Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world . Genome sequencing of B . suis and B . melitensis revealed that both are complete denitrifiers . To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrificationin the closely related B . neotomae was undertaken . In contrastto B . suis and B . melitensis, it was found that B . neotomae is a partial denitrifier that can reduce nitrate to nitritebut no further . Examination of the B . neotomae genome showedthat a deletion in the denitrification gene cluster resultedin complete loss of nirV and the partial deletion of nirK and nnrA . Even though the nor operon is intact, a norC-lacZ promoterfusion was not expressed in B . neotomae . However, the norC-lacZfusion was expressed in the related denitrifier Agrobacteriumtumefaciens, suggesting that the lack of expression in B . neotomaeis due to inactivation of NnrA . A narK-lacZ promoter fusionwas found to exhibit nitrate-dependent expression consistentwith the partial denitrifier phenotype . Complementation of thedeleted region in B . neotomae by using nirK, nirV, and nnrAfrom B . melitensis restored the ability of B . neotomae to reducenitrite . There was a significant difference in the death ofIRF-1^–/– mice when infected with B . neotomae containingnirK, nirV, and nnrA and those infected with wild-type B . neotomae.The wild-type strain killed all the infected mice, whereas mostof the mice infected with B . neotomae containing nirK, nirV, and nnrA survived.

 
Denitrification, the reduction of nitrate to gaseous nitrogenoxides, is most commonly found in eubacteria, but it has beenobserved in several archaeal species as well [37] . Among the eubacteria, denitrification is most frequently observed in membersof the -proteobacteria . Denitrification has been actively studiedin several free-living members of this group, such as Paracoccusdenitrificans and Rhodobacter sphaeroides . Denitrification occursin a number of symbiotic or pathogenic members of this groupas well, but this process has not been as heavily studied inthese bacteria . Denitrifying members of the -proteobacteria that are symbionts or pathogens include the genera Rhizobium, Sinorhizobium, Bradyrhizobium, and Agrobacterium . Recently, genomic analysis has revealed that members of the genus Brucella, which are -proteobacteria closely related to Agrobacterium andrhizobia, are denitrifiers [6, 24] . Species in this genus areunique among this group of denitrifiers, since they are animal pathogens . Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world [15].

Even though only a few pathogens have been shown to be denitrifiers, recent work suggests genes encoding nitrogen oxide reductases may be important determinants of a pathogenic lifestyle . In Neisseria, a member of the ß-proteobacterial group,nitrite and nitric oxide [NO] reductase [Nir and Nor, respectively]have been shown to be required for anaerobic growth and havebeen suggested to play a role in mitigating NO toxicity in themacrophage [1] . In Brucella suis, transposon inactivation ofthe gene encoding nitrate reductase affected growth inside themacrophage [16] . The utility of denitrification genes in pathogenesisis easy to rationalize, given the importance of NO in the hostcell's defense against infection . Pathogens able to respireNO using Nor can decrease NO levels in their surroundings andgain an additional advantage by coupling this reaction to energyconservation . NO production can increase survival of Brucellain macrophages, consistent with this compound serving as a terminalrespiratory oxidant [34].

This study was undertaken to examine the role of denitrification in the growth of Brucella in more detail . The strain chosen for the study was Brucella neotomae, isolated from desert wood rats but nonpathogenic for humans and domestic animals [23]. This bacterium's genome has not been sequenced, but initial studies suggest its genome is very similar to the genomes ofother Brucella species [8] . Brucellae are facultative intracellularpathogens, and this study was initially designed to examineregulation and activity of the various nitrogen oxide reductasesunder free-living conditions . Unexpectedly, it was found thatB . neotomae failed to grow under denitrifying conditions andaccumulated nitrite when grown under oxygen-limiting conditions in nitrate-supplemented medium . Sequence analysis revealed differences between the denitrification gene cluster of B . neotomae and that of other Brucella genomes . The extent of these differences and their effect on denitrification and in vivo growth of B. neotomae provide insight into the regulation of denitrification and its physiological role in Brucella.

 
Bacterial strains and growth conditions. The B . neotomae strain used in this study, 5K33, was lyophilizedin 1966 [5] [Table 1] . The dried material was rehydrated with Brucella broth [Difco, Detroit, Mich.] . One of the colonies arising from the rehydrated stock was saved and used for allfurther studies . Extraction of chromosomal DNA was done usingthe Wizard genomic DNA kit [Promega] . A fragment of the 16SrRNA gene was amplified and sequenced as previously describedand found to have >99% identity with the 16S rRNA gene ofother Brucella strains [7, 11] . Sequence analysis revealed it was identical to B . neotomae ATCC 23459, which is also derived from the 5K33 strain . Sequencing was carried out at the Cornell BioResource Center DNA Sequencing facility using an Applied Biosystems automated 3700 DNA analyzer.

 
B . neotomae was typically grown on brucella broth at 30°C. Microaerobic growth was achieved by growing cells in 15-ml serum vials filled with 10 ml of medium . After cells were added, thevials were sealed to prevent oxygen exchange, ensuring thatoxygen levels decreased during growth . When necessary, kanamycinor gentamicin was added to B . neotomae cultures at 25 µgml^–1 . Nitrate was added to a final concentration of 11mM . Agrobacterium tumefaciens C58 [Table 1] was grown on Sistrom's medium, and microaerobic conditions were achieved as previously described [21] . Kanamycin and tetracycline were added to A.tumefaciens cultures at 100 and 3 µg ml^–1, respectively.An A . tumefaciens strain [A011] containing an inactivated nnrRwas constructed by insertion of a streptomycin-spectinomycinresistance cassette into nnrR [S.-H . Baek and J . P . Shapleigh,unpublished data] . Escherichia coli strain DH5 was used as amaintenance strain for plasmids . E . coli S17-1 [29] was usedas a donor for conjugative transfer of plasmids . E . coli strainswere grown on Luria-Bertani medium.

Plasmids were mobilized into all strains by conjugation using standard protocols [31] . Brucella recipients were plated ontomedium containing brucella selective supplement [Sigma-Aldrich,Inc.] and any antibiotics required to select for plasmids . Exconjugantswere selected after 5 days of incubation at 30°C . Wild-typeB . neotomae was incapable of growth on this medium . Exconjugantswere maintained on medium containing selective supplement throughseveral rounds of isolation and then grown in liquid mediumcontaining selective supplement, and an aliquot of this wassaved for future use . All subsequent growth was done without selective supplement in the medium.

Construction of plasmids for sequencing, complementation, and fusions. All plasmids used in this study are shown in Table 1 . The oligonucleotidesused to amplify fragments in this study are shown in Table 2. Oligonucleotides were designed using the available Brucella genomic sequences . All amplified fragments from B . neotomae were cloned into pUC19 [36] for sequencing and to facilitate further cloning . For construction of the lacZ fusions, the fragmentswere moved into the broad-host-range vector pRK415 [12] followedby introduction of the lacZ-Kan^r cassette from pKOK6 [17] . Afterproper orientation of the lacZ cassette was confirmed, the plasmidswere transformed into S17 and then conjugated into Brucellaor other strains . For complementation analyses, fragments werecloned into pRK415 or pBBR1MCS-5 [18] and then transformed intoS17.

 
Biochemical assays. Nitrite was measured using a standard colorimetric protocol[30] . ß-Galactosidase assays were carried out as describedpreviously [19] . For all ß-galactosidase assays, cellswere grown under microaerobic conditions . Results are reportedfrom cells in late log phase, since this is when denitrifierpromoter fusions have the highest activity [2] . For experimentswith sodium nitroprusside [SNP], a 200 mM stock of SNP was preparedin Sistrom's medium . Using a syringe, SNP was added to a finalconcentration of 2 mM, cultures were incubated an additional4 h, and then cells were removed for ß-galactosidase analysis as previously described [19] . Nir activity assays wereperformed as described previously [30].

Mouse infection. IRF-1^–/– mice, originally produced by Matsuyamaet al . from C57BL/6 [H-2^b] mice, were kindly donated by TakW . Mak, Amgen Institute, Ontario Cancer Institute, Universityof Toronto, Toronto, Ontario, Canada [22] . These mouse strainswere heterozygously bred in the Department of Animal Healthand Biomedical Sciences animal care facilities, University ofWisconsin, and 6- to 9-week-old mice were used for experimentalinfection . Prior to infection, IRF-1^–/– mice weregenotyped by PCR [28] . IRF-1^–/– mice [n = 10/group]were injected intraperitoneally with 10⁷ B . neotomae or B . neotomae/pBgap.To determine CFU in livers and spleens, two mice were killedfrom each group at different time points and samples were homogenizedin phosphate-buffered saline and plated on brucella agar . Brucellacolonies were counted after 3 days of incubation at 37°Cwith 5% CO₂.

 
Absence of nirK in B . neotomae. B . neotomae was found unable to grow under anoxic conditionson solid medium supplemented with nitrate . A similar resulthas been observed with R . sphaeroides 2.4.3, which is a completedenitrifier, and so the inability of B . neotomae to grow underthese conditions might not reflect an inability to reduce nitrogenoxides [21] . Since genes for denitrification can be inducedin cells grown under oxygen-limited conditions, cells were culturedin nitrate-containing medium in sealed vials . The cells grewto an optical density at 600 nm of about 0.5 under these conditions.Analysis of the culture medium after oxygen-limited growth indicatedthat there was 332 ± 27 µg of nitrite/ml in themedium . This nitrite did not disappear even after the cellswere incubated for 5 to 7 days . Moreover, cells grown underthese conditions did not have any detectable Nir activity [Table3] . These results suggest that B . neotomae either lacks thegene coding for Nir or it is present but not expressed underthe culture conditions used in these experiments.

 
To determine if the B . neotomae genome contains nirK, the Nir structural gene, an attempt was made to amplify a fragment of the gene from B . neotomae genomic DNA using oligonucleotides that are targeted to regions encoding conserved areas of theprotein [Table 2] . No product of the correct size was amplified from B . neotomae genomic DNA . However, these oligonucleotides could be used to amplify a product of the correct size from R . sphaeroides 2.4.3 genomic DNA despite there being two mismatches in each oligonucleotide [data not shown].

The accumulation of nitrite during microaerobic growth in nitrate-supplementedmedium and the inability to amplify nirK suggest the B . neotomaegenome contains the genes for nitrate reductase [Nar] but notNir . A partial loss of denitrification genes has also been observedin other -proteobacteria, such as R . sphaeroides 2.4.1 . In strain2.4.1, nirK and nirV have been deleted but the deletion is limitedto these genes [20] . Assuming a similar type of deletion has occurred in B . neotomae, an attempt was made to amplify the closest gene downstream of nirK whose product is not involved in denitrification [Fig . 1] . Oligonucleotides targeting thisgene amplified a DNA fragment of the predicted size [data not shown] . Sequencing of this fragment revealed >99% identityto the same region of DNA from B . suis and Brucella melitensis [data not shown] . This suggests any deletion in this regionis confined to genes involved in denitrification.

 
To determine the extent of DNA loss in this region, oligonucleotides were designed to amplify a region starting about 1.3 kb upstreamof nirK and ending within the previously sequenced gene immediately downstream of nirK . Amplification gave only a single band of about 2.2 kb [data not shown] . In other Brucella species, amplificationwith these oligonucleotides resulted in a 4.4-kb fragment inlength that included nirK, nirV, and a gene encoding an Fnr/CRP-typetranscriptional regulator [data not shown] [Fig . 1] . Since thereare two genes in the Brucella denitrification gene cluster thatencode Fnr/CRP-type regulatory proteins, the one near nirK hasbeen designated nnrA and the one adjacent to the nitrous oxidesynthase [nos] gene cluster has been designated nnrB . The nnr designation was chosen since a similar terminology has beenused in related bacteria, such as P . denitrificans and R . sphaeroides, to identify genes whose products regulate nitrite and nitric oxide reductases [31, 32].

Sequencing of the 2.2-kb fragment amplified from B . neotomae revealed a deletion that affects nirK, nirV, and nnrA . Using the B . suis genome as a reference, the genes designated BRA0258 and BRA0259, which are the two genes immediately upstream of nirK, were present in their entirety in B . neotomae [Fig. 1].Downstream of the BRA0259 gene in B . neotomae is a fragmentof nirK, comprising 541 bases from the 5' region of the openreading frame [ORF] with the remaining 587 bases of nirK missing.The region between the 3' end of BRA0259 and the end of theremaining nirK fragment has >99% identity with other Brucellagenomes . The nirV ORF is completely absent in B . neotomae . The5' end of nnrA is also absent, but 229 bases of the 3' regionof nnrA are intact [Fig . 1] . As expected, the gene encodinga LacI-type regulator followed nnrA, and the sequence was >99% identical to that of other Brucella genomes.

Characterization and expression of norCB in B . neotomae. In B . suis and B . melitensis, the genes encoding all four nitrogen oxide reductases and required assembly factors are located within an 60-kb region of the small chromosome [6, 24] . The presenceof nitrate reductase and fragments of nirK and nnrA indicatesthere has been no large-scale deletion involving the entiredenitrification cluster in B . neotomae . However, a possibleconsequence of the loss of nirK is that Nor is no longer essential.Oligonucleotides targeting sequences that encode conserved regionsof NorB [Table 2] amplified a single fragment with >99% identityto the norB gene from other Brucella species [data not shown].Further sequencing of the nor region of the chromosome includingthe nor promoter region showed >99% identity to the sameregion from other sequenced Brucella species.

The organization of the nor genes in Brucella is similar to that of other denitrifiers, suggesting the genes norC through norD form an operon [26] . The region upstream of norC wouldtherefore contain all the regulatory motifs required for expressionof the operon . To determine if the nor region was expressed,a norC promoter fusion was constructed with lacZ as the reporter.There was no detectable ß-galactosidase activity inB . neotomae cells containing the fusion when cells were grownin nitrate-supplemented medium under microaerobic conditions[Table 4] . An increase in incubation temperature to 37 or 42°Cor changes in oxygen levels did not result in detectable ß-galactosidaseactivity . However, the B . neotomae norC-lacZ fusion was expressedin A . tumefaciens, indicating that the lack of expression wasnot due to the vector itself [Table 4].

 
In phenotypic tests, the only nitrogen oxide reductase shownto be expressed was nitrate reductase . Available genome sequencefrom other Brucella species suggests narK, which encodes a nitrite extrusion protein, is the first gene in an operon with othernar genes [6, 24] . Therefore, the promoter region of narK fromB . neotomae was fused to lacZ to serve as a positive controlfor promoter fusion experiments [Table 1] . As expected, ß-galactosidaseactivity was detected in B . neotomae containing the narK-lacZ fusion, and its activity showed a nitrate-dependent effect [Table 4].

Characterization of nnrB. One possible explanation for the absence of norC-lacZ expressionin B . neotomae is that the lack of Nir activity prevents endogenousNO production, which has been shown to be required for expressionof both nirK and nor in other denitrifiers [19, 32] . Since norC-lacZexpression in B . neotomae was not detected even in the presenceof SNP, an NO generator, the presence of exogenous NO is notsufficient to restore norC-lacZ expression [data not shown].Moreover, in other denitrifiers lacking Nir activity the expressionof nirK and nor does increase when the cells are grown withnitrate, due to the chemical production of NO from the highlevels of nitrite that accumulate in the medium [19] . The presence of nitrite in the medium did not lead to any detectable expressionof norC-lacZ in B . neotomae, further indicating that NO was not limiting expression.

Inactivation of nnrA may account for the lack of expression of the norC-lacZ fusion in B . neotomae . However, there is annnrA paralog, nnrB, in other Brucella species . The nnrB geneis immediately upstream of a gene encoding pseudoazurin [paz],which is involved in electron transfer to nitrogen oxide reductasesin other denitrifiers, and a gene designated nnrS, which hasbeen shown to be under NnrR control in R . sphaeroides 2.4.3[3, 25] . Amplification using oligonucleotides that targeted B . neotomae nnrB [Table 2] resulted in a product of the expectedsize which showed >99% sequence identity to the B . suis andB . melitensis sequences [data not shown] . The deduced NnrB sequenceof B . neotomae was 56 and 37% identical to NnrRs from A . tumefaciensand R . sphaeroides, respectively.

To test whether NnrB could activate norC expression, pBnnrB, which contains nnrB from B . neotomae and pBnorCZ, which carriesthe norC-lacZ promoter fusion, were mobilized into an NnrR-deficientstrain of A . tumefaciens, A011 [Table 1] . A011 carrying pBnnrBexhibited an increase in ß-galactosidase activitywhen oxygen was restricted, but there was no further increasewhen nitrate was added to the medium [Table 4] . The weak expressionof the norC-lacZ fusion could be due to NnrB not being expressed.To test if NnrB was expressed, its ability to complement anNnrR-deficient phenotype in A . tumefaciens was studied . Complementationwas assessed by monitoring nitrite accumulation and Nir activityof the nnrB-containing strain grown under limiting oxygen conditionsin nitrate-supplemented medium . Under this condition the presenceof nnrB did prevent nitrite accumulation [data not shown] . However,it was observed that this strain grew slower than the wild-typestrain upon reaching an optical density at 600 nm of about 0.50,which is the point in the growth curve where the denitrificationgenes are initially expressed [data not shown] . Cells with NnrBeventually reached a density similar to that of the wild type.

Complementation of NnrR deficiency by nnrA. Since NnrB only showed a limited ability to activate norC expression in the heterologous host A . tumefaciens, NnrA was tested under the same conditions to see if it was better able to activate expression . The nnrA from B . melitensis was amplified and cloned into a broad-host-range vector to make pBnnrA [Table 1] . Aswith nnrB-containing strains, NnrR-deficient strains of A . tumefacienswith pBnnrA in trans did not accumulate nitrite when grown microaerobicallyin nitrate-supplemented medium . However, unlike with nnrB, A011cells containing pBnnrA grew at near-wild-type rates duringall phases of growth [data not shown] . Expression of the B.neotomae norC promoter fusion in the presence of nnrA showeda nitrate-dependent increase and was three- to fivefold higherthan was measured when nnrB was present [Table 4] . The levelof ß-galactosidase activity in the A011 cells containingnnrA was similar to that measured in wild-type A . tumefacienscells containing only the B . neotomae norC-lacZ promoter fusionand grown under identical conditions [Table 4].

Restoration of denitrification in B . neotomae. Data from the complementation studies with nnrA and nnrB suggest that nnrA may be required for expression of some of the denitrificationgenes in B . neotomae . To test this, the entire nirK-nnrA regionfrom B . melitensis was cloned into a broad-host-range vectorto make pBgap, which was conjugated into B . neotomae [Table1] . When B . neotomae cells carrying pBgap were grown under oxygen-limitingconditions in nitrate-amended medium, significantly less nitriteaccumulated in the medium compared to wild-type cells grownunder identical conditions [34 ± 19 versus 332 ±27 µg/ml, respectively] . Nir assays revealed the B . neotomaecells containing pBgap had detectable Nir activity, consistentwith the decrease in nitrite accumulation in the medium [Table3] . Cells grown in medium that had not been supplemented withnitrate also had significant levels of Nir activity [Table 3].This result has been observed in other denitrifiers [19] andis likely due to residual nitrate in the unsupplemented medium.

To test whether nnrA was required for expression of nirK, pBnirKV,which contains only nirK and nirV from B . melitensis, was mobilizedinto B . neotomae . When B . neotomae with pBnirKV was grown underlimiting oxygen in medium containing nitrate, nitrite accumulationin the medium was slightly lower than with wild type but significantlyhigher than in medium from cells with pBgap [256 ± 31µg/ml, versus 332 ± 27 µg/ml with the wildtype] . Consistent with this observation, B . neotomae with pBnirKVgrown in medium with added nitrate had much lower levels of Nir activity than B . neotomae containing pBgap [Table 3], indicatingthat NnrB is a weak activator of nirK and, by extension, norexpression.

Loss of nirK and nnrA impacts in vivo growth. Given the importance of NO as part of the infection response,it is of interest to test whether the loss of the nirK-nnrA region has had an impact on the pathogenicity of B . neotomae. To test whether the deletion affects virulence, B . neotomae wild-type and pBgap-complemented strains were injected intoIRF-1^–/– mice . IRF-1^–/– mice, unlikewild-type mice, are highly susceptible to Brucella infection.Brucella infection in IRF-1^–/– mice is lethal, andthe mortality is dependent on the virulence of the Brucellaorganisms [13, 14] . Therefore, IRF-1^–/– mice serveas a rapid indicator system to assess virulence of Brucella strains . IRF-1^–/– mice were injected with eitherwild-type B . neotomae or B . neotomae/pBgap . At day 1 and every other day thereafter, two mice from each group were killed,and homogenized material from livers and spleens was culturedfor CFU . At day 5 the mice injected with the wild-type strainbegan to die, and no more mice were killed in either group.By day 10 all the mice remaining in the wild-type group haddied, while three of the four mice injected with the B . neotomae/pBgapsurvived for greater than 2 months . There was a general trendof higher CFU in both the liver and spleen in the wild-typestrain than in the strain containing pBgap . The difference inCFU was greatest on day 5, with the numbers of bacteria in theliver and spleen being at least 2 logs higher with the wild-typestrain than with B . neotomae/pBgap [Fig . 2] . The differencesin CFU were consistent with the eventual outcome of the experiment,since the mice injected with wild-type B . neotomae died soonafter day 5 . Similar levels of CFU were obtained when materialfrom the liver or spleen was plated on medium containing gentamicinto select for cells containing pBgap [data not shown] . Thisindicates that the presence of pBgap was not so detrimentalto the survival of B . neotomae as to result in its loss duringinfection.

 
A number of -proteobacteria form intimate associations withhosts, either as symbionts or as pathogens . The role of denitrificationin these associations has been poorly studied . This is the firstreport describing the role of denitrification in an -proteobacterial animal pathogen . In contrast to B . suis and B . melitensis, B.neotomae is a partial denitrifier, capable of nitrate reductionbut incapable of reducing nitrite . Nitrite cannot be reducedsince the structural gene for Nir has been partially deleted. The truncated Nir cannot form an active protein, since the remaining fragment is missing ligands for type 2 copper [9] . The deletionof nirK in B . neotomae is accompanied by the loss of nirV, agene of unknown function but likely having some role in Nirassembly or activity [10] . The genes required for Nor productionand assembly are present in B . neotomae.

Previously, the loss of portions of the denitrification pathway has been reported in some but not all strains of R . sphaeroides [20] . Analysis of genomes of denitrifying and nondenitrifyingstrains of R . sphaeroides suggests that 2.4.1 and 2.4.3 hada common ancestor that was a denitrifier but that 2.4.1, andmost other R . sphaeroides strains, lost the ability to reducenitrite [19] . However, strains did not lose the ability to reduceNO, since the nor operon and nnrR, which encodes a transcriptionalregulator required for nirK and nor expression, are presentand have been shown to be functional [20] . In contrast, B . neotomae is unlikely to reduce NO, since a gene encoding an NnrR ortholog, nnrA, has been partially deleted along with nirK and nirV . B.neotomae cells lacking nnrA but containing nirK and nirV fromB . melitensis exhibited only weak Nir activity [Table 3] . IfnnrA was present along with nirK and nirV, Nir activity increased significantly, demonstrating the importance of nnrA [Table 3].

In all brucellae characterized to date, there are two genes encoding NnrR-related proteins in the denitrification gene cluster. The occurrence of NnrR paralogs has not been previously describedin other denitrifiers . Sequence comparisons have demonstratedthat Brucella NnrA is more similar to NnrRs from other denitrifiers than is NnrB . NnrA is also a better activator of expressionof B . neotomae norC and nor genes from A . tumefaciens than is NnrB [Table 4 and data not shown] . The physiological functionof NnrB is not clear; however, NnrB could be used to activatea subset of denitrification genes since it clusters with two genes, nnrS and paz, indirectly involved in nitrogen oxide reduction.

Examination of the B . suis and B . melitensis genomes in the region where the deletion occurred in the B . neotomae genome revealed a 6-base repeat at the start and end of the region that was deleted [Fig . 1] . In pairwise comparisons of B . neotomaewith B . suis and B . melitensis genomes, the nirK ORF is conserveduntil the sequence CGTGGC, bases 536 to 541 of nirK . The remainingfragment of nnrA in B . neotomae starts at base 465, which ispreceded by the 6-bp sequence CGTGGC . The CGTGGC sequence doesnot occur between nirK and nnrA in other Brucella species, suggestingthat a recombination event across these repeated sequences mayhave caused the deletion [4].

Since other species of Brucella have no similar deletions in their denitrification gene clusters, the deletion in B . neotomae occurred after the strain diverged and adapted to its current lifestyle . While the role of denitrification in Brucella is unclear, it has been hypothesized that the ability to reduceNO would provide an effective means of dealing with the NO producedin response to infection [34] . Previous work has shown that NO production can increase long-term survival of Brucella abortus, a complete denitrifier, in macrophages [34] . However, the survivalof denitrification-compromised strains was not tested . In thisstudy, it was shown that there was a significant difference in the lethality of B . neotomae strains with and without the genes encoding Nir and NnrA . Infection with 10⁷ wild-type B. neotomae organisms proved lethal to all mice within 10 days. The majority of the mice [three out of four] survived when injected with B . neotomae carrying the nirK-nnrA region in trans . Infectionpersisted in mice injected with the nirK-nnrA strain, as indicatedby the numbers of CFU found in the spleen [Fig. 2] . The numberof hepatic CFU decreased at day 5, indicating that the micewere able to control hepatic B . neotomae . These results demonstratethat denitrification, particularly the capacity to reduce nitriteand NO, is important in modulating the interaction of Brucellawith its host . It is likely that the capacity to denitrify isphysiologically significant, because it allows Brucella to usenitrate, nitrite, and NO as terminal oxidants in the oxygen-poorenvironment inside the macrophage [27] . NO has also been reportedto inhibit oxygen respiration in intracellular pathogens, andso the capacity to reduce NO may also have the paradoxical effectof allowing cells to use both nitrogen oxides and oxygen asterminal oxidants [33] . Irrespective of the exact mechanism,utilization of nitrogen oxides by Brucella may enhance its long-termsurvival in the macrophage . In wild-type B . neotomae, the stressinduced by limiting levels of terminal oxidants in the mousemacrophage may induce a stress response that causes the cellsto have a more deleterious effect on their host . If this werethe case, then it is not clear why nirK and nnrA has been lostfrom the B . neotomae genome, since the resulting phenotypicchange would seem to be disadvantageous . One possible explanationis that wood rat macrophages may produce minimal NO . This wouldmitigate the physiological requirement for Nor, and so the phenotypicchanges arising as a result of nirK, nirV, and nnrA inactivation would not be as deleterious . The deletion of these denitrifying genes might, however, have resulted in a limitation in the hostrange of B . neotomae . Future studies are required to determine whether the Brucella denitrification genes contribute to host range.

 
We thank Sigrid Holmgren for initiating this collaboration andDavid Glover for helping with infection of mice.

This work was supported by National Institutes of Health grant R01AI048490 and BARD-US 2968-98C [to G.S.] and the Departmentof Energy grant 95ER20206 [to J.P.S.].

 
* Corresponding author . Mailing address: Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101 . Phone: [607] 255-8535 . Fax: [607] 255-3904 . E-mail: jps2@cornell.edu .

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