N-Nitrate to Study Nitrate Transport in - American Chemical Society

17 Use of N-Nitrate to Study Nitrate Transport 13

in Klebsiella pneumoniae J. R. T H A Y E R and R. C. H U F F A K E R

Downloaded by TUFTS UNIV on March 13, 2017 | http://pubs.acs.org Publication Date: March 1, 1982 | doi: 10.1021/ba-1981-0197.ch017

Plant Growth Laboratory, and Department of Agronomy and Range Science, University of California, Davis, CA 95616

Radiolabeled nitrate produced by the proton bombardment of water was purified by high-pressure liquid chromatog raphy (HPLC) over anion exchange media and then used to determine the initial rates of nitrate uptake and nitrite excretion in Klebsiella pneumoniae. Nitrite was transiently excreted when aerobically grown cells were challenged with nitrate. Labeled nitrogen was lost when cells were subjected to vacuum filtration, so an alternative method of separating cells from the radioactive medium, by sedimentation through silicone oil, was adapted. This permitted us to analyze by HPLC the methanolic extracts of the cells after exposure to radiolabeled nitrate, confirming that NO - was indeed the compound transported. Initial rate data indi cated the presence of at least one and perhaps two nitrate transport systems. 3

The goal of our study is to understand the steps involved in the conversion of N 0 ~ to N H in soil bacteria. Since transport is the first step in this process, results are presented describing N 0 " transport. The organism chosen for study is Klebsiella pneumoniae, a soil and enteric bacterium capable of anaerobic nitrogen fixation, which harbors two N 0 " reductase systems. A n assimilatory system is synthesized under conditions of nitrogen starvation (I), when N 0 ~ alone or when N 0 ~ and N gas are present as the sole sources of nitrogen (2). The presence of N 0 " under conditions in which nitrogen fixation may occur represses the synthesis of nitrogenase, apparently through the presence of the second nitrate reductase system, which permits NO3" to replace oxygen 3

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0065-2393/81/0197-0341$05.00/0 © 1981 American Chemical Society

Root and Krohn; Short-Lived Radionuclides in Chemistry and Biology Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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SHORT-LIVED RADIONUCLIDES

as the terminal electron acceptor in the electron transport chain (2). In that case, the oxidation-reduction potential of the cell is altered such that a nitrogenase redox control fails to permit its synthesis. Under anaerobic or semianaerobic conditions the respiratory nitrate reductase system (RNR) is expressed, allowing a respiratory, rather than a less efficient fermentative, metabolism to occur. The activity of the RNR is one or more orders of magnitude greater than that of the assimilatory nitrate reductase ( A N R ) and, as a consequence, high levels of nitrite are produced and excreted into the medium. The respiratory system is inactivated by oxygen, so it is present only when the cellular oxygen tension is low (J). The assimilatory system is inactivated by pure oxygen but not by air, and it may be present under either aerobic or anaerobic conditions, where it can coexist with the RNR (1). Controversy exists regarding the site of N 0 ~ reduction in enteric and denitrifying bacteria. The RNR clearly resides in the cell membrane, as preparations of lysed cells, cleared of cytoplasmic constituents, produce NOV when incubated with a suitable electron donor under anaerobic conditions (I). Thus, N 0 " may be reduced on either the inside or outside of the cell membrane. Garland and others have presented evidence that N 0 " is reduced on the outside of the cell membrane, which results in proton translocation and release of N 0 " back into the medium (3,4,5). Kristajannson et al. have evidence that N 0 " may be transported and hence reduced inside the cells (6) and that the site of proton trans location need not be the RNR itself (7). N 0 ~ is toxic to bacteria, so the internal reduction of N 0 ~ must not exceed the reduction of N 0 " or N 0 " will accumulate inside the cells. If N 0 " reduction is internal, a N 0 ~ export system must be present under anaerobic conditions (since N 0 " appears in the medium when the cells are grown in the presence of N 0 " ) . The demonstration that N 0 ~ transport occurs would have a significant impact on the status of this controversy in addition to further elucidating the process of nitrate assimilation. Definitive transport experiments in bacteria require that initial uptake rates be obtained. This is most accurately done using radioactive isotopes. The longest lived radioisotope of nitrogen, N , has a half-life of 10 min and releases positrons that annihilate to produce gamma emissions with a 511-keV energy (8). The short half-life of N requires that it be used very soon after generation, but the nature of transport in bacteria is such that most experiments can be performed in a few minutes. Using N labeled N0 ~, we present evidence that N 0 " is transported into Klebsi ella pneumoniae, is reduced to N 0 " , which is transiently excreted, and is subsequently reabsorbed. 3

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17.

THAYER AND HUFFAKER

Nitrate in Klebsiella pneumoniae

343

Experimental Microbiology. Klebsiella pneumoniae strain M 5 a l was obtained from R. C . Valentine. The medium used for growth was that used by Cohen-Bazire et al. (9), with glucose as the carbon source and K N 0 as the nitrogen source. Semiaerobic growth was obtained in 50-mL cultures placed in 250-mL flasks and shaken at 100 rpm in a New Brunswick Scientific Incorporated Model G76 shaking water bath. Aerobic growth was obtained by spreading 125 /ig dry wt of exponentially growing cells on agar plates containing the same medium and incubating them at 30°C for 3-4 h. The plate-grown cells were harvested i n agar-free growth medium containing 15 fig chloramphenicol/mL to prevent induction of the R N R . After centrifugation at 15,000 X g for 15 min, the supernatant was tested for N 0 ~ (the absence of N 0 ~ ensures the absence of significant R N R activity) by the methods of Schrader et al. (10) or Thayer and Huffaker (11). The cultures were then washed and resuspended twice i n 0.1M phosphate buffer, p H 7.0, without a nitrogen source, containing 15 ftg chloramphenicol/mL, and kept on ice until used. Transport Assays. Cells obtained as described above were diluted to 0.05-0.5 mg dry wt/mL as desired, and N 0 " was added with the radio labeled N 0 " . A t appropriate time intervals (10-150 s) after the addition of radiolabeled nitrate, 1.0 m L of cell suspension was filtered through 0.45-/mi pore size, 25-mm-diameter Millipore filters, and washed with 5-10 m L of 50mM phosphate buffer containing lOmM K N 0 . Alternatively 0.5 m L of the cells were layered over 0.5 m L of silicone oil [Dow Corning; 75% 550, 25% 510-100 cs (v/v)] in Eppendorf 1.5-mL microfuge tubes in the microfuge (12). N 0 " was added using a Gilson Pipetteman micropipet to the indicated con centrations at 15-s intervals. The microfuge was started, reaching 12,000 g's in 5 s and stopped 25 s later. The aqueous layer was sampled for H P L C analysis, and the remaining liquid was aspirated off. The pellet was extracted with methanol and counted with constant geometry in a well y counter equipped with a sodium iodide crystal. Corrections were made for elapsed time (halflife) and for N binding to filters (when used) or dead cells on filters. Preparation of N 0 ~ was as described by Parks and Krohn (13), and purification of N 0 " from other products by anion-exchange H P L C was as described by Tiedje et al. (14) except that the purified N O " was harvested by hand. The radiochemical purity of N determined by analytical anionexchange H P L C was greater than or equal to 99.5% as N O " (Figure 1). The acidic eluate from the H P L C was neutralized with 10N N a O H prior to addi tion to cells. Chemicals. A l l chemicals were of analytical grade and no further purifi cation was performed. Chloramphenicol was from Sigma Chemical Company. 3


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Results Initial studies w i t h nitrate concentrations from 10/iM-10mM using a 2.5-min incubation suggested the presence of two uptake systems w i t h K s of about 15-20/AM and l - 2 m M . That experiment was repeated w i t h semiaerobically grown cells w i t h N 0 " concentrations bracketing those two values (Table I ) . What appeared initially to be two uptake systems m

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R A D I O N U C L I D E S


NO3

Figure 1. Analysis of N-labeled compounds (A) in target solution and (B) in nitrate sample purified by high-pressure anion exchange-liquid chromatography. Conditions: SOmM sodium phosphate buffer pH 2.9 was pumped at 1.5 mL/ min through a Whatman PartisiUlO SAX column (0.46 X 25 cm). Detection was by gamma counting using a well-type sodium iodide crystal. In (A) 350 /iL of the target solution was chromatographed 1 h after production. In (B) 50jiL of purified NO ~ was chromatographed 40 min after production of N. 13

1S

s

upon correction yielded a single system for N0 ~ uptake with a K of about 8/*M and a second uptake capability that was not saturated at the N O 3 ' concentrations used. The latter capability may reflect either the ability of N O 3 " to cross the cell membrane as a permeant anion (4) or the possibility that N0 ~ is reduced outside the cell membrane and that N0 " or NH are the transported compounds. To test the latter hypoth esis, the pattern of initial N0 " transport at N0 " concentrations from 10-80/xM and from 0.67-5mM were determined. In this experiment the semiaerobically grown cells were kept aerobic prior to and during incubation with N0 " by bubbling the culture with 0 . Label was taken up rapidly but was subsequently lost (Figure 2). Several such experi ments using incubations from 5-60 s yielded similar results. We interpret m

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T H A Y E R

Table I.

A N D H U F F A K E R

Uptake Rates of

[NO,-]

1 3

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N 0 " in Semiaerobically Grown Cells" 3

K After Corrections (S Cycles)' m

ixMol/min mg dry wt

Uncorrected Apparent K

2.46 3.16 3.38 4.38

9.5 fiM

SfjM

7.3mM

not saturating

10/xM

15/iM 25fiM 80(iM 0.67mM

5.72 9.44 12.1 33.2

l.OmM



1.5mM 5mAf

m

-v

I

( )

}

f

( /

• 0.2 mg dry wt of cells was incubated with N(>3~ for 2.5 min, vacuum-filtered over 0.45-/Am Millipore filters, and washed with 10 mL H M B + 10 m M KNO3 prior to counting. Corrections for the contribution of each apparent system to the other were made using sequential approximations of the K and Vmax values and substituting them in the Lineweaver-Burk equation, 1/7 = K /Vm*x (1/s) + l/7max. After 3 cycles the values did not change significantly. 13

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this pattern to mean that a product of N 0 ~ metabolism may be released. If the A N R is located in the cell membrane, then N 0 " may be released directly back into the medium; and if A N R is cytoplasmic, then N 0 " may be rapidly exported. Alternatively, N H as N H gas may be washed from the cells during filtration. [Interestingly, Sawada and Satoh (15) have shown that N 0 " reductase from Rhodopseudomonas sphaeroides forma sp. denitrificans can be released by removing the cell wall during spheroplast formation.] 3

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Figure 2. Initial NO " transport rate studies in Klebsiella pneumoniae using the membrane filtration technique, showing the binding of Nlabeled nitrate and the subsequent release of it or its metabolic products s

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RADIONUCLIDES

Table II. N 0 " Excretion and Disappearance by Aerobic Klebsiella Cultures Incubated with 0.5mM N 99.5%)

< 2.5/Jlf

10 30 60

8.1pM 6.5pM < 5.0/JW

• 0.6 mg dry wt of cells in 2.0 mL of medium was incubated with N(>3~ and at the indicated times 0.5 mL was withdrawn through a 0.45-/*m pore size filter into a syringe. This filtrate was analyzed by H P L C to determine the N0 ~ concentration. 13

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If N 0 " or N H are released during filtration of the cells, they should be detected in the filtrates. However, under aerobic conditions no N 0 ~ or N H accumulated in cultures of growing cells. So if NOo~ or N H is released, then it either represents an artifact of filtration or is an oscillation in the metabolism of N 0 " that is later stabilized. To distin guish between these possibilities, aerobic cultures were incubated with N 0 ~ at 0.5mM, filtered at various times thereafter, and the filtrate analyzed by H P L C (Table II). The appearance of N 0 " (but not N H ) at 10 and 30 s followed by its disappearance at 60 s suggests that N 0 ~ is rapidly reduced to N 0 " , which is released or excreted back into the medium and is then taken up again. This suggests that the release of label from filtered cells represents an oscillation in metabolism rather than an artifact of filtration. The transient external appearance of N 0 " also indicates that N i R and/or N 0 " transport activation may occur. Nitrite is toxic, so the relatively high intracellular N 0 " concentra tion potentially resulting from NOjf reduction supplies a rationale for either an external site for N 0 ~ reduction or the presence of a nitrite export system. Nitrite appears to be released from the cells; therefore the transport systems described earlier (using 2.5-min incubations) may reflect either


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Table III. Initial Rates of N0 ~ Transport in K. pneumoniae Assayed by Sedimentation Through Silicone Oil" 13

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Initial Rate (imol/min mg dry wt)

[NOf]

r

0.232 0.477 0.854 1.199

lyM 3fiM WfiM 30fiM

2

0.998 0.998 0.999 0.982

"Linear regression analysis on 0-45-s time points was used to determine initial rates. K and Fmax determination from the data above. 1/7 = K /V *x (1/s) + m

m

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1/Vmax is the equation used to determine K and Tmaxt K = 4AfiM, Vmax = 124 m

m

nil//min • mg dry wt, r of Lineweaver-Burk plot = 0.997. Assays are as described in the Experimental section. 2


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T H A Y E R


A N D H U F F A K E R

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Table IV. HPLC Analysis of Radioactivity Taken Up by and Cosedimenting with Cells of K. pneumoniae in Initial Rate Experiments ... Initial [NO3-] T

7

f , . Incubation Time(s)

Percentaqe of Pellet Counts Found as: _ i N0 NOi NHf

x

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15 45

lfiM lfiM 3/tM

>99 >93

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N-Nitrate to Study Nitrate Transport in - American Chemical Society

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