BACKGROUND
the soil bacterium pseudomonas putida has to cope with diverse and variable habitat-associated stressors to ensure its survival
<cit> .
besides the exposure of p. putida to toxic pollutants and antibacterial compounds in soils, this bacterium encounters osmotic, thermal, oxidative and starvation stresses in the natural habitat
<cit> .
under certain laboratory growth conditions, p. putida exerts a filamented phenotype
<cit> .
filamentation occurs due to the lack of septation during the cell growth process and results in the formation of elongated bacteria, which is typically a consequence of dna damage or envelope stress
<cit> .
cell division inhibition is most commonly mediated by the dna-damage response system
<cit> .
dna damage  results in the exposure of single-stranded dna stretches that become covered by the reca recombinase.
in this nucleoprotein filament, reca becomes activated and stimulates the autoproteolysis of the lexa repressor, which in turn results in derepression of the sos regulon.
while most of the sos genes are involved in dna-repair, some carry out other functions, such as the inhibition of cell division.
in this context, sula  physically inhibits ftsz polymerization and causes the formation of non-septated bacterial filaments, in order to prevent transmission of damaged dna to daughter cells.
in absence of sos induction, however, direct chemical inhibition of ftsz can also lead to bacterial elongation
<cit> .
while reports describing conditions that induce p. putida filamentation are scarce, filamentation of other bacteria has been shown in response to dna damage , nutrient deprivation, low temperature, media composition, low shaking speed and high osmolarity
<cit> .
additionally, the different stages of biofilm development in p. putida have been associated with alterations in bacterial length
<cit> .
furthermore, the plant-produced alkaloid berberine was found recently to induce filamentation in escherichia coli k12
<cit> .
collectively, these studies indicate that conditions and/or products encountered by p. putida during its natural life cycle could induce filamentation.
for a variety of  pathogens, the filamentous morphology has been shown to provide survival advantages
<cit> .
more specifically, uropathogenic escherichia coli  filaments were more proficient in evading neutrophil phagocytosis compared to non-filamented upec
<cit> .
upec filamentation was presumably induced in response to effectors of the host innate immunity.
the intracellular survival of salmonella enterica serovar typhimurium in macrophages in vitro is also associated with a filamentous phenotype, which is probably induced by macrophage production of nitric oxide radicals
<cit> .
in addition, filamentation has been shown to play a role in the infection process of, among others, proteus mirabilis, legionella pneumophila, mycobacterium tuberculosis and shigella flexneri <cit> .
it remains unclear which mechanisms are at the origin of p. putida filamentation, which metabolic changes occur in p. putida filaments, and whether the p. putida filamented phenotype could confer environmentally advantageous traits.
this study is the first to assess the global proteome and stress resistance of p. putida kt <dig> when grown in conditions that induce filamentation.
RESULTS
morphologic and growth analysis of p. putida kt <dig> grown in filament and non-filament inducing conditions
the formation of filaments by p. putida kt <dig> cultures was induced by overnight shaking at low speed
<cit> , and corroborated by microscopic and flow cytometry analysis .
a bacterial culture shaken at high speed  was used as a non-filamentous control .
figure
<dig> demonstrates a clear difference in population heterogeneity between  <dig> rpm and  <dig> rpm-grown p. putida kt <dig>  with  <dig> rpm-grown bacteria showing an increased size distribution .
the increase in bacterial size for  <dig> rpm-grown p. putida is also reflected in the comparative flow cytometry histogram .
nucleic acid staining of  <dig> rpm and  <dig> rpm-grown bacteria  confirmed the size differences.
in order to rule out any effects of differences in growth phase between the two test conditions, the growth of p. putida kt <dig> as a function of shaking speed was determined .
no statistically significant  differences were found, only a slight significant increase in cell numbers was observed at  <dig> h for the  <dig> rpm-grown cultures.
in agreement with the od measurements, no statistically significant  differences were observed at  <dig> h in viable counts nor in biomass .
as differences in the dissolved oxygen concentrations are expected to occur at different shaking speeds, the dissolved oxygen was measured for  <dig> rpm and  <dig> rpm-grown bacteria as a function of culture time.
as presented in figure
<dig>   <dig> rpm cultures reached undetectable oxygen levels after approximately  <dig>  h, while this was only after  <dig> h for  <dig> rpm.
further, the maximum oxygen transfer rate at  <dig> rpm, calculated based on
<cit> , was approximately  <dig>  times higher than at  <dig> rpm.
stress resistance of p. putida kt <dig> grown in filament and non-filament inducing conditions
the stress resistance of p. putida kt <dig> grown in filament-inducing and non-filament-inducing conditions  was investigated.
p. putida kt <dig> grown at  <dig> rpm demonstrated an increased resistance to heat shock  and saline stress , when compared to cells grown at  <dig> rpm .
the acid stress resistance profile was similar for cultures grown at both tested shaking speeds.
proteomic analysis of p. putida kt <dig> grown in filament and non-filament inducing conditions
in order to investigate the molecular basis of the observed increased stress resistance of p. putida kt <dig> grown in filament-inducing conditions, differential proteomic analysis was performed on samples after  <dig> hours of growth.
this time point was chosen with the aim of obtaining an accumulation of effects associated with cultivating at different shaking speeds.
two biological replicates were analyzed, using a post-digest icpl protocol, allowing the identification of  <dig> unique proteins, of which  <dig> were quantified.
subcellular localization prediction using psortb revealed that identified proteins mainly belonged to the cytoplasmic compartment and cytoplasmic membrane .
almost  <dig> proteins could be quantified in both biological replicates and the calculated correlation between the  <dig> datasets reached  <dig> , suggesting a very high reproducibility of our observations .
finally, among the  <dig> quantified proteins,  <dig> proteins had a fold change lower than  <dig>  or higher than  <dig>  revealing that the difference in shaking speed had a major influence on the proteome of p. putida kt <dig>  the heat shock protein ibpa was induced the most in filament-inducing conditions , followed by periplasmic phosphate-binding proteins .
the reca protein was induced  <dig>  fold .
among the differentially regulated proteins, a majority was involved in metabolic activity .
altered metabolic activity in p. putida filaments was reflected in  down-regulation of a protein involved in purine/pyrimidine catabolism ,  down-regulation of proteins involved in the degradation of allantoate  and formation/downstream catabolism of urea  and glyoxylate ,  down-regulation of proteins involved in the production of atp ,  differential expression of proteins involved in the metabolism of amino acids .
in addition, proteomic analysis of p. putida filaments indicated down-regulation of formate metabolism , lipid degradation  and synthesis of polyhydroxyalkanoate .
* p-value <  <dig> .
role of reca in p. putida kt <dig> filamentation and stress resistance
the increased abundance of reca  in  <dig> rpm cultures of p. putida kt <dig>  suggested the activation of the sos response.
since only induction of reca was observed, this could indicate a mild sos response
<cit> .
in addition, the heterogeneity of the sos response at single cell level could be masked at the population level
<cit> .
this heterogeneity was also apparent in cell morphology between  <dig> rpm- and  <dig> rpm-grown p. putida kt <dig> .
in order to determine whether  <dig> rpm-induced filamentation in p. putida kt <dig> was indeed dependent on reca, an isogenic reca mutant cultured in  <dig> and  <dig> rpm conditions was examined.
intriguingly, the  <dig> rpm-grown p. putida kt <dig> reca mutant filamented at similar levels as the wild type p. putida kt <dig> .
in contrast to filamentation, the increased heat shock resistance of p. putida kt <dig> grown at  <dig> rpm was reca-dependent, since an isogenic reca mutant was equally resistant to heat shock when grown at  <dig> rpm or  <dig> rpm .
discussion
as a soil organism, p. putida recurrently encounters filament-inducing conditions during its natural life cycle.
our data indicate that filament formation of p. putida could confer environmentally advantageous traits.
indeed, p. putida kt <dig> grown at low shaking speed produced filaments and was more resistant to heat shock and saline stress.
similar observations were made for caulobacter crescentus filaments, which showed a higher resistance to oxidative, osmotic, thermal and acid stress
<cit> .
the comparative proteome profile indicated that the metabolic activity of p. putida kt <dig> grown at  <dig> rpm was significantly different from p. putida kt <dig> grown at  <dig> rpm.
the most pronounced induction occurred for the heat shock protein ibpa.
this small heat shock protein belongs to the widely conserved family of α-crystallin-type heat shock proteins.
the latter appears to play a very versatile role in the protection against different stress conditions via protein and membrane protection
<cit> .
in addition, many small heat shock proteins form oligomers, which may vary by the degree of phosphorylation or ion concentration
<cit>  .
although no observable differences in dissolved oxygen levels could be reported at the time of proteomic analysis  , this does not completely rule out the role of dissolved oxygen in the observed results as the maximum oxygen transfer rate at  <dig> rpm is approximately  <dig>  times higher than at  <dig> rpm
<cit> .
ohr, a protein of the osmc family  was  <dig> -fold down-regulated in filamented p. putida, and is involved in the resistance to oxidative stressors, such as organic peroxide, but not in osmotic stress resistance
<cit> .
in addition to a decreased ohr abundance, other proteins involved in oxidative stress resistance were present at lower levels in  <dig> rpm samples, including a catalase/peroxidase , an antioxidant ahpc , a glutaredoxin-related protein  and a putative dna binding stress protein .
the latter has recently been described as an oxidative stress-inducible dps miniferritin
<cit> , and was found up-regulated in an oxyr mutant of p. aeruginosa <cit> .
the differential abundance of proteins involved in oxidative stress resistance could potentially be explained by lower oxygen levels in  <dig> rpm cultures .
the increase of opre  and cyoa  further suggests limitations in oxygen availability in  <dig> rpm cultures
<cit> .
finally, oxygen limitation is related to bacterial filamentation and/or reca induction
<cit> .
however, jenssen and colleagues determined that, in addition to oxygen deprivation, additional factors such as exhaustion of medium components and changes in growth rate, were important for p. putida filamentation
<cit> .
while reca was more abundant in p. putida kt <dig> grown at  <dig> rpm, the p. putida kt <dig> reca mutant filamented at similar levels as the wild type.
a similar observation was reported previously, showing that an e. coli reca mutant displayed similar levels of filamentation as the wild type strain in response to growth at high pressure, despite strong evidence of reca-mediated sos response activation
<cit> .
gottesman et al.
suggested the existence of a transient filamentation phenotype in response to uv, independent of sula
<cit> , which could explain the reca-independent filamentation phenotype of  <dig> rpm-grown p. putida kt <dig> in the present study.
while the bacterial sos response and associated filamentation is typically triggered by treatments directly affecting dna integrity , a number of environmental conditions were reported to cause dna damage in an indirect manner
<cit> .
as such, high pressure-induced filamentation of e. coli was shown to stem from the activation of a cryptic type iv restriction endonuclease  endogenously present in the cell
<cit> , while β-lactam antibiotics triggered dpia to interfere with dna replication
<cit> .
even though it remains unclear which metabolic changes could indirectly lead to dna damage and sos response activation, the major changes in metabolism provide evidence for new triggers of the sos response.
CONCLUSIONS
in conclusion, our data indicate that filament-formation of p. putida kt <dig> could confer environmentally advantageous traits, by increasing its resistance to saline and heat shock.
we demonstrated that culturing at low shaking speed induced expression of reca, which plays a central role in the sos response, putatively through changes in amino acid metabolism and/or oxygen availability.
furthermore, the increased heat shock resistance was found to be reca dependent.
filamentation could thus represent an adaptive survival strategy of p. putida, allowing it to persist during times of elevated soil temperatures, increased osmolarity  and/or increased pollution.

 the soil bacterium pseudomonas putida has to cope with diverse and variable habitat-associated stressors to ensure its survival. besides the exposure of p. putida to toxic pollutants and antibacterial compounds in soils, this bacterium encounters osmotic, thermal, oxidative and starvation stresses in the natural habitat. under certain laboratory growth conditions, p. putida exerts a filamented phenotype. filamentation occurs due to the lack of septation during the cell growth process and results in the formation of elongated bacteria, which is typically a consequence of dna damage or envelope stress. cell division inhibition is most commonly mediated by the dna-damage response system. dna damage results in the exposure of single-stranded dna stretches that become covered by the reca recombinase. in this nucleoprotein filament, reca becomes activated and stimulates the autoproteolysis of the lexa repressor, which in turn results in derepression of the sos regulon. while most of the sos genes are involved in dna-repair, some carry out other functions, such as the inhibition of cell division. in this context, sula physically inhibits ftsz polymerization and causes the formation of non-septated bacterial filaments, in order to prevent transmission of damaged dna to daughter cells. in absence of sos induction, however, direct chemical inhibition of ftsz can also lead to bacterial elongation. while reports describing conditions that induce p. putida filamentation are scarce, filamentation of other bacteria has been shown in response to dna damage, nutrient deprivation, low temperature, media composition, low shaking speed and high osmolarity. additionally, the different stages of biofilm development in p. putida have been associated with alterations in bacterial length. furthermore, the plant-produced alkaloid berberine was found recently to induce filamentation in escherichia coli k12. collectively, these studies indicate that conditions and/or products encountered by p. putida during its natural life cycle could induce filamentation. for a variety of pathogens, the filamentous morphology has been shown to provide survival advantages. more specifically, uropathogenic escherichia coli filaments were more proficient in evading neutrophil phagocytosis compared to non-filamented upec. upec filamentation was presumably induced in response to effectors of the host innate immunity. the intracellular survival of salmonella enterica serovar typhimurium in macrophages in vitro is also associated with a filamentous phenotype, which is probably induced by macrophage production of nitric oxide radicals. in addition, filamentation has been shown to play a role in the infection process of, among others, proteus mirabilis, legionella pneumophila, mycobacterium tuberculosis and shigella flexneri. it remains unclear which mechanisms are at the origin of p. putida filamentation, which metabolic changes occur in p. putida filaments, and whether the p. putida filamented phenotype could confer environmentally advantageous traits. this study is the first to assess the global proteome and stress resistance of p. putida kt when grown in conditions that induce filamentation.  morphologic and growth analysis of p. putida kt grown in filament and non-filament inducing conditions the formation of filaments by p. putida kt cultures was induced by overnight shaking at low speed, and corroborated by microscopic and flow cytometry analysis. a bacterial culture shaken at high speed was used as a non-filamentous control. figure  demonstrates a clear difference in population heterogeneity between rpm and rpm-grown p. putida kt with rpm-grown bacteria showing an increased size distribution. the increase in bacterial size for rpm-grown p. putida is also reflected in the comparative flow cytometry histogram. nucleic acid staining of rpm and rpm-grown bacteria confirmed the size differences. in order to rule out any effects of differences in growth phase between the two test conditions, the growth of p. putida kt as a function of shaking speed was determined. no statistically significant differences were found, only a slight significant increase in cell numbers was observed at h for the rpm-grown cultures. in agreement with the od measurements, no statistically significant differences were observed at h in viable counts nor in biomass. as differences in the dissolved oxygen concentrations are expected to occur at different shaking speeds, the dissolved oxygen was measured for rpm and rpm-grown bacteria as a function of culture time. as presented in figure  rpm cultures reached undetectable oxygen levels after approximately h, while this was only after h for rpm. further, the maximum oxygen transfer rate at rpm, calculated based on, was approximately times higher than at rpm. stress resistance of p. putida kt grown in filament and non-filament inducing conditions the stress resistance of p. putida kt grown in filament-inducing and non-filament-inducing conditions was investigated. p. putida kt grown at rpm demonstrated an increased resistance to heat shock and saline stress, when compared to cells grown at rpm. the acid stress resistance profile was similar for cultures grown at both tested shaking speeds. proteomic analysis of p. putida kt grown in filament and non-filament inducing conditions in order to investigate the molecular basis of the observed increased stress resistance of p. putida kt grown in filament-inducing conditions, differential proteomic analysis was performed on samples after hours of growth. this time point was chosen with the aim of obtaining an accumulation of effects associated with cultivating at different shaking speeds. two biological replicates were analyzed, using a post-digest icpl protocol, allowing the identification of unique proteins, of which were quantified. subcellular localization prediction using psortb revealed that identified proteins mainly belonged to the cytoplasmic compartment and cytoplasmic membrane. almost proteins could be quantified in both biological replicates and the calculated correlation between the datasets reached, suggesting a very high reproducibility of our observations. finally, among the quantified proteins, proteins had a fold change lower than or higher than revealing that the difference in shaking speed had a major influence on the proteome of p. putida kt the heat shock protein ibpa was induced the most in filament-inducing conditions, followed by periplasmic phosphate-binding proteins. the reca protein was induced fold. among the differentially regulated proteins, a majority was involved in metabolic activity. altered metabolic activity in p. putida filaments was reflected in down-regulation of a protein involved in purine/pyrimidine catabolism, down-regulation of proteins involved in the degradation of allantoate and formation/downstream catabolism of urea and glyoxylate, down-regulation of proteins involved in the production of atp, differential expression of proteins involved in the metabolism of amino acids. in addition, proteomic analysis of p. putida filaments indicated down-regulation of formate metabolism, lipid degradation and synthesis of polyhydroxyalkanoate. * p-value <. role of reca in p. putida kt filamentation and stress resistance the increased abundance of reca in rpm cultures of p. putida kt suggested the activation of the sos response. since only induction of reca was observed, this could indicate a mild sos response. in addition, the heterogeneity of the sos response at single cell level could be masked at the population level. this heterogeneity was also apparent in cell morphology between rpm- and rpm-grown p. putida kt. in order to determine whether rpm-induced filamentation in p. putida kt was indeed dependent on reca, an isogenic reca mutant cultured in and rpm conditions was examined. intriguingly, the rpm-grown p. putida kt reca mutant filamented at similar levels as the wild type p. putida kt. in contrast to filamentation, the increased heat shock resistance of p. putida kt grown at rpm was reca-dependent, since an isogenic reca mutant was equally resistant to heat shock when grown at rpm or rpm. discussion as a soil organism, p. putida recurrently encounters filament-inducing conditions during its natural life cycle. our data indicate that filament formation of p. putida could confer environmentally advantageous traits. indeed, p. putida kt grown at low shaking speed produced filaments and was more resistant to heat shock and saline stress. similar observations were made for caulobacter crescentus filaments, which showed a higher resistance to oxidative, osmotic, thermal and acid stress. the comparative proteome profile indicated that the metabolic activity of p. putida kt grown at rpm was significantly different from p. putida kt grown at rpm. the most pronounced induction occurred for the heat shock protein ibpa. this small heat shock protein belongs to the widely conserved family of α-crystallin-type heat shock proteins. the latter appears to play a very versatile role in the protection against different stress conditions via protein and membrane protection. in addition, many small heat shock proteins form oligomers, which may vary by the degree of phosphorylation or ion concentration. although no observable differences in dissolved oxygen levels could be reported at the time of proteomic analysis, this does not completely rule out the role of dissolved oxygen in the observed results as the maximum oxygen transfer rate at rpm is approximately times higher than at rpm. ohr, a protein of the osmc family was -fold down-regulated in filamented p. putida, and is involved in the resistance to oxidative stressors, such as organic peroxide, but not in osmotic stress resistance. in addition to a decreased ohr abundance, other proteins involved in oxidative stress resistance were present at lower levels in rpm samples, including a catalase/peroxidase, an antioxidant ahpc, a glutaredoxin-related protein and a putative dna binding stress protein. the latter has recently been described as an oxidative stress-inducible dps miniferritin, and was found up-regulated in an oxyr mutant of p. aeruginosa. the differential abundance of proteins involved in oxidative stress resistance could potentially be explained by lower oxygen levels in rpm cultures. the increase of opre and cyoa further suggests limitations in oxygen availability in rpm cultures. finally, oxygen limitation is related to bacterial filamentation and/or reca induction. however, jenssen and colleagues determined that, in addition to oxygen deprivation, additional factors such as exhaustion of medium components and changes in growth rate, were important for p. putida filamentation. while reca was more abundant in p. putida kt grown at rpm, the p. putida kt reca mutant filamented at similar levels as the wild type. a similar observation was reported previously, showing that an e. coli reca mutant displayed similar levels of filamentation as the wild type strain in response to growth at high pressure, despite strong evidence of reca-mediated sos response activation. gottesman et al. suggested the existence of a transient filamentation phenotype in response to uv, independent of sula, which could explain the reca-independent filamentation phenotype of rpm-grown p. putida kt in the present study. while the bacterial sos response and associated filamentation is typically triggered by treatments directly affecting dna integrity, a number of environmental conditions were reported to cause dna damage in an indirect manner. as such, high pressure-induced filamentation of e. coli was shown to stem from the activation of a cryptic type iv restriction endonuclease endogenously present in the cell, while β-lactam antibiotics triggered dpia to interfere with dna replication. even though it remains unclear which metabolic changes could indirectly lead to dna damage and sos response activation, the major changes in metabolism provide evidence for new triggers of the sos response.  in conclusion, our data indicate that filament-formation of p. putida kt could confer environmentally advantageous traits, by increasing its resistance to saline and heat shock. we demonstrated that culturing at low shaking speed induced expression of reca, which plays a central role in the sos response, putatively through changes in amino acid metabolism and/or oxygen availability. furthermore, the increased heat shock resistance was found to be reca dependent. filamentation could thus represent an adaptive survival strategy of p. putida, allowing it to persist during times of elevated soil temperatures, increased osmolarity and/or increased pollution. 
pseudomonas putida exerts a filamentous phenotype in response to environmental stress conditions that are encountered during its natural life cycle. filamentation of p. putida was induced through culturing at low shaking speed and was compared to culturing in high shaking speed conditions, after which whole proteomic analysis and stress exposure assays were performed. proteomic analysis showed a significant metabolic change and a pronounced induction of the heat shock protein ibpa and recombinase reca in filament-inducing conditions. this study assessed whether p. putida filamentation could confer survival advantages. p. putida grown in filament-inducing conditions showed increased resistance to heat and saline stressors compared to non-filamented cultures. this study provides insights into the altered metabolism of p. putida in filament-inducing conditions, and indicates that the formation of filaments could potentially be utilized by p. putida as a survival strategy in its hostile, recurrently changing habitat. our data further indicated that the associated heat shock resistance, but not filamentation, was dependent of reca.
 pseudomonas putida exerts a filamentous phenotype in response to environmental stress conditions that are encountered during its natural life cycle. this study assessed whether p. putida filamentation could confer survival advantages. filamentation of p. putida was induced through culturing at low shaking speed and was compared to culturing in high shaking speed conditions, after which whole proteomic analysis and stress exposure assays were performed.  p. putida grown in filament-inducing conditions showed increased resistance to heat and saline stressors compared to non-filamented cultures. proteomic analysis showed a significant metabolic change and a pronounced induction of the heat shock protein ibpa and recombinase reca in filament-inducing conditions. our data further indicated that the associated heat shock resistance, but not filamentation, was dependent of reca.  this study provides insights into the altered metabolism of p. putida in filament-inducing conditions, and indicates that the formation of filaments could potentially be utilized by p. putida as a survival strategy in its hostile, recurrently changing habitat. pseudomonas putida kt2440filamentationelongationsos responserecashaking speedstress resistance 