DNA microarrays encompassing the entire genome of Yersinia pestis were used to characterize global regulatory changes during steady-state vegetative growth occurring after shift from 26 to 37°Cin the presence and absence of Ca²⁺ . Transcriptional profiles revealed that 51, 4, and 13 respective genes and open readingframes [ORFs] on pCD, pPCP, and pMT were thermoinduced and thatthe majority of these genes carried by pCD were downregulatedby Ca²⁺ . In contrast, Ca²⁺ had little effect on chromosomalgenes and ORFs, of which 235 were thermally upregulated and274 were thermally downregulated . The primary consequence ofthese regulatory events is profligate catabolism of numerousmetabolites available in the mammalian host.

 
Bubonic plague caused by Yersinia pestis is generally recognized as the most devastating acute infectious disease experiencedby mankind . It is therefore of interest that this organism hasevolved within the last 10,000 years from Yersinia pseudotuberculosis [1], known to cause chronic enteropathogenic disease . Despite their very close resemblance, plague bacilli have both lost central genes of intermediary metabolism retained in its predecessor and acquired unique genes by lateral transfer [7] . For example,even though early studies showed that Y . pestis possesses functionalEmbden-Meyerhof [28] and Entner-Doudoroff [20] pathways plusa complete tricarboxylic acid [TCA] cycle [13, 27], the species-specificabsence of detectable glucose 6-phosphate dehydrogenase [Zwf]prevents use of hexose via the pentose-phosphate pathway [21].Similarly, loss of aspartase [AspA] activity in Y . pestis butnot Y . pseudotuberculosis prevents complete catabolism of L-glutamic acid, which undergoes conversion and excretion as L-aspartate [12] . In addition, Y . pestis possesses additional species-specificmutations that cause nutritional requirements at 26°C, preventutilization of potential metabolites, and eliminate host cellinvasins and adhesins [7]; these events are now characterizedby genomic sequencing [11, 23] . The nature of nutritional requirementsat 37°C is more complex and, as noted below, dependent uponplasmid profile, the presence or absence of Ca²⁺, Na⁺, dicarboxylicamino acids, and regulatory functions addressed in this report.

Established functions unique to Y . pestis are encoded by species-specific 10-kb pPCP and 100-kb pMT . The former encodes plasminogen activator[Pla] required for tissue invasion from dermal sites infectedby fleabite whereas the structural genes for anti-phagocyticcapsular fraction 1 [Caf1] and murine toxin [MT], required forsurvival in the flea, reside on pMT [7, 25] . Plague bacilliand the enteropathogenic yersiniae share a 70-kb plasmid [pCDin Y . pestis] encoding a type III protein secretion system [TTSS]that delivers cytotoxins termed Yops to the cytosol of professionaland nonprofessional phagocytes [8] and excretes soluble LcrV[V antigen], which inhibits generation of proinflammatory cytokinesby upregulating interleukin-10 [6] . These functions providethe basis for the acute symptoms of plague as well as chronicafflictions caused by enteropathogenic Y . pseudotuberculosisand Yersinia enterocolitica . Expression of pCD-mediated activitiesare upregulated at 37°C via thermoinduction of the pCD-encodedtranscriptional activator LcrF [18] . Nevertheless, the organismsundergo bacteriostasis at this temperature in vitro unless either2.5 mM Ca²⁺ is present [16] or Na⁺ and dicarboxylic amino acids are eliminated [5, 14; R . R . Brubaker, unpublished data] . Theaddition of Ca²⁺ to culture media [but not removal of Na⁺ ordicarboxylic amino acids] downregulates LcrV, Yops, and theTTSS [7, 25; Brubaker, unpublished] . Cure of pCD causes outright avirulence, emphasizing the importance of this low calcium response [LCR] in promoting disease . Little is known about the extentor nature of the regulatory cascade initiated by Ca²⁺ or the ability of this cation or temperature to regulate chromosomalgenes.

The genome of the Y . pestis bv . Medievalis strain KIM5, used in the present study, and that of the Y . pestis bv . Orientalis strain CO92 have been sequenced [11, 23] . To identify functionsregulated by Ca²⁺ and temperature, we defined the genome-wideexpression of transcripts displaying coordinate LCR-mediatedregulation by using a high-density PCR fragment-based microarraycontaining all 4,500 chromosomal and plasmid genes of Y . pestisKIM5 . The isogenic substrain D27 lacking the deletable 100-kb chromosomal Pgm sequence was used as a source of mRNA; temperature-mediatedchanges within this region have been previously defined in detail[25].

Bacterial growth, RNA isolation, microarray analyses, and real-time reverse transcription [RT]-PCR. Sequences used to design the microarray containing the entireY . pestis genome were obtained from plasmids pMT1 [accessionno. AF074611], pCD1 [AF074612], pPCP1 [AL109969], and chromosome[AL590842] provided by GenBank . The ORF-specific primers weredesigned using the PRIMER3 program [http://www.genome.wi.mit.edu] and synthesized with a 5'-C6 amino-modification [MWG Biotech, Inc., High Point, N.C.] . After PCR amplification, the purified fragments were spotted in triplicate on SuperAldehyde substrateby TeleChem International, Inc., Sunnyvale, California . Theexact composition of the microarray primers, amplified fragments,and other supplemental information noted in the text is providedat http://bbrp.llnl.gov/microbial/Ypestis . Chemically definedBCS medium [14] was used to cultivate conditionally virulent [nonpigmented] Y . pestis KIM5 substrain D27 for two transfers at 26°C without added Ca²⁺ . The organisms were then inoculated at an optical density of 0.25 [620 nm] into parallel subcultures incubated without added Ca²⁺ at 26 and 37°C or at 37°C with 4.0 mM Ca²⁺; all three of these environments permits essentiallyfull-scale growth [14] . RNA samples were isolated at differenttime points after the temperature shift; samples containingabout 2 x 10⁹ cells were immediately mixed with an equal volumeof cold RNA stabilization solution RNAlater [Ambion, Austin,Tex.] and harvested by centrifugation . Further RNA purificationwas performed using a RNeasy Midi kit [QIAGEN, Valencia, Calif.]followed by DNase I treatment on RNeasy Mini spin columns [QIAGEN].Total RNA concentration was determined using a RiboGreen RNAquantitation kit [Molecular Probes, Inc., Eugene, Oreg.]; 20µg of RNA was labeled with either Alexa Fluor 488 or AlexaFluor 546 using ARES DNA labeling kits [Molecular Probes, Inc.].The labeled cDNAs were purified using a QIAquick PCR purificationkit [QIAGEN], dried in a SpeedVac concentrator [ThermoSavant,Holbrook, N.Y.], and resuspended in 90 µl of formamidecontaining hybridization buffer [Schleicher & Schuell, Inc.,Keene, N.H.] . Hybridizations were performed in a Hybri-Wellchamber, 21 by 41 by 0.15 mm [Sigma, St . Louis, Mo.], for 12h at 42°C, slides were washed with microarray wash buffers [TeleChem International, Inc.], and images were obtained witha ScanArray Lite 4000 confocal laser scanner [Packard BioScience, Billerica, Mass.] . RNA preparations isolated at each time pointfrom cultures grown under three distinct conditions were labeled separately with both red and green fluorescent dyes and hybridized with each other . The image for each array was captured usingthe software package Gleams v . 3.0 [NuTec Sciences, Stafford,Tex.], which produced a separate estimated intensity for thered and green signals that were subtracted for background . Theresulting intensity estimates were then normalized on each arrayseparately using intensity-dependent normalization techniques[36] . Briefly, the red and green intensities from each spot[denoted R and G] were transformed into a log ratio M [derived from log₂ R/G] and a log geometric mean intensity A [derivedfrom [log₂ M + log₂ A]/2] . A scatter plot was then used to estimatea function f[A] representing the average value of M over thechip, as a function of A [essentially zero] . The log ratio Mfor each spot was then normalized by subtracting the correspondingf[A] . The experimental approach consisted of separate loop designsat 1-, 4-, and 10-h time points after inoculation of the thirdtransfer . Each loop contained vertices for three treatmentsat a single time point [26°C without calcium added, 37°Cwithout calcium added, and 37°C with calcium added] . Eachpair of treatments in a loop was hybridized twice with dye-swapping,resulting in six hybridization intensity ratios for each timepoint . Eighteen degrees of freedom were available for estimatingin-gene 2-log ratios, or contrasts, between treatments . A linearmodel was used to estimate two contrasts at each of the threetime points in the experiment . The first contrast estimatedthe difference in log intensity between expression at 26 and37°C with no calcium added at either temperature . The secondestimated the difference in log intensity at 37°C due to the addition of calcium . Because of the design, each contrast was a weighted linear combination of all six hybridization intensities at that time point . Twelve degrees of freedom remained to estimate in-gene variability [additional statistical treatment at http://bbrp.llnl.gov/microbial/Ypestis].TaqMan assay was used to validate the microarray results [http://bbrp.llnl.gov/microbial/Ypestis].

Array quality and genome-wide transcript perspective. The average value of the log ratio of each gene plotted as afunction of intensity was essentially horizontal and had a valueof zero, indicating no residual intensity-dependent normalization.A plot of the F ratio for each gene against the estimate ofthe signal for each gene provided acceptable P value thresholds[http://bbrp.llnl.gov/microbial/Ypestis] . This approach demonstratedthat only genes carried by pCD1 mediating the TTSS were regulatedby temperature as well as Ca²⁺ [see Table 1 at http://bbrp.llnl.gov/microbial/Ypestis].In contrast, neither chromosomally carried genes nor those locatedon pPCP1 or pMT1 were affected by Ca²⁺ [P < 0.05] . Approximately10% of chromosomal genes found to be up- or downregulated bytemperature had P values of less than 0.05 and differed in expressionby at least 1.5-fold . An additional, but minor, group of genesdiffered in expression by more than twofold [at P values inthe range 0.05 to 0.1] [see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis].In total, 235 chromosomal genes were upregulated after the temperatureshift to 37°C, while 274 were downregulated . Clusteringanalysis [data not shown] revealed that nearly 80% of the chromosomalthermoregulated genes had altered transcriptional levels at1-h postshift [early response genes] while the remainder weredifferentially expressed at 4 or at 10 h [late response genes].Most of the genes that responded early to temperature shiftdid not remain differentially regulated at the later determinations,suggesting immediate roles in adapting to the change in growthconditions [Fig . 1] . The microarray expression data were evaluatedby TaqMan analysis of 12 genes representing different functionalcategories . The average n-fold changes in quantity of cDNA moleculespresent between the three growth conditions for each time pointwere determined by this real-time RT-PCR procedure and comparedto those derived from the microarray analysis . There was strongpositive correlation [r = 0.827] between results obtained bythe two techniques [Fig. 2].

 
Plasmid genes. As already noted, components of the TTSS displayed a wide rangeof thermoregulation; genes of the lcrGVHyopBD operon and theeffectors yopH, yopE, yopJ, ypkA, yopT, and yopM consistently showed the highest levels of expression [see Table 1 at http://bbrp.llnl.gov/microbial/Ypestis].The genes yopK, lcrE [yopN], tyeA, sycN, yscX, and the pseudogeneylpA were also strongly upregulated while genes of the secretionapparatus per se, including the negative regulator lcrQ [yscM],the operons yscNOPQRSTU and yscBCDEFGHIJKL, plus the individualchaperones sycE and sycT, were upregulated to a lesser extent.Thermoinduction levels for all of these genes increased progressivelyover time but were significantly reduced at postshift timesof 4 and 10 h in the presence of Ca²⁺ . The genes yscY, lcrD, lcrR, and sycH displayed the lowest levels of thermoinduction among all genes carried by pCD, and Ca²⁺ exerted little effect on their transcription . A chaperone-like protein [orf7] and a hypothetical protein [orf84] qualified as novel genes carried by pCD1 capable of undergoing upregulation by temperature and downregulation by calcium . In addition, genes encoding LcrF,the lipoprotein YscW [VirG], and a hypothetical protein [orf60] were regulated by temperature but not calcium . The latter were characterized by early induction followed by a decline in expression as opposed to orf73, orf74, and orf75 [all of unknown function],which exhibited late thermal induction . This analysis of genescarried by pCD1 is consistent with previously published results [7, 25] and now provides global, quantitative, and temporalvalues for activities required for formation and function ofthe LCR . The determination also uncovered six novel thermoregulatedgenes in pCD1 that might contribute to yersiniae virulence.

While neither pPCP1 nor pMT1 possessed genes regulated by Ca²⁺, these plasmids encoded a number of thermoregulated functions. The pPCP1 genes pla, pst, pimi, and YPPCP_08c encoding the plasminogenactivator, pesticin, pesticin immunity, and a putative transcriptionalregulator, respectively, were induced at all time points aftershift to 37°C . In contrast, the genes located on pMT1 displayeda temporal pattern of upregulation . Both the caf operon encodingcapsule components and its transcriptional regulator [caf1R]became upregulated over time, achieving a 100-fold increasein caf1 at 10 h [the strongest upregulation value encounteredin the entire genome] . This thermoinducible phenotype is infull agreement with findings established decades previously [7, 25] . The ymt gene of pMT1 encoding MT was downregulatedin agreement with previously published observations [25] . Sixof the nine newly identified thermoinduced ORFs of pMT1 werelocated in the vicinity of the capsular operon or ymt [putativegenes orf12, orf13, orf108, orf110, orf111, and orf112] . Of the remainder, orf55 and orf54 were hypothetical genes while orf38 was a putative periplasmic solute-binding protein.

Genes of carbon and energy metabolism. Numerous enzymes that directly or indirectly facilitate substratephosphorylation in cells growing in the steady state at 26°Con D-gluconate as a source of energy underwent downregulationupon shift to 37°C [Fig . 3; also see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis].Examples are 6-phosphogluconate dehydrogenase [gnd] of the pentose-phosphate pathway, 2-keto 3-deoxy 6-phosphogluconate aldolase [eda] of the Entner-Doudoroff pathway, numerous glycolytic enzymes [pfkA, fda, tpi, gapA, gmpA, eno, and pykF], carbohydrate phosphotransferasesystem components [ccr, fru, manXYZ, nagE], the six-gene clusterencoding the maltose transport system [malMBKEFG], and the relatedgenes malZPQ . The components of the pentose-phosphate pathwaywere unchanged after 6-phosphogluconate dehydrogenase promoted interconversion of 3- to 7-carbon intermediates and glucose 6-phosphate isomerase [pgi], which interconverts fructose 6-phosphate and glucose 6-phosphate . Gene zwf encoding glucose 6-phosphate dehydrogenase is evidently intact in Y . pestis [11, 23] andwe observed its constitutive expression [data not shown] despitethe absence of detectable enzymatic activity in plague bacilli[21] . In contrast to the majority of the genes involved in theutilization of carbohydrates, shift to 37°C upregulatedmembers of the D-gluconate transport system including a putativegluconokinase [YPO3953], gluconate permease [gntT], and a transcriptionfactor for the latter [gntR] . The ribose uptake genes rbsK encoding ribokinase and rbsD for ribose permease also underwent early induction at 37°C . The ABC galactose transporter operonmglBAC was similarly induced as was the entire galETKM operonof galactose metabolism . Furthermore, the genes glpF, glpK, and glpD encoding the glycerol uptake facilitator, glycerol kinase, and aerobic glycerol 3-phosphate dehydrogenase, respectively, underwent strong [10- to 25-fold] early upregulation at 37°C.

 
Concomitant changes in oxidative catabolism favoring relianceon a full TCA cycle occurred after shift to 37°C and areshown in Fig. 3 . Phosphotransacetylase [pta] and enzymes of the glyoxylate bypass [aceBAK] required for generation of acetyl-coenzymeA [CoA] and its utilization during gluconeogenesis were downregulatedafter temperature shift . In contrast, some but not all enzymesof the TCA cycle underwent at least initial induction, includingcitrate synthase [gltA], aconitases [acnA and acnB], the 2-oxyglutaratedehydrogenase complex [sucABCD], the succinate dehydrogenasecomplex [sdhCDAB], and fumarate hydratase [fumA] . Furthermore,upregulation of cytochromes [cybB and cybC] and an attendantterminal electron acceptor [katY] was dramatic [see Table 2at http://bbrp.llnl.gov/microbial/Ypestis] . These results indicate that shift to host temperature results in reliance on oxidative phosphorylation mediated by the TCA cycle and an enhanced abilityto utilize a variety of carbohydrates present in mammalian tissue.The latter are all converted to the level of pyruvate wherethey can enter the TCA cycle either after reductive decarboxylationas acetyl-CoA or as oxaloacetate or malate following carboxylationvia upregulated phosphoenolpyruvate carboxykinase [pck] or NADP⁺-dependent malic enzyme [maeB], respectively [Fig . 3] . As noted below,at 37°C the TCA cycle accommodates terminal oxidation of numerous additional sources of carbon and energy in additionto carbohydrates.

Nitrogen and amino acid metabolism. Many genes involved in nitrogen assimilation [including NTR-regulatedgenes] were strongly downregulated upon shift to 37°C, includingthe ammonium transport facilitator encoded by amtB and the neighboringnitrogen regulator glnK, glutamine synthetase [glnA], and both glutamine- [asnB] and ammonia-dependent [asnA] asparagine synthetases.Also repressed were the nitrogen regulators ntrB and ntrC [Fig.3; also see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis].In contrast, at least 11 genes required for efficient catabolismof amino acids were rapidly induced upon temperature shift,including those encoding D-amino-acid dehydrogenase [dadA], L-serine deaminase [sdaA], L-asparaginase [ansB], carbamoylphosphatesynthetase [carAB], proline/delta 1-pyrroline 5-carboxylatedehydrogenase [putA], proline permease [putP], and a putativeaminotransferase [YPO0623] . Some of these enzymes account forthe bulk of released metabolic ammonia via reactions that directlyor indirectly promote deamination during formation of -keto acids entering the TCA cycle [Fig . 3] . As previously noted,aspartase activity is cryptic in Y . pestis [12]; nevertheless,aspA transcription was upregulated following temperature shifteven though the medium lacked added L-glutamate . The mediumalso lacked L-histidine and L-serine, thus, not surprisingly,the histidine biosynthetic [hisGDCBHAFI] and transport [hisJQMP] operons underwent early postshift induction at 1 h and then became repressed as the culture approached stationary phase. L-Serine was previously shown to undergo uncontrolled reductivedeamination in Y . pestis at 37°C [12] . The biosynthesisof L-serine was downregulated at the branch point from glycolysis[gene serA] as was conversion of glycine to L-serine via serine hydroxymethyltransferase [glyA]; in addition, the oxidative cleavage of glycine via the gcvP and gcsH products was repressed. These changes serve to isolate the L-serine precursors 3-phosphoglycerateand glycine [obtained in minimal medium from threonine aldolaseencoded by ltaA], thereby minimizing loss of metabolic carbonotherwise destined for glycolysis or biosynthesis of aliphaticamino acids through L-threonine . With the exception of L-tryptophan,synthesis of other amino acids [present in abundance] was downregulatedafter shift to 37°C often at branch points or early withinspecific pathways [Fig . 3] . Examples are asd [encoding aspartate semialdehyde dehydrogenase], aroF, aroA, and aroK [facilitatingaromatic amino acid biosynthesis to chorismate], ilvC-encodedketol-acid reductoisomerase [initiating synthesis of L-isoleucineand L-valine], and the biosynthetic L-leucine operon [leuABCD].

Lipid metabolism. Shift to 37°C caused modest downregulation of fabB and fabCencoding the 3-ketoacyl synthase complex involved in lipid biosynthesis[see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis] . However,this change prompted upregulation of 3-ketoacyl thiolase [fadAand probably YPO2746], the fatty acid oxidation complex [fadBand faoA], and yafH [probable acyl-CoA dehydrogenase], as well as fadL encoding long-chain fatty acid transport . These results would be expected if shift to host temperature promotes ß-oxidation of fatty acids.

Nucleotide metabolism. Although the culture medium lacked added purines and pyrimidines,marked differences occurred between their patterns of regulationafter shift to 37°C . Purine biosynthesis was downregulatedbefore IMP at the level of GAR [phosphoribosyl glycinamide formyltransferase]transformylase [purT], phosphoribosylaminoimidazole carboxylase[purEK], and adenylosuccinate lyase [purB]; subsequent conversionof IMP to both GMP and AMP was also repressed [Fig . 3] . Nucleoside permease [nupC] mediating high-affinity transport of adenine and pyrimidine nucleosides was similarly downregulated . Transcription of all other genes in the de novo pathway, the coordinate purine repressor [purR], and genes required for purine interconversion was not influenced by temperature shift . In contrast, carbamoylphosphate synthase [carAB], aspartate carbamoyltransferase [pyrB], and its regulator [pyrI], dihydroorotate dehydrogenase [pyrD], andorotidine 5'-phosphate decarboxylase [pyrF] of the de novo pyrimidinebiosynthetic pathway were upregulated after temperature shift.Furthermore, nucleoside diphosphate kinase [ndk] that performsthe last reaction in the synthesis of nucleoside triphosphateswas induced although cytidylate kinase [cmk] was downregulated.Other than ndk, the entire set of genes encoding pyrimidinesalvage pathways and deoxyribonucleotide interconversions werenot thermoregulated.

Macromolecular synthesis. As expected, a modest number of genes mediating macromolecularsynthesis [e.g., DNA replication and ribosomal proteins] weredownregulated after 10 h at 37°C as the bacteria approachedearly stationary phase [see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis].Several genes involved in lipopolysaccharide biosynthesis werealso downregulated, including nagB and nagE concerned with glucosaminemetabolism . Similarly, fabZ, lpxA, and lpxB encoding hydroxymyristoyl-dehydratase,UDP-GlcNAc acyltransferase, and lipid A-disaccharide synthase,respectively, were downregulated; these activities are involvedin the initial steps of lipid A biosynthesis.

Unknown ORFs. A significant number of genes encoding putative exported, membrane,or unknown proteins were thermoregulated [see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis].The possible operon YPO1996-1994, which lacks significant homologyto any entry in the current version of GenBank, displayed thestrongest level of temperature-dependent upregulation withinthis category.

Pathogenicity. Determinants considered in this category constitute productsof chromosomal genes that were found by annotation to map withinputative pathogenicity islands [11, 23], result in avirulencewhen lost by mutation [7], or exist as homologues of virulenceeffectors of other species . The Mn-cofactored superoxide dismutaseencoded by sodA was reported to facilitate survival and multiplicationof Y . enterocolitica in mice [26] . However, sodA was downregulatedat all time points in the present study although sodC encodingthe Cu-Zn-cofactored enzyme displayed a pattern of prompt inductionwith later downregulation at 10 h [see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis].As noted above, the gene katY encoding Y . pestis/Y . pseudotuberculosis-specific catalase-peroxidase was strongly induced [more than 30-foldat 1 h] during the shift to 37°C in agreement with classical[9] and recent [15] observations . Although katA remained upregulatedat 10 h, thiol peroxidase [tpx] was downregulated at this time.Genes encoding the putative copper resistance proteins YPO1784and YPO1785 were induced early . Accordingly, out of 10 genes annotated as belonging to the detoxification functional group, 7 were differentially regulated, while the remainder [sodB, ahpC, and cutF] were expressed constitutively [data not shown]. Also induced was the serine protease encoded by htrA [gsrA] known to facilitate resistance to oxidative stress, growth at elevated temperature, and survival of many pathogens in macrophages including Y . pestis [35] . The two-component system PhoPQ wassimilarly upregulated: this function is a known global regulatorof virulence in yersiniae [22] and other bacterial species.

Of the chromosomally encoded fimbrial-type adhesins previously described by Parkhill et al . [23], only the psaABC locus encodingantigen 4/pH 6 antigen [4, 9] underwent strong initial upregulation[>50-fold for psaA] . This pattern is in accord with thatdefined by the initial study of thermoregulation [9] and inmore recent observations [33] . The pseudogene inv [YPO1793] encoding invasin in Y . pestis is inactivated by IS1541 [31] but underwent downregulation as reported for the ClpB protease-dependent inv of Y . enterocolitica [3] . However, ClpB [YPO2946] was notdownregulated after temperature shift in Y . pestis althoughthe close homologue YPO0506 [one of six paralogues in the genome]was repressed . YPO0506 is located within a large cluster of18 genes [YPO0499 to YPO0516] that were strongly [up to 10-fold]downregulated at all postshift time points; most ORFs comprisingthis cluster are hypothetical proteins . In contrast to inv,the gene ail [YPO2905] for attachment-invasion locus [modulatedby the distinct protease ClpP] was upregulated 10 h after shift.The clpP gene was expressed constitutively in our experimentsin accord with the finding that ail is transcribed in Y . enterocoliticaduring stationary phase at 37°C [24] . Of the additionalthree Ail-like proteins found in the genome of Y . pestis CO92[23], only YPO2505 was downregulated at 37°C although theputative invasin gene [YPO3944] containing 22 large degeneraterepeats was induced . Urease activity in Y . pestis is also crypticdue to a single base addition in ureD [30] . Nevertheless, thestructural genes of the urease locus [operon ureAB] as wellas the putative urea transporter [YPO2672] were downregulatedat 37°C.

Although not involved in mammalian pathogenesis, the seriesof genes encoding homologues of insecticidal toxin complexes[23, 34] underwent thermoregulation and are therefore considered here . The cluster of genes YPO3675-3682 was severely downregulated at 37°C . This cluster, assigned by Parkhill et al . [23] as a potential pathogenicity island, includes genes encoding putative insecticidal toxins tcaABC as well as transcriptional regulator and hypothetical ORFs of phage-related origin . Twocopies of another cryptic insecticidal toxin encoded by tccC[YPO3673 and YPO3674] are located next to this cluster but werenot thermoregulated . Genes located in other putative pathogenicity islands displaying differential expression included YPO0590, YPO0595-0597, YPO0623-0628, YPO0881, YPO1091, and YPO1242-1252.These genes are typically putative ORFs with unknown functionor are associated with degenerate bacteriophage.

Adaptive response. Genes in this category modulate responses to distinct environmentswithin the host that present unique challenges to invading pathogens.The chromosomal psp locus of Y . enterocolitica encoding phageshock proteins plays a role in virulence and was induced concomitantlywith the pYV-encoded TTSS [10] . The pspA gene of this locuswas upregulated early at 37°C as was the universal stressprotein A encoded by uspA [see Table 2 at http://bbrp.llnl.gov/microbial/Ypestis].The latter provides coupling of glucose and acetate metabolism,thus helping bacteria to effectively utilize both of these carbonsources [32] . The carbon starvation protein encoded by cstAand regulated in Escherichia coli by the cyclic AMP [cAMP] andcAMP receptor protein complex [29] was also induced following temperature shift . Also upregulated was arcB encoding the ArcAB aerobic respiration control sensor protein, cold shock proteins [genes cspH and cspE], and heat shock proteins [genes ibpA andibpB] . The later are associated with the high-level productionof certain heterologous proteins in E . coli [2] . The universalheat shock chaperonins GroEL and GroES were robustly upregulated[three- to fourfold] at all time points tested . In contrast,ORF YPO3784 [annotated as an additional carbon starvation protein]was downregulated at 37°C as was the cAMP receptor protein complex-regulated gene osmY encoding an osmotically inducible protein [19] . The oxidative stress sigma factor encoded by rpoE,known to modulate virulence in Salmonella [17], underwent notableupregulation at 37°C . Also induced was gene yfiA, a putativemodulator for the nitrogen assimilation sigma factor RpoN [sigma54] . In addition to the categories of genes described above, 8% of the Y . pestis pseudogenes [25 out of 312] are thermoregulated under the experimental conditions employed in this work [see Table 3 at http://bbrp.llnl.gov/microbial/Ypestis].

Concluding comments. Plague bacilli exist in nature at ambient temperature withinthe flea vector or in the mammalian host at 37°C in eitherCa²⁺-sufficient plasma, lymph, and interstitial fluid or inCa²⁺-deficient cytoplasm released into focal lesions . DNA microarraytechnology was used to characterize thermoregulated changesin global regulation that occur in chemically defined mediumreflecting introduction into these niches . This approach avoidedthe nutritional stepdown inherent in the LCR [37], thereby maintainingthe bacteria in a steady state and avoiding premature expressionof functions associated with entry into stationary phase . Accordingly,the observed early changes in gene expression reflect real adaptationto the host rather than changes associated with the onset of bacteriostasis . Transcriptional profiles following shift from26 to 37°C revealed 15 novel thermoregulated genes on thethree Y . pestis plasmids . Only genes carried by pCD1, includingcomponents of type III secretion [including two genes of unknownfunction], were downregulated by Ca²⁺ . In addition, approximately10% of all chromosomal genes were influenced by temperature[but not Ca²⁺] . Of these, 235 were upregulated and 274 weredownregulated upon shift from 26 to 37°C, thereby inhibitingglycolysis while favoring terminal oxidation of a variety ofsubstrates [including carbohydrates, amino acids, and fattyacids known to exist within the host] . Shift to 37°C alsorepressed Ntr-controlled genes involved in nitrogen assimilationand biosynthesis of amino acids and purines [but not pyrimidines].Numerous global regulators and transport systems were also thermoregulatedas was expression of known and putative genes associated withgeneral adaptive responses, oxidative stress, and modulatorsof innate immunity [e.g., invasins, adhesins, cytotoxins, andinhibitors of proinflammatory cytokines] . These studies indicatethat, in nature, plague bacilli favor fermentative patternsof metabolism during slow growth within the flea but exhibit pronounced oxidative catabolism during rapid proliferation in the host . Differential transcription during temperature shiftalso identified a useful list of putative virulence-associatedgenes to target as novel candidates for future research on thecontrol of this pathogen.

 
This work was performed under the auspices of the U.S . Departmentof Energy by the University of California, Lawrence LivermoreNational Laboratory, under contract no . W-7405-Eng-48 . Partialsupport was also provided by National Institutes of Health grantR21 AI53508-01 and the Region V ‘Great Lakes' RCE [NIHaward 1-U54-AI-057153].

We thank Arthur Kobayashi for designing the website for this project.

 
* Corresponding author . Mailing address: Biology and Biotechnology Research Program, L-452, 7000 East Ave., Livermore, CA 94550 . Phone: [925] 422-8002 . Fax: [925] 422-2282 . E-mail: garcia12@llnl.gov .

Present address: Departments of Pathology and Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX77555.

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