Global Gene Expression Profile of Yersinia Pestis Induced By

FEMS Microbiology Letters 243 (2005) 489–496 www.fems-microbiology.org

Global gene expression proﬁle of Yersinia pestis induced by streptomycin

Jingfu Qiu 1, Dongsheng Zhou 1, Yanping Han 1, Ling Zhang, Zongzhong Tong, Yajun Song, Erhei Dai, Bei Li, Jin Wang, Zhaobiao Guo, Junhui Zhai, Zongmin Du, Xiaoyi Wang, Ruifu Yang *

Laboratory of Analytical Microbiology, National Center for Biomedical Analysis, Army Center for Microbial Detection and Research, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing 100071, China

Received 5 November 2004; received in revised form 22 December 2004; accepted 9 January 2005

First published online 19 January 2005

Edited by A.M. George

Abstract

Plague, caused by Y ersinia pestis, is one of the most dangerous diseases that impressed a horror onto human consciousness that persists to this day. Cases of plague can be normally controlled by timely antibiotic administration. Streptomycin is the first-line antibiotic for plague treatment. In this study, a DNA microarray was used to investigate the changes in the gene expression profile of Y. pestis upon exposure to streptomycin. A total of 345 genes were identified to be differentially regulated, 144 of which were up- regulated, and 201 down-regulated. Streptomycin-induced transcriptional changes occurred in genes responsible for heat shock response, drug/analogue sensitivity, biosynthesis of the branched-chain amino acids, chemotaxis and mobility and broad regulatory functions. A wide set of genes involved in energy metabolism, biosynthesis of small macromolecules, synthesis and modification of macromoclecules and degradation of small and macro molecules were among those down-regulated. The results reveal general changes in gene expression that are consistent with known mechanisms of action of streptomycin and many new genes that are likely to play important roles in the response to streptomycin treatment, providing useful candidates for investigating the specific mechanisms of streptomycin action. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Yersinia pestis; DNA microarray; Transcriptional proﬁling; Streptomycin

1. Introduction hundreds of cases of human plague reported annually, and plague has been recognized as a re-emerging disease Plague is a zoonotic disease caused by the gram- by the World Health Organization [2]. Y. pestis has re- negative bacterium Yersinia pestis [1]. It has caused so- cently become of public interest because of its potential cial devastation on a scale unmatched by any other as an agent of bioterrorism. Since its ﬁrst development infectious agent. Plague is ever present in endemic areas, in 1944, streptomycin has been the antibiotic of choice circulating in various mammalian species. There are for the treatment of most forms of plague [3]. When administered in the early phase of the disease, it can * Corresponding author. Fax: +86 10 83820748. eﬀectively reduce the overall human mortality to 5– E-mail address: [email protected] (R. Yang). 14%, whereas untreated, the mortality rate is between 1 These authors contributed equally to this work. 50% and 90% [4]. Streptomycin is a water soluble

0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.01.018 490 J. Qiu et al. / FEMS Microbiology Letters 243 (2005) 489–496 aminoglycoside that is marketed as the sulfate salt of range from 0.1 to 128 lg/ml. Bacteria were then cultured streptomycin. Streptomycin interferes with several steps at 37 C for 20 h. The MIC was defined as the lowest of protein synthesis, its most conspicuous effects being concentration that prevents the development of visible the stimulation of translational errors and a slowing growth. down of translocation resulting in the production of faulty proteins. Streptomycin binds to the decoding cen- 2.2. Bacterial growth and RNA isolation ter of bacterial 16S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding re- Strain 201 was grown at 26 C to the middle exponen- gion against dimethyl sulfate attack [5]. tial growth phase (an A620 of about 0.6) in the TMH Although the development of antibiotics has signifi- medium. The cell cultures were 1:20 diluted in fresh cantly lowered the mortality rates, there still exists the TMH medium and the cells experienced at least 10 gen- serious threat to public health posed by pathogenic bac- erations in the medium prior to reaching to the middle teria that have developed resistance to the traditional exponential growth phase. Bacteria were then trans- antibiotics, which is also the case for plague. ferred to grow at 37 C for 1 h to be ready for antibiotic In 1995, a Y. pestis strain, resistant to streptomycin, treatment. Cells were incubated at 37 C for 30 min un- ampicillin, chloramphenicol, kanamycin, sulfonamides, der the treatment of 10· MIC (80 lg/ml) of streptomy- tetracycline and minocycline, was isolated in Madagas- cin; the control culture was allowed to continue growing car from a 16-year-old boy [6]. Therefore, it is important at 37 C for the same period of time with adding the to extensively understand the molecular mechanisms of same volume of distilled water. Immediately before har- action of the traditional antibiotics, and attempts should vesting for RNA isolation, bacterial cells were mixed be conducted as well for the development of new antibi- with RNA protect Bacteria Reagent (Qiagen) to mini- otics. The availability of the genome sequences [7–9] and mize RNA degradation. Total RNA was isolated by the subsequent development of DNA microarrays to using the MasterPure RNA Purification kits (Epicen- profile the transcriptome [10–13] have opened a window ter). RNA quality was monitored by agarose gel electro- for monitoring global changes in gene expression pat- phoresis and RNA quantity was measured by terns in Y. pestis. spectrophotometer. Two independent bacterial cultures Here we used the whole-genome DNA microarray to for each test or control condition were prepared as bio- investigate the global transcriptional response of Y. pes- logical replicates for RNA isolation. tis triggered by the treatment of streptomycin, giving an overall picture of the molecular mechanisms of action of 2.3. Probe synthesis and mciroarray hybridization the antibiotic in vanquishing this deadly pathogen. Fifteen to 20 micrograms of RNA were used to synthe- size cDNA in the presence of aminoallyl-dUTP, genome 2. Materials and methods directed primers (GDPs) and random hexamer primers with the Superscript II system (Invitrogen). The reverse 2.1. Bacterial strain, medium and MIC determination transcription of bacterial RNA by the mixture of GDPs and random hexamers has been proven to be more effec- Yersinia pestis strain 201 was used in this study. It tive and reliable than with either GDPs or random hexa- was isolated from Microtus brandti in Inner Mongolia, mers only [17]. The aminoallyl-modified cDNA was then China. It has major phenotypes as F1+ (able to produce labeled by Cy5 or Cy3 monofunctional dye (Amersham) fraction 1 antigen or the capsule), VW+ (presence of V according to the manufacturerÕ instruction. Three sepa- antigen), Pst+ (able to produce pesticin) and Pgm+ (pig- rated labeled probes were made for each RNA prepara- mentation on Congo-red media). Strain 201 has an LD50 tion as technical replicates. Pairwise comparisons were of less than 100 cells for mice by subcutaneous chal- made using dye swaps to avoid labeling bias. lenge. Strain 201 belongs to a newly established Y. pestis Glass slides spotted with PCR amplicons representing biovar, microtus [14]. Biovar microtus strains are sup- about 95% of non-redundant annotated genes or ORFs posed to be avirulent to humans, although they are of Y. pestis CO92 and 91001 were used for hybridization highly lethal to mice [14]. A chemically defined TMH [18]. Briefly, based on the genomic sequences of Y. pestis medium [15] was used for cultivating the bacteria. CO92 and 91001, a total number of 4015 annotated Minimal inhibitory concentration (MIC) was deter- open reading frames (ORFs) were selected after the mined by the broth dilution method [16]. The tests were exclusion of ORFs encoding IS protein, integrase, and performed in sterile glass tubes with an initial inoculum transposase. Specific primer pairs were designed to am- of approximately 105 CFU of Y. pestis cells (overnight plify nearly the full length of each gene. The purified cultures) in 1 ml of TMH medium per tube. Streptomy- 4005 successful amplifications were spotted on the cin sulfate (Amresco) was dissolved in each tube and CSS-1000 silylated glass slides (CEL) by using a SpotAr- dilution series were made to give a final concentration ray72 Microarray Printing System (Perkin–Elmer Life J. Qiu et al. / FEMS Microbiology Letters 243 (2005) 489–496 491

Sciences) to construct the DNA microarrays. The spot- of increasing concentration of the bactericidal agent ted slides were crosslinked by using a UV Stratalinker streptomycin to logarithmically growing cultures of Y. (Hoefer). NaBH4 was used to block the free aldehyde pestis strain 201. The MIC value of Y. pestis strain groups on the slide surface. The slides were prehybrid- 201 versus streptomycin sulfate was determined as 8 ized in a buffer containing 5· SSC, 0.1% SDS and lg/ml. In order to furthest understanding the transcrip- 0.1% BSA, and then washed and blown to dry. The tional response of Y. pestis under the treatment with two differentially labeled cDNA samples were dried streptomycin, we magnify the inhibitory drug concentra- and then resuspended in hybridization solution (50% tion to 10· MIC, meanwhile only the short-term (30 deionized formamide, 5· SSC, 0.1% SDS, 5· DenhardtÕs min) changes was examined to avoid confounding sec- solution, and 0.5 lg/ll of sheared salmon sperm DNA). ondary drug effects. The labeled DNA samples hybridized with the slides at A wide set of genes or operons was affected, both pos- 42 C for 18–20 h. After hybridization, the slides were itively and negatively, upon the exposure to streptomy- washed in 1· SSC with 0.06% SDS for 2 min, then in cin. Of all the available 2380 genes in the final 0.06· SSC for 2 min and finally in ethanol for 2 min. microarray dataset (data points of the remaining 1625 The slides were blown to dry and then were scanned genes were missed in the data filtering process, indicat- by using a GenePix Personal 4100A Microarray Scanner ing that these genes were not expressed at all in the (Axon Instruments). experimental conditions or had data with low quality), 345 were identified to be differentially regulated; among 2.4. Data analysis them, 144 genes were up-regulated, 201 down-regulated (see Supplementary Tables 1 and 2 for details). Fig. 1 The scanning images were processed and the data provides a summary of the differentially regulated genes were further analyzed by using GenePix Pro 4.1 soft- grouped by functional categories, giving an overall pic- ware (Axon Instruments) combined with Microsoft Ex- ture of the alteration of the global gene transcription cel software. Spots were analyzed by adaptive pattern of Y. pestis. Several general trends in gene tran- quantitation, and the local background was subse- scriptional response can be elicited on the basis of the quently subtracted. Spots with background-corrected differentially regulated genes with proven or annotated signal intensity (median) in both channels less than two- functions. The microarray data provide a clear cascades fold of background intensity (median) were rejected that streptomycin disturbs the metabolic pathways and from further analysis. Data normalization was per- the structuring of cell envelope in Y. pestis, which is con- formed on the remaining spots by total intensity nor- sistent with the proven or suggested mechanisms of malization methods. The normalized log2 ratio of test/ action of streptomycin. Some other interesting observa- reference signal for each spot was recorded. Genes with tions, such as the induction of stress response proteins, less than three data points were considered unreliable, the up-regulation of genes responsible for drug/analogue and their data points were discarded as well. The aver- sensitivity and the induced biosynthesis of selective aged log2 ratio for each remaining gene on the six repli- amino acids, show that Y. pestis takes several responsive cate slides was ultimately calculated. Significant changes mechanisms in confronting with the addition of strepto- of gene expression were identified with SAM software mycin. In addition, a portion of genes whose transcrip- [19] using one class mode (D = 1.01444) [the measure- tion was altered encode proteins of unknown or ment is the log(red/green) ratio from two labelled sam- unassigned functions. The detected changes in gene ples hydridized to a cDNA chip, with green denoting transcription provides seful candidates for investigating before treatment and red, after treatment]. Significance the specific mechanisms of action of this drug which is analysis of microarrays (SAM) assigns a score to each highly efficient against plague infection. Noticeably, gene on the basis of change in gene expression relative the up-regulation of many genes, especially those with to the standard deviation of repeated measurements. unknown functions, will provide novel insights into For genes with scores greater than an adjustable thresh- the unknown mechanisms of action of streptomycin old, SAM uses permutations of the repeated measure- against Y. pestis and can be useful starting points in ments to estimate the percentage of genes identified by the identification of new targets for antimicrobial chance, the false discovery rate (FDR). chemotherapy.

3.2. Genes up-regulated by treatment of streptomycin 3. Results and discussion 3.2.1. Stress response proteins 3.1. General comments on microarray profiling The category of genes with the distinct response to treatment with streptomycin was the heat shock group. In our present work, DNA microarray was used as a Bacterial heat shock response appears to a global tool to survey the global effects mediated by the addition regulatory system for effective adaptation to various 492 J. Qiu et al. / FEMS Microbiology Letters 243 (2005) 489–496

Fig. 1. Differentially regulated genes grouped by functional classification according to Y. pestis CO92 genome annotation. The differentially regulated genes on the chromosome were divided into 21 functional categories, and those on the three plasmids were listed additionally. The number of genes up-regulated and down-regulated for each functional group was represented. environmental changes including elevation in tempera- itively controls the expression of the r32 regulon, HtrA ture, the addition of ethanol and heavy metals, high and FkpA [20]. Our previous study showed that the sud- osmolarity, and starvation [20,21]. It involves the induc- den upshift of temperature from 37 to 45 C for 10 min tion of the major heat shock proteins (MHSPs), includ- (heat shock) significantly enhanced the transcription of ing chaperones, proteases, transcriptional regulators rpoH in Y. pestis, but rpoE was not differentially regu- and other structural proteins, that act by repairing and lated [12]. In this study, both rpoH and rpoE were up- preventing damages caused by an accumulation of un- regulated by the addition of streptomycin. folded proteins [21]. Previous studies on Escherichia coli The psp FABCD locus encoding the phage shock pro- have shown that the addition of streptomycin or kana- teins, initially identified in E. coli, was found to help to mycin (another aminoglycoside antibiotics) results in ensure survival of the bacterium in late stationary phase the induction of the MHSPs [22,23]. The microarray at alkaline pH, and protect the cell against dissipation of analysis showed that streptomycin induces the heat its proton-motive force against challenge [28]. Until shock regulon in Streptococcus pneumoniae [24]. The now, the precise biochemical and physiological func- heat shock response to aminoglycosides may be a com- tions of the psp genes are not understood. In this study, mon stress response for bacteria. In this study five heat transcription of pspABC (pspD was not represented on shock genes (rpoH, rpoE, ibpA, ibpB, and htrA) were in- the microarray) was up-regulated by 4- to 7-fold after duced by the treatment of streptomycin. HtrA is a pro- the treatment of streptomycin. Interestingly, transcrip- tease that digests abnormal proteins in the periplasm tion of Y. pestis pspA was also up-regulated as well upon [25]. Mutation in the htrA gene leads to decreased sur- shift from 26 to 37 C during steady-state vegetative vival of Y. pestis in mice and/or macrophages [26]. The growth [10]. The psp genes likely play some roles in heat shock genes ibpA and ibpB, encoding chaperones, the accommodation of Y. pestis to environmental play roles in assisting the folding of newly synthesized perturbations. proteins, preventing aggregation of proteins, and repairing proteins that have been damaged or misfolded by 3.2.2. Drug/analogue sensitivity heat shock and other stresses [27]. The heat shock re- In E. coli, the plasmid-borne terZABCDE operon sponse in Gram-negative bacteria is positively con- encoding tellurium resistance proteins was associated trolled at transcriptional level by two sigma factors, with resistance to tellurium, bacteriophage and colicins r32 (RpoH) and rE (RpoE). In E. coli, r32 controls [29]. These genes are part of a pathogenicity island that the expression of many MHSPs including FtsH, DnaKJ, contains additional integrase, phage and urease genes GroELS, Lon, ClpAP, HslVU and GrpE, while rE pos- and appear to be up-regulated in the presence of tellurite J. Qiu et al. / FEMS Microbiology Letters 243 (2005) 489–496 493 that has a long history as an antimicrobial agent and is 3.2.5. Chemotaxis and mobility often employed in selective media for the isolation of a Motile bacteria can swim toward or away from spe- wide range of pathogens [29]. Four genes (terZABC) cific environmental perturbations, such as nutrient- were up-regulated in this study, suggesting that the ter limiting or stress conditions; this behavior, called che- genes are associated with protection from other forms motaxis, is mediated by the bacteria changing direction of stress or agents causing cellular damages. Two genes by briefly reversing the direction of rotation of the fla- (YP0976-0977 according to the genome annotation of Y. gellar motors [34]. Surprisingly, four genes (YPO0704, pestis 91001 [9]) encoding an ATPase and an permeases flhB, fliR/mopE and motB/lafU) involved in chemotaxis of the ABC-type multidrug transport system and the and flagellar assembly were up-regulated by 2.0- to 4.0- acrB/acrE gene encoding a multidrug efflux protein were fold in this study, although Y. pestis is uniformly non- down-regulated by streptomycin treatment, indicating motile. We also found that seven flagellar genes were that these genes do not play an important role in con- greatly up-regulated after heat shock (sudden tempera- fronting with streptomycin. ture upshift from 37 to 45 C) [12]. Genome annotation of Y. pestis CO92 revealed a total of 86 chemotaxis- or 3.2.3. Induced biosynthesis of selective amino acids flagellar-related genes that constitute several distinct Transcription of three (thrA, dapB and dapF) and six clusters in chromosome, and mutations exist in most (YPO1687 and ilvADEMG) genes, responsible for bio- of these gene clusters, but it was still suggested that some synthesis of amino acids of aspartate and pyruvate fam- form of motility might be possible in Y. pestis under ilies respectively, was greatly elevated in response to atypical conditions [7]. streptomycin treatment. Noticeably, the ilvADEMG operon encodes four of the enzymes for the biosynthesis of 3.3. Streptomycin-repressed cellular pathways the branched-chain amino acids, isoleucine, leucine and valine in E coli; when the bacterium grows in a chemi- The addition of streptomycin repressed the transcrip- cally defined medium, limitation of one of these three tion of a large number of genes involved in energy branched-chain amino acids caused an increase in metabolism, biosynthesis of small macromolecules, syn- expression of the enzymes, while the concomitant addi- thesis and modification of macromolecules, degradation tion of all three amion acids cause a decrease in expres- of small and macro molecules and transport/binding sion [30,31]. Streptomycin dramatically disturbs the cell functions, suggesting that most of the metabolic path- envelope structure and trans-membrane transporting ways in Y. pestis were retarded after the drug treatment. functions (see below). Therefore, it can be hypothesized This general repression of metabolic processes is likely that transport of some amino acids is disturbed, thus to be a common response reflecting growth arrest, rather introducing a deprivation condition, which will induce than organism- or drug-specific. the expression of the corresponding biosynthesis genes. 3.3.1. Energy metabolism 3.2.4. Broad regulatory functions The highest number of genes which are regulated by Transcriptional change of a gene encoding global reg- streptomycin is found in the energy metabolism func- ulatory protein may have pleiotropic effects. Nine genes tional category, and that this regulation is always a de- involved in broad regulatory functions were up- crease of the transcription rate. Fifty-two (39%) of the regulated by treatment with streptomycin (see totally 132 energy-metabolism-related genes on Supplementary Table 1). The most notable one is the the microarray were down-regulated, accounting for Lrp gene encoding the leucine-responsive regulatory the overall slowing down of cellular energy generation. protein. In E. coli, Lrp regulates, both positively and These genes or operons fall into the functional catego- negatively, the expression of more than 40 genes and ries of aerobic respiration (16 genes), ATP-proton mo- proteins involved in amino acid biosynthesis, pili bio- tive force (8), electron transport (9), fermentation (2), synthesis, ammonia assimilation, amino acid catabo- glycolysis (3), non-oxidative branch (1), pyruvate dehy- lism, and peptide transport [32,33]. A substantial drogenase (3) and tricarboxylic acid cycle (10), showing fraction of operons regulated by Lrp are also regulated the wide inhibition of energy metabolisms in Y. pestis by by leucine, and the effect of leucine on expression of streptomycin treatment. these operons requires a functional Lrp protein [33]. Therefore, the induced transcription of the Y. pestis 3.3.2. Biosynthesis of small macromolecules lrp gene in response to streptomycin is consistent with The proteins encoded by tauABC constitute a system the elevated expression of the ilv genes responsible for for uptaking taurine as a source of sulfur and the prod- the biosynthesis of isoleucine and leucine. Together with uct of tauD is involved in the oxygenolytic release of sul- the results above, we can postulate that some amino fite from taurine [35]. The cys genes involved in uptake acids will play an important role in facing the presence of sulfite, reduction of sulfite to sulfide and synthesis of streptomycin by unknown mechanisms. of cysteine from serine, and these three processes are 494 J. Qiu et al. / FEMS Microbiology Letters 243 (2005) 489–496 classified into the cysteine biosynthetic pathway [36]. wall, and some sorts of surface structure; the later Addition of the cysteine thiols to the vinyl groups of three coin a descriptive term ‘‘cell envelope’’. The heme occurs in the oxidative environment of the bacte- cytoplasm is surrounded by the plasma membrane, rial periplasm [37]. This heme-binding process is crucial while the cell wall contribute to prevent damage to for the maturation pathway of cytochrome c that is the the underlying protoplast. Outside the cell wall, there indispensable component of electron transport. In this is the surface structure composed of polysaccharides study, the transcription of three tau genes (tauACD) or proteins. In this study, at least 25 genes encoding and nine cys genes (cysA, cysG, cysH, cysI, cysJ, cysK/ membrane components (membrane proteins, lipopro- cysZ, cysP, cysT and cysW) was repressed. It is specu- teins, porins and exported proteins) were up-regulated lated that the reduced rate of energy metabolism (elec- by the streptomycin treatment. The induced expression tron transport) may result in a deceased requirement of selective membrane components indicates the of dissociative cysteine that acts as a signal inhibiting remodeling of cell membrane of Y. pestis upon expo- the transcription of these genes involved in organosulfur sure to streptomycin. utilization and cysteine biosynthesis The cell wall of Gram-negative bacterium consists of Some genes involved in biosynthesis of pyrimidine a thin peptidoglycan sheet between the plasma (inner) ribonucleotides (nadE/adgA) and purine ribonucleotides membrane and an outer membrane. Peptidoglycan is a (purA, purB, purF, purL, purI and guaA) and also salvage giant macromolecule of periodic structure that forms of nucleosides and nucleotides (upp/uraP) were down- the polymer to constitute the shape-maintaining struc- regulated, indicating a generally reduced cellular pool of ture of cell wall [39]. The Y. pestis mur operon responsi- nucleotides resulted from the retardance of cell growth. ble for peptidoglycan synthesis was down-regulated by the streptomycin treatment. Lipopolysaccharide (LPS) 3.3.3. Synthesis and modification of macromoclecules is a major component of the outer membrane. Y. pestis Among the down-regulated genes is another group expresses rough type of LPS without O-antigen polysac- genes involved in synthesis and modification of macro- charide due to the inactivation of the O-antigen gene moclecules. These genes could be mainly assigned into cluster [40]. Our microarray analysis identified nine the functional categories of DNA replication, restric- genes, encoding enzymes for LPS synthesis, whose tran- tion/modification and repair (7 genes), RNA and amino- scription was down-regulated. acyl tRNA synthesis (10), and biosynthesis of An essential feature of living organisms is the ability cytoplasmic glycogen (4), lipopolysaccharide (9) and to accumulate nutrients against a concentration gradient peptidoglycan (7). It was not surprising that there was and to excrete the various end products of metabolism. the general retardance of DNA and RNA synthesis. The main cellular structure responsible for nutrient Our interest was primarily focused on the reduced bio- transport is the plasma membrane and the outer mem- synthesis of cytoplasmic and surface polysaccharides, brane in Gram-negative bacteria. Under streptomycin and of the cell wall component of peptidoglycan (see be- treatment, at least 29 Y. pestis genes encoding trans- low). The glgPACXB operon responsible for glycogen port/binding proteins were differently regulated; among synthesis was down-regulated; the reduced accumula- them, 12 genes were up-regulated and 17 down- tion of cytoplasmic glycogen should destroy the original regulated. Noticeably, the down-regulated genes mainly volume of the Y. pestis cytoplasm. fall into the functional categories of transport of amino acids and amines, cations, and carbohydrates, organic 3.3.4. Degradation of small and macro molecules acids and alcohols. This showed that streptomycin could The RecBCD enzyme, encoded by recB, recC, and slowdown the ability of Y. pestis to acquire amino, cat- recD genes, is a potent large ATP-dependent exodeoxy- ion and carbohydrate through affecting the relevant ribonuclease enzyme that is active on either ds or ss transporters. DNA and a weak ATP-stimulated endonuclease activity As a member of the aminoglycoside class of antibac- that acts only on ss DNA [38]. The reduced transcrip- terial agents, streptomycin irreversibly binds to the 30S tion of the three rec genes in this study indicated a re- ribosomal subunit to inhibit protein synthesis and cause strained metabolic activity in the degradation of misreading of mRNA [5]. However, additional hypoth- DNAs. In addition, transcription of 13 genes involved eses have been proposed to suggest the Ôlethal mecha- in amino acids metabolism, carbon compounds metabo- nismÕ of aminoglycosides, including changes in the cell lism, degradation of RNAs, or degradation of proteins membrane, mistranslation of critical proteins, and other and peptides was repressed. uncharacterized secondary effects [41]. In this study, the detecting transcriptional changes in the genes responsi- 3.4. Action of streptomycin on cell envelope ble for cell envelope function categories supports the no- tion that streptomycin has the profound detrimental A bacterial cell has the four major structural com- effects on the normal structuring of Y. pestis cell ponents: a cytoplasmic region, a cell membrane, a cell envelope. J. Qiu et al. / FEMS Microbiology Letters 243 (2005) 489–496 495

3.5. Differentially regulated plasmid-borne genes [6] Galimand, M., Guiyoule, A., Gerbaud, G., Rasoamanana, B., Chanteau, S., Carniel, E. and Courvalin, P. (1997) Multidrug Yersinia pestis normally carries three prototypical vir- resistance in Yersinia pestis mediated by a transferable plasmid. N. Engl. J. Med. 337, 677–680. ulence plasmids: a 9.5-kb plasmid pPCP1, a 110-kb [7] Parkhill, J., Wren, B.W., Thomson, N.R., Titball, R.W., Holden, pMT1 and a 70-kb pCD1. At least 23 plasmid genes M.T., Prentice, M.B., Sebaihia, M., James, K.D., Churcher, C., were differentially regulated after streptomycin treat- Mungall, K.L., Baker, S., Basham, D., Bentley, S.D., Brooks, K., ment. Y. pestis has acquired two unique plasmids, Cerdeno-Tarraga, A.M., Chillingworth, T., Cronin, A., Davies, pPCP1 and pMT1; both of them contribute to the ex- R.M., Davis, P., Dougan, G., Feltwell, T., Hamlin, N., Holroyd, S., Jagels, K., Karlyshev, A.V., Leather, S., Moule, S., Oyston, treme pathogenicity of Y. pestis. All the three patho- P.C., Quail, M., Rutherford, K., Simmonds, M., Skelton, J., genic yersiniae, Y. pestis, Y. pseudotuberculosis, and Y. Stevens, K., Whitehead, S. and Barrell, B.G. (2001) Genome enterocolitica, carry the pCD1 plasmid that encode a sequence of Yersinia pestis, the causative agent of plague. Nature type III secretion system (T3SS). The T3SS allows extra- 413, 523–527. cellular Yersinia that is docked at the surface of cells of [8] Deng, W., Burland, V., Plunkett, G., Boutin, A., Mayhew, G.F., Liss, P., Perna, N.T., Rose, D.J., Mau, B., Zhou, S., Schwartz, the immune system to deliver Yop effectors into the D.C., Fetherston, J.D., Lindler, L.E., Brubaker, R.R., Plano, cytosol of these cells. These Yops could prevent phago- G.V., Straley, S.C., McDonough, K.A., Nilles, M.L., Matson, cytosis of the bacteria and inhibit the host immune re- J.S., Blattner, F.R. and Perry, R.D. (2002) Genome sequence of sponse. The T3SS composes of four elements: (i) type Yersinia pestis KIM. J. Bacteriol. 184, 4601–4611. 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