synthetic lethality , a phenomenon in which the combination of two nonlethal mutations causes death , is a powerful genetic tool that can , in an unbiased fashion , identify novel connections between cellular processes that function together to permit survival in a stressful environment . however , because the double mutant dies , investigating the process by which death occurs can be difficult . if , however , some permissive condition exists that permits survival of the double mutant , then the study of the death process is greatly facilitated , because genetic manipulations can be performed under permissive conditions and the consequences of those manipulations studied at nonpermissive conditions . here , we explore one environmental condition ( exposure to glycolytic carbon sources ) that permits survival of the previously reported synthetic lethal mutant ompr malt     , which lacks the response regulator ompr whilst harboring a constitutively active malt protein .     as osmolality increases , the two - component response regulator ompr becomes activated by the receipt of a phosphoryl group from its cognate sensor kinase envz [ 2 , 3 ] . upon phosphorylation , ompr controls more than 100 genes associated with outer membrane biogenesis , osmoregulation , and envelope stress     [ 4 , 5 ] . malt is the central regulator of all mal genes     [ 6 , 7 ] ( figure 1 ) . the outer membrane porin lamb facilitates the uptake of maltose and maltodextrins into the periplasm , where these sugars are bound by the maltose - binding protein male and delivered to the malfgk2 transporter . following transport into the cytoplasm malt itself is activated by the maltose metabolite maltotriose and inhibited by malk , maly , aes , and glucokinase     [ 912 ] . the nucleoid proteins h - ns and stpa positively regulate malt translation     [ 13 , 14 ] . malt transcription is activated by the camp - crp complex , which renders it subject to catabolite repression     [ 15 , 16 ] . finally , mlc represses malt transcription     . under non - permissive conditions , the ompr malt mutant displays a set of striking phenotypes . the culture loses turbidity upon entry into late exponential phase     , because the inner membrane disintegrates . the main cause for these phenotypes is elevated lamb expression in the absence of ompr : deletion of lamb permits survival     , while genetic suppressors that reduce lamb expression also permit survival . similar to genetic suppressors , any environmental condition that reduces lamb levels should permit cell survival . for example , the ompr malt mutant survives on minimal medium supplemented with glucose as the sole carbon source     , almost certainly , because glucose causes catabolite repression of transcription from the maltand malk promoters and thus reduced expression of lamb     [ 15 , 19 ] . in this study , we identified an additional permissive environmental condition that supports survival of the ompr malt mutant : the presence of noncatabolite repressing glycolytic carbon sources in the growth medium . characterization of this condition allowed us to unveil a new regulatory layer of the malt regulon . the transcription fusion malep92-lac was a generous gift from winfried boos ( universitt konstanz , germany ) and was described previously . the malt allele ( maltc-1 ) used in this study was a generous gift from linda kenney ( university of illinois at chicago , il , usa ) . unless otherwise mentioned , deletion alleles were derived from the keio collection . because the ompr malt mutant is conditionally lethal , cells were grown overnight under a permissive condition : 22c in m63 minimal salts   with 22  mm sorbitol as the sole carbon source and supplemented with 100  g  ml l - threonine , l - histidine , l - leucine , l - methionine , l - tryptophan , and 10  g thiamine ml . whenever required , kanamycin ( 40  g  ml ) , chloramphenicol ( 25 g  ml ) , ampicillin ( 100 g  ml ) , or tetracycline ( 15  g  ml ) was added .   for tests of lethality , an inoculum from the overnight culture was subcultured at 37c in lb ( 1% ( w / v ) tryptone , 0.5% ( w / v ) yeast extract , 0.5% ( w / v ) nacl ) . cell growth was monitored spectrophotometrically ( beckman instruments du640 ) at 600  nm ( od600 ) . to monitor malep92-lac promoter activity , cells were grown aerobically with 250  rpm agitation at 37c . at various time points during growth , 50  l aliquots were harvested and added to 50  l all - in - one -galactosidase reagent ( pierce biotechnology ) . -galactosidase activity was determined quantitatively in a microtiter format , as described previously . to avoid misleading results caused by lysing cells that spill -galactosidase into the growth medium , we only considered -galactosidase measurements before the onset of cell death . to obtain nonpolar deletions , resistance cassettes were removed using flp - recombinase , according to the previously described protocol . outer membrane proteins were separated using 12% sds - page containing 4.8  m urea and stained with coomassie brilliant blue . to compare malt transcript levels , dna contamination was removed by treatment with 5  u of rq1 rnase - free dnase ( promega ) in 1x rq1 dnase buffer for 1  h at 37c , followed by phenol - chloroform extraction and ethanol precipitation . the subsequent reverse transcription reaction was performed using the revertaid first strand cdna synthesis kit ( fermentas ) . to exclude dna contamination the resulting cdna was diluted and pcr amplified in a reaction mixture containing 2  l cdna product , 1x pcr buffer , 0.2  m dntps , 4  mm  mgcl2 , 0.5  m malt - specific primer maltfor2 ( 5-actcagcccataagtcggc-3 ) , 0.5  m malt - specific primer maltrev2 ( 5-caagacttcaatcccgctag-3 ) , and 1  u taq polymerase in a total volume of 25  l . amplification conditions were 95c for 30 sec , 54c for 30  sec , and 72c for 60  sec ( 30 cycles ) , followed by 72c for 5  min . we previously reported that the ompr malt double mutant ( strain ajw3098 , table 1 ) exhibits a synthetic lethal phenotype caused by the increased expression of lamb . glucose catabolite represses mal transcription , and thus reduces lamb expression [ 15 , 16 ] . therefore , it should not be surprising that exposure to glucose promoted survival of the ompr malt mutant . in contrast , the effect of maltose on mal gene expression can vary . depending on expression or activity levels of the malt protein , maltose can either enhance or inhibit malt regulon expression [ 6 , 2729 ] .   here , we tested if exposure to maltose enhances or suppresses lethality of the ompr maltmutant by growing it in lb at 37c in the presence or absence of maltose and found that exposure to maltose suppressed death ( figure 2(a ) ) . to test if this behavior is a general characteristic of malt alleles , we tested if other malt alleles behaved similarly . several ompr malt double mutants harboring a set of representative malt alleles ( strains ajw3732-ajw3741 ) [ 1 , 11 ] were grown in the presence or absence of maltose . as reported previously , each of these double mutants died in the absence of maltose . we conclude that maltose can promote viability and that this behavior is a general characteristic of malt alleles . our finding that all the ompr malt double mutants survived when exposed to maltose , combined with our previous report that disruption of lamb permits survival , supports the earlier observation that maltose can reduce mal gene expression in cells carrying malt alleles . first , we monitored malt regulon expression of an ompr malt double mutant carrying a transcriptional malep92-lac fusion . we , then , directly monitored lamb expression using outer membrane preparations . exposure of the ompr malt   double mutant to maltose resulted in repressed malep92-lac transcription ( figure 2(b ) ) and reduced lamb expression ( figure 2(c ) and supplemental figure 1(a ) which is available at doi:10.1155/2011/107023 ) . similarly , the malt single mutant displayed reduced lamb expression in the presence of maltose ( supplemental figure 1(b ) ) , indicating that this effect is independent of ompr . in contrast , wt cells and the ompr single mutant showed an increase in lamb expression when maltose was present ( supplemental figure 2 ) . we conclude that maltose supports survival of the ompr malt double mutant by downregulating lamb expression . the observation that maltose , a noncatabolite - repressing sugar , could reduce mal gene expression prompted us to ask if other noncatabolite - repressing sugars exert the same effect . we , therefore , grew the ompr malt mutant in lb at 37c in the presence or absence of diverse carbon sources . as expected , strong catabolite - repressing sugars ( i.e. , glucose , fructose , and mannitol ) enabled survival ( figure 2(a ) and supplemental table 1 ) , whilst many noncatabolite - repressing carbon sources did not ( figure 3(a ) and supplemental table 1 ) . surprisingly , some weaker or noncatabolite repressing carbon sources ( i.e. , sorbitol , serine , pyruvate , and mannose ) promoted cell survival ( figures 2(a ) and 3(a ) and supplemental table 1 ) . all the survival - supporting carbon sources are metabolized via glycolysis , whilst all the nonsurvival supporting carbon sources are not . thus , some glycolysis - associated mechanism , in addition to catabolite repression , must be able to promote cell survival . to determine why carbon sources like sorbitol promoted cell survival , we first tested if sorbitol exerts its effect by influencing mal gene expression . we monitored both malep92 transcription and lamb expression in the ompr malt double mutant supplemented with sorbitol and found them to be reduced ( figures 2(b ) and 2(c ) ) . since sorbitol requires a transport mechanism that is different from maltose or glucose , [ 7 , 8 , 31 ] , we asked whether survival requires transport or metabolism of the sugar . we , therefore , constructed an ompr malt srld triple mutant ( strain ajw3927 ) , which can transport sorbitol but not metabolize it . we also constructed an ompr malt srla triple mutant ( strain ajw3926 ) , which can neither transport nor metabolize sorbitol due to a polar effect of the srla deletion on srld . exposure to sorbitol did not permit survival of either mutant ( figure 3(b ) ) . these results support the argument that the effect of sorbitol on the viability of the ompr malt double mutant requires metabolism of the sugar . the mechanism by which sorbitol or a metabolite exerts its effect could require either the known regulators of the maltose regulon ( figure 1 ) , or the outer membrane porin phoe , which has been shown to promote viability when expressed at high levels . our aim was to test whether any of these factors are required for sorbitol - promoted survival . if none of these regulators is involved , we reasoned that sorbitol must operate through a currently unknown mechanism . in a previous report , we found that the increased expression of the phob regulon member phoe , an outer membrane porin , can promote viability of the ompr malt double mutant . since sugar metabolism is known to de - repress the phob regulon , we tested whether sorbitol exerts its effect by increasing phoe abundance in the outer membrane . however , the ompr malt   pstc phoe mutant ( strain ajw4197 ) , which dies in lb , survived when we supplemented lb with sorbitol ( data not shown ) . we conclude that sorbitol acts independently of phoe . to exert its effect on viability , products of sorbitol metabolism could act through a variety of regulators known to control malt expression or activity ( figure 1 ) . for example , mlc and crp - camp affect malt transcription [ 1517 ] , h - ns and stpa stimulate malt translation [ 13 , 14 ] , and the binding of maltotriose , malk , maly , aes , or glucokinase modulates malt activity [ 6 , 7 , 912 , 28 ] . the death of the ompr malt mlc triple mutant ( strain ajw3936 ) in the absence of sorbitol and its survival when sorbitol was present ( supplemental figure 3(a ) ) shows that sorbitol does not exert its effect by activating the transcriptional repressor mlc ( figure 1 )   or by increasing its concentration and thereby repressing malt transcription . that sorbitol did not influence malt transcription was confirmed by semiquantitative rt - pcr , which showed that exposure to sorbitol did not reduce malt mrna ( figure 4 ) . exposure to maltose also did not reduce malt mrna , confirming earlier reports that maltose affects maltactivity rather than affecting malt transcription [ 6 , 28 ] . glucose , on the other hand , caused a reduction of malt mrna ( figure 4 ) , which can be explained by its catabolite - repressing effect on malt transcription [ 15 , 16 ] . thus , sorbitol permits viability of the ompr malt double mutant by a mechanism that does not involve altered transcription of malt . since sorbitol does not affect malt transcription , we asked if it exerts its effect through any of the regulators that affect malt activity . stpa is reported to exert a weak , activating effect on malt regulon expression by modulating malt translation . accordingly , we found that deletion of stpa in the ompr malt mutant ( strain ajw4028 ) did not promote survival ( supplemental figure 3(b ) ) . we further determined that sorbitol did not exert its effect through stpa ( supplemental figure 3(b ) ) . since deletion of hns in the ompr malt double mutant promoted viability we presumed that the excess carbon might be converted to glycogen and that the subsequent degradation of that glycogen might increase the intracellular maltotriose concentration , and cause endogenous induction of malt ( figure 1 ) [ 33 , 34 ] . whilst glycogen phosphorylase ( glgp ) is instrumental in the production of maltotriose from glycogen , maltodextrin glucosidase ( malz ) has been reported to remove maltotriose by hydrolyzing it to maltose and glucose . deletion of either malz or glgp in the ompr malt background ( strains ajw3888 and ajw3902 , respectively ) did not rescue viability and did not diminish the ability of sorbitol to support growth ( figure 5 ) . we conclude that the ompr malt mutant is largely insensitive to maltotriose and that sorbitol likely does not suppress lethality by altering maltotriose concentrations . maly , aes , malk , and glucokinase inhibit malt activity ( figure 1 ) [ 912 ] . we , therefore , constructed the triple mutants ompr malt maly ( strain ajw3943 ) and ompr malt aes ( strain ajw3947 ) , and an ompr malt malk triple mutant carrying a nonpolar malk allele ( strain ajw3967 ) to avoid disruption of lamb expression . we monitored growth of the first two mutants under non - permissive conditions in the presence or absence of glucose , maltose , or sorbitol . since null mutations of malk and/or glk cause defects in the importation or metabolism of maltose , mutants carrying these alleles were only grown in the presence of glucose or sorbitol . in response to all tested sugars , the mutants survived ( supplemental figures 4(a)4(d ) ) . thus , none of the sugars , including sorbitol , act through maly , aes , malk , or glucokinase . since sorbitol - dependent survival of the ompr malt mutant depends on none of the known regulatory mechanisms , we hypothesize that a novel regulatory mechanism exists , which involves posttranscriptional modulation of malt activity . a highly complex network integrates numerous diverse signals to precisely regulate the expression and function of the maltose transport and metabolism system     [ 6 , 7 , 35 ] ( figure 1 ) . we base this hypothesis on the observation that the synthetic lethality of the ompr malt mutant can be inhibited by growth in the presence of several glycolysis - associated carbon sources ( figures 2(a ) and 3(a ) and supplemental table 1 ) . that a sugar like glucose or fructose can inhibit lethality is easily explained by its capacity to catabolite - repress maltp and malkp transcription and , hence , limit lamb expression     [ 15 , 16 ] . that sugars like maltose and sorbitol also can inhibit lethality , however , is both surprising and telling . depending on malt levels or malt activity , the effect of maltose on malt regulon expression can vary . in cells expressing wt malt in large excess , it is reported that exposure to maltose causes a slight reduction ( ~2-fold ) in mal gene expression . however , in cells moderately overexpressing wt malt , exposure to maltose has been reported to induce malt regulon expression . the same is true when cells express wt malt from the endogenous gene     [ 6 , 28 ] . we confirmed this observation by showing that lamb levels increase in wt cells and ompr mutants when maltose is present ( supplemental figure 2 ) . if the same were true of cells that carry the malt allele , then the resulting increase in lamb levels would be expected to lead to an even more premature death of the ompr malt double mutant . instead , maltose reduced male transcription and lamb expression , and thus permitted survival ( figures 2(b ) and 2(c ) and supplemental figure 1 ) .   in cells carrying malt alleles , exposure to maltose has been reported to induce expression at the male promoter , with the notable exception of highly constitutive malt proteins     [ 27 , 29 ] . in contrast , we found that exposure to maltose causes reduced male promoter activity in the ompr malt double mutant ( figure 2(b ) ) and reduced lamb levels in both the malt single and ompr malt double mutants ( figure 2(c ) and supplemental figure 1 ) , permitting the ompr malt double mutant to survive . since survival was observed in all 10 ompr malt mutants tested , representing each location cluster and inhibition class , it is likely that the observed inhibitory response is a general characteristic of malt proteins . the discrepancy between this and the previous reports could be due to utilization of different strain backgrounds ( mc4100 versus ajw678 ) or of different media ( minimal medium with glycerol as the base carbon source and supplemented with maltose versus lb supplemented with maltose ) . in a previous study , exposure to maltose in the context of high expression of malt to explain this result , a model was proposed in which overproduction of malt results in a large number of malt oligomers that substitute for crp . this would result in formation at the malep and malkp promoters of a less active nucleoprotein complex containing only malt . a further development of this model proposed unlimited aggregation of malt to be responsible for the inhibition of malep transcription at high concentrations of malt . for these models to explain our observations , the native gene would have to express enough malt to permit successful competition with crp for dna binding . furthermore , those constitutively active proteins would have to become more active in response to maltose . finally , the combination would have to be able to exert a 2-fold larger effect ( 4-fold inhibition ) than did the wild - type protein expressed from a multicopy plasmid ( 2-fold inhibition ) . we think it is more likely that maltose acts upon malt in a manner similar to that of sorbitol . sorbitol has never been reported to influence the maltose system or inhibit malt regulon transcription ; thus , the inhibitory mechanism through which it operates must be novel . with the notable exceptions of h - ns and the crp - camp complex , we tested the involvement of each known malt regulator ( figure 1 ) and found that none are required ( figures 4 and 5 and supplemental figures 3   and   4 ) . we ruled out the crp - camp complex , because sorbitol causes only weak catabolite repression     [ 8 , 38 ] . this argument is further supported by the observation that exposure to sorbitol did not alter malt mrna levels ( figure 4 ) . we also excluded h - ns , since , to our knowledge , the global regulator has never been reported to respond to glycolysis . thus , we hypothesize that sorbitol exerts its effect on mal gene transcription through a novel mechanism that is independent of the currently reported regulators and signals . our studies show that the lethal phentoype caused by the malt protein used in this study is insensitive to both the inducer maltotriose ( figure 5 ) and the inhibitor malk     [ 1 , 39 ] ( data not shown ) . although malk can exert a small effect on the activity of this malt protein , an observation made when we tested media that does not contain the malt inducer trehalose , this small effect was insufficient to influence the lethal phenotype ( reimann and wolfe , unpublished data ) . thus , stripped of the two primary layers of regulation provided by maltotriose and malk , the ompr   malt double mutant exposes an otherwise undetectable layer of regulation . that sorbitol must be metabolized to inhibit malt regulon transcription ( figure 3(b ) ) however , recent reports that crp and other transcription factors can become acetylated     [ 4042 ] coupled with the knowledge that the protein deacetylase cobb depends on nad for its function      raises the exciting possibility that increased glycolytic flux due to metabolism of the excess sorbitol results in acetylation of crp , malt , or some other component of the nucleoprotein complex that modulates male and malk transcription , resulting in inhibition and thus survival . the concept of a glycolytic metabolite opens up the possibility that maltose , glucose , fructose , and other glycolytic carbon sources could work through the same mechanism . in the case of glucose and fructose , however , the effect is normally concealed by their strong catabolite - repressing effect . likewise , in cells harboring a malt protein , the strong regulatory effects of maltotriose and malk would normally counterbalance the regulatory effect of maltose metabolism .
bacteria adapt to changing environments by means of tightly coordinated regulatory circuits . 
 the use of synthetic lethality , a genetic phenomenon in which the combination of two nonlethal mutations causes cell death , facilitates identification and study of such circuitry . in this study , we show that the e. coli ompr maltcon double mutant exhibits a synthetic lethal phenotype that is environmentally conditional . 
 maltcon , the constitutively active form of the maltose system regulator malt , causes elevated expression of the outer membrane porin lamb , which leads to death in the absence of the osmoregulator ompr . however , the presence and metabolism of glycolytic carbon sources , such as sorbitol , promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism .