RAPD ANALYSIS OF GENETIC DIVERSITY AND QUALITATIVE ASSESSMENT OF HYDROLYTIC ACTIVITIES IN A COLLECTION OF BACILLUS SP . ISOLATE

Genetic diversity and production of hydrolytic enzymes of 205 Bacillus isolates from different geographical and ecological niches in Serbia were studied. Combining RAPD analysis and 16S DNA sequencing, we determined 13 different groups of RAPD profiles within four (five) species: B. subtilis, B. cereus/B. thuringiensis, B. pumilus, and B. firmus. Screening for production of hydrolytic enzymes showed that there was no correlation of enzyme production with species. Most of the isolates from all habitats produced amylase, protease, lipase, mannanase, and xylanase to some extent at 25oC and 37oC. The number of isolates that retained enzyme production ability at 55oC is considerably lower and they predominantly came from manure.


INTRODUCTION
Bacillus species are known as producers of different exoenzymes, a trait that is linked with their way of life and places where they thrive.In addition, these bacilli often grow at temperatures that are higher than mesophilic temperatures.Extracellular hydrolytic enzymes that retain their activity at higher temperatures have a great potential for commercial application.Amylase degrades starch components of different waste material into simple sugars that find many uses in the feed and fermentation industries (Hyun and Zeikus, 1985).Proteases are highly exploited enzymes in the food, leather, detergent, and pharmaceutical industries, as well as for diagnostic purposes and waste management.The protease enzymes constitute two thirds of all enzymes used in various industries (Gupta et al., 2002).Lipase catalyzes a variety of biotechnologically relevant reactions such as production of free fatty acids, interesterification of oils and fats, and synthesis of esters (Macrae and Hammond, 1985;Harwood, 1989).Mannanases have potential use in the paper industry for complete hydrolysis of soft-wood sulfite liquors to fermentable sugar (Ratto and Poutanen, 1988).Recently, interest in the xylanases has markedly increased due to potential application in pulping and bleaching processing using cellulose free preparations, in the food and feed industries, in textile processes, in the enzymatic saccharification of lignocellulosic materials, and in waste treatment (Van der Broeck et al., 1990;Gilbert et al., 1992;Mechaly et al., 1997).
It was reported that high metabolic activities of Bacillus strains often correspond to their specific genotypes (Pinchuk et al., 2002).This genetic heterogeneity is interesting in view of the production of extracellular enzymes.Use of the random amplified polymorphic DNA (RAPD) method has extensive application for rapid typing of microorganisms at the species and subspecies levels (Stephan et al., 1994;Ronimus et al., 1997;Pinchuk et al., 2002).In this study, the RAPD method was used to investigate the genetic heterogeneity of Bacillus strains isolated from different habitats in relation to production of extracellular enzymes.

RAPD ANALYSIS OF GENETIC DIVERSITY AND QUALITATIVE ASSESSMENT OF HYDROLYTIC ACTIVITIES IN A COLLECTION OF BACILLUS SP. ISOLATE
From various habitats (straw and hay, soil, and manure) in Serbia, we isolated 205 different Grampositive, spore-forming bacilli on the basis of colony formation (Stanković and Lazarević, 2001).Our goals were to determine the genetic diversity of this large collection of Bacillus sp.isolates and screen it for production of amylase, protease, lipase, mannanase, and xylanase at 25, 37, and 55ºC on solid media.

Isolation and preliminary characterization of Bacillus sp. strains
The method of isolating various Bacillus strains was based solely on the resistance of their endospores to elevated temperatures.Test tubes containing a mixture of app. 1 g of sample (straw, soil, or manure) and 1 ml of nutrient broth were placed in a water bath at 80°C for 10 min so that endospores would be separated from vegetative cells (Walker et al., 1998).Concentrated samples, as well as two following decimal dilution, were spread on the surface of LA plates and incubated at 30°C for 48 h.Distinct single colonies were subcultured onto fresh LA plates.The subcultured bacterial isolates were preliminarily characterized on the basis of microscopic appearance and the results of Gram staining and the catalase test.

DNA extraction
Genomic DNA from Bacillus strains was prepared as described earlier (Le Marrec et al., 2000).In short, after centrifugation and two washes in TE buffer (10 mmol/l Tris-HCl, pH 8.0; 1 mmol/l EDTA), cells were resuspended in 1 ml of a lysis buffer (50 mmol/ l Tris, pH 8.0; 1 mmol/l EDTA; 25% saccharose) containing 20 μgm/l of lysozyme (Merck) and incubated for 45 min at 37°C.The reaction was stopped using 1ml of EDTA (250 mmol/l, pH 8.0) for 5 min.The samples were then treated with 400 μl of 20% (w/v) SDS and 20 μl of a 20 mg/ml proteinase K (Sigma) solution.The mixture was incubated at 65°C until it became clear and less viscous, after which phenolchloroform extraction was performed.The DNA was precipitated in ethanol and resuspended in 100 μl of TE buffer with 10 μl of RNase (10 mg/ml).

RAPD analysis
Arbitrary

Amplification of 16S rDNA by PCR and sequence determination
Genomic DNA from Bacillus strains was prepared.The 16S rDNA gene fragments were amplified by PCR using the universal primers pU1 (5'-TGTTCCCATCCCAGATTCC-3') and pU3 (5'-GCGTGGCTGCGGGTCCCT-3'), which amplify the maximum number of nucleotides in 16S rDNA from a wide variety of bacterial taxa.The following thermocycling program was used: 5-min denaturation at 95°C; 35 subsequent cycles of 1-min denaturation at 95°C, 40-s annealing at 46°C; 1-min extension at 72°; and a final extension step of 3 min at 72°C.

Testing for hydrolase activities
Hydrolase activities were screened on plates containing the substrate for each particular enzyme.Plates containing 30 ml of growth medium were used throughout the work.Bacillus isolates were applied to plates as 5-μl drops of overnight culture.
Appearance of a clearing zone around colonies that produce the enzyme was noted.
For each isolate, three replicates were prepared.Plates were incubated at 25, 37, and 55°C for 24 h.For each of the three replicates, the diameter of each colony and activity zone was measured in two dimensions at 90° to each other and the values averaged.The index of relative enzyme activity (RA) for each isolate, substrate, and temperature combination was calculated by dividing the total area of activity (area of the clearing zone minus area of the colony) by area of the colony (Bradner et al., 1999).

Isolation and preliminary characterization of Bacillus sp. isolates
Using the feature of members of the genus Bacillus to form endospores resistant to elevated temperatures, 205 strains were isolated from 33 localities and from three ecological niches in Serbia: from straw and hay, 22; from soil, 126; and from manure, 57.Colonies with different morphology were subcultured on LA agar plates.All 205 isolates proved to be Gram-positive, endospore forming, catalasepositive rods.

Analysis of hydrolytic activity
The results of screening for hydrolytic activities of Bacillus isolates are shown in Table 2.As expected, most strains, produced amylase at 25 and 37°C.
The percent of producing strains in each particular habitat ranged from 73 (hay and straw at 25°C) to 100 (manure at 25°C, and soil and manure at 37°C).At 55°C, production of amylase was retained in four strains isolated from straw and hay, 22 from soil, and 10 from manure.
Similar results were obtained for production of protease.However, fewer strains isolated from manure were positive for protease (68% at 25°C and 65% at 37°C), but as much as 37% of these strains kept their activity at 55°C.All strains that thrived in straw produced protease at 25 and 37°C, but none of them retained activity at 55°C.As much as 90% (at 37°C) and 93% (at 25°C) of strains isolated from soil produced protease to some extent, but only 14% of these strains still produced the enzyme when incubated at 55°C.Hydrolysis of oil was observed for considerably fewer strains and only at 25 and 37°C: at 25°C, 23, 18, and 5%, from straw and hay, soil, and manure; respectively.Slightly more producers were found at 37°C: 27% from straw, 25% from soil, and 13% from manure.
The number of mannanase-producing strains isolated from straw and hay ranged from 95% at 25°C to 91% at 37°C.None of the strains produced the enzyme at 55°C.Eighty-five percent of strains isolated from soil produced mannanase at 25°C and 81% retained that ability at 37°C.Only two strains produced the enzyme at 55°C.From manure, 79% of strains produced mannanase at 25°C, 84% at 37°C, and as much as 33% at 55°C.
Xylanase at 25°C was produced by 81% of strains from straw, 65% from soil, and 63% from manure.At 37°C, there were 91% of producers from straw, 73% from soil, and 59% from manure.Only one strain, isolated from manure, produced xylanase at 55°C.The results of measuring relative activity of enzyme-producing isolates at different temperatures are shown in Fig. 3.For all enzymes tested, most producing isolates had low to medium hydrolytic activities (RA of 0-5) and only a small portion (or for some enzyme none) of isolates showed a high production rate (RA of 5-10).

DISCUSSION
Bacteria of the genus Bacillus are among the most widespread microorganisms in nature.In this work, as a source for isolation of strains we used straw and hay, soil, and manure from different locations in Serbia.Two hundred and five strains were preliminarily identified as members of the genus Bacillus (Stanković et al., 2007).Analysis by RAPD revealed considerable genetic diversity, represented by 13 distinctive groups (and six subgroups) of band profiles.Using sequencing of 16S rDNA, we established that all the observed diversity was from only four (five) species of Bacillus.More than half of the strains isolated belonged to B. subtilis (110) and, as statistically expected, this cluster of groups had considerable diversity.Nevertheless, B. cereus/B.thuringiensis, with 60 corresponding isolates has also considerable genetic heterogeneity, with five distinctive groups of profiles (but only one subgroup).Bacillus cereus and B. thuringiensis are essentially identical (Carlson et al., 1994) and can not be separated by 16S rDNA analysis.Bacillus thuringiensis can be distinguished only by the presence of intracellular protein crystals during sporulation (Hegason et al., 1998).The fact that some B. thuringiensis and B. cereus strains have a very efficient conjugation system (Gonzales et al., 1982;Jensen et al., 1996) facilitates the exchange of genetic material between strains (Hegason et al., 1998), making separation of DNA on the molecular level impossible.High local genotypic diversity in the cases of B. subtilis and B. cereus/B.thuringiensis can be attributed to recombination, which appeared to be frequent (Istock et al., 1992).Bacillus pumilus was represented by 34 isolates with three groups and one subgroup and less pronounced heterogeneity than in the other species.Finally, B. firmus was rep-  resented with only one strain.As can be seen from Fig. 2, the dominant species in straw was B. pumilus, while B. subtilis was dominant in soil and manure.Manure was the habitat harboring the greatest diversity, with representatives of all four species.This is in accordance with previous findings that bacteria are the predominant microorganisms cultured during the entire composting process.They accounted for 80-90% of the microorganisms typically found in a gram of compost (Trautmann and Olynciw, 2000).
Examination of hydrolytic activities at temperatures of 25 and 37ºC revealed no correlation with the specific habitat, i.e., straw and hay, soil, and manure were equally good sources for isolating producing strains.However, when monitoring hydrolytic activity at 55ºC, manure stood out as the best source of enzyme-producing isolates (51) and soil as the second best (41).No correlation was perceived between enzyme production at 55°C and the particular species of Bacillus.
Regarding the efficacy of enzyme production (Fig. 3), for all enzymes and for all temperatures tested, most producing isolates had low to medium hydrolytic activities (RA of 0-5) and only a small portion (or for some enzymes none) of isolates showed a high production rate (RA of 5-10).In addition, no correlation was detected between the efficacy of enzyme production and the particular Bacillus species.

Table 1 .
Characteristics of Bacillus strains isolated from different ecological niches.
with the corresponding species, four in total.Bacillus pumilus was represented with three groups of differ-ent RAPD profiles (I, II, and III) with subgroups (IIa and IIb), 34 strains overall.Bacillus subtilis was represented with five groups of distinctive profiles (IV, V, VI, VII, and VIII) with subgroups (Va, Vb, Vc, VIIa, and VIIb), 110 strains in total.Sixty strains of B. cereus/B.thuringiensisisolatesfellintofivegroups(IX, X, XI, XII, and XIII) with subgroups (XIa and XIb).Bacillus firmus had only one representative, strain 28.2.Distribution of the determined species of Bacillus in different habitats that were the source of isolation is represented in Fig.2.From straw, 64% of isolates were B. pumilus, 27% B. subtilis, and 9% B. cereus/B.thuringiensis.From soil, the predominant species was B. subtilis (65%), followed by B. cereus/B.thuringiensiswith23%andB. pumilus with 12%.Among strains from manure, B. subtilis was Fig.1.Fingerprints obtained by RAPD analysis with OPL12 primer of Bacillus strains represented in Table1.One strain of each group is represented.Lanes: 2

Table 2 .
Screening for hydrolytic activity in a Bacillus collection.