Alkalophilic BACTERIA

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Annu. Rev. Microbial. 1989. 43:435-{;3
Copyright © 1'989 by Annual Reviews Inc. All rights reserved
ALKl\LOPHILIC BACTERIA
Terry Ann Krulwich and Arthur A. Guffanti

Annu. Rev. Microbiol. 1989.43:435-463. Downloaded from www.annualreviews.org
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Department of Biochemistry, Mount Sinai School of Medicine of the City University

of New York, New York, New York 10029
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
BIODIVER SITY OF ALKALOPHILE S . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
COMMON PROPERTIES OF EXTREME A LKALOPHILES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Sodium Cycle Facilitating pH Homeostasis and Solute Uptake . . . . . . . . . . . . . . . . . . . . . . '" 440
Requirement of an Electrochemical Sodium Gradient for Motility . . . . . . . . . . . . . . . . . . . . . . 443
Use of Energy From a Proton-Extruding Respiratory Chain and a Proton-
Translocating F/-Fo ATPase for Oxidative Phosphorylation . . . . . . . . . . . . . . . 444
Properties of Cell Components That are Exposed to the Exterior . . . . . . . . . . . . . . . . . . . . . . 451
BIOTECHNOLOGY OF ALKALOPHILE S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Enzymes and Other A lkalophile Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
GENETIC E NGINEERING USING ALKALOPHILE GENE S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
PRO SPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 457
INTRODUCTION
Microorganisms occupy almost every conceivable habitat on Earth, from

subzero climates (59, 107) to hot springs (9), the ocean depths (l08), and
hypersaline waters (116). Most microorganisms are found in less exotic
environmelllts, and the vast majority of all these species grow best in the
neutral pH range; these are referred to as neutrophiles. There is, however, a
diverse group of bacteria that thrive in highly alkaline environments. They
can be separated into two broad categories: alkaline-tolerant organisms,
which show optimal growth in the pH range of 7. 0--9.0 but cannot grow above
pH 9. 5, and alkalophilic organisms, which show optimal growth between pH
10. 0 and 12. 0. The extreme alkalophiles can be further subdivided into
facultative alkalophiles, which show optimal growth at 10.0 or above but
435
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436 KRULWICH & GUFFANTI
which can grow well in the neutral pH range, and obligate alkalophiles , which
show optimal growth above pH 10.0 but cannot grow below pH 8. 5-9.0.
The facultative alkalophiles are of particular interest because they offer an
experimental opportunity to use pH-conditional mutants to investigate the
structural and functional features that enable a cell to thrive at very high pH.
Several properties are shared by all the extreme alkalophiles studied to date.
These include the composition of membrane lipids and the membrane lipid!
protein ratio, very high levels of respiratory-chain components in the mem
brane, a generally more acidic amino acid composition of proteins that are
exposed to or excreted into the external milieu, and a Na + cycle that facili
tates solute uptake and pH homeostasis (88). Any or all of these properties
could be prerequisites of alkalophily. Studies with some nonalkalophilic

mutant strains have supported the conclusion that at least the Na+ IH+ antipor
ter that is involved in pH homeostasis is a necessary function for life at high
pH (79, 88) . A major focus of future work will be to ascertain how many
other gene products are specifically required for alkalophily and whether the
genes encoding such products may be coordinately regulated in ways that are
related to the need of many of these organisms to survive large swings in
external pH.
The ultimate success of the investigations outlined above will depend in
part upon the growing availability of genetically accessible alkalophiles.
Fortunately, the alkalophiles are as interesting from the point of view of
applied microbiology as they are from that of bioenergetics and membrane
physiology. They produce many enzymes of potential industrial use. Thus,
the first molecular-biological initiatives and initial genetic transformations
have been undertaken in connection with applied biotechnological interests.
We therefore focus on the diversity of the organisms, the fundamental
bioenergetic and biochemical puzzles of alkalophily, and the biotechnology of
the alkalophilic bacteria.
BIODIVERSITY OF ALKALOPHILES
The first reports of microorganisms that grow in a highly alkaline environ

ment appeared over 60 years ago, when alkaline-tolerant nitrifying bacteria
(104) and an "alkalophilic" Streptococcusjaecalis strain (18) were described.
We now know that the alkalophilic bacteria are a diverse group, ranging from
eubacteria, such as Bacillus spp. to archaebacteria, such as Natronobacterium
spp. Representative strains of alkalophilic and alkaline-tolerant bacteria are
listed in Table 1. Gram-negative and gram-positive non-spore-formers have
been described. Even more highly represented are strains of gram-positive
endosporeformers, particularly Bacillus strains. Most of the alkalophilic
organisms are aerobic or facultatively anaerobic, but a few strictly anaerobic
ALKALOPHILIC BACTERIA 437
Clostridium and Methanobacterium strains that are at least alkaline-tolerant

have been described. Phototrophic cyanobacteria and purple sulfur bacteria
are also represented. During the past several years, the growth and morpho
logical characteristics of the major groups of alkalophiles , especially the
halophilic species (28 , 1 3 1) , have been extensively reviewed (53 , 88). We
therefore restrict this discussion to some alkalophilic organisms that have
been isolated very recently and that seem to illustrate generalities with respect
to the biodiversity of alkalophiles .
Much of the interest that the alkalophiles have evoked centers around the
expected alkalostability of their extracellular enzymes. Enzymes from
thermophilic alkalophiles might be both alkalostable and thermostable. Thus

far thermotolerant alkalophiles have been used for production of enzymes, but
a full characterization of a highly thermophilic and alkalophilic strain has
been repOlted only recently. In 1978, Kitada et al (75) described a thermophil
ic alkalophile that they designated Bacillus strain IC and identified as a strain
of Bacillus licheniformis. Bacillus strain IC grew optimally at 58°C and grew
well between pH 8.0 and 10.0, with some growth at neutral pH. One
intracellular enzyme, enolase, proved to be stable up to 60°C and was most
active in the neutral pH range. Extracellular enzymes have not yet been
characterized. A less alkalophilic species, Methanobacterium thermoalca
liphilum (7), appears to be the first thermophilic methanogen exhibiting good
growth in the high pH range. It grows up to pH 9.0-9 . 5 , with optimal growth
between pH 7 . 5 and 8 . 5 . This alkaline-tolerant anaerobe shows optimal
growth at 60°C. At the other end of the temperature spectrum, several as yet
unidentifie:d bacterial strains that grow well at pH 10.0 and at temperatures as
low as O°C have been isolated (72).
Marine environments abound in alkaline-tolerant bacteria and will general
ly yield small numbers of true alkalophiles from suitable enrichment cultures.
These species usually belong to the major alkalophile genus, i.e. Bacillus. By
contrast, the large number of gram-positive and gram-negative alkaline
tolerant and halotolerant species fall into many genera. Most recently, an
alkaline-tolerant coryneform bacterium was isolated from seawater ( 1 09). Its
growth was optimal between 0.5 and 2.0 M NaCl or KCI and occurred over a
broad pH range, from below 7.0 up to 9 . 5 . Interestingly, although most
extreme alkalophiles are Bacillus species, extensive studies of highly alkaline
saline lakes (28 , 142) have yielded only one possible member of this group.
There may be special biological problems associated with the combined
stresses of halophilicity and alkalophily.
Most of the truly alkalophilic microorganisms have either been isolated
from specific, enriched environments such as indigo dye balls ( 1 30), potato
processing-plant effluents ( 1 3) , or alkaline lakes (29, 1 24, 1 32) or have been
isolated upon suitable enrichment culturing of soil. The eubacterial alka-
..,.
w
Table 1 Diversity of alkalophilic bacteria 00
Group pH range for growth Natural habitat Comments Ref.

;:0:::
:;.::I
Eubacteria c::
Gram-negative photo-
autotrophs
�
-
n
Spirulina sp. 8.0-11.0 Alkaline soda lakes, Rift Val- Halotolerant cyanobacterium 29 :I:
ley, pH 10.5 ?:o
Synechococcus sp. 6.5-10.0, optimum Yellowstone, 55°C, pH 5.5 Alkali-tolerant, halotolerant 61 0
c::
pH 8.0 cyanobacterium
Ectothiorhodospira sp. 8.0-10.0 Mud from alkaline salt lakes, Anoxygenic purple sulfur bacterium 27
�
>
pH 11.0 Z
::l
Gram-negative
nonendosporeformers
Aeromonas sp. 7.0-10.0 Soil Facultative anaerobe 53
Flavobacterium sp. 7.0-1 1.2 Soil Facultative anaerobe 56
Vibrio alginolyticus 6.0-9.2 Seawater Halotolerant, alkali-tolerant faculta- 1 35
tive anaerobe
Gram-positive
nonendosporeformers
Exiguobacterium sp. 7.0-11.5, optimum Potato-processing effluent Facultative anaerobe 13
8.5-9.5
Actinomyces sp. 8.0-1 1.5, optimum Soil Heterotrophic facultative 105
9.0-9.5 anaerobe
Streptococcus sp. 5.0-11.0, optimum Alkaline potato processing Fermentative facultative 26
8.0-9.0 effluent anaerobe
Coryneform bacteria 6.6-9.5 Seawater Halotolerant facultative 109
anaerobes
Gram-positive
endosporeformers
Clostridium sp. 8.0-11.3, optimum Alkaline springs Strict anaerobe 124

9.5
Bacillus (alcalophilus) 8.5-II.5 optimum Soil (acid-alkaline) Aerobic heterotroph 141
10.6
Bacillus strain A007 9.0-11.0 Soil Aerobic heterotroph 2
Bacillus strain WNl 3 8.0-11.5, optimum Soil Aerobic, heterotrophic halophile 142
9.0-9.5
Archaebacteria �
�
Aerobes
Natronobacterium
Natronococcus sp.
sp. optimum 9.0-10.0 Alkaline saline lakes Halophilic (3 M NaCI) 132
[;
:i!
Anaerobes E
n
Methanobacterium 6.5-10.0 Biogas plant, optimum Thermophilic autotroph 7 t:::tl
thermoalcaliphilum pH 7.5-&.5 :.>
q
trl
�
:.>
.j>.
W
\0
lophiles isolated from soil are usually, but not always, Bacillus species.
Facultative alkalophiles have recently been isolated from soil and assigned to
the genus Flavobacterium (56). A group of these strains grew at pH 7. 1 - 1 1 . 2,
with better initial growth at pH 10.2 than at pH 7 . 2; a-galactosidase produc
tion was also better at very high pH. Another category of alkalophilic soil
organisms, the alkalophilic actinomycetes, has recently been reviewed ( 105) .
Several isolates have been shown to b e obligate alkalophiles, exhibiting
excellent growth up to pH 1 1.0. Notably, the alkalophilic actinomycetes and
many of the alkalophilic Bacillus species have been isolated from soils that
are not particularly alkaline. Although the organisms are more prevalent in
alkaline soils ( 1 29), true alkalophiles have been isolated even from acidic
soils. Presumably there are microenvironments that allow the growth of even
extreme, obligate alkalophiles.
COMMON PROPERTIES OF EXTREME ALKALOPHILES
Sodium Cycle Facilitating pH Homeostasis and Solute Uptake
+ +
Na /H ANTIPORTER AND OTHER POSSIBLE MECHANISMS FOR NET H+
ACCUMULATION As has been reviewed previously (85 , 89, 9 1 ) , extreme

alkalophiles that grow optimally at pH 10. 0-- 1 1 .0 maintain a cytoplasmic pH
that is typically at least two pH units below the external pH. The aerobic
alkalophilic bacilli all exhibit primary proton pumping out of the cell during
respiration (98 , 100). The net accumulation of protons by the cells requires
Na+ (73 , 87 , 100). Suspension of cells of extreme alkalophiles in the absence
of added Na + results in an immediate alkalinization of the cytoplasm to equal
the pHout (73, 87). A pH gradient (dpH) with the cell interior more acidic
(acid in) is maintained by an electrogenic Na+ /H+ antiporter that exchanges
intracellular Na+ (or Li+) for external H+. The antiporter is probably an
abundant membrane protein, and it catalyzes rapid cation exchange. The
stoichiometry of the exchange is not precisely known, but is such that the
number of protons translocated in is greater than the number of sodium ions
translocated out. The Ill/! set up by proton pumping during respiration ener
gizes the electrogenic antiport (88, 99). A pH gradient, acid in, and an
inwardly directed chemical gradient of Na+ (llpNa+) are formed, and a
transmembrane electrical potential, cell interior negative, is retained. The
ultimate steady-state electrochemical proton gradient (llflwlF of the extreme
alkolophiles, in the presence of an energy source, is quite low (e.g. -50 mY
at pH 1 0 . 5 to about -25 mY at pH 1 1 .5). Skulachev ( 1 22, 1 23) has
questioned the central role of Na t /H+ antiport function in extreme alka
lophiles. His concern apparently arose from graphic presentations of early
data (35 , 1 14) on (Ilflw patterns in unenergized cells. The apparent (llflwlF
ALKALOPHILIC BACTERIA 44 1
values at the upper pH limit for growth were near zero (-1 5 mV i n that study,
and higher when malate was included in the buffer). How could the antiport
be energized to achieve the proton gradient found if the ajj,wlF were as low
as it seemed to be? At ajj,H+IF 0, an electrogenic antiporter could not be the
=
sole underlying mechanism. The real values, however, in energized cells (and
even cells suspended in buffer) are never zero and could be achieved at very
reasonable: H+/Na+ stoichiometries.
The cmcial involvement of the Na+ /H+ antiporter is supported by the
isolation of nonalkalophilic mutant strains that can no longer grow well above
pH 9.0 and have lost Na+ /H+ antiporter activity (79, 85 , 89). Also important
are experiments in which extreme alkalophiles that were growing or equili

brated at moderate pH (e.g. pH 8 . 0-8 .5) were subjected to a sudden upward

shift in the external pH (e.g. to pH 10.0-10.5) in either the presence or
absence of Na+. McLaggan et al ( 1 02) first demonstrated that in the absence
of Na+ , the internal pH of the facultatively alkalophilic bacterium Ex
iguobacterium aurantiacum rapidly rose to equal the new external pH. In the
presence of Na+ , the pH inside the cell (pHin) was maintained at a new
steady-state level that was well below the pH of the suspending medium
(pHout). Similar experiments with alkalophilic Bacillus firmus RAB (87)
confirmed those with E. aurantiacum; in B. firmus RAB, inclusion of the
protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was
shown to impair or abolish the Na+-dependent homeostasis (87). This is
consistent with a secondary Na+/H+ antiporter that catalyzes coupled move
ment of the two ions , but it would not be consistent with a model in which the
inward proton movements were the secondary but passive response to the
membrane potential set up by primary Na + pumping. In the latter case,
protonophores would have enhanced the inward proton movements. Finally,
the role of the Na+/H+ antiporter in pH homeostasis is supported by the
finding that extremely alkalophilic bacilli possess mechanisms that allow the
generation of genetic variants with altered levels of at least some gene
products that are important in alkalophily (90). When cells of B.firmus RAB
were plated at pH 10.5 on media containing suboptimal concentrations of N a +
(3 mM), variants were isolated that exhibited growth at unusually low con
centrations of added Na+, growth at even higher pH values than usual (up to
pH 1 1 . 5 Olr 12.0), and an accompanying increase in Na+/H+ antiport activity.
Many of the properties of the alkalophile Na+/H+ antiporter have been
elucidated (9 1 ) . The general properties include the use of Li+ or Na+, linear
dependence upon the dl/!, and inhibition by a high internal proton concentra
tion (consiistent with the role of the antiporter in acidifying the cell interior).
The assays used have measured Na +-dependent proton movements, apH
induced Na+ movements, and Na+ fluxes induced either by the initiation of
respiration or by imposition of a valinomycin-mediated potassium diffusion
potential in cells, vesicles, and proteoliposomes (85, 89, 91). When the same
methods were used at somewhat lower pH values, many aerobic eubacteria
seemed to possess a comparable activity, e.g. Vibrio alginolyticus (110),
Escherichia coli (5, 120), and even the archaebacterium Halobacterium
halobium (95). It is probable that in many organisms the antiporter is required
for growth under moderately alkaline conditions, such as at pH 8.5-9. 0. This
idea was proposed some time ago for E. coli (114, 146), and compelling
recent evidence in its favor has emerged (60, 103). Less clear is whether
there are other ion fluxes or mechanisms that have an important role in
pH homeostasis in the alkaline range. Nakamura et al (110) have suggested

that in the marine vibrios, which grow in a pH range whose upper limit is
not very different from that for E. coli growth, a K+/H+ antiporter has a
role in maintaining a constant pHin at high pH, even though a Na+/H+ anti
porter is still apparently required for major tuning. Koyama et al (83,
84) have proposed, by contrast, that fluxes of K+ are involved in alkaliniz
ing rather than in acidifying the cytoplasm of extremely alkalophilic bacilli.
Perhaps K+ fluxes are more relevant, then, to pH homeostasis at the
low end of the pH range for growth. The studies of Koyama et al (84) in
dicated that K+ uptake was electrogenic at neutral pH and electroneutral
at high pH. They correlated this apparent difference with a greatly en
hanced permeability of the alkalophile membrane to K+ at neutral ver
sus high pH. It is notable that Matsukura & Imae (101) found that the
permeability to K + and the K + -dependent diminution of the 11r/J of several
different alkalophilic bacilli at high pH was greatly enhanced when Na+
was omitted. This effect also seemed pronounced at pH 7. 5 (101) and
may thus underlie the observations of Koyama et al (84). The mecha
nism of the Na+ effect on the permeability to K+ is unknown, but it will be
of interest to determine whether it is a global effect on the bulk membrane,
a Na + -dependent exchange phenomenon, or some other more specific
effect.
Finally, with respect to activities that catalyze or facilitate net proton
accumulation by extreme alkalophiles, Koyama et al (82) recently reported a
facultatively anaerobic alkalophile that maintains a cytoplasmic pH below the
external pH value even though (a) the total l1[.tw is positive, since the I1pH is
reversed and there is either no 11r/J or a very small 11r/J, positive out; (b) the
I1pH is only partially Na+ dependent; (c) the organism contains no
cytochromes; and (d) the I1pH is not dissipated by protonophores. Koyama et
al (82) proposed that a Donnan potential allows these bacteria to acidify the
interior even in the absence of Na+. It is not clear whether the N a+-dependent
component might result from an antiport activity, perhaps energized suf
ficiently by the very small Ar/J, which could arise, for example, by ATP
hydrolysis.
Na+ -COUPLED SOLUTE PORTERS THAT COMPLETE THE SODIUM CY

CLE Tht! use of Na+ as the coupling ion for active transport, which is a
major theme in eukaryotes and marine and halophilic organisms (40), resolves
two specific problems for extreme alkalophiles (85 , 91). First, if the extrusion
of Na+ is a prerequisite for pH homeostasis , then the operation of a multitude
of Na+-coupled solute porters may provide the means for Na+ reentry .
Second, since the totalll{.tw of the alkalophiles is very low, the use of Na+
rather than H+ as a coupling ion for many solute uptake systems can bypass
the bioenergetic problem.
The demonstration that most solutes are actively transported by alkalophilic
bacteria in symport with Na+ began with findings in the mid-1 970s (74, 80).
Interestingly, at least some of the solute porters cannot substitute Li + for

Na+ , as can the Na+/H+ antiporter of the same organisms ( 1 27). Studies of
solute uptake, passive efflux , and exchange have provided some insights into
the characteristics of the coupled transport (85). As yet, however, none of the
alkalophile symporters has been purified and studied in a reconstituted
system.
Evidence that the Na + -solute symporters constitute a physiologically im
portant route for Na+ reentry for pH homeostasis has come from studies in
which cells of B. firmus RAB were equilibrated at pH 8.5 in the presence of
Na+ and subjected to a shift in pHOU! to pH 10.5 in either the presence or
absence of the nonmetabolizable amino acid analog a-aminoisobutyric acid
(AlB). The presence of AlB markedly enhanced the ability of the cells to
maintain a relatively acidified and constant pHin upon such a shift in pH OU!
(87). Because both the relevant antiporter and the Na+-AlB cotransport would
be consumers of the Ill/! (and AlB would not produce energy) , this result
strongly supports the view that the symporters are part of the pH homeostasis
mechanism and presumably act by recycling the Na+ . AlB would not have
had the observed effect if there were channels that responded to pHin or pH OU!
to allow alll the requisite Na + entry. There is no evidence for the existence of
such channels that are independent of the symporters . We may therefore
expect that the Na + -solute symporters will exhibit strong regulation by pH
and that symporters for ions and other omnipresent solutes will ensure the
constant illlward flow of Na+ . Little is known about the cation and anion
transport systems in alkalophiles, and little is known about the few carbo
hydrate transport systems that are Na+ independent and seem to depend upon
ATP (85, 91).
Requirement of an Electrochemical Sodium -Gradient for

Motility
The motihty of the extreme alkalophiles (as well as that of at least some
moderately alkaline-tolerant marine bacteria) depends upon the presence of
Na+ and appears to be energized by an electrochemical gradient of Na+ (45,

91). Na+ is required for motility of alkalophilic bacilli; swimming speed
increased in a linear fashion with logarithmic increases in the Na+ concentra
tion up to 100 mM Na+ (44, 45). Motility also depends upon the membrane
potential; at a constant Na+ concentration but no gradient of Na+, motility
was abolished when the membrane potential was dropped to about -90 mV
(45). In addition, amiloride, which is an inhibitor of various Na+ fluxes and
of the electroneutral Na + IH+ antiporter of eukaryotes (6), inhibited the
motility of an alkalophilic Bacillus strain (126). The inhibition by amiloride
may represent an experimental opportunity to probe the details of the Na+
dependent flagellar rotor, because the inhibition appeared to be competitive

and motility was highly susceptible to some amiloride analogs that are potent
Na+ channel inhibitors (126).
Thus far the motility of the alkalophilic bacilli has appeared to be de
pendent exclusively upon Na+, with neither substitution by Li+ at high pH
(44) nor any energization by a dpH at near-neutral pH (128). To date, even
facultative strains that grow over a broad pH range have shown this Na+
specificity for both motility and Na+-coupled solute symporters (128). This
rigid specificity is unexpected, because solute symporters in other organisms
at least sometimes accommodate Na+, Li +, or H+ as the coupling ion (139).
Some nonalkalophilic mutants of alkalophiles may use H+ as the coupling ion
for solute symport (88). In addition, a growing literature exists on the
modification of coupling-ion specificity for Na + -coupled porters upon various
mutational alterations (8, 68, 112).
Although Na+ appears to be an obligatory component of the energization
system for alkalophile motility, a chemical gradient of Na + alone may not
energize motility in the absence of a dl/l in untethered cells. Only for the
modestly alkaline-tolerant marine vibrios has it been possible to demonstrate
motility upon imposition of an artificial apNa+ upon freely swimming cells.
Cells of V. alginolyticus were rendered immotile by treatment with arsenate
and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) before imposition of
the Na+ gradient (16); however, because the levels of HQNO were IO-fold
lower than those shown to abolish the dl/l totally in the same study, it is not
completely clear whether a residual dl/l was present during the imposition of
the dpNa+.
Use of Energy From a Proton-Extruding Respiratory Chain

and a Proton-Translocating FIFo-ATPase for Oxidative
Phosphorylation
The most extremely alkalophilic bacteria are aerobic bacilli that grow op
timally on nonfermentable carbon sources in a pH range in which their d[.tw
levels are low (85, 91). This represents a bioenergetic dilemma with respect to
ATP synthesis, as schematically presented in the box in Figure 1. Although

the ilrjJ is substantial, the total ilfLw is too low to account easily for the
amount of ATP synthesized by oxidative phosphorylation by a chemiosmotic
mechanism (106) in which a conventional FIFo-ATPase is used at con
ventional ratios of H+ translocated per ATP molecule synthesized. If ATP
synthesis occurred via a synthase that used 3 protons per ATP molecule, and
if the synthesis were completely coupled to a bulk ilfLw, then the ilfLwiF
would h,we to be more than -160 mV to account for the observed
phosphorylation potential (ilGp) of -499 mY (91).
This quantitative dilemma is compounded by striking qualitative observa
tions with respect to the energization of ATP synthesis in extreme alka

lophiles. Energization of ATP synthesis by starved cells or ADP + Pi-loaded
A
H+(2-3/ATP)
C.pH>+120mV
c.'I' =-170mV Solutes
+ C.}lH·�- 50mV
C.Gp =-499mV
No'
SOhJleS
Figure 1 Box: Schematic representation of the bioenergetic dilemma posed by extremely

alkalophilic aerobic bacteria vis-ii-vis oxidative phosphorylation. Primary proton pumping by the
alkalophile respiratory chain and the secondary, electrogenic Na+ /H+ antiport together result in a
quantitative discrepancy between the magnitudes of the Jl.jLH+IF and Jl.Gp (see text). A-C: Three
possible solutions to the dilemma. A, thc presence of intracellular organelles on which ATP
synthesis oc(:urs; B, the use of a Jl.jLH+ to energize ATP synthesis (either by the FIFo-ATPase or
by an independent Na+-coupled ATP synthase), perhaps accompanied by the presence of
primary, respiration-dependent Na+ pumping; C, the use of a H+ -translocating FIFo-ATPase that
translocates many protons per ATP molecule synthesized. The solid circles are membrane
proteins of an unspecified nature.
membrane vesicles from B. firmus RAB or OF4 can be initiated by the

addition of an electron donor, e.g. malate or ascorbate-phenazine methosul
fate (PMS) (30, 33). With starved cells that are equilibrated at pH up to 1 0 . 2,
it is also possible to initiate ATP synthesis by the sudden imposition of a apH,
acid out, of 1-3 pH units (34). Under neutral or moderately alkaline con
ditions (up to about pH 9.0), the imposition of a valinomycin-mediated
potassium diffusion potential is yet another way to energize ATP synthesis
(30, 33, 34). However, the establishment of this bulk transmembrane diffu
sion potential at very high pH (i.e. the optimal pH for growth of the organ
isms) does not result in ATP synthesis (30, 33, 34). Although the calculated
magnitudes of the chemiosmotic driving force derived from respiration, an
imposed dpH, and a potassium diffusion potential could be made compara

ble, only the K+ diffusion potential does not involve the actual provision of
protons. No methodological errors in the measurements of the forces are large
enough to explain the quantitative discrepancy, and none can explain the
apparent need for either natural proton pumping or proton addition to achieve
ATP synthesis by alkalophilic cells at very high (but not at neutral) pH.
What, then, might be the solution to the dilemma? Three cogent initial
possibilities, which now appear to have been eliminated, are outlined in
Figure 1 . First, it was proposed that the trans--cytoplasmic membrane gra
dients might be irrelevant and that ATP synthesis might occur on an in
tracytoplasmic membrane system. Then the set of gradients across the in
tracellular organelle, not the cytoplasmic membrane, would be relevant (Fig
ure 1, solution A). Extensive studies of thin sections and freeze-fracture
preparations of alkalophilic bacilli have shown no hint of such organelles
(9 1 ) . Moreover, in recent studies, immunoelectron-microscopic techniques
have shown that the F I Fo-ATPase is clearly associated with the cytoplasmic
membrane .
The second possible solution (35) was that the extreme alkalophiles might
utilize an ATP synthase that was energized by the aiLNa+ (Figure I, solution
B). This solution would have bypassed the 10w-diLw problem by using the
same approach that is used for most solute porters and motility. Results of
early experiments did not support this kind of solution to the bioenergetic
dilemma, in that ATP synthesis neither required nor was stimulated by added
Na+ (85, 88) . Moreover, no evidence for a Na+-stimulated or vanadate
sensitive ATPase was found during a characterization of the membrane
associated ATPase activity of B. firmus RAB (43). The purified F)-ATPase
from B. firmus RAB is a typical five-subunit enzyme that shows im
munological cross-reactivity with antibody raised against the ,B-subunit of the
E. coli enzyme. The F 1-ATPase from B. firmus RAB, like another F )-ATPase
purified earlier from a different alkalophile (8 1 ) , exhibits Ca2+ - and Mg2+_
dependent activity, but the latter (and presumably physiological) activity is
latent. In B. firmus RAB, the Mg2+ -dependent hydrolytic activity of both

membram:s and purified F)-ATPase was greatly stimulated by octylglucoside
or methanol but not by sulfhydryl reagents or mild proteolysis (43). Im
portantly, antibody raised against the purified FI-ATPase inhibited most of
the ATPase activity of membranes from B. firmus RAB (43). Therefore, the
F I-ATPase is certainly the major and possibly the only ATPase in the
membram:. The alkalophile F)-ATPase is not stimulated by Na+, but in the
one well-documented FIFo-type ATPase that does translocate Na+, that from
the nonalkalophilic anaerobe Propionigenium modestum, the holoenzyme and
not the F)-ATPase is stimulated by Na+ (96, 97) . However, in recent studies
(D. B . Hicks, unpublished) the FIFo-ATPase from B . firmus RAB has been
solubilized, partially purified, and reconstituted into proteoliposomes in

which uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide (DCCD)
inhibited ATPase activity as well as CCCP- and DCCD-inhibited ATP_32Pj
exchange could be demonstrated. Neither activity required or was stimulated
by Na+.
Still, it might have been argued that a Na+-coupled synthase with little
hydrolytic activity remained in the membrane and was missed because it was
saturated at low N a + concentrations . However, large imposed gradients of
Na+, even in the presence of large diffusion potentials at high pH, failed to
energize ATP synthesis by starved cells (34). Both the imposed AjLNa+ and the
imposed ApNa+ were bioenergetically competent; the imposed Na+ gra
dients, ApNa + alone, energized sustained accumulation of AlB up to 20-fold
concentration gradients . Unless the phantom Na+ -ATPase required an even
higher threshold AjLNa+, it should have functioned. In the experiments in
which a VI!ry large force composed of a combined �pNa+ and K+ diffusion
potential was imposed, even a Na+-coupled ATP synthase that is normally
saturated and has a AjLNa+IF requirement of -250 mY would have produced
ATP (30, 33, 34). The membranes evidently contain no such enzyme.
Not only do the most extreme aerobic alkalophiles lack a Na+ -coupled ATP
synthase, they also lack primary, respiration-coupled Na+ pumps . Such
pumps , together with a Na + -coupled ATP synthase, were proposed to con
stitute a Na+ cycle (as shown in Figure IB) that allows the growth of
facultatively anaerobic, marine V. alginolyticus at moderately high pH ( 17,
122, 123) V. alginolyticus and at least some other marine bacteria indeed
possess primary Na+-translocating respiratory-chain segments (70, 1 34,
1 36) . As indicated above, Na+ extrusion by the extreme alkalophiles can be
entirely accounted for by a secondary Na + IH+ antiporter. No Na + -induced
stimulation of either oxygen consumption or NADH oxidation in the extreme
alkalophiks could be demonstrated (32). Moreover, in recent experiments (A.
A. Guffanti, unpublished), ascorbate-PMS-dependent or malate-dependent
extrusion of 22Na+ from B. firmus cells that had been starved or briefly made
anaerobic was shown to be completely inhibited by valinomycin in the

presence of added K+ (at concentrations that were shown to have no effect
upon respiration). This inhibition of Na+ extrusion by conditions that dis
sipate the ill/! is expected for a ill/!-dependent Na+JH+ antiporter; were a
primary Na+ pump present, however, the dissipation of the ill/! should have
been stimulatory. Perhaps the role that a primary Na+ pump evidently has in
providing a boost to the ill/! of marine vibrios during growth at the alkaline
end of their pH range ( 1 33) is one of the functions of the extraordinary
cytochrome content found in all the extreme alkalophiles (85, 91). As dis
cussed below, this striking property of the alkalophiles may also have a more
specific relationship to the problem of oxidative phosphorylation. Clearly, the

presence of high concentrations of respiratory-chain components is not a

reflection of poor respiratory-chain function at very high pH. On the contrary,
the respiratory chain exhibits high H+/0 ratios as well as high respiratory
rates at high pH (98).
Even if the most extreme alkalophiles examined to date do not use a Na+
solution to the problem of oxidative pllosphorylation, a marine organism still
might use such a strategy to achieve modest alkaline tolerance. V. alginolyti
cus grows very poorly on nonfermentative carbon sources at pH 8.5 ( 1 33). It
does possess a Na+-translocating ATPase that can be made to synthesize ATP
under specific experimental conditions ( 17), e.g. a large imposed ilpNa+, but
it is not clear that this enzyme functions as a synthase under physiological
conditions. Unless independent H+- and Na+-coupled ATP synthases both
function at low and high pH, it should be possible to resolve the question by
isolating mutants similar in phenotype to E. coli unc mutants at pH 7 that have
lost the FIFo-ATPase activity that V. alginolyticus also has ( 1 7). If the Na+
and H+-coupled enzymes are distinct entities, such mutants should lose
ilpH-induced ATP synthesis, retain imposed ilpNa+-dependent ATP synthe
sis, and (if the Na+-ATPase is truly a synthase) still show growth on malate
and other nonfermentable carbon sources at high pH. If the Na + -translocating
activity is an activity of the FIFo-ATPase that also translocates protons, then
unc mutants should lose ilpH- and ilpNa+-dependent ATP synthesis and lose
the ability to grow on nonfermentable carbon sources at both pH 6.5 and 8.5.
Until this kind of rigorous test of the Na+-coupled synthase model is com
pleted, it is at least as plausible that the Na+-ATPase of V. alginolyticus
functions as a Na+ pump, rather than a synthase, under the anaerobic or
fermentative conditions that are optimal for growth.
For the extreme alkalophiles that neither possess cytoplasmic organelles
nor utilize a Na+-coupled synthase, the bioenergetic dilemma also seems not
to be bypassed by a H+:ATP stoichiometry of 10 or more (Figure Ie). In
experiments in which imposed pH gradients were used to energize ATP
synthesis by starved, cyanide-treated whole cells of alkalophilic B. firmus
OF4, the amount of ATP synthesized was linear with the magnitude of the
imposed j�pH over the range studied (34). However, the total amount of ATP
synthesizl�d at steady state upon imposition of a dpH of one unit was much
smaller than that observed in respiring whole cells whose d{Lw/F of 50 mV
-
is a smaller bulk driving force (34). Were the ATPase to translocate a large
number of protons per ATP molecule synthesize, it should have been capable
of doing so in the experiments with the imposed dpH. Even more strikingly,
if the extreme alkalophiles used a high H+:ATP stoichiometry to resolve the
problem of low-bulk d{Lw, then a high stoichiometry should have allowed
the organisms to synthesize ATP at pH 9.0 upon energization by a K+
diffusion potential.
A different solution (Figure 2) to the problem of oxidative phosphorylation

by extreme alkalophiles is suggested by the apparent requirement for proton
addition or the operation of natural proton pumps at very high pH. The model
accommodates the finding that the most extreme aerobic alkalophiles possess
very high concentrations of the respiratory-chain components. It takes into
account models of localized energy coupling. Such models have been widely
discussed and have a variety of forms (20, 1 17 , 143, 1 44) . Those that retain
the proton as a key intermediate in energy coupling posit that at least some of
the protons that move from pumps to sinks such as the FIFo-ATPase may do
so without equilibration with the bulk phase. In one model ( 1 1 8) the proton
pumping dements of the respiratory-chain polypeptides can directly interact
with the proton-binding elements (e.g. the c polypeptide) of the Fo-ATPase
within the membrane, so that the proton is sometimes transferred directly
before being able to escape into the bulk. A variety of direct or indirect roles
for membrane lipids could be envisioned in this process (20, 117). Other
protons do escape into the bulk, establishing a d{LH+ that may serve as a
parallel mode of energization. Importantly, this bulk d{Lw also makes local
ized coupling possible by forming a back pressure that inhibits the outward
leak of protons from intramembrane binding sites on the pumps and sinks. In
this type of model, the frequency with which the localized transfer occurs
would depend upon the proximity of the pumps and sinks. The ATPase and a
proton-pumping respiratory-chain component might be trapped together in
transient membrane domains that arise from specific properties of the particu
lar coupling membrane. Alternatively or in addition, the frequency of local
ized proton transfer might be envisioned as similar to the multicollisional
events described by Hackenbrock and colleagues ( 1 0, 36) to account for the
rates of mitochondrial electron transport. In such a description, the frequency
of proton "handoff' directly between pumps and the ATPase within the
membrane core might depend upon the concentration of the pumps and the
ATPase (or even the ratio of the proton-binding c subunit to the other
polypeptides in the Fo-ATPase) in the membrane; the relative concentration of
obstructive proteins that would simply get in the way; the fluidity of the
membrane or other lipid characteristics that would influence protein diffusion
rates; and the juxtaposition of the requisite residues or the proton leak into the
bulk.
This type of model accounts for the following observations about the
extreme alkalophiles. ATP synthesis at low /1jLwlF, but never at /1jLH+IP 0 =
would be consistent with a model in which the bulk force resisted outward
leakage of protons but was not the primary, direct energetic force . The
primary force would be provided by directly moving protons that are not
measured when the bulk /1jLH+ measurements are made. The localized flow
would require an actual membrane-associated proton pump. Since the K+

diffusion potential does not deliver protons, it would understandably be

incompetent at the very high pH values at which localized flow may be
essential . Moreover, the failure of an imposed LlpH to energize ATP synthesis
as effectively as native, intramembrane pumps could reflect the relatively
high resistance of the pathway for proton flow to and from the bulk phase; this
Figure 2 Schematic representation of A hypothesis that accounts for the bioenergetic properties
of extreme alkalophiles. Energy coupling at very high pH is proposed to occur largely (perhaps
even exclusively) through actual collisions between the proton-binding polypeptides of the
respiratory chain and proton-binding residues of the FIFo-ATPase. The frequency of those
collisions is high, and the path of proton flow to and from the exterior is of relatively high
resistance, so that localized proton flow is facilitated. The high collision frequency would be
favored by the very high concentration of respiratory-chain components (hatched areas) that is
characteristic of these organisms, by a somewhat high lipid/protein ratio in the membrane [i.e.
fewer obstructing proteins (solid circles) than in some other coupling membranes], and by high
membrane fluidity to allow the protein diffusion that would be required for collisions to occur.
Some components of the membrane might also enhance proton binding or favor the formation of
domains within the membrane lipids such that respiratory-chain components and FIFo-ATPase
are trapped together. This system would work only with natural H+ pumps. It may be dependent
upon an especially high concentration of proton pumps and sinks (or H+ -binding segments
thereof). Other aspects of the model are discussed in the text.
high resistance would assist in retention of protons in the localized pathway.

The very high concentration of respiratory-chain components may provide the
mechanism by which the alkalophiles maximize productive, proton
transferring collisions between respiratory-chain components and the
ATPase. In addition, the alkalophiles possess somewhat high membrane
lipid/membrane protein ratios ( 1 2), illustrated as a low concentration of
obstructive proteins in Figure 2. The alkalophile membrane also has high
cardiolipin concentrations and a fatty acid composition that is consistent with
a very fluid membrane ( 1 2, 78). These properties might be needed to
maximize diffusion-based collisions. It is also possible that phase changes of
the membJrane lipids and the possession of polar isoprenoid lipids that may
allow domain formation are related to energy coupling (12, 91). Importantly,
the model should be testable through genetic and biochemical approaches.
Properties of Cell Components That are Exposed to the

Exterior
Both the e:xternal and internal buffering capacities of extremely alkalophilic
bacilli are high under alkaline conditions (86), but the specific compounds
responsibk:, especially for the high internal buffering, are unknown. The
cytoplasmic membranes of all the extreme alkalophiles possess high con
centrations of cardiolipin, have phosphatidylethanolamine and phosphatidyl
glycerol as the other major phospholipids, and have squalene and squalene
derivatives as well as C40 and Cso isoprenoids among the neutral lipids (12,
78). Preliminary evidence indicates that they have a polar Cso isoprenoid
fraction. Recent studies ( 1 1) suggest that the obligate alkalophiles fail to grow
at neutral pH because their membranes become leaky. This explanation
accounts for early observations that even though respiration proceeds well in
the obligate alkalophiles at neutral pH, the �P.w generated is low. By
contrast, dosely related facultative strains have alterations in the membrane
lipids that result in a tighter coupling membrane at neutral pH, and they
generate substantial �{LH+ (9 1 ) .
The alkalophilic bacilli are sensitive to lysozyme treatment and t o cefoxitin
treatment; this behavior is consistent with the presence of a peptidoglycan cell
wall layer. Aono & Uramoto (3, 4) demonstrated the presence of a peptido
glycan, a fucosamine-containing teichuronic acid, and a polymer of glucuron
ate and gllutamate in cell walls of alkalophilic Bacillus strain C-1 25. The
fucosamine level showed positive correlation with elevated growth pH (4).
Presumably, proteins exposed to a very alkaline milieu have specific
properties that permit catalytic or other functions. Perhaps proper conforma
tion depends, for example, on the restriction of the content or exposed
position of residues whose pK values are close to the pH of the medium. The
flagellin from B. firmus RAB is acidic relative to its neutrophilic bacterial
counterpart (31), and a cytochrome c that may well have an exposed localiza
tion is an acidic protein (14).
BIOTECHNOLOGY OF ALKALOPHILES
Horikoshi & Akiba (51-53) have reviewed the industrial applications of

alkalophiles and the development of neutrophile excretion systems for alka
lophile enzymes. Sharp & Munster (121) have reviewed more generally the
biotechnological uses of microorganisms from several extreme environments.
Therefore, we again concentrate on the most recent advances.

Enzymes and Other Alkalophile Products

There are numerous applications for enzymes or pharmacologically active
chemicals that are stable at high pH. As the investigations fostered by
potential applications become increasingly molecular, it may be possible to
discern the properties that are the basis for stability under alkaline conditions.
For example, are extracellular enzymes from alkalophiles generally more
acidic than the corresponding enzymes produced by neutrophiles? It is at least
tantalizing that a carboxymethylcellulase (CMCase) from alkalophilic Bacil
lus strain 1139 possesses an abundance of aspartic acid residues compared
with similar enzymes from several neutrophiles (21).
CELLULASES The degradation of cellulose is an increasingly important

problem in waste treatment. Extracellular cellulase from alkalophilic Bacillus
strain N-4 has been characterized, and the gene encoding the enzyme has been
cloned into E. coli and sequenced (119). Two alkaline CMCases were partial
ly purified and exhibited pH optima of 10.0 and molecular masses of 54 and
46 kd. The enzymes were stable up to 60 and 80°C, respectively (54). The
two genes for these enzymes have been sequenced; they have a high degree of
homology with one another (25) but show little homology to the ,8-glucanase
gene of Bacillus subtilis. It appears that the genes are tandernly located on the
Bacillus strain N-4 chromosome and may have arisen via gene duplication by
homologous recombination (24). Horikoshi's group has characterized another
cellulase from alkalophilic Bacillus strain 1139. It exhibited a pH optimum of
9.0 and still showed much activity at pH 10.5 (21). As noted above, this
extracellular enzyme had a particularly high content of aspartic acid. Cloned
into E. coli, the enzyme had the same properties as the cellulase from Bacillus
strain 1139, but its molecular mass was 94 rather than 92 kd, perhaps
indicating processing at a different site(s) from that in the Bacillus strain of
origin (23). An insertional terminator cartridge was used to produce several
truncated products of the alkaline cellulase (22). One of them, of 46 kd, had
similar enzyme activity to the original 92-kd protein, indicating that the large
C-terminal part of the enzyme is not needed for activity. The Bacillus strain
1 1 39 cellulase showed strong nucleotide sequence homology in the N
tenninal re:gion toward the other two smaller cellulases from Bacillus strain
N-4.
PROTEASES Alkaline proteases are important as additives to detergents, in

food proc{�ssing, and in the leather-tanning industry ( 1 2 1 ) . Tsuchida et al
( 1 38) have isolated an extracellular alkaline serine protease from alkalophilic
Bacillus strain NKS-2 1 . The 3 1 -kd enzyme had an isoelectric point (pI) of 8 . 2
and showed optimal growth on casein at p H 10.0-1 1 .0. A 25-kd alkaline
elastase from alkalophilic Bacillus strain Ya-B ( 1 37) had a pI of 10.6 and
showed optimal activity at pH 1 1 .75; it was stable at pH 5 .0-10.0 in the

presence of Ca2 + . It exhibited extensive amino acid sequence homology with
subtilisin BPN ' (47%) and subtilisin Carlsberg (55%) but had different sub
strate specificities.
Three alkaline proteases from facultatively alkalophilic Bacillus strain
GX6638 have been purified ( 1 9) . Two of the enzymes, HS and AS, showed
optimal acltivities between pH 8.0 and 12.0 and temperature optima at 65°C.
HS was very stable at 60°C at pH 9.5, whereas AS was stable at 60°C above
pH 9.5 in the presence of Ca2+ . Interestingly, and unlike most other alkaline
proteases , HS and AS are acidic proteins with pIs of 4.2 and 5 . 2 , respec
tively .
I3-LACTAMASES J3-Lactamase, which cleaves the amide bond of the 13-

lactam ring of penicillin or cephalosporin, is used, among other applications,
in the development of new semisynthetic antibiotics that are resistant to
cleavage by the enzyme. The J3-lactamase from alkalophilic Bacillus strain
1 70 was cloned into E. coli (66). The nucleotide sequence was determined
and shown to have an open reading frame coding for 257 amino acids, 30 of
which represent the signal peptide. The sequence showed partial homology
with BaciUus cereus J3-1actamase II. Most of the plasmid-encoded penicillin
ase was excreted into the culture medium by E. coli, along with other
normally periplasmic proteins such as alkaline phosphatase (77) . An interest
ing observation was the activation of the kit gene that is nonnally present in,
but unexpressed by, the plasmid used in the cloning; the kit gene is responsi
ble for colicin release. The activation of the gene (albeit without the usual cell
lysis) is apparently mediated by the promoter in the insertion fragment
containing the alkalophile penicillinase (77) .
CYCLOMALTODEXTRIN GLUCANOTRANSFERASE Cyclodextrins are ring

structures consisting of six to eight glucose molecules linked by a- I ,4 bonds.
They are used in the food, pharmaceutical, and cosmetic industries for
stabilizing flavors , protecting against ultraviolet (UV)-induced degradation,

and inhibiting oxidation ( 1 2 1 ) . Cyclodextrins are produced from starch by
cyclomaltodextrin glucanotransferase (CGTase) . The. activity of this ex
tracellular enzyme from alkalophilic Bacillus strain 38-2 exhibited three pH
optima: pH 4 . 5 , 6.0, and 8 . 5 (63), i.e. not particularly alkaline conditions . In
fact, Hamamoto and colleagues (39, 62) cloned the CGTase gene into E. coli
and determined that its nucleotide sequence had high homology with the gene
encoding the CGTase from neutrophilic Bacillus macerans. Nevertheless , the
enzyme from Bacillus strain 38-2 is useful industrially because of its
thermostability and its production of cyclodextrin in high yield (53 , 1 2 1 ) .
a-AMYLASES a-Amylases have industrial and pharmaceutical uses, particu

larly as detergent additives at high pH. Extracellular a-amylase production
from alkalophilic Bacillus strain A-40-2 was found to be stimulated by the
addition of D,L-norvaline, l3-alanine, and D-methionine to the culture
medium; it was not known whether synthesis or excretion was the site of
stimulation (58) . Facultatively alkalophilic Bacillus strain H- 1 67 , which is
able to grow at pH 7 . 0-1 2.0, produces three a-amylases with different
molecular weights but with optimal activity at similar pH , i.e. 1 0 . 5 (4 1 , 42) .
All three were stable at 55°C and up to pH 1 2 .0. The enzymes , of 73, 59, and
80 kd, had pIs of 4. 1 , 3 . 5 , and 4.3, respectively. Maltohexaose, the main
product of the action of these enzymes on starch, has applications in both the
health and food industries (4 1 ) .
Another alkalophile, Bacillus strain 707, produces a maltohexaose amylase
whose amino acid sequence has been deduced from the cloned gene, which
was expressed in both E . coli and B. subtilis . The amino acid sequence
indicated homology with the a-amylases from Bacillus amyloliquefaciens, B.
licheniformis, and Bacillus stearothermophilus ( 1 40) , although the signal
peptides and N-terminal amino acids in all four enzymes differed. Therefore,
it has been suggested that the four genes have a common ancestry ( 1 40) .
Both the a-amylases and the CGTases discussed above cleave a- l ,4-
glucosidic bonds, although they yield different products . The amino acid
sequence deduced from the nucleotide sequence of a I3-CGTase gene from
alkalophilic Bacillus strain 10 1 1 exhibited homology with a-amylases from
human salivary gland, Aspergillus oryzae, B. amyloliquefaciens, and B.
subtilis (7 1 ) , especially in the regions near the N-terminus that encompass the
active sites of the amylases. The C-terminus of the {3-CGTase shows no
homology with those of the a-amylases. Kimura et al (7 1 ) proposed that
I3-CGTase may have two domains; the N-terminus cleaves a- I ,4-glucosides,
whereas the domain at the C-terminus may catalyze the relinkage of a- l ,4-
glucosidic bonds in the cyclization process .
XYLANASES Xylanases hydrolyze (3-1 ,4 linkages in xylan polysaccharides

found in many plants, particularly crops . Thus, xylanase is of importance to
any industrial process that requires degradation of plant material . The
alkalophilic Aeromonas strain 2 1 2 produces three types of extracellular xyla
nases (xylanase L, xylanase M, and xylanase S) of 145 , 37, and 23 kd,
respectively ( 1 1 1 ) . Xylanase L, M, and S activities were optimal at pH
7 .0-8 .0, 6.0-8.0, and 5 . 0-7.0, respectively, and the enzymes were stable at
pH 5 . 0-12.0, 5 .0-10.0, and 3 . 0-10.0, respectively. Xylanase L has been
cloned into E. coli by using pBR322 (92) . About 40% of the xylanase was
found in the periplasmic space; the rest was cytoplasmic. The cloned xylanase
had the same enzymatic properties as xylanase L but a molecular mass of 1 35

rather than 145 kd. It was speculated that this difference might be due to
glycosylation occurring in Aeromonas spp. but not in E. coli. More recently,
Kato et al (65) cloned the xylanase L gene into E. coli by using a pEAP37
or pEAP2 excretion vector and achieved extracellular enzyme production
(67) .
Alkalophilic Bacillus strain C- I 25 produces two xylanases , xylanase A and
xylanase N, of 43 and 1 6 kd, respectively (46 , 50) . Xylanase N was most
active at pH 6.0-7 .0, whereas xylanase A was most active at pH 6.0-10.0;
the latter was active even up to pH 12.0, with a temperature optimum of 70°C.
When cloned into E. coli, xylanase A was produced extracellularly (38 , 48,
49) and exhibited similar properties to the enzyme from Bacillus strain C- I 25
(46, 47) , Ibut the E. coli borne enzyme was slightly less stable at low pH
-
values. Interestingly, E. coli yielded a much larger amount of enzyme activity

than did Bacillus strain C- I25, presumably because of the derepression of the
xylanase A gene in E. coli (49). Glucose, at high concentration, totally
inhibited the cloned xylanase A expression , probably by catabolite repression
(49) . At an intermediate concentration of glucose ( 1 g liter- I ) little ex
tracellular xylanase A was produced, but it did accumulate in the cytoplasm
and periplasm. Tlms , the production of the enzyme has been separated from
its subsequent excretion . Moreover, xylanase A in E . coli was, unlike that in
Bacillus strain C- I25, constitutively expressed whether or not the inducer
(xylan) was present. The production of xylanase A by E. coli also appeared to
be regulated by osmotic pressure (49) .
Alkalophilic Bacillus strain C- I 25 was recently shown to produce more
xylanase when either glycine or D,L-norvaline was added to the culture
medium, perhaps altering cell permeability (57). In addition, these amino
acids inhibited extracellular protease production, thereby enhancing xylanase
stability. The N-terminus of xylanase A, determined by peptide sequencing or
deduced from the nucleotide sequence (37) , has a signal peptide of 28 amino
acids that iis cleaved during secretion. Hamamoto et al (37) have identified a
ribosome-binding sequence, initiation site, and possible translation termina

tion site in the nucleotide sequence.
Two xylanases from alkalophilic thermophilic Bacillus strains W I and W2
have also been purified and characterized (113). A xylanase of 22 kd showed
optimal activity at pH 6.0 and 65°C and had a pI of 8.4. The second (50 kd)
showed optimal activity at pH 7 . 0--9.5 and 70°C and had a pI of 3 . 6 . Again, it
is interesting that the enzyme with the much higher pH optimum (and
considerable industrial potential) had a low pI.
Using glycine
MISCELLANEOUS ALKALOPHILE ENZYMES A N D PRODUCTS
to "loosen" the cell surface components , Ikura & Horikoshi (55) caused the
extracellular production of two intracellular enzymes by several alkalophilic

Bacilli strains without lysing the cells. Kelly et al (69) have shown that
a-glucosidase is released from the alkalophilic Bacillus strain ATCC 2 1591
when the external pH drops from 9.7 to the neutral range; this finding is
consistent with the previously discussed loss of membrane integrity by obli
gate alkalophiles at near-neutral pH. Indeed, the enzyme had a pH optimum
of 7 .0 and might have been an intracellular enzyme.
The gene encoding oligo- l ,6-glucosidase from an alkalophilic Bacillus
strain has been cloned and expressed in E. coli ( 145). D-Xylose isomerase
(94) and p-mannosidase ( 1 ) from other alkalophilic bacilli have been purified
and characterized. The optimal pH for the extracellular xylose isomerase was
7 .0-10.0, whereas that of the intracellular p-mannosidase was 6.0, probably
reflecting the very different pH outside and inside the cell. Polygalacturonate
lyase from the thermophilic, alkalophilic Thermomonospora fusca ( 1 25) , an
actinomycete able to grow between pH 6.0 and 1 2 .0, showed optimal activity
at pH 1 0.45 and 60°C. Alkalophilic actinomycetes may prove to be a new
source of enzymes and biological substances with useful properties ( 105) .
GENETIC ENGINEERING USING ALKALOPHILIC

GENES
Recent studies have involved the use of alkalophile-derived genes to regulate

the expression or secretion of gene products in other bacteria. A genetic
element from an alkalophilic Bacillus strain that acted as promoter for the
chloramphenicol acetyltransferase gene (cat) on plasmid pGR7 1 has been
cloned into B. subtilis by Kudo et al (93). Expression of the cat gene was
shown to be developmentally regulated, being expressed only in the sporula
tion phase.
Kitai et al (76) , taking advantage of an observation mentioned above in
which the dormant kif gene of pMB9 was activated by the penicillinase
promoter from the alkalophilic Bacillus strain 1 70, constructed a recombinant
plasmid containing the human immunoglobulin G-Fc region and kil gene. E .
coli carrying the plasmid excreted about 40% of the human immunoglobulin
G-Fc region into the culture medium (76); the rest remained in the periplasm
and cytoplasm. The same protocol was used to introduce the gene for human
growth hormone (hGH) into E. coli (64); 55% of the total hGH was excreted
into the medium and 42% into the periplasm. The mature hGH was processed
normally and exhibited authentic hGH activity. Thus, activation of the kil
gene by an alkalophilic promoter opens up interesting opportunities for
establishing excretion of appropriate gene products by E. coli.
PROSPECTS
The enomlOUS progress in studies of the biology, bioenergetics, and

biotechnology of alkalophilic bacteria brings into focus the many key ques
tions that remain. To approach many of these questions, it will be essential to
develop a genetic system for the alkalophiles. Moreover, since at least some
extreme alkalophiles generate variants at high frequency (90), these ex
tremophiles may possess a system of transposable elements that alters the
expression of alkali-related genes analogous to that described for the
halophiles ( 1 5 , 1 1 5). Once a genetic system is established for the alka
lophiles , it should be possible to identify the genes and gene products that are
specifically required for life at very high pH. Within that task are in
vestigations of physiological subareas that include the still unclear pathways
of K+ fluxes , K+ -Na+ interplay, localized versus delocalized energy cou
pling, the iron-sequestering mechanisms that these cells must possess , and the
entire area of anaerobic alkalophiles. Finally, it should ultimately be possible
to define the features that underlie enzyme alkalostability and then to put
those insights to use.
ADDENDUM
Sakai et al ( 1 1 8a) have isolated a pleiotropic mutant of Vibrio parahaemolyti
cus that lacks H+ -ATPase activity and F I -ATPase subunits as well as suc
cinate transport. The mutant retains the ability to synthesize ATP in response
to an artificially imposed gradient of Na+ . Moreover, Na+ -coupled synthesis
may support some growth of the mutant on lactate at an unspecified but
presumably modestly alkaline pH, since some growth of the mutant is re
ported , albeit at "a slower rate than the parent. " Thus this nonalkalophile may
utilize a Na + -coupled enzyme to address partially a bioenergetic problem at
the extreme edge of its biological spectrum, just as Propionigenium mod
estum, another nonalkalophile, appears to have used the Na+ strategy. By
contrast, the true alkalophiles that are well adapted to very high pH values are
strikingly more mitochondrial in properties and retain a H+ -cycle for oxida
tive phosphorylation. The basis for this proton preference will be of interest.
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Alkalophilic BACTERIA

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Alkalophilic BACTERIA

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ANNUAL

Terry Ann Krulwich and Arthur A. Guffanti

Department of Biochemistry, Mount Sinai School of Medicine of the City University

Microorganisms occupy almost every conceivable habitat on Earth, from

could be prerequisites of alkalophily. Studies with some nonalkalophilic

The first reports of microorganisms that grow in a highly alkaline environ­

Clostridium and Methanobacterium strains that are at least alkaline-tolerant

thermophilic alkalophiles might be both alkalostable and thermostable. Thus

Group pH range for growth Natural habitat Comments Ref.

Clostridium sp. 8.0-11.3, optimum Alkaline springs Strict anaerobe 124

COMMON PROPERTIES OF EXTREME ALKALOPHILES

Sodium Cycle Facilitating pH Homeostasis and Solute Uptake

ACCUMULATION As has been reviewed previously (85 , 89, 9 1 ) , extreme

are experiments in which extreme alkalophiles that were growing or equili­

brated at moderate pH (e.g. pH 8 . 0-8 .5) were subjected to a sudden upward

pH homeostasis in the alkaline range. Nakamura et al (110) have suggested

Na+ -COUPLED SOLUTE PORTERS THAT COMPLETE THE SODIUM CY­

Interestingly, at least some of the solute porters cannot substitute Li + for

Requirement of an Electrochemical Sodium -Gradient for

Na+ and appears to be energized by an electrochemical gradient of Na+ (45,

dependent flagellar rotor, because the inhibition appeared to be competitive

Use of Energy From a Proton-Extruding Respiratory Chain

ATP synthesis, as schematically presented in the box in Figure 1. Although

tions with respect to the energization of ATP synthesis in extreme alka­

lophiles. Energization of ATP synthesis by starved cells or ADP + Pi-loaded

Figure 1 Box: Schematic representation of the bioenergetic dilemma posed by extremely

membrane vesicles from B. firmus RAB or OF4 can be initiated by the

imposed dpH, and a potassium diffusion potential could be made compara­

latent. In B. firmus RAB, the Mg2+ -dependent hydrolytic activity of both

solubilized, partially purified, and reconstituted into proteoliposomes in

anaerobic was shown to be completely inhibited by valinomycin in the

specific relationship to the problem of oxidative phosphorylation. Clearly, the

presence of high concentrations of respiratory-chain components is not a

A different solution (Figure 2) to the problem of oxidative phosphorylation

would require an actual membrane-associated proton pump. Since the K+

diffusion potential does not deliver protons, it would understandably be

high resistance would assist in retention of protons in the localized pathway.

Properties of Cell Components That are Exposed to the

Horikoshi & Akiba (51-53) have reviewed the industrial applications of

Therefore, we again concentrate on the most recent advances.

Enzymes and Other Alkalophile Products

CELLULASES The degradation of cellulose is an increasingly important

PROTEASES Alkaline proteases are important as additives to detergents, in

showed optimal activity at pH 1 1 .75; it was stable at pH 5 .0-10.0 in the

I3-LACTAMASES J3-Lactamase, which cleaves the amide bond of the 13-

CYCLOMALTODEXTRIN GLUCANOTRANSFERASE Cyclodextrins are ring

stabilizing flavors , protecting against ultraviolet (UV)-induced degradation,

a-AMYLASES a-Amylases have industrial and pharmaceutical uses, particu­

XYLANASES Xylanases hydrolyze (3-1 ,4 linkages in xylan polysaccharides

had the same enzymatic properties as xylanase L but a molecular mass of 1 35

values. Interestingly, E. coli yielded a much larger amount of enzyme activity

ribosome-binding sequence, initiation site, and possible translation termina­

MISCELLANEOUS ALKALOPHILE ENZYMES A N D PRODUCTS

extracellular production of two intracellular enzymes by several alkalophilic

GENETIC ENGINEERING USING ALKALOPHILIC

Recent studies have involved the use of alkalophile-derived genes to regulate

The enomlOUS progress in studies of the biology, bioenergetics, and

1. Akino, T., Nakamura, N., Horikoshi, 1 3. Collins, M. D., Lund, B. M . , Farrow, 1.

lus strain C-125. J. Gen. Microbiol. 933:470--77

of fucosamine in teichuronic acid of the spontaneous mutation in the bacterio­

effect of alkaline pH on growth and 38. Hamamoto, T. , Horikoshi, K. 1987.

pigmented Streptococcus sp. 1. Gen . cretable protein through outer membrane

15 excretion vector: extracellular produc­

content of the medium. J. Bacteriol. fanti, A. A. 1984. Presence of a non­

The first reports of microorganisms that grow in a highly alkaline environ

are experiments in which extreme alkalophiles that were growing or equili

Na+ -COUPLED SOLUTE PORTERS THAT COMPLETE THE SODIUM CY

tions with respect to the energization of ATP synthesis in extreme alka

imposed dpH, and a potassium diffusion potential could be made compara

a-AMYLASES a-Amylases have industrial and pharmaceutical uses, particu

ribosome-binding sequence, initiation site, and possible translation termina

of fucosamine in teichuronic acid of the spontaneous mutation in the bacterio

15 excretion vector: extracellular produc

content of the medium. J. Bacteriol. fanti, A. A. 1984. Presence of a non

growth of Escherichia coli at alkaline oxidative phosphorylation . Eur. Bio

fermentation vat (VII). 1. Ferment. tide sequence of the maltohexaose