APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1994, p. 12-180099-2240/94/$04.00+0Copyright ) 1994, American Society for Microbiology

Characterization of Psychrotrophic MicroorganismsProducing 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 environmentof the Earth is cold and enzymes with high activities at low temperatures could have commercial uses. We haveisolated 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 electronmicroscope appeared as rods during the early stages ofgrowth but became coccoid during the stationary phase.An analysis of the amino acid composition of the cell walls demonstrated the presence of lysine as thepredominant diamino acid in all three isolates. The cell cycle morphology and cell wall composition suggest thatthe three isolates are members of the genus Arthrobacter. The 13-galactosidase activities in whole cells werelabile when incubated at 40°C and had temperature optima about 20°C below that of the enzyme encoded bythe lacZ gene ofEscherichia coli. Electrophoresis of extracts from the isolates in nondenaturing polyacrylamidegels 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 andits oceans is below 20°C, we know little about the types andphysiology of microorganisms growing at these tempera-tures. Psychrotrophs, or cold-tolerant microorganisms, mustoften adapt to temperatures ranging from subzero to above30°C. Research examining mechanisms that might permitlow-temperature growth, such as changes in membranefluidity, regulation of protein synthesis, and production ofheat and cold shock proteins, has been reviewed (4, 5, 7, 8,17, 18). However, it is amazing that so little is known aboutthe diversity and metabolism of psychrotrophic microorgan-isms considering that vast regions of the globe are subject tocold climates and that temperature controls virtually everycell reaction. The study of these microorganisms could addconsiderably to our understanding of microbial diversity andlead 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 catalyticactivities at temperatures below 20°C. The properties ofthese enzymes would then be compared with those fromcorresponding proteins from mesophiles and thermophiles,and the information would be used to determine whichfeatures are important for functions at different tempera-tures. In addition, cold-active enzymes could have importantapplications in biotechnology, food processing, biomassconversion, molecular biology, etc. To initiate this work, wehave isolated psychrotrophic microorganisms with 13-galac-tosidase activities. We selected 3-galactosidase as a modelenzyme because it is possible to screen numerous coloniesby using chromogenic substrates and there are well-studiedcounterparts from mesophiles and thermophiles that makestructural comparisons possible. Cold-active 3-galactosi-

* Corresponding author. Mailing address: Department of Bio-chemistry and Molecular Biology, 209 S. Frear, The PennsylvaniaState University, University Park, PA 16802. Phone: (814) 863-7794.Fax: (814) 863-7024. Electronic mail address: JEB7@psu.edu.

dases could also be used to remove lactose from refrigeratedmilk so that it could be consumed by people who are lactoseintolerant and to convert the lactose in whey from a pollutantto the more readily fermentable glucose and galactose.

In this study we isolated psychrotrophic microorganismsfrom fields that had been spread with whey during thewinter. We screened for microorganisms growing at lowtemperature and producing 3-galactosidase activities. Wedescribe here the characterization of three isolates whichhave a rod-coccus cycle typical ofArthrobacter species. Wehave examined the growth and physiology of these isolatesand the effect of temperature on the activity and stability oftheir 3-galactosidase activities. We found that these isolatescontain 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 isolatedfrom Pennsylvania farmlands. Soil samples giving rise toisolates D2 and D5 were fr6m adjacent fields on the samefarm, and B7 was isolated from a nearby farm. The siteswere selected because the fields had been spread with whey,which might have served as an enrichment for lactose-utilizing microorganisms. The samples were collected inearly spring just after the snow had melted. All samples andmedia were kept cold during transport and the isolation andselection procedures. The soil samples were diluted with0.9% NaCl and spread onto tryptic soy agar (TSA) with orwithout 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 7days, colonies which appeared blue were picked andstreaked onto fresh medium. Once the isolates were purified,the 3-galactosidase activities were determined as describedbelow.

Other strains used in the study were Arthrobacter globi-

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formis ATCC 8010 (type strain), obtained from L. E. Casida,Jr., and Escherichia coli and Bacillus subtilis, obtained fromMary 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) withdifferent carbon sources added at 0.2%. Unless otherwisenoted, isolates B7 and D5 andA. globiformis were cultivatedin M9 with no additional supplementation. Isolate D2 had avitamin requirement, and 1 ml of Basal Medium EagleVitamin Solution (GIBCO BRL, Gaithersburg, Md.} wasroutinely added per 100 ml of M9. Thiamine (1 ,ug ml- ) wasadded to M9 for growth of E. coli. Tryptic soy broth (TSB)and TSA with no added carbohydrate were used for growthrate and growth range determinations, respectively. Culturesof isolates D2, D5, and B7 and A. globifonnis were incu-bated at 25°C on a platform shaker or in a shaking water bathat 200 rpm. E. coli cultures were incubated in a shakingwater bath at 37°C at 300 rpm.

Characterization of isolates. Initial observations of the cellsduring the tests for the Gram stain reaction suggested thatthey were pleomorphic. To further examine this and todetermine 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 aKlett-Summerson colorimeter (Manostat, New York, N.Y.)to harvest the cells at early exponential and stationaryphases. The cells were prepared for scanning electron mi-croscopy by the procedures of Cole (2), which includedcollection of the cells by filtration through a MilliporeSwinney disk holder containing a polycarbonate filter (poresize, 0.2 ,um; Nuclepore Corp., Pleasanton, Calif.). Thesamples were then prepared for microscopy by conventionalmethods (13) and examined under a JEOL JSM 5400 scan-ning electron microscope.

Cell wall extracts from isolates D2, D5, and B7 and fromBacillus 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 performedat The Hershey Medical Center of The Pennsylvania StateUniversity.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 weredone twice, and if the results were marginal, they wererepeated a third time. Urease production was detected byusing API strips and urease test broth (BBL, Cockeysville,Md.). To detect gelatin hydrolysis, strains were inoculatedonto plates of nutrient agar containing 5% gelatin and theplates were flooded with saturated ammonium sulfate aftergrowth to indicate zones of hydrolysis (3). DNA degradationwas determined by growing the organisms on nutrient agarwith 0.2% DNA and toluidine blue by a method presented inthe Manual of Methods for General Bacteriology (20).Strains were inoculated onto M9 agar which contained, inaddition 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 weredetected with 1% Congo red, and the excess dye was washedoff with 1 M NaCl (1). The medium used for carbon sourceutilization was M9 containing 0.2% carbohydrate. Growthwas assessed after 18, 42, 66, and 116 h.

1-Galactosidase assay. Cells were grown in M9 containing0.2% lactose, harvested by centrifugation, washed with 0.9%

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 wassecreted, 0.5 ml of the culture medium was added to 0.5 mlof buffer and assayed by the procedure of Miller (16). Thespecific 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 isolatesproduced more than one ,B-galactosidase isozyme. Cultureswere grown in M9 medium with 0.2% lactose, except for B7,for which 1% TSB (10 ml of TSB to 1 liter of minimalmedium) was added to the M9 medium. Cultures wereharvested at mid- to late exponential phase. After harvest,the cells were broken in a French pressure cell at 18,000lb/in2 and the extracts were concentrated with an Ul-trafree-MC filter unit (nominal molecular weight limit,100,000; Millipore, Bedford, Mass.). Concentrated extractswere then subjected to electrophoresis in 7.5% nondenatur-ing polyacrylamide gels by the procedure of Laemmli (15)without the addition of sodium dodecyl sulfate. Followingelectrophoresis, the gels were incubated in an assay buffer inwhich 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 andhydrolyzed the chromogen X-Gal were screened for strainsthat made I-galactosidases with temperature optima below35°C. From these we selected three, B7, D2, and D5, forfurther characterization. The isolates formed smooth, circu-lar, yellow to cream colonies. The cells were gram positivebut also appeared gram negative during the growth cycle andwere easily decolorized late in growth. Samples taken atdifferent times during growth and examined under a scanningelectron microscope showed elongated, club-shaped cellsduring exponential growth which fragmented into short rodsand coccoid cells later in growth (Fig. 1). The average celllengths 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 strictaerobes, 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 physiologicaltraits are typical of Arthrobacter species, we analyzed thecell walls of the isolates to determine whether lysine, whichis the characteristic diamino acid present in Arthrobacterpeptidoglycan, was present (Table 1). The results show thatthe isolates and an A. globiformis type strain containedlysine as the dominant diamino acid, whereas B. subtilis,included as a control, contained diaminopimelic acid asexpected.

In addition, these isolates were sent to MIDI (MicrobialID, Inc., Newark, Del.) and to IEA (Industrial and Environ-mental Analysts, Inc., Essex Junction, Vt.) to determinetheir fatty acid compositions. Both groups reported that thethree strains are similar to each other, with D2 and D5possibly more closely related to each other than to B7.However, the testing services differed in their first choices ofgenera: IEA suggested Arthrobacter for isolate B7 andCurtobacterium for D2 and D5, whereas MIDI suggestedMicrococcus as the most likely genus for all three with

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I II

A

B

C

FIG. 1. Scanning electron micrographs of the three isolates showing the morphological changes. Isolates B7 (A), D2 (B), and D5 (C) weregrown 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 Curtobactenumfor isolates D2 and D5. Our results showing that the isolateshad 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 notchange with medium or culture age (11). Cell pleomorphismis also not distinctive for members of the genus Curtobacte-rium, although cells can become shorter in older cultures.

Curtobacterium strains are generally motile, produce acidslowly from carbohydrates, and contain ornithine in theinterpeptide bridge of the cell wall (12). Our results (Table 1)showed that ornithine was not present in the cell walls of theisolates. On the basis of the combination of these results, theisolates had the characteristics associated with the genusArthrobacter.

Growth was tested on minimal medium to determine

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TABLE 1. Comparison of the amino acid composition in cellwalls of isolates B7, D2, and D5 and control strains

Amino acid composition (% of total pmol)aStrain

Lys Dpm Om Ala Thr Ser Gly Glu Leu

B. subtilis 4.0 15.4 0.2 27.1 1.7 4.7 4.3 18.7 5.3A. globiformis 14.0 1.6 0.4 45.7 1.9 3.4 5.0 9.9 4.1Isolate B7 14.8 1.4 0.07 50.0 5.1 3.7 4.1 10.7 2.5Isolate D5 13.3 1.4 0.01 50.1 5.6 4.0 4.0 10.4 2.5Isolate D2 13.9 1.8 0.06 46.4 6.0 3.8 4.7 10.8 3.1

a The values for the isolates are averages of two amino acid determinations.The total picomoles were 34,089 for B. subtilis; 19,610 for A. globifornis;21,079 for B7; 31,697 for D5; and 33,519 for D2. Minor amounts of other aminoacids are not reported.

whether the isolates had any additional requirements. Iso-lates D5 and B7 grew without added supplements, althoughthe lag phase for B7 was shortened by the addition of 1%TSB. Isolate D2 had a vitamin requirement and grew onlywhen the minimal medium was supplemented with BasalMedium Eagle Vitamin Solution. The growth rates weredetermined for cells growing in TSB at 10 and 25°C (Table 2).All the isolates grew more rapidly at both temperatures thanA. globiformis did. In addition, the ability to form colonieson TSA at different temperatures was examined. The iso-lates grew at 0WC, the lowest temperature tested. Isolatedcolonies did not form above 30°C. A. globiformis had agrowth 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 producedDNase, as did D2. Isolate D2, which differed from B7 and D5because it had a vitamin requirement, also reduced nitrateand had alkaline phosphatase and urease activities. All threeisolates had 0-glucuronidase and gelatinase activities,whereas the A. globiformis ATCC 8010 type strain lacksthese activities. The carbohydrate utilization patterns weresimilar, 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 notshown). These results demonstrated that the isolates differedfrom 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 culturemedia and the whole cells. All the activity was associated

TABLE 2. Comparison of the growth characteristics of isolatesB7, D2, and D5 and A. globifonnis

Strain Generation timea (h) at: Growth range1°oc 25°C (o)

A. globifonnis > 13c 2.4 10-37Isolate B7 4.8 1.5 0-30Isolate D2 5.3 1.5 0-30Isolate D5 5.2 1.5 0-30

a Generation times were determined during exponential growth at theindicated temperatures in TSB containing no additional carbohydrate.

b Temperature ranges for growth were determined by examining TSA platesfor colony formation at 0, 5, 10, 25, 30, 31, 35, 37, and 38°C. The plates wereincubated for 1 month at temperatures below 30'C and 7 to 10 days at 300C andabove before they were discarded. The results give the minimum andmaximum temperatures at which isolated colonies were observed.

c Generation times were difficult to determine for A. globiformis at 100Cbecause this is at its lower limit for growth.

TABLE 3. Comparison of enzyme production among isolates B7,D2, and D5 and A. globiformis

Production by:Enzyme

A. globiformis B7 D5 D2

Nitrate reductiona - - - +Alkaline phosphatasea + - - +l-Glucuronidasea - + + +ot-Glucosidasea + + + +P-Glucosidasea + + + +N-Acetylglucosaminidasea - - - +Ureasea,b - _ _ +Amylase + + + +Gelatinase - + + +DNase - - + +

a Production of these enzymes was determined by using the API Corynetest strip. Symbols: +, clear positive; +, weak reaction; -, representsnegative reaction.

b Production of urease was also determined by using urease test broth(BBL).

with the cells for the three isolates (data not shown). Theassay could have detected 0.0001 U of activity per ml in themedium if it had been present. Cells grown at 25°C were thenassayed at increasing temperatures from 5 to 50°C to obtaina temperature profile of the P-galactosidase activities (Fig.2). The results show that the peak activity for isolate D2 wasat 25°C and that the activities for isolates B7 and D5 wereoptimal around 30 to 35°C. Activities from cells grown at 5and 10°C showed similar optima (data not shown). In con-trast, the highest activity for the E. coli enzyme was above50°C, and it had little activity below 20°C, where the en-zymes from the isolates retained about half their highestactivity. 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 growthconditions might further induce P-galactosidase synthesis inthe isolates or the cloning of the gene could be used toincrease the specific activity.The stability of the P-galactosidase activities was exam-

ined by incubating cells at set temperatures for differingtimes and then transferring a sample to be assayed at therespective temperature optimum (Fig. 3). The activities from

100 1

~80-

o60-

~)40-

20-

0-0 10 2~0 30 0 50 60

Temperature (°C)FIG. 2. Measurement of the 3-galactosidase activities from the

three isolates and E. coli at different temperatures. E. coli (I) wasgrown at 37°C. Isolates B7 (-), D2 (0), and D5 (A) were grown at25°C. The specific activities (micromoles per minute per milligram ofprotein) at the 100% values were 6.7 for E. coli, 0.51 for B7, 0.26 forD2, and 1.2 for D5.

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0

0

.)

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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. Thecell suspensions were incubated at 10°C (-), 35°C (-), 40°C (A),45°C (*), and 50°C (O) for the'indicated times and then assayed forP-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 fewminutes when incubated at 50°C. The activity from D2appeared slightly more stable, with about 50% remainingafter incubation at 40°C for 120 min.

Detection of 13-galactosidase activities following polyacryl-amide gel electrophoresis. The results in Fig. 3 showed thatwhen cells were incubated at the intermediate temperatures,the 3-galactosidase activity remained relatively constantafter an initial loss. One explanation for this result is that theisolates contain more than one enzyme with 3-galactosidaseactivity and that these isozymes have different temperaturelabilities. Bacillus stearothermophilus, for example, has twostructural 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 wereinterested in determining whether any of our isolates mightmake more than one form of 3-galactosidase. To examinethis possibility, extracts were subjected to nondenaturingpolyacrylamide gel electrophoresis and the gels were incu-bated with X-Gal to detect 3-galactosidase activity. The

1 2 3 4

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) weresubjected 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 ofthe gel. The scanned image was generated by using an Apple ColorOneScanner and the application Ofoto, version 2.0. The file wassaved in the TIFF format.

results (Fig. 4) show that the E. coli control (lane 1) formedone 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 sameprotein, we examined the effects of changing the polyacryl-amide concentrations on the migration distance of the bandsor the effects of the pH of the in situ assay buffers on theintensity of the bands. The intensities of the bands variedindependently of each other (data not shown), suggestingthat the activities were from different proteins. Furthermore,the intensity of the upper band for isolate B7 was influencedby growth conditions whereas the lower band was presenteven when glucose or cellobiose was substituted for lactoseas a carbon source. These results were consistent with theactivity bands representing different proteins that hydrolyzethe X-Gal chromogen.The most convincing evidence that isolate B7 can synthe-

size different isozymes was provided by the cloning of threeunique genes encoding P-galactosidase activities (21). Oneclone encodes a protein that migrates during nondenaturingpolyacrylamide gel electrophoresis in a position correspond-ing to the upper band shown in Fig. 4, while a second cloneencodes a protein migrating with the lower band. The thirdgene produces a third protein that was not observed forisolate B7 during growth on lactose.

DISCUSSION

TheX-galactosidase gene from E. ccli has been so wellstudied and exploited as a reporter gene that the need forenzymes with other properties is often overlooked. How-ever, new ed-galactosidases, such as ones with high activitylevels at low temperatures, might prove useful for removinglactose from refrigerated milk to be consumed by lactose-intolerant individuals and for converting the lactose in wheyinto glucose and galactose to be used as carbon sources infermentation broths. This report demonstrates that it is

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possible to select and isolate psychrotrophs that make cold-active 0-galactosidases. Our isolates have ,B-galactosidaseswith optimal activities about 20°C below that of the E. colienzyme. These isolates are classified as new Arthrobacterspecies on the basis of their rod-to-sphere morphogenesis,their physiological traits, and the presence of lysine as theprimary diamino acid in their cell walls.The genus Arthrobacter comprises a heterogeneous group

of soil bacteria with the rod-to-coccus morphological cycleas their major distinguishing feature (9, 10). Comparisons ofthe 16S rRNA (9, 22) suggest that the genus Arthrobacter isrelated to the other coryneform genera Aureobacterium,Cellulomonas, Curtobacterium, and Microbacterium and ismore distantly related to the genus Brevibacterium. Thesegenera are representatives of the high-G+C actinomycetegroup of gram-positive bacteria, and distinctions among

them may be minor. The isolates differ fromAureobacteriumand Curtobacterium species, which contain D-ornithine in-stead of lysine in the cell wall peptidoglycan (Table 1).Microbacterium species also contain lysine as the diaminoacid. However, Microbacterium strains produce acid fromglucose and have a group B peptidoglycan containing glycineand lysine in the interpeptide bridge (19). Thus, the Mi-crobacterium cell wall has two to three times more glycinethan glutamate. Since the isolates had only the backgroundlevels of glycine observed for A. globiformis and B. subtilis,their cell wall structures do not resemble that typically foundfor Microbactenum cells.

It is interesting that the fatty acid profiles determined forour isolates suggested Micrococcus as a possible genus.

Members of the genus Micrococcus would seem to beunrelated to Arthrobacter on the basis of cell shape. How-ever, Kocur (11) suggests that the genus Micrococcus ismore closely related to the genus Arthrobacter than to othercoccoid genera and that Micrococcus species may be degen-erate forms of arthrobacters which are locked in the coccoidstage. These similarities are reflected in the fatty acidprofiles. Both the MIDI and IEA testing groups indepen-dently identified the same predominant fatty acids. Thevariations in their results may reflect the different data basesused for comparisons. Our results illustrate the need toincorporate results from morphological and physiologicaltests when identifying atypical strains.We found no studies of P-galactosidases from Arthrobac-

ter species, and our work with the type strain,A. globifonnisATCC 8010, shows that it does not use lactose as a carbonsource. We did observe, however, thatA. globiformis ATCC8010 tested faintly positive for ,B-galactosidase on the APICoryne test strip even though it cannot use lactose as a

carbon source. This could be explained if the strain containslow levels of some other enzyme that hydrolyzes the chro-mogen but does not hydrolyze sufficient lactose for growthor if it lacks the permease for transporting lactose. Ourfinding that our isolates contain two to three isozymescapable of hydrolyzing ONPG or X-Gal suggests that thesesensitive chromogens detect enzymes that might be involvedin the use of other sugars or polysaccharides. Although thesewould not be true ,B-galactosidases, they might be glucosi-dases or other enzymes with sufficiently broad specificitiesto hydrolyze the chromogenic substrates slowly. Theseresults illustrate that physiological traits cannot be deter-mined by using test strips and chromogens alone and shouldbe substantiated with enzyme assays and growth studies.

Since lactose utilization by Arthrobacter species has notbeen reported, we are obtaining other strains for comparisonwith our isolates. Preliminary results show that another A.

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 determinewhether our isolates are new species and will also helpexplain the function of their different 3-galactosidaseisozymes.The results of ,B-galactosidase assays show that these

isolates produce at least one enzyme with peak activities atabout 20°C below that for the E. coli enzyme (Fig. 2).Activities for the isolates are significant below 20°C, atwhich less than 10% of the E. coli enzyme activity remains.Thermostability studies of the activities for the isolates showthat they are stable at low temperatures but labile whenincubated above about 40°C for 120 min (Fig. 4). Very littleis known about the survival of psychrotrophs in climateswith wide temperature ranges. One mechanism that micro-organisms could use to adapt to widely varying temperaturesis 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 aredesigned to function at different temperatures. It is alsopossible that these activities have other, yet to be discoveredfunctions independent of growth on lactose. The discoveryof these Arthrobacter strains containing ,-galactosidaseisozymes opens new questions and opportunities for explor-ing the regulation and function of these enzymes. We havecloned three genes encoding P-galactosidase activities forisolate B7 and are purifying these enzymes to determinewhich substrates they use and their temperature optima. Weare also preparing antibodies to each isozyme synthesized byisolate B7 to be used to measure the amounts of eachP-galactosidase synthesized in cells grown at different tem-peratures to determine whether their expression is regulatedby temperature.

ACKNOWLEDGMENTS

Scanning electron microscopy was performed at the ElectronMicroscope Facility for the Life Sciences in the Penn State Biotech-nology Institute. We thank R. Walsh for support and help inpreparing materials for microscopy. We thank M. J. Tershak andL. 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.

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