CURRENT MICROBIOLOGYVol. 31 (1995), pp.

1%22
Current
Microbiology
An International Journal
9 Springer-Verlag New York Inc. 1995

Rapid Screening of Leuconostoc mesenteroides Mutants for

Elevated Proportions of Alternan to Dextran
Timothy D. Leathers, G. Thomas Hayman,* Gregory L. Cote
Biopolymer Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service,
U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA

Abstract. Certain isolates of the bacterium Leuconostoc mesenteroides, such as strain N R R L

B-1355, have been found to produce alternan, a polysaccharide with unique properties of
potentially high commercial value. However, all of these isolates also produce significant amounts
of the polysaccharide dextran, which would be costly to separate from alternan on a commercial
basis. We developed a rapid screening method for the isolation of L. mesenteroides mutants that
produce elevated proportions of alternan to dextran. With this technique, a set of mutants of strain
N R R L B-1355 was isolated, including strain N R R L B-21138, which produced a high proportion of
alternan to dextran and showed complete genetic stability after more than 60 generations.

Dextrans produced by strains of Leuconostoc mesen-
teroides are homopolysaccharides, high-molecular-weight
a-o-glucans containing predominantly e~-(1~ 6) link-
ages, with variable (strain-specific) degrees of branch-
ing [2]. Dextrans have found numerous industrial and
pharmaceutical applications, including uses in paper
and metal-plating processes, and as food syrup stabi-
lizers, blood volume extenders, molecular sieves, etc.
[1].
As early as 1954, L. mesenteroides strain N R R L
B-1355 was described as producing an additional
unique polysaccharide, termed fraction S [5, 13].
Numerous studies have contributed to the conclusion
that fraction S consists of an eL-D-glucan with the
unique structure of alternating oL-(1 ~ 6), 0~(1 ~ 3)
linkages, consequently termed "alternan" by Cote
and Robyt [4, 9, 10]. Alternan and alternan deriva-
tives (sonicated and limit alternan) have unique
The use of brand or trade names may be necessary to report
factually on available data. The USDA neither guarantees nor
warrants the standard of the product, and the use of the name by
USDA implies no approval of the product to the exclusion of
others that may also be suitable. All programs and services of the
USDA are offered on a nondiscriminatory basis without regard to
race, color, national origin, religion, sex, age, marital status, or
handicap.
* Present address': Phytoproducts Research Unit, NCAUR, ARS,
USDA, Peoria, IL 61604, USA.
Correspondence to. T.D. Leathers
properties that resemble certain functional character-
istics of gum arabic, maltodextrins, or Polydextrose@
[3]. Alternan thus has potential commercial applica-
tions as a low-viscosity bulking agent, extender, etc. in
foods and cosmetics. A domestic substitute for gum
arabic would be particularly attractive, since gum
arabic is an imported commodity with unpredictable
supply, quality, and price. In addition, the valuable
sweetener fructose is a by-product of the enzymatic
synthesis of alternan. However, no naturally occur-
ring strains of L. mesenteroides are known that pro-
duce only alternan, and the separation of dextran
from alternan is a relatively complicated and expen-
sive process [3, 4].
Alternan is synthesized from sucrose by the
enzyme alternansucrase, demonstrated to be distinct
from dextransucrase, the enzyme that produces dex-
tran [4, 8]. Presumably, the structural genes for
alternansucrase and dextransucrase are also distinct,
although they have not been characterized on a
genetic level. If so, it should be possible to isolate
stable derivative strains in which the dextransucrase
gene or genes have been inactivated. Recently, su-
crase-deficient mutants of strain N R R L B-1355 have
been described, identified through labor-intensive
methods [6, 12]. We describe here a convenient, rapid
screening procedure for the isolation ofL. mesenteroi-
des mutants that produce a high proportion of alter-
nan to dextran.
20
UV Mutagenesis
99 % Kill
Plate Survivors
Dark Recovery
Transfer Colonies
Master Plate
Replicate to Liquid
I Sucrose-=.- Dextran
Dextranase, DNSA
Dextran-~- Sugars
/
Purify from Master Plate
Retest
Fig. 1. Schematic diagram illustrating the rapid screening proce-
dure used for the isolation of L. mesenteroides mutants that
produce elevated proportions of alternan to dextran.
Materials and Methods
Culture maintenance and mutagenesis. Leuconostoc mesenteroides
strain NRRL B-1355, obtained from the ARS Culture Collection
(Peoria, Illinois), was routinely maintained on solid medium
containing 2.0% sucrose, 0.15% polypeptone, 0.15% beef extract,
0.15% yeast extract, 0.1% Tween 80, 0.2% ammonium citrate,
0.5% sodium acetate, 0.01% MgSO4 97H20, 0.005% MnSO4 9Hz0,
and 0.2% KzPO4, pH 6.5. For mutagenesis, liquid cultures of the
same medium were grown to midlog phase (OD600 of 0.2-0.7) at
28~ with shaking at 100 rpm. Harvested cells were resuspended in
10 mM MgSO4 and exposed to approximately 330 W/cm2 ultravio-
let radiation for 10-30 s. Time course aliquots were stored at
-20~ in 40% glycerol. Samples showing approximately 99%
mortality (usually exposed for 20 s) were diluted and spread onto
solid medium (described above) to produce isolated colonies.
Plates were incubated at 28~ in the dark for 1-2 days.
Analytical methods. Total glucansucrase activity was measured as
previously described [4], based on the incorporation of [U-14C]su-
crose into methanol-precipitable polysaccharide. Alternan purity
was estimated as the percentage of total precipitable radioactivity
after exhaustive digestion by dextranase [4]. Alternan authenticity
was judged by the methylation procedure described by Slodki et al.
[111.
Results and Discussion
Rapid screening of mutants. T h e developed m e t h o d
took advantage of microtiter plate techniques for the
rapid handling and microculture of potential mu-
tants. Microcultures were then directly treated with
an e n z y m e that specifically hydrolyzes dextran but not
alternan. T h e s e hydrolysates were then directly tested
by a colorimetric microassay to detect reducing sugars
p r o d u c e d from the b r e a k d o w n of dextran, In this
CURRENTMICROBIOLOGYVol. 31 (1995)
Table 1. Mutants ofLeuconostoc mesenteroides
strain NRRL B-1355
Strain number Dextran production Genetic stability
NRRL B-21138 Low Stable a
569-D6 Low Revertible b
571-F5 Intermediate Revertible
572-B9 Low Revertible
572-F9 Low Revertible
574-E6 Intermediate Revertible
599-Hll Intermediate Stable
608-F8 Low Revertible
616-A1 Intermediate Revertible
618-D1 Low Revertible
a Less than 1.0% revertants after more than 60 generations.
b Greater than 1.0% revertants after more than 60 generations.
manner, potential mutants that p r o d u c e d a high
p r o p o r t i o n of alternan to dextran could rapidly be
identified (Fig. 1).
Specifically, m u t a g e n i z e d colonies were trans-
ferred onto fresh plates (sucrose m e d i u m described
above) in a grid pattern corresponding to 96-well
microtiter dishes. Wild-type (unmutagenized) colo-
nies were included on each plate as controls. After
growth, these master plates were used to inoculate
liquid cultures (same m e d i u m ) in sterile microtiter
dishes (0.1 ml/well), with a 48-peg inoculator. These
plates were incubated statically for 2 days at 28~
Approximately 0.2 unit of crude dextranase (1,6-~-D-
glucan-6-glucanohydrolase, E C 3.2.1.11, from Penicil-
l i u m sp. (Sigma Chemical Co., St. Louis, Missouri)
were then a d d e d to each well, except for wells
designated as negative controls. Plates were mixed
briefly and incubated for 2 h at 37~ Plates were then
developed to reveal reducing sugar content, with a
microassay similar to one previously described [7].
Specifically, 0.1 ml of a solution consisting of 30%
sodium potassium tartrate, 1.6% sodium hydroxide,
and 1.0% dinitrosalicylic acid ( D N S A ) was a d d e d to
the culture in each well. After mixing, plates were
incubated at 80~ for 15-30 min, until clear distinc-
tions were a p p a r e n t by eye between positive control
wells (wild-type cultures receiving dextranase) and
negative controls (wild-type cultures to which no
dextranase had b e e n added). Typically, positive con-
trol wells were as m u c h as 1.0 OD540 darker than
negative c o n t r o l wells. Putative m u t a n t s corre-
s p o n d e d to wells exhibiting color densities intermedi-
ate b e t w e e n those of positive and negative control
wells. Typically, "low dextran" mutants (Table 1)
a p p e a r e d by eye only slightly darker than negative
controls, typically corresponding to 0 . 1 - 0 . 3 0 D s ~ 0
T.D. Leathers et al.: Screening of L. mesenteroides
unit. Since color development varied among indi-
vidual plates by 0.3 OD540 unit or more, it was
important to include both positive and negative
controls on each plate.
Unexpectedly, we found that crude rather than
purified dextranase was advantageous for the method.
Crude enzyme preparations apparently included en-
zymes able to hydrolyze residual sucrose, a nonreduc-
ing sugar. As a result, wells corresponding to poor
growth mutants, which failed to completely utilize
sucrose in the growth medium, were at least as dark
as positive controls, and were not misidentified as low
dextran producers.
Mutants isolated by the method. Most mutagenized
survivor colonies exhibited a wild-type (mucoid) mor-
phology, suggesting that alternan a n d / o r dextran was
being produced. Rare morphology mutant colonies
with nonmucoid (usually wrinkled) appearance were
all found to be wild type with respect to dextran
production in liquid medium assays. This observation
underscores the general unreliability of colonial mor-
phology as an indicator of polysaccharide production
capacity.
Approximately 5280 mutagenized survivor colo-
nies were screened by the described method. Putative
mutants were recovered from master plates, single-
colony purified twice, and retested with the method.
Mutants fell into two classes with respect to apparent
dextran production (Table 1). One class, termed "low
dextran," exhibited test wells only slightly darker than
negative controls. A second class appeared to pro-
duce intermediate levels of dextran. It has recently
been proposed that N R R L B-1355 carries two genes
for dextransucrase rather than one as generally sup-
posed [12]. While this theory provides a ready expla-
nation for "intermediate" dextran producers, it also
predicts that "low dextran" mutants should be far
more rare, contrary to our results. Alternatively,
mutants exhibiting a variety of phenotypes might
result from mutations in regulatory elements.
To determine the genetic stability of the mutants
obtained, we cultured isolates continuously by sequen-
tial transfer in liquid medium, for at least 60 genera-
tions. Two mutants ( N R R L B-21138, low dextran;
and 599-Hll, intermediate dextran) showed com-
plete stability after more than 60 generations (desig-
nated "stable" in Table 1). These mutants may thus
have resulted from ultraviolet-induced deletions. The
remaining mutants (designated "revertible" in Table
1) showed from 16% to 100% reversion after 60
generations, suggestive that these mutants bear ultra-
violet-induced base substitutions (point mutations).
21
Table 2. Methylationanalysis of polysaccharidefrom
Leuconostoc mesenteroidesmutant NRRL B-21138
Mole percentage of methylated PAANa
glucose derivative
2,3,4,6- 2,4,6- 2,3,4,- 2,4-
tetra-O-Me tri-O-Me tri-O-Me di-O-Me
NRRL B-21138
polysaccharide 9 34 46 10
Purified alternan
from NRRL B-1355 10 35 45 10
a per-O-acetylatedaldononitrile.
Characterization of alternan from mutant strain
NRRL B-21138. Stable mutant strain N R R L B-21138
was cultured in volumes of up to 10 L and confirmed
to produce polysaccharide in yields similar to wild-
type strain N R R L B-1355. By radioassay (Materials
and Methods), total glucansucrase activity from
N R R L B-21138 was about half that of N R R L B-1355
and was estimated to include 88 + 8% alternansu-
crase activity. By comparison, alternansucrase activity
from wild-type strain N R R L B-1355 was estimated to
be 69 - 8% of total glucansucrase activity by this
assay. Polysaccharide from mutant strain N R R L
B-21138 was characterized by the methylation proce-
dure of Slodki et al. [11]. Results, shown in Table 2,
supported the conclusion that polysaccharide from
N R R L B-21138 was identical to alternan purified
from wild-type strain N R R L B-1355. Because of its
genetically stable production of polysaccharide with a
high proportion of alternan to dextran, N R R L B-21138
or similar strains may have potential for commercial
production of alternan.
ACKNOWLEDGMENTS
The authors are grateful for the expert technical assistance of Kelly
Orrick and Heidi Hefner, and thank Jim Nicholsonfor carryingout
the alternan methylationanalysis.
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