of
Protein tyrosine nitration has been described as irreversible ( 3 ) , and E. coli cell extracts show no evidence of an ability to repair nitrated proteins ( 23 ) . 
On the other hand , there is evidence for a protein tyrosine `` denitrase '' activity in rat tissues and in mitochondria ( 21 , 22 ) , and nitrated proteins may be subject to more rapid turnover than their native counterparts ( 15 , 36 ) . 
Despite these observations , the fate of nitrated proteins remains poorly understood . 
The degradation of nitrated proteins ( whether or not it is selective ) would liberate free 3-NTyr , and so there is some interest in the biochemical fate of this molecule in both host cells and invading pathogens . 
In rat PC12 cells , 3-NTyr can be converted to 4-hydroxy-3-nitrophe-nylacetate by the sequential action of an aromatic amino acid decarboxylase , an amine oxidase , and a NAD-linked dehydrogenase ( 4 ) . 
The intermediates in this pathway are 3-nitrotyra-mine and 4-hydroxy-3-nitrophenylacetaldehyde ( Fig. 1 ) . 
Bacteria isolated on the basis of their ability to use 3-NTyr as a carbon and energy source convert 3-NTyr to 4-hydroxy-3-ni-trophenylacetate via 4-hydroxy-3-nitrophenylpyruvate through the sequential action of a deaminase and a decarboxylase . 
The nitro group is then removed from 4-hydroxy-3-nitrophenyl-acetate by a novel denitrase activity ( 29 ) . 
The responses of bacteria to oxygen and nitrogen radicals attract considerable interest , in part because of their roles in the innate immune response ( 12 ) . 
In the case of NO , diverse bacteria express several different NO detoxification enzymes , and there are numerous regulatory systems that have been reported to respond to NO ( 12 , 38 ) . 
In the context of the current work , the regulator of interest is NsrR , a transcriptional repressor from the Rrf2 family , which probably contains 
* Corresponding author . 
Mailing address : Department of Molecular and Cell Biology , the University of Texas at Dallas , 800 W Campbell Road , Richardson , TX 75080 . 
Phone : (972) 883-6896 . 
Fax : (972) 883-2409 . 
E-mail : stephen.spiro@utdallas.edu . 
† Supplemental material for this article may be found at http://jb . 
asm.org / . 
‡ Present address : 1 Coca Cola Plaza , TEC 434C , Atlanta , Georgia 30313 . 
Published ahead of print on 25 July 2008 . 
an iron-sulfur cluster and is sensitive to sources of NO . 
NsrR has been shown to act as an NO-sensitive regulator of gene expression in several organisms ( 1 , 2 , 5 , 14 , 17 , 19 , 28 , 31 , 33 ) , and targets for NsrR regulation have been predicted ( 34 ) . 
In E. coli , NsrR regulates expression of the NO-detoxifying flavohemoglobin , along with several other genes and operons , some of which are of unknown or poorly understood function ( 5 , 13 , 24 , 40 ) . 
In this report , we show that 3-nitrotyramine can be used as a nitrogen source by cultures of E. coli , supporting growth at slow rates . 
We present evidence that the pathway of 3-nitro-tyramine degradation to 4-hydroxy-3-nitrophenylacetate is similar to that found in rat cells ( 4 ) , involving a periplasmic amine oxidase ( TynA , also known as MaoA ) and a cytosolic NAD-linked dehydrogenase ( FeaB , also known as PadA ) . 
The tynA and feaB promoters are bound by NsrR in vivo , and NsrR exerts a weak , though significant , degree of control on both promoters . 
Overexpression of NsrR represses the tynA and feaB promoters and severely retards growth on phenylethyl-amine ( PEA ) , catabolism of which requires TynA and FeaB activities . 
Expression of the tynA and feaB genes is upregulated by growth on PEA and 3-nitrotyramine , regulation that requires an AraC-type regulator encoded by the feaR gene . 
We speculate that one physiological function of TynA and FeaB is to metabolize nitrated aromatic compounds that may accumulate in cells exposed to NO and superoxide . 
MATERIALS AND METHODS
Bacterial strains , media , and growth conditions . 
The strains and plasmids used in this work are listed in Table 1 . 
Transposon insertions in the tynA and feaB genes of E. coli MG1655 were obtained by P1 transduction , using strains JD22473 ( tynA : : Tn10 ) and JD22470 ( feaB : : Tn10 ) from the National Bio-Resource Project ( Japan ) as the donors . 
To construct an unmarked deletion in the lacZ gene of MG1655 , we first introduced a lacZ : : kan mutation from strain VJS8363 ( a gift from Valley Stewart ) and then removed the kanamycin resistance cartridge by site-specific recombination with pCP20 ( 8 ) . 
The nsrR gene was disrupted by replacing the coding region with a kanamycin resistance cassette , using the red recombinase method , with pKD4 as the template and primers designed to generate a nonpolar mutation ( 8 ) . 
The mutation was transferred to other strains by P1 transduction . 
To convert the insertion mutation to an unmarked nonpolar deletion , we transformed the strains with pCP20 , and kana-mycin/ampicillin-sensitive transformants were identified after colony purification at 43 °C ( 8 ) . 
Reporter strains with feaR : : kan mutations were constructed by P1 transduction using JW1379 ( from the National BioResource Project , Japan ) as the donor . 
The structures of all insertion and deletion mutants were confirmed at each step by PCR . 
The rich medium for routine propagation of E. coli strains was L broth ( tryptone , 10 g liter 1 ; yeast extract , 5 g liter 1 ; NaCl , 5 g liter 1 ) . 
For growth tests and enzyme determinations , a defined medium ( 37 ) was used , supplemented with the indicated carbon and nitrogen sources and with Casamino acids ( 0.05 % [ wt/vol ] ) , as needed . 
For growth with alternative nitrogen sources , the ammonium sulfate in this medium was substituted with sodium sulfate . 
PEA has limited solubility in water , so it was added directly to the bulk medium , which was then sterilized by filtration . 
Growth on PEA is temperature sensitive ( 32 ) and is significantly improved by the addition of Casamino acids to growth media . 
Therefore , all PEA cultures were grown at 30 °C in the presence of 0.05 % ( wt/vol ) Casamino acids ( in 250-ml flasks shaken at 250 rpm ) and were inoculated with precultures grown in glucose minimal medium . 
For cultures grown on glucose with 3-nitrotyramine as the nitrogen source , we found that growth was improved by restricting the oxygen supply ( which perhaps alleviated the oxidative stress associated with the production of hydrogen peroxide by the amine oxidase ) . 
Precultures were grown at 30 °C in 5 ml of medium in 16-mm culture tubes rotated at 50 rpm . 
Experimental cultures were grown at 30 °C in 20 to 50 ml of medium , in 250-ml flasks shaken at 60 to 70 rpm . 
Genetic manipulations . 
The tynA and feaB promoter regions ( on 279 - and 247-bp fragments , respectively ) were amplified by PCR ( primer sequences for these and other procedures are listed in Table S1 in the supplemental material ) and cloned into pSTBlue-1 , using methods similar to those described previously ( 5 ) . 
Promoter fusions to lacZ were then constructed in pRS415 , transferred to RS45 , and integrated into the chromosome as described previously ( 5 , 35 ) . 
The plasmid pJP07 contains the nsrR gene ( with its own promoter ) modified at the 3 end by the addition of sequences encoding the 3XFlag epitope tag ( 41 ) . 
The modified nsrR gene was amplified from the chromosome of strain JOEY135 ( 10 ) and cloned into p2795 , a high-copy number plasmid derived from pBluescript ( 18 ) . 
The C-terminal epitope tag has no detectable effect on the activity of NsrR , either in vivo or in vitro ( unpublished work ) . 
The same modification was used to identify NsrR binding sites by chromatin immunoprecipitation and microarray analysis ( ChIP-chip ) . 
For ChIP-chip experiments , published procedures were followed for strain constructions , growth of cultures , chromatin extraction , DNA labeling , array hybridization , and data analysis ( 10 ) . 
Enzyme assays . 
Extracts for the TynA and FeaB activity assays were prepared from 50-ml cultures grown to late exponential phase . 
Cell pellets were washed three times and resuspended in 1 ml of basal minimal medium ( with no carbon or nitrogen source ) . 
Cells were disrupted by sonication and then centrifuged at 
16,000 g at 4 °C for 20 min . 
To remove membrane fragments , extracts were centrifuged at 100,000 g at 4 °C for 1 h. To assay the amine oxidase TynA , we measured oxygen uptake rates at 30 °C , using a Clark-type electrode ( Hansatech Instruments , King 's Lynn , Norfolk , England ) in a 0.1 M phosphate buffer ( pH 7.0 ) , 1.5 mM Na2SO4 . 
Reactions were started by the addition of 100 M substrate , a concentration chosen to avoid substrate inhibition by PEA . 
FeaB activities were assayed at 30 °C in 50 mM potassium phosphate ( pH 7.0 ) containing 2 mM NAD . 
Reactions were initiated by the addition of 50 M PEA or 100 M 3-nitrotyramine , and the absorbance at 340 nm was followed with a Cary 50 spectrophotometer ( Varian , Palo Alto , CA ) . 
Enzyme kinetic data were analyzed by direct curve fitting using Kaleidagraph ( Synergy Software , Reading , PA ) software . 
Where substrate inhibition was evident , data were fitted to the Haldane equation ( equation 1 ) ; otherwise data were fitted to the Michaelis-Menten equation . 
V Vmax S2 Km S Ki
- Galactosidase activities were measured according to published protocols ( 25 ) . 
All enzyme activities were measured in duplicate with samples from three independently grown cultures . 
Chemicals and analytical methods . 
3-Nitrotyramine was purchased from Apin Chemicals ( Abingdon , United Kingdom ) . 
Concentrations of stock solutions of 3-nitrotyramine were determined spectrophotometrically . 
The molar extinction coefficient of 3-nitrotyramine ( 422 nm ) at pH 7.5 is 2,800 M 1 cm 1 ( 26 ) . 
Using this value , we determined the extinction coefficient to be 1,973 M 1 cm 1 at pH 7.0 and used this latter value for measuring the concentrations of stock solutions . 
Diethylenetriamine ( DETA ) - NONOate was purchased from Cayman Chemicals ( Ann Arbor , MI ) . 
This compound decomposes at pH 7.4 , with a half-life ( t0 .5 ) of 20 h at 37 °C and 56 h at 22 to 25 °C , and releases two equivalents of NO ( Cayman Chemicals ) . 
The half-life of DETA-NONOate under the conditions of our experiments ( pH 7.0 ; 30 °C ) is not known , but we assume that it is between 20 and 56 h , and the interpretation of results is not affected by the exact half-life of the compound . 
DETA-NONOate was added to cultures at the time of inoculation and was present throughout growth . 
3-Nitrotyramine and 4-hydroxy-3-nitrophenylacetate concentrations were measured in filtered culture supernatants , using previously published methods ( 29 ) , except that a 100-mm column was used for high-performance liquid chromatography ( HPLC ) . 
RESULTS
Regulation of tynA and feaB by NsrR . 
We have recently used transcriptomics ( 13 ) and ChIP-chip analysis ( unpublished data ) to identify E. coli genes regulated by NsrR . 
The ChIP-chip approach identified binding sites for NsrR in the intergenic region between feaB and the regulatory gene feaR and in the promoter region of the adjacent tynA gene ( Fig. 2 ) . 
Full results of the genome-wide mapping of NsrR binding sites using ChIP-chip will be published elsewhere . 
The tynA and feaB genes encode the first two enzymes of a pathway ( Fig. 1 ) that is required for the utilization of PEA as a carbon and energy source or utilization of tyramine or dopamine as a nitrogen source ( 9 ) . 
To determine whether there is any regulation of these genes by NsrR , we fused the feaR , feaB , and tynA promoters to lacZ and transferred the reporter fusions in single copies to the chromosome . 
We found no evidence for NsrR regulation of the feaR promoter ( data not shown ) . 
In exponential-phase cultures growing in complex medium ( not shown ) or in defined medium with glycerol as the carbon source , the tynA and feaB promoters had low activities in both a wild-type strain and an nsrR mutant ( Table 2 ) . 
However , in cultures grown on PEA as the sole source of carbon and energy , the activities of both promoters increased substantially ( Table 2 ) . 
Regulation of the tynA and feaB genes by PEA ( and tyramine ) has been observed previously ( 16 , 42 ) and is presumed to involve FeaR , a predicted regulatory protein with an AraC-type DNA binding domain , though this regulation has not been confirmed biochemically ( 9 ) . 
Accordingly , feaR mutants grew very poorly on 
PEA , and the tynA and feaB promoters were not induced by PEA in a feaR mutant ( Table 2 ) . 
In cultures of the nsrR mutant grown on PEA , we consistently observed small , though significant ( 20 to 50 % ) , increases in feaB and tynA promoter activities ( representative data are shown in Table 2 ) . 
The low feaB promoter activities observed in a feaR mutant were also derepressed to a small extent in the feaR nsrR double mutant . 
Thus , the magnitude of the repression exerted by NsrR on the feaB promoter is similar in both the presence and the absence of FeaR . 
In contrast to the small effects of the nsrR mutation , we found that overexpression of nsrR ( by increasing the gene copy number ) had large effects . 
Transformation with a high-copy number plasmid carrying a cloned nsrR gene resulted in se-verely impaired growth on PEA ( Fig. 3 ) and 17 - and 6-fold reductions in the activities of the tynA and feaB promoters , respectively ( Table 3 ) . 
Addition of a compound ( DETA-NONOate ) that releases NO very slowly ( t 20 to 56 h ) in 0.5 these slow-growing cultures restored both growth on PEA and maximal promoter activities ( Fig. 3 and Table 3 ) . 
This suggests that increasing the copy number of the nsrR gene causes re-a Activities are shown for the tynA and feaB promoters in reporter strains containing multiple copies of the nsrR gene and exposed ( ) or not ( ) to NO . 
Cultures grown with PEA as the carbon source were supplemented with 100 M DETA-NONOate , which decomposes with a t0 .5 of between 20 and 56 h under the conditions of this experiment , to release 2 equivalents of NO . 
b Numbers in parentheses are 1 standard deviation . 
Units are as defined by Miller ( 25 ) . 
pression of the tynA and feaB promoters and , therefore , impaired growth on PEA . 
Under these conditions , the activities of both promoters can be regulated by NO . 
This repression by NsrR that can be alleviated by NO presumably involves NsrR binding to the sites identified by ChIP-chip analysis ( Fig. 2 ) . 
The cellular concentration of NsrR ( under the conditions used for these experiments ) seems to be poised such that its removal ( by mutation ) has small effects on these promoters , but overexpression causes severe repression . 
We were interested in determining the physiological properties of the feaB and tynA gene products that might provide a rationale for the inclusion of these genes in the NsrR regulon . 
Utilization of 3-nitrotyramine as a nitrogen source . 
TynA and FeaB have broad substrate specificities and , besides PEA , can also oxidize tyramine to 4-hydroxyphenylacetate . 
E. coli K-12 strains can not further oxidize 4-hydroxyphenylacetate and so use tyramine only as a nitrogen source . 
Dopamine may also be used as a substrate by this pathway and is oxidized to dihydroxyphenylacetate ( 9 ) . 
Decarboxylation of 3-NTyr yields 3-nitrotyramine ( 4 ) , and so we wondered whether 3-nitrotyra-mine might be a substrate for TynA and 4-hydroxy-3-nitrophe-nylacetaldehyde a substrate for FeaB . 
Assuming the presence of an as-yet-unidentified 3-NTyr decarboxylase , TynA and FeaB would provide a pathway for the conversion of 3-NTyr to 4-hydroxy-3-nitrophenylacetate ( Fig. 1 ) , a pathway similar to that described for rat PC12 cells ( 4 ) . 
E. coli MG1655 grew slowly in a defined medium containing 3-nitrotyramine as the sole source of nitrogen ( Fig. 4 ) . 
The growth yield ( as estimated by the final culture density per mole of substrate ) of cultures grown on 3-nitrotyramine was 59 % of that of cultures grown on ( NH4 ) 2SO4 ( Fig. 4 ) . 
Although we can not exclude other physiological explanations for the reduced growth yield , it is at least consistent with the notion that only one nitrogen of 3-ni-trotyramine can be assimilated . 
A tynA mutant of MG1655 failed to grow on 3-nitrotyramine ( data not shown ) , whereas a feaB mutant grew with the same yield as that of the wild-type strain ( Fig. 4 ) . 
The phenotypes of the tynA and feaB mutants are consistent with the pathway shown in Fig. 1 , and , together with the growth yield data suggest that growth on 3-nitrotyra-mine is at the expense of the amino group . 
Growth on 3-nitrotyramine as the sole source of nitrogen induced the tynA and feaB promoters in a wild-type strain and in an nsrR mutant , the - galactosidase activities being about 40 % of those observed for cultures grown on PEA ( Table 2 ) . 
FeaR is thought to mediate substrate inducibility of the tynA and feaB genes ( 9 ) , and a feaR mutant can not grow on 3-ni-trotyramine . 
Thus , upregulation of the two promoters by 3-ni-trotyramine is independent of NsrR and probably requires FeaR . 
The identity of the ligand for FeaR has not been established ; it may not be PEA ( or tyramine ) , given that TynA is located in the periplasm and that its substrate is , therefore , presumably not transported into the cell . 
In any case , the simplest explanation for our results is that 3-nitrotyramine , or a molecule related to 3-nitrotyramine ( perhaps 4-hydroxy-3-nitro-phenylacetaldehyde ) , can function with FeaR to control the activity of the tynA and feaB promoters . 
In nsrR mutants grown on 3-nitrotyramine , the tynA and feaB promoters showed activities that were modestly increased compared to those of the wild-type strains , as was the case for cultures grown on PEA ( Table 2 ) . 
The pathway for 3-nitrotyramine catabolism . 
To test the prediction ( Fig. 1 ) that 3-nitrotyramine is a substrate for TynA , we assayed substrate-dependent amine oxidase activity by following oxygen uptake by cell extracts in a Clark-type oxygen electrode . 
Using PEA as the substrate , we found evidence for substrate inhibition ( Fig. 5 ) , as has been reported previously ( 39 ) . 
The activity data fitted well to the Haldane equation ( equation 1 ) for substrate inhibition , with estimates of apparent K 5.5 1.4 M and K 690 109 M. With m i 3-nitrotyramine as the substrate , oxygen uptake rates in the same cell extracts were somewhat lower ( Vmax 29.6 0.7 versus 55.5 2.9 nmol/min/mg protein for PEA ) but followed Michaelis-Menten kinetics , with an estimated apparent Km value of 7.2 1.3 M ( Fig. 5 ) . 
The tynA mutant of E. coli MG1655 does not grow on PEA ( conditions which are required to induce activity ) ; therefore , we were unable to assay 3-nitrotyramine-dependent oxygen uptake in a tynA mutant ( to provide direct proof that TynA is responsible for the measured activity ) . 
Nevertheless , other data we present in this paper lend confidence to the idea that TynA is the enzyme responsible for oxidizing 3-nitrotyramine . 
We measured substrate-dependent oxygen uptake activities in cultures of MG1655 and in an nsrR mutant culture grown under a range of conditions that were similar to those used for assays of reporter fusions . 
The enzyme had extremely low activity or was undetectable in cells grown on glycerol ( Table 4 ) , which is consistent with the assays of tynA promoter activity ( Table 2 ) . 
TynA activity was detected in cells grown on PEA or 3-nitrotyramine ( Table 4 ) , which is again consistent with the reporter fusion assays . 
TynA activities ( measured with either substrate ) were 10-fold higher in PEA - versus 3-nitrotyra-mine-grown cells ( Table 4 ) , whereas the tynA promoter was only 2.5-fold more active in PEA-grown cells ( Table 2 ) . 
This discrepancy may be indicative of some posttranscriptional control of the tynA gene . 
Importantly , the activity assays provide additional confirmation of the suggestion that 3-nitrotyramine acts as an inducer of the catabolic pathway . 
We were unable to test the role of FeaB in 3-nitrotyramine catabolism directly , since the postulated substrate ( 4-hydroxy-3-nitrophenylacetaldehyde ) is not commercially available . 
Therefore , we developed a coupled assay in which FeaB activity can be measured in cell extracts in the physiological direction by adding the substrate for TynA , which is oxidized in situ to generate the FeaB substrate . 
FeaB activity was measured by following the reduction of NAD to NADH . 
A feaB mutant can utilize both PEA ( 32 ) and 3-nitrotyramine ( Fig. 4 ) as nitrogen sources , presumably because the mutant can liberate the amino group of PEA and 3-nitrotyramine through the activity of TynA . 
The feaB mutant grown on PEA as a nitrogen source showed no PEA - or 3-nitrotyramine-dependent reduction of NAD with the FeaB assay , confirming that FeaB is responsible for the measured activity . 
Phenylethylamine 66 (5) 41 (3) 73 (11) 44 (7) Glycerol ND ND ND 3 (1) 3-Nitrotyramine 6 (3) 4 (2) 7 (2) 4 (1)
a Phenylethylamine - and 3-nitrotyramine-dependent O2 uptake in extracts of cells grown on phenylethylamine or glycerol ( as the carbon source ) or on 3-ni-trotyramine ( as the nitrogen source ) . 
Cultures were grown at 30 °C in defined medium containing 5 mM phenylethylamine or 40 mM glycerol as the carbon source or containing 11.1 mM glucose as the carbon source with 2.56 mM nitrotyramine as the nitrogen source . 
b Numbers in parentheses are 1 standard deviation ( SD ) . 
ND , not detectable ( 2 nmol min 1 mg protein 1 ) . 
Phenylethylamine 38 ( 2.3 ) 18 ( 1.6 ) 35 ( 2.8 ) 15 ( 2.2 ) Glycerol ND 2.4 ( 1.0 ) 1.0 ( 0.7 ) 1.3 ( 0.3 ) 3-Nitrotyramine 54 ( 2.5 ) 20 ( 6.4 ) 42 ( 4.4 ) 14 ( 4.0 ) a Data show phenylethylamine - and 3-nitrotyramine-dependent NAD reduction in extracts of cells grown on phenylethylamine or glycerol ( as the carbon source ) or on nitrotyramine ( as the nitrogen source ) . 
Cultures were grown at 30 °C in defined medium containing 5 mM phenylethylamine or 40 mM glycerol as the carbon source or containing 11.1 mM glucose as the carbon source with 1.3 mM nitrotyramine as the nitrogen source . 
b Numbers in parentheses are 1 standard deviation ( SD ) . 
ND , not detectable ( 1 nmol min 1 mg protein 1 ) . 
Using the coupled NAD - linked assay , we could detect FeaB activity in cell extracts with PEA as the substrate for the assay ( Table 5 ) , activities that did not differ significantly from those measured with the known FeaB substrate phenylacetal-dehyde ( data not shown ) . 
FeaB activity was low or undetect-able in cells grown on glycerol , though this may be a reflection of the absence of TynA activity under these conditions . 
In cells grown on 3-nitrotyramine as the nitrogen source , FeaB activity was detectable at levels similar to those seen with cells grown on PEA ( Table 5 ) . 
The major conclusion that can be drawn from the results is that oxidation of 3-nitrotyramine by TynA generates an intermediate ( presumably 4-hydroxy-3-nitrophe-nylacetaldehyde ) that can be further oxidized by FeaB . 
Thus , the predicted product of the pathway is 4-hydroxy-3-nitrophe-nylacetate ( Fig. 1 ) . 
This hypothesis was tested by determination of 3-nitrotyramine and 4-hydroxy-3-nitrophenylacetate in culture supernatants , using HPLC . 
After the growth ( of MG1655 and its nsrR mutant ) on a limiting concentration ( 1 mM ; Fig. 4 ) , 3-nitrotyramine was undetectable in culture supernatants , and there was almost stoichiometric ( 88 to 90 % ) accumulation of 4-hydroxy-3-nitrophenylacetate ( data not shown ) . 
Thus , 4-hydroxy-3-nitrophenylacetate is the likely end product of 3-nitrotyramine metabolism in E. coli . 
DISCUSSION
The starting point for the work described in this paper was the discovery , using ChIP-chip analysis , of NsrR binding sites in the tynA and feaB promoter regions . 
We went on to show that NsrR can function as a regulator of tynA and feaB expression and that the enzymes encoded by these genes can oxidize 3-nitrotyramine , in addition to the previously described substrates . 
These findings illustrate one advantage of the ChIP-chip approach as a means of identifying regulon members . 
We could not have discovered NsrR regulation of tynA and feaB by comparing the transcriptomes ( or proteomes ) of a wild-type strain and an nsrR mutant , because ( i ) regulation requires cultures to be grown on PEA , and there would be no a priori reason to choose those growth conditions for a transcriptomics experiment ; and ( ii ) revealing the full extent of NsrR regulation requires overexpression rather than deletion of nsrR ; again , it is unlikely we would have chosen to use those conditions in a transcriptomics experiment . 
The tynA and feaB promoters have not been well characterized , and the nature of the DNA sequence recognized by NsrR is incompletely understood , although a consensus sequence has been proposed , which is a long inverted repeat ( 5 , 34 ) . 
Analysis of the ChIP-chip targets ( unpublished work ) suggests that NsrR can bind to half of the inverted repeat sequence , but , in the absence of in vitro data , we can not reach firm conclusions about the locations of the NsrR binding sites in the tynA and feaB promoters . 
Deletion of nsrR has very small effects on the tynA and feaB promoters , while overexpression of nsrR causes severe repression . 
We have observed similar effects at some other NsrR-regulated promoters ( unpublished work ) and believe that the concentration of NsrR is typically poised in a range that is insufficient to repress some promoters that are potentially controlled by NsrR . 
In this case , understanding the factors that regulate expression of the nsrR gene becomes especially important , since conditions that lead to the upregulation of nsrR would potentially lead to the regulation of promoters ( such as tynA and feaB ) that may otherwise escape repression . 
In this context , we have found that the nsrR promoter is twofold more active in cultures grown in minimal medium than in medium supplemented with amino acids ( unpublished data ) . 
This effect , albeit small , may provide an explanation for our observation that good growth on PEA requires the addition of amino acids to growth media . 
The amine oxidase ( TynA ) and phenylacetaldehyde dehydrogenase ( FeaB ) enzymes of E. coli K-12 strains have been viewed as providing a straightforward pathway for the catabo-lism of PEA , tyramine , and dopamine ( 9 ) . 
Two recently published observations suggest that these enzymes might have alternative and/or additional physiological roles . 
First , tynA mutants express the SOS response constitutively , which has been interpreted as indicating that the amine oxidase is responsible for removing an endogenously generated genotoxic compound ( 30 ) . 
Second , feaB was identified in a screen for genes important for survival under planktonic ( versus biofilm ) growth conditions ( 20 ) . 
This observation implies that a substrate for FeaB was present in the minimal growth medium used or can be generated endogenously . 
Thus , there is circumstantial evidence from independent studies to suggest that TynA and FeaB might have roles in catabolizing endogenously generated substrates . 
The substrate inhibition of TynA ( Fig. 5 ) indicates that the enzyme is significantly inhibited by the concentrations of PEA typically used in growth media ( 1 mM ) . 
Since TynA is located in the periplasm ( 32 ) , it is exposed to the medium concentration of PEA . 
The inhibition of TynA by physiologically relevant concentrations of PEA suggests that the enzyme is not optimally suited to a major role in PEA catabolism , which may account for the very slow growth on PEA ( Fig. 3 ) . 
Our results clearly show that TynA and FeaB also provide a pathway for the metabolism of 3-nitrotyramine ( which does not exert substrate inhibition on TynA ) and that the corresponding genes are regulated by NsrR and by NO . 
A rationale for this regulatory pattern may be provided if 3-ni-trotyramine accumulates , and must be disposed of , in cells exposed to NO. 3-Nitrotyramine can be generated by the decarboxylation of 3-NTyr ( 4 ) , which may accumulate in cells exposed to NO and superoxide ( 12 ) . 
However , E. coli strains are not known to express or encode an aromatic amino acid decarboxylase , and there is therefore no known pathway from 3-NTyr to 3-nitrotyramine . 
Accordingly , 3-NTyr can not be used as a nitrogen source by E. coli MG1655 , and there is no 3-NTyr-mediated stimulation of oxygen uptake or NAD reduction in cell extracts ( L. D. Rankin , and S. Spiro , unpublished observations ) . 
It remains to be seen whether 3-nitrotyra-mine is a physiologically significant substrate for TynA , either endogenously generated or encountered in natural environments . 
The wider significance of these observations also remains to be established . 
Homologs of tynA and feaB have restricted distributions in sequenced genomes and are found in the same organism quite infrequently . 
The feaR-feaB-tynA region of E. coli strain MG1655 is absent from several other E. coli strains , including some clinical isolates . 
Thus , the metab-olism we have identified may not be ubiquitously important . 
On the other hand , we would predict that organisms capable of expressing tyrosine decarboxylase along with homologs of tynA and feaB are potentially capable of degrading 3-nitrotyrosine . 
ACKNOWLEDGMENTS We thank Valley Stewart , Barry Wanner , Michael Hensel , and the National Bioresource Project ( Japan ) for gifts of strains and plasmids . 
This work was supported by grant MCB-0702858 from the National Science Foundation ( to S.S. ) and by grant AB07CBT002 from the Army Research Office and the Defense Threat Reduction Agency ( to J.C.S. ) .