APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1994, p. 12-18 Vol. 60, No.

1
0099-2240/94/$04.00+0
Copyright ) 1994, American Society for Microbiology

Characterization of Psychrotrophic Microorganisms

Producing 3-Galactosidase Activities
JENNIFER LOVELAND, KEVIN GUTSHALL, JODIE KASMIR, P. PREMA,
AND JEAN E. BRENCHLEY*
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, Pennsylvania 16802
Received 17 August 1993/Accepted 13 October 1993

Investigations of psychrotrophic microorganisms have been limited even though the dominant environment
of the Earth is cold and enzymes with high activities at low temperatures could have commercial uses. We have

Downloaded from http://aem.asm.org/ on April 5, 2019 by guest

isolated and characterized three psychrotrophic strains with 13-galactosidase activities. The isolates, B7, D2,
and D5, were gram-positive, catalase-positive, obligate aerobes. Cells observed with a scanning electron
microscope appeared as rods during the early stages of growth but became coccoid during the stationary phase.
An analysis of the amino acid composition of the cell walls demonstrated the presence of lysine as the
predominant diamino acid in all three isolates. The cell cycle morphology and cell wall composition suggest that
the three isolates are members of the genus Arthrobacter. The 13-galactosidase activities in whole cells were
labile when incubated at 40°C and had temperature optima about 20°C below that of the enzyme encoded by
the lacZ gene of Escherichia coli. Electrophoresis of extracts from the isolates in nondenaturing polyacrylamide
gels detected at least two protein bands that hydrolyzed 5-bromo-4-chloro-3-indolyl-13-D-galactopyranoside
(X-Gal), suggesting the presence of 13-galactosidase isozymes.

Even though the dominant environment of the Earth and
its oceans is below 20°C, we know little about the types and
physiology of microorganisms growing at these tempera-
tures. Psychrotrophs, or cold-tolerant microorganisms, must
often adapt to temperatures ranging from subzero to above
30°C. Research examining mechanisms that might permit
low-temperature growth, such as changes in membrane
fluidity, regulation of protein synthesis, and production of
heat and cold shock proteins, has been reviewed (4, 5, 7, 8,
17, 18). However, it is amazing that so little is known about
the diversity and metabolism of psychrotrophic microorgan-
isms considering that vast regions of the globe are subject to
cold climates and that temperature controls virtually every
cell reaction. The study of these microorganisms could add
considerably to our understanding of microbial diversity and
lead to the discovery of important new enzymes, antibiotics,
and other metabolites.
A long-term objective of our work is to discover novel and
potentially useful cold-active enzymes with high catalytic
activities at temperatures below 20°C. The properties of
these enzymes would then be compared with those from
corresponding proteins from mesophiles and thermophiles,
and the information would be used to determine which
features are important for functions at different tempera-
tures. In addition, cold-active enzymes could have important
applications in biotechnology, food processing, biomass
conversion, molecular biology, etc. To initiate this work, we
have isolated psychrotrophic microorganisms with 13-galac-
tosidase activities. We selected 3-galactosidase as a model
enzyme because it is possible to screen numerous colonies
by using chromogenic substrates and there are well-studied
counterparts from mesophiles and thermophiles that make
structural comparisons possible. Cold-active 3-galactosi-
*
Corresponding author. Mailing address: Department of Bio-
chemistry and Molecular Biology, 209 S. Frear, The Pennsylvania
State University, University Park, PA 16802. Phone: (814) 863-7794.
Fax: (814) 863-7024. Electronic mail address: JEB7@psu.edu.
12
dases could also be used to remove lactose from refrigerated
milk so that it could be consumed by people who are lactose
intolerant and to convert the lactose in whey from a pollutant
to the more readily fermentable glucose and galactose.
In this study we isolated psychrotrophic microorganisms
from fields that had been spread with whey during the
winter. We screened for microorganisms growing at low
temperature and producing 3-galactosidase activities. We
describe here the characterization of three isolates which
have a rod-coccus cycle typical of Arthrobacter species. We
have examined the growth and physiology of these isolates
and the effect of temperature on the activity and stability of
their 3-galactosidase activities. We found that these isolates
contain two or more ,-galactosidase isozymes which sepa-
rate from one another during electrophoresis in nondenatur-
ing polyacrylamide gels.
MATERUILS AND METHODS
Bacterial strains. Strains D2, B7, and D5 were isolated
from Pennsylvania farmlands. Soil samples giving rise to
isolates D2 and D5 were fr6m adjacent fields on the same
farm, and B7 was isolated from a nearby farm. The sites
were selected because the fields had been spread with whey,
which might have served as an enrichment for lactose-
utilizing microorganisms. The samples were collected in
early spring just after the snow had melted. All samples and
media were kept cold during transport and the isolation and
selection procedures. The soil samples were diluted with
0.9% NaCl and spread onto tryptic soy agar (TSA) with or
without 2% lactose and containing 0.13 mg of 5-bromo-4-
chloro-3-indolyl-3-D-galactopyranoside (X-Gal) ml-'. Incu-
bation was carried out at 5°C. After approximately 4 to 7
days, colonies which appeared blue were picked and
streaked onto fresh medium. Once the isolates were purified,
the 3-galactosidase activities were determined as described
below.
Other strains used in the study were Arthrobacter globi-
VOL. 60, 1994
formis ATCC 8010 (type strain), obtained from L. E. Casida,
Jr., and Escherichia coli and Bacillus subtilis, obtained from
Mary Jane Tershak, The Pennsylvania State University.
Growth media and cultivation. M9 served as the minimal
medium and was prepared by the method of Miller (16) with
different carbon sources added at 0.2%. Unless otherwise
noted, isolates B7 and D5 and A. globiformis were cultivated
in M9 with no additional supplementation. Isolate D2 had a
vitamin requirement, and 1 ml of Basal Medium Eagle
Vitamin Solution (GIBCO BRL, Gaithersburg, Md.} was
routinely added per 100 ml of M9. Thiamine (1 ,ug ml- ) was
added to M9 for growth of E. coli. Tryptic soy broth (TSB)
and TSA with no added carbohydrate were used for growth
rate and growth range determinations, respectively. Cultures
of isolates D2, D5, and B7 and A. globifonnis were incu-
bated at 25°C on a platform shaker or in a shaking water bath
at 200 rpm. E. coli cultures were incubated in a shaking
water bath at 37°C at 300 rpm.
Characterization of isolates. Initial observations of the cells
during the tests for the Gram stain reaction suggested that
they were pleomorphic. To further examine this and to
determine whether the isolates undergo a rod-coccus cycle,
cells were prepared for observation under a scanning elec-
tron microscope. The growth of the isolates in TSB contain-
ing no added carbohydrate at 25°C was monitored by using a
Klett-Summerson colorimeter (Manostat, New York, N.Y.)
to harvest the cells at early exponential and stationary
phases. The cells were prepared for scanning electron mi-
croscopy by the procedures of Cole (2), which included
collection of the cells by filtration through a Millipore
Swinney disk holder containing a polycarbonate filter (pore
size, 0.2 ,um; Nuclepore Corp., Pleasanton, Calif.). The
samples were then prepared for microscopy by conventional
methods (13) and examined under a JEOL JSM 5400 scan-
ning electron microscope.
Cell wall extracts from isolates D2, D5, and B7 and from
Bacillus subtilis and A. globiformis were prepared by the
"rapid screening method" presented in the review by Schle-
ifer and Kandler (19). The extracts were completely hydro-
lyzed and a quantitative amino acid analysis was performed
at The Hershey Medical Center of The Pennsylvania State
University.
The production of a variety of other enzymes was deter-
mined by using API Coryne test strips (Bio-Merieux Vitek,
Inc., Hazelwood, Mo.) and standard assays. Assays were
done twice, and if the results were marginal, they were
repeated a third time. Urease production was detected by
using API strips and urease test broth (BBL, Cockeysville,
Md.). To detect gelatin hydrolysis, strains were inoculated
onto plates of nutrient agar containing 5% gelatin and the
plates were flooded with saturated ammonium sulfate after
growth to indicate zones of hydrolysis (3). DNA degradation
was determined by growing the organisms on nutrient agar
with 0.2% DNA and toluidine blue by a method presented in
the Manual of Methods for General Bacteriology (20).
Strains were inoculated onto M9 agar which contained, in
addition to 0.5% ax-cellulose (Sigma Chemical Co., St. Louis,
Mo.), 0.2% Casamino Acids, vitamin solution (GIBCO),
trace element mixture (14), and FeSO4 7H20 (0.01 mg/ml)
to screen for cellulase activity. Zones of hydrolysis were
detected with 1% Congo red, and the excess dye was washed
off with 1 M NaCl (1). The medium used for carbon source
utilization was M9 containing 0.2% carbohydrate. Growth
was assessed after 18, 42, 66, and 116 h.
1-Galactosidase assay. Cells were grown in M9 containing
0.2% lactose, harvested by centrifugation, washed with 0.9%
PSYCHROTROPHIC ISOLATES WITH 3-GALACTOSIDASES 13
NaCl or Z buffer (16) without P-mercaptoethanol, and as-
sayed for 3-galactosidase activity with ortho-nitrophenyl-p-
D-galactopyranoside (ONPG) as the substrate by the proce-
dure of Miller (16). To determine whether any enzyme was
secreted, 0.5 ml of the culture medium was added to 0.5 ml
of buffer and assayed by the procedure of Miller (16). The
specific activity was expressed as micromoles of ortho-
nitrophenyl produced per minute per milligram of protein.
Separation of 13-galactosidase activities. Extracts were sub-
jected to electrophoresis to determine whether the isolates
produced more than one ,B-galactosidase isozyme. Cultures
were grown in M9 medium with 0.2% lactose, except for B7,
for which 1% TSB (10 ml of TSB to 1 liter of minimal
medium) was added to the M9 medium. Cultures were
harvested at mid- to late exponential phase. After harvest,
Downloaded from http://aem.asm.org/ on April 5, 2019 by guest
the cells were broken in a French pressure cell at 18,000
lb/in2 and the extracts were concentrated with an Ul-
trafree-MC filter unit (nominal molecular weight limit,
100,000; Millipore, Bedford, Mass.). Concentrated extracts
were then subjected to electrophoresis in 7.5% nondenatur-
ing polyacrylamide gels by the procedure of Laemmli (15)
without the addition of sodium dodecyl sulfate. Following
electrophoresis, the gels were incubated in an assay buffer in
which X-Gal was substituted for ONPG to detect P-galacto-
sidase activity in situ. Hydrolysis of X-Gal by B-galactosi-
dase activity formed blue bands within the acrylamide gels.
RESULTS
Characterization of isolates. Colonies that grew at 5°C and
hydrolyzed the chromogen X-Gal were screened for strains
that made I-galactosidases with temperature optima below
35°C. From these we selected three, B7, D2, and D5, for
further characterization. The isolates formed smooth, circu-
lar, yellow to cream colonies. The cells were gram positive
but also appeared gram negative during the growth cycle and
were easily decolorized late in growth. Samples taken at
different times during growth and examined under a scanning
electron microscope showed elongated, club-shaped cells
during exponential growth which fragmented into short rods
and coccoid cells later in growth (Fig. 1). The average cell
lengths for isolates B7, D2, and D5 were 1.4, 1.2, and 1.2
,um, respectively, during the early stage of growth and 0.5,
0.6, and 0.6 ,um, respectively, during the stationary phase.
Other tests demonstrated that the isolates were strict
aerobes, did not make acid with glucose as a carbon source,
produced catalase, and were nonmotile. Since the rod-to-
sphere morphological change and the other physiological
traits are typical of Arthrobacter species, we analyzed the
cell walls of the isolates to determine whether lysine, which
is the characteristic diamino acid present in Arthrobacter
peptidoglycan, was present (Table 1). The results show that
the isolates and an A. globiformis type strain contained
lysine as the dominant diamino acid, whereas B. subtilis,
included as a control, contained diaminopimelic acid as
expected.
In addition, these isolates were sent to MIDI (Microbial
ID, Inc., Newark, Del.) and to IEA (Industrial and Environ-
mental Analysts, Inc., Essex Junction, Vt.) to determine
their fatty acid compositions. Both groups reported that the
three strains are similar to each other, with D2 and D5
possibly more closely related to each other than to B7.
However, the testing services differed in their first choices of
genera: IEA suggested Arthrobacter for isolate B7 and
Curtobacterium for D2 and D5, whereas MIDI suggested
Micrococcus as the most likely genus for all three with
14 LOVELAND ET AL. APPL. ENvIRON. MICROBIOL.

I II

Downloaded from http://aem.asm.org/ on April 5, 2019 by guest

B

FIG. 1. Scanning electron micrographs of the three isolates showing the morphological changes. Isolates B7 (A), D2 (B), and D5 (C) were
grown in TSB at 25'C and harvested during exponential (panel I) or stationary (panel II) phase. Magnification, x7,500.

second choices of Arthrobacter for B7 and Curtobactenum
for isolates D2 and D5. Our results showing that the isolates
had a rod-coccus cycle (Fig. 1) eliminated the genus Micro-
coccus, in which the cells are spheres arranged predomi-
nantly in tetrads or diplococci and the cell shape does not
change with medium or culture age (11). Cell pleomorphism
is also not distinctive for members of the genus Curtobacte-
rium, although cells can become shorter in older cultures.
Curtobacterium strains are generally motile, produce acid
slowly from carbohydrates, and contain ornithine in the
interpeptide bridge of the cell wall (12). Our results (Table 1)
showed that ornithine was not present in the cell walls of the
isolates. On the basis of the combination of these results, the
isolates had the characteristics associated with the genus
Arthrobacter.
Growth was tested on minimal medium to determine
VOL. 60, 1994 PSYCHROTROPHIC ISOLATES WITH 1-GALACTOSIDASES 15

TABLE 1. Comparison of the amino acid composition in cell TABLE 3. Comparison of enzyme production among isolates B7,
walls of isolates B7, D2, and D5 and control strains D2, and D5 and A. globiformis
Amino acid composition (% of total pmol)a Production by:
Strain Enzyme
Lys Dpm Om Ala Thr Ser Gly Glu Leu A. globiformis B7 D5 D2
B. subtilis 4.0 15.4 0.2 27.1 1.7 4.7 4.3 18.7 5.3 Nitrate reductiona - - - +
A. globiformis 14.0 1.6 0.4 45.7 1.9 3.4 5.0 9.9 4.1 Alkaline phosphatasea + - - +
Isolate B7 14.8 1.4 0.07 50.0 5.1 3.7 4.1 10.7 2.5 l-Glucuronidasea - + + +
Isolate D5 13.3 1.4 0.01 50.1 5.6 4.0 4.0 10.4 2.5 ot-Glucosidasea + + + +
Isolate D2 13.9 1.8 0.06 46.4 6.0 3.8 4.7 10.8 3.1 P-Glucosidasea + + + +
a The values for the isolates are averages of two amino acid determinations. N-Acetylglucosaminidasea - - - +
The total picomoles were 34,089 for B. subtilis; 19,610 for A. globifornis;
Ureasea, b - _ _ +
21,079 for B7; 31,697 for D5; and 33,519 for D2. Minor amounts of other amino Amylase + + + +
acids are not reported. Gelatinase - + + +
DNase - - + +

Downloaded from http://aem.asm.org/ on April 5, 2019 by guest

whether the isolates had any additional requirements. Iso-
lates D5 and B7 grew without added supplements, although
the lag phase for B7 was shortened by the addition of 1%
TSB. Isolate D2 had a vitamin requirement and grew only
when the minimal medium was supplemented with Basal
Medium Eagle Vitamin Solution. The growth rates were
determined for cells growing in TSB at 10 and 25°C (Table 2).
All the isolates grew more rapidly at both temperatures than
A. globiformis did. In addition, the ability to form colonies
on TSA at different temperatures was examined. The iso-
lates grew at 0WC, the lowest temperature tested. Isolated
colonies did not form above 30°C. A. globiformis had a
growth range of 10 to 37°C (Table 2).
The production of different enzymes was compared by
using API Coryne test strips and indicator plates (Table 3).
Isolates B7 and D5 were similar, except that D5 produced
DNase, as did D2. Isolate D2, which differed from B7 and D5
because it had a vitamin requirement, also reduced nitrate
and had alkaline phosphatase and urease activities. All three
isolates had 0-glucuronidase and gelatinase activities,
whereas the A. globiformis ATCC 8010 type strain lacks
these activities. The carbohydrate utilization patterns were
similar, except that the isolates grew on lactose whereas A.
globiformis ATCC 8010 did not and B7 did not use trehalose.
The isolates grew on cellobiose but not cellulose (data not
shown). These results demonstrated that the isolates differed
from A. globiformis and from each other.
Characterization of the 13-galactosidase activities. To deter-
mine whether the 1-galactosidase activities were intracellu-
lar or secreted into the medium, we assayed both the culture
media and the whole cells. All the activity was associated
a Production of these enzymes was determined by using the API Coryne
test strip. Symbols: +, clear positive; +, weak reaction; -, represents
negative reaction.
b Production of urease was also determined by using urease test broth
(BBL).
with the cells for the three isolates (data not shown). The
assay could have detected 0.0001 U of activity per ml in the
medium if it had been present. Cells grown at 25°C were then
assayed at increasing temperatures from 5 to 50°C to obtain
a temperature profile of the P-galactosidase activities (Fig.
2). The results show that the peak activity for isolate D2 was
at 25°C and that the activities for isolates B7 and D5 were
optimal around 30 to 35°C. Activities from cells grown at 5
and 10°C showed similar optima (data not shown). In con-
trast, the highest activity for the E. coli enzyme was above
50°C, and it had little activity below 20°C, where the en-
zymes from the isolates retained about half their highest
activity. Although E. coli cells have higher specific activi-
ties, this simply reflects their ability to express more ,-ga-
lactosidase relative to the total cell protein. Other growth
conditions might further induce P-galactosidase synthesis in
the isolates or the cloning of the gene could be used to
increase the specific activity.
The stability of the P-galactosidase activities was exam-
ined by incubating cells at set temperatures for differing
times and then transferring a sample to be assayed at the
respective temperature optimum (Fig. 3). The activities from
100 1

TABLE 2. Comparison of the growth characteristics of isolates ~80-

B7, D2, and D5 and A. globifonnis
o60-
Strain Generation timea (h) at: Growth range
1°oc 25°C (o) ~)40-
A. globifonnis > 13c 2.4 10-37
Isolate B7 4.8 1.5 0-30 20-
Isolate D2 5.3 1.5 0-30
Isolate D5 5.2 1.5 0-30 0-
a Generation times were determined during exponential growth at the
0 10 2~0 30 0 50 60
indicated temperatures in TSB containing no additional carbohydrate. Temperature (°C)
b
Temperature ranges for growth were determined by examining TSA plates FIG. 2. Measurement of the 3-galactosidase activities from the
for colony formation at 0, 5, 10, 25, 30, 31, 35, 37, and 38°C. The plates were three isolates and E. coli at different temperatures. E. coli (I) was
incubated for 1 month at temperatures below 30'C and 7 to 10 days at 300C and
above before they were discarded. The results give the minimum and grown at 37°C. Isolates B7 (-), D2 (0), and D5 (A) were grown at
maximum temperatures at which isolated colonies were observed. 25°C. The specific activities (micromoles per minute per milligram of
c Generation times were difficult to determine for A. globiformis at 100C protein) at the 100% values were 6.7 for E. coli, 0.51 for B7, 0.26 for
because this is at its lower limit for growth. D2, and 1.2 for D5.
16 LOVELAND ET AL.
0~
0
;>
.)
Time (min)
FIG. 3. Thermostability of the ,3-galactosidase activities found in
isolates B7 (A), D2 (B), and D5 (C). Cells were grown at 10°C,
harvested, and assayed as deslribed in Materials and Methods. The
cell suspensions were incubated at 10°C (-), 35°C (-), 40°C (A),
45°C (*), and 50°C (O) for the'indicated times and then assayed for
P-galactosidase activity at 25°C for D2 and B7 and 30°C for D5.
all isolates were stable at 10°C but decreased within a few
minutes when incubated at 50°C. The activity from D2
appeared slightly more stable, with about 50% remaining
after incubation at 40°C for 120 min.
Detection of 13-galactosidase activities following polyacryl-
amide gel electrophoresis. The results in Fig. 3 showed that
when cells were incubated at the intermediate temperatures,
the 3-galactosidase activity remained relatively constant
after an initial loss. One explanation for this result is that the
isolates contain more than one enzyme with 3-galactosidase
activity and that these isozymes have different temperature
labilities. Bacillus stearothermophilus, for example, has two
structural genes for 3-galactosidase. The bgaA gene pro-
duces an enzyme that is less thermostable than the 3-galac-
tosidase encoded by the bgaB gene (6). Therefore we were
interested in determining whether any of our isolates might
make more than one form of 3-galactosidase. To examine
this possibility, extracts were subjected to nondenaturing
polyacrylamide gel electrophoresis and the gels were incu-
bated with X-Gal to detect 3-galactosidase activity. The
APPL. ENvIRON. MICROBIOL.
1 2 3 4
Downloaded from http://aem.asm.org/ on April 5, 2019 by guest
FIG. 4. Migration of 13-galactosidase activities during nondena-
turing polyacrylamide gel electrophoresis. The extracts from E. coli
(lane 1) and isolates B7 (lane 2), D5 (lane 3), and D2 (lane 4) were
subjected to electrophoresis on a 7.5% nondenaturing polyacryl-
amide gel, and the gel was incubated with X-Gal to detect P-galac-
tosidase activity in situ. The image is a scan of a color photograph of
the gel. The scanned image was generated by using an Apple Color
OneScanner and the application Ofoto, version 2.0. The file was
saved in the TIFF format.
results (Fig. 4) show that the E. coli control (lane 1) formed
one band representing 13-galactosidase hydrolysis of X-Gal.
The extracts from the isolates (lanes 2, 3, and 4), however,
gave rise to more than one activity band for each isolate.
To determine whether the activity bands represented
different isozymes or were simply aggregates of the same
protein, we examined the effects of changing the polyacryl-
amide concentrations on the migration distance of the bands
or the effects of the pH of the in situ assay buffers on the
intensity of the bands. The intensities of the bands varied
independently of each other (data not shown), suggesting
that the activities were from different proteins. Furthermore,
the intensity of the upper band for isolate B7 was influenced
by growth conditions whereas the lower band was present
even when glucose or cellobiose was substituted for lactose
as a carbon source. These results were consistent with the
activity bands representing different proteins that hydrolyze
the X-Gal chromogen.
The most convincing evidence that isolate B7 can synthe-
size different isozymes was provided by the cloning of three
unique genes encoding P-galactosidase activities (21). One
clone encodes a protein that migrates during nondenaturing
polyacrylamide gel electrophoresis in a position correspond-
ing to the upper band shown in Fig. 4, while a second clone
encodes a protein migrating with the lower band. The third
gene produces a third protein that was not observed for
isolate B7 during growth on lactose.
DISCUSSION
X-galactosidase gene from E. ccli has been so well
The
studied and exploited as a reporter gene that the need for
enzymes with other properties is often overlooked. How-
ever, new ed-galactosidases, such as ones with high activity
levels at low temperatures, might prove useful for removing
lactose from refrigerated milk to be consumed by lactose-
intolerant individuals and for converting the lactose in whey
into glucose and galactose to be used as carbon sources in
fermentation broths. This report demonstrates that it is
VOL. 60, 1994 PSYCHROTROPHIC ISOLATES WITH P-GALACTOSIDASES
possible to select and isolate psychrotrophs that make cold-
active 0-galactosidases. Our isolates have ,B-galactosidases
with optimal activities about 20°C below that of the E. coli
enzyme. These isolates are classified as new Arthrobacter
species on the basis of their rod-to-sphere morphogenesis,
their physiological traits, and the presence of lysine as the
primary diamino acid in their cell walls.
The genus Arthrobacter comprises a heterogeneous group
of soil bacteria with the rod-to-coccus morphological cycle
as their major distinguishing feature (9, 10). Comparisons of
the 16S rRNA (9, 22) suggest that the genus Arthrobacter is
related to the other coryneform genera Aureobacterium,
Cellulomonas, Curtobacterium, and Microbacterium and is
more distantly related to the genus Brevibacterium. These
genera are representatives of the high-G+C actinomycete
group of gram-positive bacteria, and distinctions among
them may be minor. The isolates differ fromAureobacterium
and Curtobacterium species, which contain D-ornithine in-
stead of lysine in the cell wall peptidoglycan (Table 1).
Microbacterium species also contain lysine as the diamino
acid. However, Microbacterium strains produce acid from
glucose and have a group B peptidoglycan containing glycine
and lysine in the interpeptide bridge (19). Thus, the Mi-
crobacterium cell wall has two to three times more glycine
than glutamate. Since the isolates had only the background
levels of glycine observed for A. globiformis and B. subtilis,
their cell wall structures do not resemble that typically found
for Microbactenum cells.
It is interesting that the fatty acid profiles determined for
our isolates suggested Micrococcus as a possible genus.
Members of the genus Micrococcus would seem to be
unrelated to Arthrobacter on the basis of cell shape. How-
ever, Kocur (11) suggests that the genus Micrococcus is
more closely related to the genus Arthrobacter than to other
coccoid genera and that Micrococcus species may be degen-
erate forms of arthrobacters which are locked in the coccoid
stage. These similarities are reflected in the fatty acid
profiles. Both the MIDI and IEA testing groups indepen-
dently identified the same predominant fatty acids. The
variations in their results may reflect the different data bases
used for comparisons. Our results illustrate the need to
incorporate results from morphological and physiological
tests when identifying atypical strains.
We found no studies of P-galactosidases from Arthrobac-
ter species, and our work with the type strain,A. globifonnis
ATCC 8010, shows that it does not use lactose as a carbon
source. We did observe, however, thatA. globiformis ATCC
8010 tested faintly positive for ,B-galactosidase on the API
Coryne test strip even though it cannot use lactose as a
carbon source. This could be explained if the strain contains
low levels of some other enzyme that hydrolyzes the chro-
mogen but does not hydrolyze sufficient lactose for growth
or if it lacks the permease for transporting lactose. Our
finding that our isolates contain two to three isozymes
capable of hydrolyzing ONPG or X-Gal suggests that these
sensitive chromogens detect enzymes that might be involved
in the use of other sugars or polysaccharides. Although these
would not be true ,B-galactosidases, they might be glucosi-
dases or other enzymes with sufficiently broad specificities
to hydrolyze the chromogenic substrates slowly. These
results illustrate that physiological traits cannot be deter-
mined by using test strips and chromogens alone and should
be substantiated with enzyme assays and growth studies.
Since lactose utilization by Arthrobacter species has not
been reported, we are obtaining other strains for comparison
with our isolates. Preliminary results show that another A.
17
globiformis strain (NRRL B-2979) and an A. citreus strain
(NRRL B-14091) do not use lactose as a carbon source.
Further study of these and other strains will help determine
whether our isolates are new species and will also help
explain the function of their different 3-galactosidase
isozymes.
The results of ,B-galactosidase assays show that these
isolates produce at least one enzyme with peak activities at
about 20°C below that for the E. coli enzyme (Fig. 2).
Activities for the isolates are significant below 20°C, at
which less than 10% of the E. coli enzyme activity remains.
Thermostability studies of the activities for the isolates show
that they are stable at low temperatures but labile when
incubated above about 40°C for 120 min (Fig. 4). Very little
is known about the survival of psychrotrophs in climates
Downloaded from http://aem.asm.org/ on April 5, 2019 by guest
with wide temperature ranges. One mechanism that micro-
organisms could use to adapt to widely varying temperatures
is production of isozymes with different temperature optima.
Since the isolates synthesize more than one protein with
,B-galactosidase activity, it is possible that these enzymes are
designed to function at different temperatures. It is also
possible that these activities have other, yet to be discovered
functions independent of growth on lactose. The discovery
of these Arthrobacter strains containing ,-galactosidase
isozymes opens new questions and opportunities for explor-
ing the regulation and function of these enzymes. We have
cloned three genes encoding P-galactosidase activities for
isolate B7 and are purifying these enzymes to determine
which substrates they use and their temperature optima. We
are also preparing antibodies to each isozyme synthesized by
isolate B7 to be used to measure the amounts of each
P-galactosidase synthesized in cells grown at different tem-
peratures to determine whether their expression is regulated
by temperature.
ACKNOWLEDGMENTS
Scanning electron microscopy was performed at the Electron
Microscope Facility for the Life Sciences in the Penn State Biotech-
nology Institute. We thank R. Walsh for support and help in
preparing materials for microscopy. We thank M. J. Tershak and
L. E. Casida, Jr., for providing strains and D. Trimbur, K. Miller,
and A. Phillips for helpful discussions.
P. Prema was supported by a Biotechnology Overseas Associate-
ship from the Department of Biotechnology, India.
REFERENCES
1. Beguin, P., N. R. Gilkes, D. G. Kilburn, R. C. J. Miller, G. P.
O'Neill, and R. A. J. Warren. 1987. Cloning of cellulase genes.
Crit. Rev. Biotechnol. 6:129-162.
2. Cole, G. T. 1986. Preparation of microfungi for scanning elec-
tron microscopy, p. 1-44. In H. C. Aldrich and W. J. Todd (ed.),
Ultrastructure techniques for microorganisms. Plenum Press,
New York.
3. Collins, C. H., P. M. Lyne, and J. M. Grange (ed.). 1991. Collins
and Lyne's microbiological methods, 6th ed. Butterworth-
Heinemann, Oxford. (Reprint of 1989 edition.)
4. Gounot, A.-M. 1991. Bacterial life at low temperature: physio-
logical aspects and biotechnological implications. J. Appl. Bac-
teriol. 71:386-397.
5. Herbert, R. A. 1986. The ecology and physiology of psychro-
philic microorganisms. Spec. Pub. Soc. Gen. Microbiol. 17:1-
23.
6. Hirata, H., S. Megoro, and H. Okada. 1984. Molecular basis of
isozyme formation of 3-galactosidases in Bacillus stearothermo-
philus: isolation of two ,3-galactosidase genes, bgaA and bgaB.
J. Bacteriol. 160:9-14.
7. Ingraham, J. L. 1958. Growth of psychrophilic bacteria. J.
Bacteriol. 76:75-80.
18 LOVELAND ET AL.
8. Inniss, W. E. 1975. Interaction of temperature and psychrophilic
microorganisms. Annu. Rev. Microbiol. 29:445-465.
9. Jones, D., and R. M. Keddie. 1992. The genus Arthrobacter, p.
1283-1299. In A. Balows, H. Truper, M. Dworkin, W. Harder,
and K.-H. Schleifer (ed.), The prokaryotes, 2nd ed, vol. II.
Springer-Verlag, New York.
10. Keddie, R. M., M. D. Collins, and D. Jones. 1986. Genus
Arthrobacter Conn and Dimmick 1947, 300AL, p. 1288-1301. In
P. H. A. Sneath, N. S. Mair, E. M. Sharpe, and J. G. Holt (ed.),
Bergey's manual of systematic bacteriology, vol. 2. The Wil-
liams & Wilkins Co., Baltimore.
11. Kocur, M. 1986. Genus I Micrococcus Cohn 1872, 151AL, p.
1004-1008. In P. H. A. Sneath, N. S. Mair, E. M. Sharpe, and
J. G. Holt (ed.), Bergey's manual of systematic bacteriology,
vol. 2. The Williams & Wilkins Co., Baltimore.
12. Komagata, K., and K. I. Suzuki. 1986. Genus Curtobacterium
Yamada and Komagata 1972, 425AL, p. 1313-1316. In P. H. A.
Sneath, N. S. Mair, E. M. Sharpe, and J. G. Holt (ed.), Bergey's
manual of systematic bacteriology, vol. 2. The Williams &
Wilkins Co., Baltimore.
13. Kormendy, A. C. 1975. Microorganisms, p. 82-108. In M. A.
Hayat (ed.), Principles and techniques of scanning electron
microscopy. Van Nostrand Reinhold Co., New York.
14. Krieg, N. R. 1981. Enrichment and isolation, p. 139. In P.
Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester,
W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of
methods for general bacteriology. American Society for Micro-
APPL. ENvIRON. MICROBIOL.
biology, Washington, D.C.
15. Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (London)
227:680-685.
16. Miller, J. H. 1972. Experiments in molecular genetics. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
17. Morita, R. Y. 1975. Psychrophilic bacteria. Bacteriol. Rev.
39:144-167.
18. Neidhardt, F. C., R. VanBogelen, and V. Vaughn. 1984. The
genetics and regulations of heat shock proteins. Annu. Rev.
Genet. 18:295-329.
19. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types of
bacterial cell walls and their taxonomic implications. Bacteriol.
Rev. 36:407-477.
20. Smibert, R. M., and N. R. Krieg. 1981. General characteriza-
tion, p. 407-443. In P. Gerhardt, R. G. E. Murray, R. N.
Downloaded from http://aem.asm.org/ on April 5, 2019 by guest
Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B.
Phillips (ed.), Manual of methods for general bacteriology.
American Society for Microbiology, Washington, D.C.
21. Trimbur, D., K. Gutshall, and J. Brenchley. 1993. Characteriza-
tion and cloning of 1-galactosidases from a psychrotrophic
isolate, abstr. K-138, p. 284. Abstr. 93rd Gen. Meet. Am. Soc.
Microbiol. 1993. American Society for Microbiology, Washing-
ton, D.C.
22. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-
271.