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1 Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA
2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA
3 Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA
Correspondence
Shawn R. Starkenburg
starkens{at}onid.orst.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and fixes carbon dioxide (CO2) as its sole source of carbon. The availability of the N. hamburgensis X14 genome sequence initiated a re-examination of its mixotrophic and organotrophic potential, as genes encoding three flavin-dependent oxidases were identified that may function to oxidize lactate, providing energy and carbon for growth. The response of N. hamburgensis to D- and L-lactate in the presence (mixotrophy) and absence (organotrophy) of was examined. L-Lactate did not support organotrophic growth or stimulate mixotrophic growth. In contrast, D-lactate enhanced the growth rate and yield of N. hamburgensis in the presence of , and served as the sole carbon and energy source for growth in the absence of with ammonium as the sole nitrogen source. Lithoautotrophically grown cells immediately consumed D-lactate, suggesting that a lactate metabolic pathway is constitutively expressed. Nevertheless, a physiological adaptation to lactate occurred, as D-lactate-grown cells consumed and assimilated lactate at a faster rate than -grown cells, and the D-lactate-dependent O2 uptake rate was significantly greater in cells grown either organotrophically or mixotrophically compared with cells grown lithoautotrophically. Although D-lactate was assimilated and metabolized to CO2 in the presence or absence of , exposure to atmospheric CO2 or the addition of 0.75 mM sodium carbonate was required for mixotrophic growth and for optimum organotrophic growth on D-lactate.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Nitrite-oxidizing bacteria (NOB) participate in the second step of nitrification by converting nitrite (
) into . NOB use as their primary energy source to generate reductant, assimilate carbon dioxide (CO2), and drive oxidative phophorylation. is a relatively poor energy substrate due to the positive midpoint potential of the – couple (
=+420 mV). Since it is estimated that 85–115 moles are required to fix 1 mole CO2 (Bock et al., 1986
, 1991), it is not surprising that some NOB use organic carbon as a carbon and energy source to reduce the burden of relying solely on to meet the energy demands of biosynthesis.
Although several phylogenetically distinct genera carry out oxidation (Nitrospira, Nitrobacter, Nitrococcus and Nitrospina), most of what is known about the organic carbon metabolism of NOB has been derived from studies of the genus Nitrobacter. Several reports using strains of Nitrobacter winogradskyi have shown that simple organic substrates, such as acetate, pyruvate and glycerol, can support organotrophic growth (Bock, 1976; Delwiche & Feinstein, 1965; Smith & Hoare, 1968; Steinmuller & Bock, 1976, 1977). Nevertheless, the bias of N. winogradskyi towards a lithoautotrophic lifestyle is supported by the fact that organotrophic growth rates are much slower than lithoautotrophic growth rates, and when CO2 is stripped from cultures containing organic carbon and (mixotrophy), these organic carbon sources cannot serve as the sole carbon source (Delwiche & Feinstein, 1965; Ida & Alexander, 1965).
Nitrobacter hamburgensis, a more recent isolate (circa 1983) of the Nitrobacter genus, has been described as having a greater organotrophic potential than N. winogradskyi, as an early description reported (without presenting data) that mixotrophic and organotrophic growth rates were faster than lithoautotrophic growth rates (Bock et al., 1983). Many investigations of N. hamburgensis have been conducted with cells grown mixotrophically (Harris et al., 1988; Kirstein et al., 1986; Laanbroek et al., 1994; Spieck et al., 1996), although it is not well understood how this bacterium utilizes, or adapts to, organic carbon. Some evidence of a physiological adaptation to organic carbon by N. hamburgensis exists, as cell membranes from mixotrophic and organotrophic cultures have been shown to contain different b-type cytochromes (Kirstein et al., 1986). Nevertheless, detailed studies of how organic carbon is processed in N. hamburgensis and how its metabolism is influenced by have not been completed. Furthermore, it is not known whether organic carbon can be used as the sole carbon source in the presence of (lithoheterotrophy), or whether organic carbon positively or negatively influences the rate of oxidation in this species.
The recent availability of the N. hamburgensis X14 genome sequence prompted a re-examination of mixotrophy and organotrophy in this bacterium, as three genes that could encode lactate dehydrogenases (LDHs) have been identified (Starkenburg et al., 2008). In contrast to the well-studied NAD-dependent LDHs, these genes encode homologues of flavin-dependent LDHs (iLDHs), which oxidize lactate to pyruvate and could provide energy and/or carbon to the cell (Garvie, 1980). In this study, the ability of N. hamburgensis to metabolize lactate was examined and its affect on lithoautotrophy was explored.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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was measured spectrophotometrically or colorimetrically, as described elsewhere (Hageman & Hucklesby, 1971). Whole-cell protein content was assessed using a modified Bradford method (Nelson et al., 1982). Cultures were routinely checked for contamination by plating 100 µl samples on Luria agar plates or by inoculating 1/10 nutrient broth (Difco) containing glucose (1 g l–1). Culture purity of lactate-containing cultures was additionally verified by visualization of cells at x25 000 magnification with a transmission electron microscope.
O2-uptake measurements.
N. hamburgensis cells were harvested by centrifugation from late-exponential phase cultures, washed, and resuspended in 50 mM potassium phosphate buffer, pH 7.5. Rates of lactate- or -dependent O2 uptake by cell suspensions were measured with a Clark-type O2 electrode (Yellow Springs Instrument) mounted in an all-glass, water-jacketed reaction vessel (1.8 ml volume) held at 30 °C.
Lactate consumption assay.
Lithotrophically, mixotrophically and organotrophically grown cells of N. hamburgensis were harvested by centrifugation from late-exponential phase cultures, washed twice, and resuspended to OD600 0.3–1.0 in sterile phosphate-buffered base medium (pH 7.5). Aliquots (5 ml) of cell suspensions were added to 38 ml culture bottles and sealed with Teflon-faced grey butyl rubber stoppers (Supelco) and fastened with aluminium crimp seals. CO2 in the headspace was measured with a thermal conductivity gas chromatograph (model GC-8A, Shimadzu) equipped with a 90 cm Porapak T column (Waters Associates) with the column temperature set to 150 °C and the detector set to 220 °C. The concentration of D-lactate was measured with a D-lactate assay kit (Megazyme International) according to the manufacturer's instructions.
[14C]lactate incorporation.
Resting cells of lithotrophically, mixotrophically and organotrophically grown N. hamburgensis were harvested by centrifugation from late-exponential phase cultures and resuspended in sealed 38 ml bottles containing 5 ml phosphate-buffered medium with 2 mM (NH4)2SO4, 1 mM D-lactate and 1.4 µCi (518 Mbq) DL-[1-14C]lactate (Sigma-Aldrich). N. hamburgensis was inoculated into these sealed bottles to OD600 0.4–0.6. Each culture bottle contained a 1.5 ml plastic tube containing a piece of Whatman filter paper soaked in 100 µl 18 M KOH to trap evolved CO2. At the end of the experiment (t=4 h), the bottles were opened, and the KOH-soaked filter paper was removed and added to a vial with 3.5 ml Ecolume scintillation fluid (ICN), and 14CO2 was measured on a Beckman 6500 multi-purpose scintillation counter. A 1 ml aliquot of the cell-free supernatant was added to another sealed vial containing a KOH trap and was acidified with 20 µl 12.1 M hydrochloric acid to measure the residual 14CO2 dissolved in the incubation medium. This quantity was added to the radiolabel measured in the first KOH trap to determine the total 14CO2 released from lactate. Cellular incorporation of radioactive 14C from D-lactate was measured in cells that were harvested by centrifugation, washed twice, and resuspended in 1 ml phosphate-buffered base medium. A 200 µl aliquot of the cell suspension was added to a vial containing 3.5 ml scintillation fluid, and cellular 14C incorporation was measured as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, to assess its mixotrophic and organotrophic potential. When N. hamburgensis was grown in medium supplemented with D-lactate and , the growth rate and final cell yield increased 50 and 60 %, respectively, compared with cultures grown lithoautotrophically on medium containing and CO2 (Fig. 1a). Concomitantly, an increase in the total protein content was also observed in N. hamburgensis cells grown mixotrophically with D-lactate and (Fig. 1c). Because D-lactate did not affect the gross amount of consumed (Fig. 1b), the increased growth rate and cell yield indicated that less was consumed per OD600 unit under mixotrophic conditions. Growth was also observed in N. hamburgensis cultures incubated with D-lactate as the only source of energy (organotrophy) (Fig. 1a). Despite the fact that equal quantities of D-lactate were consumed by both mixotrophic and organotrophic cultures, the cell yield of organotrophically grown cultures was 50 % less than that of mixotrophic cultures containing D-lactate and . Similarly, when the amount of protein recovered from these cultures was normalized to OD600, organotrophically grown cells contained 30 % less protein per OD600 unit than lithoautotrophically or mixotrophically grown cells.
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and CO2, and growth was not detected when L-lactate was added in the absence of . Furthermore, the optical densities of cultures amended with a combination of L- and D-lactate were not significantly different from those of cultures containing only D-lactate (data not shown), indicating that L-lactate does not inhibit (or enhance) the consumption of D-lactate.
The effect of on lactate consumption was measured in resting cells harvested from both lithoautotrophic and mixotrophic cultures (Table 1). Cells harvested from lithoautotrophic cultures readily consumed lactate at 1.17 µmol D-lactate (mg protein)–1 h–1. This short-term rate of lactate consumption by lithoautotrophically grown cells was not significantly affected by the addition of . Mixotrophically grown cells consumed lactate at a faster rate [1.67 µmol D-lactate (mg protein)–1 h–1] than lithoautotrophically grown cells, suggesting that N. hamburgensis had adapted to growth on lactate. In the presence of , the short-term rate of lactate consumption by mixotrophically grown cells decreased by 15 %.
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). D-Lactate supported significantly higher rates of respiration in organotrophically and mixotrophically grown cells compared with cells grown autotrophically. Additionally, -dependent respiration was reduced after organotrophic growth on lactate, whereas mixotrophically grown cells maintained high rates of both - and lactate-dependent O2 uptake. When the rate of lactate consumption and the rate of lactate-dependent oxygen uptake were compared, mixotrophically grown cells appeared to be more effective at using lactate to support respiration, as lactate-dependent oxygen uptake in mixotrophically grown cells was threefold higher than the rate of lactate-dependent oxygen consumption by lithoautotrophically grown cells (Table 2), despite the fact that mixotrophically grown cells consumed only 1.5-fold more lactate than lithoautotrophically grown cells (Table 1
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and in the absence of CO2. Growth and consumption were monitored in a closed system (preventing the influx of atmospheric CO2) during treatment with sodium carbonate (Na2CO3) and/or D-lactate as carbon sources (Table 3). In the absence of an added carbon source, growth did not occur in the closed system, despite 
14 mmol being consumed during the experiment (4 days). When 0.5 mM D-lactate was provided as the sole source of carbon, measurable growth did not occur and consumption was similar to that of the negative control cultures without an added carbon source. When a limiting amount of Na2CO3 (0.19 mM) was added, in the presence or absence of D-lactate, similar amounts of growth and consumption were measured (Table 3). When Na2CO3 was provided in excess (0.75 mM), the OD600 increased twofold over cultures containing 0.19 mM Na2CO3, and nearly all of the was consumed. In contrast to the lack of stimulation observed when D-lactate was added to CO2-limited cultures, the growth yield of cultures containing both D-lactate and 0.75 mM sodium carbonate increased significantly (45 %) compared with cultures containing 0.75 mM Na2CO3 alone. Growth and consumption were not affected when a higher concentration (1.5 mM) of Na2CO3 was added, or if parallel culture bottles were exposed to atmospheric CO2 (data not shown), indicating that 0.75 mM Na2CO3 was saturating lithoautotrophic growth.
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on lactate and CO2 metabolism slightly affected lactate consumption in mixotrophically grown cells under CO2-replete conditions (Table 1), it was possible that was affecting lactate consumption and organotrophic growth when CO2 was limiting. Therefore, growth, net CO2 production and lactate consumption were measured in a closed system in cultures containing D-lactate amended with 0, 7.5, 15 or 30 mM initially containing an atmospheric concentration of CO2 (Fig. 2). In cultures amended with , the ambient CO2 was quickly fixed and remained undetectable until after the was consumed (Fig. 2c). Although lactate was initially consumed in all treatments, lactate consumption increased in cultures without and as the was depleted in others (Fig. 2b, d). In the presence of 7.5 mM , the same amount of lactate was consumed and growth was enhanced by 20 % compared with cultures containing lactate alone. In contrast, in cultures amended with higher concentrations of (15 and 30 mM), growth was almost completely inhibited, CO2 remained undetectable, and lactate consumption was suppressed until after depletion of . The presence of also negatively affected the rate of lactate consumption. For example, when the lactate consumption values (Fig. 2d) were normalized to changes in OD600 (Fig. 2a), the initial rates of lactate consumed per cell in all treatments ranged from 39 to 130 µg lactate-C (OD600 unit)–1 h–1. Lactate consumption increased to a maximum rate of 284 µg lactate-C (OD600 unit)–1 h–1 by 66 h in cultures not amended with . Yet, in cultures amended with 15 mM , a low rate of lactate consumption [69 µg lactate-C (OD600 unit)–1 h–1] was sustained until after the was depleted, reaching a maximum rate [182 µg lactate-C (OD600 unit)–1 h–1] by 118 h. Taken together, these results suggest that is suppressing lactate consumption and that CO2 fixation continues to occur in the presence of and lactate. Nevertheless, because lactate was consumed constitutively at a low rate, once the was depleted (or the -induced CO2 limitation was relieved) growth was able to occur with all treatments.
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on lactate and CO2 assimilation, the consumption and distribution of [1-14C]D-lactate were measured in cells harvested from lithoautotrophically, mixotrophically or organotrophically grown cultures of N. hamburgensis (Table 4). Regardless of how N. hamburgensis was grown, when cells were incubated with lactate in the absence of , most 14C from lactate was retrieved as CO2, and only 31–42 % of the 14C was incorporated into cells. In contrast, in the presence of , a greater percentage of lactate-C was assimilated (61–68 %) and the percentage of 14C retrieved as CO2 decreased significantly. More [14C]lactate was consumed by organotrophically grown cells than by autotrophic and mixotrophically grown cells, further suggesting a physiological adaptation to growth on lactate as a sole energy source. In the presence of , more 14C was incorporated into cell material, regardless of how cells were grown, yet the proportional distribution and amount of 14C in cellular material versus CO2 did not shift as drastically in cells that had been exposed to lactate previously. Furthermore, the total amount of [14C]lactate consumed by lithoautotrophically grown cells was unaffected by the presence of , whereas lactate consumption decreased 18 and 25 % in mixotrophically and organotrophically grown N. hamburgensis, respectively.
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and CO2, and (2) CO2 fixation appeared to be induced by , we wanted to determine whether the suppressive effect of on lactate metabolism was caused indirectly by a CO2 limitation. N. hamburgensis was grown organotrophically on lactate in sealed culture bottles with and without a CO2 trap. In organotrophic cultures without a CO2 trap, almost 90 % of the lactate (0.5 mM) was consumed by 96 h (Fig. 3b). When CO2 was stripped from replicate cultures, the initial rates of growth and lactate consumption were not significantly altered. By 72 h, the rate of growth and lactate consumption slowed, leading to a 20 % decrease in growth yield (Fig. 3a, b). In contrast, when was added in lieu of a CO2 trap, N. hamburgensis did not grow at all and 75 % of the initial concentration of lactate was not metabolized.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The reason for the lack of growth on L-lactate is unclear as the putative L-isomer-specific iLDH (EC 1.1.2.3, Nham_1112) shares 78 % protein sequence identity with an iLDH in the close alphaproteobacterial relative of N. hamburgensis, Rhodopseudomonas, which can metabolize and grow on L-lactate (Horikiri et al., 2004; Markwell & Lascelles, 1978). Candidate genes for D-lactate metabolism include Nham_4010, a gene which encodes an FAD/FMN-dependent oxidase (EC 1.1.2.4) with an unspecified substrate range, and a putative glycolate oxidase (Nham_3202-4), which has been shown to turn over D-lactate in Escherichia coli (Lord, 1972). Transport may be a limiting factor for metabolism of both D- and L-lactate, since the only identified lactate permease gene (Nham_2174) is likely a pseudogene, given that a translated protein product would span two reading frames. Other candidate lactate transporters include a putative tellurite-resistance/dicarboxylate transporter (TDT)-family transporter gene located in the operon that encodes the oxidoreductase. Similar TDT-family members have been shown to transport dicarboxylic acids (Grobler et al., 1995). Further investigation is required to determine which genes encode the products that are responsible for both the uptake and the initial metabolism of D-lactate.
Our results for the metabolism of organic carbon by N. hamburgensis appear to differ from published literature regarding organotrophy in both N. winogradskyi and N. hamburgensis. First, although stimulates acetate assimilation by both lithoautotrophically and organotrophically grown cells of N. winogradskyi (Smith & Hoare, 1968), did not affect lactate consumption by lithoautotrophically grown cells of N. hamburgensis, and even slightly reduced the rate of lactate consumption in mixotrophically and organotrophically grown cells. Second, although organic compounds in general positively affect the rate of oxidation in other Nitrobacter species (Steinmuller & Bock, 1976; Tandon & Mishra, 1968), our data suggest that lactate does not increase the rate of oxidation in N. hamburgensis, but instead reduces the requirement of the cell, as evidenced by a faster growth rate and an increase in growth yield in mixotrophic versus lithoautotrophic conditions. Third, an early description of the growth phenotype of N. hamburgensis indicated that organotrophic growth rates on pyruvate, acetate or glycerol were faster than lithoautotrophic growth (Bock et al., 1983). In our laboratory, the generation time of D-lactate-grown cells was threefold slower than that of lithoautotrophic cultures, and growth rates on pyruvate or acetate were also consistently slower than either lithoautotrophic or mixotrophic growth (our unpublished results). The discrepancy in organotrophic growth rates between our studies and other reports may be explained by the fact that a chemically defined minimal medium was used in our experiments, whereas in earlier investigations, N. hamburgensis was grown in an undefined, complex medium containing yeast extract and peptone (Bock et al., 1983; Harris et al., 1988; Kirstein & Bock, 1993; Sundermeyer-Klinger et al., 1984). We have observed that ammonium amendment of -containing cultures enhances the growth rate and cell yield of lithoautotrophically grown N. hamburgensis (our unpublished results), implying that reduced forms of organic nitrogen could have stimulated the faster mixotrophic and organotrophic growth rates of N. hamburgensis reported in the earlier studies.
Although a lactate metabolic pathway in N. hamburgensis appears to be constitutively expressed, our results indicate that the physiology of N. hamburgensis changes in response to lactate, in both the presence and absence of . The rates of D-lactate consumption and D-lactate-dependent O2 uptake were faster in organotrophically compared to lithoautotrophically grown cells, and others have shown that a shift occurs in the cytochrome and FMN content of organotrophically grown N. hamburgensis (Bock et al., 1986; Kirstein et al., 1986). These adaptive changes also complement our observation that lactate-dependent O2 uptake rates were threefold higher in organotrophically and mixotrophically grown cells compared with cells harvested from lithoautotrophic cultures.
Despite these physiological adaptations, N. hamburgensis could only use D-lactate as the sole carbon source in the absence, but not the presence, of , suggesting (but not proving conclusively) that lithoheterotrophic growth is not possible. The apparent inability of N. hamburgensis to grow on and D-lactate (a C3 molecule) as the sole energy and carbon source, respectively, could be the result of an insufficient supply of the CO2 required for biosynthetic carboxylations. However, this hypothesis is not consistent with findings from an earlier study in which various C4–C6 compounds did not rescue growth of N. winogradskyi in the absence of CO2 (Delwiche & Feinstein, 1965). Alternatively, the lack of growth and the reduced rate of lactate metabolism in the presence of during CO2 limitation may be explained by a redox imbalance. Since reductant for biosynthesis is thought to be generated via an energetically unfavourable reverse flow of electrons from to complex I (Sewell & Aleem, 1969), continuous consumption of reductant by CO2 fixation (and the concomitant maintenance of an oxidized pool of electron carriers) may be required to sustain reverse electron flow from . Use of CO2 fixation as a ‘reductant sink’ is not unprecedented, as other alphaproteobacteria maintain redox balance by fixing CO2 to regenerate oxidized electron carriers during photoheterotrophic growth (Dubbs & Tabita, 2004; Shively et al., 1998). Similarly, if oxidized electron carriers cannot be regenerated efficiently by N. hamburgensis in the absence of CO2, they would be unavailable to serve as electron acceptors to oxidize lactate. It is possible that N. hamburgensis is able to overcome this redox imbalance after a more prolonged adaptation to organotrophic growth, since reduced electron carriers generated from lactate oxidation must be driving a forward flow of electrons to oxygen to generate ATP. When other facultative lithoautotrophs encounter organic carbon, in many cases a complete repression of autotrophic CO2 fixation occurs (Shively et al., 1998; Tabita, 1988). On the other hand, in both Thiobacillus intermedius and Ralstonia eutropha, RuBisCO and other Calvin cycle enzymes are only partially repressed by the presence of some organic carbon sources and fully repressed by others (Shively et al., 1998; Tabita, 1988). At least some repression of autotrophy occurs in Nitrobacter, because RuBisCO activity has been reported to be suppressed 50–99 % in organotrophically grown cells of N. winogradskyi strain ‘agilis’ (Smith & Hoare, 1968; Steinmuller & Bock, 1977) and our results indicate that -dependent O2 uptake was also suppressed by 40 % after growth solely on lactate. Undoubtedly, further experimentation is required to determine the details of how the redox state of a -driven autotrophic metabolism in N. hamburgensis is affected by D-lactate.
In summary, our data suggest that Nitrobacter can take full advantage of D-lactate when it is the sole source of energy, yet if is present, the organism's heterotrophic and mixotrophic potential is hampered by an inability to stop fixing CO2 and efficiently switch to an organic carbon source. Further investigations of the obligate requirement of Nitrobacter for CO2 during growth on may help elucidate the physiological constraints of utilizing relatively poor inorganic energy sources, and aid in understanding how N. hamburgensis and other facultative lithoautotrophs regulate the metabolism of mixed energy and carbon sources.
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ACKNOWLEDGEMENTS |
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Edited by: H. L. Drake
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Bock, E., Sundermeyer-Klinger, H. & Stackebrandt, E. (1983). New facultative lithoautotrophic nitrite-oxidizing bacteria. Arch Microbiol 136, 281–284.[CrossRef]
Bock, E., Koops, H. P. & Harms, H. (1986). Cell biology of nitrifiers. In Nitrification, pp. 17–38. Edited by J. I. Prosser. Oxford: IRL Press.
Bock, E., Koops, H. P., Harms, H. & Ahlers, B. (1991). The Biochemistry of nitrifying organisms. In Variations in Autotrophic Life, pp. 171–200. Edited by J. M. Shively and L. L. Barton. San Diego, CA: Academic Press.
Delwiche, C. C. & Feinstein, M. S. (1965). Carbon and energy sources for the nitrifying autotroph Nitrobacter. J Bacteriol 90, 102–107.
Dubbs, J. M. & Tabita, F. R. (2004). Regulators of nonsulfur purple phototrophic bacteria and the interactive control of CO2 assimilation, nitrogen fixation, hydrogen metabolism and energy generation. FEMS Microbiol Rev 28, 353–376.[CrossRef][Medline]
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Grobler, J., Bauer, F., Subden, R. E. & Van Vuuren, H. J. (1995). The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids. Yeast 11, 1485–1491.[CrossRef][Medline]
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Laanbroek, H. J., Bodelier, P. & Gerards, S. (1994). Oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter hamburgensis grown in mixed continuous cultures at different oxygen concentrations. Arch Microbiol 161, 156–162.[CrossRef]
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Markwell, J. P. & Lascelles, J. (1978). Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides. J Bacteriol 133, 593–600.
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Shively, J. M., van Keulen, G. & Meijer, W. G. (1998). Something from almost nothing: carbon dioxide fixation in chemoautotrophs. Annu Rev Microbiol 52, 191–230.[CrossRef][Medline]
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Spieck, E., Muller, S., Engel, A., Mandelkow, E., Patel, H. & Bock, E. (1996). Two-dimensional structure of membrane-bound nitrite oxidoreductase from Nitrobacter hamburgensis. J Struct Biol 117, 117–123.[CrossRef]
Starkenburg, S. R., Larimer, F. W., Stein, L. Y., Klotz, M. G., Chain, P. S., Sayavedra-Soto, L. A., Poret-Peterson, A. T., Gentry, M. E., Arp, D. J. & other authors (2008). Complete genome sequence of Nitrobacter hamburgensis X14 and a comparative genomic analysis of species within the genus Nitrobacter. Appl Environ Microbiol 74, 2852–2863.
Steinmuller, W. & Bock, E. (1976). Growth of Nitrobacter in the presence of organic matter. I. Mixotrophic growth. Arch Microbiol 108, 299–304.[CrossRef][Medline]
Steinmuller, W. & Bock, E. (1977). Enzymatic studies on autotrophically, mixotrophically and heterotrophically grown Nitrobacter agilis with special reference to nitrite oxidase. Arch Microbiol 115, 51–54.[CrossRef][Medline]
Sundermeyer-Klinger, H., Meyer, W., Warninghoff, B. & Bock, E. (1984). Membrane-bound nitrite-oxidoreductase of Nitrobacter: evidence for a nitrate reductase system. Arch Microbiol 140, 153–158.[CrossRef]
Tabita, F. R. (1988). Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms. Microbiol Rev 52, 155–189.
Tandon, S. P. & Mishra, M. M. (1968). Effect of some organic acids on nitrification by Nitrobacter agilis. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg 122, 401–404.[Medline]
Received 29 February 2008;
revised 10 April 2008;
accepted 14 April 2008.
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. Turbidometric growth, 
), 1 mM L-lactate (
), 45 mM 
), 45 mM 
), or 45 mM 
). Measurements depicted in (c) are from a representative experiment harvested at 100 h; filled and chequered bars represent OD600 and protein content, respectively.
consumption by N. hamburgensis in response to the presence of carbonate and D-lactate
) and 30 mM 
). Growth (a), concentration of 