Pyridine Nucleotide Metabolism in Escherichia coli

SUMMARY The effect of niacin starvation has been studied in a niacin-requiring auxotroph of Escherichia coli. If a culture is totally deprived of niacin, cells continue to divide until the total pyridine nucleotide content has fallen from 1.9 X lo6 to 1.2 X 10” molecules per cell. During starvation, the relative proportion of the pyridine nucleotides changes greatly: the TPN: DPN ratio increases from 0.30 to over 2.0 and nicotinic acid mononucleotide accumulates until it is present at concentrations comparable to DPN. The changes in the distribution of the pyridine nucleotides suggest that DPN is turning over during niacin starvation and that the normally observed breakdown of TPN to DPN during exponential growth is inhibited. If cells are starved for niacin by balanced growth under limiting concentrations of niacin, less disproportion in the TPN : DPN ratio is observed, and growth occurs with a pyridine nucleotide content as low as 4 x lo4 molecules per cell. The fate of the niacin ring in Escherichia coli during normal exponential growth is metabolically

requiring auxotroph of Escherichia coli. If a culture is totally deprived of niacin, cells continue to divide until the total pyridine nucleotide content has fallen from 1.9 X lo6 to 1.2 X 10" molecules per cell. During starvation, the relative proportion of the pyridine nucleotides changes greatly: the TPN: DPN ratio increases from 0.30 to over 2.0 and nicotinic acid mononucleotide accumulates until it is present at concentrations comparable to DPN.
The changes in the distribution of the pyridine nucleotides suggest that DPN is turning over during niacin starvation and that the normally observed breakdown of TPN to DPN during exponential growth is inhibited.
If cells are starved for niacin by balanced growth under limiting concentrations of niacin, less disproportion in the TPN : DPN ratio is observed, and growth occurs with a pyridine nucleotide content as low as 4 x lo4 molecules per cell.
The fate of the niacin ring in Escherichia coli during normal exponential growth is metabolically limited.
Once the niacin ring is taken up, it is not excreted into the medium.
Inside the bacterial cell over 95% is found as pyridine nucleotides DPN and TPN even after many generations (1).
In the studies described in this series of papers, we have attempted to define the various pathways through which the niacin ring can flow in E. coli and to understand the factors which regulate the flow of the niacin ring through the various pathways. Our particular interest in these studies sterns from the discovery that T1PN is the cofactor for DNA ligase in bacterial cells (2,4), a molecule of DPN being broken down to nicotinamide mononucleotide for each DNA-joining event. It is the ultimate aim of our work to examine DNA ligase activity under different physiological conditions by measuring the kinetic parameters of niacin metabolism in Go. In this paper, we have studied the effect of niacin starvation on pyridine nucleobide metabolism in E. co&. Niacin starvation * This investigation was supported by Nat.ional Science Foundation Grants GB 8610 and GB 22869X, and by the Natural Science Research Council, University of the Philippines.
causes an obvious pertubation on pyridine nucleotide metabolism: we have examined how this pertubation affects other reactions involving the niacin ring within the cell. For example, in our previous studies, we found that the TPN:DPN ratio was maintained largely by the rates of interconversion of TPN and DPN.
In these studies we show that the rates of these interconversion reactions appear to change with the total pyridine nucleotide concentration.
Niacin starvation has also been used by other workers to examine DNA-joining activity (4). These earlier studies examined the fate of the DNA during the starvation but did not examine the effect on the pyridine nucleotides.
We are undertaking a more detailed analysis of this physiological condition here.
In the following paper (III) we show how the regulation of endogenous synthesis and exogenous uptake of the niacin ring are regulated in vivo to yield a relatively constant level of total pyridine nucleotide (5).
In Paper IV of this series, the turnover cycles of pyridine IW cleotide metabolism arc defined and measured (6). Biochemicals. All other biochcmicals and enzymes were obtained from Sigma.
Other materials arc described in the previous paper (1). Starvation Experiments-Sicotinic acid-requiring strains of E. coli were starved for niacin in the following way. The strain was grown in an M9 medium containing 8.1 X lop6 M (1 pg per ml) [%]niacin to a cell density of about 3 x lo* cells per ml and then filtered rapidly on a Millipore filter (Bact-T-Flex B6, Schleicher and Schuell), washed with M9 salts (with no glucose), and resuspended and grown in a 20.fold larger volume of M9 medium containing no niacin.
The number of cells per ml, the absorbance at 595 nm, and the upt,ake of 14C were followed as a function of time.
Niacin-limited Growth Experiments-Cultures were grown under conditions of limiting niacin as follows.
A culture of E. coli 15T-nit-was grown in M9 medium containing 4.1 X 5137 1OP M (0.5 pg per ml) of unlabeled niacin.
Each culture was allowed to grow for at least five generations in this medium before being diluted at least 50.fold into an identical medium.
The absorbance at 595 nm, cell density, and [14C]niacin uptake was then followed.
This series of dilutions was necessary to make sure that balanced growth was occurring at the limiting niacin concentrations.
Measurement of Bacterial Volume-Cell volumes were measured at various stages of niacin starvation.
Electron microscopy was performed as described by Chai and Lark (7) on an RCA-2 electron microscope.
Cells were also examined by phase contrast illumination in a Zeiss p:~otomicroscope using an apochromatic oil immersion objet ive (100 X) Micrographs were made using ADOXKI314 film. Volumes were estimated from well focused cells on positive prints at x 3000 magnification.
For exponentially growing cultures, the cells were assumed to be cylindrical with two half spheres at both ends of t'he cylinder. Measurements were then taken of the length and width of the cylindrical portion of the cell and of the radii of the hemispheric ends. The cells that had been severely starved for niacin were more nearly spherically shaped, and the diameter of the sphere was measured on the photographs.
We arc indebted to Dr. David Wolstenholme for advice regarding these measurements, and for taking the photographs of the bacteria.
Other Methods-Methods for measurements of radioactivity taken up by the cell, for preparation of ccl1 extracts, and for chromatography of the pyridine nucleotides are described in the previous paper (1).
Bacterial Strains-All niacin-requiring strains of E. coli used were described previously (1).

General EJects of Starvation for Niacin-The
behavior of niacin auxotrophs of B. coli durin, g niacin starvation is shown in Fig. 1. In this experiment, cells are grown in a medium containing ['%]niacin, and at the time indicated, the culture is filtered, washed, and transferred to a medium without niacin. After transfer, the cells continue to divide with an unchanged or slightly faster growth rate for over 2 hours, after which cell division stops. During late starvation, substantial leakage of the intracellular radioactivity into the medium takes place. This is not due to cell lysis as there is no measurable decrease in viability.
The leakage is a specific function of niacin starvation which stops when unlabeled niacin is added to the medium.
Cells deprived of niacin were examined by electron and photomicroscopy.
Normal cells are more elongate than the almost spherical starved cells. Normal cells and starved cells were measured as described under "Experiment'al Procedures" and a continuous decrease in cell volume was found during starvation. Cells that were no longer dividing were found to be 30% of normal volume.
Electron micrographs show that while cxponentially growing cells generally have two nuclear regions, starved cells have one. Nozawa and Mizuno (4) have reported the formation of unusually long forms during niacin starvation in the auxotroph they have investigated. These forms do not occur to any significant extent upon starvation of E. coli 15Tnit-.
The growth characteristics of E. coli during niacin starvation are generally reproducible in the four niacin-requiring strains (15T-nit-, nit A, nit B, and nit C) which we have tested al-though a certain amount of variation in the amount of growth after transfer to the medium lacking niacin, as well as in the rate of leakage of the radioactivity into the medium has been observed.
However, in all cases? for 2 hours after deprivation of niacin, the rate of cell division was equal to or greater than the normal rate.
Changes in Intracellular Distribution OJ" Pyridine Nucleotides during Niacin Starvation- Fig.  2 shows the intracellular distribution of pyridine nucleotides at various times after transfer to a medium lacking niacin.
A marked transition takes place 1. Niacin starvation of Escherichia coli 15T-nit-. A culture of E. coli 15'lY nit-was grown in an M9 medium containing 8 X lo-" M [14C]niacin (59.1 Ci per mole). At the time indicated by the arrow an aliquot of the culture was filtered on a Millipore membrane filter, washed with M9 salts solution without glucose (M9-G), and transferred to an MS medium without niacin at a 15.fold dilution.
The cell density was determined at regular intervals by counting aliquots of the culture on a Coulter counter.
Uptake of [14C]niacin was measured by filtering a O.lml aliquot of the culture on a membrane filter and washing with M9-G containing 40 rg per ml of unlabeled niacin.
The open circles and triangles are measurements of [14C]niacin uptake in the culture with and without niacin, respectively.
The closed circles and triangles are the cell density in the culture with and without niacin, respectively. In addition, a new peak of radioactivity is found (traveling between DPN and TPN on DEAE-paper). Three hours after the cells have been deprived of niacin, the We have identified the new peak of radioactivity as nicotinic acid mononucleotide &MN). The radioactivity isolated from the new peak traveled with authentic nicotinic acid mono-amount of this new material and of DPN are approximately nucleotide on DEAE-paper chromatography.
When both the radioactivity in the new peak and authentic carrier NaMN were equal.
treated with bacterial alkaline phosphatase, the optical density and the radioactivity still traveled together upon chromatography, in a spot which moved just behind the solvent front (presumably nicotinic acid ribose) . On treatment with alkali, most of the radioactivity, as well as the ultraviolet absorbing material derived from authentic N,MN, traveled with the RF of nicotinic acid (Fig. 3).
As shown in Fig. 1, a substantial  medium. This radioactivity which is excreted into the medium has been examined by CEAE-paper chromatography (Fig. 4), and it is seen to travel just behind the solvent front as a single peak.
The relative distribution of the [r4C]niacin label as a function of the time of starvation is shown on Fig. 5.

This material has been tentatively identified as nicotinic
The intracellular acid ribonucleoside since it travels on chromatography with the and extracellular levels of the pyridine nucleotides at various times after starvation are summarized on Table I. RF of authentic nicotinic acid ribonucleoside.
Recovery from Starvation-The effect of adding back niacin to a culture of starved cells has been examined.
In Fig. 6, it is seen that 30 min after niacin is added, normal intracellular levels are reached and cell division begins to take place. Cells return to their normal size during this 30.min lag period.
The results of chromatography of the pyridine nucleotides during starvation and after niacin has been added back are shown in Fig. 7 Under the growth conditions used, the niacin content per cell was found to be from 2 to 20% of normal levels. These cells divide with a significantly longer generation time than do normally growing cells (Table II) The data were calculated from niacin uptake curves (Fig. l), and chromatographic analysis of the intracellular pyridine nucleotides (see Fig. 2). distribution during starvation Eseherichia coli 15T-nit-was grown in an M9 medium containing [WJniacin, and then starved as described under "Experimental Procedures" and in the legend to Fig. 1. Cell extracts were prepared (Fig. 2), and the intracellular pyridine nucleotide distribution calculated from the chromatographic data. Intracellular niacin and percentage of original niacin found extracellularly were determined from uptake and cell density curves. This shows that although the total niacin per cell may be the same in cultures starved in different ways, the resulting distribution of the niacin is different.
A decrease in cell size was observed under all niacin-limited conditions of growth.
A cell growing in 0.05 pg per ml of niacin with a generation time of 70 min had an average cell volume 50% smaller than cells growing with the normal generation time for this medium (40 min).

Recovery from niacin starvation.
A culture of Escherichia coli 151' nit-was grown in 8 X 10-C M [**C]niacin and then starved for niacin as described under "Experimental Procedures." After 400 min of starvation, cells were transferred to an M9 medium containing 8 X 1Om6 M PHIniacin (787 mCi per mmole).
Uptake of radioactivity and cell density were followed at regular intervals throughout the experiment.
The open circles are 1% uptake points; closed circles, cell density; and triangles, [sH]niacin uptake.

DISCUSSION
In previous studies (l), we found that the relative levels of DPN and TPN in E. coli were maintained by a steady state balance between the de 1u)vo synthesis of DPN, the synthesis of TPN from DPN, and the breakdown of TPN to DPN.
It could be predicted that by altering the relative contribution of any one of the three factors, a change in the TPN :DPN ratio would occur. Thus, inhibiting the de nova synthesis of DPN should not only lower the levels of the total pyridine nucleotide in the cell, but change the proportion of TPN to DPN as well. We have inhibited de rwvo DPN synthesis by imposing niacin starvation on a mutant of E. coli which is unable to synthesize niacin endogenously.
Our previous studies (1) lead to the prediction that if the rates of DPN and TPN interconversion remain constant the TPN:DPN ratio should increase from 0.30 to 0.57 during starvation.
We in fact, observe that the TPN:DPN ratio increases continuously beyond a value of 0.57. There is also a net conversion of DPN to nicotinic acid mononucleotide and nicotinic acid ribonucleoside.
The conversion of DPN to N,MN and nicotinic acid ribonucleoside may be a consequence of normal pyridine nucleotide turnover.
The results suggest that niacin starvation causes the inhibition of a metabolic reaction believed to be involved in pyridine nucleotide turnover (8), the N,AD pyrophosphorylase reaction, N,MN + ATP -+ N,AD + PPi.
If this reaction were differentially affected by starvation possibly because of the direct requirement for ATP, the normal turnover of DPN would then result in conversion of DPN to N&W.
In addition, if N,MN accumulated during starvation, there is a possibility that it would be acted on by a phosphatase and be transformed to nicotinic acid ribonucleoside.
Since the 7 P latter compound is not phosphorylated, it could leak out of the cell.
The data suggest, however, that during starvation, there is also an inhibition of TPN breakdown. The plots in Fig. 5 indicate that although after 100 min of starvation, DPN and TPN are present in approximately equal amounts, by 250 min, the total TPPI; radioactivity has decreased only slightly (from 37 to 327, of the total radioactivity) despite the much larger decrease in DPN (from 36 to 1170). This would suggest that the TPN present at 150 min is quite stable; if it were breaking down at t,he normal rate (140 molecules per s (l)), then the TPN radioactivity should remain roughly proportional to DPN radioactivity.
These experimental results suggest that TPN levels increase with respect to DPN because: (a) DPN is turning over in the cell but is not re-formed efficiently due to inhibition of the N,AD pyrophosphorylase reaction, and (b) the breakdown of  figure  are derived  from  Tables  I and II. TPS molecules to DPN is inhibited as the pyridine nucleot,ide concentration falls. Two methods have been used for starvation, i.e. total deprivation of niacin or balanced growth under conditions of limiting niacin in the medium.
This allows a comparison of two cells with the same amount of total pyridine nucleotide per cell, but which have been deprived of niacin in different ways. The comparison cannot be regarded as rigorous, since on the one hand cells are growing under balanced growth conditions, and on the other hand they are growing under unbalanced growth conditions.
There is not as great a disproportionation of the TPN :DPN ratio in niacinlimited cells as there is in the totally starved cells, comparing cells that have the same total pyridine nucleotidc content.
This would be expected since the former cells are able to synthesize a little bit of DPN from the medium and therefore should have a relatively higher DPN level. An additional difference is the fact that the totally starved cells continue to divide at a normal rate (see Fig. l), and then do not divide at all when the total pyridine nucleotide content has fallen below 120,000 molecules per cell. On the other hand, cells grooving under limited niacin conditions have a lower rate of cell division but even cells with as low as 40,000 molecules per cell of total pyridine nucleotide still have the capacity to grow and divide.
The unbalanced growth conditions and possibly the greater disproportion between TPN and DPN in the tot.ally starved cells may account for the latter physiological observation.
Our results are consistent with the postulate that severe niacin starvation is accompanied by an ATP starvation. The accumulation of N,MN under these conditions is of some interest in view of the unusual discovery by Friedmann and Cagen (9) that N,MN is a specific ribosyl donor in l-12 coenzyme biosynthesis.
Since N,MN is not detectable in normally growing E. coli, our results raised the mteresting possibility that &MN may accumulate under other metabolic conditions related to an ATP deficiency, and therefore serve as a switch for 13-12 biosynthesis.
The results above indicate that some care should be taken in interpreting experiments involving severe niacin starvation, since niacin starvation probably is accompanied by an ATP deficiency.
Thus, the effects on DNA joining which have been reported due to niacin starvation (4) or the addition of cyanide (10) may not be due directly to lowering of DPN levels but due to the ATP deficiency.
Recent studies suggest that optimal sealing of discontinuous chains requires ATP, in addition to the deoxynucleoside triphosphates and DPN.