Contribution to Phenotypic and Genotypic Characterization of Bacillus licheniformis and Description of new Genomovars

System. App!. Microbio!. 21, 520-529 (1998) _©_G_us_ta_vF_is_ch_er_V_er_lag_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

SYSTEMATIC AND APPLIED MICROBIOLOGY

Contribution to Phenotypic and Genotypic Characterization of Bacillus licheniformis and Description of new Genomovars PIER

L.

MANACHINI, MARIA G. FORTINA, LAURA LEVATI, and CARLO PARINI

Universia degli Studi di Milano, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia Industriale, Milano, Italy Received September 21,1998

Summary A study of phenotypic and genotypic characteristics was carried out on 182 strains isolated from soil of different geographical areas; the type strains were B. licheniformis, B. subtilis, B. pumilus, B. cereus and B. coagulans. The results showed, primarily on the basis of phenotypic features, that all the isolates belonged to the B. licheniformis species, however DNA relatedness studies revealed only 161 to be genetically related to B. licheniformis, the DNA relatedness levels ranging from 66 to 100%. The other 21 isolates appeared to be genetically distinct not only from B. licheniformis but also from B. subtilis and B. pumilus, where there were low levels of DNA relatedness (from 4 to 37%). Nevertheless the ARDRA results indicate that the 21 atypical isolates were phylogenetic ally related to B. licheniformis. Our data and the phenotypic homogeneity found suggest the presence of three different genomovars. Key words: Bacillus licheniformis - wild isolates - phenotypic characteristics - DNA relatedness ARDRA - genomovar

Introduction Microbial biodiversity, that characterizes strains of the same species isolated from different natural habitats, is an important tool in understanding the peculiarity of microorganisms (BULL et al., 1992). Wild strains from different environmental conditions often differ phenotypically from the reference strain itself, and even differ among themselves due to their specialized living niches. This means that it is possible to find biotypes that have evolved with minor or major changes of the genotype. Thus, an analysis of a bacterial population from natural habitats is of scientific and practical significance in that it leads to a better definition of a microbial species, evaluating the range of phenotypic variability in genotypically closely related strains, and thus offering the opportunity to have a collection of selected strains that can be better exploited in specific biotechnological processes. Within the genus Bacillus, an industrially important species of bacteria is Bacillus licheni(ormis. This species is a source of carbohydrase and protease whose enzyme preparations are used in food processing and the preparation of laundry detergent (MANACHINI et al., 1987a, 1988; MANACHINI and FORTINA, 1994); furthermore the species has also been safely used for large-scale industrial fermentation (ZUKOWSKI, 1992; FIECHTER, 1992). Thus B. licheni(armis is an attractive host for recombinant DNA and

there are several reports describing the cloning and expression of industrially relevant products (ANDREOLI et al., 1988; DIDERICHSEN et al., 1991; LEEN et al., 1991; QUAX et al., 1993; DE BOER et al., 1994). B. licheni(armis could be also considered an interesting source of vectors (PARINI et al., 1989, 1991) and site-specific restriction endonucleases (MANACHINI et al., 1987b; PARINI and FORTINA, 1995). Given all this we were prompted to characterize, phenotypically and genotypically, strains of B. licheniformis isolated from different geographical areas. The intent of the study was to determine, on the basis of physiological reactions, the presence of extrachromosomal elements, DNA base composition and DNA reassociation, whether this species is genetically homogeneous and what features can be considered typical of the species.

Materials and methods Bacterial strains and growth conditions: The B. licheniformis isolates tested and their history are listed in Table 1. Reference strains B. licheniformis DSMZ 13 T , B. subtilis DSMZ lOT, B. pumilus DSMZ 27\ B. cereus DSMZ 3F and B. coagulans DSMZ 1T were used for comparative studies. All strains routinely grew on Luria-Bertani broth (LB) at 40 DC on an alter-

Bacillus licheniformis genomovars

521

Table 1. List of strains used in this study. Laboratory No.

History

1.1.A, 1.1.B, 1.l.C, 1.2, 1.3, 1.4, 1.5.A, 1.5.B, 1.6, 1.7,2.1,2.3,2.4,2.6,3.2, 3.4,3.5,3.5.1,3.6,3.7,4.1,4.2,4.4,4.5,4.6,4.7,5.1, 5.3, 5.4, 5.5, 5.6

People's Republic of China; from a south-east region of Peking

6.1,6.1.1,6.2,6.3,8.1.1,8.2,8.4,11,12,16.1,16.2,17.1

People's Republic of China; from a north region of Peking

35.2,35.3,35.4 36.1,36.2,36.3,36.4 61,61.1 62.4

Island of Bali (Indonesia) Island of Nios (Indonesia) Island of Java (Indonesia) Island of Sumatra (Indonesia)

75.2,75.3,75.4,75.5,75.6,75.7,75.8 73.6,76.6,76.7

Osaka (Japan) Tokyo (Japan)

51.1,51.2,51.4,51.5,51.7 52.2, 52.3, 52.4, 52.5 53.2,53.3,53.4,53.6

Harvey Bay (Australia) Australia Australia

18.1,18.2, 18.2.A, 18.3, 18.5A, 18.5.B

Montreal (Canada)

19 39.1,41.1,41.2 42.1,42.2 49.1,49.3 64.1,64.2,64.3,64.4,64.6 80.3,80.4,81.1,81.2, 81.3.A, 81.3.B, 81.3.C, 81.4, 82.1, 82.2, 82.3, 82.4

St. Barbara California (USA) Yellowstone park (USA) North Dakota (USA) Montana (USA) New York (USA) Arizona (USA)

57,57.6

Lima (Peru)

58.1,58.2,58.5,58.6

Ecuador

59.1,59.2,59.3,59.4,59.5

Galapagos

56,56.5,56.6

Buenos Ayres (Argentina)

20.3,21.1 23.1, 23.l.A, 23.1.B, 23.2.B 24.2.1,24.3,24.3.4,24.4 25.1,25.2 26.2.B, 27.1.A, 27.3 28.2.A, 28.2.B, 28.4.l.B, 28.4.2.B, 28.5.2 29.1,29.2,29.3,29.4,29.5

Cairo (Egypt) Luxor (Egypt) Esna (Egypt) Kom Ombo (Egypt) Aswan (Egypt) Abu Simbel (Egypt) Giza (Egypt)

47.5,47.6,47.8

Tunisia

60.1

Madagascar

74.3

Israel

32,33.1,33.3 65.1,65.2 9.1,9.2,50.2,50.3,68.5 69.2,72,73

Sardinia (Italy) Sicily (Italy) North of Italy South of Italy

37 54,55

Berlin (Germany) Munich (Germany)

46

Austria

45,45.5

Czech Republic

63,63.1,63.3

Paris (France)

48.1,48.2,48.3,48.6

Wales (UK)

43.1,43.2

Croatia

77.2,78.2, 78.4

Norway

B. licheniformis

DSMZ 13 T DSMZ lOT DSMZ 27T DSMZ 31 T DSMZ 1T

B. subtilis

B. pumilus B. cereus B. coagulans

DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

522

P. L. MANACHINI et al.

native shaker (spm 100; 5 em run). Working stock cultures were grown on LB agar supplemented with 10 mg of MnS0 4 • H 2 0 per liter until sporulation occurred. Screening for strains with the B. licheniformis phenotype: Isolates of this species were selectively obtained from soil samples by the anaerobic enrichment procedure suggested by CLAUS and BERKELEY (1986). A suspension of soil was heated at 80°C for 10 min to kill vegetative cells and induce spore germination. 1 ml of the pasteurized soil suspension was inoculated into Nutrient broth (Difco) containing (g per liter) KN0 3 80.0, adjusted to pH 7.0. Before use the medium was pre reduced in a gas pack system to drive off the free oxygen and then anaerobic conditions were provided, capping the tubes with 10-15 mm of sterile melted vaspar (equal parts of vaseline and paraffin). After 48 h incubation at 40°C the cultures from the tubes showing turbidity and gas production were streaked on glucose mineral base agar consisting of (g per liter in distilled water): K2HP0 4, 0.8; KH 2P0 4 , 0.2; CaS0 4 • H 2 0, 0.05; FeS0 4 . 7H2 0, 0.01; MgS04 . 7H2 0, 0.5; (NH4hS04' 1.0; glucose, 10.0; agar, 12.0; pH 6.8. The emergent colonies were subsequently streaked for isolation, the isolates then being screened for their ability to utilize propionate, reduce nitrate in aerobic conditions, grow anaerobically in glucose broth and to produce gas from nitrate in anaerobic conditions. Each isolate that was positive for all these metabolic tests was considered a B. licheniformis isolate. Physiological tests: The isolates were examined to identify the Bacillus species. Conventional tests, as described by GORDON et a!. (1973), were used, as well as: catalase production; Voges-Proskauer reaction; hydrolysis of gelatin, casein and starch; utilization of citrate and succinate, degradation of tyrosine; egg-yolk lecithinase. Carbon source utilization was performed using the "API-50CHB" system (API system, bioMerieux) according to the instructions of the suppliers. Sugar tests were conducted at 40°C in aerobic conditions and readings were taken from 4 to 24 h and at 48 h. Tolerance to saline was determined by inoculating tubes containing Nutrient broth (Difco) supplemented with 5 to 18% (wt/vol) NaCI. For the detection of the maximum temperature range of growth, the cultures were streaked onto plates of Tryptic Soy agar (TSA) (Difco) and incubated at a range of temperatures (from 35 to 70°C). The plates were examined daily up to 3 days. Antibiotic activity production and microbiological assay: The medium used for antibiotic activity production was as follows (g per liter in distilled water): soy tone (Difco), 20.0; peptone (Difco), 10.0; glucose, 10.0; K2 S0 4 , 1.0; MnS0 4 . H 2 0, 0.01. The pH was adjusted to 7.0 before sterilization. Growth occurred at 40°C on a alternative shaker (spm 100; 5 cm run). After 24 and 48 h incubation, 1 ml of culture broth was centrifuged and 10-20 pI of subernatant used for antibiotic activity detection. Microbiological assay was performed by an agar diffusion method as described by HAAVIK and THOMASSEN (1973) employing Micrococcus luteus DSMZ 1790 as the test strain. Plasmid detection: For detection of plasmid DNA, the alkaline extraction procedure described by SAMBROOK et al. (1989) was followed. Molecular weight of the plasmids obtained was determined from standard curves with a supercoiled DNA ladder (Gibco-BRL, UK). For further analyses of CCC and OC forms of plasmids, second dimension electrophoresis was performed according to HINTERMANN et al. (1981). DNA preparation, G+C content, and DNA reassociation: For DNA preparation the organisms were grown in LB broth under agitation and at 40°C, and then harvested by centrifugation, at 5 °C in the mid- to late-logarithmic growth phase when microscopic examination revealed the absence of sporulation.

DNA was extracted and purified as described by MARMUR (1961) but with minor modification (MANACHINI et a!., 1985). The required purity and quality of each DNA preparation was achieved according to MANDEL and MARMUR (1968). The G+C content was estimated by the thermal melting procedure described by MARMUR and DoTY (1962) using a "Gilford Response" spectrophotometer (Ciba Corning Diagnostics Corp., OH). In determining the Tm, all the experiments were repeated at least 3 times. The mol% G+C content was calculated from the equation of OWEN and HILL (1979). DNA from Escherichia coli strain B (Sigma) with a G+C content of 50.9 mol % was used as internal standard. The extent of DNA reassociation was calculated on the basis of renaturation rates determined spectrophotometric ally, with the same spectrophotometer mentioned above, according to the procedures of SEIDLER and MANDEL (1971) and KURTZMAN et a!. (1979). The reaction was carried out under optimal conditions (25°C below the Tm) in 5X SSC containing 20% dimethylsulfoxide (DMSO). ARDRA analysis: Cells from 600 pI of an overnight cell culture (LB broth, 40°C) were collected by centrifugation, washed in 1 ml of sterile washing solution (0.5% SDS, 50 mM EDTA), then washed in 1 ml of sterile water and centrifuged. For the cell lysis the pellet was resuspended in 600 pI of Chelex suspension (15% Chelex 100, 1 % (wt/vol) SDS, 1 % (vol/vol) Tween 20, 1 % (vol/vol) Nonidet P40) and incubated for 15 min at 100 dc. After centrifugation the supernatant was used for the polymerase chain reaction (PCR). Amplification of 16S rDNA was performed in a volume of 100 pI containing: 3 pI of bacterial genomic DNA solution obtained as above, 10 pI of lOX PCR reaction buffer (500 mM KCI, 100 mM Tris-HCl [pH 8.5 at 25°C], 0.5% (vol/vol) Tween 20), 200 pM of each deoxynucleoside triphosphate (dNTP), 2.5 mM of MgCI 2 , 0.5 pM of each primer, 2 U of Taq polymerase (Perkin-Elmer, USA). Primer set: forward primer 5'AGAGTTTGATCCTGGCTCAG-3' and reverse primer 5'CTACGGCTACCTTGTTACGA-3' were used for the amplification of the 16S rDNA gene. All amplification reactions were performed in a Gene Amp PCR System 2400 (Perkin-Elmer). The temperature profile was the following: after a denaturation step of 94°C for 40 s to increase the specificity of the amplification the annealing temperature was set at 70°C (10°C above the calculated Tm of the primers) then decreased 1 °C each second cycle until a touchdown of 55°C, at which temperature 25 additional cycles were carried out. Extension was carried out at 72 °C for 1 min and the final cycle was followed by an additional 7 min at 72 dc. The presence and amount of the specific PCR product (length, about 1500 bp for the 16S rDNA) was evaluated by 1.4% (wt/vol) agarose gel electrophoresis. Restriction digestion of the amplified 16S rDNA was carried out for 4 h at 37°C in 20 pI reaction mixture containing 15 pI of the PCR product solution, 2 pI of lOX incubation buffer and 15 U of one of the following restriction enzymes, Alu I, Ava II, Ban II, Hae III, Hha I, Hpa II, Nde II, Rsa I and Taq I (Boehringer). Restriction digests were subsequently analyzed by 3% (wt/vol) agarose gel electrophoresis at 5 V/cm in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8). The gel was stained in a solution containing 0.5 pg of ethidium bromide per ml and photographed in UV light.

Results Phenotypic characteristics Through the anaerobic enrichment described above, we selected 182 isolates from 44 different soil samples.

Bacillus licheniformis genomovars

Their ability to grow anaerobically in glucose broth, reduce nitrate in aerobic conditions, produce gas from nitrate in anaerobic conditions and use propionate as the source of carbon and energy inicate that the isolates were similar to the type strain of B. licheniformis. Further physiological tests excluded their assignment to any other documented Bacillus species (CLAUS and BERKELEY, 1986). Indeed the isolates themselves had common physiological

and biochemical traits: all the strains were positive for the production of catalase, the Votes-Proskauer reaction, the utilization of citrate and succinate as the sole carbon source and the hydrolysis of gelatin, casein and starch. Moreover none of the strains degraded tyrosine, and all were negative with respect to egg-yolk reaction. The information provided by the carbohydrate tests (Table 2) shows that all the isolates were glycerol, L-ara-

Table 2. Number and percentage of isolates studied giving positive reactions in API 50CHB test. Carbohydrates

Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol ~- Methylxyloside Galactose D-Glucose D-Fructose D-Mannose L-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol a- Methyl-D-mannoside a-Methyl-D-glucoside N-Acetyl-Glucosamine Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inulin Melezitose D-Raffinose Starch Glycogen Xylitol ~-Gentiobiose

D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2-Ketogluconate 5 -Ketogl ucona te

Our results

523

Logan and Berkeley results

n° of positive

% of positive

n° of positive

% of positive

182 0 0 182 182 150 0 0 0 96 182 182 182 0 96 0 182 182 182 0 182 173 182 182 182 182 182 182 97 72 182 182 59 15 98 182 182 0 136 160 0 182 0 0 0 0 0 0 0

100 0 0 100 100 82 0 0 0 53 100 100 100 0 53 0 100 100 100 0 100 95 100 100 100 100 100 100 53 40 100 100 32 8 53 100 100 0 75 88 0 100 0 0 0 0 0 0 0

81 0 0 81 81 80 0 0 0 81 81 81 80 5 67 0 62 73

100 0 0 100 100 99 0 0 0 100 100 100 99 7 83 0 76 90 95 0 100 89 100 100 100 100 100 100 89 45 100 100 68 1 79 99 96 1 89 100 0 96 0 0 0 0 2 0 0

77

0 81 72 81 81 81 81 81 81 72 36 81 81 55 1 64 80 78 1 72 81 0 78 0 0 0 0 2 0 0

524

P. L. MANACHINI et al.

binose, ribose, D-glucose, D-fructose, D-mannose, inositol, mannitol, sorbitol, a-methyl-D-glucoside, arbutin, exculin, salicin, cellobiose, maltose, sucrose, trehalose, starch, glycogen and D-tagatose positive within 24 hand amygdalin positive within 48 h. Typically, no isolate was able to utilize erythritol, D. arabinose, L-xylose, L-sorbose, adonitol, ~-methyl-xylo­ side, dulcitol, a-methyl-D-mannoside, xylitol, D-lyxose, D and L-fucose, D and L-arabitol, gluconate or 2-keto and 5-ketogluconate. A certain degree of variability was observed in the assimilation of D-xylose, galactose, rhamnose, N-acetyl-Dglucosamine, lactose, melibiose, inulin, melezitose, Draffinose, ~-gentiobiose and D-turanose. When assimilated, D-xylose, galactose, rhamnose, melibiose, inulin and D-raffinose were positive within 42 h. With regard to these last 11 carbon sources an inter-strain phenotypic variability was observed. Considering the ability to assimilate these carbon sources it has been possible to note 98 isolates, some which typical of specific geographical areas (Table 3). In particular most of the isolates from Northern China (biotype b) differed from those isolated from the South East region (biotype a) in their abilit to utilize inulin. The isolates from Japan and New York (biotype c) were the only ones able to utilize melezitose; those from Sicily, Norway and the Czech Republic (biotype d) were unable to use xylose; N-acetyl glucosamine (NAG) was assimilated by all the strains with the exception of those isolated from the island of Nios (Indonesia) and Abu Simbel (Egypt) samples (biotype f). Moreover, as shown in table 3 the isolates from China and Egypt (biotype a) seemed to be able to utilize a very high number of different carbon sources, while the isolates from

Table 3. Carbohydrate assimilation biotypes of Bacillus licheniformis isolates.

Wales, Paris, and the desert of Arizona (biotype e) utilize only xylose, NAG and turanose. Table 2 shows the distribution of our 182 isolates with respect to the assimilated carbon source, also in comparison with the findings recorded by LOGAN and BERKELEY (1984) on 81 isolates of B.lichenifarmis. With regard to saline tolerance, all the strains grew in 6% and 8% NaCl, as reported for the species B. lichen ifarmis (CLAUS and BERKELY, 1986). Moreover, we found 75 biotypes able to grow in 12% NaCI and 10 biotypes that grew in 15% NaC!. These halotolerant variants were isolated from different geographical areas and did not seem to be a feature peculiar to isolates from brackish habitats. Maximum growth temperature for all isolates was 55°C, considered characteristic of the species (CLAUS and BERKELEY, 1986). Moreover, also in this case we found 103 biotypes able to grow at up to 60°C, and 33 biotypes which showed visible growth at 65°C after 48 h. As B. lichenifarmis is a known producer of bacitracin, a peptide antibiotic, not synthesized ribosomally (ZuKOWSKI, 1992), the 182 isolates were tested for antibiotic activity; after 24-48 h of incubation a bacitracin-like activity was detected in 70% of the isolates tested. The remaining isolates showed no such ability.

Plasmid profiling Several papers report the presence of extrachromosomal elements in several strains of Bacillus but to our knowledge only a few papers refer to B. lichenifarmis (YOSHIMURA et aI., 1983; PARINI et aI., 1989, 1991; ZWADZKI et aI., 1996). Our findings appear to be in agreement with these observations. Plasmid DNA was checked in each of the 182 isolates; plasmids were detected in only 44 cases (Table 4). The isolates from China, Australia, Bali, Sumatra, Osaka, USA (with the exception of strains from Arizona), Ecuador, Egypt (Kom

biotypes

D-Xylose Galactose Rhamnose N-Acetyl-Glucosamine Lactose Melibiose Inulin Melezitose D-Raffinose p-Gentiobiose D-Turanose Number of strains

a

b

c

d

+ + + + + + +

+ + + + + +

+ +

+

+ + +

+ + +

36

12

+ +

+

e

f

Table 4. Plasmid profiling of Bacillus licheniformis isolates.

+

+ + +

Laboratory No

Number of plasmids

Molecular weight (kb)

24.3.4

3

>30 - 9.5 -7.7

8.4; 20.3 8.1.1; 21.1; 23.1

2 2

>30 -7.6 >30 - 6.0

5.1; 6.3; 18.3; 23.1.A; 23.1.B; 23.2.B; 53.2; 53.3; 57; 59.1; 59.2; 61; 76.6; 77.2; 78.4; 80.4; 81.2; 81.3.A; 81.3.C; 82.2; 82.4

1

>30

73; 81.4 5.3; 36.3; 45.5; 65.1; 65.2; 72

1 1

9.5 7.6

28.4.1.B; 28.4.2.B.; 28.5.2; 45; 37; 59.5

1

6.0

63.1; 82.1

1

3.6

56.6

1

2.6

+ + + +

+

+ +

+

+ 15

+ 7

+ +

+ 19

+ 9

a - Isolates from south-east region of Peking and from Egypt (with the exception of Abu Simbel). b - Isolates from a north region of Peking. c - Isolates from Japan and New York. d - Isolates from Sicily, Norway and Czech Republic. e - Isolates from Wales, Paris and Arizona. f - Isolates from Abu Simbel and island of Nios.

6

7

8

9

10 11

12 13

14

15

16

17

18 19 20

21

22

23 24

17 20

25 10

17

22 - B. licheni

10 8 18

13

28 16 23

21

24

25 23 15 18

17

9

11

6

14

32

20 28 21

12

15 29

34

23

12

26

32

30 37 29

(100) 83 (100) 91 93 (100) 94 (100) 100 84 88 92 92

(100)

d

13

4

23

19 10

25

20

12

37 18

28

5

25 18

26

14

25

19 3

19

7

33

12

35

15

25

8

18

21 6

31

"- Reassociation values are average of two determinations, the maximum difference noted between determination was 7%. b _ Values in parentheses indicate that, by definition, the reassociation value was 100%. C _ Type strain DSM 13 T d _ Type strain DSM lOT '- Type strain DSM 27T

23 - B. subtilis 15 24 - B. pumilus'

formis c 13

17

32

12

24

33

10

10

8

4

9

7

22 (100) (100) 15

(100)

---------------------------------------------------------------------------------------------------------------------------------

22 26

23

16 18

17-43.1 18-51.1 19 - 51.2 20 - 51.5 21 - 68.5

--------------------------------------- - -----------------------------------------------------------------------------------------

5

(100)b 100 (100) 100 98 (100) 89 (100) 85 92 100 (100) 87 93 (100) 89 94 (100) 87 90 100 85 82 (100) 88 91 90 100 (100) 95 96 95 86 99 90 (100) 97 98 95 88 (100) 93 86 98 91 97 90 91 84 92 87 97 (100) 97 85 100 93 (100) 96 93 100 100 84 87 91 87 91 94 89 (100) 82 88 94 92 86 90 89 87 90 98 (100) 91 84 94 90 83 92 81 (100) 92 92 80 80 95

4

1 - loLA 2 - 1.1.B 3 - 1.1.C 4 -1.6 5 -1.7 6-3.4 7-3.5 8-3.5.1 9-4.1 10 -4.2 11 - 4.4 12 - 4.5 13 - 4.6 14 - 5.4 15 - 5.5 16 - 51.4

3

1

Strains

2

Table 5. Levels of DNA-DNA reassociation (%)" among reference strains and some atypical isolates.

V.

N

v.

'"

.... '"

<

0

;3

0

~

(1)

CJq

~ ;;:.

~ ...

'";:;

~

~

'"

~

b:l

'"~

526

P. L. MANACHINI et al.

Ombo, Aswan and Giza), Tunisia, Madagascar, Israel, Sardinia, northern Italy, Wales and Croatia were plasmid-free. DNA could be detected in one to three plasmid bands of the 44 plasmid harbouring isolates and plasmid size ranged from 2.6 to 30 kb; from these strains 6 different plasmid molecules (2.6; 3.6; 6.0; 7.6; 9.5 and >30 kb) were identified. Plasmids of molecular size of 6.0, 7.6 and >30 kb seemed to be the most representative. In fact, 9 isolates harboured a plasmid of 6.0 kb, another 9 possessed one of 7.6 kb and the largest plasmid (>30 kb) was found in 27 isolates. Genotypic characteristics An analysis of the DNA of 182 isolates ascribed to the B. licheni{ormis species revealed a G+C content ranging from 45.7 to 49.8 mol%, including the G+C content (46.7) of the type strain. Our results are in agreement with the data reported in literature (CLAUS and BERGELEY, 1986). The levels of DNA relatedness between DSMZ 13 T (the type strain of B. licheni{ormis), and the isolates were evaluated. For 161 isolates they ranged from 66 to 100%. Particularly high DNA relatedness values (91 to 100%) were found for 48 isolates, showing that the isolates were genetically closely related to the type strain. Another 102 isolates exhibited values ranging from 81 to 90% (50 isolates) and 70 to 80% (52 isolates). The remaining 11 showed moderate levels of DNA relatedness (66 to 69%); also these isolates could be considered members of the B. licheniformis species according to the recommendations of the Ad Hoc Committee on Recon-

ciliation of Approaches to Bacterial Systematics and other authors (WAYNE et aI., 1987; STACKEBRANDT and GOEBEL, 1994). Unexpectedly much lower levels of relatedness were found for 21 isolates (about 11.4%) obtained mainly from a south-east region of China and Australia. These isolates proved to be related phenotypically, but not genetically to B. licheni{ormis, exhibiting an extent of DNA realtedness ranging from 4 to 37%. DNA-DNA reassociation experiments among these isolates were also carried out. The results obtained allowed the grouping of these isolates into two clusters of homology (A and B consisting of 16 and 5 isolates respectively) within which the isolates were closely related genetically with high DNA relatedness values (above 84%). Table 5 shows the results of DNA relatedness obtained for the isolates of each cluster with B. lichen i{ormis type strains and some reference strains. The DNA relatedness values range from 6 to 37%, indicating that the strains belong to different species. The levels of DNA relatedness with two other type strains, B. subtilis DSMZ lOT and B. pumilus DSMZ 2?T, phylogenetically related to B. licheni{ormis, were found to be between 3 and 21 % (also shown in Table 5), indicating that the strains of the two clusters were not related genetically to these reference strains. With the aim of obtaining further evidence for the correct identification of these 21 isolates, a comparison was made of the restriction digestion patterns of amplified 16S rDNA (ARDRA) and those of the reference strains. The B. coagulans type strain (DSMZ IT) and B. cereus type strain (DSMZ 3F) were chosen as the out-group

Fig. 1. ARDRA patterns of 16S rDNAs from the atypical B. licheniformis strains and some Bacillus reference strains. Patterns obtained with the restriction enzyme Nde II. M: molecular weight marker VI (Boehringer) 2176,1766, 1230, 1033, 653, 517, 453, 394, 298, 234/220, 154 bp; lanes 1 to 18: isolates 1.1.B, 1.6,3.4,3.5,3.5.1,4.1,4.2, 4.4,4.5,4.6,5.4,5.5,43.1,51.1,51.2,51.4,51.5,68.5; lane 19: B. coagulans DSMZ IT; lane 20: B. subtilis DSMZ lOT; lane 21; B. licheniformis DSMZ 13 T; lane 22: B. pumilus DSMZ 2?T; lane 23: B. cereus DSMZ 31T.

Fig. 2. ARDRA patterns of 16S rDNAs from the atypical B. licheniformis strains and some Bacillus reference strains. Patterns obtained with the restriction enzyme Taq I. M: molecular weight marker VI (Boehringer) 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234/220, 154 bp; lanes 1 to 18: isolates 1.1.B, 1.6, 3.4, 3.5, 3.5.1, 4.1, 4.2, 4.4,4.5,4.6,5.4,5.5,43.1,51.1,51.2,51.4,51.5,68.5, lane 19: B. coagulans DSMZ IT; lane 20: B. subtilis DSMZ lOT; lane 21: B. licheniformis DSMZ 13 T; lane 22: B. pumilus DSMZ 27T; lane 23: B. cereus DSMZ 31 T.

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Fig. 3. ARDRA pattern of 16S rDNAs from the atypical B. licheniformis strains and some Bacillus reference strains. Patterns obtained with the restriction enzyme Ban II. M: molecular weight marker VI (Boehringer) 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234/220, 154 bpi lanes 1 to 18: isolates 1.1.B, 1.6,3.4,3.5,3.5.1,4.1,4.2, 4.4,4.5,4.6,5.4,5.5,43.1,51.1,51.2,51.4,51.5,68.5; lane 19: B. coagulans DSMZ IT; lane 20: B. subtilis DSMZ lOT; lane 21: B. licheniformis DSMZ 13 T; lane 22; B. pumilus DSMZ 27T; lane 23: B. cereus DSMZ 31 T

Fig. 4. ARDRA patterns of 16S rDNAs from the atypical B. licheniformis strains and some Bacillus reference strains. Patterns obtained with the restriction enzyme Hhal. M: molecular weight marker VI (Boehringer) 2176, 1766, 1230, 653, 517, 453, 394, 298, 234/220, 154 bpi lanes 1 to 18; isolates 1.1.B, 1.6, 3.4, 3.5, 3.5.1, 4.1, 4.2, 4.4,4.5,4.6,5.4,5.5,43.1,51.1,51.2,51.4,51.5,68.5; lane 19: B. coagulans DSMZ fI; lane 20: B. subtilis DSMZ lOT; lane 21: B. licheniformis DSMZ 13 T; lane 22: B. pumilus DSMZ 27T; lane 23: B. cereus DSMZ 3F.

Fig. 5. ARDRA patterns of 16S rDNAs from the atypical B. licheniformis strains and some Bacillus reference strains. Patterns obtained with the restriction enzyme Alu I. M: molecular weight marker VI (Boehringer) 2176, 1766, 1230,1033,653,517,453,394,298,234/220,154 bPi lanes 1 to 18: isolates 1.1.B, 1.6, 3.4, 3.5, 3.5.1, 4.1, 4.2, 4.4,4.5,4.6,5.4,5.5,43.1,51.1,51.2,51.4,51.5,68.5; lane 19: B. coagulans DSMZ IT; lane 20: B. subtilis DSMZ lOT; lane 21: B. licheniformis DSMZ 13\ lane 22: B. pumilus DSMZ 27T; lane 23: B. cereus DSMZ 3F.

strains. A series of four-base cutting restriction endonucleases were tested. In all cases the 21 isolates showed the identical pattern of B. licheni{ormis. Nde II (Fig. 1) and Hae III grouped all the 21 isolates together B. subtilis and B. pumilus, while Hpa II, Rsa I and Taq I (Fig. 2) showed an identical pattern only among the isolates, B. licheni{ormis and B. subtilis. Ban II and Hha I (Fig. 3-4) were able to discriminate all the isolates and B. lichen i{ormis from B. subtilis and B. pumilus showing a similar fingerprinting to the one of B. cereus and B. coagulans, respectively. On the contrary, Alu I (Fig. 5) and Ava II were the only endonucleases that clearly evidenced B. licheni{ormis and all the isolates from the reference strains tested. Among the isolates the one labelled 43.1 had a characteristic pattern due to the absence of the 145 bp band when Alu I was employed (Fig. 5).

Discussion The present data provide information concerning the levels of phenotypic and genotypic relatedness within the species B. licheni{ormis. This bacterial species appears to be very homogeneous phenotypically, exhibiting a characteristic and stable carbohydrate fermentation pattern and biochemical traits. Our results and those of LOGAN and BERKELEY (1984) appear substantially in agreement even if it has been possible to evidence some discrepancies. We believe that some of these, such as the different percentage in the assimilation of D-xylose, rhamnose and ~-gentiobiose, or of lactose, inulin and D-raffinose are not significant, as these carbohydrates are variable characters, and therefore of no value in identification tests. On the contrary, more significant discrepancies were

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found for galactose and turanose. Our results show that only 53 % of our isolates were able to utilize galactose and 87% turanose whereas inositol mannitol and sorbitol were utilized by all 182 isolates. Bergey's Manual of Systematic Bacteriology describes B. lichenifarmis as a species able to grow in 7% NaCI and at a maximum temperature of 55°C. Among our isolates we found that, regardless of the sample origin, 46% of the strains showed growth in 12% NaCI, and 64% were able to grow at up to 60°C; these features led us to consider B. lichenifarmis as a species including moderate halophilic and thermotolerant biotypes. With regard to the plasmids that appeared to be the most representative, as they were found in strains from samples collected in widely different geographical areas, we believe it is most unlikely that there exists any relationship between the presence of a particular plasmid and the environmental conditions of the habitat where the carrying strains were isolated. However, further molecular characterization studies are in progress to better define their structural molecular organization, in order to understand which biological role is carried out by these plasmids. The findings concerning the levels of genotypic relatedness reveal a certain degree of heterogeneity within this bacterial species. Our results show the presence of atypical isolates phenotypically indistinguishable but with significatively different levels of DNA relatedness. A phylogenetic relationship among these atypical strains and some reference strains was investigated using ARDRA analysis according to HEYNDRICKX et al. (1996). These authors have shown that the ARDRA technique based on the combination of five selected restriction enzymes is reliable and valuable for phylogenetic and taxonomic studies. Our results obtained from testing nine selected restriction endonucleases revealed that these isolates are phylogenetically related among themselves, to B. lichenifarmis and in many cases to B. subtilis. This fact could be explained by the fact that the latter two bacterial species are phylogenetic ally related (ASH et aI., 1991) even if they show distinctive phenotypic characteristics such as: anaerobic growth in glucose broth, production of gas from nitrate in anaerobic conditions and the ability to use propionate as the source of carbon and energy. Through the very low DNA-DNA reassociation values preclude the classification of any of the strains of the two clusters to the reference species considered, the absence of distinctive phenotypic traits with B. lichenifarmis rules out their being considered strains of a new species. Our conclusion is that these 21 isolates should be considered members of different genomovars of the species B. lichenifarmis (URSING et aI., 1995). The largest genomovar included the type strain and the 161 isolates genotypically related to it, the second genomovar (cluster A) consisted of 16 strains principally isolated from a south-east region of China. The last genomovar (cluster B) included the remaining 5 strains isolated from different areas. This is the first time that, within the B. lichenifarmis species, three different genomovars have been identified,

and this was done by means of DNA/DNA reassociation experiments. A genotypic heterogeneity within the B. lichenifarmis species was also reported by DUNCAN et al. (1994) but through genetic analysis using multilocus enzyme electrophoresis, which pointed out two different clusters in which the two groups appeared closely related phenotypically. The selective procedure of isolation based on anaerobic growth in nitrate broth, and on the ability to utilize propionate, can be used to successfully isolate B. lichenifarmis strains. However, for the moment, DNA-DNA hybridization experiments are needed to recognize possible genotypic variants that cannot be distinguished by the above mentioned selective phenotypic tests. Our laboratory is presently carrying out further experiments to develop a PCR assay that can readily, and unambiguously, discriminate between the different B. lichenifarmis genomovars. The representative strains of the new genomovars are 1.1.B for the second genomovar (cluster A) and 51.1 for the third genomovar (cluster B) deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany as DSMZ 12369 and DSMZ 12370 respectively.

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Corresponding author: CARLO PARINI, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche Sezione di Microbiologia Industriale, Via Celoria n° 2, 20133 Milano Italy Tel.: 00 39-2-2 39 55 81; Fax: 00 39-2-70 63 08 29; E-mail: [email protected]