chloramphenicol ( cm ) has been used as an broad - spectrum antibiotic in human and veterinary medicine since the 1950s , but the use of chloramphenicol in humans is now rather limited . in animals , the structure of chloramphenicol is relatively simple , and it was the first chemically synthesized antibiotic on the market .chloramphenicol inhibits translation in bacteria , by inhibition of the peptidyl transferase reaction of the large subunit of the ribosome . the inhibition of the peptidyl transferase activity is mediated by binding to several proteins in the 50s ribosomal subunit . a number of resistance mechanisms to chloramphenicol in bacteria has been described , of which the most common is enzymatic inactivation by acetylation of chloramphenicol via chloramphenicol transferases ( cats ) ; chloramphenicol acetyltransferases ( cats ) have been described in both gram - positive and gram - negative bacteria . the inactivation of chloramphenicol can also be performed by xenobiotic acetyltransferases [ 1 , 3 ] . several examples of chloramphenicol resistance or lowered sensitivity to chloramphenicol due to efflux pumps ( specific or multidrug transporters ) have been described , mainly in gram - negative bacteria . finally , chloramphenicol resistance may be due to mutations in 23s rrna , thereby changing the binding site of chloramphenicol in the cells . in this paper we present the transcriptional profile of enterococcus faecalis v583 ( v583 ) treated with chloramphenicol . enterococci are known to be inherently resistant to many antibiotics , while acquired resistance to several other antibiotics has been described ( see e.g. , review by franz et al . ) . these days , e. faecalis is recognized as a notorious opportunistic pathogen , which frequently acquires antibiotic resistance determinants and potential virulence factors . v583 is a multiresistant isolate , but no specific chloramphenicol resistance genes have been identified in its genome sequence . v583 survives treatment with chloramphenicol , but the growth is decreased , which shows that chloramphenicol induces stress on the cells . we used dna microarrays to obtain a profile of the transcriptional events in chloramphenicol treated v583 cells , to identify mechanisms / genes behind the tolerance to chloramphenicol . in this paper we have , in addition to  traditional  analysis of microarray images , used a prototype method for automated analysis of microarray images , which reduce the manual interference with image analysis . the use of dna microarrays to monitor transcription in bacteria is very useful , since it may help to identify specific as well as general mechanisms of stress tolerance / adaptation . it also adds crucial information to the growing amount of genomic data on bacterial behavior . e. faecalis v583 was grown aerobically overnight in flasks with gm17 medium ( difco ) at 37c on a rotary shaker ( 300  rpm ) . to determine the level of chloramphenicol to be used in cultures prior to the microarray experiments , cells were grown in gm17 medium containing 10 , 7.5 , 5.0 , 2.5 , and 0  g / ml chloramphenicol . v583 cells were treated with 2.5  g / ml chloramphenicol . for the microarray experiments , cultures grown overnight were diluted 50 and grown in gm17 as above , for approximately 1 hour , to od600 ~0.2 . then , cultures were split in two , and chloramphenicol ( sigma ) was added to one of the cultures . the final concentration of chloramphenicol was 2.5  g / ml . the two cultures ( gm17 and gm17 plus chloramphenicol ) were then incubated further and 5  ml samples of each culture were collected at 0 ( t0 ) , 90 ( t90 ) , and 180 ( t180 ) min after addition of chloramphenicol . cell harvesting , rna extraction , determination of integrity , and concentration of rna were performed as described previously . , cdna synthesis , fluorescent labeling , hybridization , and image acquisition were performed as described previously . microarray experiments were performed as dye - swap experiments , to avoid bias introduced by differential labeling of the cdna . the microarray images were analyzed twice ; with the genepix pro 6.0 ( see ) and with a prototype program for microarray image analysis ( see [ 8 , 9 ] ) . a prototype program competitive to established methodology on dna microarray analysis was used [ 8 , 9 ] . central aspects in development of this program were ( 1 ) spot segmentation , ( 2 ) intensity modeling and calculation of gene expression values , and ( 3 ) quality assessment of the spots on the microarray images .   for each spot box ( grid cell ) the solution of ( 1 ) included estimation of bivariate gaussians , rearrangement of the pixels into a one - dimensional sequence defined by the gaussian and solution of the spot segmentation problem ( identification of the foreground spot pixels ) by maximizing associated two - sample t - statistics [ 8 , 9 ] . the solution of ( 2 ) used the rearranged sequence of pixels as a basis for logistic modeling to estimate fore- and background levels ( cy3- and cy5 channels were considered separately ) . traditional calculations of associated log - ratios were then based on these estimated fore- and background levels . quality assessment ( 3 ) was based on a set of decision rules , which involved 12 distinct quality parameters defined in close association with the estimated gaussians ( i.e. , shape , size , and position of the spots ) and the logistic models , that is , features of the fitted curves and model residuals [ 8 , 9 ] . downstream analysis was done by the limma package ( www.bioconductor.org ) in the r computing environment ( www.r-project.org ) . preprocessing and normalization followed a standard procedure using methods described by smyth and speed . testing for differentially expressed genes was done in a linear model context as described by smyth . a mixed - model approach was chosen to describe within - array variation between replicates ( 5 replicates of each probe in each array ) , and an empirical bayes smoothing of gene - wise variances was conducted according to smyth et al . . for each gene , p - values were adjusted to control the false discovery rate , hence all p - values displayed are adjusted for false discovery rates ( fdr - adjusted ; often referred to as q - values in the literature ) . to confirm independently the differential gene expression observed by microarray experiments , the following genes were selected for analysis by real - time quantitative reverse transcription pcr ( rtq ) : ef0633 ( trys-1 , encoding tyrosyl - trna synthetases ) , ef2653 ( encoding a transcriptional regulator of the cro / ci family ) , and ef0105 ( argf-1 , encoding ornithine carbamoyltransferase ) . ef1964 ( gap-2 , encoding glyceraldehydes-3-phosphate dehydrogenase ) , which is constitutively expressed , was used to normalize the taqman data . the microarray data have been deposited in the arrayexpress database ( http://www.ebi.ac.uk/arrayexpress/ ) with the series accession number ( not yet available ) . to determine suitable concentrations of chloramphenicol in the transcription profiling experiments , v583 was treated with various concentrations ( 2.5  g10  g  per  ml ) of chloramphenicol . after 90 min od600 of all chloramphenicol - treated cultures was < 50% of the od600 of the untreated control ( figure 1 ) . for the microarray experiments , it was decided to treat the v583 cells with 2.5  g / ml chloramphenicol . for transcriptional profiling experiments using microarrays , it has been recommended to use low inhibitor concentrations , since higher concentrations may induce secondary responses [ 13 , 14 ] . the microarray experiments were performed as time course experiments , where cell samples for rna extraction and further hybridizations were collected three times after addition of chloramphenicol to one of the v583 cultures . cell samples were collected immediately after addition of chloramphenicol ( t0 ) , after 90 min ( t90 ) and after 180 min ( t180 ) . corresponding cdna samples for each time point were mixed and hybridized to microarray slides . for determination of differential transcription , genes were  scored  as significantly differentially transcribed , with threshold p < .05 . in the present context , where results from the use of two different methods for image analysis were to be compared , it was useful to have a consistent method to determine whether each gene was differentially transcribed or not . when the transcription of genes was higher in the presence of chloramphenicol , the gene expression is denoted upregulated ( induced ) , and when the transcription was lower , the gene expression was denoted downregulated ( repressed ) . we applied two different methods for analysis of the microarray images obtained during the experiments : the genepix software and a prototype program . the prototype microarray image analysis method analyses the spots on the images automatically , based on numerous quality parameters , which reduces the manual interference with the analysis considerably . most genes that were scored as significantly differentially expressed using genepix were also found significantly differentially expressed by the prototype program , and vice versa ; 609 genes were scored significantly up- or downregulated at one or more time points both in the data based on genepix and the prototype analyses . of these , 23 were plasmid encoded . by the use of the prototype program and p < .05 as a threshold for significance , 694 chromosomal ( 27 plasmid ) genes were scored as significantly up- or downregulated at one or more of the three time points . contrary , based on the genepix data , 672 ( 28 plasmid ) genes were scored as significantly differentially expressed . those genes that were scored as significantly up- or downregulated by only one of the methods were mainly represented on the microarrays by spots with weak signal intensities . the p - value associated with the expression data of these genes were , for most genes , close to the threshold value for significance ( p > .05 ) . we decided to go on with the genes ( 609 ) that were scored as significantly differentially expressed in both datasets . the transcriptional patterns of plasmid genes ( ptef1 , ptef2 , and ptef3 ) are dealt with in a separate section ( see below ) . chloramphenicol treatment appeared to have a significant effect on transcriptional events in the v583 cells , as seen by the high number ( 609 ) of genes that were differentially transcribed at one or more time points . 301 genes were downregulated , 336 genes were upregulated at one or more time points . common patterns of regulation were seen ; 15 genes were differentially transcribed at all three time points , while 121 genes were differentially transcribed at two time points . the number of differentially transcribed genes was highest at t90 ( lowest at t0 ) . the differential transcription is probably due to ( 1 ) the chloramphenicol treatment and ( 2 ) differences in growth rate between the treated and untreated cultures . for 25 genes we found that the transcription level varied ( up and down ) during the time course .   among the genes with altered transcription , at one or more time points , genes encoding hypothetical proteins constituted the largest group , but these genes also constitute the largest group in the v583 genome . relative to the number of genes encoding hypothetical proteins among the v583 genes ( > 1/3 ) , the number of differentially transcribed genes ( both upregulated and downregulated ) belonging to this group is rather low ( data not shown ) . genes related to transport and binding processes also constitute a large group among the differentially transcribed genes , but the number of downregulated genes in this category is higher than the number of upregulated genes : the genes encoding transport and binding proteins constitute nearly 12% of the v583 genes , 12% of the downregulated genes , and 6% of the upregulated genes . among the upregulated genes , were genes belonging to the categories protein synthesis ; purines , pyrimidines , nucleosides , and nucleotides ; as well as fatty acid and phospholipid metabolism were enriched . among the downregulated genes were amino acid biosynthesis genes , and energy metabolism genes . as mentioned briefly above , 15 genes showed significant expression at all three time points examined ( see table 1 ) . most of these genes ( 12 ) were upregulated at all time points . the cystathionine beta - lyase gene ( ef0290 ) the chaperonin encoding genes ( ef2633 and ef2634 , groe operon ) were downregulated at t0 and t180 and upregulated at t90 . this operon ( groe ) was the only operon , in which genes were regulated at all points during the time course . an important characteristic of the v583 genome sequence is the high amount of probable mobile genetic elements and exogenously acquired dna ( mge ) . these genes constitute about 1/4 of the v583 genome sequence ; 54 of the mge genes ( out of approx 680 chromosomal mge genes ) were differentially transcribed . see   for designation ) were upregulated in the chloramphenicol - treated cells . among these genes from the phage05 , phage06 , and phage07 were also differentially transcribed ; the phage05 genes were downregulated , while the phage06 and phage07 genes were upregulated ( except for ef2822 , which was downregulated at t0 ) . another endolysin - coding gene ( ef2802 ) is located in the phage06 region , and this gene was among the upregulated phage06 genes . in the phage02 ( ef1276ef1293 ) no genes were differentially transcribed . the phage02 is found in many different e. faecalis strains [ 1618 ] . several genes from the pathogenicity island ( pai ; see [ 15 , 19 ] ) and from the region comprising the vanb resistance determinants ( see ) were differentially transcribed . the pai genes were mainly repressed ( 7 out of 9 ) , while most of the differentially transcribed genes from the vanb region were induced ( 8 out of 11 ) . among the induced vanb region genes were the genes encoding the histidine kinase vansb and its cognate response regulator , vanrb .   to be better able to determine which genes were more important in the response of v583 to chloramphenicol treatment , we examined differentially transcribed operons ( see http://www.microbesonline.org/operons/gnc226185.html and ) with differential transcription . in the operon ef0205ef0234 , encoding ribosomal proteins ( r - proteins ) , eight genes were upregulated . other putative operons encoding ribosomal proteins were also upregulated ; ef0915ef0916 , and ef2715ef2716 . among the differentially transcribed genes , which encode r - proteins , only one ( ef0820 ) was repressed .   in the operon containing genes for v - type atpase ( ef1492ef1500 ) the upregulation of genes in this operon was also seen in the study by solheim et al . in v583 treated with bovine bile and in v583 treated with bovine bile and sds . another induced operon was the pyr operon ( ef1712ef1721 ) , which was induced in v583 treated with bovine bile as well . most of the genes in the operon ef2875ef2885 , which encodes genes involved in fatty acid biosynthesis , were induced ( t90 and t180 ) . the ef1732 and ef1733 genes encode two potential transporters of chloramphenicol out of the v583 cells ; these two genes were both strongly induced in the v583 cells ( t90 ) . the three plasmids of the v583 genome are all similar to well - known plasmids . antibiotic resistance genes ( e.g. , ermb ) and some putative virulence factors ( e.g. , aggregation substance ) are among the genes that are specified by the plasmids . in the chloramphenicol - treated v583 cells , only a few of the plasmid encoded genes were differentially transcribed : eleven genes ( one up , ten down regulated ) from ptef1 , eight genes ( three upregulated , five/5 downregulated ) from ptef2 and four genes from ptef3 ( one downregulated , three upregulated ) were differentially transcribed . among the differentially transcribed ( downregulated ) plasmid genes was one gene coding for drug resistance or transport ( efa0010 ) . in this paper , we report the transcriptional patterns of e. faecalis v583 treated with chloramphenicol . e. faecalis v583 is able to grow in media containing chloramphenicol , but compared to optimal growth , it then grows slowly . no specific genes coding for chloramphenicol resistance have been identified in v583 , and the ability to grow in the presence of chloramphenicol is not due to mutations in the 23s rrna gene . to study the transcriptional patterns of v583 cells treated with chloramphenicol , a time course experiment was chosen , since such experiments might provide clues on how the bacterium adapts to the drug . we used two different methods for image analysis prior to estimation of transcription levels , and results were consistent . for example , the differential regulation of numerous operons corroborates the consistency and reliability of the results from the microarray experiments . moreover , the number of induced and repressed genes was similar , as expected in experiments like this . for example , genes encoding ribosomal proteins were upregulated , and genes for trna synthetases were downregulated . genes involved in fatty acid biosynthesis were also induced , as well as genes coding for a v - type atpase , which was seen also in another study of stress response in v583 . the induction of the genes for the v - type atpase points to the importance of optimal redox conditions in the stressed ( treated ) cultures , as discussed also by solheim et al . . functional groupsfor annotation of genome sequences the classification of genes into functional groups , is useful . the largest group of genes in v583 ( and most other genomes ) are genes encoding hypothetical proteins and conserved hypothetical proteins . they constitute more than 1/3 of the genome , and , not surprising , these genes are a major group of regulated genes in transcription profiles [ 6 , 7 , 21 , 22 ] . the importance of the genes for hypothetical proteins has been discussed in many papers , see for example [ 2325 ] . in our work , we found that the genes encoding hypothetical proteins actually constituted a smaller group of the regulated genes than what was expected based on the pure number of these genes in the genome sequence . genes specifying proteins involved in protein synthesis , fatty acid , and phospholipid metabolism were enriched among the regulated genes , compared to their fraction in the genome sequence . although the functional classes of genes are defined broadly , this may reflect which genes and groups are specifically important in the response to chloramphenicol treatment in v583.the pyrimidine biosynthesis operon ( pyr ; ef1721ef1712 ) is regulated by transcriptional attenuation , where pyrr ( ef1721 , first gene in the operon ) encodes the regulator causing transcription termination . carbamoyl phosphate is the precursor for pyrimidine biosynthesis and for arginine biosynthesis . in the chloramphenicol - treated v583 cells , pyr was upregulated ( t180 , mainly ) , while genes specifying arginine biosynthesis were downregulated ( t90 and t180 ) . differential transcription of these genes has been observed also in previous papers [ 6 , 7 ] . since the complete pyr operon was upregulated in the chloramphenicol - treated v583 cells , we may assume that this is related specifically to the chloramphenicol addition in the cultures.genes involved in fatty acid biosynthesis ( e.g. , ef0282ef0284 and ef2875ef2885 ) were enriched among the induced genes ( t180 , mainly ) . the induction of these genes points to alteration of the cell membrane as one mechanism for adaptation of v583 to stress during time courses . an altered membrane may change the transport capabilities of the cell , which may partly explain the relatively low number of induced genes specifying transport and binding proteins ( low fraction compared to the size of this group in the genome ) . 
  for annotation of genome sequences the classification of genes into functional groups , the largest group of genes in v583 ( and most other genomes ) are genes encoding hypothetical proteins and conserved hypothetical proteins . they constitute more than 1/3 of the genome , and , not surprising , these genes are a major group of regulated genes in transcription profiles [ 6 , 7 , 21 , 22 ] . the importance of the genes for hypothetical proteins has been discussed in many papers , see for example [ 2325 ] . in our work , we found that the genes encoding hypothetical proteins actually constituted a smaller group of the regulated genes than what was expected based on the pure number of these genes in the genome sequence . genes specifying proteins involved in protein synthesis , fatty acid , and phospholipid metabolism were enriched among the regulated genes , compared to their fraction in the genome sequence . although the functional classes of genes are defined broadly , this may reflect which genes and groups are specifically important in the response to chloramphenicol treatment in v583 . the pyrimidine biosynthesis operon ( pyr ; ef1721ef1712 ) is regulated by transcriptional attenuation , where pyrr ( ef1721 , first gene in the operon ) encodes the regulator causing transcription termination . carbamoyl phosphate is the precursor for pyrimidine biosynthesis and for arginine biosynthesis . in the chloramphenicol - treated v583 cells , pyr was upregulated ( t180 , mainly ) , while genes specifying arginine biosynthesis were downregulated ( t90 and t180 ) differential transcription of these genes has been observed also in previous papers [ 6 , 7 ] . since the complete pyr operon was upregulated in the chloramphenicol - treated v583 cells , we may assume that this is related specifically to the chloramphenicol addition in the cultures . genes involved in fatty acid biosynthesis ( e.g. , ef0282ef0284 and ef2875ef2885 ) were enriched among the induced genes ( t180 , mainly ) . the induction of these genes points to alteration of the cell membrane as one mechanism for adaptation of v583 to stress during time courses . an altered membrane may change the transport capabilities of the cell , which may partly explain the relatively low number of induced genes specifying transport and binding proteins ( low fraction compared to the size of this group in the genome ) . mge genescompared to previously published transcription profiles of v583 [ 6 , 7 ] , we observed that a substantial amount of the exogenously acquired genes of v583 was expressed in this study . the induction of phage genes ( e.g. , genes coding for endolysin ) may be an indication of a certain extent of lysis of the chloramphenicol - treated cells . 
  compared to previously published transcription profiles of v583 [ 6 , 7 ] , we observed that a substantial amount of the exogenously acquired genes of v583 was expressed in this study . the induction of phage genes ( e.g. , genes coding for endolysin ) may be an indication of a certain extent of lysis of the chloramphenicol - treated cells . differential transcription of genes involved in protein synthesissince rrna is the cellular target of chloramphenicol , a transcriptional response of genes encoding ribosomal proteins ( r - proteins ) was expected . indeed , several genes coding for r - proteins were induced , and the overproduction of these proteins may partly explain the tolerance of v583 to chloramphenicol . however , the synthesis of many r - proteins is regulated at the translational level , and , therefore microarray analyses may not reflect the actual level of r - proteins in the chloramphenicol treated cells.genes coding for aminoacyl trna synthetases were downregulated ( t90 ) , while the prolyl trna synthetase gene was upregulated . the repression of aminoacyl trna synthetase genes was seen also in the study by ng et al . , where the transcription patterns of streptococcus pneumoniae treated with different translation inhibitors were described . the repression of the trna synthetase genes may express lower rate of protein synthesis in the treated cells . the untreated cells , which grew faster , need to synthesize proteins faster than the untreated cells . the repression of the trna synthetase genes , may , though , appear peculiar , since genes for other parts of the translation apparatus ( genes encoding ribosomal proteins and elongation factors ) were induced . antitermination regulates expression of most aminoacyl trna synthetases and other enzymes involved in amino acid biosynthesis ( review by ryckelynck et al . ) . 
  since rrna is the cellular target of chloramphenicol , a transcriptional response of genes encoding ribosomal proteins ( r - proteins ) was expected . indeed , several genes coding for r - proteins were induced , and the overproduction of these proteins may partly explain the tolerance of v583 to chloramphenicol . however , the synthesis of many r - proteins is regulated at the translational level , and , therefore microarray analyses may not reflect the actual level of r - proteins in the chloramphenicol treated cells . genes coding for aminoacyl trna synthetases were downregulated ( t90 ) , while the prolyl trna synthetase gene was upregulated . the repression of aminoacyl trna synthetase genes was seen also in the study by ng et al . , where the transcription patterns of streptococcus pneumoniae treated with different translation inhibitors were described . the repression of the trna synthetase genes may express lower rate of protein synthesis in the treated cells . the untreated cells , which grew faster , need to synthesize proteins faster than the untreated cells . the repression of the trna synthetase genes , may , though , appear peculiar , since genes for other parts of the translation apparatus ( genes encoding ribosomal proteins and elongation factors ) were induced . antitermination regulates expression of most aminoacyl trna synthetases and other enzymes involved in amino acid biosynthesis ( review by ryckelynck et al . ) . transcription of genes for drug transportno chloramphenicol resistance genes have been identified in the v583 genome . in the v583 genome sequence , the ef1732 and ef1733 genes , however , which encode abc transporters in the mdr family , were upregulated ( t90 ( ef1732 ) ; all time points ( ef1733 ) ) . these two genes were induced in v583 cells treated with erythromycin , also , which points to them as potentially important drug transporters in v583 . they are , thus , obvious targets for future detailed experiments , like knockout studies . genes corresponding to ef1732 and ef1733 appear widespread in e. faecalis , and their function in other strains than v583 should also be further elucidated . efflux of chloramphenicol appears as one of the mechanisms that may confer the resistance in v583 . v583 cells treated with chloramphenicol grow very slowly immediately after addition of the drug , while the growth rate increases later ( figure 1 ) . the changed growth rate supports the hypothesis on efflux of chloramphenicol as one resistance mechanism.chloramphenicol binds to the 23s rrna by hydrogen bonding , and probably , an equilibrium between bound and unbound chloramphenicol in the cells are established . the efflux of free - drug molecules will then lead to release of bound chloramphenicol from the ribosome . no chloramphenicol resistance genes have been identified in the v583 genome . in the v583 genome sequence , the ef1732 and ef1733 genes , however , which encode abc transporters in the mdr family , were upregulated ( t90 ( ef1732 ) ; all time points ( ef1733 ) ) . these two genes were induced in v583 cells treated with erythromycin , also , which points to them as potentially important drug transporters in v583 . they are , thus , obvious targets for future detailed experiments , like knockout studies . genes corresponding to ef1732 and ef1733 appear widespread in e. faecalis , and their function in other strains than v583 should also be further elucidated . efflux of chloramphenicol appears as one of the mechanisms that may confer the resistance in v583 . v583 cells treated with chloramphenicol grow very slowly immediately after addition of the drug , while the growth rate increases later ( figure 1 ) . the changed growth rate supports the hypothesis on efflux of chloramphenicol as one resistance mechanism . chloramphenicol binds to the 23s rrna by hydrogen bonding , and probably , an equilibrium between bound and unbound chloramphenicol in the cells are established . the efflux of free - drug molecules will then lead to release of bound chloramphenicol from the ribosome .
many enterococcus faecalis strains display tolerance or resistance to many antibiotics , but genes that contribute to the resistance can not be specified . 
 the multiresistant e. faecalis v583 , for which the complete genome sequence is available , survives and grows in media containing relatively high levels of chloramphenicol . 
 no specific genes coding for chloramphenicol resistance has been recognized in v583 . 
 we used microarrays to identify genes and mechanisms behind the tolerance to chloramphenicol in v583 , by comparison of cells treated with subinhibitory concentrations of chloramphenicol and untreated v583 cells . during a time course experiment , 
 more than 600 genes were significantly differentially transcribed . 
 since chloramphenicol affects protein synthesis in bacteria , many genes involved in protein synthesis , for example , genes for ribosomal proteins , were induced . 
 genes involved in amino acid biosynthesis , for example , genes for trna synthetases and energy metabolism were downregulated , mainly . among the upregulated genes were ef1732 and ef1733 , which code for potential chloramphenicol transporters . 
 efflux of drug out of the cells may be one mechanism used by v583 to overcome the effect of chloramphenicol .