BACKGROUND
burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis  <cit> .
this serious and often fatal disease of humans and animals such as horses, sheep, goats, pigs and cows is endemic in southeast asia and northern australia  <cit> .
b. pseudomallei is intrinsically resistant to several antibiotics and treatment typically involves an initial parenteral phase of therapy, followed by a prolonged course of oral antibiotics  <cit> .
no melioidosis vaccine is currently available.
in endemic areas, b. pseudomallei can be found in soil and in stagnant waters  <cit> .
in the natural environment, this saprophytic bacterium is able to survive a wide range of conditions, including fluctuating temperatures, ph levels, oxygen levels, osmotic pressures and nutritional stresses.
during infection, the bacterium is able to survive and replicate in phagocytic or non-phagocytic cells.
within these cells b. pseudomallei may be exposed to free radicals, reactive oxygen intermediates and high osmolality.
to survive exposure to stressful environments, b. pseudomallei must be able to activate the appropriate genes and regulate their expression.
many of these genes are organized into regulons which are under the control of sigma factors.
rpoe  is a member of the extra-cytoplasmic function  subfamily of sigma factors  <cit>  and has been characterized to be one of the most important gene regulatory systems in response to extracellular stress in gram-negative bacteria.
in escherichia coli k <dig>  the inhibition of σe resulted in increased sensitivity to bacterial cell wall disruption  <cit>  and in vibrio vulnificus, deletion of σe resulted in increased sensitivity to membrane-perturbing agents such as ethanol, peroxide and sds  <cit> .
inactivation of σe in salmonella enterica serovar typhimurium  resulted in attenuation in a mouse model of infection  <cit> .
in addition, microarray analysis of a s. typhimurium σe mutant identified the σe regulon and virulence factors that contributed to disease  <cit> .
a b. pseudomallei rpoe insertional inactivation mutant has previously been constructed and showed increased susceptibility to hydrogen peroxide , suggesting a role for σe in the oxidative stress response  <cit> .
furthermore, inactivation of b. pseudomallei σe resulted in reduced survival in j774a.
<dig> macrophages and the mutant was attenuated in a murine model of infection  <cit> .
a proteomic comparison of b. pseudomallei wild type and the σe insertional mutant revealed the differential levels of proteins that may contribute to the stress tolerance and survival of b. pseudomallei
<cit>  but this study was unable to identify all the proteins involved in this response, because of the limitations of the proteomic platform.
recently, the development of a tiling microarray for b. pseudomallei enabled comprehensive transcriptional profiling, providing global snapshots of regulons in response to various stimuli.
analyses of transcriptional profiles of σe would lead to a better understanding of the mechanisms that bacteria use to circumvent environmental stresses.
such microarray studies will also complement our previous proteomic data and is likely to provide new insights to gene members and regulation of these genes under stress.
in this study, global transcriptional profiles of b. pseudomallei in response to h2o2-induced oxidative stress were analyzed.
we compared the transcriptional profiles of b. pseudomallei wild type and its isogenic σe mutant under oxidative stress.
in addition, the transcriptional profiles also revealed a novel gene member of the σe regulon, speg, that is involved in maintaining the physiological balance of the polyamine spermidine in bacterial cells during oxidative stress.
this is the first report to demonstrate the direct and indirect roles of σe contributing to b. pseudomallei survival in the environment.
RESULTS
comparative transcriptional profiles of b. pseudomalleiwild type with and without oxidative stress
the transcriptional profiles of b. pseudomallei in the presence or absence of  <dig> μm h2o <dig> for  <dig> min were first determined.
analyses of the profiles revealed a total of  <dig> genes  that were differentially regulated  representing approximately  <dig> % of all b. pseudomallei k <dig> genes.
these differentially regulated genes were found on chromosome  <dig>  and chromosome  <dig> .
among the  <dig> genes,  <dig> genes were up regulated  and  <dig> genes  were down regulated under oxidative stress.
since the objective of this study was to identify b. pseudomallei gene expression in response to oxidative stress, we focused on the analysis of the up-regulated genes.
among  <dig> up-regulated genes,  <dig>  could be classified into  <dig> major functional groups according to the cluster of orthologous groups of proteins  database.
these included genes involved in cell wall/membrane biosynthesis; energy and metabolism; regulatory, signal transduction and post-translational modification; intracellular trafficking/secretion system.
the remaining  <dig> genes  had unknown functions .figure  <dig>
differentially expressed genes.
a) up-regulated genes under oxidative stress in wild type b. pseudomallei k <dig>  b) down-regulated genes under control condition  in the σe mutant.
c) comparison of microarray profiles from σe mutant under control and oxidative stress conditions shows  <dig> genes as σe-dependent oxidative-stress-responsive genes.
d) functional classification of the  <dig> σe-dependent oxidative-stress-responsive genes.
of the  <dig> up-regulated genes,  <dig> genes  were involved in cell wall/cell membrane biosynthesis  including bpsl <dig> , bpsl <dig> , bpsl <dig> , bpss <dig>  and bpss <dig> .
we also found that genes involved in the transport of lipopolysaccharide  and capsular polysaccharide  across the cell membrane to the bacterial cell surface were up-regulated.
we found increased expression of mreb  gene after oxidative stress.
mreb is a bacterial ortholog of actin and mreb is reported to be important in maintaining the shape of bacteria  <cit> .
mreb is thought to organize the incorporation of cell wall precursors into the side-wall  <cit> .
the up-regulation of genes involved in cell wall/cell membrane synthesis may reflect their roles in the repair of the cell wall after oxidative stress damage.
in addition to genes involved in cell wall and membrane biosynthesis,  <dig> genes involved in energy production and metabolism were up-regulated .
these included three genes belonging to the sugar transporter superfamily , and two genes of the entner-doudoroff pathway .
in addition, many genes related to amino acid utilization  and amino acid biosynthesis  were up-regulated when b. pseudomallei was exposed to oxidative stress.
we found an increased expression of b. pseudomallei bpsl <dig>  which plays a role in inorganic iron transport.
a previous study  <cit>  reported that the bioavailability of iron decreases under oxidative stress conditions, and the up-regulation of bpsl <dig> is consistent with this observation.
the increased expression of b. pseudomallei fis-regulatory gene  suggests that bpsl <dig> interacts with σ <dig> , with the consequential transcription of genes involved in the metabolism and transportation of nitrogen and carbon and genes involved in alginate and flagella synthesis  <cit> .
we observed an increase in the expression of peroxide scavenging enzymes including katg , katb  and ahpc  during exposure to h2o2-induced oxidative stress .
the expression of b. pseudomallei katg and ahpc is regulated through a global h2o <dig> sensor and the oxyr transcriptional regulator .
the increased expression of katg and ahpc after exposure to oxidative stress is consistent with the findings from previous studies  <cit> .
the role of katg may be to enable b. pseudomallei survival within phagocytes through the detoxification of antibacterial reactive oxygen species.table  <dig>
selected differentially regulated genes of
b. pseudomallei
k <dig> and σ
e
mutant under h
2
o
2
–induced oxidative stress
*
oxidative stress responsive gene
bpss0993
bpss0492
bpsl2865
bpss0238
bpss0281
bpss0711
bpsl3142
bpsl2285
bpsl0497
bpsl2286
bpsl1787
bpsl0785
bpsl0963
bpsl2806
bpss0585
bpsl1527
bpsl0186
bpsl3312
σ
e
-dependent and osr gene
bpss1837
bpss1839
bpss1838
bpsl2931
bpsl2932
bpsl2605
bpsl1799
bpsl1800
bpss1437
bpss1251
bpss1434
bpsl3419
bpss1252
bpsl2300
bpsl2301
bpss0175-0184
bpsl2933
bpsl1042
bpsl1806
bpsl1983
bpss0796a
bpsl0609
bpsl0320
bpss0016
bpss0124
bpsl0785
bpss1133
bpss2053
bpsl1043
bpsl3216
bpsl1577
bpsl1893
σ
e
-dependent but not osr gene
bpsl0096
bpsl0224
bpsl0327
bpsl2289
bpss1944
bpss1945
bpss1946
ns; not significant different.
*b. pseudomallei cultured in lb broth without h2o <dig>
the differential transcription profile of b. pseudomallei under oxidative stress revealed that  <dig> genes  were predicted to encode regulatory, signal transduction or post-translational modification-related proteins .
these genes included bpsl <dig>  encoding a gntr family regulatory protein and bpsl <dig>  encoding an ecf sigma factor.
several genes involved in transcription regulation such as tex , nrdr  and an arac family gene  were up-regulated.
in streptococcus pneumoniae and pseudomonas aeruginosa the transcription factor tex is important for bacterial fitness  <cit> .
the nrdr transcription regulator is reported to control the expression of a ribonucleotide reductase involved in deoxyribonucleotide biosynthesis, which is required for dna replication and repair  <cit> .
many members of the arac family transcription regulator have been proven to play critical roles in regulating bacterial virulence factors in response to environmental stress  <cit> .
the high number of regulatory genes up-regulated after exposure to oxidative stress may indicate that b. pseudomallei employs multiple regulation systems in response to oxidative stress.
in addition to genes involved in transcription, a number of chaperone-encoding genes were up-regulated after expsore of the bacteria to oxidative stress including hsca/hscb , and groes <dig> .
hsca is a specialized member of the hsp <dig> family of molecular chaperones that plays a role in the biosynthesis of several iron-sulfur proteins  <cit> .
previous studies indicated the essential roles of iron-sulfur proteins in the adaptation of bacteria to iron starvation  <cit> .
chaperonin groes <dig> binds to heat shock protein groel to facilitate protein folding in response to environmental stresses  <cit> .
oxidative stress can cause to protein misfolding, and as a result, the bacterial cells are unable to maintain their protein functions.
the up-regulation of genes involved in protein folding may reflect the fact that under oxidative stress conditions, b. pseudomallei proteins are likely to become damaged.
the smallest functional group of proteins that were up-regulated under oxidative stress included  <dig> genes  encoding proteins related to intracellular trafficking and secretion .
increased expression of proteins in this group, such as type ii/iv and vi secretion systems implies that the virulence of b. pseudomallei is likely to be affected by oxidative stress.
comparative analysis of transcription profiles of b. pseudomalleiwild type and σemutant without oxidative stress
we have previously reported the construction of b. pseudomallei σe mutant.
the mutant shows increased susceptibility to killing by h2o <dig>  indicating the role of σe in regulating resistance to oxidative stress  <cit> .
to identify the σe regulon under oxidative stress conditions, we first investigated the transcriptional profiles of b. pseudomallei wild type and the σe mutant grown in lb medium without antibiotic supplementation.
analysis of the transcription profiles revealed that a total of  <dig> genes  were differentially regulated , representing approximately  <dig> % of the total b. pseudomallei k <dig> genes.
these differentially regulated genes were distributed on both chromosome  <dig>  and chromosome  <dig> .
in total,  <dig> genes were up-regulated  and  <dig> genes  were down-regulated in the σe mutant.
the down-regulation of genes may indicate either direct or indirect regulation by σe.
among the down-regulated genes,  <dig>  could be classified into  <dig> major cog functional groups, including  <dig> genes  predicted to be involved in cell wall/cell membrane biosynthesis,  <dig> genes  involved in energy production/metabolisms,  <dig> genes  involved in regulatory/signal transduction/post-translational modification and repair, and  <dig> genes  involved in intracellular trafficking/secretion .
the remaining  <dig> genes  have unknown functions.
comparative analysis of transcription profiles of b. pseudomalleiwild type and σemutant under oxidative stress
to identify σe-dependent genes that are differentially expressed under oxidative stress conditions, we compared the transcriptome profiles of the σe mutant and wild type which had been exposed to oxidative stress.
the bacteria were treated with h2o <dig> for  <dig> min before rna extraction and microarray analysis.
a total of  <dig> genes  were differentially regulated  representing approximately  <dig> % of the total b. pseudomallei k <dig> genes.
of these,  <dig> genes were up-regulated in the σe mutant  and were located on either chromosome  <dig>  or chromosome  <dig> .
among the  <dig> down-regulated genes in the σe mutant,  <dig> genes were also down-regulated in the mutant under normal growth conditions.
by excluding these genes, we identified  <dig> genes defined as the σe-dependent oxidative stress regulon .
these  <dig> genes were distributed on both chromosome  <dig>  and chromosome  <dig> .
two genes  were predicted to be involved in cell wall/cell membrane biosynthesis,  <dig> genes  in energy production/metabolisms,  <dig> genes  in regulatory/signal transduction/post-translational modification and repair,  <dig> genes  in intracellular trafficking/secretion.
the remaining  <dig> genes  had unknown functions .
amongst the  <dig> genes making up the σe-dependent oxidative stress regulon, bpsl <dig> is predicted to be involved in cell wall/cell membrane biosynthesis and bpss <dig> is predicted to encode a membrane protein related to metalloendopeptidases and porins.
genes involved in energy production and metabolism included bpsl0320- <dig> , bpsl <dig>  and bpsl2300-l <dig> .
the absence of a functional σe under oxidative stress affected the expression of bpss1838- <dig> , genes that play important roles in increasing tolerance and resistance to oxidative stress  <cit> .
we identified  <dig>  σe-regulated genes involved in regulatory, signal transduction and post-translational modification after oxidative stress including bpsl <dig> , bpsl <dig> , bpsl <dig> , bpss <dig>  and bpsl <dig> .
the latter encodes thioredoxin reductase which functions in post-translational modification.
in addition, site-specific recombinase , which is involved in dna replication, recombination and repair, was also under σe regulation.
intracellular trafficking and secretion genes accounted for  <dig> % of σe-dependent oxidative stress responsive genes.
these included genes of the type ii secretion system , fimbrial proteins  and membrane-anchored cell surface protein .
b. pseudomallei contains six clusters type vi secretion system   <cit> .
the expression of t6ss genes has been reported to be induced in vivo
.
a previous study reported that the t6ss- <dig> cluster is important for host adaptation of b. pseudomallei within phagocytes, and that the expression of genes in this cluster is significantly elevated after infection of murine macrophages  <cit> .
we found the increased expression of ten genes  belonging to t6ss- <dig> under oxidative stress conditions suggesting that t6ss- <dig> may play a role in combating oxidative stress.
rt-pcr analysis of genes under normal and oxidative stress conditions
to validate the results from our microarray analysis, rt-pcr was performed.
figure  <dig> shows the increased expression of the bpsl <dig> and bpss <dig> genes in b. pseudomallei wild type after h2o <dig> treatment.
we did not observe a significant difference in expression of bpsl <dig> .
these results are consistent with the microarray data which indicated that bpsl <dig> and bpss <dig>  but not bpsl <dig>  were up-regulated in response to oxidative stress .
in the b. pseudomallei σe mutant exposed to h2o <dig> treatment, the expression of the bpsl <dig>  bpss <dig> and bpsl <dig> genes was down-regulated compared to the wild type, indicating that these genes are under σe control.
these results are also consistent with our microarray results .figure  <dig>
rt-pcr analysis of genes under normal and oxidative stress conditions.
b. pseudomallei wild type or the isogenic σe mutant was incubated for  <dig> min in the presence or absence of  <dig> μm h2o <dig> and rt-pcr analysis carried out.
each row represents an individual gene  and normalized against 23s rrna expression.
in addition to rt-pcr, the differential transcription profiles were analyzed to ensure the quality of our transcription profile data.
for example, we found that  <dig> genes  encoding b. pseudomallei t6ss- <dig> were all up-regulated, suggesting that these genes may be co-expressed as an operon and support the validity of our transcriptional profiling results .
other studies have shown that the katg  and ahpc  genes are up-regulated following the exposure of b. pseudomallei to oxidative stress  <cit> .
our transcriptional data  also reveals these patterns of gene expression.
collectively, these results indicate that our data is robust and reliable.
b. pseudomalleiσeindirectly regulates spermidine levels during oxidative stress
previous studies have suggested that sigma factors regulate speg and consequently spermidine levels  <cit> .
spermidine is one of the predominant polyamines in gram-negative bacteria, widely distributed in the environment, and is involved in various biological processes including gene regulation, protein translation and stress resistance  <cit> .
during oxidative stress, spermidine functions as a free radical scavenger and plays an important adjunctive role in protecting bacterial cells from the toxic effects of reactive oxygen species  <cit> .
the intracellular level of spermidine in bacteria is reported to range from 1- <dig> mm  <cit> .
high concentrations of spermidine are toxic for bacteria.
excess spermidine can be a result of de-regulated bacterial biosynthesis/metabolism or from environmental exposure, inhibiting bacterial growth and even killing the bacterial cells  <cit> .
therefore, in bacteria, the maintenance of an appropriate intracellular level of spermidine is critical.
excess spermidine can be converted into the physiologically inert acetylspermidine by the spermidine-acetyltransferase .
a recent study revealed that the speg gene has been silenced by convergent evolution in shigella and this resulted in elevated levesl of intracellular spermidine.
as a result, the survival of shigella under oxidative stress is enhanced, contributing to its successful pathogenic lifestyle  <cit> .
we observed expression of speg  in b. pseudomallei wild type under both control and oxidative stress conditions .
however, the speg gene was down-regulated in the σe mutant , indicating that the inactivation of rpoe effected the expression of speg.
this suggests that speg is regulated by σe.
the decreased gene expression we have observed corroborates our previous proteomic study  <cit> .
to our knowledge, this is the first report on the regulation of speg by σe.
we hypothesised that under high concentrations of spermidine, σe positively regulate expression of speg gene in order to prevent spermidine accumulation, the failure of which, will result in inhibition of b. pseudomallei growth and even cytotoxicity.
to test this hypothesis, cultures of b. pseudomallei wild type or σe mutant grown in lb broth were exposed to  <dig> mm spermidine and the number of viable bacteria determined.
the number of wild type bacteria was not affected by the addition of spermidine.
this is likely due to the presence of a functional σe gene in the wild type, activating the expression of speg gene.
in contrast, in the b. pseudomallei σe mutant, the number of viable bacteria was significantly reduced by the addition of spermidine, indicating the accumulation of spermidine to toxic levels .figure  <dig>
effect of spermidine on the viability of
b. pseudomallei
wild type or σ
e
mutant.
the numbers of b. pseudomallei wild type or the σe mutant cells were grown in the presence or absence of  <dig> mm spermidine.
after  <dig> h, the numbers of viable bacteria  were determined after plating onto lb agar.
the viability of wild type and σe mutant in the presence of spermidine was calculated from cfu count divided by the cfu count of control condition and multiplied by  <dig>  values shown are the mean of three independent experiments.
asterisks indicate significant differences .
this result corroborates the findings from a previous study in e. coli, where excess spermidine was shown to be toxic to bacteria and speg was shown to be important for bacterial viability  <cit> .
our study provides further evidence that the regulation of speg is affected by σe; speg is a novel member of the σe regulon and σe plays an indirect but important role in the regulation of polyamine levels in bacterial cells to protect the cells during oxidative stress.
CONCLUSIONS
dna tiling arrays were employed to identify global transcriptional profile changes in b. pseudomallei k <dig> exposed to oxidative stress induced by h2o <dig>  we have identified not only genes involved in repairing cell wall/membrane biosynthesis but also genes involved in energy and metabolism, regulatory and signal transduction, post translational modification, and intracellular trafficking/secretion genes, which are directly regulated by σe during oxidative stress.
we found the increased expression of the b. pseudomallei t6ss- <dig> under oxidative stress.
more importantly, we provided evidence that σe also indirectly regulates the polyamine levels in b. pseudomallei, to protect the cells from oxidative stress.

 burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis. this serious and often fatal disease of humans and animals such as horses, sheep, goats, pigs and cows is endemic in southeast asia and northern australia. b. pseudomallei is intrinsically resistant to several antibiotics and treatment typically involves an initial parenteral phase of therapy, followed by a prolonged course of oral antibiotics. no melioidosis vaccine is currently available. in endemic areas, b. pseudomallei can be found in soil and in stagnant waters. in the natural environment, this saprophytic bacterium is able to survive a wide range of conditions, including fluctuating temperatures, ph levels, oxygen levels, osmotic pressures and nutritional stresses. during infection, the bacterium is able to survive and replicate in phagocytic or non-phagocytic cells. within these cells b. pseudomallei may be exposed to free radicals, reactive oxygen intermediates and high osmolality. to survive exposure to stressful environments, b. pseudomallei must be able to activate the appropriate genes and regulate their expression. many of these genes are organized into regulons which are under the control of sigma factors. rpoe is a member of the extra-cytoplasmic function subfamily of sigma factors and has been characterized to be one of the most important gene regulatory systems in response to extracellular stress in gram-negative bacteria. in escherichia coli k the inhibition of σe resulted in increased sensitivity to bacterial cell wall disruption and in vibrio vulnificus, deletion of σe resulted in increased sensitivity to membrane-perturbing agents such as ethanol, peroxide and sds. inactivation of σe in salmonella enterica serovar typhimurium resulted in attenuation in a mouse model of infection. in addition, microarray analysis of a s. typhimurium σe mutant identified the σe regulon and virulence factors that contributed to disease. a b. pseudomallei rpoe insertional inactivation mutant has previously been constructed and showed increased susceptibility to hydrogen peroxide, suggesting a role for σe in the oxidative stress response. furthermore, inactivation of b. pseudomallei σe resulted in reduced survival in j774a. macrophages and the mutant was attenuated in a murine model of infection. a proteomic comparison of b. pseudomallei wild type and the σe insertional mutant revealed the differential levels of proteins that may contribute to the stress tolerance and survival of b. pseudomallei but this study was unable to identify all the proteins involved in this response, because of the limitations of the proteomic platform. recently, the development of a tiling microarray for b. pseudomallei enabled comprehensive transcriptional profiling, providing global snapshots of regulons in response to various stimuli. analyses of transcriptional profiles of σe would lead to a better understanding of the mechanisms that bacteria use to circumvent environmental stresses. such microarray studies will also complement our previous proteomic data and is likely to provide new insights to gene members and regulation of these genes under stress. in this study, global transcriptional profiles of b. pseudomallei in response to h2o2-induced oxidative stress were analyzed. we compared the transcriptional profiles of b. pseudomallei wild type and its isogenic σe mutant under oxidative stress. in addition, the transcriptional profiles also revealed a novel gene member of the σe regulon, speg, that is involved in maintaining the physiological balance of the polyamine spermidine in bacterial cells during oxidative stress. this is the first report to demonstrate the direct and indirect roles of σe contributing to b. pseudomallei survival in the environment.  comparative transcriptional profiles of b. pseudomalleiwild type with and without oxidative stress the transcriptional profiles of b. pseudomallei in the presence or absence of μm h2o for min were first determined. analyses of the profiles revealed a total of genes that were differentially regulated representing approximately % of all b. pseudomallei k genes. these differentially regulated genes were found on chromosome and chromosome. among the genes, genes were up regulated and genes were down regulated under oxidative stress. since the objective of this study was to identify b. pseudomallei gene expression in response to oxidative stress, we focused on the analysis of the up-regulated genes. among up-regulated genes, could be classified into major functional groups according to the cluster of orthologous groups of proteins database. these included genes involved in cell wall/membrane biosynthesis; energy and metabolism; regulatory, signal transduction and post-translational modification; intracellular trafficking/secretion system. the remaining genes had unknown functions.figure differentially expressed genes. a) up-regulated genes under oxidative stress in wild type b. pseudomallei k b) down-regulated genes under control condition in the σe mutant. c) comparison of microarray profiles from σe mutant under control and oxidative stress conditions shows genes as σe-dependent oxidative-stress-responsive genes. d) functional classification of the σe-dependent oxidative-stress-responsive genes. of the up-regulated genes, genes were involved in cell wall/cell membrane biosynthesis including bpsl, bpsl, bpsl, bpss and bpss. we also found that genes involved in the transport of lipopolysaccharide and capsular polysaccharide across the cell membrane to the bacterial cell surface were up-regulated. we found increased expression of mreb gene after oxidative stress. mreb is a bacterial ortholog of actin and mreb is reported to be important in maintaining the shape of bacteria. mreb is thought to organize the incorporation of cell wall precursors into the side-wall. the up-regulation of genes involved in cell wall/cell membrane synthesis may reflect their roles in the repair of the cell wall after oxidative stress damage. in addition to genes involved in cell wall and membrane biosynthesis, genes involved in energy production and metabolism were up-regulated. these included three genes belonging to the sugar transporter superfamily, and two genes of the entner-doudoroff pathway. in addition, many genes related to amino acid utilization and amino acid biosynthesis were up-regulated when b. pseudomallei was exposed to oxidative stress. we found an increased expression of b. pseudomallei bpsl which plays a role in inorganic iron transport. a previous study reported that the bioavailability of iron decreases under oxidative stress conditions, and the up-regulation of bpsl is consistent with this observation. the increased expression of b. pseudomallei fis-regulatory gene suggests that bpsl interacts with σ, with the consequential transcription of genes involved in the metabolism and transportation of nitrogen and carbon and genes involved in alginate and flagella synthesis. we observed an increase in the expression of peroxide scavenging enzymes including katg, katb and ahpc during exposure to h2o2-induced oxidative stress. the expression of b. pseudomallei katg and ahpc is regulated through a global h2o sensor and the oxyr transcriptional regulator. the increased expression of katg and ahpc after exposure to oxidative stress is consistent with the findings from previous studies. the role of katg may be to enable b. pseudomallei survival within phagocytes through the detoxification of antibacterial reactive oxygen species.table selected differentially regulated genes of b. pseudomallei k and σ e mutant under h 2 o 2 –induced oxidative stress * oxidative stress responsive gene bpss0993 bpss0492 bpsl2865 bpss0238 bpss0281 bpss0711 bpsl3142 bpsl2285 bpsl0497 bpsl2286 bpsl1787 bpsl0785 bpsl0963 bpsl2806 bpss0585 bpsl1527 bpsl0186 bpsl3312 σ e -dependent and osr gene bpss1837 bpss1839 bpss1838 bpsl2931 bpsl2932 bpsl2605 bpsl1799 bpsl1800 bpss1437 bpss1251 bpss1434 bpsl3419 bpss1252 bpsl2300 bpsl2301 bpss0175-0184 bpsl2933 bpsl1042 bpsl1806 bpsl1983 bpss0796a bpsl0609 bpsl0320 bpss0016 bpss0124 bpsl0785 bpss1133 bpss2053 bpsl1043 bpsl3216 bpsl1577 bpsl1893 σ e -dependent but not osr gene bpsl0096 bpsl0224 bpsl0327 bpsl2289 bpss1944 bpss1945 bpss1946 ns; not significant different. *b. pseudomallei cultured in lb broth without h2o the differential transcription profile of b. pseudomallei under oxidative stress revealed that genes were predicted to encode regulatory, signal transduction or post-translational modification-related proteins. these genes included bpsl encoding a gntr family regulatory protein and bpsl encoding an ecf sigma factor. several genes involved in transcription regulation such as tex, nrdr and an arac family gene were up-regulated. in streptococcus pneumoniae and pseudomonas aeruginosa the transcription factor tex is important for bacterial fitness. the nrdr transcription regulator is reported to control the expression of a ribonucleotide reductase involved in deoxyribonucleotide biosynthesis, which is required for dna replication and repair. many members of the arac family transcription regulator have been proven to play critical roles in regulating bacterial virulence factors in response to environmental stress. the high number of regulatory genes up-regulated after exposure to oxidative stress may indicate that b. pseudomallei employs multiple regulation systems in response to oxidative stress. in addition to genes involved in transcription, a number of chaperone-encoding genes were up-regulated after expsore of the bacteria to oxidative stress including hsca/hscb, and groes. hsca is a specialized member of the hsp family of molecular chaperones that plays a role in the biosynthesis of several iron-sulfur proteins. previous studies indicated the essential roles of iron-sulfur proteins in the adaptation of bacteria to iron starvation. chaperonin groes binds to heat shock protein groel to facilitate protein folding in response to environmental stresses. oxidative stress can cause to protein misfolding, and as a result, the bacterial cells are unable to maintain their protein functions. the up-regulation of genes involved in protein folding may reflect the fact that under oxidative stress conditions, b. pseudomallei proteins are likely to become damaged. the smallest functional group of proteins that were up-regulated under oxidative stress included genes encoding proteins related to intracellular trafficking and secretion. increased expression of proteins in this group, such as type ii/iv and vi secretion systems implies that the virulence of b. pseudomallei is likely to be affected by oxidative stress. comparative analysis of transcription profiles of b. pseudomalleiwild type and σemutant without oxidative stress we have previously reported the construction of b. pseudomallei σe mutant. the mutant shows increased susceptibility to killing by h2o indicating the role of σe in regulating resistance to oxidative stress. to identify the σe regulon under oxidative stress conditions, we first investigated the transcriptional profiles of b. pseudomallei wild type and the σe mutant grown in lb medium without antibiotic supplementation. analysis of the transcription profiles revealed that a total of genes were differentially regulated, representing approximately % of the total b. pseudomallei k genes. these differentially regulated genes were distributed on both chromosome and chromosome. in total, genes were up-regulated and genes were down-regulated in the σe mutant. the down-regulation of genes may indicate either direct or indirect regulation by σe. among the down-regulated genes, could be classified into major cog functional groups, including genes predicted to be involved in cell wall/cell membrane biosynthesis, genes involved in energy production/metabolisms, genes involved in regulatory/signal transduction/post-translational modification and repair, and genes involved in intracellular trafficking/secretion. the remaining genes have unknown functions. comparative analysis of transcription profiles of b. pseudomalleiwild type and σemutant under oxidative stress to identify σe-dependent genes that are differentially expressed under oxidative stress conditions, we compared the transcriptome profiles of the σe mutant and wild type which had been exposed to oxidative stress. the bacteria were treated with h2o for min before rna extraction and microarray analysis. a total of genes were differentially regulated representing approximately % of the total b. pseudomallei k genes. of these, genes were up-regulated in the σe mutant and were located on either chromosome or chromosome. among the down-regulated genes in the σe mutant, genes were also down-regulated in the mutant under normal growth conditions. by excluding these genes, we identified genes defined as the σe-dependent oxidative stress regulon. these genes were distributed on both chromosome and chromosome. two genes were predicted to be involved in cell wall/cell membrane biosynthesis, genes in energy production/metabolisms, genes in regulatory/signal transduction/post-translational modification and repair, genes in intracellular trafficking/secretion. the remaining genes had unknown functions. amongst the genes making up the σe-dependent oxidative stress regulon, bpsl is predicted to be involved in cell wall/cell membrane biosynthesis and bpss is predicted to encode a membrane protein related to metalloendopeptidases and porins. genes involved in energy production and metabolism included bpsl0320-, bpsl and bpsl2300-l. the absence of a functional σe under oxidative stress affected the expression of bpss1838-, genes that play important roles in increasing tolerance and resistance to oxidative stress. we identified σe-regulated genes involved in regulatory, signal transduction and post-translational modification after oxidative stress including bpsl, bpsl, bpsl, bpss and bpsl. the latter encodes thioredoxin reductase which functions in post-translational modification. in addition, site-specific recombinase, which is involved in dna replication, recombination and repair, was also under σe regulation. intracellular trafficking and secretion genes accounted for % of σe-dependent oxidative stress responsive genes. these included genes of the type ii secretion system, fimbrial proteins and membrane-anchored cell surface protein. b. pseudomallei contains six clusters type vi secretion system. the expression of t6ss genes has been reported to be induced in vivo. a previous study reported that the t6ss- cluster is important for host adaptation of b. pseudomallei within phagocytes, and that the expression of genes in this cluster is significantly elevated after infection of murine macrophages. we found the increased expression of ten genes belonging to t6ss- under oxidative stress conditions suggesting that t6ss- may play a role in combating oxidative stress. rt-pcr analysis of genes under normal and oxidative stress conditions to validate the results from our microarray analysis, rt-pcr was performed. figure  shows the increased expression of the bpsl and bpss genes in b. pseudomallei wild type after h2o treatment. we did not observe a significant difference in expression of bpsl. these results are consistent with the microarray data which indicated that bpsl and bpss but not bpsl were up-regulated in response to oxidative stress. in the b. pseudomallei σe mutant exposed to h2o treatment, the expression of the bpsl bpss and bpsl genes was down-regulated compared to the wild type, indicating that these genes are under σe control. these results are also consistent with our microarray results.figure rt-pcr analysis of genes under normal and oxidative stress conditions. b. pseudomallei wild type or the isogenic σe mutant was incubated for min in the presence or absence of μm h2o and rt-pcr analysis carried out. each row represents an individual gene and normalized against 23s rrna expression. in addition to rt-pcr, the differential transcription profiles were analyzed to ensure the quality of our transcription profile data. for example, we found that genes encoding b. pseudomallei t6ss- were all up-regulated, suggesting that these genes may be co-expressed as an operon and support the validity of our transcriptional profiling results. other studies have shown that the katg and ahpc genes are up-regulated following the exposure of b. pseudomallei to oxidative stress. our transcriptional data also reveals these patterns of gene expression. collectively, these results indicate that our data is robust and reliable. b. pseudomalleiσeindirectly regulates spermidine levels during oxidative stress previous studies have suggested that sigma factors regulate speg and consequently spermidine levels. spermidine is one of the predominant polyamines in gram-negative bacteria, widely distributed in the environment, and is involved in various biological processes including gene regulation, protein translation and stress resistance. during oxidative stress, spermidine functions as a free radical scavenger and plays an important adjunctive role in protecting bacterial cells from the toxic effects of reactive oxygen species. the intracellular level of spermidine in bacteria is reported to range from 1- mm. high concentrations of spermidine are toxic for bacteria. excess spermidine can be a result of de-regulated bacterial biosynthesis/metabolism or from environmental exposure, inhibiting bacterial growth and even killing the bacterial cells. therefore, in bacteria, the maintenance of an appropriate intracellular level of spermidine is critical. excess spermidine can be converted into the physiologically inert acetylspermidine by the spermidine-acetyltransferase. a recent study revealed that the speg gene has been silenced by convergent evolution in shigella and this resulted in elevated levesl of intracellular spermidine. as a result, the survival of shigella under oxidative stress is enhanced, contributing to its successful pathogenic lifestyle. we observed expression of speg in b. pseudomallei wild type under both control and oxidative stress conditions. however, the speg gene was down-regulated in the σe mutant, indicating that the inactivation of rpoe effected the expression of speg. this suggests that speg is regulated by σe. the decreased gene expression we have observed corroborates our previous proteomic study. to our knowledge, this is the first report on the regulation of speg by σe. we hypothesised that under high concentrations of spermidine, σe positively regulate expression of speg gene in order to prevent spermidine accumulation, the failure of which, will result in inhibition of b. pseudomallei growth and even cytotoxicity. to test this hypothesis, cultures of b. pseudomallei wild type or σe mutant grown in lb broth were exposed to mm spermidine and the number of viable bacteria determined. the number of wild type bacteria was not affected by the addition of spermidine. this is likely due to the presence of a functional σe gene in the wild type, activating the expression of speg gene. in contrast, in the b. pseudomallei σe mutant, the number of viable bacteria was significantly reduced by the addition of spermidine, indicating the accumulation of spermidine to toxic levels.figure effect of spermidine on the viability of b. pseudomallei wild type or σ e mutant. the numbers of b. pseudomallei wild type or the σe mutant cells were grown in the presence or absence of mm spermidine. after h, the numbers of viable bacteria were determined after plating onto lb agar. the viability of wild type and σe mutant in the presence of spermidine was calculated from cfu count divided by the cfu count of control condition and multiplied by values shown are the mean of three independent experiments. asterisks indicate significant differences. this result corroborates the findings from a previous study in e. coli, where excess spermidine was shown to be toxic to bacteria and speg was shown to be important for bacterial viability. our study provides further evidence that the regulation of speg is affected by σe; speg is a novel member of the σe regulon and σe plays an indirect but important role in the regulation of polyamine levels in bacterial cells to protect the cells during oxidative stress.  dna tiling arrays were employed to identify global transcriptional profile changes in b. pseudomallei k exposed to oxidative stress induced by h2o we have identified not only genes involved in repairing cell wall/membrane biosynthesis but also genes involved in energy and metabolism, regulatory and signal transduction, post translational modification, and intracellular trafficking/secretion genes, which are directly regulated by σe during oxidative stress. we found the increased expression of the b. pseudomallei t6ss- under oxidative stress. more importantly, we provided evidence that σe also indirectly regulates the polyamine levels in b. pseudomallei, to protect the cells from oxidative stress. 
in this study, we identified the b. pseudomallei σe regulon and characterized the indirect role that σe plays in the regulation of spermidine, contributing to the successful survival of b. pseudomallei in stressful environments. changes in the global transcriptional profiles of b. pseudomallei wild type and σe mutant under physiological and oxidative stress conditions were determined. comparison of the transcriptional profiles of b. pseudomallei wild type and σe mutant under control or oxidative stress conditions identified oxidative-responsive genes regulated by σe, including genes involved in cell membrane repair, maintenance of protein folding and oxidative stress response and potential virulence factors such as a type vi secretion system. importantly, we identified that the speg gene, encoding spermidine-acetyltransferase, is a novel member of the b. pseudomallei σe regulon. this study identified b. pseudomallei genes directly regulated by σe in response to oxidative stress and revealed the indirect role of σe in the regulation of the polyamine spermidine for bacterial cell protection during oxidative stress. this study provides new insights into the regulatory mechanisms by which σe contributes to the survival of b. pseudomallei under stressful conditions. b. pseudomalleitranscription profilesigma espegoxidative stressissue-copyright-statement© the author 2014 sigma factor e is a key regulator of extra-cytoplasmic stress response in gram-negative bacteria. burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium widely distributed in soil and water in endemic areas. we identified up-regulated genes under oxidative stress condition. the expression of speg was regulated by σe, implying that σe plays an indirect role in the regulation of physiological level of spermidine to protect the bacteria during oxidative stress.
 burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium widely distributed in soil and water in endemic areas. this soil saprophyte can survive harsh environmental conditions, even in soils where herbicides are abundant. sigma factor e is a key regulator of extra-cytoplasmic stress response in gram-negative bacteria. in this study, we identified the b. pseudomallei σe regulon and characterized the indirect role that σe plays in the regulation of spermidine, contributing to the successful survival of b. pseudomallei in stressful environments.  changes in the global transcriptional profiles of b. pseudomallei wild type and σe mutant under physiological and oxidative stress conditions were determined. we identified up-regulated genes under oxidative stress condition. comparison of the transcriptional profiles of b. pseudomallei wild type and σe mutant under control or oxidative stress conditions identified oxidative-responsive genes regulated by σe, including genes involved in cell membrane repair, maintenance of protein folding and oxidative stress response and potential virulence factors such as a type vi secretion system. importantly, we identified that the speg gene, encoding spermidine-acetyltransferase, is a novel member of the b. pseudomallei σe regulon. the expression of speg was regulated by σe, implying that σe plays an indirect role in the regulation of physiological level of spermidine to protect the bacteria during oxidative stress.  this study identified b. pseudomallei genes directly regulated by σe in response to oxidative stress and revealed the indirect role of σe in the regulation of the polyamine spermidine for bacterial cell protection during oxidative stress. this study provides new insights into the regulatory mechanisms by which σe contributes to the survival of b. pseudomallei under stressful conditions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords b. pseudomalleitranscription profilesigma espegoxidative stressissue-copyright-statement© the author 2014 