BACKGROUND
the burkholderia cepacia complex is a group of gram-negative β-proteobacteria that comprises at least  <dig> species  <cit> .
originally identified as plant pathogens  <cit> , these versatile microorganisms have emerged as notorious pathogens in cystic fibrosis  patients  <cit> .
one of the best-studied aspects of these opportunistic pathogens is their high level of resistance to a broad range of antimicrobial agents.
due to this multi-drug resistance, treatment of b. cepacia complex infected cf patients is problematic  <cit>  and a b. cepacia complex positive status is often associated with increased morbidity and mortality  <cit> .
as several outbreaks with genetically distinct b. cepacia complex strains have occurred, stringent infection control guidelines were brought into force; these guidelines not only aim to segregate infected patients from non-infected ones, but also emphasize the importance of good hand hygiene practices and frequent disinfection of environmental surfaces and respiratory equipment  <cit> .
in a recent study, we showed that some of the current disinfection protocols are inadequate to effectively remove and kill biofilm-grown burkholderia cenocepacia cells  <cit> .
biofilm formation has been described for several b. cepacia complex strains and is considered to be an important virulence trait  <cit> .
the observation that these sessile cells are highly resistant against h2o <dig> and naocl is worrying since these oxidative agents are also very important in the endogenous defence against microorganisms.
during infection, neutrophils normally produce a variety of reactive oxygen species , including superoxide, h2o <dig> and hypochlorite  <cit> .
h2o <dig> and naocl can react with intracellular iron via the fenton reaction and the resulting highly reactive hydroxyl radicals will damage lipids, proteins and dna  <cit> .
in addition, b. cepacia complex bacteria are one of the most common contaminants of pharmaceutical and industrial products  <cit>  and this feature is probably highly dependent on their ability to form persistent biofilms.
the fact that b. cenocepacia cells can survive high doses of exogenous ros, suggests that this microorganism possesses various protective mechanisms involved in the scavenging and neutralization of ros.
thus far, the role of kata and katb in the survival of planktonic b. cenocepacia cells has been studied  <cit>  and the presence of genes encoding catalases and alkyl hydroperoxide reductases within the b. cenocepacia j <dig> genome has been described  <cit> .
a recent transcriptomic study of b. cenocepacia planktonic growth in cf sputum, an environment that is rich in ros, demonstrated that an organic hydroperoxide resistance gene  and an osmc-like oxidative stress gene  were highly upregulated  <cit> .
however, no data are available yet regarding the wider molecular mechanisms used by sessile b. cenocepacia cells to survive acute exposure to h2o <dig> or naocl.
in view of the large fractions of sessile cells that can survive high levels of ros, a better insight into the genomic basis of this major virulence trait is required.
over the last decade, many studies have been performed on the molecular mechanisms involved in bacterial resistance against ros.
these studies include microarray analyses in which the transcriptional response in untreated planktonic cells was compared with the transcriptional response in ros-exposed planktonic cells  <cit> .
in addition, the effect of various mutations on the survival of bacteria in the presence of ros and oxygen has been evaluated in a number of other studies  <cit> .
although the biofilm mode of growth is the prevailing microbial lifestyle  <cit> , no microarray analyses focussing on the transcriptional response of sessile cells exposed to ros have been performed thus far.
in the present study, we compared gene expression in treated and untreated b. cenocepacia j <dig> biofilms, using microarray analysis and qpcr.
in addition, the role of several genes was further examined using mutant strains.
RESULTS
treatment of b. cenocepacia j <dig> biofilms with h2o <dig> and naocl
previous research indicated that disinfection protocols based on the use of h2o <dig>  and naocl   have insufficient activity against b. cenocepacia biofilms  <cit> .
based on these disinfection protocols, we determined the h2o2- and naocl-concentrations  that resulted in a reduction in the number of viable cells of approximately 50% compared to untreated biofilms.
using this approach, we ensured that a sufficient number of viable cells would remain as a source of mrna for microarray analyses.
a resazurin based viability staining performed on treated and untreated b. cenocepacia j <dig> cells revealed that exposure to  <dig> % h2o <dig>  or  <dig> % naocl  killed approximately half the sessile b. cenocepacia j <dig> population .
transcriptomic response to high levels of oxidative stress: microarray data analysis
compared to the normalized signal intensities obtained for the untreated b. cenocepacia j <dig> sessile cells, the normalized signal intensity for  <dig>  and  <dig>  probes was significantly altered after treatments with h2o <dig> and naocl, respectively .
in response to the treatment with h2o <dig>  using a fold change cut off of  <dig> , the expression of  <dig>  genes was significantly higher , while the expression of  <dig>  genes was lower .
using the same data analysis parameters, treatment of the biofilms with  <dig> % naocl resulted in a significant upregulation of  <dig>  genes  and downregulation of  <dig>  genes .
a total of  <dig>  and  <dig>  genes showed a higher or lower expression in response to both treatments.
the fraction of up- and downregulated genes is comparable to the fraction of genes showing a significantly altered expression in planktonic pseudomonas aeruginosa pao <dig> cells exposed to sublethal concentrations of h2o <dig> or naocl   <cit> .
the number of probes on the array that correspond to b. cenocepacia j <dig> protein encoding sequences , intergenic regions  and trna- or rrna-encoding sequences are shown for each category .
among the upregulated genes, many were known to prevent, counteract or repair damage resulting from oxidative stress.
furthermore, the expression of various genes involved in the synthesis and the assembly of the flagellum was also increased.
in addition to the changes in expression of a variety of protein-encoding genes and trna-encoding sequences, a change in expression was observed for many intergenic regions , which might contain non-coding regulatory rna .
the transcription of several genes belonging to a bcepmu prophage was also significantly increased in biofilms exposed to h2o <dig> for  <dig> or  <dig> min.
these gene sets with significantly altered expression are discussed in more detail below.
genes involved in protection against oxidative stress
a large proportion of the upregulated genes were known to be involved in the protection against oxidative stress .
in other gram-negative microorganisms such as escherichia coli and p. aeruginosa, coordinated regulation of many genes associated with oxidative stress is mediated by the transcriptional regulator oxyr  <cit> .
in e. coli, oxidation of oxyr leads to an increased expression of various genes, including genes coding for alkyl hydroperoxide reductases c and f  and hydroperoxidase i   <cit> .
in p. aeruginosa, oxidized oxyr increases the expression of ahpcf and katb   <cit> .
a putative oxyr-binding site was found upstream of all genes encoding homologues of the latter proteins in b. cenocepacia j <dig> .
furthermore, a putative oxyr box was also found upstream of the highly upregulated gene bcal <dig>  which encodes a hypothetical protein .
the results represent the fold change in gene expression in the treated biofilms compared to the gene expression in the untreated biofilms.
-: no qpcr experiments were performed.
ns: no significant change in expression was observed between the treated and the untreated biofilms .
although the transcription of both ahpcf  and bcam <dig> was markedly increased after treatment with h2o <dig> , the increased transcription of bcal <dig>  was less pronounced: the microarray data revealed a statistically significant  <dig> -fold upregulation following treatment with h2o <dig>  which was confirmed by qpcr .
the importance of the ahpc protein in the protection against oxidative stress has been reported previously  <cit>  and the high upregulation of both ahpc and ahpf confirms these data.
yet, the limited upregulation of katb was surprising, as katb is supposed to be the major catalase/peroxidase in b. cenocepacia and hypersensitivity of planktonic b. cenocepacia mdl <dig>  cells to h2o <dig> has been reported  <cit> .
as expected, the expression of the minor catalase/peroxidase encoding gene kata  was not altered in response to exogenous oxidative stress  <cit> .
in order to further clarify the importance of katb for the survival of b. cenocepacia biofilms exposed to h2o <dig>  biofilms of two wild type strains  and of two mutant strains  were treated with  <dig> % h2o <dig> and the fraction of surviving sessile cells was determined using a resazurin based viability staining  <cit> .
in addition, the remaining fraction of h2o <dig> was determined in the supernatants of these biofilms after treating them for  <dig>   <dig> or  <dig> min.
it should be noted that strains j <dig> and c <dig> are not identical, although they both belong to the et <dig> lineage; it is at present unclear whether this has an impact on our results.
these results show that, in contrast to the observations for the wild type strains and for the kata mutant strain, less than 5% and  <dig> % of all sessile cells present in the b. cenocepacia katb mutant biofilms survived  <dig> and  <dig> min treatments with  <dig> % h2o <dig>  respectively .
in addition, approximately 90% of all initially added h2o <dig> was still present in the supernatant of the b. cenocepacia katb mutant biofilms, whereas only trace amounts of h2o <dig> were recovered in the supernatants of the biofilms of the wild type strains and of the kata mutant .
in keeping with these observations, strong effervescence was observed when h2o <dig> was added to the biofilms of both wild type strains and of the kata mutant, whereas no effervescence was observed for the b. cenocepacia katb mutant biofilms.
the data obtained in these experiments clearly indicate that katb is not only essential for the survival of planktonic b. cenocepacia cells exposed to h2o <dig>  but that this catalase/peroxidase is also crucial for the protection of sessile b. cenocepacia cells against exogenous h2o <dig>  conforming with previous data for stationary phase planktonic cultures  <cit> , we observed a substantial expression of katb in untreated biofilms, which suggests the involvement of other factors in the regulation of the expression of this particular gene during sessile growth.
the transcriptional regulation by various transcription factors might also explain the limited upregulation of katb under conditions of oxidative stress  <cit> .
in addition to the upregulation of katb by these sessile populations, an increased transcription of two other catalase encoding genes  was observed in the h2o2-treated biofilms.
these two catalase encoding genes possessed differential expression patterns in relation to the length of h2o <dig> exposure: while bcam <dig> showed the highest upregulation in the biofilms treated for  <dig> min, the transcription of bcal <dig> was much more pronounced in the  <dig> min treated biofilms .
as only trace amounts of h2o <dig> were still present after a  <dig> min treatment, the increased transcription of bcal <dig> in biofilms treated for longer periods suggests that it does not participate in the direct neutralization of exogenous h2o <dig> and that the expression of this catalase is triggered indirectly.
we also observed that the increased transcription of bcam <dig> was accompanied by the increased expression of its flanking gene ankb .
in p. aeruginosa, these two genes are part of a small operon and the lack of a functional ankb protein results in a decreased katb activity and an increased sensitivity to h2o <dig>  <cit> .
the results represent the fold change in gene expression in the treated biofilms compared to the gene expression in the untreated biofilms .
ns: no significant change in expression was observed between the treated and the untreated biofilms .
in e. coli the oxyr regulon also comprises genes encoding glutaredoxin  <dig>  and a glutathione reductase   <cit> , yet no significant upregulation of gora or of any of the glutaredoxin encoding genes was observed in the ros exposed sessile b. cenocepacia j <dig> cells.
we did observe a significant upregulation of the expression of a thioredoxin reductase , of thioredoxin a  and of a putative thioredoxin , although no oxyr-boxes are present upstream of these genes.
since thioredoxins are capable of reducing oxidized proteins and of scavenging hydroxyl radicals  <cit> , their upregulated expression in h2o2-treated b. cenocepacia cells was not unexpected.
in addition, the strong upregulation of a carboxymuconolactone decarboxylase encoding gene  in response to naocl exposure and the significant upregulation of a second carboxymuconolactone decarboxylase encoding gene  in response to exposure to both h2o <dig> and naocl, suggest that both these proteins are also involved in the neutralization of exogenous ros in b. cenocepacia j <dig> biofilms .
treatments with h2o <dig> or naocl resulted in the increased transcription of three organic hydroperoxide resistance  genes: bcas <dig>  bcam <dig> and bcal <dig>  although the latter gene encodes a protein that is annotated as an osmc-like protein, the protein has the typical characteristics of the ohr subfamily .
furthermore, bcal <dig> and bcam <dig> are both located in close proximity of putative ohrr encoding genes .
in a recent study by drevinek et al.
<cit> , the upregulation of a further ohr family gene, bcam <dig>  was observed in planktonic cells exposed to sub-inhibitory concentrations of h2o <dig> and organic hydroperoxides; however, no such upregulation was observed in h2o2-treated sessile cells.
overall, such a strong upregulation of the expression of ohr genes in response to h2o <dig> or naocl treatments has not been reported thus far  <cit> .
ohr expression in p. aeruginosa pao <dig> appears to be triggered specifically by organic hydroperoxides  <cit>  and ohr proteins preferably reduce organic, rather than inorganic hydroperoxides  <cit> .
in addition to the upregulation of bcas <dig> and bcal <dig>  similar expression patterns were observed for their neighbouring genes, suggesting that the genes in both clusters are co-transcribed .
overall, our results suggest that ohr proteins are very important for the protection of biofilm-grown b. cenocepacia cells against ros and that it can draw on multiple genes associated with this family of stress proteins.
in addition to the upregulation of bcas <dig>  we also observed a marked increase in the expression of bcas <dig>  which encodes an exported lipase.
this overexpression is probably the result of the cotranscription of this gene with bcas <dig>  in order to determine whether this overexpression results in an increased extracellular lipase activity, the cleavage of three fluorogenic 4-methylumbelliferyl substrates  was determined in the supernatants of treated and untreated biofilms  <cit> .
for all substrates used, the fluorescent signal was higher in the treated biofilms compared to the untreated biofilms, confirming the increased extracellular lipase activity for the treated biofilms .
within the genomes of the sequenced b. cepacia complex strains, orthologues of bcas <dig> and bcas <dig> are organised in a similar operon-like fashion and therefore we examined the induction of lipase expression by h2o <dig> in biofilms of six b. cepacia complex reference strains.
for b. cenocepacia c <dig>  hi <dig>  burkholderia ambifaria lmg  <dig> and burkholderia dolosa au <dig>  an increased lipase activity was observed in the supernatant of all treated biofilms.
similarly, lipase activity was increased in the supernatant of b. cenocepacia au <dig> biofilms treated for  <dig> and  <dig> min, and in the supernatant of a  <dig> min treated burkholderia multivorans lmg  <dig> biofilm .
these data confirmed that lipase activity in the supernatant of b. cepacia complex biofilms following exposure to oxidative stress is increased, which is probably due to the co-transcription of bcas <dig> orthologues with bcas <dig> orthologues.
the transcription of a cytochrome b <dig> encoding gene  and of its adjacent genes  was also highly increased when the biofilms were exposed to exogenous ros .
the qpcr data clearly indicate that prolonged exposure to h2o <dig> results in an increasing overexpression of bcal <dig> .
yet, even after a  <dig> min exposure to naocl, a considerable overexpression of all three genes was observed .
drevinek et al.
<cit>  recently reported a high upregulation of these genes in b. cenocepacia j <dig> cells grown in cf sputum and they suggested that this may result from the presence of ros in cf sputum.
the microarray data also revealed that the transcription of bcal <dig>  a gene encoding a non-heme chloroperoxidase increased drastically in biofilms exposed to h2o <dig>  this finding was confirmed by qpcr and an increased expression was observed in response to prolonged treatments .
although four genes encoding a non-heme chloroperoxidase are present in the b. cenocepacia j <dig> genome , overexpression was only observed for bcal <dig>  indicating the ability of our microarray-based strategy to highlight the most active gene in burkholderia genomes where multiple paralogous functional pathways exist  <cit> .
upregulation of reca
h2o <dig> and naocl can both damage nucleic acids.
in the fenton reaction, h2o <dig> reacts with fe2+ resulting in the formation of highly reactive hydroxyl radicals  <cit> .
similarly, naocl can also generate hydroxyl radicals via a fenton-type reaction  <cit> .
because positively charged fe2+ ions are often localized along the negatively charged phosphodiester backbone of nucleic acids, dna is a vulnerable target for harmful reactions with these radicals  <cit> .
as exposure to ros can result in extensive dna damage, processes for dna protection and dna repair are vital  <cit> .
the microarray analysis revealed a significant increase in the transcription of reca  in response to a  <dig> min treatment with naocl and a  <dig> min treatment with h2o <dig> .
in the qpcr experiments, no significant increase in transcription of reca was observed in the biofilms treated with h2o <dig> for  <dig> min .
in contrast, in the  <dig> min treated biofilms, the transcription of this gene was significantly increased.
we also observed a slight upregulation of reca following a  <dig> min treatment  but this was not statistically significant .
our hypothesis is that, although most of the h2o <dig> has disappeared after  <dig> min, the transcriptional response to dna damage caused by h2o <dig> is delayed.
upregulation of the reca gene has previously already been described for planktonic p. aeruginosa cultures treated with oxidizing agents  <cit> .
no increased transcription of dps , encoding the non-specific dna-binding protein dps, was neither observed in the h2o2- nor in the naocl-treated biofilms; these data are similar to previous observations in burkholderia pseudomallei  <cit> .
in e. coli, the transcription of dps is regulated by oxyr in exponentially growing planktonic cultures, whereas in stationary phase cultures, dps is transcribed in an rpos-dependent manner  <cit> .
the high expression of dps in the untreated biofilms and the absence of an increased expression in the ros-treated biofilms suggest that rpos rather than oxyr regulates the transcription of dps in mature b. cenocepacia biofilms  <cit> .
upregulation of the iscrsua-hscab-fdx-iscx gene cluster
iron-sulfur cluster-containing proteins are highly sensitive to the cellular redox status and they are very vulnerable to oxidants such as h2o <dig>  <cit> .
in order to restore their function, iron-sulfur clusters are either repaired or they can be assembled de novo  <cit> .
in e. coli, apo-iscr will increase the transcription of the isc gene cluster, and together with oxyr it will also stimulate the production of oxidation-resistant suf proteins  <cit> .
as no homologues of the e. coli suf gene cluster are found within the b. cepacia complex, the transcriptional response in the h2o2-treated biofilms only involves the increased transcription of the iscsua-hscab-fdx-iscx gene cluster .
in addition, an increase in the number of transcripts for iscr  was also observed .
the latter gene has been identified in a recent screening of  <dig> mutants of p. aeruginosa pao <dig>  which also lacks the suf operon, as an essential gene in the resistance to h2o <dig>  <cit> .
our data suggest that the increased transcription of the iscrsua-hscab-fdx-iscx gene cluster is important to regain intact iron-sulfur clusters in the ros-treated b. cenocepacia j <dig> cells.
as a consequence of the damage by oxidants to the iron-sulfur clusters, 2+ and 2+ containing enzymes, including aconitase b , are inactivated  <cit> .
although acnb is the major aconitase during normal growth conditions, it loses all activity in response to strong oxidants  <cit> .
usually, this drop in aconitase activity is compensated by the increased production of aconitase a , which is invulnerable to oxidative inactivation in vivo  <cit> .
however, in the present study no increased expression of acna  was observed, while the number of mrna transcripts of acnb  increased both in the h2o2- and in the naocl-treated biofilms; the data obtained for the h2o2-treated biofilms were confirmed by qpcr .
this observation is puzzling, yet it may be the result of an increased stability of acnb mrna by the accumulating apo-aconitases, as previously observed in e. coli  <cit> .
identification of ncrna
the custom made  <dig> × 44k b. cenocepacia j <dig> microarray does not only contain probes for protein encoding genes, but also  <dig>  probes for selected igs.
a significant upregulation was observed in  <dig>  and  <dig>  of these igs after treatments with h2o <dig> or naocl, respectively, whereas the expression of  <dig>  and  <dig>  igs was significantly downregulated in response to these treatments .
a closer inspection of these igs revealed that many are located in close proximity of genes with a similar expression pattern, suggesting that co-transcription of the igs occurs synchronously with their adjacent genes.
however, certain igs demonstrated markedly different expression patterns compared to their flanking genes, and the basal expression levels of several of these igs were also high.
of the  <dig> igs, which showed a significant upregulation after exposure to h2o <dig>   <dig> displayed an expression pattern different from their flanking genes.
these igs include seven previously shown to be either significantly up- or downregulated in b. cenocepacia j <dig> cells grown in sputum  <cit> .
two igs, ig1_ <dig> and ig1_ <dig> identified by yoder-himes et al.
<cit>  to contain putative ncrna, were also overexpressed.
finally, the transcription of two igs, ig1_ <dig>  and ig1_ <dig>  which have been identified previously to contain putative ncrna in a computational search  <cit> , was also increased in response to exogenous oxidative stress.
of the  <dig> igs significantly upregulated in response to a naocl-treatment,  <dig> displayed a different expression pattern than their flanking genes.
again, a significant upregulation of the expression of ig1_ <dig> and ig1_ <dig> as well as ig1_ <dig> was observed.
in order to confirm these microarray data, we performed qpcr experiments for ig1_ <dig> and ig1_ <dig> as well as for their flanking genes.
the expression of ig1_ <dig> was clearly higher when the biofilms were treated with h2o <dig>  whereas no such upregulation was observed either for bcal <dig>  or for bcal <dig>  .
in addition, the transcription of bcal <dig> and bcal <dig> in the untreated biofilms was clearly lower compared to the transcription of ig1_ <dig> .
the secondary structure for the mrna transcript  has a marked similarity to the 6s rna consensus structure   <cit> .
the qpcr results also revealed a significant upregulation of ig1_ <dig> in the h2o2-treated biofilms .
however, the transcription of the neighbouring genes bcal <dig>  and bcal <dig>  was not altered to the same extent and the ct values obtained for ig1_ <dig> in the untreated biofilms were markedly lower than the corresponding ct values of bcal <dig> and bcal <dig>  although the presence of putative ncrna in ig1_ <dig> has been reported previously  <cit> , no ncrna was identified in this ig using the rfam database  <cit> .
three igs  in which putative ncrnas were identified in a computational search  <cit>  were downregulated in the h2o2- as well as in the naocl-treated biofilms.
sigma factors
as the present study focussed on the changes in gene expression in sessile cells following treatment with high concentrations of ros, major  stress responses were observed and the expression of multiple σ factors was increased .
these σ factors included an rna polymerase σ <dig> factor , an ecf  σ <dig> factor orbs , a putative σ factor  and an ecf σ <dig> factor feci .
the ecf σ <dig> factor encoded by bcal <dig> has been annotated as orbs or ecfi, a regulatory σ factor located adjacent to the ornibactin gene cluster in b. cenocepacia  <cit> .
the transcription of orbs is regulated by the fur repressor, which represses the orbs promoter when cells are growing under iron-replete conditions  <cit> .
in correspondence to the high increase of the transcription of orbs, two of the genes belonging to the ornibactin gene cluster, orbh  and orbg , were also found to be upregulated .
the ig sequence between orbs and orbh also showed elevated expression .
however, none of the other genes present in the ornibactin gene cluster  were upregulated.
other studies have shown that orbh and orbg are cotranscribed with orbs  <cit> , correlating to the increases we observed in these two genes and the intervening ig region.
the lack of transcription of the other orb genes suggests that the levels of orbs under our experimental conditions were insufficient to induce the transcription of orbe and orbi-orbl  <cit> .
alternatively, the transcription of orbe and orbi-orbl may depend on a post-transcriptional regulation of orbs activity  <cit> .
this posttranscriptional regulation might result from the interaction of orbs with 6s rna  <cit> .
an increased transcription of a second σ <dig> factor involved in iron-uptake, feci , was also observed.
similar observations have been described for h2o2-treated p. aeruginosa cells  <cit> .
the increased transcription of orbs as well as of feci suggests that the cells apparently suffer from some degree of iron starvation or that the fur repressor function is transiently lost  <cit> .
the exact roles of the σ factors, encoded by bcal <dig> and bcal <dig>  in relation to these conditions of oxidative stress remain to be elucidated.
remarkably, the expression pattern of bcal <dig> is highly similar to that of its divergently transcribed neighbouring gene bcal <dig> .
neither rpon , nor rpoe  showed an increased transcription following exposure to exogenous ros, corroborating previous data that neither rpon nor rpoe are involved in the resistance towards h2o <dig>  <cit> .
genes involved in motility and chemotaxis
b. cepacia complex bacteria are motile rods, equipped with flagella.
these flagella are multi-component structures, which are synthesized and assembled in a complex process involving over  <dig> hierarchically expressed genes  <cit> .
flagella are not only important in the formation of biofilms, they also play a key role in the adherence to and the invasion into host cells  <cit> .
in addition, intact flagella evoke host immune responses  <cit> .
the data obtained in the microarray analysis reveal that many b. cenocepacia j <dig> flagellum-related genes and genes involved in chemotaxis show an increased expression in biofilms exposed to exogenous ros.
the overexpressed genes in the h2o2-treated biofilms include those that belong to two major gene clusters:  bcal0520-bcal <dig> and bcal0526-bcal <dig>  and  bcal0564-bcal <dig>  bcal <dig> and bcal0576-bcal <dig>  the expression of the majority of these genes was also significantly increased in response to a short exposure to naocl .
in addition, the increased transcription of the flagellar motor protein encoding gene mota was also observed .
furthermore, the transcription of five and nine genes coding for  methyl-accepting chemotaxis proteins was increased in the h2o2- and naocl-treated biofilms, respectively.
although our data clearly indicate that sessile b. cenocepacia j <dig> cells exposed to exogenous oxidative stress overexpress many flagellar and chemotaxis genes, previous experiments demonstrated that treatments with  <dig> % h2o <dig> did not result in significant decreases in total biofilm biomass  <cit> .
the latter observation suggests that the overexpression of these genes does not result in a global dispersal event.
further research is required to elucidate the role of the increased transcription of these genes in response to exogenous ros.
phage-related genes
the microarray analysis revealed a significant upregulation of multiple phage-related genes in sessile cells after exposure to h2o <dig> .
bcepmu is a mu-like prophage that is present in many b. cenocepacia et <dig> strains and it has been annotated bcengi <dig> .
the prophage sequence was found to encode  <dig> proteins and it has been divided in three functional clusters, including a module involved in replication, regulation and pathogenesis  <cit> .
the transcription of these genes probably results in a monocistronic mrna as a similar upregulation of multiple igs within this cluster was observed .
this prophage module contains many genes coding for small hypothetical proteins.
in addition, it contains two genes  that show high similarities to pseudomonas phage b <dig> transposase b and a subunits   <cit> .
the results of the qpcr analysis for three selected genes  indicated that none of these genes was upregulated after a  <dig> min treatment with h2o2; yet, a significant upregulation was observed when b. cenocepacia j <dig> biofilms were treated for  <dig> or  <dig> min .
thus far, the transcriptional mechanism resulting in the upregulation of these bcepmu prophage genes has not been elucidated.
the results represent the fold change in expression in the treated biofilms compared to the expression in the untreated biofilms .
downregulated genes in the h2o2- and naocl-treated biofilms
although  <dig>  genes showed a significant downregulation of the transcription in biofilms exposed to h2o <dig>  a more than five fold change in expression was only observed for  <dig> genes.
similarly, for  <dig> of the  <dig>  downregulated genes in the naocl-treated biofilms, transcription was decreased more than five fold.
a total of  <dig>  genes was downregulated in response to both treatments.
despite the large numbers of genes showing a decreased expression in response to these ros, no obvious patterns were observed.
qpcr experiments were performed for two selected genes: bcam <dig>  and bcam <dig>  and they confirmed that both genes were significantly downregulated in response to exposure to h2o <dig> .
fold change correlation between qpcr experiments and microarray analysis
in order to determine the correlation between the fold changes in gene expression observed in the microarray analysis and the qpcr experiments, a regression analysis was performed.
the results of the regression analysis between the log <dig> fold changes obtained in the qpcr experiments and the microarray analysis showed a good linear correlation between both datasets, with a correlation coefficient of  <dig>   and a slope of  <dig> .
this suggests that the custom made  <dig> × 44k microarray for b. cenocepacia is highly reliable to evaluate changes in global gene expression in b. cenocepacia j <dig>
CONCLUSIONS
our data show that the successful defence of sessile b. cenocepacia j <dig> cells against high doses of ros involves a plethora of protective mechanisms.
these include mechanisms that allow cells to neutralize and scavenge ros and mechanisms that are involved in the repair of the cellular damage resulting from the exposure to ros.
several results in the present study corroborate data from previous studies that focussed on the transcriptional response to sublethal doses of exogenous ros  <cit> .
rather unexpected observations included the high upregulation of three ohr encoding genes in h2o2-treated sessile b. cenocepacia j <dig> cells and the increased extracellular lipase activity in several h2o2-treated b. cepacia complex biofilms.
in addition, our data revealed an increased transcription of multiple phage-related genes, igs and chemotaxis- and motility-related genes in the treated biofilms.
these observations contribute to a better insight in the molecular mechanisms involved in the resistance of sessile b. cenocepacia j <dig> cells against exogenous ros.

 the burkholderia cepacia complex is a group of gram-negative β-proteobacteria that comprises at least species. originally identified as plant pathogens, these versatile microorganisms have emerged as notorious pathogens in cystic fibrosis patients. one of the best-studied aspects of these opportunistic pathogens is their high level of resistance to a broad range of antimicrobial agents. due to this multi-drug resistance, treatment of b. cepacia complex infected cf patients is problematic and a b. cepacia complex positive status is often associated with increased morbidity and mortality. as several outbreaks with genetically distinct b. cepacia complex strains have occurred, stringent infection control guidelines were brought into force; these guidelines not only aim to segregate infected patients from non-infected ones, but also emphasize the importance of good hand hygiene practices and frequent disinfection of environmental surfaces and respiratory equipment. in a recent study, we showed that some of the current disinfection protocols are inadequate to effectively remove and kill biofilm-grown burkholderia cenocepacia cells. biofilm formation has been described for several b. cepacia complex strains and is considered to be an important virulence trait. the observation that these sessile cells are highly resistant against h2o and naocl is worrying since these oxidative agents are also very important in the endogenous defence against microorganisms. during infection, neutrophils normally produce a variety of reactive oxygen species, including superoxide, h2o and hypochlorite. h2o and naocl can react with intracellular iron via the fenton reaction and the resulting highly reactive hydroxyl radicals will damage lipids, proteins and dna. in addition, b. cepacia complex bacteria are one of the most common contaminants of pharmaceutical and industrial products and this feature is probably highly dependent on their ability to form persistent biofilms. the fact that b. cenocepacia cells can survive high doses of exogenous ros, suggests that this microorganism possesses various protective mechanisms involved in the scavenging and neutralization of ros. thus far, the role of kata and katb in the survival of planktonic b. cenocepacia cells has been studied and the presence of genes encoding catalases and alkyl hydroperoxide reductases within the b. cenocepacia j genome has been described. a recent transcriptomic study of b. cenocepacia planktonic growth in cf sputum, an environment that is rich in ros, demonstrated that an organic hydroperoxide resistance gene and an osmc-like oxidative stress gene were highly upregulated. however, no data are available yet regarding the wider molecular mechanisms used by sessile b. cenocepacia cells to survive acute exposure to h2o or naocl. in view of the large fractions of sessile cells that can survive high levels of ros, a better insight into the genomic basis of this major virulence trait is required. over the last decade, many studies have been performed on the molecular mechanisms involved in bacterial resistance against ros. these studies include microarray analyses in which the transcriptional response in untreated planktonic cells was compared with the transcriptional response in ros-exposed planktonic cells. in addition, the effect of various mutations on the survival of bacteria in the presence of ros and oxygen has been evaluated in a number of other studies. although the biofilm mode of growth is the prevailing microbial lifestyle, no microarray analyses focussing on the transcriptional response of sessile cells exposed to ros have been performed thus far. in the present study, we compared gene expression in treated and untreated b. cenocepacia j biofilms, using microarray analysis and qpcr. in addition, the role of several genes was further examined using mutant strains.  treatment of b. cenocepacia j biofilms with h2o and naocl previous research indicated that disinfection protocols based on the use of h2o and naocl have insufficient activity against b. cenocepacia biofilms. based on these disinfection protocols, we determined the h2o2- and naocl-concentrations that resulted in a reduction in the number of viable cells of approximately 50% compared to untreated biofilms. using this approach, we ensured that a sufficient number of viable cells would remain as a source of mrna for microarray analyses. a resazurin based viability staining performed on treated and untreated b. cenocepacia j cells revealed that exposure to % h2o or % naocl killed approximately half the sessile b. cenocepacia j population. transcriptomic response to high levels of oxidative stress: microarray data analysis compared to the normalized signal intensities obtained for the untreated b. cenocepacia j sessile cells, the normalized signal intensity for and probes was significantly altered after treatments with h2o and naocl, respectively. in response to the treatment with h2o using a fold change cut off of, the expression of genes was significantly higher, while the expression of genes was lower. using the same data analysis parameters, treatment of the biofilms with % naocl resulted in a significant upregulation of genes and downregulation of genes. a total of and genes showed a higher or lower expression in response to both treatments. the fraction of up- and downregulated genes is comparable to the fraction of genes showing a significantly altered expression in planktonic pseudomonas aeruginosa pao cells exposed to sublethal concentrations of h2o or naocl. the number of probes on the array that correspond to b. cenocepacia j protein encoding sequences, intergenic regions and trna- or rrna-encoding sequences are shown for each category. among the upregulated genes, many were known to prevent, counteract or repair damage resulting from oxidative stress. furthermore, the expression of various genes involved in the synthesis and the assembly of the flagellum was also increased. in addition to the changes in expression of a variety of protein-encoding genes and trna-encoding sequences, a change in expression was observed for many intergenic regions, which might contain non-coding regulatory rna. the transcription of several genes belonging to a bcepmu prophage was also significantly increased in biofilms exposed to h2o for or min. these gene sets with significantly altered expression are discussed in more detail below. genes involved in protection against oxidative stress a large proportion of the upregulated genes were known to be involved in the protection against oxidative stress. in other gram-negative microorganisms such as escherichia coli and p. aeruginosa, coordinated regulation of many genes associated with oxidative stress is mediated by the transcriptional regulator oxyr. in e. coli, oxidation of oxyr leads to an increased expression of various genes, including genes coding for alkyl hydroperoxide reductases c and f and hydroperoxidase i. in p. aeruginosa, oxidized oxyr increases the expression of ahpcf and katb. a putative oxyr-binding site was found upstream of all genes encoding homologues of the latter proteins in b. cenocepacia j. furthermore, a putative oxyr box was also found upstream of the highly upregulated gene bcal which encodes a hypothetical protein. the results represent the fold change in gene expression in the treated biofilms compared to the gene expression in the untreated biofilms. -: no qpcr experiments were performed. ns: no significant change in expression was observed between the treated and the untreated biofilms. although the transcription of both ahpcf and bcam was markedly increased after treatment with h2o, the increased transcription of bcal was less pronounced: the microarray data revealed a statistically significant -fold upregulation following treatment with h2o which was confirmed by qpcr. the importance of the ahpc protein in the protection against oxidative stress has been reported previously and the high upregulation of both ahpc and ahpf confirms these data. yet, the limited upregulation of katb was surprising, as katb is supposed to be the major catalase/peroxidase in b. cenocepacia and hypersensitivity of planktonic b. cenocepacia mdl cells to h2o has been reported. as expected, the expression of the minor catalase/peroxidase encoding gene kata was not altered in response to exogenous oxidative stress. in order to further clarify the importance of katb for the survival of b. cenocepacia biofilms exposed to h2o biofilms of two wild type strains and of two mutant strains were treated with % h2o and the fraction of surviving sessile cells was determined using a resazurin based viability staining. in addition, the remaining fraction of h2o was determined in the supernatants of these biofilms after treating them for or min. it should be noted that strains j and c are not identical, although they both belong to the et lineage; it is at present unclear whether this has an impact on our results. these results show that, in contrast to the observations for the wild type strains and for the kata mutant strain, less than 5% and % of all sessile cells present in the b. cenocepacia katb mutant biofilms survived and min treatments with % h2o respectively. in addition, approximately 90% of all initially added h2o was still present in the supernatant of the b. cenocepacia katb mutant biofilms, whereas only trace amounts of h2o were recovered in the supernatants of the biofilms of the wild type strains and of the kata mutant. in keeping with these observations, strong effervescence was observed when h2o was added to the biofilms of both wild type strains and of the kata mutant, whereas no effervescence was observed for the b. cenocepacia katb mutant biofilms. the data obtained in these experiments clearly indicate that katb is not only essential for the survival of planktonic b. cenocepacia cells exposed to h2o but that this catalase/peroxidase is also crucial for the protection of sessile b. cenocepacia cells against exogenous h2o conforming with previous data for stationary phase planktonic cultures, we observed a substantial expression of katb in untreated biofilms, which suggests the involvement of other factors in the regulation of the expression of this particular gene during sessile growth. the transcriptional regulation by various transcription factors might also explain the limited upregulation of katb under conditions of oxidative stress. in addition to the upregulation of katb by these sessile populations, an increased transcription of two other catalase encoding genes was observed in the h2o2-treated biofilms. these two catalase encoding genes possessed differential expression patterns in relation to the length of h2o exposure: while bcam showed the highest upregulation in the biofilms treated for min, the transcription of bcal was much more pronounced in the min treated biofilms. as only trace amounts of h2o were still present after a min treatment, the increased transcription of bcal in biofilms treated for longer periods suggests that it does not participate in the direct neutralization of exogenous h2o and that the expression of this catalase is triggered indirectly. we also observed that the increased transcription of bcam was accompanied by the increased expression of its flanking gene ankb. in p. aeruginosa, these two genes are part of a small operon and the lack of a functional ankb protein results in a decreased katb activity and an increased sensitivity to h2o. the results represent the fold change in gene expression in the treated biofilms compared to the gene expression in the untreated biofilms. ns: no significant change in expression was observed between the treated and the untreated biofilms. in e. coli the oxyr regulon also comprises genes encoding glutaredoxin and a glutathione reductase, yet no significant upregulation of gora or of any of the glutaredoxin encoding genes was observed in the ros exposed sessile b. cenocepacia j cells. we did observe a significant upregulation of the expression of a thioredoxin reductase, of thioredoxin a and of a putative thioredoxin, although no oxyr-boxes are present upstream of these genes. since thioredoxins are capable of reducing oxidized proteins and of scavenging hydroxyl radicals, their upregulated expression in h2o2-treated b. cenocepacia cells was not unexpected. in addition, the strong upregulation of a carboxymuconolactone decarboxylase encoding gene in response to naocl exposure and the significant upregulation of a second carboxymuconolactone decarboxylase encoding gene in response to exposure to both h2o and naocl, suggest that both these proteins are also involved in the neutralization of exogenous ros in b. cenocepacia j biofilms. treatments with h2o or naocl resulted in the increased transcription of three organic hydroperoxide resistance genes: bcas bcam and bcal although the latter gene encodes a protein that is annotated as an osmc-like protein, the protein has the typical characteristics of the ohr subfamily. furthermore, bcal and bcam are both located in close proximity of putative ohrr encoding genes. in a recent study by drevinek et al., the upregulation of a further ohr family gene, bcam was observed in planktonic cells exposed to sub-inhibitory concentrations of h2o and organic hydroperoxides; however, no such upregulation was observed in h2o2-treated sessile cells. overall, such a strong upregulation of the expression of ohr genes in response to h2o or naocl treatments has not been reported thus far. ohr expression in p. aeruginosa pao appears to be triggered specifically by organic hydroperoxides and ohr proteins preferably reduce organic, rather than inorganic hydroperoxides. in addition to the upregulation of bcas and bcal similar expression patterns were observed for their neighbouring genes, suggesting that the genes in both clusters are co-transcribed. overall, our results suggest that ohr proteins are very important for the protection of biofilm-grown b. cenocepacia cells against ros and that it can draw on multiple genes associated with this family of stress proteins. in addition to the upregulation of bcas we also observed a marked increase in the expression of bcas which encodes an exported lipase. this overexpression is probably the result of the cotranscription of this gene with bcas in order to determine whether this overexpression results in an increased extracellular lipase activity, the cleavage of three fluorogenic 4-methylumbelliferyl substrates was determined in the supernatants of treated and untreated biofilms. for all substrates used, the fluorescent signal was higher in the treated biofilms compared to the untreated biofilms, confirming the increased extracellular lipase activity for the treated biofilms. within the genomes of the sequenced b. cepacia complex strains, orthologues of bcas and bcas are organised in a similar operon-like fashion and therefore we examined the induction of lipase expression by h2o in biofilms of six b. cepacia complex reference strains. for b. cenocepacia c hi burkholderia ambifaria lmg and burkholderia dolosa au an increased lipase activity was observed in the supernatant of all treated biofilms. similarly, lipase activity was increased in the supernatant of b. cenocepacia au biofilms treated for and min, and in the supernatant of a min treated burkholderia multivorans lmg biofilm. these data confirmed that lipase activity in the supernatant of b. cepacia complex biofilms following exposure to oxidative stress is increased, which is probably due to the co-transcription of bcas orthologues with bcas orthologues. the transcription of a cytochrome b encoding gene and of its adjacent genes was also highly increased when the biofilms were exposed to exogenous ros. the qpcr data clearly indicate that prolonged exposure to h2o results in an increasing overexpression of bcal. yet, even after a min exposure to naocl, a considerable overexpression of all three genes was observed. drevinek et al. recently reported a high upregulation of these genes in b. cenocepacia j cells grown in cf sputum and they suggested that this may result from the presence of ros in cf sputum. the microarray data also revealed that the transcription of bcal a gene encoding a non-heme chloroperoxidase increased drastically in biofilms exposed to h2o this finding was confirmed by qpcr and an increased expression was observed in response to prolonged treatments. although four genes encoding a non-heme chloroperoxidase are present in the b. cenocepacia j genome, overexpression was only observed for bcal indicating the ability of our microarray-based strategy to highlight the most active gene in burkholderia genomes where multiple paralogous functional pathways exist. upregulation of reca h2o and naocl can both damage nucleic acids. in the fenton reaction, h2o reacts with fe2+ resulting in the formation of highly reactive hydroxyl radicals. similarly, naocl can also generate hydroxyl radicals via a fenton-type reaction. because positively charged fe2+ ions are often localized along the negatively charged phosphodiester backbone of nucleic acids, dna is a vulnerable target for harmful reactions with these radicals. as exposure to ros can result in extensive dna damage, processes for dna protection and dna repair are vital. the microarray analysis revealed a significant increase in the transcription of reca in response to a min treatment with naocl and a min treatment with h2o. in the qpcr experiments, no significant increase in transcription of reca was observed in the biofilms treated with h2o for min. in contrast, in the min treated biofilms, the transcription of this gene was significantly increased. we also observed a slight upregulation of reca following a min treatment but this was not statistically significant. our hypothesis is that, although most of the h2o has disappeared after min, the transcriptional response to dna damage caused by h2o is delayed. upregulation of the reca gene has previously already been described for planktonic p. aeruginosa cultures treated with oxidizing agents. no increased transcription of dps, encoding the non-specific dna-binding protein dps, was neither observed in the h2o2- nor in the naocl-treated biofilms; these data are similar to previous observations in burkholderia pseudomallei. in e. coli, the transcription of dps is regulated by oxyr in exponentially growing planktonic cultures, whereas in stationary phase cultures, dps is transcribed in an rpos-dependent manner. the high expression of dps in the untreated biofilms and the absence of an increased expression in the ros-treated biofilms suggest that rpos rather than oxyr regulates the transcription of dps in mature b. cenocepacia biofilms. upregulation of the iscrsua-hscab-fdx-iscx gene cluster iron-sulfur cluster-containing proteins are highly sensitive to the cellular redox status and they are very vulnerable to oxidants such as h2o. in order to restore their function, iron-sulfur clusters are either repaired or they can be assembled de novo. in e. coli, apo-iscr will increase the transcription of the isc gene cluster, and together with oxyr it will also stimulate the production of oxidation-resistant suf proteins. as no homologues of the e. coli suf gene cluster are found within the b. cepacia complex, the transcriptional response in the h2o2-treated biofilms only involves the increased transcription of the iscsua-hscab-fdx-iscx gene cluster. in addition, an increase in the number of transcripts for iscr was also observed. the latter gene has been identified in a recent screening of mutants of p. aeruginosa pao which also lacks the suf operon, as an essential gene in the resistance to h2o. our data suggest that the increased transcription of the iscrsua-hscab-fdx-iscx gene cluster is important to regain intact iron-sulfur clusters in the ros-treated b. cenocepacia j cells. as a consequence of the damage by oxidants to the iron-sulfur clusters, 2+ and 2+ containing enzymes, including aconitase b, are inactivated. although acnb is the major aconitase during normal growth conditions, it loses all activity in response to strong oxidants. usually, this drop in aconitase activity is compensated by the increased production of aconitase a, which is invulnerable to oxidative inactivation in vivo. however, in the present study no increased expression of acna was observed, while the number of mrna transcripts of acnb increased both in the h2o2- and in the naocl-treated biofilms; the data obtained for the h2o2-treated biofilms were confirmed by qpcr. this observation is puzzling, yet it may be the result of an increased stability of acnb mrna by the accumulating apo-aconitases, as previously observed in e. coli. identification of ncrna the custom made × 44k b. cenocepacia j microarray does not only contain probes for protein encoding genes, but also probes for selected igs. a significant upregulation was observed in and of these igs after treatments with h2o or naocl, respectively, whereas the expression of and igs was significantly downregulated in response to these treatments. a closer inspection of these igs revealed that many are located in close proximity of genes with a similar expression pattern, suggesting that co-transcription of the igs occurs synchronously with their adjacent genes. however, certain igs demonstrated markedly different expression patterns compared to their flanking genes, and the basal expression levels of several of these igs were also high. of the igs, which showed a significant upregulation after exposure to h2o displayed an expression pattern different from their flanking genes. these igs include seven previously shown to be either significantly up- or downregulated in b. cenocepacia j cells grown in sputum. two igs, ig1_ and ig1_ identified by yoder-himes et al. to contain putative ncrna, were also overexpressed. finally, the transcription of two igs, ig1_ and ig1_ which have been identified previously to contain putative ncrna in a computational search, was also increased in response to exogenous oxidative stress. of the igs significantly upregulated in response to a naocl-treatment, displayed a different expression pattern than their flanking genes. again, a significant upregulation of the expression of ig1_ and ig1_ as well as ig1_ was observed. in order to confirm these microarray data, we performed qpcr experiments for ig1_ and ig1_ as well as for their flanking genes. the expression of ig1_ was clearly higher when the biofilms were treated with h2o whereas no such upregulation was observed either for bcal or for bcal. in addition, the transcription of bcal and bcal in the untreated biofilms was clearly lower compared to the transcription of ig1_. the secondary structure for the mrna transcript has a marked similarity to the 6s rna consensus structure. the qpcr results also revealed a significant upregulation of ig1_ in the h2o2-treated biofilms. however, the transcription of the neighbouring genes bcal and bcal was not altered to the same extent and the ct values obtained for ig1_ in the untreated biofilms were markedly lower than the corresponding ct values of bcal and bcal although the presence of putative ncrna in ig1_ has been reported previously, no ncrna was identified in this ig using the rfam database. three igs in which putative ncrnas were identified in a computational search were downregulated in the h2o2- as well as in the naocl-treated biofilms. sigma factors as the present study focussed on the changes in gene expression in sessile cells following treatment with high concentrations of ros, major stress responses were observed and the expression of multiple σ factors was increased. these σ factors included an rna polymerase σ factor, an ecf σ factor orbs, a putative σ factor and an ecf σ factor feci. the ecf σ factor encoded by bcal has been annotated as orbs or ecfi, a regulatory σ factor located adjacent to the ornibactin gene cluster in b. cenocepacia. the transcription of orbs is regulated by the fur repressor, which represses the orbs promoter when cells are growing under iron-replete conditions. in correspondence to the high increase of the transcription of orbs, two of the genes belonging to the ornibactin gene cluster, orbh and orbg, were also found to be upregulated. the ig sequence between orbs and orbh also showed elevated expression. however, none of the other genes present in the ornibactin gene cluster were upregulated. other studies have shown that orbh and orbg are cotranscribed with orbs, correlating to the increases we observed in these two genes and the intervening ig region. the lack of transcription of the other orb genes suggests that the levels of orbs under our experimental conditions were insufficient to induce the transcription of orbe and orbi-orbl. alternatively, the transcription of orbe and orbi-orbl may depend on a post-transcriptional regulation of orbs activity. this posttranscriptional regulation might result from the interaction of orbs with 6s rna. an increased transcription of a second σ factor involved in iron-uptake, feci, was also observed. similar observations have been described for h2o2-treated p. aeruginosa cells. the increased transcription of orbs as well as of feci suggests that the cells apparently suffer from some degree of iron starvation or that the fur repressor function is transiently lost. the exact roles of the σ factors, encoded by bcal and bcal in relation to these conditions of oxidative stress remain to be elucidated. remarkably, the expression pattern of bcal is highly similar to that of its divergently transcribed neighbouring gene bcal. neither rpon, nor rpoe showed an increased transcription following exposure to exogenous ros, corroborating previous data that neither rpon nor rpoe are involved in the resistance towards h2o. genes involved in motility and chemotaxis b. cepacia complex bacteria are motile rods, equipped with flagella. these flagella are multi-component structures, which are synthesized and assembled in a complex process involving over hierarchically expressed genes. flagella are not only important in the formation of biofilms, they also play a key role in the adherence to and the invasion into host cells. in addition, intact flagella evoke host immune responses. the data obtained in the microarray analysis reveal that many b. cenocepacia j flagellum-related genes and genes involved in chemotaxis show an increased expression in biofilms exposed to exogenous ros. the overexpressed genes in the h2o2-treated biofilms include those that belong to two major gene clusters: bcal0520-bcal and bcal0526-bcal and bcal0564-bcal bcal and bcal0576-bcal the expression of the majority of these genes was also significantly increased in response to a short exposure to naocl. in addition, the increased transcription of the flagellar motor protein encoding gene mota was also observed. furthermore, the transcription of five and nine genes coding for methyl-accepting chemotaxis proteins was increased in the h2o2- and naocl-treated biofilms, respectively. although our data clearly indicate that sessile b. cenocepacia j cells exposed to exogenous oxidative stress overexpress many flagellar and chemotaxis genes, previous experiments demonstrated that treatments with % h2o did not result in significant decreases in total biofilm biomass. the latter observation suggests that the overexpression of these genes does not result in a global dispersal event. further research is required to elucidate the role of the increased transcription of these genes in response to exogenous ros. phage-related genes the microarray analysis revealed a significant upregulation of multiple phage-related genes in sessile cells after exposure to h2o. bcepmu is a mu-like prophage that is present in many b. cenocepacia et strains and it has been annotated bcengi. the prophage sequence was found to encode proteins and it has been divided in three functional clusters, including a module involved in replication, regulation and pathogenesis. the transcription of these genes probably results in a monocistronic mrna as a similar upregulation of multiple igs within this cluster was observed. this prophage module contains many genes coding for small hypothetical proteins. in addition, it contains two genes that show high similarities to pseudomonas phage b transposase b and a subunits. the results of the qpcr analysis for three selected genes indicated that none of these genes was upregulated after a min treatment with h2o2; yet, a significant upregulation was observed when b. cenocepacia j biofilms were treated for or min. thus far, the transcriptional mechanism resulting in the upregulation of these bcepmu prophage genes has not been elucidated. the results represent the fold change in expression in the treated biofilms compared to the expression in the untreated biofilms. downregulated genes in the h2o2- and naocl-treated biofilms although genes showed a significant downregulation of the transcription in biofilms exposed to h2o a more than five fold change in expression was only observed for genes. similarly, for of the downregulated genes in the naocl-treated biofilms, transcription was decreased more than five fold. a total of genes was downregulated in response to both treatments. despite the large numbers of genes showing a decreased expression in response to these ros, no obvious patterns were observed. qpcr experiments were performed for two selected genes: bcam and bcam and they confirmed that both genes were significantly downregulated in response to exposure to h2o. fold change correlation between qpcr experiments and microarray analysis in order to determine the correlation between the fold changes in gene expression observed in the microarray analysis and the qpcr experiments, a regression analysis was performed. the results of the regression analysis between the log fold changes obtained in the qpcr experiments and the microarray analysis showed a good linear correlation between both datasets, with a correlation coefficient of and a slope of. this suggests that the custom made × 44k microarray for b. cenocepacia is highly reliable to evaluate changes in global gene expression in b. cenocepacia j  our data show that the successful defence of sessile b. cenocepacia j cells against high doses of ros involves a plethora of protective mechanisms. these include mechanisms that allow cells to neutralize and scavenge ros and mechanisms that are involved in the repair of the cellular damage resulting from the exposure to ros. several results in the present study corroborate data from previous studies that focussed on the transcriptional response to sublethal doses of exogenous ros. rather unexpected observations included the high upregulation of three ohr encoding genes in h2o2-treated sessile b. cenocepacia j cells and the increased extracellular lipase activity in several h2o2-treated b. cepacia complex biofilms. in addition, our data revealed an increased transcription of multiple phage-related genes, igs and chemotaxis- and motility-related genes in the treated biofilms. these observations contribute to a better insight in the molecular mechanisms involved in the resistance of sessile b. cenocepacia j cells against exogenous ros. 
our results reveal that the transcription of a large fraction of b. cenocepacia j genes is altered upon exposure of sessile cells to ros. in addition, multiple upregulated genes encode proteins that are necessary to repair ros-induced cellular damage. a closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6s rna. burkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis. however, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of b. cepacia complex bacteria have been reported. in order to unravel the molecular mechanisms involved in the resistance of biofilm-grown burkholderia cenocepacia cells against high concentrations of reactive oxygen species, the present study focussed on the transcriptional response in sessile b. cenocepacia j cells following exposure to high levels of h2o or naocl. as treatment of infected cf patients is problematic, multiple preventive measures are taken to reduce the infection risk. these observations have highlighted that b. cenocepacia may alter several pathways in response to exposure to ros and they have led to the identification of many genes not previously implicated in the stress response of this pathogen. many of the upregulated genes in the naocl- and h2o2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ros.
 burkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis. as treatment of infected cf patients is problematic, multiple preventive measures are taken to reduce the infection risk. besides a stringent segregation policy to prevent patient-to-patient transmission, clinicians also advise patients to clean and disinfect their respiratory equipment on a regular basis. however, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of b. cepacia complex bacteria have been reported. in order to unravel the molecular mechanisms involved in the resistance of biofilm-grown burkholderia cenocepacia cells against high concentrations of reactive oxygen species, the present study focussed on the transcriptional response in sessile b. cenocepacia j cells following exposure to high levels of h2o or naocl.  the exposure to h2o and naocl resulted in an upregulation of the transcription of and genes, respectively. transcription of and genes was decreased in response to the respective treatments. many of the upregulated genes in the naocl- and h2o2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ros. in addition, multiple upregulated genes encode proteins that are necessary to repair ros-induced cellular damage. unexpectedly, a prolonged treatment with h2o also resulted in an increased transcription of multiple phage-related genes. a closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6s rna.  our results reveal that the transcription of a large fraction of b. cenocepacia j genes is altered upon exposure of sessile cells to ros. these observations have highlighted that b. cenocepacia may alter several pathways in response to exposure to ros and they have led to the identification of many genes not previously implicated in the stress response of this pathogen. 