Evidence for ammonia as an inhibitor of heterocyst and nitrogenase formation in the cyanobacterium Anabaena cycadeae

Growth and regulation of heterocyst and nitrogenase by fixed nitrogen sources were studied comparatively in parent and glutamine auxotrophic mutant of Anabaena cycadeae. The parent strain grew well on N2, NH+4 or glutamine while the mutant strain grew on glutamine but not on N2 or NH+4. The total lack of active glutamine synthetase in the mutant strain thus appears to be the reason for its observed lack of growth in N2 or NH+4, which explains why it is a glutamine auxotroph and at the same time shows glutamine synthetase to be the sole primary ammonia assimilating enzyme. NH+4 repression of heterocyst and nitrogenase in the mutant and the parental strains and their derepression by L-methionine-DL-sulfoximine suggest that NH+4 per se and not glutamine synthetase mediated pathway of ammonia assimilation is the initial repressor signal of heterocyst and nitrogenase in A. cycadeae.     

Potential roles for the glnB and ntrYX genes in Azospirillum brasilense

Three Azospirillum brasilense mutants constitutive for nitrogen fixation (Nif(C)) in the presence of NH4(+) and deficient in nitrate-dependent growth were used as tools to define the roles of the glnB and ntrYX genes in this organism. Mutant HM14 was complemented for nitrate-dependent growth and NH4(+) regulation of nitrogenase by plasmid pL46 which contains the ntrYX genes of A. brasilense. Mutant HM26 was restored for NH4(+) regulation and nitrate-dependent growth by plasmid pJC1, carrying the A. brasilense glnB gene expressed from a constitutive promoter. Mutant HM053, on the other hand, was not complemented for NH4(+) regulation of nitrogenase and nitrate-dependent growth by both plasmids pJCI and pL46. The levels and control of glutamine synthetase activity of all mutants were not affected by both plasmids pL46 (ntrYX) and pJC1 (glnB). These results support the characterization of strains HM14 as an ntrYX mutant and strain HM26 as a glnB mutant and the involvement of ntrYX and glnB in the regulation of the general nitrogen metabolism in A. brasilense.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium.

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH+4 is in equiblibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in gamma-glutamyl transferase assays containing 60 mM Mg2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of gamma-glutamyl transferase activities without and with addition of 60 mM Mg2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH+4, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH+4 produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Regulation of Nitrogen Fixation in Klebsiella pneumoniae: Evidence for a Role of Glutamine Synthetase as a Regulator of Nitrogenase Synthesis

Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae.

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.     

Photoproduction of ammonium ion from N2 in Rhodospirillum rubrum.

NH+4 excretion was undetectable in N2-fixing cultures of Rhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH+4 to the medium. The glutamate analog, L-methionine-DL-sulfoximine (MSX), derepressed N2 fixation even in the presence of 10 mM extracellular NH+4. When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N2 as NH+4. Nitrogenase activities and NH+4 production from fixed N2 were increased considerably when a combined nitrogen source, NH+4 (greater than 40 mumoles NH+4/mg cell protein in 6 days) or L-glutamate (greater than 60 mumoles NH+4/ mg cell protein in 6 days) was added to the cultures together with MSX. Biochemical analysis revealed that R. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH+4 as well as by glutamate. The results demonstrate that utilization of solar energy to photoproduce large quantities of NH+4 from N2 is possible with photosynthetic bacteria by interfering with their regulatory control of N2 fixation.     

An azide-resistant mutant of the blue-green algaNostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source

The N₂, NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine growing cultures of parentNostoc muscorum are found more or less equally sensitive to azide inhibition of growth. A mutant strain resistant to sodium azide was isolated from the parent strain in NO ₃ ^– medium and the two strains were compared with regard to their heterocyst formation and nitrogenase activity in NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine media. While the parent strain stops production of both heterocyst and nitrogenase in all the fixed nitrogen media, the azide resistant strain forms both in the fixed inorganic nitrogen media but only heterocyst and no nitrogenase in the glutamine medium. Clearly a single genetic determinant of regulatory nature appears to mediate azide-resistance as well as relief of heterocyst and nitrogenase formation from inhibition by the fixed inorganic nitrogen source. The results of glutamine effect on the heterocyst and nitrogenase formation of the two strains indicate the operation of two levels of glutamine-sensitive regulation, one which operates through the common genetic determinant of heterocyst and nitrogenase regulation and the other exclusive to nitrogenase regulation. The in vivo functional nitrogenase does not appear to be the reason for azide-resistance and neither ammonia nor glutamine or its close metabolic product seems to function in the control of heterocyst spacing.     

Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae.

Nitrogenase biosynthesis in Klebsiella pneumoniae including mutant strains, which produce nitrogenase in the presence of NH+4 (Shanmugam, K.T., Chan, Irene, and Morandi, C. (1975) Biochim. Biophys. Acta 408, 101--111) is repressed by a mixture of L-amino acids. Biochemical analysis shows that glutamine synthetase activity in strains SK-24, SK-28, and SK-29 is also repressed by amino acids, with no detectable effect on glutamate dehydrogenase. Among the various amino acids, L-glutamine in combination with L-aspartate was found to repress nitrogenase biosynthesis completely. In the presence of high concentrations of glutamine (1 mg/ml) even NH+4 repressed nitrogenase biosynthesis in the strains SK-27, SK-37, SK-55 and SK-56. Under these conditions, increased glutamate dehydrogenase activity was also detected. Physiological studies show that nitrogenase derepressed strains are unable to utilize NH+4 as sole source of nitrogen for biosynthesis of glutamate for biosynthesis of glutamate, whereas back mutations leading to NH+4 utilization results in sensitivity to repression by NH+4. These findings suggest that amino acids play an important role as regulators of nitrogen fixation.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Mutants of the Cyanobacterium Anabaena sp. PCC 7120 Altered in Nitrate Transport and Reduction

Nitrate assimilation-defective mutants SP7, SP9, and SP17 of the cyanobacterium Anabaena sp. PCC 7120 were isolated by use of transposon mutagenesis and screened on medium containing chlorate. SP7 and SP17 represented nitrate reductase-defective nature, while mutant SP9 appeared to be a regulatory mutant exhibiting pleiotropic behavior. Kinetics of nitrate uptake system exhibited K _s values of 31–38 μM for parent, SP7, and SP17 strains; however, mutant SP9 exhibited a high K _s value of 109.5 μM. Defective nitrate reductase was apparent in mutant SP7 and SP9, while mutant SP17 exhibited partial defective nature. Methyl viologen-dependent NR activity in parent strain presented a biphasic nature with K _m values of 0.13 and 2.47 mM, whereas a single K _m value (2.96 mM) was observed for mutant SP17. Mutant SP9 was also defective in nitrite uptake and reduction. Mutant strains exhibited derepressed nitrogenase activity in the presence of nitrate, while glutamine synthetase activity remained unaltered. Received: 20 April 1999 / Accepted: 22 May 1999     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het+ nif-) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N2-fixing blue-green algae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

The photoproduction of H2 and NH4 fixed from N2 by a derepressed mutant of Rhodospirillum rubrum.

A mutant of Rhodospirillum rubrum has been isolated, after mutagenesis with nitrosoguanidine, which is characterized by its inability to grow in the light on malate-minimal media with exogenous ammonia or alanine, poor growth on glutamine and vigorous growth on glutamate. This mutant produces low levels of a key NH+4 assimilation enzyme, glutamate synthase (NADPH-dependent). It also exhibits significant derepression of nitrogenase biosynthesis in the presence of ammonia or alanine, being 15% derepressed for the former and about 70% derepressed for the latter. Some of this mutant's fixed N2 is excreted into the medium as NH+4 (1 mumol NH+4 per mg cell protein in 50 h). Nitrogenase-mediated H2 production by this strain is considerable (42 mumol H2 per mg cell protein in 50 h), approximately twice that of the wild type assayed under similar conditions. These results demonstrate that genetic alteration of the photosynthetic N2-fixer's NH+4 assimilation system disrupts the tight coupling of N2 fixation and NH+4 assimilation normally observed in these organisms, enabling photochemical conversion steps to be utilized for the photoproduction of NH+4 and H2.     

Ineffective and non-nodulating mutant strains of Rhizobium japonicum.

Mutant strains of Rhizobium japonicum that were unable to allow the Corsoy cultivar of soybean to reduce acetylene or fix N2 were isolated. These strains grow as well as the wild type in a variety of media. Mutant strains SM1 and SM2 did not form nodules on the host plant; however, they reduced acetylene in the nonsymbiotic assay. Strains SM3 and SM4 produced nodules that did not have the characteristic pink pigment caused by leghemoglobin. The nodules formed by these strains also were small. One mutant strain, SM5, produced large pink nodules. The lesion in this strain seems to be in the gene that specifies nitrogenase component II.     

Ammonium uptake by nitrogen fixing bacteria I. Azotobacter vinelandii.

Both the changes in the activities of nitrogenase, glutamine synthetase and glutamate dehydrogenase and in the extracellular and intracellular NH4+ concentrations were investigated during the transition from an NH4+ free medium to one containing NH4+ ions for a continuous culture of Azotobacter vinelandii. If added in amounts causing 80-100% repression of nitrogenase, ammonium acetate, lactate and phosphate are absorbed completely, whereas chloride, sulfate and citrate are only taken up to about 80%. After about 1-2 hrs the NH4+ remaining in the medium is absorbed too, indicating the induction or activation of a new NH4+ transport system. One of the new permeases allows the uptake of citrate in the presence of sucrose. Addition of inorganic NH4+ level leads to a reversible rise in the glutamine synthetase activity which is not prevented by chloramphenicol, and to a reversible decrease in nitrogenase activity. During these measurements glutamate dehydrogenase activity remains close to zero. The intracellular NH4+ level of about 0.6 mM does not change when extracellular NH4+ is taken up and repression of nitrogenase starts.     

Growth of Pseudomonas aeruginosa mutants lacking glutamate synthase activity.

Mutant strains SU1, SU4, and US1 lacking glutamate synthase (GOGAT) activity were isolated from strains of P. aeruginosa for which histidine is a growth rate-limiting source of nitrogen. Strains SU1 and SU4 were unable to grow when a low concentration of ammonia and a variety of compounds, including histidine, were supplied as sole sources of nitrogen. A revertant of strain SU1, strain 39, produced no GOGAT but high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase and had restored ability to grow on a limited number of nitrogen sources. Strain US1 grew at the same rate in histidine medium as did its parent; it was derepressed for glutamine synthase synthesis, and histidase was less sensitive to repression by ammonia than in the parent strain. We conclude that GOGAT is not essential for growth on histidine but high levels of glutamine synthase are required nd high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase can sustain growth at low concentrations of ammonia in the absence of GOGAT.     

Nitrogenase synthesis in Klebsiella pneumoniae: comparison of ammonium and oxygen regulation.

Rates of nitrogenase synthesis by Klebsiella pneumoniae were measured by pulse-labelling organisms with a mixture of 14C-labelled amino acids followed by sodium dodecyl sulphate gel electrophoresis and autoradiography. Populations from an NH4+-repressed, SO42--limited chemostat (0.46 mg dry wt ml-1), when released from NH4+ repression, simultaneously synthesized detectable quantities of the three nitrogenase polypeptides 45 min before acetylene-reducing activity was observed. Exposure of populations synthesizing nitrogenase to air or NH4+ (200 microgram N ml-1) repressed synthesis of both component proteins simultaneously, the rate initially decreasing by half in 11 to 12 min; in the presence of NH4+ a second slower phase with an approximate half-life of 30 min was observed. With 5% O2 in N2 the half-lives for the decreases in the rates of synthesis were 30 min for the Fe protein and 33 min for the Mo-Fe protein. Oxygen also repressed nitrogenase in a glutamine synthetase constitutive derivative of K. pneumoniae (strain SK24) which escapes NH4+ repression. Regulation of nitrogenase by O2 may therefore be independent of glutamine synthetase.     

Nitrogenase synthesis in Klebsiella pneumoniae: enhanced nif expression without accumulation of guanosine 5'-diphosphate 3'-diphosphate

Derepression of nitrogen fixation (nif) genes in Klebsiella pneumoniae following transfer from NH+4-sufficiency to N-free medium was preceded by rapid expansion of the guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool. When derepressed in N-free medium supplemented with glutamine (600 micrograms ml-1), expression from the nifH and nifL promoters, determined as beta-galactosidase activity in nif::lac merodiploid strains, was stimulated 7-fold and nitrogenase activity 26-fold; ppGpp did not accumulate, remaining at the levels found in NH+4-repressed populations. The relaxed mutant K. pneumoniae relA40, which accumulates only very low levels of ppGpp, showed partial derepression of nitrogenase activity in the presence of glutamine, thus ppGpp is unlikely to be an effector of nif expression. ATP and GTP levels were elevated under conditions where nif expression was enhanced, consistent with previous data suggesting that maintenance of ATP levels is a prerequisite for the expression of nif genes in K. pneumoniae.     

Ammonium and methylammonium transport in Rhodobacter sphaeroides.

Rhodobacter sphaeroides maintained intracellular ammonium pools of 1.1 to 2.6 mM during growth in several fixed nitrogen sources as well as during diazotrophic growth. Addition of 0.15 mM NH4+ to washed, nitrogen-free cell suspensions was followed by linear uptake of NH4+ from the medium and transient formation of intracellular pools of 0.9 to 1.5 mM NH4+. Transport of NH4+ was shown to be independent of assimilation by glutamine synthetase because intracellular pools of over 1 mM represented NH4+ concentration gradients of at least 100-fold across the cytoplasmic membrane. Ammonium pools of over 1 mM were also found in non-growing cell suspensions in nitrogen-free medium after glutamine synthetase was inhibited with methionine sulfoximine. In NH4+-free cell suspensions, methylammonium (14CH3NH3+) was taken up rapidly, and intracellular concentrations of 0.4 to 0.5 mM were maintained. The 14CH3NH3+ pool was not affected by methionine sulfoximine. Unlike NH4+ uptake, 14CH3NH3+ uptake in nitrogen-free cell suspensions was repressed by growth in NH4+. These results suggest that R. sphaeroides may produce an NH4+-specific transport system in addition to the NH4+/14CH3NH3+ transporter. This second transporter is able to produce normal-size NH4+ pools but has very little affinity for 14CH3NH3+ and is not repressed by growth in high concentrations of NH4+.     

Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth

By employing serial enrichment, a derivative of Clostridium acetobutylicum ATCC 824 was obtained which grew at concentrations of butanol that prevented growth of the wild-type strain. The parent strain demonstrated a negative growth rate at 15 g of butanol/liter, whereas the SA-1 mutant was still able to grow at a rate which was 66% of the uninhibited control. SA-1 produced consistently higher concentrations of butanol (from 5 to 14%) and lower concentrations of acetone (12.5 to 40%) than the wild-type strain in 4 to 20% extruded corn broth (ECB). Although the highest concentration of butanol was produced by SA-1 and the wild-type strain in 14% ECB, the best solvent ratio with respect to optimizing butanol and decreasing acetone occurred between 4 and 8% ECB for SA-1. SA-1 demonstrated higher conversion efficiency to butanol than the wild-type strain at every concentration of ECB tested. Characterization of the wild-type and SA-1 strain in 6% ECB demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, carbohydrate utilization, pH resistance, and final butanol concentration in the fermentation broth.