BACKGROUND
biomass-based bioenergy is crucial to meet national goals of making cellulosic ethanol cost-competitive with gasoline.
a core challenge in fermenting cellulosic material to ethanol is the recalcitrance of biomass to breakdown.
severe biomass pretreatments are therefore required to release the sugars, which along with by-products of fermentation can create inhibitors including sugar degradation products such as furfural and hydroxymethylfurfural ; weak acids such as acetic, formic, and levulinic acids; lignin degradation products such as the substituted phenolics vanillin and lignin monomers  <cit> .
in addition, the metabolic byproducts such as ethanol, lactate, and acetate also influence the fermentation by slowing and potentially stopping the fermentation prematurely.
the increased lag phase and slower growth increases the ethanol cost due to both ethanol production rate and total ethanol yield decreases  <cit> .
one approach to overcome the issue of inhibition caused by pretreatment processes is to remove the inhibitor after pretreatment from the biomass physically or chemically, which requires extra equipment and time leading to increased costs.
a second approach utilizes inhibitor tolerant microorganisms for efficient fermentation of lignocellulosic material to ethanol and their utility is considered an industrial requirement  <cit> .
z. mobilis are gram-negative facultative anaerobic bacteria with a number of desirable industrial characteristics, such as high-specific productivity and ethanol yield, unique anaerobic use of the entner-doudoroff pathway that results in low cell mass formation, high ethanol tolerance , ph  <dig> - <dig>  range for ethanol production and has a generally regarded as safe  status  <cit> .
z. mobilis strains have been engineered to ferment pentose sugars such as xylose, arabinose and other substrates with high yields, but a low tolerance to acetic acid and decreased ethanol tolerance have been reported in recombinant strains  <cit> .
z. mobilis mutant strains tolerant to a pretreatment inhibitor such as acetate have been generated by chemical mutagenesis with n-methyl n'-nitro n-nitrosoguanidine and selection in continuous culture with a progressively increasing concentration of sodium acetate in the medium feed  <cit> .
acr is capable of efficient ethanol production in the presence of  <dig> g/l naac, while the parent zm <dig> is inhibited significantly above  <dig> g/l naac under the same conditions  <cit> .
we have investigated z. mobilis zm <dig> gene expression and metabolomic profiles during aerobic and anaerobic conditions and found that aerobic, stationary phase conditions produced a number of inhibitory secondary metabolites  <cit>  and the expression of a putative hfq gene zmo <dig> was greater in anaerobic stationary phase compared to that of aerobic conditions  <cit> .
hfq is a global regulator that acts as an rna chaperone and is involved in coordinating regulatory responses to multiple stresses  <cit> .
however, little is known about z. mobilis hfq.
the aim of this study was to investigate the role of a putative hfq gene zmo <dig> on multiple pretreatment inhibitor tolerances.
z. mobilis genetic modification has been reported previously with the sacc, adhb, and ndh targets for mutagenesis  <cit> .
however, the existence of native plasmids  <cit>  and intrinsic antibiotic resistance impedes the use of many broad-host-range plasmids  <cit> .
in this work, we identified appropriate antibiotics for z. mobilis genetic studies, created an expression plasmid vector, and utilized the pknock-km suicide plasmid  <cit>  to create an hfq mutant in z. mobilis acetate tolerant strain acr.
we demonstrate that the z. mobilis hfq is important for z. mobilis tolerance to several classes of lignocellulosic pretreatment inhibitors.
hfq is part of an ancient family of proteins termed sm and sm-like  proteins that are conserved among bacteria, archaea, and eukaryotes such as yeast s. cerevisiae  <cit> .
seven core yeast sm proteins form a heteroheptameric ring with a small central hole and are essential  <cit> .
eight lsm proteins  in s. cerevisiae form two different heteroheptameric rings containing either lsm1p or lsm8p with common lsm2p-7p components  <cit> .
the complex containing lsm2-8p localizes to the nucleus and is involved in nuclear rna processing, and the complex containing lsm1-7p contributes to cytoplasmic rna processing  <cit> .
in addition, lsm <dig>  has also been reported to contain a sm domain, as well as other proteins such as lsm <dig> , lsm <dig> , and lsm <dig>   <cit> .
in this study, we also show that s. cerevisiae lsm <dig>   <dig>  and  <dig> proteins contribute to yeast pretreatment inhibitor tolerance.
RESULTS
bioinformatics analysis of a putative z. mobilis hfq and related s. cerevisiae proteins
to assess whether z. mobilis zmo <dig> was similar to other known members of the hfq family of regulators, the zmo <dig> protein sequence was used in a blastp analysis  <cit> .
the blastp result indicates that zmo <dig> is similar to the e. coli global regulator hfq protein , and to eukaryotic homologues such as sm or lsm proteins exist in other microorganisms like s. cerevisiae .
these analyses suggest that zmo <dig> is likely an hfq regulator family protein in z. mobilis.
interestingly, the z. mobilis zmo <dig>  protein possesses two sm-like family domains, two intra-hexamer interaction sites, two inter-hexamer interaction sites, two nucleotide binding pockets, and has an extra sm-like domain near the c-terminus  which is unlike most of the bacterial hfq protein sequences that have only one sm-like domain .
s. cerevisiae has nineteen proteins with a sm or sm-like domain, and although examples like sm protein  and lsm protein   contain sm-like domains, significant sequence similarity was not observed by blastp analysis.
z. mobilis acr strain hfq mutant construction and complementation
intrinsic z. mobilis antibiotic resistance has been reported previously  <cit> , which restricts the use of the available broad-host-range plasmids.
we tested the antibiotic sensitivities of zm <dig> and acr as an initial step for genetic studies with these strains.
each strain was tested against the following antibiotics; chloramphenicol , gentamicin , kanamycin , streptomycin , and tetracycline .
each assay was conducted under aerobic and anaerobic conditions and similar growth results were observed under the respective conditions for the different doses.
z. mobilis was tolerant to streptomycin at concentration of  <dig> μg/ml and gentamicin at  <dig> μg/ml.
z. mobilis was able to grow slightly at  <dig> μg/ml kanamycin and  <dig> μg/ml gentamicin, and was sensitive to tetracycline and chloramphenicol at concentrations above  <dig> μg/ml .
we generated an hfq insertion mutant in a z. mobilis acetate tolerant strain  background using the pknock-km suicide plasmid system  <cit> , and designated it as strain acrim <dig> .
since many mutagenesis systems use either chloramphenicol or kanamycin markers, tetracycline resistance was used as an expression plasmid antibiotic marker for new gateway entry vector pbbr3dest <dig> construction , which was then used to generate plasmid p42- <dig> to express hfq gene zmo <dig>  the nucleotide sequence for plasmid p42- <dig> was verified by sanger sequencing, and the expression of hfq from plasmid p42- <dig> in e. coli was confirmed by western blot analysis .
plasmid p42- <dig> was then introduced into the wild-type strain zm <dig>  acetate tolerant mutant acr, and hfq mutant acrim <dig> background by conjugation and selection.
the presence of correct p42- <dig> construct in ex-conjugants was confirmed by pcr and sanger sequencing analysis.
the oligonucleotide primers used to generate hfq mutant acrim <dig> and complement hfq mutation in acrim <dig> are listed in table  <dig>
hfq_mf and hfq_mr are primers used for insertional mutant construction using pknock mutagenesis system.
hfq_ocf and hfq_ocr are primers for mutant confirmation.
hfq_cf and hfq_cr are primers used to clone the hfq gene into low copy number gate-way compatible plasmid pbbr3dest <dig> for complementation, which results in a plasmid called p42- <dig>
z. mobilis hfq contributes to pretreatment inhibitor tolerance
pretreatment inhibitors had negative effects on z. mobilis growth: the growth of z. mobilis strains was reduced in the presence of acetate, vanillin, furfural, or hmf with increased lag phases and/or slower growth rates and/or final bacterial cell densities depending on the respective condition and strain .
among the different forms of acetate counter-ions tested, sodium acetate had the most inhibitory effect on wild-type z. mobilis growth.
this was followed by potassium acetate and ammonium acetate and sodium chloride had the least negative influence on wild-type z. mobilis growth .
wild-type zm <dig> growth was completely inhibited when rm medium was amended with  <dig> mm sodium acetate  in keeping with previous reports  <cit> .
among the pretreatment inhibitors of vanillin, furfural, and hmf, vanillin had the most inhibitory effect on z. mobilis and hmf the least .
z. mobilis took longer to complete active growth and reach the stationary phase, which was about  <dig>   <dig> or  <dig> h in the presence of hmf, furfural or vanillin, respectively, compared to  <dig> h without any inhibitor present in the medium .
"na" indicates that the data are not available due to the lack of growth in that condition.
the concentration for all the chemicals  supplemented into the rm is  <dig> mm.
nacl: sodium chloride, nh4oac: ammonium acetate, kac: potassium acetate, naac: sodium acetate.
strains included in this study are: zm4: zymomonas mobilis zm <dig> wild-type; acr: previously described zm <dig> acetate tolerant mutant; zm <dig> : zm <dig> containing a gateway plasmid p42- <dig> to express zm <dig> gene zmo0347; acrim0347: acr insertional mutant of zmo0347; acrim <dig> : acrim <dig> containing gateway plasmid p42- <dig>  this experiment has been repeated at least three times with similar result.
duplicate biological replicates were used for each condition.
the concentration for the inhibitor supplemented into the rm is: hmf:  <dig>  g/l, furfural, or vanillin:  <dig> g/l.
strains included in this study are: zm4: zymomonas mobilis zm <dig> wild-type; acr: previously described zm <dig> acetate tolerant mutant; acrim0347: acr insertional mutant of zmo0347; acrim <dig> : acrim <dig> containing gateway plasmid p42- <dig>  this experiment has been repeated at least three times with similar result.
duplicate biological replicates were used for each condition.
hfq contributes to sodium and acetate ion tolerances: although the final cell density of hfq mutant acrim <dig> is similar to that of acr parental strain , the growth rate of acrim <dig> was reduced about one-fifth even without any inhibitor in the rm, which indicates hfq plays a central role in normal z. mobilis physiology.
wild-type zm <dig> that contained p42- <dig> was able to grow in the presence of  <dig> mm sodium acetate and had a similar growth rate and final cell density to that of acetate tolerant strain acr .
the wild-type zm <dig> was unable to grow under this condition.
the inactivation of the hfq gene in acr decreased this acetate tolerant strain's resistance to both sodium ion  and acetate ion  .
hfq mutant acrim <dig> was unable to grow in the presence of  <dig> mm ammonium acetate or potassium acetate .
both the growth rate and final cell density of hfq mutant acrim <dig> were reduced by at least a quarter in the presence of  <dig> mm sodium chloride, and about 60% in the presence of  <dig> mm sodium acetate compared to that of the parental strain acr .
the acrim <dig> hfq mutation was complemented by the introduction of an hfq-expressing plasmid  into the strain.
the complemented mutant strain recovered at least half of the parental strains growth rate and 70% of its final cell density in the presence of  <dig> mm acetate ion  .
hfq contributes to vanillin, furfural and hmf tolerances: acrim <dig> growth rates were lower than that of zm <dig> and acr under all conditions tests, and except for growth in rm broth .
acrim <dig> also achieved lower final cell densities compared to zm <dig> and acr .
when acrim <dig> was provided functional z. mobilis hfq via p42- <dig>  growth rates under all conditions were largely unchanged .
however, shorter lag phases were observed for acrim <dig>  grown with vanillin, furfural or hmf and increases in final cell densities were also observed under these conditions .
these data indicate that hfq is important for optimal z. mobilis growth and its ability to resist furfural, hmf and vanillin toxicity.
yeast lsm proteins contribute to pretreatment inhibitor tolerance
lsm protein and yeast tolerance to sodium and acetate ions
s. cerevisiae sm and sm-like  proteins are similar to z. mobilis hfq at the level of protein sequence .
growth of yeast lsm deletion mutants and lsm over-expressing strains in  <dig> mm ammonium acetate, potassium acetate, or sodium acetate was assessed to test whether s. cerevisiae lsm proteins and zm <dig> hfq had functionally similar roles.
these studies included seven lsm deletion mutants affecting three lsm heteroheptameric ring components  and four other lsm proteins containing an sm domain , as well as six lsm protein over-expressing strains .
we present growth data for those genes that gave clear phenotypic differences for the sake of clarity and a list of all strains tested in this study can be found in table  <dig>
growth differences between the lsm mutants and yeast wild-type by <dig> in the cm broth without the addition of acetate or with  <dig> mm nacl were not observed .
s. cerevisiae lsm proteins involved in rna processing ring complex formation , especially lsm <dig>  played a role in acetate tolerance .
lsm protein deletion mutants lsm <dig>   <dig>  and  <dig> showed decreased acetate tolerance compared to the wild-type control strain, especially in early growth stages for acetate with sodium, ammonium and potassium counter-ions .
the lsm overexpression strains grew similarly to wild-type by <dig> without the addition of acetate or with  <dig> mm nacl , but each of the lsm protein overexpression strains showed enhanced acetate tolerance compared to the wild-type strain with sodium, ammonium or potassium counter-ions .
lsm proteins and yeast tolerance to vanillin, furfural and hmf
the effect of lsm proteins on s. cerevisiae tolerance to pretreatment inhibitors vanillin, furfural, and hmf was also investigated using the seven lsm deletion mutants and six lsm overexpression strains described above.
each yeast deletion mutant and each overexpression strain showed similar growth profiles compared to wild-type strain by <dig> in the absence of inhibitors .
deletion mutants for lsm <dig>   <dig> and  <dig> proteins were unable to grow or showed extended lag phases before recovery from the inhibitory effects of pretreatment inhibitors .
overexpression of lsm proteins provided a slight growth advantage in the presence of  <dig>  g/l hmf and furfural .
however, a detrimental effect on growth was observed for overexpression strains when cultured in the presence of  <dig>  g/l vanillin .
the data indicated that lsm proteins lsm <dig>   <dig>  and  <dig> especially lsm <dig>  which are the components of yeast rna processing ring complex, play a role in tolerance to the model inhibitors used in this study.
discussion
a previous study indicated that z. mobilis zm <dig> hfq was less abundant in aerobic, stationary phase fermentations compared to the equivalent anaerobic condition and that rpoh was induced under the aerobic condition  <cit> .
the role of z. mobilis regulators like hfq and extent of cross talk between regulatory networks remains to be elucidated.
this study indicated that hfq also plays a role in z. mobilis resistance to both acetate  and sodium ions  .
a recent study has identified that nhaa overexpression  conferred the previously reported acr  mutant phenotype  <cit> .
constitutive nhaa over-expression in strain acrim <dig>  is a likely possibility for it being unable to survive with  <dig> mm ammonium acetate or potassium acetate, while the same concentration of sodium acetate only partially repressed its growth.
hfq or nhaa each contribute to sodium acetate tolerance   <cit> , but there is no additive benefit for increased inhibitor tolerance for hfq and nhaa if both were over-expressed at the same time .
in addition, the overexpression of nhaa gene in z. mobilis had no advantage over other physiological stress responses for model pretreatment inhibitors such as vanillin, furfural, and hmf  <cit> .
while z. mobilis hfq contributes to the tolerance of these inhibitors as shown by increased hfq mutant acrim <dig> lag phases and slower growth rates during early logarithmic growth phase compared to acr strain .
these separate studies indicate there may often be more than one pathway for industrial strain development.
the majority of proteins similar to z. mobilis hfq contained one sm-like superfamily domain , with the exception of those from six other species also within the sphingomonadales.
future structural studies are required to define the role for z. mobilis and other microorganisms with two sm-like family domains, to elucidate hfq subunit interactions, and to test whether only three hfq proteins would be needed for z. mobilis to form the active homo-hexameric ring structure.
we assayed growth phenotypes for s. cerevisiae lsm protein mutant and overexpression phenotypes.
lsm <dig>   <dig>  and  <dig> mutants showed reduced tolerance to acetate and other pretreatment inhibitors .
the s. cerevisiae lsm over-expression studies showed these strains had increased acetate and hmf resistance compared to the wild-type strain, while the overexpression strains were more inhibited under vanillin stress conditions .
the conserved nature of sm-like proteins, the involvement of zm <dig> hfq and s. cerevisiae sm-like proteins in pretreatment inhibitor tolerance offers the possibility of manipulating hfq and members of the lsm family for strain development purposes.
CONCLUSIONS
we created a plasmid for gene expression and mutation complementation in z. mobilis and used the pknock system to create an hfq mutant in z. mobilis acetate tolerant strain acr.
we showed that z. mobilis hfq played a role in tolerance to multiple biomass pretreatment inhibitors including acetate, vanillin, furfural, and hmf.
in addition, hfq homologues of yeast lsm proteins lsm <dig>   <dig>  and  <dig> involving in the rna processing heteroheptameric ring complex formation, especially lsm <dig>  contribute to multiple pretreatment inhibitor tolerance in s. cerevisiae.
however, further studies such as systems biology studies and chip-seq are required to elucidate the hfq stress response regulon in z. mobilis and the yeast inhibitor tolerance genes affected by the rna processing lsm complexes.

 biomass-based bioenergy is crucial to meet national goals of making cellulosic ethanol cost-competitive with gasoline. a core challenge in fermenting cellulosic material to ethanol is the recalcitrance of biomass to breakdown. severe biomass pretreatments are therefore required to release the sugars, which along with by-products of fermentation can create inhibitors including sugar degradation products such as furfural and hydroxymethylfurfural; weak acids such as acetic, formic, and levulinic acids; lignin degradation products such as the substituted phenolics vanillin and lignin monomers. in addition, the metabolic byproducts such as ethanol, lactate, and acetate also influence the fermentation by slowing and potentially stopping the fermentation prematurely. the increased lag phase and slower growth increases the ethanol cost due to both ethanol production rate and total ethanol yield decreases. one approach to overcome the issue of inhibition caused by pretreatment processes is to remove the inhibitor after pretreatment from the biomass physically or chemically, which requires extra equipment and time leading to increased costs. a second approach utilizes inhibitor tolerant microorganisms for efficient fermentation of lignocellulosic material to ethanol and their utility is considered an industrial requirement. z. mobilis are gram-negative facultative anaerobic bacteria with a number of desirable industrial characteristics, such as high-specific productivity and ethanol yield, unique anaerobic use of the entner-doudoroff pathway that results in low cell mass formation, high ethanol tolerance, ph - range for ethanol production and has a generally regarded as safe status. z. mobilis strains have been engineered to ferment pentose sugars such as xylose, arabinose and other substrates with high yields, but a low tolerance to acetic acid and decreased ethanol tolerance have been reported in recombinant strains. z. mobilis mutant strains tolerant to a pretreatment inhibitor such as acetate have been generated by chemical mutagenesis with n-methyl n'-nitro n-nitrosoguanidine and selection in continuous culture with a progressively increasing concentration of sodium acetate in the medium feed. acr is capable of efficient ethanol production in the presence of g/l naac, while the parent zm is inhibited significantly above g/l naac under the same conditions. we have investigated z. mobilis zm gene expression and metabolomic profiles during aerobic and anaerobic conditions and found that aerobic, stationary phase conditions produced a number of inhibitory secondary metabolites and the expression of a putative hfq gene zmo was greater in anaerobic stationary phase compared to that of aerobic conditions. hfq is a global regulator that acts as an rna chaperone and is involved in coordinating regulatory responses to multiple stresses. however, little is known about z. mobilis hfq. the aim of this study was to investigate the role of a putative hfq gene zmo on multiple pretreatment inhibitor tolerances. z. mobilis genetic modification has been reported previously with the sacc, adhb, and ndh targets for mutagenesis. however, the existence of native plasmids and intrinsic antibiotic resistance impedes the use of many broad-host-range plasmids. in this work, we identified appropriate antibiotics for z. mobilis genetic studies, created an expression plasmid vector, and utilized the pknock-km suicide plasmid to create an hfq mutant in z. mobilis acetate tolerant strain acr. we demonstrate that the z. mobilis hfq is important for z. mobilis tolerance to several classes of lignocellulosic pretreatment inhibitors. hfq is part of an ancient family of proteins termed sm and sm-like proteins that are conserved among bacteria, archaea, and eukaryotes such as yeast s. cerevisiae. seven core yeast sm proteins form a heteroheptameric ring with a small central hole and are essential. eight lsm proteins in s. cerevisiae form two different heteroheptameric rings containing either lsm1p or lsm8p with common lsm2p-7p components. the complex containing lsm2-8p localizes to the nucleus and is involved in nuclear rna processing, and the complex containing lsm1-7p contributes to cytoplasmic rna processing. in addition, lsm has also been reported to contain a sm domain, as well as other proteins such as lsm, lsm, and lsm. in this study, we also show that s. cerevisiae lsm and proteins contribute to yeast pretreatment inhibitor tolerance.  bioinformatics analysis of a putative z. mobilis hfq and related s. cerevisiae proteins to assess whether z. mobilis zmo was similar to other known members of the hfq family of regulators, the zmo protein sequence was used in a blastp analysis. the blastp result indicates that zmo is similar to the e. coli global regulator hfq protein, and to eukaryotic homologues such as sm or lsm proteins exist in other microorganisms like s. cerevisiae. these analyses suggest that zmo is likely an hfq regulator family protein in z. mobilis. interestingly, the z. mobilis zmo protein possesses two sm-like family domains, two intra-hexamer interaction sites, two inter-hexamer interaction sites, two nucleotide binding pockets, and has an extra sm-like domain near the c-terminus which is unlike most of the bacterial hfq protein sequences that have only one sm-like domain. s. cerevisiae has nineteen proteins with a sm or sm-like domain, and although examples like sm protein and lsm protein contain sm-like domains, significant sequence similarity was not observed by blastp analysis. z. mobilis acr strain hfq mutant construction and complementation intrinsic z. mobilis antibiotic resistance has been reported previously, which restricts the use of the available broad-host-range plasmids. we tested the antibiotic sensitivities of zm and acr as an initial step for genetic studies with these strains. each strain was tested against the following antibiotics; chloramphenicol, gentamicin, kanamycin, streptomycin, and tetracycline. each assay was conducted under aerobic and anaerobic conditions and similar growth results were observed under the respective conditions for the different doses. z. mobilis was tolerant to streptomycin at concentration of μg/ml and gentamicin at μg/ml. z. mobilis was able to grow slightly at μg/ml kanamycin and μg/ml gentamicin, and was sensitive to tetracycline and chloramphenicol at concentrations above μg/ml. we generated an hfq insertion mutant in a z. mobilis acetate tolerant strain background using the pknock-km suicide plasmid system, and designated it as strain acrim. since many mutagenesis systems use either chloramphenicol or kanamycin markers, tetracycline resistance was used as an expression plasmid antibiotic marker for new gateway entry vector pbbr3dest construction, which was then used to generate plasmid p42- to express hfq gene zmo the nucleotide sequence for plasmid p42- was verified by sanger sequencing, and the expression of hfq from plasmid p42- in e. coli was confirmed by western blot analysis. plasmid p42- was then introduced into the wild-type strain zm acetate tolerant mutant acr, and hfq mutant acrim background by conjugation and selection. the presence of correct p42- construct in ex-conjugants was confirmed by pcr and sanger sequencing analysis. the oligonucleotide primers used to generate hfq mutant acrim and complement hfq mutation in acrim are listed in table hfq_mf and hfq_mr are primers used for insertional mutant construction using pknock mutagenesis system. hfq_ocf and hfq_ocr are primers for mutant confirmation. hfq_cf and hfq_cr are primers used to clone the hfq gene into low copy number gate-way compatible plasmid pbbr3dest for complementation, which results in a plasmid called p42- z. mobilis hfq contributes to pretreatment inhibitor tolerance pretreatment inhibitors had negative effects on z. mobilis growth: the growth of z. mobilis strains was reduced in the presence of acetate, vanillin, furfural, or hmf with increased lag phases and/or slower growth rates and/or final bacterial cell densities depending on the respective condition and strain. among the different forms of acetate counter-ions tested, sodium acetate had the most inhibitory effect on wild-type z. mobilis growth. this was followed by potassium acetate and ammonium acetate and sodium chloride had the least negative influence on wild-type z. mobilis growth. wild-type zm growth was completely inhibited when rm medium was amended with mm sodium acetate in keeping with previous reports. among the pretreatment inhibitors of vanillin, furfural, and hmf, vanillin had the most inhibitory effect on z. mobilis and hmf the least. z. mobilis took longer to complete active growth and reach the stationary phase, which was about or h in the presence of hmf, furfural or vanillin, respectively, compared to h without any inhibitor present in the medium. "na" indicates that the data are not available due to the lack of growth in that condition. the concentration for all the chemicals supplemented into the rm is mm. nacl: sodium chloride, nh4oac: ammonium acetate, kac: potassium acetate, naac: sodium acetate. strains included in this study are: zm4: zymomonas mobilis zm wild-type; acr: previously described zm acetate tolerant mutant; zm: zm containing a gateway plasmid p42- to express zm gene zmo0347; acrim0347: acr insertional mutant of zmo0347; acrim: acrim containing gateway plasmid p42- this experiment has been repeated at least three times with similar result. duplicate biological replicates were used for each condition. the concentration for the inhibitor supplemented into the rm is: hmf: g/l, furfural, or vanillin: g/l. strains included in this study are: zm4: zymomonas mobilis zm wild-type; acr: previously described zm acetate tolerant mutant; acrim0347: acr insertional mutant of zmo0347; acrim: acrim containing gateway plasmid p42- this experiment has been repeated at least three times with similar result. duplicate biological replicates were used for each condition. hfq contributes to sodium and acetate ion tolerances: although the final cell density of hfq mutant acrim is similar to that of acr parental strain, the growth rate of acrim was reduced about one-fifth even without any inhibitor in the rm, which indicates hfq plays a central role in normal z. mobilis physiology. wild-type zm that contained p42- was able to grow in the presence of mm sodium acetate and had a similar growth rate and final cell density to that of acetate tolerant strain acr. the wild-type zm was unable to grow under this condition. the inactivation of the hfq gene in acr decreased this acetate tolerant strain's resistance to both sodium ion and acetate ion. hfq mutant acrim was unable to grow in the presence of mm ammonium acetate or potassium acetate. both the growth rate and final cell density of hfq mutant acrim were reduced by at least a quarter in the presence of mm sodium chloride, and about 60% in the presence of mm sodium acetate compared to that of the parental strain acr. the acrim hfq mutation was complemented by the introduction of an hfq-expressing plasmid into the strain. the complemented mutant strain recovered at least half of the parental strains growth rate and 70% of its final cell density in the presence of mm acetate ion. hfq contributes to vanillin, furfural and hmf tolerances: acrim growth rates were lower than that of zm and acr under all conditions tests, and except for growth in rm broth. acrim also achieved lower final cell densities compared to zm and acr. when acrim was provided functional z. mobilis hfq via p42- growth rates under all conditions were largely unchanged. however, shorter lag phases were observed for acrim grown with vanillin, furfural or hmf and increases in final cell densities were also observed under these conditions. these data indicate that hfq is important for optimal z. mobilis growth and its ability to resist furfural, hmf and vanillin toxicity. yeast lsm proteins contribute to pretreatment inhibitor tolerance lsm protein and yeast tolerance to sodium and acetate ions s. cerevisiae sm and sm-like proteins are similar to z. mobilis hfq at the level of protein sequence. growth of yeast lsm deletion mutants and lsm over-expressing strains in mm ammonium acetate, potassium acetate, or sodium acetate was assessed to test whether s. cerevisiae lsm proteins and zm hfq had functionally similar roles. these studies included seven lsm deletion mutants affecting three lsm heteroheptameric ring components and four other lsm proteins containing an sm domain, as well as six lsm protein over-expressing strains. we present growth data for those genes that gave clear phenotypic differences for the sake of clarity and a list of all strains tested in this study can be found in table growth differences between the lsm mutants and yeast wild-type by in the cm broth without the addition of acetate or with mm nacl were not observed. s. cerevisiae lsm proteins involved in rna processing ring complex formation, especially lsm played a role in acetate tolerance. lsm protein deletion mutants lsm and showed decreased acetate tolerance compared to the wild-type control strain, especially in early growth stages for acetate with sodium, ammonium and potassium counter-ions. the lsm overexpression strains grew similarly to wild-type by without the addition of acetate or with mm nacl, but each of the lsm protein overexpression strains showed enhanced acetate tolerance compared to the wild-type strain with sodium, ammonium or potassium counter-ions. lsm proteins and yeast tolerance to vanillin, furfural and hmf the effect of lsm proteins on s. cerevisiae tolerance to pretreatment inhibitors vanillin, furfural, and hmf was also investigated using the seven lsm deletion mutants and six lsm overexpression strains described above. each yeast deletion mutant and each overexpression strain showed similar growth profiles compared to wild-type strain by in the absence of inhibitors. deletion mutants for lsm and proteins were unable to grow or showed extended lag phases before recovery from the inhibitory effects of pretreatment inhibitors. overexpression of lsm proteins provided a slight growth advantage in the presence of g/l hmf and furfural. however, a detrimental effect on growth was observed for overexpression strains when cultured in the presence of g/l vanillin. the data indicated that lsm proteins lsm and especially lsm which are the components of yeast rna processing ring complex, play a role in tolerance to the model inhibitors used in this study. discussion a previous study indicated that z. mobilis zm hfq was less abundant in aerobic, stationary phase fermentations compared to the equivalent anaerobic condition and that rpoh was induced under the aerobic condition. the role of z. mobilis regulators like hfq and extent of cross talk between regulatory networks remains to be elucidated. this study indicated that hfq also plays a role in z. mobilis resistance to both acetate and sodium ions. a recent study has identified that nhaa overexpression conferred the previously reported acr mutant phenotype. constitutive nhaa over-expression in strain acrim is a likely possibility for it being unable to survive with mm ammonium acetate or potassium acetate, while the same concentration of sodium acetate only partially repressed its growth. hfq or nhaa each contribute to sodium acetate tolerance, but there is no additive benefit for increased inhibitor tolerance for hfq and nhaa if both were over-expressed at the same time. in addition, the overexpression of nhaa gene in z. mobilis had no advantage over other physiological stress responses for model pretreatment inhibitors such as vanillin, furfural, and hmf. while z. mobilis hfq contributes to the tolerance of these inhibitors as shown by increased hfq mutant acrim lag phases and slower growth rates during early logarithmic growth phase compared to acr strain. these separate studies indicate there may often be more than one pathway for industrial strain development. the majority of proteins similar to z. mobilis hfq contained one sm-like superfamily domain, with the exception of those from six other species also within the sphingomonadales. future structural studies are required to define the role for z. mobilis and other microorganisms with two sm-like family domains, to elucidate hfq subunit interactions, and to test whether only three hfq proteins would be needed for z. mobilis to form the active homo-hexameric ring structure. we assayed growth phenotypes for s. cerevisiae lsm protein mutant and overexpression phenotypes. lsm and mutants showed reduced tolerance to acetate and other pretreatment inhibitors. the s. cerevisiae lsm over-expression studies showed these strains had increased acetate and hmf resistance compared to the wild-type strain, while the overexpression strains were more inhibited under vanillin stress conditions. the conserved nature of sm-like proteins, the involvement of zm hfq and s. cerevisiae sm-like proteins in pretreatment inhibitor tolerance offers the possibility of manipulating hfq and members of the lsm family for strain development purposes.  we created a plasmid for gene expression and mutation complementation in z. mobilis and used the pknock system to create an hfq mutant in z. mobilis acetate tolerant strain acr. we showed that z. mobilis hfq played a role in tolerance to multiple biomass pretreatment inhibitors including acetate, vanillin, furfural, and hmf. in addition, hfq homologues of yeast lsm proteins lsm and involving in the rna processing heteroheptameric ring complex formation, especially lsm contribute to multiple pretreatment inhibitor tolerance in s. cerevisiae. however, further studies such as systems biology studies and chip-seq are required to elucidate the hfq stress response regulon in z. mobilis and the yeast inhibitor tolerance genes affected by the rna processing lsm complexes. 
our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both z. mobilis and s. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then hmf for both z. mobilis and s. cerevisiae. saccharomyces cerevisiae lsm protein mutants and lsm protein overexpression strains were also assayed for their inhibitor phenotypes. s. cerevisiae lsm protein overexpression strains showed increased acetate and hmf resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions. we also assayed growth phenotypes for s. cerevisiae lsm protein mutant and overexpression phenotypes. in a previous study, we observed z. mobilis zm gene zmo showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions. the conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development. we generated a z. mobilis hfq insertion mutant acrim in an acetate tolerant strain background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural. we have also used genetics to show z. mobilis hfq and s. cerevisiae lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. we have shown the utility of the pknock suicide plasmid for mutant construction in z. mobilis, and constructed a gateway compatible expression plasmid for use in z. mobilis for the first time. lsm and mutants showed reduced tolerance to acetate and other pretreatment inhibitors.
 zymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. to date, few studies have examined its responses to various stresses at the gene level. hfq is a conserved bacterial member of the sm-like family of rna-binding proteins, coordinating a broad array of responses including multiple stress responses. in a previous study, we observed z. mobilis zm gene zmo showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions.  we generated a z. mobilis hfq insertion mutant acrim in an acetate tolerant strain background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural. saccharomyces cerevisiae lsm protein mutants and lsm protein overexpression strains were also assayed for their inhibitor phenotypes. our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both z. mobilis and s. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then hmf for both z. mobilis and s. cerevisiae. acrim was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. the hfq mutation in acrim was complemented partially by trans-acting hfq gene expression. we also assayed growth phenotypes for s. cerevisiae lsm protein mutant and overexpression phenotypes. lsm and mutants showed reduced tolerance to acetate and other pretreatment inhibitors. s. cerevisiae lsm protein overexpression strains showed increased acetate and hmf resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions.  we have shown the utility of the pknock suicide plasmid for mutant construction in z. mobilis, and constructed a gateway compatible expression plasmid for use in z. mobilis for the first time. we have also used genetics to show z. mobilis hfq and s. cerevisiae lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. the conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development. 