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ENHANCED TREHALOSE PRODUCTION
IMPROVES GROWTH OF Escherichia coli
UNDER OSMOTIC STRESS
JEREMY E. PURVIS
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
Jeremy E. Purvis
Long after the details of this work have faded from my memory, I will still be
obliged to commend the few outstanding individuals who toiled alongside me during
these past two years. I am particularly thankful to my advisor, Lonnie Ingram, for his
instruction and encouragements, which have extended well beyond the boundaries of our
laboratory; to my committee members, James Preston and Keelnatham Shanmugam, for
their open resource and discussion; to my parents, for rej oicing in my successes as avidly
as during my childhood; and to Joy, my future wife, for giving me something far better
than a master' s degree to look forward to.
TABLE OF CONTENTS
ACKNOWLEDGMENT S .........__.. ..... .__. .............._ iii..
LIST OF TABLES ........._.___..... .__. ...............v....
LIST OF FIGURES .............. ....................vi
AB STRAC T ................ .............. vii
1 INTRODUCTION ................. ...............1.......... ......
Osmoregulation in Escherichia coli .............. ...............1.....
Role of Trehalose in Stress Tolerance ................. ...............1...............
2 MATERIALS AND METHODS .............. ...............5.....
Bacteria, Plasmids, and Culture Conditions .............. ...............5.....
Genetic M ethods s................. ............... ...............5.......
Measurement of Intracellular Trehalose .....___.....__.___ .......____ ...........8
Tolerance As say s ........._.. ..... ._ ...............8.....
3 RE SULT S .............. ...............9.....
Native Trehalose Production Provided a Small Benefit for Growth ........._.................9
Construction of Strains for Increased Production of Trehalose. .............. ...............11
Comparison of Integrants ............... .... ......_ .. ..... ..... .. ...............12
Optimization of Trehalose Expression for Salt and Sugar Stress............... ............... 13
Elevated Trehalose Production Increased Cell Growth after 24 h in the Presence or
Absence of Osmotic Stress Agent. ....__ ......_____ .......___ ...........1
4 DI SCUS SSION ............ ..... .._ ............... 18..
LIST OF REFERENCES ............_ ..... ..__ ...............22...
BIOGRAPHICAL SKETCH .............. ...............29....
LIST OF TABLES
2-1 Strain sources and characteristics............... .............
2-2 Plasmid sources and characteristics .............. ...............7.....
3-1 Stress tolerance of W3 110 strains ................ .............15............ .
LIST OF FIGURES
1-1 Trehalose metabolism in E. coli ................. ...............2...............
3-1 Growth of E coli strains under stress .............. ...............10....
3-2 Plasmid constructions ................. ...............11......___ ....
3-3 Intracellular trehalose accumulated by JP10 harboring pLOI3607 and
derivatives .............. ...............12....
3-4 Incremental growth of wild type and otsA' otsB' integrants under salt stress .........13
3-5 Optimization of otsBA expression during salt and sugar stresses ..........................14
3-6 Intracellular trehalose accumulated by unstressed W3110 strains ................... ........16
3-7 Growth of E. coli strains in the absence or presence of osmotic stress. ...................1 7
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
ENHANCED TREHALOSE PRODUCTION
IMPROVES GROWTH OF Escherichia coli
UNDER OSMOTIC STRESS
Jeremy E. Purvis
Chair: Lonnie O. Ingram
Major Department: Microbiology and Cell Science
The disaccharide trehalose protects bacterial cells against multiple environmental
stresses. Escherichia coli synthesizes trehalose in response to osmotic stress, heat shock,
extreme cold, desiccation, and entry of cells into stationary phase. Previous studies have
shown that mutants that cannot produce trehalose are sensitive to elevated osmolarity,
oxidation, and heat.
Here we examined the potential benefit of trehalose for growth in glucose-mineral
salts medium containing a series of concentrations of salts and sugars. Strain W31 10
(wild type) was compared to an isogenic strain in which all genes for trehalose synthesis
and degradation were deleted, and to a derivative in which a single chromosomal copy of
the otsA otsB operon was provided under LacI regulation. Modest differences in growth
between wild type and mutant strains suggested a direct protective role for trehalose.
These differences in growth were increased when trehalose production was elevated
above that of wild-type cells. Our results demonstrate that production of high intracellular
trehalose levels can be used to increase cell growth under salt and sugar stress. This
finding should aid the development of efficient microbial bioconversion processes that
demand high substrate concentrations and greater tolerance to products.
Osmoregulation in Escherichia coli
Bacteria have a remarkable ability to sense and respond to environmental stress
(Storz and Hengge-Aronis 2000). A part of this natural defense system involves the
intracellular accumulation of protective compounds that shield macromolecules and
membranes from damage (Csonka 1989; Kempf and Bremer 1998). For this purpose,
Escherichia coli can use a variety of compounds including glutamate, proline, trehalose,
betaine, and dimethyl sulfoniopropionate (Gouesbet et al. 1994; Landfald and Strarm
1986; Measures 1975; Perroud and Le Rudulier 1985; Underwood et al. 2004). Although
glutamate and proline provide transient relief from osmotic stress (Dinnbier et al. 1988),
allosteric control of proline synthesis and the negative charge of glutamate limit their
effectiveness at high concentrations (Richey et al. 1987). In recent studies, adding
betaine was shown to stimulate growth and ethanol production in recombinant E. coli
(Underwood et al. 2004) and to increase thermal tolerance in Bacillus subtilis (Holtmann
and Bremer 2004 ). However, neither betaine nor dimethylsulfoniopropionate can be
synthesized de novo by E. coli. In the absence of these supplements, trehalose is
produced as the primary protective osmolyte (Ishida et al. 1996).
Role of Trehalose in Stress Tolerance
Trehalose is a nonreducing disaccharide that has proven very useful for stabilizing
proteins and enhancing cell survival during dessication (Sola-Penna et al. 1998). Genes
encoding trehalose biosynthesis are widely distributed in nature (Elbein 1974; Elbein et
al. 2003; Richards et al. 2002) and have been extensively studied in E. coli and
Saccharomyces. Escherichia coli regulates trehalose production at the transcriptional
level (otsAB operon) with induction in response to osmotic shock (Gi~ever et al. 1988),
extreme heat (de Virgilio et al. 1994), extreme cold (Kandror et al. 2002), dessication
(Van Laere 1989), and entry into stationary phase (Hengge-Aronis et al. 1991). Two
enzymes are unique to trehalose biosynthesis: trehalose-6-phosphate synthase (otsA) and
trehalose-6-phosphate phosphatase (otsB) (Figure 1) (Kaasen et al. 1992). Previous
studies showed that mutations in either otsA, otsB, galU (glucose-6-phosphate uridylyl
transferase), or rpoS (Hengge-Aronis et al. 1991) (038 required for stationary phase
induction) are sufficient to prevent trehalose synthesis (Elbein et al. 2003).
Glucose 2 Glucose Trehalose
PEP pyr uvate
A ots4 A ots3
Glurcose-6-P Trehalose-6-P )~ Trehalose
UDP-Glucose UDP H0PP H2
1p~ g l P UTP PP teG lucose + C I 'e
Glucose- 1-P Glucose- 6- P 2 Gluc ose
Figure 1-1. Trehalose metabolism in E. coli. Bold arrows denote synthetic route by
trehalose-overproducing strain, JP20. Reactions that have been blocked by
gene deletions are marked with filled triangles. A) Glucose enters the cell as
glucose-6-phosphate via a PEP-dependent phosphotransferase enzyme
complex. B) The OtsA synthase condenses trehalose-6-phosphate with
UDP-glucose to form the precursor trehalose-6-phosphate, which is
dephosphorylated by a specific OtsB phosphatase. C) Intracellular trehalose
can be degraded by the cytosolic trehalase TreF or by hydrolysis of
trehalose-6-phosphate by a specific hydrolase TreC.
Inability to synthesize trehalose results in poor growth under salt stress (Ginever
et al. 1988) and decreased survival during storage at high or low temperatures (Kandror
et al. 2002; Hengge-Aronis et al. 1993), at low pH (Hengge-Aronis et al. 1991), during
desiccation (Welsh and Herbert 1999), and under oxidative stress (Banaroudj et al. 2001).
Although these effects are not reversed by the external addition of trehalose because of
periplasmic catabolic enzymes in E. coli (Boos et al. 1990), the addition of trehalose has
been shown to restore salt growth in an otsA mutant of Thermus themophihts (Silva et al.
2003). A combination of otsA and otsB genes from E. coli has been used to genetically
engineer increased stress tolerance in plants (Garg et al. 2002) and in mammalian cells
(Guo et al. 2000; Tunnacliffe et al. 2001).
Prior studies with trehalose have focused primarily on cell survival under stress
conditions. Recent interest in the development of microbial biocatalysts for the
production of high concentrations of commodity chemicals (Causey et al. 2003; Zhou et
al. 2003) implies a potential need for increased tolerance to high concentrations of sugar
feedstocks and mineral nutrients during growth; and a need to minimize the effect of high
product concentration. Previous studies by Billi et al. (2000) used the Synechocystis sp.
spsA gene to show that the intracellular production of sucrose, a nonreducing sugar dimer
with some of the properties of trehalose (Crowe 2002), dramatically increased the
desiccation resistance of E. coli. Their results indicate that suboptimal levels of trehalose
are produced by native control systems.
Our study examined the importance of trehalose for growth in glucose-mineral salts
containing a series of concentrations of salts and sugars. Tolerance was evaluated by
measuring final cell density after 24 h in the presence of osmotic stress. Strain W31 10
(wild type) was compared to an isogenic strain in which all genes for trehalose synthesis
and degradation were deleted, and to a derivative in which a single chromosomal copy of
the otsA otsB operon was provided under LacI regulation. These studies demonstrate that
native levels of trehalose synthesized in the parent strain are of limited benefit for growth
under osmotic stress, at high temperature, or at pH extremes. However, increasing
trehalose production above that produced by the native regulatory system improved
growth substantially under both salt and sugar stress.
MATERIALS AND METHODS
Bacteria, Plasmids, and Culture Conditions
Strains and plasmids used in our study are listed in Table 2-1. Strains DH~a and
TOPO 10F' were used as hosts for plasmid constructions. For constructions, cultures were
grown at 370C in Luria-Burtani medium (LB) (Ausubel et al. 1987) or on LB solidified
with 1.5% agar. Ampicillin (50 Cpg/mL), kanamycin (50 Cpg/mL), and tetracycline
(12.5 Cpg/mL) were added as appropriate for selection. For stress studies, cultures were
maintained on M9 plates (Miller 1992) containing 2% glucose.
Isopropyl-P-D-thiogalactopyranoside (IPTG) was used to induce expression of otsBA in
JP20. Inducer was added at time of inoculation. Growth was monitored
spectrophotometrically at 550 nm with a Spectronic 70 spectrophotometer
(Bausch & Lomb, Inc., Rochester, NY).
Standard methods were used for plasmid construction and analyses (Ausubel, et al.
1987). Coding regions for treA, treC and treF were amplified using ORFmer primers
(Sigma-Genosys, The Woodlands, TX) and cloned initially into pCR2. 1-TOPO
(Invitrogen). Chromosomal integration of mutated genes was facilitated by pKD46
containing an arabinose-inducible Red Recombinase (Datsenko and Wanner 2000).
Mutants were screened for appropriate antibiotic resistance and verified by analysis of
PCR products. Coding regions for otsBA genes were amplified by PCR using W3110
geneomic DNA as the template for the primer pair: N terminus
Table 2-1. Strain sources and characteristics
Strains Relevant Characteristics
DH~a lacZAM15 recA
W3110 wild type ATCC 27325
TOP 10F' lacP (episome) Invitrogen
S17-11pir thi pro hsdR hsdllf recA RP4-2-Tc::Mu-Km:: Tn7 Simon 1983
JP10 W3110, AotsBA::FRT AtreA AtreC AtreF This study
JPl5 W3110, AotsBA::FRT AtreA::FRT-tet-FRT This study
JP20 W3110, AotsBA::F'RT AtreA::FRT AtreC::F'RT This study
AtreF: :FRT OampH:: 1acP4-Pta-otsBA-FRT
JP21 W31 10, AotsBA::FRT AtreA::FRT AtreC::FRT This study
AtreF: :FRT OalsA:: 1acP-Ptac-otsBA-FRT
JP22 W3110, AotsBA::FRT AtreA::FRT AtreC::FRT This study
AtreF: :FRT lacP-Ptac-otsBA-FRT
JP23 W3110, AotsBA::F'RT AtreA::FRT AtreC::F'RT This study
AtreF: :FRT lacP4-Ptac-otsBA-FRT
JP24 W3110, AotsBA::F'RT AtreA::FRT AtreC::F'RT This study
AtreF: :FRT lacP-Ptac-otsBA-FRT
5'AAGGAGGAGAACCGGGTGACA3 and C terminus
5'ACGCAGCGTGATGCATGAAG3 '. A 6. 1 kb fragment containing the inducible ots
operon (Ptac-otsBA-FRT-kank~-FRT) was integrated into JPl5 by conjugation with donor
strain S17-1h containing the xn-dependent transposase vector, pLOI3650. Kanomycin-
resistant exconjugates (sensitive to ampicillin) were selected. Integration was confirmed
by PCR. The FRT (FLP recognition target)-flanked antibiotic resistance genes were
deleted by FLP recombinase (Posfai et al. 1997; Martinez-Morales et al. 1999).
Chromosomal DNA adj acent to the Ptac-otsBA-FRT insertion was amplified using
arbitrarily primed PCR (Gibson and Silhavy 1999; Caetano-Annoles 1993; Wang et al.
2004). Sequences of primers used in the first (ARB 1 and OUT-OTS) and second rounds
Table 2-2. Plasmid sources and characteristics
Plasmid Relevant Characteristics Reference
pCR2.1-TOPO bla kan, TOPOTM TA cloning vector Invitrogen
pFT-A bla flp low-copy vector containing recombinase and 28
temperature-conditional pSC101 replicon
pKD46 bla y P exo low-copy vector containing red 26
recombinase and temperature-conditional pSC101
pFLAG-CTC bla Ptae controlled expression vector Sigma
pLOI2065 bla, Smal fragment with FRT-flanked tet gene Underwood 2002
pLOI2511 bla, Smal fragment with FRT-flanked kan gene Underwood 2002
pLOI3469 bla tnp, hpir-dependent Tn5 transposase vector This study
pLOI3601 bla kan otsBA This study
pLOI3604 bla otsBA This study
pLOI3605 bla otsBA-FRT-kan-FRT This study
pLOI3607 bla Ptac- otsBA-FRT-kan-FRT This study
pLOI3617 pLOI3607, AotsA (M~lul, Klenow) This study
pLOI3618 pLOI3607, AotsB (BglII, Klenow) This study
pLOI3619 pLOI3617, AotsB (BglII, Klenow) This study
pLOI3 62 1 bla kan treA This study
pLOI3625 bla kan treA ::FRT-tet-FRT This study
pLOI3631 bla kan treF This study
pLOI3635 bla kan treF::FRT-tet-FRT This study
pLOI3 64 1 bla kan treC This study
pLOI3645 bla kan treC::FRT-tet-FRT This study
pLOI3650 bla tnp Apir-dependent vector containing This study
transposable Tn5 element [ Ptac- otsBA-FRT-kan-
(ARB2 and IN-OTS) of amplification are listed below. Resulting products were
gel-purified and used as a template for DNA sequencing.
* ARB1 5'GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT3'
* OUT-OTS 5'TGGCAGATGCACGGTTACGA3'
* ARB2 5'GGCCACGCGTCGACTAGTAC3'
* IN-OTS 5 'C TATGC GGC ATC AGAGCAG3 '
Measurement of Intracellular Trehalose
Sufficient culture volume was harvested (10000 x g, 250C) to provide 2.0 mg dry
cell weight (1 OD5sonm = 0.33 g liter ~1dry cell weight). Cells were permeabilized with
50% methanol and extracted for 30 min on ice. The mixture was vortexed briefly and
centrifuged at 10,000 x g for 1 min. The supernatant was assayed for trehalose by
thin-layer chromatography as described previously (Zhou and Ingram 2001). After
visualizing with N-(1 -naphthyl)ethylenediamine reagent (Bounias 1980), relative
amounts of trehalose were determined by densitometry using Quantity One Software
(BioRad). For estimates of intracellular trehalose concentrations, an aqueous volume of
1 mL was assumed per gram of dry cells.
Tolerance was evaluated by measuring growth (defined as final cell mass after 24 h
of incubation) in M9 minimal medium containing 2% glucose (without antibiotics).
Ignoring dissociation effects, basal medium contained 93 mM mineral salts and 111 mM
glucose. For each stress condition, a range of concentrations (or temperatures, or initial
pH) was selected that caused a gradual, near complete inhibition of growth (defined as
less than 2 doublings). Cells from a fresh plate were resuspended in M9 medium
containing 2% glucose and used as inocula (initial level of 0.030 OD5sonm). Cultures were
incubated in 13 x 100 mm capped tubes (370C water bath, 50 rpm reciprocating shaker,
24 h) and tested in triplicate. Results are presented as average values with standard errors
(bars) from three or more separate experiments, or as an average of replicates from one or
two experiments (without error bars). All compounds tested were purchased from either
the Sigma Chemical Company (St. Louis, MO) or from Fisher Scientific.
Native Trehalose Production Provided a Small Benefit for Growth
A mutant of W3 110 was constructed (strain JP 10) in which both biosynthetic genes
for trehalose were deleted (otsA, otsB) as well as genes encoding cytoplasmic (treC, treF)
and periplasmic (treA) trehalase (Figure 1-1). Although native trehalose production has
been shown to be highly beneficial for survival under many conditions (Kandror et al.
2002; Hengge-Aronis et al. 1993), loss of trehalose synthesis in JP10 resulted in only
modest decreases in final cell density during osmotic stress from salts and sugars
(glucose, mannose, xylose, and arabinose) (Figure 3-1). Differences were most evident at
the higher levels in which growth was reduced by more than half, decreasing the
minimum inhibitory level for (NaC1, KC1, and KH2PO4) and increasing the
concentrations of salts and hexose sugars (glucose and mannose) that permitted growth
equivalent to half that of the unstressed parent (1L50).
Inactivation of trehalose biosynthesis had no effect on tolerance to osmotic stress
from pentose sugars (arabinose and xylose) or on tolerance to pH and elevated
temperature. For glucose, mannose, arabinose, and the salts (assuming 2 particles per
KH2PO4 at pH 7), growth inhibition was roughly the same at equivalent osmolalities.
Xylose was two-fold more toxic than other osmolytes and caused an abrupt inhibition of
growth at concentrations above 120 mM (Figure 3-1).
Most added osmolytes caused a progressive, dose-dependent reduction in growth,
which began even with small additions (Figure 3-1). Both pentose sugars and KH2PO4
SA B C
[KCl] (mM) [KH2PO~ (mM) [NaCl] (mM)
[Ikabmnose] (mM) [Glucose] (mM) [Ma nose] (mM)
[X/osee] (mM) Tempematume (oC) pH
Figure 3-1. Growth of E coli strains under stress. Plotted concentrations represent the
osmolar contribution of each compound in addition to basal medium
osmolarity of 204 mM. The wild type strain W3 110 (0) and trehalose-
deficient mutant JP10 (A) were grown in a defined medium containing
increasing concentrations of osmolytes. A) KC1. B) KH2PO4. C) NaC1.
D) Arabinose. E) Glucose. F) Mannose. G) Xylose. JP20 (described below)
was grown under the same conditions with () and without (0) 0.1 mM
IPTG. The same strains were compared for tolerance to physical stress.
H) Heat. I) pH. Results are presented as average values with standard
deviations (bars) from three or more separate experiments.
were exceptions in which 100 mM additions resulted in an increase in final cell density.
In contrast to glucose and mannose, small additions of xylose and arabinose increased the
final cell density. The largest increase was caused by KH2PO4 and appears to result from
pH buffering. All cultures that reached final densities of over 1.0 Assonm were
approximately pH 4.6, below that permitting growth. The addition of MOPS buffer
(100 mM) to M9-glucose medium resulted in a similar increase in growth (data not
Construction of Strains for Increased Production of Trehalose
A medium-copy number expression vector containing an inducible otsBA operon
(pLOI3607) was originally used to investigate trehalose overproduction (Figure 3-2).
Derivatives of this plasmid were constructed in which frameshift mutations were inserted
at unique sites in the coding regions of otsA, otsB, or both genes (Table 2-1).
Trehalose production in JP10 cells was compared for strains harboring each
plasmid. Although a small increase was observed for plasmids containing a defect in
either gene (1 to 6%), expression of both genes increased intracellular trehalose levels by
more than 100-fold based on densitometry (Figure 3-3). Using trehalose standards, the
intracellular concentrations in JP10 harboring pLOI3607 (both genes functional) and
pLOI3619 (both genes deleted) were estimated to be 180 mM and <1 mM, respectively.
Plasmid pLOI3607 and derivatives were quite unstable in W3110 and JP10 during
growth in M9-glucose medium. To eliminate this problem, a single copy of the
.5348 bps 5291 bps
EcoRI t Pacl (T4) / ta
kan bp Ecdl Psi lo 8868 bps mr Snal Tntp 11366 bps
EcoRI~ FRknma sI R
Figure 3-2. Plasmid constructions.
pLOI3607 pLOI3617 pLOI3618 pLOI3619
Figure 3-3. Intracellular trehalose accumulated by JP10 harboring pLOI3607 and
derivatives. Cultures were grown to mid-log phase and induced with 0.1 mM
IPTG for 2 h. A) Section from thin-layer plate used for densitometry
calculation. B) Intracellular trehalose levels.
Lacl-regulated ots operon was transposed into the chromosome of JP 10. Five resulting
integrants were chosen at random for deletion of the kankkkkkk~~~~~~~kkkkkk gene used for selection of the
transposition. Chromosomal insertion of the modified ots casette was determined using
arbitrarily-primed PCR to map the site of integration in two strains:
JP20 (QampH: :1acl-Pta-otsBA-FR T), JP21 (QalsA: :1acl-Ptac-otsBA-FR T).
Comparison of Integrants
All integrants were similar and exhibited 6-fold to 10-fold increase in cell growth
after 24 h in M9-glucose containing NaCl (300, 400, and 500 mM) in comparison to the
parent containing otsAB deletions (JP10) and the wild type, W3110 (Figure 3-4).
Addition of inducer (0. 1 mM IPTG) to the medium of these integrants resulted in further
doubling of cell growth. One integrant, strain JP20, was selected for further study.
JP20 JP21 JP22 JP23 JP24
Figure 3-4. Incremental growth of wild type and otsA+ otsB+ integrants under salt stress.
Tops of bars mark final cell density after 24 h of incubation with 300 (black
bars), 400 (hatched bars), and 500 (open bars) mM NaC1. Basal medium
osmolarity equaled 204 mM. Integrants were treated with (+) or without (-)
0.1 mM IPTG. Results are presented as average values with standard
deviations (bars) from three or more separate experiments.
Intracellular trehalose levels in JP20 with 0.1 mM IPTG (952 mM) and without IPTG
(74 mM) were much higher than in W3110 (<1 mM) and JP10 (<1 mM). These results
are consistent with incomplete repression of the ots casette in JP20 by the adj acent lacl.
In this JP10 background devoid of periplasmic and cytoplasmic trehalase activity, even
low levels of otsAB expression could lead to substantial intracellular accumulation of
Optimization of Trehalose Expression for Salt and Sugar Stress
Concentrations of individual salts and sugars were selected near the minimal
inhibitory level (ILmin) for the parental strain, JP10. With each stress agent, JP20 growth
was evaluated with a series of IPTG concentrations to determine the optimal level for
induction (Figure 3-5). Results for all sugars and salts tested were essentially the same
with an optimum at 0.1 mM IPTG. At this concentration, cell growth after 24 h was
2-fold to 4-fold that of the uninduced culture. With the exception of glucose, further
increases in IPTG were detrimental for cell growth in the presence of other osmotic stress
Elevated Trehalose Production Increased Cell Growth After 24 h in the Presence or
Absence of Osmotic Stress Agent
The growth of JP20 was compared after 24 h to that of the trehalose-deficient strain
(JP 10) and wild type (W3110) under all stress conditions. Results are summarized in
Table 3-1. Induced expression of otsBA was marked by both increased accumulation of
trehalose (Figure 3-6) and improved stress tolerance (Table 3-2). Comparison of growth
patterns for JP20 during exposure to KCl and NaCl stress revealed a 5-fold improvement
in growth for trehalose-overproducing cells at salt concentrations near IL5o (Figure 3-1).
During KH2PO4 Stress, induced JP20 grew 6-fold better than the trehalose-free strain,
reaching a maximum ODssonm Of 2.0 at 200 mM KH2PO4. ILmin increased similarly for all
D E "
Iog [IPTU~~m log [IPTG] (mM) lg[PG(M
Figure 3-5. Optimization of otsBA expression during salt and sugar stresses. JP20 was
treated with varying doses of IPTG during growth in the presence of each
osmolyte. A) KCl (400 mM). B) KH2PO4 (400 mM). C) NaCl (400 mM).
D) Glucose (600 mM). E) Mannose (400 mM). F) Xylose (140 mM). Basal
medium osmolarity equaled 204 mM. Results are presented as average values
with standard deviations (bars) from three or more separate experiments.
Table 3-1. Stress tolerance of W3 110 strains
Minimum Inhibitory Level (ILmm,)
SStrains were grown at 370C for 24 h in M9 media containing 2% glucose and indicated additives. Millimolar values represent the osmolar contribution of each
compound to total media osmolarity. Ignoring dissociation effects, basal medium contained 93 mM mineral salts and 111 mM glucose.
2 ILmm, equals the lowest osmolyte concentration, growth temperature, or initial pH necessary to restrict growth to less than two doublings (OD55onm < 0.012).
SIL5o equals the lowest osmolyte concentration, growth temperature, or initial pH necessary to restrict growth to half the OD55onm of the wild type culture grown
SIL,, equals the concentration at which growth is reduced to a density equivalent to the maximal growth of the wild type in medium containing no additives.
T (oC) pH
Median InhibitorY Level (IL5o) 3
Arabinose Glucose Mannose
333 26() 247
325 314 324
321 51() 5)8
354 632 573
Density Equivalent to Unstressed Control (IL, 4)
T (oC) pH
44 () 6 26
T (oC) pH
salts (40 to 60%). JP20 also exhibited higher tolerance than either parent strain under
sugar stress (Figure 3-1). With 600 mM glucose present in the media, induced JP20
achieved a 24-fold higher cell mass than JP10. Trehalose overproduction improved
absolute glucose tolerance by 35% (ILmin = 800 mM). Growth of JP20 in 500 mM
mannose was improved 20-fold compared to JP10. Growth improved less dramatically
for arabinose and xylose, with the largest increases in final density (2-fold) occurring
near the IL5o values for each pentose. Overproduction of trehalose was not beneficial to
heat- or pH-stressed cell.
Time-course growth of wild type and JP20 was measured in aerobic shake flasks
in the absence and presence of osmotic stress (Figure 3-7). Although growth in basal
media was similar for all strains, final cell densities for wild type and uninduced JP20
were 15% higher than for induced JP20. Growth rate was 2-fold higher for JP20 when
grown in either 400 mM NaCl or 600 mM glucose.
JP10 W3110 JP20 (-) JP20 (+)
Figure 3-6. Intracellular trehalose accumulated by unstressed W3110 strains. Cells were
grown for 24 h in M9 medium containing 2% glucose. JP20 was treated with
(+) or without (-) 0.1 mM IPTG. A) Section from thin-layer plate used for
densitometry calculation. B) Intracellular trehalose levels.
r P P
16 20 24
0 0625j m f-
Figure 3-7. Growth ofE. coli strains in the absence or presence of osmotic stress.
A) W3110 and JP20 were grown for 24 h in basal media containing no
additives. B) Growth in 400 mM NaC1. C) Growth in 600 mM glucose. Basal
media (M9, 2% glucose) osmolarity equaled 204 mM. Symbols: O, W3110;
O, JP20; JP20 + 100CLM IPTG.
Czonka (1989) has estimated that E. coli maintains an intracellular trehalose level
equivalent to 20% of the osmolar concentration of solutes in the growth medium. The
results presented in this paper show that this native regulation of trehalose synthesis is not
optimal at high osmotic strength. The consequence of high external osmolarity is the
collapse of turgor pressure, which is generally thought to be necessary for growth by
stretching the cell envelope during division (Koch 1991; Norris and Manners 1993). In
minimal media, cells counteract this loss of turgor by adjusting the intracellular solute
pool, increasing glutamate levels first followed by sustained synthesis of trehalose
(Dinnbier et al. 1988). Weak turgor resulting from high external osmolarity may have
contributed to the lower growth rates observed for wild type cells during salt or sugar
challenge (Figure 3-7). This interpretation is consistent with the observation that cells
that rapidly produced trehalose under osmotic stress exhibited higher growth rates and
shorter fermentation times than the wild type, implying a more favorable balance of
osmotic pressure. Conversely, excessive trehalose production was also detrimental to the
growth of trehalose-overproducing cells, which actually benefited from mild salt
concentrations (Figure 3-1). Over the range of concentrations tested for each condition,
the largest growth advantage for JP20 always occurred near the concentration at which
expression was optimized, suggesting that a single level of intracellular trehalose
provides the optimal osmotic balance at a given medium osmolarity.
These results demonstrate that unnaturally high trehalose production can
substantially improve growth of E coli under osmotic stress. Preliminary experiments
revealed small differences in growth between wild type and mutant strains during
osmotic stress, which suggested a direct protective role for trehalose; these differences in
growth were increased when trehalose production was elevated to levels beyond the
capability of wild type cells (Figure 3-1). Under elevated osmolarity, synthesis of
trehalose benefited cells in a dose-dependent fashion (Figure 3-5), and analysis of cell
extracts revealed that intracellular trehalose levels (Figure 3-6) corresponded well with
osmotic tolerance. Even in the absence of inducer, JP20 was more tolerant to osmotic
stress than the wild type. This result was corroborated by analysis of cell extracts from
JP20, which were found to contain significant amounts of trehalose after 24 h growth.
Accumulation by uninduced cells probably reflects a low level of unregulated gene
expression, which has an amplified effect in JP20 since this strain lacks the ability to
degrade trehalose once it has been synthesized. Hence, even low rates of production by
these cells are likely to boost tolerance significantly. In contrast, wild type E. coli
moderates intracellular trehalose levels with a cytoplasmic trehalase (Horlacher et al.
1996) and a trehalose-6-phosphate hydrolase (Rimmele and Boos 1994). Externally, a
periplasmic trehalase (TreA) functions under high osmolarity to break down extracellular
trehalose for subsequent transport and resynthesis (Boos et al. 1987). Under hypoosmotic
conditions, E. coli can excrete trehalose via a family of stretch-activated channels that
allow rapid efflux of osmoprotectants (Sleator and Hill 2001; Schleyer et al. 1993;
Sukharev et al. 1997). Trehalose released through these channels is retained in treA
strains (Styrvold and Strarm 1991).
All salts used in this study produced similar effects on growth, suggesting a
common mechanism of toxicity and relief by trehalose. Differences in final cell mass
were greatest for growth experiments involving moderately high salinity (approximately
half of ILmin). Cultures exposed to mild KH2PO4 COncentrations (100-3 00 mM) reached
higher cell masses than those observed for NaCl and KC land higher than unstressed
cultures), presumably due to the buffering capacity of the phosphate anion. Cells
responded similarly to sugars with the same molecular weight and showed greater
tolerance to hexoses than pentoses. When compared on the basis of total media
osmolarity, inhibitory levels for glucose and mannose were nearly identical (2% glucose
provides an additional 111 mM to total osmolarity). Comparatively slow growth on
xylose is characteristic of the wild type strain (results not shown), and may reflect
catabolite repression systems.
Trehalose occurs naturally in a variety of plants, yeast, fungi, bacteria, insects, and
some invertebrates. The molecule's unusual effectiveness under diverse conditions has
been attributed to its unique physical properties. Trehalose is a nonreducing sugar; the
[1-1] glucosyl bond formed by trehalose-6-phosphate synthase conceals the most reactive
end of each glucose monomer (Gibson et al. 2002). The resulting chemical inertness
allows cells to accumulate high concentrations of trehalose without disturbing
biochemical processes. Additionally, trehalose has an unusually high glass transition
temperature, which effectively slows kinetic processes in solutions by making
macromolecular movement difficult (Crowe 2002). Under dehydrating conditions, the
sugar protects cells by replacing water at the surface of macromolecules, holding proteins
and membranes in their native conformations until water content is restored (Crowe et al.
1984). Sola-Penna et al. (1998) illustrated this water-structuring behavior by relating the
stabilizing effect of trehalose to its large hydrated volume. Their results showed that other
sugars protect enzyme activity equally well only after the solution viscosity is increased
to match that of trehalose.
Endogenous synthesis of trehalose has potential to improve stress tolerance in
genetically engineered organisms. There is considerable interest in the development of
microbial biocatalysts for the production of chemicals that are medically or commercially
valuable (Burton et al., 2002). High product yields require robust organisms capable of
tolerating high levels of substrate and toxic byproducts. These attributes are scarcely
present in wild-type organisms, whose native stress response systems are adapted to
conditions routinely encountered in nature. The use of microbes for industrial purposes
demands a new breed of organisms that must be engineered for optimal growth under
specific physical and chemical parameters. Our work presents a simplified approach to
this task in which we have amplified a stress response system that is already present in
the target organism.
LIST OF REFERENCES
Alakomi, H.-L., E. Skytta, M. Saarela, T. Mattila-Sandholm, K. Latva-Kala, and I. M.
Helander. 2000. Lactic acid permeabilizes gram-negative bacteria by disrupting the
outer membrane. Appl. Environ. Microbiol. 66:2001-2005.
Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Deidman, J. A. Smith, and
K. Struhl (ed.). 1987. Current protocols in molecular biology. John Wiley & Sons,
Inc., New York, N.Y.
Bachmann, B. 1972. Pedigrees of Some Mutant Strains of Escherichi coli K-12.
Bacteriological Reviews. 36:525-557.
Banaroudj, N., D. H. Lee, and A. L. Goldberg. 2001. Trehalose accumulation during
cellular stress protects cells and cellular proteins from damage by oxygen radicals.
J. Biol. Chem. 276:24261-24267.
Billi, D., Wright, D. J., Helm, R. F., Prickett, T., Potts, M., and J. H. Crowe. 2000.
Engineering Dessication Tolerance in Escherichia coli. Appl. Environ. Microbiol.
Blazquez, M. A., R. Lagunas, C. Gancedo, and J. M. Gancedo. 1993.
Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits
hexokinases. FEBS 329:51-54.
Bonini, M. B., Van Dij ck, P., and J. M. Thevelein. 2003. Uncoupling of the glucose
growth defect and the deregulation of glycolysis in Saccharomyces cerevisiae tps1
mutants expressing trehalose-6-phosphate-insensitive hexokinase from
Schizosacch aromyces~~hh~~hh~~ pombe. Biochim. Biophys. Acta. 1606:83-93.
Boos, W., U. Ehmann, E. Bremer, A. Middendorf, and P. Postma. 1987. Trehalase of
Escherichia coli: mapping and cloning of its structural gene and identification of
the enzyme as a periplasmic protein induced under high osmolarity conditions. J.
Biol. Chem. 262:13212-13218.
Boos, W., U. Ehmann, H. Forkyl, W. Klein, M. Rimemele, and P. Postma. 1990.
Trehalose Transport and Metabolism in Echerichia coli. J. Bacteriol. 172:3450-
Bounias M. N-(1 -naphthyl)ethylenediamine dihydrochloride as a new reagent for
nanomole quantification of sugars on thin-layer plates by a mathematical
calibration process. 1980. Anal. Biochem. 106:291-295.
Burton, S. G., D. A. Cowen, and J. M. Woodley. The search for the ideal biocatalyst.
2002. Nature Biotechnol. 20:37-45.
Caetano-Annoles, G. 1993. Amplifying DNA with arbitrary oligonucleotide primers.
PCR Methods Appl. 3:85-92.
Caliskan G., A. Kisliuk, A. M. Tsai, C. L. Soles, and A. P. Sokolov. 2003. Protein
dynamics in viscous solvents. J. Chem. Phys. 118:4230-4236.
Canovas, D., S. A. Fletcher, M. Hayashi, and L. N. Csonka. 2001. Role of Trehalose in
Growth at High Temperature of Salmonella enterica Serovar Typhimurium. J.
Causey, T. B., S. Zhou, K. T. Shanmugam, and L. O. Ingram. 2003. Engineering the
metabolism of Escherichia coli W3 110 for the conversion of sugar to redox-neutral
and oxidized products: Homoacetate production. Proc. Natl. Acad. Sci. USA.
Cheville, A. M., K. W. Arnold, C. Buchrieser, C. M. Cheng, and C. W. Kaspar. 1996.
rpoS Regulation fo Acid, Heat, and Salt Tolerance in Escherichia coli Il57:H7. J.
Bacteriol. 62: 1822-1824.
Crowe, J. H., L. M. Crowe, and D. Chapman. 1984. Preservation of membranes in
anhydrobiotic organisms: the role of trehalose. Science. 223:701-703.
Crowe, J. H., F. A. Hoekstra, and L. M. Crowe 1992. Anhydrobiosis. Annu. Rev. Physiol.
Crowe, L. M. 2002. Lessons from nature: the role of sugars in anhydrobiosis. Comp.
Biochem. and Physiol. 131:505-513.
Csonka, L. N. 1989. Physiological and Genetic Responses of Bacteria to Osmotic Stress.
Micriobiol. Rev. 53:121-147.
Csonka, L. N. and W. Epstein. Osmoregulation, p. 1210-1223. In F. C. Neidhard. (ed.),
Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology.
ASM Press, Washington, DC.
Datsenko K. A. and B. L. Wanner. One-step inactivation of chromosomal genes in
Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA. 2000
de Castro, A. G., H. Bredholt, A. R. Stream, and A. Tunnacliffe. 2000. Anhydrobiotic
Engineering of Gram-Negative Bacteria. Appl. Environ. Microbiol. 66:4142-4144.
de Virgilio, C., T. Hottinger, J. Dominguez, T. Boiler, and A. Wiemken. 1994. The role
of trehalose synthesis for the acquisition of thermotolerance in yeast: I. Genetic
evidence that trehalose is a thermoprotectant. Eur. J. Biochem. 219: 179-186.
Diamant S., N. Eliahu, D. Rosenthal, and P. Goloubinoff. Chemical chaperones regulate
molecular chaperones in vitro and in cells under combined salt and heat stresses. J.
Biol. Chem. 276:39586-39591.
Dinnbier, U., E. Limpinsel, R. Schmid, and E. P. Bakker. 1988. Transient accumulation
of potassium glutamate and its replacement by trehalose during adaptation of
growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations.
Arch. Microbiol. 150:348-357.
Dulaney, E. L., D. D. Dulaney, and E. L. Rickes. 1968. Factors in yeast extract which
relieve growth inhibition of bacteria in defined medium of high osmolarity. Dev.
Ind. Microbiol. 9:260-269.
Elbein, A. D. 1974. The metabolism of a,a-trehalose. Adv. Carbohyd. Chem. Biochem.
Elbein, A. D., Y. T. Pan, I. Pastuszak, and D. Carroll. 2003. New insights on trehalose: a
multifunctional molecule. Glycobiology. 13:17R-27R.
Garg, A. K., J. K. Kim, T. G. Owens, A. P. Ranwala, Y. D. Choi, L. V. Kochian, and R.
J. Wu. 2002. Trehalose accumulation in rice plants confers high tolerance levels to
different abiotic stresses. Proc. Natl. Acad. Sci. U S A. 99: 15898-15903.
Ginever H. M., O. B. Styrvold, I. Kaasen, and A. R. Stream. 1988. Biochemical and
genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli.
J Bacteriol. 170:2841-2849.
Gibson, K. E., and T. J. Silhavy. 1999. The LysR homolog LrhA promotes RpoS
degradation by modulating activity of the response regulator SprE. J. Bacteriol.
Gibson R. P., J. P. Turkenburg, S. J. Charnock, R. Lloyd, and G. J. Davies. 2002. Insights
into Trehalose Synthesis Provided by the Structure of the Retaining
Glucosyltransferase OtsA. Chem. Biol. 9:1337-1346.
Goddijn O. and K. van Dun. 1999. Trehalose metabolism in plants. Trends Plant Sci.
Gouesbet, G., M. Jebbar, R. Talibart, T. Bernard, and C. Blanco. 1994. Pipecolic acid is
an osmoprotectant for Escherichia coli taken up by the general osmoporters ProU
and ProP. Microbiol. UK. 140:2415-2422.
Guo, N., Puhley, I. Brown, D. R., Manbridge, J., Levine, F. 2000. Trehalose expression
confers dessication tolerane on human cells. Nature Biotechnol. 18:167-171.
Hars, U., R. Horlacher, W. Boos, W. Welte, and K. Diederichs. 1998. Crystal structure of
the effector-binding domain of the trehalose-repressor of Escherichia coli, a
member of the LacI family, in its complexes with inducer trehalose-6-phosphate
and noninducer trehalose. Protein Sci. 7:2511-2521.
Hengge-Aronis, R., R. Lange, N. Henneberg, and D. Fischer. 1993. Osmotic regulation of
the rpoS-dependent genes in Escherichia coli. J. Bacteriol. 175:259-265.
Hengge-Aronis, R., K. Wolfgang, R. Lange, M. Rimmele, and W. Boos. 1991. Trehalose
synthesis genes are controlled by the putative sigma factor encoded by rpoS and are
involved in stationary-phase thermotolerance in Escherichia coli. J. Bacteriol.
Holtmann, G. and E. Bremer. 2004. Thermoprotection of Bacillus subtilis by
exogenously provided glycine betaine and structurally related compatible solutes:
involvement of Opu transporters. J. Bacteriol. 186: 1683-1693.
Horlacher, R., K. Uhland, W. Klein, M. Ehrmann, and W. Boos. 1996. Characterization
of a cytoplasmic trehalase ofEscherichia coli. J. Bacteriol. 178:6250-6257.
Ishida, A., N. Otsuka, S. Nagata, K. Adachi, and H. Sano. 1996. The effect of salinity
stress on the accumulation of compatible solutes related to the induction of salt-
tolerance in Escherichia coli. J. Gen. Appl. Microbiol. 42:331-336.
Kaasen, I., P. Falkenberg, O. B. Styrvold, and A. R. Stream. 1992. Molecular cloning and
physical mapping of the otsBA genes, which encode the osmoregulatory trehalose
pathway of Escherichia coli: evidence that transcription is activated by katF
(AppR). J Bacteriol. 1992 174:889-898.
Kaasen, I., J. McDougall, and A. R. Stream. 1994. Analysis of the otsBA operon for
osmoregulatory trehalose synthesis in Escherichia coli and homology of the OtsA
and OtsB proteins to the yeast trehalose-6-phosphate synthase/phosphatase
complex. Gene 145:9-15.
Kandror, O., A. DeLeon, and A. L. Goldberg. 2002. Trehalose synthesis is induced upon
exposure ofEscherichia coli to cold and is essential for viability at low
temperatures. Proc. Natl. Acad. Sci. USA. 99:9727-9732.
Karp, P. D., M. Riley, M. Saier, I. T. Paulsen, S. Paley, and A. Pellegrini-Toole. 2002.
The Ecocyc Database. Nucleic Acids Res. 30:56.
Kempf, B. and E. Bremer. 1998. Uptake and synthesis of compatible solutes as microbial
stress responses to high-osmolality environments. Arch Microbiol. 170:319-330.
Koch, A. 1991. Effective growth by the simplest means: the bacterial way. ASM News.
Landfald, D. and A. R. Stream. 1986. Choline-glycinebetaine pathway confers a high level
of osmotic tolerance in Escherichia coli. J. Bacteriol. 165:849-855.
Leslie, S. B., E. Israeli, B. Lighthart, J. H. Crowe, and L. M. Crowe. 1995. Trehalose and
sucrose protect both membranes and proteins in intact bacteria during drying. Appl
Environ Microbiol. 61:3592-3597.
Loewen, P. C. and R. Hengge-Aronis. 1994. The regulation of the sigma factor as (KatF)
in bacterial global regulation. Annu. Rev. Microbiol. 48:53-80.
Mansure, J. J., A. D. Panek, L. M. Crowe, and J. H. Crowe. 1994. Trehalose inhibits
ethanol effects on intact yeast cells and liposomes. Biochim. Biophys. Acta.
Martinez-Morales, F., Borges, A. G., Martinez, A., Shanmugam, K. T., and L. O. Ingram.
1999. Chromosomal integration of heterologous DNA in Escherichia coli with
precise removal of markers and replicons used during construction. J. Bacteriol.
Measures, J. C. 1975. Role of amino acids in osmoregulation of nonhalophilic bacteria.
Miller, J. H. 1992. A short course in bacterial genetics: a laboratory manual and
handbook for Escherichia coli and related bacteria. Cold Spring Harbor Press, Cold
Spring Harbor, N.Y.
Norris, V., and B. Manners. 1993. Deformations in the cytoplasmic membrane of
Escherichia coli direct the synthesis of peptidoglycan: the herni model. Biophys. J.
Padilla, L. R. Kramer, G. Stephanopulos, and E. Agosin. 2004. Overproduction of
trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate
synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum.tt~~~tt~~~ttt~~
Appl. Environ. Microbiol. 70:370-376.
Perroud, B. and D. Le Rudulier. 1985. Glycine betaine transport in Escherichia coli:
Osmotic Modulation. J. Bacteriol. 161:393-401.
Poolman, B. and E. Glaasker. 1998. Regulation of compatible solute accumulation in
bacteria. Mol. Microbiol. 29:397-407.
Posfai, G., M. D. Koob, H. A. Kirkpatrick, and F. C. Blattner. 1997. Versatile insertion
plasmids for targeted genome manipulations in bacteria: isolation, deletion, and
rescue of the pathogenicity island LEE of the Escherichia coli 0157:H7 genome. J.
Richards A. B., S. Krakowka, L. B. Dexter, H. Schmid, A. P. M. Wolterbeek, D. H.
Waalkens-Berendsen, A. Shigoyuki, and M. Kurimoto. 2002. Trehalose: a review
of properties, history of use and human tolerance, and results of multiple safety
studies. Food Chem. Toxicol. 40:871-898.
Richey, B. D., S. Cayley, M. C. Mossing, C. Kolka, C. F. Anderson, T. C. Farrar, and M.
T. Record, Jr. 1987. Variability in the intracellular ionic environment of
Escherichia coli: differences between in vitro and in vivo effects of ion
concentrations on protein-DNA interactions and gene expression. J. Biol. Chem.
Rimmele, M. and W. Boos. 1994. Trehalose-6-phosphate hydrolase of Escherichia coli.
J. Bacteriol. 176:5654-5664.
Rudolph, B. R., I. Chandrasekhar, B. P. Gaber, and M. Nagumo. 1990. Molecular
modeling of saccharide-lipid interactions. Chem. Phys. Lipids. 53:243-261.
Schleyer, M. R. Schmidt, and E. O. Bakker. 1993. Transient, specific and extremely rapid
release of osmolytes from growing cells of Escherichia coli K-12 exposed to
hypoosmotic shock. Arch. Microbiol. 160:424-431.
Silva, Z., S. Alarico, A. Nobre, R. Horlacher, J. Marugg, W. Boos, A. I. Mingote, and M.
S. da Costa. 2003. Osmotic adaptation of Thermus thermophihts RQ-1: lesson from
a mutant deficient in synthesis oftrehalose. J. Bacteriol. 185:5943-5952.
Simon, R., J. Quandt, and W. Klipp. 1983. A broad host range mobilization system for in
vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria.
Singer, M. A. and S. Lindquist. 1998. Multiple effects of trehalose on protein folding in
vitro and in vivo. Mol. Cell. 1:639-638.
Sleator, R. D. and C. Hill. 2001. Bacterial osmoadaptation: the role of osmolytes in
bacterial stress and virulence. FEMS Microbiol. Rev. 26:49-71.
Sola-Penna, M. and J. R. Meyer-Fernandes. 1998. Stabilization against thermal
inactivation promoted by sugars on enzyme structure and function: why is trehalose
more effective than other sugars? Arch. Biochem. Biophys. 360:10-14.
Storz, G., and R. Hengge-Aronis. 2000. Bacterial Stress Responses. ASM Press,
Stream, A. R. 1998. Osmoregulation in the model organism Escherichia coli: genes
governing the sythesis of glycine betaine and trehalose and their use in metabolic
engineering of stress tolerance. J. Biosci. 23:43 7-445.
Stream, A. R. and I. Kaasen. 1993. Trehalose metabolism in Escherichia coli: stress
protection and stress regulation of gene expression. Mol. Microbiol. 8:205-210.
Styrvold, O. B. and A. R. Stream. 1991. Synthesis, accumulation, and excretion of
trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber
suppressors and function of the periplasmic trehalase. J. Bacteriol. 173:1 187-1 192.
Sukharev, S.I., P. Blount, B. Martinac, and C. Kung. 1997. Mechanosensitive channels of
Escherichia coli: the MscL gene, protein and activities. Annu. Rev. Physiol.
Tunnacliffe, A., A. G. de Castro, and M. Manzanera. 2001. Anhydrobiotic engineering of
bacterial and mammalian cells: is intracellular trehalose sufficient? Cryobiology.
Van Laere, A. 1989. Trehalose, reserve and/or stress metabolite? FEMS Microbiol. Rev.
Underwood, S. A., M. L. Buszko, K. T. Shanmugam, and L.O. Ingram. 2004. Lack of
protective osmolytes limits cell growth and volumetric productivity of
ethanologenic Escherichia coli KO11 during xylose fermentation. Appl. Environ.
Microbiol., in press.*
Wang, X., J. F. Preston, and T. Romeo. 2004. The pgaABCD Locus of Escherichia coli
Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm
Formation. J. Bateriol., in press.*
Welsh, D. T. and R. A Herbert. 1999. Osmotically induced trehalose, but not glycine
betaine accumulation promotes desiccation tolerance in Escherichia coli. FEM
Microbiol. Lett. 174:57-63.
Yilmaz, J. L. and L. Btillow. 2002. Enhanced stress tolerance in Escherichia coli and
Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline
dehydrogenase fusion protein. Biotechnol. Prog. 18:1176-1182.
Zhou, S. D., T. B. Causey, A. Hasona, K. T. Shanmugam, and L. O. Ingram. 2003.
Production of optically pure D-lactic acid in mineral salts medium by metabolically
engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69:399-407.
Zhou, S. D. and L. O. Ingram. 2001. Simultaneous saccharification and fermentation of
amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without
supplemental cellulase. Biotechnol. Lett. 23:1455-1462.
On August 15th, 1979, Jeremy Edward Purvis was born in Sarasota, Florida, to a
red-haired mechanic and a coupon-clipping schoolteacher who delighted themselves
through selfless outpour of time, strength, and hard-earned dollars on behalf of two lucky
children. From kindergarten, Jeremy was formally educated by Florida' s public school
system, receiving a bachelor' s degree in microbiology from the University of Florida in
May, 2002. He plans to devote his future research efforts to computational and
experimental approaches to understanding biomolecular structure and function.