System. App!. Microbio!. 20, 559-565 (1997) © Gustav Fischer Verlag

Taxonomic Comparison of Different Thermophilic Sugar Beet Isolates with Glycosylated Surface Layer (S-Layer) Proteins and their Affiliation to Bacillus smithii

PAUL MESSNER\ ANDREA SCHEBERL\ WOLFGANG SCHWEIGKOFLER2, FRIEDRICH HOLLAUS3, FREDERICK A. RAINEy 4;\

lUTIA BURGHARDT4 and HANS]0RG PRILLINGER2

IZentrum fur Ultrastrukrurforschung und Ludwig Boltzmann-Institut fur Molekulare Nanotechnologie, Universitiit fur Bodenkultur, Wien, Austria 2Institut fur Angewandte Mikrobiologie, Universitiit fur Bodenkultur, Wien, Austria 3Zuckerforschung Tulln Ges. m. b. H., Tulln, Austria 4Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany

Received June 5,1997

Summary

During the beet sugar campaign 199111992 we have characterized thermophilic bacteria from the extraction plant of an Austrian beet sugar factory in a polyphasic approach. Sodium dodecyl sulfate­polyacrylamide gel electrophoresis (SDS-PAGE) and freeze-fracture electron microscopy revealed the presence of weakly glycosylated, oblique crystalline cell surface layers (S-layers) on all sugar factory iso­lates. By partial 16S rDNA sequencing and DNA-DNA hybridisation it was demonstrated that the strains belong to the species Bacillus smithii. In the course of the campaign, however, different strains of B. smithii became dominant, which was demonstrated by fingerprinting using SDS-PAGE, polar lipid analysis, random amplified polymorphic DNA (RAPD) assays, fluorophore-assisted carbohydrate elec­trophoresis (FACE). Possible explanations for this divergence in strain development are discussed from the technological point of view.

Key words: Bacillus smithii - surface layer (S-layer) - prokaryotic glycoprotein - taxonomy - polar lipids - RAPD analysis - 16S rDNA sequencing - DNA-DNA hybridisation

Introduction

Many thermophilic bacterial strains have been isolat­ed from extraction plants of beet sugar factories (for re­view see DUBOURG and DEVILLER, 1953; KLAUSHOFER and HOLLAUS, 1970; HOLLAUS and KLAUSHOFER, 1973). They belong mainly to the species Bacillus stearothermophilus, Thermoanaerobacter (formerly Clostridium) thermohy­drosulfuricus, and Thermoanaerobacterium (formerly Clostridium) thermosaccharolyticum. Just recently we have identified in sugar factory juices new thermophilic organisms such as Bacillus thermoaerophilus, Bacillus smithii (MEIER-STAUFFER et al., 1996), and Thermus sp. (HOLLAUS et al., 1997). Most of the organisms are cov­ered with crystalline cell surface layers (S-layers) for re­view see MESSNER and SLEYTR, 1992; BEVERIDGE, 1994; SLEYTR et al., 1996). Of particular interest for us were

5 Present address: Department of Microbiology, Louisiana State University Baton Rouge, LA, U.S.A.

those strains which possess glycosylated S-layer proteins (for review see MESSNER, 1996, 1997).

The results of a precharacterization study of isolates from the beet sugar campaign 199111992 of the Austrian beet sugar factory Leopoldsdorf by electron microscopy of freeze-etched and thin-sectioned whole cells and SDS­PAGE of SDS extracts of the biomass had indicated that isolates L400-91, L401-91, L402-91, L403-91, L428-91, and L434-91 belong to the same group and possess s­layers (c. NEUNINGER, unpublished). In the course of the taxonomic characterization of Bacillus thermoaerophilus

Abbreviations S-layer - surface layer; SDS-PAGE - sodium dodecyl sulfate­polyacrylamide gel electrophoresis; RAPD - random amplyfied polymorphic DNA; PCR - polymerase chain reaction; FACE -fluorophore-assisted carbohydrate electrophoresis; AMAC - 2-aminoacridon; TLC - thin layer chromatography

560 P. MESSNER et al.

L420-91 (DSM 10154) isolate L400-91, as a typical rep­resentative of the previous group, was found to belong to the species Bacillus smithii (MEIER-STAUFFER et aI., 1996).

Recently RAPD-PCR was used for identifying ther­mophilic and mesophilic Bacillus species, including Bacillus smith ii, in factory samples (RONIMUS et aI., 1997). It has been proven a versatile method for produc­ing unique genetic fingerprints of new bacterial isolates (WILLIAMS et aI., 1990). Therefore RAPD-PCR, together with other chemotaxonomic and genetic methods, was chosen for the analysis of the new sugar factory isolates. Here we report on our efforts to verify the taxonomic af­filiation of the different isolates which had been shown high similarity to the thermophilic species Bacillus smithii (NAKAMURA et aI., 1988).

Materials and Methods

Bacterial strains and cultivation: The strains from the sugar beet campaign 199111992 were isolated from enrichment cultures by incubation of sugar beet raw juice from the extrac­tion plant of the beet sugar factory Leopoldsdorf, Austria, in a fermenter at 70 0c. During the drop of the pH value, indicating bacterial activity by acid formation, fractional smears of the juice were made on PBYS- and TYG agar plates [PBYS agar: 1 % peptone (Oxoid), 0.5% meat extract (Oxoid), 0.5% yeast extract (Oxoid) 0.05% glucose, 0.1% KH2P04, 0.01 % MgS04· 7H20, 0.001 % FeS04· 7H20, 2% Bacto agar (Difco), pH 6.8-7.0; TYG agar: 0.5% Bacto tryptone (Difco), 0.25% yeast extract (Oxoid) 0.1 % glucose, 2 % Bacto agar (Difco), pH 6.8-7.0]. The plates were incubated at 60 0c. From isolated colonies again fractionated smears were carried out and the procedure was repeated until pure cultures were obtained (HoL­LAUS and POLLACH, 1993). These cultures were maintained on TYG agar slopes (MEIER-STAUFFER et aI., 1996).

The new isolates and reference strains used in this study are listed in Table 1.

Growth temperature: Growth tests were carried out in TYG or PBYS medium at temperatures of 35, 37, 55, 60, 65, and 68 °C, respectively. After precultivation of the strains on TYG agar plates and TYG broth (Erlenmeyer flasks in a rotary shak­ing bath, 250 rpm) for 16 h at 55 °C, preheated flasks contain­ing either TYG or PBYS medium were inoculated with 1 ml of the TYG broth preculture and incubated at 55 0c. After incuba­tion for 16 to 24 h at the respective temperatures (60 and 65 0c) these cultures served as inoculum for the next assay step. The ambient temperature range (35 and 37 0c) was tested directly with inoculates from the 55 °C culture.

Morphology: Light microscopy, preparation of the samples for electron microscopy and electron microscopy were carried out as previously described (SLEYTR et aI., 1988; MEIER-STAUF­FER et aI., 1996).

Electrophoresis of whole-cell protein and FACE analysis of sugars: SDS-PAGE according to LAEMMLI (1970) of SDS-soluble whole-cell extracts was described previously (MEIER-STAUFFER et aI., 1996).

Analysis of covalently attached carbohydrate residues was carried out after hydrolysis and fluorescence labelling according to JACKSON (1991) in amodified form. Neutral sugars were treated with 8 N hydrochloric aqd for 2 h at 100 0c. After la­belling with AMAC (JACKSON, 1991) the samples were separat­ed by PAGE on an 20% acrylamide gel. The fluorescence-la­belled sugar residues were visualized on a UV screen at 302 nm and documented using a Polaroid camera and a Wratten 8 filter.

Polar lipid analysis: After solvent extraction as described by BLIGH and DYER (1959) the extracts were analyzed by one­dimensional TLC. Phosphate groups were detected with the molybdenum blue reagent and amino groups were detected with ninhydrin as described previously (MEYER-STAUFFER et aI., 1996).

RAPD analysis: DNA for the RAPD analysis was prepared from cells in the logarithmic phase as described by MAZURIER et al. (1992).5 to 50 ng of DNA (ODZ60 nm - 0.15) were used in the PCR assay. DNA amplification was performed using the Phar­macia Ready-To-Go® RAPD analysis kit (Pharmacia Biotech) ac­cording to the instruction manual. The samples were placed in a thermocycler (Trio-Thermoblock TB1, Biometra, Gi::ittingen, Germany) and the following cycles were applied: 1 cycle at 95 °C for 5 min followed by 45 cycles at 95 °C for 1 min, 36 °C for 1 min and 72 °C for 2 min. Both Pharmacia primers (10-mers, included in the Ready-To-Go® RAPD analysis kit) and Biomol primers (10-mers, Kit 8, Prod. No. 52607; Biomol Fein­chemikalien, Hamburg, Germany) were used. E. coli DNA was used as a positive control. The reaction products were compared on a 7.5-19% vertical gradient acrylamide gel and visualzed by silver staining according to BASSAM et al. (1991).

16S rDNA sequence analysis and DNA-DNA hybridisation: Both methods followed exactly previously described techniques as summarized in MEIER-STAUFFER et al. (1996).

Results

Isolation

We investigated sugar beet raw juice samples that had been collected in the beet sugar factory Leopoldsdorf, to monitor the bacterial population in the extraction plant throughout the sugar beet campaign 199111992 with re­gard to bacterial monosaccharide fermentation in the presence of high concentrations of sucrose (HOLLAUS and POLLACH, 1993). Pure cultures of bacteria were obtained as described in Materials and Methods and assigned isola­tion numbers (Table 1) . Due to investigations in other sugar factories sampling of new isolates from one of the extraction towers in Leopoldsdorf was only possible in the second half of the sugar beet campaign 199111992, start­ing at campaign day 47. At this day only four strains (L400-91, L401-91, L402-91, and L403-91; Table 2) had been isolated which, later on, were identified as B. smithii. One week later (day 54) in the isolation series thirteen

Table 1. Bacterial strains used in this study.

Strain

L400-91 L401-91 L402-91 L403-91 L428-91 L434-91 Bacillus smithii DSM 4216T

Source1

New isolate New isolate New isolate New isolate New isolate New isolate DSM

1 The new isolates were enriched from sugar beet raw juices ob­tained from the beet sugar factory Leopoldsdorf, Austria; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

Taxonomic Comparison of Different Thermophilic Sugar Beet Isolates 561

Table 2. Distribution of Bacillus smithii strains among the ex­traction juice isolates.

Date of isolation Campaign Total number Isolates of day of isolates l Bacillus smithii

11/21/1991 1112811991 12/3/1991 12/6/1991 1/4/1992

47 54 59 62 91

4 13 2 7

12

4 1 o o 1

1 Not all isolates that were collected at different days during the campaign were taxonomically characterized. Among them were isolates of Bacillus stearothermophilus (HOLLAUS and POLLACH, 1993) and B. thermoaerophilus and strains closely related to B. subtilis, B. licheniformis and Bacillus sp. (NEU"lINCER, unpub­lished; MEIER-STAUFFER et al., 1996).

thermophilic bacilli had been collected. However, only one isolate among them (L434-91) could be affiliated to B. smithii. Interestingly, L434-91 couldn't be distiguished from the other isolates L400-91, L401-91, L402-91, and L403-91 when analysed either by chemotaxonomic or ge­nomic methods (Figs. 1-4). Among the thermophilic bacil­li isolated at days 59 and 62 there were no B. smithii strains. Only at day 91 one out of twelve bacilli could be assigned to B. smithii (L428-91) (Table 2). This isolate, however, showed significant differences to the strains of the same species isolated before (Figs. 2-4).

Growth temperatures

All strains grew well at 55°C. The type strain of B. smithii (DSM 421e) didn't grow in PBYS medium. Thus,

Fig. 1. Electron micrographs of freeze-etched (a) and ultra-thin sectioned (b) cells of typical rep­resentatives of sugar factory iso­lates (e. g. isolate L403-91), showing the oblique S-layer lat­tice and the Gram-positive cell wall profile. S = S-layer; Bar = 100 nm.

it was only tested in TYG both. In this medium, however, this strain grew only sparsely at 60°C and showed no growth at 65 0C. All other strains were active in PBYS medium up to 65°C but did not grow at all at 68°C. The effect of growth temperature and culture medium on the investigated strains is summarized in Table 3.

Morphological and chemical characterization

Since we are interested in glycosylated bacterial S-layer proteins we have chosen those isolates for further charac­terization which have shown a positive glycosylation reac­tion in the previous study (MEIER-STAUFFER et ai., 1996). Freeze-etching (Fig. 1a) and thin sectioning (Fig. 1b) of in­tact cells showed that all isolates were covered by oblique S-layers with base vectors of the S-layer lattice in the range of a "" 11.2 nm and b "" 1 0.3 nm and an angle y between the base vectors of approximately 85°. Isolate L400-91 as a typical representative of the extraction juice isolates (see Table 1) had been identified in a previous study by partial 16S rDNA sequencing as a member of the species Bacillus smithii (MEIER-STAUFFER et ai., 1996). All isolates from the sugar factory investigated in the present work, including isolate L400-91 but with the exception of isolate L428-91, showed identical banding pattern in SDS-PAGE (Fig. 2). However, there was a difference in the high-molecular­weight protein bands to the type strain Bacillus smithii DSM 42161 . The molecular masses of the identical pro­tomers of the S-layer lattices are in the range of approxi­mately 132,000 (L428-91) to 138,000 (all other isolates). After blotting to a nitrocellulose membrane and staining with the DIG glycan detection method, a weak positive glycosylation reaction was observed with the extraction juice isolates. The type strain, however, didn't show a car­bohydrate staining reaction at ali.

562 P. MESSNER et al.

1 2345678 1

- 200,000

_ 116,300 97,400 66,300 55,400

36,500

31,000

21,500

14,400

6,000

Fig. 2. SDS-PAGE of SDS-soluble whole cells ex­tracts of the beet sugar factory isolates. Lanes (1) molecular mass standard; (2) B. smithii DSM 4216T; (3) L400-91; (4) L401-91; (5) L402-91; (6) L403-91; (7) L428-91; (8) L434-91.

1 2 3 4 5 6 7 1 2 3 4 5 6 7

Fig. 3. FACE analysis for demonstrating carbohydrate constituents on the beet sugar factory isolates. Neutral sugars (a) and amino sugars (b) are shown. Lanes (1) B. smithii DSM 4216T ; (2) L400-91; (3) L401-91; (4) L402-91; (5) L403-91; (6) L428-91; (7) L434-91.

FACE analysis for demonstrating carbohydrate con­stituents on S-layers and other cell wall polymers (e. g. peptidoglycan) yielded the same result. Identical banding patterns were obtained for the neutral sugars as well as the amino sugars (Fig. 3). Even isolate L428-91 didn't show significant differences.

Analysis of polar lipids confirmed the previous results that all organisms appeared to be almost identical (not shown).

Genomic analysis

To determine whether the similarity among the differ­ent extraction juice isolates, which was observed by chemotaxonomic methods, is manifested at the DNA level we compared the strains by RAPD fingerprinting. Twelve different primers were used (Fig. 4). Visualization of DNA by silver staining instead of the common ethidi­um bromide method increased the number of bands con-

Taxonomic Comparison of Different Thermophilic Sugar Beet Isolates 563

23456781234567812345678

~ _2,323 ;,...,J -1,929

-1,371 -1,264

702

224

I primer no. 2 1 ..... I __ p_ri_m_e_r_n_o_. _4_ ..... 1 I-I_p_r_i m_e_r_n_o_._10_ ......

Fig. 4. RAPD-PCR analysis of the beet sugar factory isolates. As a typical example the banding patterns obtained with Biomol primers no. 2, no. 4, and no. 10 are demonstrated (for details see Materials and Methods). Lanes (1) DNA size standard (bp); (2) B. smithii DSM 421e; (3) L400-91; (4) L401-91; (5) L402-91; (6) L403-91; (7) L428-91; (8 ) L434-91.

Table 3. Temperature range for growth of Bacillus smithii strains.

Temperature (0C) Growth in TYG medium

Strain

DSM 4216T

L400-91 L428-91

NI not investigated

35

NI NI NI

++ OD600 nm > 0.4; acid formation + OD600 nm < 0.3; acid formation (+) OD600 nm < 0.1; acid formation

no growth

37 55

NI ++ NI + NI +

60 65

(+)

siderably. With all primers there was complete similarity of the banding patterns of strains L400-91, L401 -91, L402-91, L403-91, and L434-91. The calculated similar­ity values (NEI and LI, 1979) between type strain B. smithii DSM 4216T and isolate L400-91 as a typical rep­resentative of the previously mentioned group of organ-

Growth in PBYS medium

68 35 37 55 60 65 68

does not grow in this medium ++ ++ ++ +

++ ++ ++

isms varied, dependent on the primer, between 88 and 26%. Between L400-91 and L428-91, however, similari­ty values between 86 and 42% were determined. The cal­culated values between B. smithii 4216T and isolate L428-91 were in the range of 92 to 36%. Three indepen­dent sets of RAPD analyses resulted in completely repro-

564 P. MESSNER et al.

ducible data. These analyses confirmed the results of the chemotaxonomical analyses and showed that the type strain B. smithii DSM 4216T and isolate L428-91 were different to the other strains.

To verify that isolate L428-91 belongs to the species B. smithii partial 16S rDNA sequence comparison was performed. The determined similarity coefficient to the type strain B. smithii DSM 4216T was 99.8%. DNA­DNA hybridisation between both strains resulted in 95% similarity, confirming that isolate L428-91 indeed is a member of the species B. smithii.

Based on the results from the chemotaxonomic char­acterizations and the data of the 16S rDNA sequence analysis and the DNA-DNA hybridisation experiment, the isolates from the beet sugar factory should be as­signed to B. smithii (NAKAMURA et al., 1988). The ob­served differences among some of the strains of this group (Table 3) obviously reflect strainspecific variations within the species B. smithii.

Discussion

In the course of our systematic survey to indentify bacteria with glycosylated S-layer proteins (MESSNER, 1996) we have compared different thermophilic strains of the domain Bacteria which had been isolated in beet sugar factories (MESSNER et al., 1984; MEIER-STAUFFER et al., 1996). Although the similarity among these strains, for example, B. stearothermophilus strains, was higher than 80%, identical isolates usually were rare (MESSNER et al., 1984). Therefore, we were surprised to see in this study almost no strain-specific differences among the in­vestigated sugar factory strains. All strains were covered with obliquely arranged, weakly glycosylated S-layer proteins. Fingerprinting of isolates L400-91, L40 1-91, L402-91, L403-91, L428-91, and L434-91 by different techniques resulted always in identical results (Fig. 1-4), irrespective of the feature investigated. These results sup­port the notion that, despite the observed diversity of thermophilic bacteria in extraction plants of beet sugar factories (DUBOURG and DEVILLER, 1953; KLAUSHOFER and HOLLAUS, 1970; HOLLAUS and KLAUSHOFER, 1973), a certain strain or species can find, at least temporarily, conditions to dominate the bacterial population in the plant (Table 2). This assumption is supported by another line of evidence because HOLLAUS and POLLACH (1993) occasionally observed the predominance of monosaccha­ride-degrading bacteria in extraction plants of Austrian beet sugar factories. Since no disinfectants were used during the extraction process the overall development of a bacterial population in the plant was not influenced. The temperature in the middle section of the extraction tower is in the range of 70-72 DC, which is close to the upper temperature limit of most thermophilic Bacillus strains. Thus, small temperature shifts of ±1 to 2 °C may affect composition and activity of the existing bacterial population.

To demonstrate the influence of environmental condi­tions onto the growth of microorganisms we have ana-

lyzed in our study the dependence of growth temperature and composition of growth medium. Two different media, namely PBYS and TYG medium, were used for cultivation. All strains possess a significantly higher tem­perature tolerance in PBYS medium compared to TYG medium (Table 3). The connection between upper tem­perature limit for growth and composition of the culture medium can also be seen from the fact that all sugar fac­tory strains which were isolated from the complex sugar beet raw juice at 70°C were not able to grow in the PBYS test medium at 68°C. The reduced growth at 37 °C and the complete inability to grow at 35°C indicate that all strains are obligate thermophiles. Additionally, the growth differences at 37°C between L428-91 and the other B. smithii strains (Table 3) indicate that L428-91 is indeed a different strain within the species B. smithii.

The previously discussed selection pressure is further enhanced by acid formation, mainly L-lactic acid, as a re­sult of microbial activity (HOLLAUS and POLLACH, 1993). In the central section of the extraction tower pH values as low as 4.5 can be reached. Thus, acid tolerance could be an additional selection criterion for the development of a given strain.

In conclusion, the dominance of specific strains of B. smithii in extraction juices at different periods of the sugar beet campaign might reflect such situations. There­fore it is not surprising that isolates from different sam­plings are indistinguishable (e. g. L400-91, L401-91, L402-91, L403-91, and L434-91; Table 2). At different situations, however, new strains of the same species (e. g. B. smithii strain L428-91) may become dominant.

Acknowledgement We thank the Zuckerfabrik Leopoldsdorf, Austria for the

kind cooperation. The work was supported by grants from the Austrian Science Foundation, project SnOl-MOB (to P. M.), and the Federal Ministry of Science and Transportation.

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Corresponding author: Dr. Paul Messner, Zentrum fur Ultrastrukturforschung, Univer­sitat fur Bodenkultur, Gregor-Mendel-Str. 33, A-1180 Wien, Austria. Tel.: +43-1-476 54 ext. 2202; Fax: +43-1-478 9112; e-mail: pmessner@edv1.boku.ac.at