Journal of Bacteriology, March 2004, p . 1388-1397, Vol . 186, No . 5

Transcriptional Organization and Regulation of the L-Idonic Acid Pathway [GntII System] in Escherichia coli

Christoph Bausch, Matthew Ramsey, and Tyrrell Conway^*

Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019

Received 23 July 2003/ Accepted 19 November 2003

 
The genetic organization of the idn genes that encode the pathway for L-idonate catabolism was characterized . The monocistronicidnK gene is transcribed divergently from the idnDOTR genes,which were shown to form an operon . The 215-bp regulatory regionbetween the idnK and idnD genes contains promoters in oppositeorientation with transcription start sites that mapped to positions-26 and -29 with respect to the start codons . The regulatoryregion also contains a single putative IdnR/GntR binding sitecentered between the two promoters, a CRP binding site upstreamof idnD, and an UP element upstream of idnK . The genes of theL-idonate pathway were shown to be under catabolite repressioncontrol . Analysis of idnD- and idnK-lacZ fusions in a nonpolar idnD mutant that is unable to interconvert L-idonate and 5-ketogluconateindicated that either compound could induce the pathway . TheL-idonate pathway was first characterized as a subsidiary pathwayfor D-gluconate catabolism [GntII], which is induced by D-gluconate in a GntI [primary gluconate system] mutant . Here we showed that the idnK and idnD operons are induced by D-gluconate ina GntI system mutant, presumably by endogenous formation of 5-ketogluconate from D-gluconate . Thus, the regulation of theGntII system is appropriate for this pathway, which is primarilyinvolved in L-idonate catabolism; the GntII system can be inducedby D-gluconate under conditions that block the GntI system.

 
For three decades, there was thought to be two systems for D-gluconate catabolism, GntI and GntII [1] . The GntI system consists ofgntT, gntU, and gntK, which encode high- and low-affinity D-gluconatetransporters and a thermoresistant gluconate kinase, respectively[19-21, 29] . GntR negatively controls the GntI genes, as well as edd and eda of the Entner-Doudoroff pathway . The GntII systemis composed of a thermosensitive gluconate kinase and a gluconatetransporter, which provide for subsidiary catabolism of gluconatein GntI mutants [3, 16] . Recently, we discovered that the GntIIsystem is, in fact, a pathway for catabolism of L-idonate, whichproceeds via a D-gluconate intermediate [3] . The discovery ofthis novel pathway solved the longstanding question of why thereare two pathways for gluconate; GntI is primarily involved ingluconate catabolism, and GntII is responsible for idonate catabolism.

The catabolic sequence for L-idonate is as follows: L-idonateis transported by the L-idonate transporter, IdnT; L-idonate is oxidized to 5-keto-gluconate [5KG] by L-idonate 5-dehydrogenase,IdnD; 5KG is reduced to D-gluconate by 5-keto-D-gluconate 5-reductase,IdnO; and D-gluconate is phosphorylated by a thermosensitive gluconate kinase, IdnK, to make 6-phosphogluconate [6PG], which is further catabolized via the Entner-Doudoroff pathway . Thus,IdnD and IdnO allow for the redox-coupled interconversion of L-idonate to D-gluconate via 5KG . The L-idonate catabolic pathwayoverlaps D-gluconate catabolism through the common intermediates D-gluconate and 6PG.

While the biochemistry of the L-idonate pathway is firmly established,the organization and regulation of the corresponding genes havenot been characterized . Sequence annotation indicates that idnKis monocistronic and is divergently transcribed from a putativeoperon consisting of idnD, idnO, and idnT along with idnR, whichencodes a repressor of the GalR-LacI family . In this report,we confirm the operon arrangement, transcription start sites,and regulation of the L-idonate genes . The results indicatethat both L-idonate and 5KG act as inducers of the idonate pathway.Furthermore, the subsidiary role of the GntII system for gluconatecatabolism was investigated in a GntI system mutant and shownto result from induction of the idnD operon and idnK by gluconate,presumably caused by accumulation of an endogenous inducer [e.g.,5KG] . Lastly, functional genomic analyses with DNA arrays andtwo-dimensional [2-D] protein gels were used to characterizethe global gene expression—and hence the physiology—of cells grown with L-idonate as the sole carbon source.

 
Bacterial strains and growth conditions. The Escherichia coli strains used in this study are listed inTable 1 . E . coli W1485 was the wild-type strain . All mutantand chromosomal lacZ fusion strains were derived from E . coli W1485 [2] . E . coli DH5 and XL1-Blue were used for propagationof plasmids . Strains were grown at 37°C in Luria broth [LB][13] with or without added carbohydrate [0.4%], in morpholinepropanesulfonicacid [MOPS] minimal medium with added carbohydrate [0.2%] [17], or in MOPS complete medium with added carbohydrate [0.2%] [32]. MOPS complete medium contains amino acid, vitamin, purine, and pyrimidine supplements . When appropriate, ampicillin [100 µg/ml]and kanamycin [25 µg/ml] were included in the growth medium.All cultures [50-ml volume] were grown in 250-ml Erlenmeyerflasks and aerated by gyratory shaking at 300 rpm . Cell growthwas monitored spectrophotometrically at 600 nm with a DU 530Life Science UV/Vis spectrophotometer [Beckman Coulter, Inc.,Fullerton, Calif.] . Cultures in the early and late logarithmicphases of growth were harvested at optical densities of 0.3and 0.7, respectively . Phenotypes of E . coli strains were determinedon MacConkey indicator medium [14], tryptone-yeast extract agar [6], or LB plates [13].

 
Plasmid construction, DNA modification, and transformation. Standard methods were used for DNA restrictions, ligations,and transformations and other DNA manipulations [23] . PCR amplificationwas performed with Platinum high-fidelity Taq DNA polymerase[Invitrogen Life Technologies, Carlsbad, Calif.] . The plasmidsused in this study are listed in Table 2 . Primers specific tothis study are listed as supplementary material found on ourwebsite [http://www.ou.edu/microarray] . Gene-specific deletionswere carried out with E . coli W1485 by the method reported byDatsenko and Wanner [6] . PCR-generated products were purifiedwith a QIAquick PCR Purification Kit [Qiagen Inc., Valencia,Calif.] . Electroporation was performed on a Gene Pulser II with0.2-cm-gap cuvettes [Bio-Rad Laboratories, Hercules, Calif.].Colony PCR was accomplished by scraping cells from agar plates,thoroughly washing the cells five times with water, and amplifyingPCR products by using sequence-specific primers and HotStarTaqDNA polymerase [Qiagen Inc., Valencia, Calif.].

 
Construction of idnK-lacZ and idnD-lacZ gene fusions. Single-copy idnK-lacZ and idnD-lacZ gene fusions were constructed by a recombinase-assisted lacZ fusion system developed for this work by combining the lacZ fusion system described by Simons et al . [27] and the system for allele replacement describedby Wanner et al . [32] . The fusion vectors used for recombinase-assistedlacZ fusion included pCB551, pCB552, and pCB577 [Table 2] . These plasmids contain the ori and bla genes from pSP72, 542 nucleotides of the 3' end of the E . coli W1485 lacI gene cloned from pCB108,and functional elements common to pRS551, pRS552, and pRS577 , including a selectable kanamycin resistance gene; four tandem copies of the T1 terminator from the E . coli rrnB operon; the unique multiple cloning site [MCS] containing BamHI, SmaI, andEcoRI; and the 5' region of the lacZ gene . A DNA fragment containingthe untranslated region between the idnK and idnD genes anda terminal BamHI or EcoRI site located upstream of the idnKand idnD start codons, respectively, was amplified by PCR . ThisDNA fragment was cloned into the BamHI-EcoRI sites of the proteinfusion vector pCB552 and the transcription fusion vector pCB551,creating idnK-lacZ fusion plasmids pCB120 and pCB220, respectively.The same fragment was cloned in the opposite orientation intothe protein fusion vector pCB577 at the EcoRI-BamHI site, generating an idnD-lacZ fusion [pCB121].

Integration of the lacZ fusions into the chromosome of E . coli W1485 was achieved by allelic replacement by homologous recombination of the fusion construct into the lacI-lacZ region of the genome.Linear DNA fragments for allelic replacement were amplified by PCR with pCB120 and pCB121 as the templates . This method eliminated native lacZ regulation and generated a lacZ fusion in one step . Bacterial colonies with the desired phenotype on tryptone-yeast extract agar-kanamycin plates were transferredto MacConkey plates, and the cells were screened for the lactose-negative phenotype . PCR was used to verify correct allelic replacement of the native lacZ regulatory region with the lacZ fusion; all of the lacZ fusion constructions were confirmed by DNA sequence analysis [25].

ß-Galactosidase measurements. ß-Galactosidase activity was measured with the yeastß-galactosidase assay kit from Pierce Biotechnology,Inc . [Rockford, Ill.] . Cell cultures were grown in triplicate,and each culture was assayed in triplicate . A 70-µl aliquotwas taken from each culture, mixed with an equal volume of ß-galactosidaseassay solution, placed in individual wells of a 96-well assayplate [Falcon Software, Inc., Wellesley, Mass.], and then heldat 4°C until the assay was performed . The ß-galactosidase assay solution was a 1:1 mixture of Y-PER [yeast protein extraction reagent] and 2x ß-galactosidase assay buffer . Beforeinitiation of the assay, spectrophotometric measurements at590 nm were made with a PowerWave X 96-well Microplate Spectrophotometer[Bio-Tek Instruments, Inc., Winooski, Vt.] to determine relativecell densities . The 96-well plate was incubated in the platereader at 37°C . Measurements were made spectrophotometricallyat 420 nm every 4 min for 1 h, and the data were analyzed withthe KC4 kinetics software package [Bio-Tek Instruments, Inc.].ß-Galactosidase activity was calculated when the reactionwas linear and expressed in Miller units [15] . The values reportedfor each sample are the means ± the standard deviationsfor nine independent measurements.

RNA isolation. Total RNA for Northern blot assays and primer extension analysiswas isolated by the hot-phenol method as described previously[21] . Total RNA for gene expression profiling and reverse transcriptasePCR [RT-PCR] was isolated by pipetting an equal volume of anactively growing cell culture into ice-cold RNAlater [Ambion,Inc., Austin, Tex.] . The RNA was then purified and treated withDNase with RNeasy mini kits and RNase-free DNase kits [QiagenInc.] . RNA concentrations were determined by spectrophotometricmeasurements at 260 nm . RNA was stored in ethanol at -80°C.

Primer extension analysis. Oligonucleotides complementary to the mRNA sequences upstreamof the idnK and idnD start codons were end labeled by usingT4 polynucleotide kinase [Invitrogen Life Technologies] and[-³²P]ATP [>5,000 Ci mmol^-1] as previously described [23]. Each 5'-end-labeled primer [0.5 pmol [1.5 x 10⁶ cpm]] was annealed to 30 µg of total RNA in a 10-µl reaction mixtureby heating to 94°C for 2 min, followed by slow cooling to42°C . The primers were then extended at 42°C for 5 hby using Moloney murine leukemia virus RT [Ambion, Inc.] . Thereaction was stopped by addition of 10 µl of sequenceloading buffer . The reaction mixtures were boiled for 3 min, and 4-µl aliquots were run on 6% polyacrylamide gels withsize reference ladders generated by dideoxy sequencing of pNP204with the same primers used for primer extension.

Northern blot analysis. Total cellular RNA [5 µg] was denatured by incubationfor 10 min at 68°C in formaldehyde-MOPS gel loading buffer[Ambion, Inc.] and electrophoresed through a 1.5% agarose gelcontaining formaldehyde and MOPS buffer . RNA was transferredto Nytran SuPerCharged superior nylon transfer membranes [Schleicher& Schuell, Inc., Keene, N.H.] by using a rapid downward transfer system . Antisense RNA probes were generated by reverse transcription from plasmids pCB92, pCB100, pCB200, pCB620, pCB700, and pCB900, containing the truncated genes idnK', idnD', idnO', idnT', yjgR', and idnR', respectively . These plasmids were constructedby cloning PCR products generated with nested gene-specificprimers into pBluescript II SK+ [Table 2] . All plasmids werelinearized at the 3' end of the truncated gene at the BamHIsite, and a ³²P-labeled RNA probe was synthesized by transcriptionwith T7 RNA polymerase [Cloned; Ambion, Inc.] in the presenceof [-³²P]UTP [23] . Probe hybridization to the membrane-bound RNA and stripping from the membranes were done as described previously [29] . Hybridized membranes were visualized by exposureto X-ray film or phosphorimaging screens, which were scanned with a STORM 820 PhosphorImager [Molecular Dynamics, Sunnyvale, Calif.].

RT-PCR. RT-PCR products were prepared by using the SuperScript One-StepRT-PCR system with Platinum Taq DNA polymerase [Invitrogen LifeTechnologies] as instructed by the manufacturer . Total RNA wasisolated at an optical density of 0.7 from E . coli W1485 grownin MOPS complete medium containing 0.2% L-idonic acid . The primerswere checked for performance in PCRs by using E . coli W1485genomic DNA as the template . RNA samples were tested for contaminatinggenomic DNA by using each RNA sample as a template for PCR;RNA samples contaminated with DNA were not used . The RT-PCRproducts were separated by electrophoresis through 1% agarosegels stained with ethidium bromide and documented with an EpiChemi II Darkroom [UVP, Inc., Upland, Calif.].

Transcriptome profiling and treatment of data. The methods used to handle whole-genome E . coli arrays and dataanalysis are described in detail on our website [http://www.ou.edu/microarray] and by Conway et al . [5] . The C-terminal primer set [Sigma-GenoSys,The Woodlands, Tex.] was used to transcribe radioactively labeledcDNA [first-strand synthesis] with [-³²P]dCTP and SuperScriptII RNase H^- RT [Invitrogen Life Technologies] from samples oftotal cellular RNA . Duplicate Panorama E . coli Gene Array membranes[Sigma-GenoSys] from consecutive printings were used . Hybridizationand stripping of membranes were done as described previously[28] . Phosphorimages of hybridized membranes were analyzed withArrayVision [Imaging Research Inc., St . Catharines, Ontario,Canada] to obtain raw spot intensity data . The raw data werenormalized by expressing individual spot intensities as a fractionof the sum of all gene-specific spot intensities in each image,and the data were analyzed as previously described by usingsemiautomated Microsoft Visual Basic programs in Microsoft Excel[5].

2-D polyacrylamide gel electrophoresis [PAGE]. Cells were harvested by centrifugation and washed twice in a10 mM MgCl-50 mM HEPES solution at pH 6.5 and then transferredto a lysis buffer that contained 9 M urea, 40 mM Tris-HCl, 4% 3-[[cholamidopropyl]-dimethylammonio]-1-propanesulfonate [CHAPS],and 1% dithiothreitol [DTT] . After sonication on ice for 5 x1 min with 30-s cooling intervals, cell debris was removed bycentrifugation at 3,000 x g for 10 min at 4°C . The protein concentration of the supernatant was determined by the Bradford assay [4].

A 200-µg sample of cell extract was loaded onto 7-cm immobilized pH gradient strips that had a nonlinear pH range of 3 to 10[Amersham Biosciences, Uppsala, Sweden] . A rehydration solutionthat contained 8 M urea, 2% CHAPS, 1% DTT, and 0.5% immobilizedpH gradient buffer [Amersham Biosciences] was added to the extractto a final volume of 120 µl . Rehydration was carried outfor 10 h at 20°C as described by Sanchez et al . [24] . Isoelectricfocusing [IEF] was carried out with an Ettan IPGphor IEF unit[Amersham Biosciences] for 1 h at 100 V, 30 min at 500 V, 30min at 1 kV, 1 h at 3 kV, 1 h at 5 kV, and 2 h at 8 kV . Thetemperature was held at 20°C throughout IEF . After IEF,the strips were incubated in a 50 mM Tris-HCl solution [pH 8.8]that contained 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate,and 1% DTT for 30 min . The strips were then placed on top of15% PAGE gels containing 2% sodium dodecyl sulfate and attachedwith a 0.5% agarose solution that contained a trace amount ofbromophenol blue [American Bioanalytical, Natick, Mass.] . Electrophoresiswas then carried out with a Mini-PROTEAN II system [Bio-RadLaboratories] at 20 mA for 4 h, until the bromophenol blue frontreached the bottom of the gel, and the gel was then stainedwith Coomassie brilliant blue.

In situ digestion, nano-electrospray MS-MS/MS, and data analysis. The stained gels were compared visually, and differentiallyexpressed spots of interest were excised and prepared by trypsindigestion in accordance with the protocol of Devreese et al.[7] . Nano-electrospray mass spectrometry [MS] and tandem mass spectrophotometry [MS/MS] were carried out on a Q-Tof mass spectrometer [Micromass, Manchester, United Kingdom] under conditions similar to these described by Devreese et al . [7] . In situ digests werewashed with C₁₈ ZipTip pipette tips [Millipore Corp., Bedford,Mass.] . Extracts thus prepared were loaded into a coated fused-silicacapillary tip [New Objective, Inc., Woburn, Mass.] and thenplaced into the nanospray source on the mass spectrometer . Thecapillary tube voltage was held at 0.9 kV, and spraying wasinitiated with a flow of N₂ [3 lb/in²] at the back of the capillarytubing . Spectra were taken in the 100-to-2,000 mass range with2-s scans, and data were collected for 2 min . Several of themost prominent doubly and triply charged molecular ions weremanually identified and selected for collision-induced dissociationfragmentation with Ar as the collision gas, with the collisionenergy adjusted between 22 to 33 eV, depending on the optimumfor fragmentation of the peptide.

The MS/MS spectra were interpreted with MassLynx 4.0 softwareas described by the manufacturer [Micromass] . The MaxEnt3 toolwas used to convert multiply charged fragment ions to singlycharged species, and the PepSeq tool was used to determine theamino acid sequence after finding the fragment ion series . Sequenceswere matched to an E . coli protein database with version 3.4of the FASTA search program [18].

Chemicals and enzymes. Restriction enzymes and DNA-modifying enzymes were purchasedfrom Invitrogen Life Technologies, Qiagen Inc., and PromegaCorp . [Madison, Wis.] . The T7 Sequenase version 2.0 kit andradioactive [-³²P]UTP and [-³²P]ATP were purchased from AmershamBiosciences, Inc . [Piscataway, N.J.] . Biochemicals were purchasedfrom Sigma-Aldrich Corp . [St . Louis, Mo.] . Panorama E . coligene arrays were obtained from Sigma-GenoSys . Sodium L-idonatewas received as a generous gift from Alisha S . Jarnagin [GenencorInternational, Inc., Palo Alto, Calif.].

 
Annotation of the idn promoter region. Examination of the 215-bp sequence between the idnD and idnKgenes revealed two putative -10 and -35 RNA polymerase bindingsites on opposite strands . Both genes contain conserved Shine-Dalgarno sequences located 4 and 7 bp upstream from the IdnD and IdnO translation start sites, respectively [26] . In addition, a singleputative cyclic AMP [cAMP] receptor protein [CRP] binding site [ATTTGTGA-TGAAGA-TCACGTCA] was identified upstream of the idnD gene . A putative IdnR operator site [ATGTTA-CGCA-TAACGT] with homology to the GntR consensus binding sequence [ATGTTA-[N₄]-TAACAT] [21] is centered between the two promoters, -78.5 and -83.5,with respect to the idnK and idnD transcription start sites,respectively, suggesting that this site may function as a regulatoryelement for both promoters . The position of the putative IdnRbinding site is interesting because this location is atypical of negative control, despite the fact that IdnR and GntR belong to the GalR-LacI family of negative regulators [29] . Slightly upstream of the idnK gene [-38 to -59] is a putative A-T-rich UP element sequence [8] that could be involved in stabilizationof RNA polymerase-promoter interactions.

Transcription start sites for the idn genes. Primer extension analysis was used to map the transcriptionstart sites for idnK and idnD with RNA extracted from cellsgrown in the presence of L-idonate [Fig . 1] . Growth on 5KG resultedin the same transcription start sites [data not shown] . TheidnD transcript start site [PD1] was located 29 bp upstreamof the idnD start codon [Fig . 1A], and the idnK transcript startsite [PK1] was located 26 bp upstream of the idnK start codon[Fig . 1B] . These transcription start sites are consistent withthe locations of the putative idnD and idnK promoter sequenceelements.

 
Organization of idn transcription. The organization of the idn genes suggested that idnD, -O, -T, and -R might be transcribed as a polycistronic message . The idnK transcript is monocistronic, as indicated by a 0.8-kb band of the expected length [Fig . 2] . Northern blot analysis alsosuggested that idnD, -O, -T, and -R are cotranscribed [Fig.2] . Transcripts that hybridized with the idnD and idnO probeswere observed at 1.9, 3.3, and 4.3 kb, although the latter transcripthybridized with very low intensity . An individual transcriptfor idnD was not observed, but there was an idnO-specific transcriptof 0.8 kb . The most abundant transcript for idnD and idnO was 1.9 kb . The idnT probe hybridized to a 3.3-kb transcript, suggesting cotranscription with idnD and idnO . In addition, Northern hybridizationrevealed a 1.5-kb idnT transcript of sufficient length to encodeidnT alone . The 4.3-kb transcript that hybridized to all fouridnD, idnO, idnT, and idnR probes is consistent with the predictedtranscript length of the idnDOTR operon . However, this transcriptwas apparently unstable and only a very faint band was observed.Overall, the results of Northern blot analysis supported thehypothesis that idnD, idnO, idnT, and idnR are cotranscribed and that the primary transcript is processed to form several gene-specific transcripts, which are more stable than the primary idnDOTR message.

 
Computer analysis of predicted mRNA secondary structures inthe idn regulatory region suggested the presence of stem-loop terminator-like structures at the 3' ends of idnO, idnT, and idnK, but not idnR [data not shown] . The strong intensity of the putative 1.9-kb idnD-idnO transcript in Northern blot assaysimplies that the predicted stem-loop structure at the end of idnO functions as a terminator . It is also likely that the stem-loopstructures positioned after idnT and idnK function as transcriptionterminators, since transcripts ending after the predicted codingregion of both genes were resolved in Northern blot assays.

RT-PCR with RNA obtained from cells grown on L-idonate confirmedcotranscription of the idnDOTR operon [lanes 2 to 5, 8 and 9,Fig . 3] . The monocistronic idnK transcript observed by Northernblot analysis was also confirmed by RT-PCR [lanes 6 and 7, Fig.3] . RT-PCR indicated that transcription did not terminate immediatelydownstream of idnR, but rather extended at least 500 bp intothe yjgR gene [lanes 10 and 11, Fig . 3] . However, this transcript did not appear to extend beyond the carboxy terminus of the yjgR structural gene, as downstream primers failed to yielda product [lane 12, Fig . 3] . Further, Northern hybridization with a probe specific for yjgR revealed a 1.5-kb transcript that was not induced by L-idonate or D-gluconate [data not shown].A yjgR knockout grew well on L-idonate, confirming that YjgR is not required for L-idonate catabolism [data not shown].

 
Transcription regulation of the idn genes. The enzymes of the L-idonate pathway are induced by L-idonate [3] . To confirm that the idn transcripts are similarly induced,we measured carbon source-dependent transcription of the L-idonatepathway genes [Fig. 2] . Northern blot hybridization analysisindicated strong induction of idn transcripts in the presenceof L-idonate and no induction with D-glucose . This result suggeststhat L-idonate functions to induce the idn genes.

To determine if 5KG also acts as an inducer, we tested induction of idn transcription in a strain containing a nonpolar idnD mutation that blocks the interconversion of L-idonate and 5KGwithout affecting expression of the other idn genes . An idnD-lacZfusion in the idnD nonpolar mutant strain [CB361Z] was inducedby 5KG and L-idonate, suggesting that both sugars can inducethe L-idonate pathway [Table 3] . This result was confirmed by Northern analysis, which showed that transcription of idnO was induced by growth on either 5KG or L-idonate in CB361Z [datanot shown].

 
Transcriptional regulation of the idn regulon was further analyzed with lacZ gene fusions . Because the idnD and idnK genes aredivergently transcribed from the same 215-bp region of DNA, gene fusions were constructed with the same promoter-containing fragment cloned in opposite orientations—one in the directionof idnK transcription and the other in the direction of idnD transcription . These fusions were integrated into the genomeas single copies, because multicopy fusions expressed from plasmidsdid not appropriately reflect regulation . The idnD- and idnK-lacZ fusions were remarkably similar in expression, suggesting that regulation of the two promoters is coordinated [Table 4] . Both fusions were induced by L-idonate and 5KG and slightly inducedby D-gluconate, whereas D-glycerol, D-glucose, and succinatedid not cause induction . Sugars related to the L-idonate pathwayin other eubacteria [30], 2-ketogluconate, 2,5-diketogluconate,iduronate, and 2-ketogulonate, did not cause induction of theidn genes in E . coli W1485.

 
Catabolite repression of the idn genes was observed in cells growing on a combination of L-idonate and D-gluconate or L-idonateand D-glucose; greater repression was observed with the additionof D-glucose [Fig . 2 and Table 4] . Moreover, addition of cAMP[4 mM] to cells harboring the lacZ reporter fusions caused a2.5-fold increase in reporter activity when the cells were grownon L-idonate, and a similar response was also observed for cellsgrown on D-gluconate [Table 4] . A crp mutant strain [CB370]was unable to grow on MOPS minimal medium containing L-idonate [Table 5] . As reported previously, the crp mutant demonstratedvery poor growth on D-gluconate [19] . Taken together, theseresults indicate that the idn promoters are subject to cAMP-CRP-dependentcatabolite repression.

 
Growth physiology of GntI and GntII system mutants. To understand the role of the GntI and GntII systems in growthon sugar acids, we used mutational analysis to evaluate growthon MOPS minimal medium supplemented with either D-glucose, D-gluconate,5-ketogluconate, or L-idonate [Table 5] . The wild-type E . colistrain, W1485, grew well on all of the carbon sources used except5KG, which has been described previously [3] . The idnR mutant[CB366] was unable to grow on L-idonate . By comparison, thegntR mutant [CB371] was unaffected for growth on L-idonate.Failure of the idnK mutant [MD5] to grow on MOPS minimal medium containing L-idonate is consistent with its role in phosphorylationof D-gluconate, an intermediate of the L-idonate pathway . The idnK gntK double mutant [MDE5] failed to grow on D-gluconate, as well as L-idonate . Interestingly, a gntRKU deletion mutant[NP202] can grow on D-gluconate after a lag phase of 24 h . Northernblot analysis of the gntRKU mutant, NP202, revealed that theidnD and idnK transcripts were fully induced when cells weregrown on D-gluconate [Fig . 4] . In the wild-type strain, inductionof the idn genes by D-gluconate is minimal compared to thatby L-idonate [Table 4] . This result suggests that growth ofthe gntK mutant on D-gluconate causes endogenous accumulationof the inducer of idnK and idnD.

 
Functional genomic analysis of cells grown on idonate. Very little is known about the physiology of cells growing onrarely studied sugar acids, such as L-idonate . Therefore, we used whole-genome DNA arrays to identify genes induced by growth on MOPS complete medium containing L-idonate and D-glucose.These data sets are available on the Internet [http://www.ou.edu/microarray]. The five idn genes were among the most strongly induced genes in cells grown on L-idonate compared to D-glucose, includingidnD and idnO, which topped the list [Table 6] . The expression profile of the idn genes in cells grown on L-idonate was qualitativelysimilar to the relative induction observed in Northern blotassays [compare Fig . 2 and Table 6] . When cells were grown onL-idonate, the percentage of total transcripts in the cellswas highest for idnD, followed by idnO, idnT, idnR, and idnK [Table 6] . Of all of the transcripts in E . coli cells grownon L-idonate, the idnD and idnO transcripts were the 36th and58th most highly expressed, respectively [data not shown] . Theselevels are typical of highly expressed genes in fast-growingbacteria [12] . To confirm that changes in the transcript levelsof idnD and idnO directly correlated with the changes in theprotein levels, proteins found to be specifically induced bygrowth on L-idonate were cut out of 2-D gels [Fig . 5], digestedwith trypsin, and identified by MS/MS . Four spots thus analyzedwere identified as being IdnD and IdnO . Two modified forms ofeach protein were present on the gels.

 
The 50 genes most highly induced on L-idonate compared to D-glucoseare shown in Table 6 . Only 19 of these genes encode proteinswith known functions, 5 of which belong to the idn operon . Theremaining 31 significantly induced genes encode products withunknown functions . The induction of these genes was not confirmedby other methods used for monitoring transcription, and it isnot clear that their induction is relevant to growth on L-idonate.Thus, expression profiling did not shed any additional lighton the physiology of growth on L-idonate.

 
The organization of the genes of the L-idonate pathway, whichis suggested by the arrangement of the pathway genes around a divergent regulatory region, was confirmed in these studies. Transcription start sites for the divergent promoters are positioned at -29 and -26 relative to the idnD and idnK start codons, respectively,consistent with the predicted -10 and -35 promoter elements[Fig . 1] . The pathway genes are arranged in two transcriptionunits, the idnDOTR operon, and the divergently transcribed,monocistronic idnK gene [Fig. 2 and 3] . The putative regulatory elements identified within the idn regulatory region provide some interesting clues regarding the regulation of the idn genes. A putative CRP binding site is positioned at -41.5 relativeto the idnD transcription start site, suggesting a CRP-dependent class II promoter [33] . The UP element at -42.5 bp relative to the idnK transcription start site is in a position expected to improve transcription initiation at the idnK promoter [22]. The location of the putative IdnR binding site centered between the idnD and idnK transcription start sites suggests that IdnR may coordinately regulate both promoters.

L-Idonate and 5KG both induced the L-idonate pathway, as indicatedby induction of idnD and idnK reporter fusions in an idnD nonpolarmutant that cannot interconvert L-idonate and 5KG [Table 3]. The induction ratios of the idnD and idnK promoters were remarkablysimilar, indicating that transcription from the divergent promotersis, in fact, coordinated [Table 4] . This coordinated expressionapparently provides a mechanism by which to balance flux throughthe L-idonate pathway and maintain concentrations of the pathwayintermediates at levels required for induction of the pathwaygenes and for appropriate regulation of the closely associatedGntI pathway.

The relative order of idn transcript abundance in the Northern blot and DNA array experiments [Fig . 2 and Table 6, respectively]indicates that idnD and idnO are the most highly expressed idntranscripts [in that order], followed by idnT, idnR, and idnK. Thus, their relative expression levels are correlated with their proximity to the promoters . The low level of idnR expression is consistent with the known expression level of most regulators[9] . The lower level of idnT and idnK expression suggests that flux through the pathway could be limited by L-idonate transportand phosphorylation . In addition to being highly induced by growth on L-idonate, idnD and idnO were among the most highlyexpressed genes in the E . coli transcriptome [Table 6] and theirproducts were among the most abundant proteins [Fig . 5].

The relative levels of gene-specific idn gene transcripts appear to be controlled by posttranscriptional processing and/or mRNA secondary structures that could act as terminators . Under inducing conditions, there was a low level of the full-length idnDOTR transcript and shorter gene-specific transcripts were observed.The relatively high abundance of 1.9-kb idnDO and 3.3-kb idnDOT transcripts suggests that the predicted mRNA stem-loop structures located at the 3' ends of the idnO and idnT genes may functionas transcriptional terminators . The alternative possibility that the gene-specific transcripts correspond to promoters within the idnDOTR operon was not tested . The 3' end of the idnDOTR transcript does not appear to contain any secondary structure indicative of a terminator, and transcription of the operonwas found to extend into the 5' end of the downstream yjgR gene[Fig. 3] . However, yjgR knockout mutants grew normally on L-idonateand yjgR was not induced in cells grown on L-idonate, indicatingthat YjgR is not involved in L-idonate catabolism.

Catabolite repression of the L-idonate pathway indicates thatglucose and D-gluconate are preferred over L-idonate [Table 4]; the slower growth rate of cells on L-idonate seems to explainthis hierarchy of nutrient choice [Table 5] . Hogema et al . [10] demonstrated that D-gluconate is catabolite repressing becauseit lowers the intracellular cAMP and CRP concentrations througha mechanism that does not involve the phosphotransferase system[PTS] EIIA^Glu enzyme . This explains why the addition of cAMPdid not fully relieve the repression of the idn genes causedby D-gluconate [Table 4] . In the presence of catabolite-repressingsugars such as D-glucose and D-gluconate, cAMP and CRP levelsare low and transcription of the idn genes is not induced . Onlyin the absence of catabolite-repressing sugars, when L-idonateor 5KG is present, are the idn genes fully expressed.

Failure of the idnK mutant [MDE5] to grow on L-idonate indicatesthat the presumed intracellular accumulation of D-gluconateformed by IdnD and IdnO did not reach levels high enough toinduce the GntI system for D-gluconate catabolism, specificallygntK, the idnK paralog . This result suggests that transcriptionof the GntI and GntII systems is tuned to the concentrationsof inducers such that the D-gluconate and L-idonate pathwaysare regulated appropriately [i.e., GntI is induced by gluconateand GntII is induced by L-idonate] . This possibility is beingexplored.

The operation of GntII as a subsidiary gluconate pathway was examined in a gntRKU mutant [Fig . 4 and Table 5] that exhibitsa lag before initiating growth on D-gluconate [11] . It was previously suggested that the physiological reason why 5KG functions asan inducer of the L-idonate pathway could be to act as an endogenousinducer of the GntII system for subsidiary D-gluconate catabolism[31] . Since cells grow poorly on 5KG, it is unlikely that 5KGis physiologically relevant as a growth substrate . Inductionof the GntII system in the GntI mutant can be attributed toaccumulation of D-gluconate in mutants blocked in gluconatekinase [e.g., gntK]; in turn, the accumulated D-gluconate couldbe converted to 5KG by the basal level of IdnO, a freely reversibleenzyme that converts D-gluconate to 5KG with NAD as a cofactor [3] . As 5KG accumulates, it would induce the subsidiary D-gluconatekinase encoded by idnK, which can functionally substitute forGntK of the GntI pathway for D-gluconate catabolism . This samemechanism would also be expected to substitute for GntT in agntT mutant by inducing the subsidiary D-gluconate transporter IdnT.

We used functional genomic tools to ensure that nothing was overlooked regarding the physiology of growth on L-idonate. As predicted, the genes of the L-idonate pathway were inducedby growth on idonate [Table 6] . What was not anticipated wasthe induction of genes such as araD, narW, thiM, hyaF, and nrfE.The induction of these genes has not been confirmed by othermethods used to monitor transcription, and it is not clear thattheir induction is relevant to growth on L-idonate . Thus, expression profiling failed to shed any additional light on the physiology of growth on L-idonate.

We investigated the translation of the idnD and idnO transcriptsand determined that the protein level directly correlated withthe transcript level, suggesting little, if any, translation control in expression of the idnDOTR transcript . The 2-D gel analysis revealed duplicate spots for both IdnD and IdnO, suggesting that a charged group had modified these proteins and altered their mobility in the gel [Fig . 5] . The only protein-modifying enzyme that was induced by growth on L-idonate was rimJ, whichencodes an N-terminal acetyltransferase that modifies ribosomalprotein S5 [Table 6] . It is unlikely that RimJ modifies IdnDor IdnO, since acetyl groups are generally neutral in charge.Alternatively, the negatively charged molecules L-idonate and5KG may have remained bound to the catalytic sites of theseproteins during extraction, thereby changing their overall charge.

In summary, the results presented here indicate that the idn genes are organized in two coordinately regulated operons, idnDOTR and idnK . The idn genes are specifically induced by L-idonateand 5KG and are catabolite repressed by glucose and gluconate.Whole-genome expression profiling of cells growing on L-idonateindicated that the majority of the genes induced code for proteinsof unknown function and thus reveal little about the physiologyof growth on L-idonate . Lastly, D-gluconate does not normallyinduce the idn [GntII] genes unless the GntI system is nonfunctionaland does so apparently by formation of the endogenous inducer5KG.

 
We thank April Anderson for critical reading of the manuscript.

Work on this project was supported by grants from the NSF [MCB-9723593] and NIH [AI48945], as well as a generous gift from Genencor International.

 
* Corresponding author . Mailing address: Advanced Center for Genome Technology, OU Microarray Core Facility, Department of Botany and Microbiology, 770 Van Vleet Oval, University of Oklahoma, Norman, OK 73019-0245 . Phone: [405] 325-1683 . Fax: [405] 325-7619 . E-mail: tconway@ou.edu .

Present address: Stowers Institute for Medical Research, KansasCity, MO 64110.

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