INTRODUCTION

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Interconnections among biochemical pathways remain an understudied question in modern biology. Currently, this problem is being addressed in several different bacterial systems. For example, Frodyma et al. (28) and Tsang et al. (77) investigated the metabolic integration (23) of vitamin synthetic pathways which are thought to have a rather low flux since cofactors are used in catalytic rather than structural quantities. We have chosen to explore metabolic integration by focusing on the action of an inhibitor, acivicin (Fig. 1), since it is believed to interact with glutamine amidotransferases (61, 62, 74, 84) and its antibacterial activity toward Bacillus subtilis is antagonized by histidine and purine nucleosides (35). These enzymes extract ammonia from glutamine prior to attaching it to an organic backbone.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Structures of some amino acids. Only R groups are depicted.

In Escherichia coli there are at least 12 distinct glutamine amidotransferases (58, 83) involved in biosynthesis, underscoring the importance of ammonia assimilation by processes in addition to transamination. Five are involved in amino acid biosynthesis: anthranilate synthase (EC 4.1.3.27, TrpE), asparagine synthase (EC 6.3.5.4, AsnB), carbamoyl phosphate synthetase (EC 6.3.5.5, CarAB [used in arginine as well as pyrimidine nucleotide production]), glutamate synthase (EC 1.4.1.13, GltBD), and imidazole glycerol phosphate (IGP) synthase (HisHF). Along with CarAB, another four (EC 6.3.4.2, PyrG, CTP synthetase; EC 2.4.2.14, PurF, glutamine 5-phospho-D-ribosyl--1-pyrophosphate [PRPP] amidotransferase; EC 6.3.5.3, PurL, 5^'-phosphoribosyl-N-formyl glycinamide [FGAM] synthetase; and EC 6.3.4.1, GuaA, GMP synthetase) are enzymes of nucleotide biosynthesis. Two, PabAB (4-amino-4-deoxychorismate synthase, a component of the folate pathway) and NadE (EC 6.3.5.1, NAD synthetase), function in cofactor synthesis, while one, GlmS (EC 2.6.1.13), is involved in production of the cell wall precursor N-acetylglucosamine phosphate. Thus, antagonism of this enzyme family might be most revealing.

One glutamine amidotransferase, IGP synthase, is encoded by hisH and hisF; these two genes are components of the histidine operon, hisGDCBHAFI (Fig. 2A) (88). This amidotransferase occupies a central position in the eight-enzyme pathway from PRPP and ATP to histidine (Fig. 3). If this reaction or the immediately preceding HisA (pro-phosphoribosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide [PROFAR] isomerase)-catalyzed reaction is blocked, ATP is still condensed with PRPP and undergoes subsequent modification, including opening of its six-membered ring. Such blockages drain the purine nucleotide pools, effectively causing the metabolic economy to grind to a halt due to a lack of "currency," presumably in the form of adenylates. Normally the amidotransferase reaction of the histidine biosynthetic pathway liberates 5-aminoimidazole-4-carboxamido-1--D-ribofuranosyl 5'-monophosphate (AICAR) as a by-product. The latter molecule, a purine biosynthetic intermediate, is salvaged in a process that leads to the resynthesis of ATP. This combined histidine-purine cycle is hence critical for cellular function, as demonstrated by the studies of Hartman et al. (36), Shedlovsky and Magasanik (70, 71), Johnston and Roth (44), and Taylor et al. (29, 42, 72, 73). Moreover, overproduction of HisHF has other deleterious consequences for cell division (3, 27, 57) independent of the above-mentioned adenylate drain. Thus, the HisHF enzyme is an attractive site for the study of metabolic integration.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   (A) The histidine operon. his genes are indicated by boxes. Promoters are indicated by filled dots with arrows denoting direction of transcriptions. Sites of transcriptional termination are denoted by lollipops. (B) Plasmids that complement his point mutants, denoted by lines.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Histidine biosynthesis. Also shown is the reaction (b) catalyzed by yeast inorganic pyrophosphatase that drives reaction a to the right in a coupled in vitro system.

Due to the arrangement of the his genes within an operon (Fig. 2A) (88), it is difficult to eliminate function of an individual gene due to the polar nature of many his mutations. Furthermore, draining of adenylates by such mutants might provide a strong selective pressure for true reversion or pseudo-reversion. Hence, the ability to transiently compromise HisHF or HisA activity by the addition of a specific inhibitor is desirable. We demonstrate that acivicin has such HisHF-directed antagonism. The nutrients that prevent its inhibitory action, its specificity, and the consequences of its administration are investigated by the genetic, biochemical, and enzymological analyses of E. coli reported here.

	MATERIALS AND METHODS

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Abbreviations and nomenclature. Standard bacterial nomenclature (8) is used. Biosynthetic intermediates are abbreviated as follows: PRFAR, N¹-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide; IAP, imidazole acetol phosphate; HOL-P, L-histidinol phosphate; HOL, L-histidinol; and 2-KG, 2-ketoglutarate. Polypeptide nomenclature includes HisG (ATP phosphoribosyl transferase), the HisI (the bifunctional phosphoribosyl-ATP pyrophosphorohydrolase/phosphoribosyl-AMP cyclohydrolase), the HisH (glutamine amidotransferase) domain, the HisF (cyclase) domain, HisB (the bifunctional IGP dehydratase/HOL-P phosphatase), HisC (HOL-P aminotransferase), HisD (histidinol dehydrogenase), and YIP (yeast inorganic pyrophosphatase).

Chemicals and biochemical reagents. Acivicin, glutamine, PRPP, and yeast inorganic pyrophosphatase were purchased from Sigma (St. Louis, Mo.). Purified E. coli HisHF enzyme (0.4 mg/ml, 7 U/mg) was a gift from V. J. Davisson, Purdue University.

Strains and plasmids. Plasmids are described in Table 1. E. coli strains FB1 (hisGDCBHAFI750) (12) and FB1/phisAGIE-tac were obtained from V. J. Davisson. The set of his operon point mutants was obtained from P. E. Hartman and has been described previously (30, 31). Salmonella enterica serovar Typhimurium Tn10 mutations were backcrossed into the wild type, selecting for tetracycline resistance as described elsewhere (20).

Inhibition assays. Disk diffusion was performed as described for sulfometuron methyl (47, 50), a modification of a previously described scheme (75). An alternative bioluminescent technique was also used (26). Briefly, an insertion of a recA promoter-Photorhabdus luminescens luxCDABE fusion within lacZ was crossed into strain DPD1692, selecting for kanamycin resistance. This strain, DPD1718, produces a high, baseline bioluminescence that is induced by DNA- damaging agents (82) and dampened by a wide range of metabolic inhibitors (11). Details of the construction have been described elsewhere (25). Both techniques are amenable to auxanography, a means to determine the pathway blocked by either mutation (20) or inhibitor action (47) through the supplementation with pools of nutrients. This method was used to determine those nutrients that allow metabolic function, be it growth or bioluminescence, in the presence of the inhibitor.

Genetic titration and complementation. The method used has been described in general for E. coli (15). The specific plasmid libraries containing random segments of the E. coli W3110 genome have been described (26) and used (87; Z. Xue, D. R. Smulski, D. Delduco, S.-Y. Soon-Yong Choi, M. H. Jia, and R. A. LaRossa, unpublished data) elsewhere. The MIC of acivicin for strain DPD1675 was 1 µg/ml on E (20) minimal agar medium supplemented with 0.2% glucose, thiamine, and proline. Selection was for those few transformants (59) of pBR322- and pUC18-based genomic libraries of E. coli that would support growth on the above-described medium supplemented with ampicillin (100 µg/ml) and acivicin (3 µg/ml). Plasmid DNA was isolated from resistant clones, backcrossed by transformation (59) into strain DPD1675 to ascertain that resistance was a plasmid-specified phenotype, and sequenced as described previously (26, 87; Xue et al., unpublished).

Substrate preparation. PRFAR was synthesized using E. coli strain FB1/phisAGIE-tac based on published protocols of Davisson et al. (21) and Deras (22), with some modifications. The reaction scheme is shown in Fig. 3. PRPP (270 µmol) was reacted with excess ATP (400 µmol) in the presence of 72 U of inorganic pyrophosphatase and 9.6 mg of FB1/phisAGIE-tac extract in 86 mM potassium phosphate (pH 7.5)-28 mM MgCl₂-3.5 mM EDTA. After the reaction flask was shaken at 30°C for 1 h, an additional 4.8 mg of FB1/phisAGIE-tac extract was added, and the reaction mixture was incubated for another 2 h. PRFAR was purified by applying the reaction mixture to a Q-Sepharose column (2.5 by 14 cm; Pharmacia) equilibrated with 60 mM NH₄HCO₃, and eluting with an NH₄HCO₃ gradient (60 to 300 mM over 300 ml). The fractionated eluant was analyzed by UV-visible light spectroscopy, and fractions in which A₂₉₀/A₂₆₀ was >1.0 were pooled and dried by lyophilization. The lyophilized product was redissolved in 0.1 M tetraethylammonium acetate (pH 7.0) and further purified by reverse-phase high-pressure liquid chromatography on a C₁₈ column (Supelco LC18 column; preparative scale; 2.5 by 25 cm). PRFAR was eluted isocratically by 0.1 M tetraethylammonium acetate, monitored by optical density at 300 nm. Peak fractions containing PRFAR were pooled, lyophilized, and stored at 80°C. Purified PRFAR was analyzed by UV-visible light spectroscopy and gave characteristic absorbance peaks at A₂₂₀ and A₃₀₀.

HisHF enzyme assay. The standard assay for E. coli HisHF (55) was performed as described elsewhere (45). The assay basis is the initial rate of substrate PRFAR disappearance as monitored by A₃₀₀. Briefly 100 µM PRFAR is mixed with 5 mM glutamine in 50 mM Tris-HCl (pH 8.0), and the reaction is initiated by addition of purified HisHF at 0.02 U/ml (62 nM). The mixture was incubated at 25°C for 2 to 5 min. One unit of activity is defined as formation of 1 µmol of product per minute under the specified reaction conditions.

K_i determination. To determine the inhibition constant (K_i) for acivicin, the HisHF assay was performed in the presence of 400 or 625 nM acivicin at glutamine concentrations of 0.125, 0.25, 0.5, 1, and 2 mM. The K_i for acivicin was determined from the reciprocal plot of 1/V versus 1/[glutamine].

HisHF inactivation by acivicin. HisHF (1.0 µM) was preincubated at 25°C in 100 µl of 50 mM Tris-HCl (pH 8.0) with (i) 10 µM acivicin, (ii) 100 µM PRFAR, (iii) 10 µM acivicin and 100 µM PRFAR, and (iv) buffer only. An aliquot (12 µl) was removed at 0, 10, 20, 40, and 60 min and diluted into a reaction mixture (288 µl) containing 100 µM PRFAR, 5 mM glutamine, and 50 mM Tris-HCl (pH 8.0), and the initial rate of the reaction was measured as described above.

Gene expression profiling. The basic dual-label, fluorescence-based method has been described in detail elsewhere (86). These experiments differed from those described previously in that the genes were spotted at a higher density, i.e., 9,000 spots/per slide, using a generation III DNA spotter (Molecular Dynamics, Sunnyvale, Calif.) such that an entire genome was spotted in duplicate on a single slide, negating the need for slide-to-slide correction. Genes were categorized into the functional groups of Riley and Labedan (67) as has been used in other transcript profiling exercises (85, 86).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Nutritional supplementation. Acivicin inhibited the growth of many E. coli K-12 and S. enterica serovar Typhimurium strains when grown on solidified minimal, but not rich, media. The presence of histidine or purines (guanine plus adenine), but not tryptophan, glutamate, or glucosamine-6-phosphate, significantly lessened the inhibition as monitored by disk diffusion assays (data not shown) with E. coli strain CS1562 (69) and S. enterica serovar Typhimurium strain TV101. Similar results were obtained when nutrient pools were used in auxanography (20) with a bioluminescent tester strain, DPD1718 (data not shown). The mixture of adenosine, histidine, phenylalanine, glutamine, and thymine was completely effective at preventing inhibition by acivicin, while supplementation with histidine, lysine, and the three branched chain amino acids was almost as effective an antidote. Other pools were incapable of preventing the inhibitory action. Histidine alone was not quite as effective an antidote as either mixture. This, together with the structural similarity between acivicin and glutamine, suggested that the target of acivicin in E. coli might be the HisHF enzyme.

Acivicin is a competitive inhibitor of glutamine for HisHF. HisHF catalyzes the reaction of PRFAR and glutamine to form of IGP, AICAR, and glutamate (88). The k_cat of E. coli HisHF is 8.5 s¹, the K_m for glutamine is 240 µM, and the K_m for PRFAR is 1.5 µM (45). We assayed the activity of HisHF in the presence of 100 or 400 nM acivicin, with glutamine concentrations ranging from 0.125 to 2 mM, while keeping PRFAR at 100 µM, a vast excess. A reciprocal plot of 1/V versus 1/[glutamine] was generated (Fig. 4), resulting in an estimate of 290 µM for the glutamine K_m. This value was consistent with another determination noted above. Moreover, the K_i of acivicin was determined to be 140 nM. This indicates that acivicin is at least an order of magnitude more inhibitory in vitro toward HisHF than those enzymes that have been tested by others. Thus, acivicin was a potent inhibitor of HisHF in vitro.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Inhibition of E. coli IGP synthase (HisHF) by acivicin.

The inactivation of HisHF by acivicin is accelerated by PRFAR. A possible mechanism of acivicin action is that it binds competitively to the glutamine binding site on HisH and inactivates the enzyme by covalent modification of an active site cysteine residue essential for glutamine amidotransferase activity. HisH and HisF of E. coli are isolated as a single heterodimer (45). It thus is possible that both the glutamine amidotransferase activity of HisH and the cyclase activity of HisF are carried out at one active site shared between the two polypeptides and that the binding of glutamine and PRFAR is cooperative. A second possibility is that separate substrate binding sites exist on the HisH and HisF polypeptides. After the glutamine amidotransferase reaction is carried out on the HisH domain, NH₃ might be transferred to the PRFAR binding site on the HisF domain. To test this hypothesis, acivicin was used to probe the active site of HisHF together with PRFAR. HisHF was preincubated with 10 µM acivicin alone (1:10 ratio of enzyme to inhibitor), 100 µM PRFAR alone (1:100 ratio of enzyme to substrate), or both 10 µM acivicin and 100 µM PRFAR. The preincubation time varied from 0 to 60 min. At each time point, an aliquot of the mixture was removed and assayed for remaining activity. The fraction of residual activity was plotted versus preincubation time (Fig. 5). In the presence of either acivicin or PRFAR alone, there was a time-dependent loss of HisHF activity. This indicates that both acivicin and PRFAR were irreversible inhibitors of the reaction when incubated with HisHF alone. It is rather interesting that PRFAR inhibited the reaction irreversibly when it was added to the enzyme before the addition of glutamine. Even more interesting, when both acivicin and PRFAR were preincubated with HisHF, the enzyme activity at the first time point (10 min) decreased dramatically to almost the background level. We have not yet investigated if inactivation is more than additive. In addition, binding of PRFAR to the active site may promote covalent bond formation between glutamine and the catalytic cysteinyl residue of the HisH domain. These results were consistent with the proposed catalytic mechanism (66) and our hypothesis that the HisH domain and HisF domain share one active site that carries out both the glutamine amidotransferase and cyclase steps of the reaction. Moreover, they raise the possibility that the dominant factor contributing to the growth inhibition of a variety of microbial and eukaryotic cells could be the inactivation rates of various glutamine amidotransferases by acivicin as well as each enzyme's K_i for the small molecule.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Inactivation of E. coli IGP synthase (HisHF) by acivicin (triangles), PRFAR (diamonds), and acivicin plus PRFAR (open circles). The untreated enzyme is indicated by closed circles.

Reconstruction experiments. Several salient features of his operon expression must be noted to aid in the interpretation of the following experiments. Three promoters (12) (Fig. 2A) are present in the operon although the primary promoter (hisp1) is more than 10-fold stronger than hisp2, which in turn is far stronger than hisp3. Furthermore, transcription from hisp1 occludes usage of hisp2 (88). Initiation at hisp1 is stimulated by the product of RelA, ppGpp (89), produced in response to starvation for any amino acid (13). Transcriptional read-through past att (43), the attenuator site, occurs when the in vivo level of histidyl-tRNA^his is low (88). Thus, the operon is subject to both global and specific regulatory circuitry.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Titration of a bioluminescent response by acivicin. The bioluminescent host strain DPD1718 was transformed with either pUC18 or pDEW327. Squares, the strain contained pUC18 and the medium was not supplemented with histidine; diamonds, the strain contained pUC18 and the medium was supplemented with histidine; crosses, the strain contained pDEW327 (his'BHAFI) and the medium was not supplemented with histidine; triangles, the strain contained pDEW327 (his'BHAFI) and the medium was supplemented with histidine.

Genetic titration. Libraries of random fragments of the E. coli genome in either pUC18 or pBR322, harbored in strain DPD1675, served as a source of genetic variation. Clones resistant to acivicin were selected, plasmids conferring the resistance phenotype were sequenced, and the precise locations of the resistance elements were thus determined. These resistance elements mapped to two regions distinct from his.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   yedA region of the E. coli chromosome. Lines below the map show the extents of various plasmids that confer acivicin resistance. Directions of transcription are indicated by arrowheads on symbols denoting genes; dots indicate promoters.

View larger version (14K): [in this window] [in a new window]   FIG. 8.   iciA region of the E. coli chromosome, denoted as described for Fig. 7.

Gene expression profiling. The growth of E. coli strain MG1655 in minimal medium with glucose as a carbon source was inhibited about 80% by 0.5 µg of acivicin per ml; such inhibition was completely prevented by coexposure to histidine. Histidine also reversed a greater acivicin challenge (2 µg/ml, 90% inhibition); the culture exposed to both the antagonist and the antidote grew at about 90% of the uninhibited rate (data not shown). Results from a representative DNA microarray experiment, in which E. coli MG1655 was challenged with acivicin at 2 µg/ml in minimal medium for 60 min, are presented in Tables 2 and 3. This treatment lowered the growth rate by about 85%. The structural similarity among acivicin, glutamine, and asparagine is illustrated in Fig. 1. This similarity was reflected in the gene expression profile described below.

Indications that acivicin serves as an imposter of certain natural amino acids. Transcription of glnA, encoding glutamine synthetase, was lowered more than sixfold. Expression of asnS, coding for asparaginyl-tRNA synthetase, was decreased more than fivefold; unfortunately, the spotted glnS PCR product was of poor quality (Y. Wei and R. LaRossa, unpublished data), precluding insight into its transcriptional response to acivicin.

Evidence that HisHF is the major target in vivo. If the HisHF enzyme is inhibited by acivicin in vivo, then transcription should initiate frequently at the his promoter (75, 89), pass through the leader/attenuator (43), and traverse the structural genes. This expectation was indeed realized, as the eight structural genes, hisGDCBHAFI, and the leader hisL were up-regulated 6- to 16-fold by administration of 2 µg of acivicin per ml. Ranking of open reading frames (ORFs) by fold induction with acivicin placed his operon genes in the 4th (hisC), 5th (hisL), 6th (hisB), 7th (hisI), 8th (hisG), 9th (hisD), 11th (hisH), 12th (hisF), and 28th (hisA) positions.

Metabolic mayhem. The term "metabolic mayhem" has been applied to the action of sulfonylurea herbicides in S. enterica serovar Typhimurium, which causes the cell to (i) signal methionine sufficiency (10) in the face of methionine limitation (51) and (ii) skew the ratios of 2-ketoacids (52) and acyl coenzyme A's (78), two classes of central precursor metabolites. Together, about 60% of the organic content of E. coli is derived from these two sets of central building blocks (48). A similar case may be evident when HisHF activity was limiting. The ATP pool was compromised (29, 42). The cell, however, did not respond by elevating the F₀-F₁ ATP synthase-specifying transcripts; rather, the atp operon was mildly repressed (Table 3), indicating that the capacity to convert ADP to ATP was not enhanced. Most purine biosynthetic transcripts were not affected appreciably by the acivicin administration, suggesting that the PurR regulon (92) was indifferent to this treatment. Evidence for modulating expression of two purine-related operons, however, was found. The bicistronic guaBA operon was down-regulated about twofold (Table 3), while the purA transcript was elevated more than sevenfold (Table 2). The latter result contradicted the thought (37) that the PurR-independent regulation of purA is posttranscriptional. Both the purA and guaBA operons are regulated by multiple regulatory circuits (92). guaBA is responsive to PurR (92), cyclic AMP receptor protein (39), and DnaA (92), while purA is controlled by both PurR and an adenine-dependent mechanism (92). These transcriptional data suggested that flux from IMP to AMP was being encouraged at the expense of forming GMP from the common intermediate IMP. Thus, the apparent inconsistencies in the transcriptional regulation of the purine regulon might be indicative of a baroque regulatory mechanism designed to maintain balance between the GTP and ATP pools.

Acivicin triggers the stringent response. The just-mentioned induction and repression of gene expression suggest that the in vivo levels of histidyl-tRNA, glutaminyl-tRNA, and/or asparaginyl-tRNA may be lowered by acivicin treatment. Any such a drop would trigger the stringent response. This response has two basic elements, conservation of amino acid reserves by shutting off synthesis of ribosomes and other translational machinery (13) and a redirection of resources toward increasing amino acid biosynthesis (75, 89). Aspects of each are apparent in the gene expression profile of acivicin-treated cells. As expected for a treatment resulting in amino acid starvation, expression of the translational apparatus was decreased; 49 distinct ORFs encoding proteins involved in translation were down-regulated by a factor of 2 or more (Table 3).

Other stress responses triggered by the acivicin challenge. The two most highly induced genes were hslS and hslT, heat shock loci (65), whose transcripts were elevated 20- to 30-fold (Table 2). Other stress-responsive transcripts (Table 2) were highly induced, including clpB (8.6-fold), appA (4.3-fold), uspA (4.2-fold), ahpC (3.4-fold), rpoH (3.2-fold), slp (3-fold), rob (2.8-fold), cspE (2.8-fold), soxS (2.8-fold), htpX (2.6-fold), osmE (2.6-fold), sugE (2.5-fold), grpE (2.5-fold), sohA (2.4-fold), and slyD (2.1-fold). Also within this group of induced mRNA species were transcripts involved in osmotolerance, including betT (8-fold), otsA (4.4-fold), betI (4.3-fold), osmY (3.8-fold), otsB (2.7-fold), osmE (2.6-fold), treF (2.4-fold), betA (2.4-fold), and betB (2.4-fold). Expression of some genes involved in DNA and RNA metabolism was also heightened; elevated levels of hepA, rna, nfo, recN, and xseB transcripts were observed.

Unanticipated repression by acivicin. Many changes were observed that were not predictable. Strikingly, the level of several amino acid biosynthetic transcripts was not up-regulated but rather reduced by histidine starvation (Table 3), including transcripts for genes specifying components of the aromatic pathways (aroA, aroF, aroH, pheA, trpL, and tyrB), the pyruvate family (avt, ilvB, ilvC, ilvD, ilvE, ilvG, ilvM, leuC, and leuD), sulfur amino acids (cysA, cysC, cysD, cysK, cysM, cysN, and metE), and the aspartate family (lysC). Greater than fivefold repression was observed for aroA, ilvC, ilvG, and metE. Each of these genes is distinctive; aroA (16) and ilvG (47) specify enzymes targeted by commercially important herbicides, while ilvC and metE encode enzymes that must be highly expressed (86) due to their poor performance as catalysts (32, 60, 90). Interestingly, expression of another most highly expressed, pyrimidine biosynthetic operon (86) was down-regulated by the acivicin challenge; pyrBI (specifying aspartate transcarbamylase) transcripts were greatly reduced. Surprisingly, genes implicated in uptake of amino acids or their precursors were often down-regulated. Included in this category were cysP, livJK, and proX. Certain carbon utilization transcripts were down-regulated. Repression of six gat and three uid genes was observed.

Unanticipated elevation of gene expression by acivicin administration. Expression of three genes, ack (78), pta (78), and mrp (D. R. Smulski and R. A. LaRossa, unpublished data), whose inactivation leads to a sulfometuron methyl-sensitive phenotype in S. enterica serovar Typhimurium LT2 (52), was elevated after acivicin treatment. Other transcripts increased by this treatment were specified by the tdh-kbl operon. The corresponding gene products specified by this operon degrade threonine to pyruvate and ammonia through glycine and serine. Thus, the overall pathway involves tdh, kbl, gcvTHP, lpdA, glyA, and sdaA or metC (64). The lpdA and sdaA mRNA titers, as well as the tdh-kbl transcript levels, were elevated by acivicin treatment, suggesting increased flux through this pathway.

Summation. Acivicin is a natural product produced by streptomycetes. Its ecological role may be to defend the home turf of the producing species, encouraging other microbes to emigrate toward less hostile environments. Such use in antibacterial warfare may be explained by theories concerning the evolution of translation (18, 19) and biosynthetic pathways. Hence, adoption of acivicin for use in cancer therapy represents its introduction into a novel niche, one occupied by cells lacking its intended target, HisHF. Thus, studies of the action of acivicin with other glutamine amidotransferases could represent more general aspects of the binding of glutamine to the amidotransferase enzyme family. In contrast, acivicin interacted much more avidly with HisHF from E. coli. This specific interaction is not limited to the E. coli enzyme; inhibition of the homologous eukaryotic enzyme, His7 from yeast, has been found (M. McCluskey and L. Huang, unpublished data). The relative contributions of acivicin's competition with glutamine and its inactivation of glutamine amidotransferases to the observed in vivo effects are worthy of further study.