavidus is capable of denitrification is
16
supported by work [46] that noted that members of the genus were among the most frequently
17
isolated denitrifiers obtained from a luvisol, that the type species of the genus (C. necator) was
18
specifically identified as a denitrifier and that a number of previously recognized genera
19
capable of denitrification are now part of the Cupriavidus genus. GenBank lists 20 Cupriavidus
20
nirS entries, 9 norB entries and 6 nosZ entries and Schwartz et al. [47] noted that Ralstonia
21
eutropha (C. necator) H16 contained the megaplasmid pHG1, which had a gene cluster for
15
1
denitrification. The cloned sequences of C. pauculus UM1’s nitrite reductase gene were good
2
matches (87-95%) to known nirS nucleotide sequences [87%-Ralstonia eutropha JMP134
3
chromosome 2, complete sequence, 95%-Pseudomonas chloritidismutans partial nirS gene for
4
nitrite reductase, isolate AW-1]. As denitrifiers contain a single type of nitrite reductase
5
[3,32,33], and since our isolate contained a nirS-like gene, we conclude that C. pauculus UM1
6
denitrifies via its cytochrome cd1 nitrite reductase. While an earlier study [48] reported that R.
7
paucula (now C. pauculus) was unable to denitrify, considering our isolate’s resistance to Cu
8
and Ni (see below), its sharing a denitrification capacity with other members of the genus
9
[46,47,49], its 16S rRNA gene homology to C. pauculus KPS201 and other Cupriavidus spp.
10
and the above G+C content and phenotypic similarities, the data support that C. pauculus UM1
11
denitrifies via the use of a nirS-like gene. Of particular interest, and revealed in this study, is
12
that even though C. pauculus UM1 spp. contains a nirS-like gene which it uses to denitrify [6],
13
the organism did not invoke its use when heterotrophically nitrifying as evidenced by the lack
14
of either N2O or N2 synthesis under the heterotrophic nitrification or heterotrophic nitrification-
15
aerobic denitrification conditions of the current study.
16
Makkar and Casida [50] were the first to characterize the genus Cupriavidus. They
17
noted that the type strain (C. necator) grew more quickly in media containing 0.4, 0.6 or 0.8
18
mM Cu2+ than with no copper. The genus is noted for its metal resistance [43] and members
19
have been studied for their ability to tolerate metals [51,52]. Like C. necator, C. pauculus
20
UM1 tolerated copper (and nickel) well, demonstrating robust growth in media containing 1
21
mM Cu2+ and 0.5 mM Ni2+ (Tables 1 and 2 , Figure 2). MIC values were determined as 3 mM
16
2+
and 1 mM Ni
2+
1
Cu
(data not shown). C. pauculus UM1 also grew readily, and
2
heterotrophically nitrified well, when in NB-PO with either 1 mM Cu
3
1). Confirmation of C. pauculus UM1’s ability to withstand Cu2+ was noted as it grew readily
4
in the 1 and 2 mM Cu2+ synthetic medium used to study the related bacterium Cupriavidus
5
metallidurans CH34 [51].
2+
or 0.5 mM Ni
2+
(Table
6
The ability of C. pauculus UM1 to heterotrophically nitrify PO in the absence of other
7
carbon sources (no NB present) when either Cu2+ or Ni2+ was present was investigated. When
8
in the presence of either 1 mM Cu
9
C source, nitrifying it vigorously (Table 1). The relative ability of the bacterium to use PO and
10
to nitrify it with and without the metals was also examined (Figure 2). C. pauculus UM1 grew
11
well and nitrified readily under these conditions, demonstrating growth and nitrification when
12
in the presence of either 1 mM Cu
13
being absent. The higher Abs600 of either the control or experimental samples containing Cu2+
14
was due to the blue color imparted to the clear medium by the 1 mM Cu2+.
15
2+
2+
2+
or 0.5 mM Ni , the bacterium readily used PO as its sole
or 0.5 mM Ni
2+
that was commensurate with the metals
C. pauculus KPS 201 [22] was able to withstand notably high concentrations of nickel
16
(MIC of 28.9 mM). More common metal resistance values of approximately 0.5-12 mM Cu
17
[53,54,55] and 2-25 mM Ni [53,54,56,57] have been noted for a number of metal-resistant
18
microbes, with Ni-sensitivity cited as tolerating no more than 0.1 mM Ni [57]. C. pauculus
19
UM1 readily grew in media containing either 1 mM Cu
20
Figure 2) and tolerated higher copper than nickel concentrations (see above). C. pauculus
17
2+
or 0.5 mM Ni
2+
(Tables 1 and 2,
1
UM1 is thus Cu and Ni tolerant when compared to other microbes [53,54,55,56]. Of interest is
2
the observation that C. pauculus UM1’s ability to use PO, as both a carbon and heterotrophic
3
nitrification source, was not impeded by the metals (Table 1 and Figure 2). When an inoculum
4
was grown in a nutrient-rich medium (NB) containing either 1 mM Cu
5
was used to inoculate media containing either NB-PO, NB-PO-1 mM Cu
6
Ni , C. pauculus UM1 grew as fast in the presence of either 1 mM Cu
7
without the metals being present (data not shown) and yielded higher amounts of cells (Table
8
1). When C. pauculus UM1 was grown in a PO-mineral salts medium and used to inoculate
9
either PO, PO-1 mM Cu
2+
or 0.5 mM Ni
2+
2+
2+
or PO-0.5 mM Ni
2+
2+
2+
or NB-PO-0.5 mM
or 0.5 mM Ni
2+
as
mineral salts media, the organism grew as
10
readily in the metal-containing media and nitrified to the same extent, producing equal
11
amounts of NO2- (Figure 2).
12
and
Xenobiotics metabolized and degraded by C. pauculus include benzene, toluene [58],
13
3-nitrophenol [59], the herbicide 2,4-dichlorophenoxyacetic acid [60,61] and the pesticides
14
chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl)phosphorothionate) and TCP (3,5,6-
15
trichloro-2-pyridinol) [62,63]. As a representative xenobiotic, phenol (2 mM) supported the
16
growth of C. pauculus UM1 (Figure 3), using virtually all of the phenol by the end of the
17
experiment. A shorter induction (about 2-3 days) was observed if the concentration of phenol
18
was reduced to 1 mM (data not shown). C. pauculus UM1 also demonstrated the ability to use
19
phenol in the presence of either Cu2+ or Ni2+ (Table 2) which suggests that it may be an
20
effective bioremediation agent in environments where these metals are present. Preliminary
18
1
experiments show that C. pauculus UM1 grows in media with catechol as the sole C source
2
(data not shown). That C. pauculus UM1 uses phenol as a sole carbon source (Table 2, Figure
3
3) suggests that catechol is an intermediate as phenol is known to be metabolized via one of
4
two catechol-dependent pathways [64,65].
5
This report is the first, to our knowledge, to describe a member of the genus
6
Cupriavidus as being a heterotrophic nitrifier. That C. pauculus UM1 is a potent heterotrophic
7
nitrifier and denitrifier, is able to resist the toxic effects of copper and nickel while
8
heterotrophically nitrifying (Table 1, Figure 2 and MIC values of 3 mM Cu
9
Ni ) and can use phenol as a carbon source further expands the remarkable metabolic abilities
2+
and 1.0 mM
2+
10
of the genus and the species. Given C. pauculus UM1’s metabolic capacities, it will be of
11
interest to determine whether the microbe contains the metal resistance genes noted in other
12
Cupriavidus spp. as well as the genes necessary to metabolize xenobiotics such as those noted
13
above. We are investigating the use of such xenobiotics by C. pauculus UM1. If C. pauculus
14
UM1 does contain such genes, it may be employed as a bioremediation agent. Given the
15
bacterium’s resistance to copper and nickel, its metabolism of such xenobiotics may be
16
beneficial in those environments where metal tolerance is either an advantage or a necessity.
17
18
Conclusions
19
A soil microbe was examined for its ability to produce nitrogenous gasses while
20
heterotrophically nitrifying. The organism nitrified the organic substrate pyruvic oxime to
21
nitrite well but produced neither N2 nor N2O. Nucleotide and phylogenetic analyses resulted in
19
1
classifying the bacterium as a member of the genus Cupriavidus, thus establishing that this
2
genus can heterotrophically nitrify. The microbe, C. pauculus UM1, performed heterotrophic
3
nitrification in the presence of Cu
4
mM Ni
5
substrate for growth either in the absence or presence of 1 mM Cu2+ or 0.5 mM Ni2+.
2+
2+
2+
and Ni , nitrifying well when either 1mM Cu
2+
or 0.5
was present. C. pauculus UM1 also metabolized the xenobiotic phenol, using it as
6
7
Conflicts of Interest-The authors declare that there is no conflict of interest regarding the
8
publication of this paper.
9
10
Note Added in Proof-Subsequent to the submission of this manuscript, 16S rRNA gene
11
cloning of C. pauculus UM1 was performed using the primer pair of Yu et. al. (Yu, C,
12
Hongwei, G, Yanming, Z, Mingjun, D, Zhenxying, W, Laihua, Z, Qing, D, Biao, X, Chengzhu,
13
L, Zhiquin, Y, Xizhi X. (2012) Analysis of the bacterial diversity existing on animal hide and
14
wool: development of a preliminary pcr-restriction fragment length polymorphism fingerprint
15
database for identifying isolates. Journal of AOAC International. 95(6):1750-1754). These
16
primers are designed to clone from bp 27-1492 of the E. coli 16S rRNA gene. With respect to
17
base pairs 906-926, the sequence obtained was a perfect match to that previously obtained for
18
C. pauculus UM1’s 16S rRNA gene, further supporting the conclusion that the match between
19
nucleotides 906-926 was not the result of primer sequence resulting in errant sequence data.
20
21
Acknowledgments - The authors are indebted to Martin Berg, John Kelly, Catherine Putonti
20
1
and Samantha Lindemann for their helpful discussions and assistance concerning this work.
2
The support of a Loyola University Chicago Provost Award and of Loyola University Chicago
3
Mulcahy Scholarships are acknowledged as funding part of this work.
4
5
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32
1
Figure Legends:
2
Figure 1 Phylogenetic analysis of the bacterium of this study based on 16 S rRNA gene
3
homologies.a
4
a
5
100 bootstrap replicates. The dissimilarity bar, accession numbers of the sequences and the
6
species/strain designations used for comparison are included in the figure. Alcaligenes faecalis
7
IITRM4 served as the out-group.
Phylogenetic tree construction was done via the SeaView program using the NJ algorithm and
8
9
Figure 2 Growth (a) and nitrification (b) of C. pauculus UM1 in PO-mineral salts medium with
10
or without either 1.0 mM Cu2+ or 0.5 mM Ni2+ abcdef. Error bars represent the standard deviation
11
of a point.
12
a
13
logarithmic growth and used at 0.5% (v/v).
14
b
15
(p<0.01), or without Cu2+ or Ni2+ (p<0.01) were significantly different from their
16
corresponding uninoculated controls. Similarly, the NO2- synthesis curves with either 1.0 mM
17
Cu2+, 0.5 mM Ni2+ or without either Cu2+ or Ni2+ were significantly different (p<0.01) from
18
their corresponding uninoculated controls.
19
c
20
significantly different (p<0.01) than the corresponding uninoculated controls. In addition, at T
21
= 120 and 167 hours, C. pauculus UM1 cultures grown either without Cu2+ or Ni2+ or with 0.5
The inoculum for the cultures was grown in 0.2% PO-mineral salts medium until mid to late
The growth curves for C. pauculus UM1 with either 1.0 mM Cu2+ (p<0.05), 0.5 mM Ni2+
At the T = 24 and 72 hours, growth of all inoculated C. pauculus UM1 cultures was
33
1
mM Ni2+ were significantly different (p<0.01) than their corresponding uninoculated controls.
2
d
3
(p<0.01) than C. pauculus UM1 grown without Cu2+ or Ni2+.
4
e
5
different than the uninoculated 1 mM Cu2+ control.
6
f
7
inoculated cultures and their corresponding uninoculated controls. It is presumed that at the
8
early time points (T = 0 and perhaps 6 hours), this was due to NO2- carry-over with the inocula.
9
g
10
At T = 24 hours, the growth of C. pauculus UM1 with 0.5 mM Ni2+ was significantly less
At T = 120 and 167 hours, the growth of all C. pauculus UM1 cultures was not significantly
At all measurements, NO2- concentrations were significantly different (p<0.01) between the
At T = 24 hours, NO2- concentrations were significantly different (p<0.05) between the
inoculated PO-0.5 mM Ni+ cultures and inoculated PO cultures without either Cu2+ or Ni2+.
11
12
Figure 3 Growth of C. pauculus UM1 with phenol as the sole carbon sourcea. Error bars
13
represent the standard deviation of a point.
14
a
15
significant differences (p < 0.01) were noted for 244 and 288 hours.
For both the growth of C. pauculus UM1 and the amount of phenol remaining in the media,
16
34
1
2
3
4
5
6
7
8
9
10
11
Table 1. Growth and nitrification of PO by C. pauculus UM1 in the presence of either 1 mM
Cu2+ or 0.5 mM Ni2+ in either NB or PO-mineral salts medium.a,b
Time (d)
7
Growth (Abs600)
Growth Condition
0
Growth
(Abs600)
0
NO2 (mM)
NB-1 mM Cu2+
NB-1 mM Cu2+-PO
0.01 (0.00)
0.07 (0.00)
2.04
2.50
(0.09)
(0.26)
0.00 (0.00)
0.00 (0.00)
0.01 (0.02)
13.62 d (0.48)
NB-0.5 mM Ni2+
NB-0.5 mM Ni2+-PO
0.00 (0.00)
0.01 (0.00)
1.77 (0.04)
2.07d (0.06)
0.00 (0.00)
0.00 (0.00)
0.03 (0.01)
6.61d (1.29)
PO-1 mM Cu2+ (Uninoculated)
PO-1 mM Cu2+
0.01 (0.01)
0.00 (0.00)
0.00 (0.00)
0.14d (0.01)
0.00 (0.00)
0.03d (0.01)
-0.04 (0.00)
10.39d (0.32)
-
7
NO2 (mM)
-
PO-0.5 mM Ni2+ (Uninoculated)
0.00 (0.00)
0.00 (0.00)
-0.01 (0.01) -0.01 (0.00)
PO-0.5 mM Ni2+
0.00 (0.00)
0.23c (0.06)
0.12d (0.01) 12.36d (0.98)
a
Inocula for the NB-1 mM Cu2+ and the NB-0.5 mM Ni2+ experiments were grown in the NB1 mM Cu2+ and NB-0.5 mM Ni2+ medium, respectively, until late logarithmic growth and used
at 0.5% (v/v). Inocula for the PO-1.0 mM Cu2+ medium and for the PO-0.5 mM Ni2+ medium
were grown in 0.2% PO medium until late logarithmic growth and used at 0.5% (v/v).
b
Standard deviation values are in parentheses.
c
Statistically different from the corresponding control at p < 0.05.
d
Statistically different from the corresponding control at p < 0.01.
35
1
2
3
Table 2. Growth of C. pauculus UM1when in the presence of either no metals, 1 mM Cu2+ or
0.5 mM Ni2+ in the phenol-mineral salts medium.a,b
0
Growth
(Abs600)
3
Growth
(Abs600)
Time (d)
6
Growth
(Abs600)
Phenol
0.0018
(0.0005)
0.0800d
(0.0028)
0.0715d,e
(0.0038)
1.89
(0.12)
0.27d
(0.01)
0.20d,e
(0.01)
Phenol with
1 mM Cu2+
0.0920
(0.0094)
0.1417d
(0.0118)
0.1385
(0.0324)
1.89
(0.12)
0.27d
(0.02)
0.22d,e
(0.00)
Growth
Condition
4
5
6
7
8
9
10
11
0
Phenol
(mM)
3
Phenol
(mM)
6
Phenol
(mM)
Phenol with
0.0025
0.0769c
0.1793c
1.93
1.30
0.20d
2+
0.5 mM Ni
(0.0005)
(0.0252)
(0.0616)
(0.18)
(0.57)
(0.00)
a
Inocula was a 1:100 dilution of C. pauculus UM1 grown to mid-logarithmic phase in 2 mM
phenol-mineral salts medium. The relatively large t = 0 Abs600 value of the phenol with 1 mM
Cu2+ cultures was due to the blue color imparted to the medium by Cu2+.
b
Standard deviation values are in parentheses.
c
Statistically different from the initial (time 0) measurement at p < 0.05.
d
Statistically different from the initial (time 0) measurement at p < 0.01.
e
Statistically different from the day 3 measurement at p < 0.05.
36
1
Figure 1
2
3
4
37
1
Figure 2
2
3
4
38
1
Figure 3
2
39