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An improved ARDRA method of microorganism identification and its application in identifying thermolabile alkaline phosphatase strains-producers

 

This email address is being protected from spambots. You need JavaScript enabled to view it. , M.A.Abdurashitov, S.Kh. Degtyarev

Translated from Biotechnologia (russ.). 2005, N 6, pp 3-11

 

Based on the analysis of amplified ribosomal DNA fragmentation by restriction endonucleases, an improved method for microorganism identification (called ARDRA) has been suggested. A set of 6 restriction endonucleases (Sse9I, Tru9I, BsuRI, MspI, BstMBI and RsaI) was used to get corresponding patterns of DNA cleavage. DNA samples were isolated from four strain-producers of thermolabile alkaline phosphatase, obtained from sea water. Carrying out of ARDRA, followed by a comparison with the calculated cleavage patterns of DNA from several referenced microorganisms, allowed us to conclude that the new strains-producers belong to the Alteromonas genus.

 

There are traditional methods of microorganism identification, which are based on studies of cultural and morphological characteristics of these strains, as well as their chemical and biochemical reactions [1]. In the last years, two other identification methods are becoming more widely used. One method is based on the comparison of the microorganisms gene primary structures [2-4] and the other is based on the analysis of polymorphism of DNA fragments length; which are obtained as a result of DNA cleavage [5, 6] . Genes, which encode 16S and 23S ribosomal RNA, are the most suitable for this identification because they are present in all bacterial cells and are specific for most microorganisms [7-9]. Identification based on a long DNA fragment, which contains genes 16S and 23S RNA, and variable spacer located between them, allows to distinguish between closely related types and subtypes of microorganisms [10].
This paper presents ARDRA results of a 1500 bp DNA fragment of various microorganisms and shows that using 6 restriction endonucleases (Sse9I, Tru9I, BsuRI, MspI, BstMBI and RsaI) allows to fairly accurately identify most microorganisms. Four new strains-producers of thermolabile alkaline phosphatase were discovered and characterized by the improved ARDRA method. All strains belong to the Alteromonas genus.

 

MATERIALS AND METHODS

In order to isolate the thermolabile alkaline phosphatase producers, seawater samples (50 μ l) were smeared onto a nutrient agar surface and analyzed as described previously [11]. Producers were grown at 20°C, in flasks containing 200 ml of a medium (1% trypton, 0.5% yeast extract) and sea salt (NaCl - 27.5, MgCl2 - 5, MgSO4 - 2, CaCl2 - 0.5, KCl - 1, FeSO4 - 0.001 g/L [12]), pH 7.2 - 7.7, in a G-25 shaker (New Brunswick, USA). The cells were grown for 16 hours.
DNA was extracted from bacterial cells, as described previously [13].
PCR amplification of 16S rDNA was also performed as described in [14].
The amplified DNA was digested with 2 U of the restriction endonucleases Sse9I, Tru9I, BsuRI, MspI, RsaI or BstMBI respectively, (restriction endonucleases provided by SibEnzyme Ltd. Novosibirsk) in a total volume 20 μl of reaction mixture, at 37 °C, for 4 h. Reactions were stopped by adding 5 μl of stop solution containing 0.1 M EDTA, 0.05% bromfenol blue, and 40% sucrose.
Cleavage products were analyzed by 2% agarose (Sigma) gel electrophoresis, in a TAE buffer containing ethidium bromide (0.5 mg/l) at 120 V for 4 h.
DNA molecular weight markers (100bp +1.5 Kb Ladder) were produced at SibEnzyme Ltd, Novosibirsk.
Lengths of DNA fragments were determined using computer program Gel Pro Analyzer Version 4.0.00.001. Percentage of fragments with identical lengths was calculated for each pair of microorganisms, by comparing electrophoretic pictures of each restriction endonuclease cleavage product. Fragments lengths were considered identical, when their lengths varied by no more than 5%.
For comparison of experimental data with the published sequences of 16S RNA genes, we used genetic sequences databank (GEN BANK).

 

RESULTS AND DISCUSSION

Four strains-producers of thermolabile alkaline phosphatase were isolated from the natural sea water isolates, and three were marked as strains 20, 27, 48. The last strain was characterized previously [11], using traditional methods, as Alteromonas undina.
For the identification of microbial strains, chromosomal DNA was isolated from a biomass, which was grown in a liquid nutrient medium with the addition of sea salt.
Chromosomal DNA was used in the polymerase chain reaction amplification of the gene ribosomal 16S RNA.
PCR amplification products were cleaved independently by each of the six different restriction endonucleases. All used restriction endonucleases have a tetranucleotide recognition site. They produce anywhere from 3 to 8 DNA fragments from the amplified product with length of 1,500 bp. Restriction endonucleases Sse9I and Tru9I cleave AATT and TTAA recognithion sites, respectively, while enzymes BsuRI and MspI split DNA at sites GGCC and CCGG. Recognition sites of restriction endonucleases BstMBI and RsaI are GATC and GTAC, respectively, and contain four different nucleotides. In our view, using this set of restriction endonucleases should be universal for identification of microorganisms, which have AT- rich or GC- rich genomes. We believe that the use of six different restriction endonucleases is the optimal quantity for sufficient identification. Using 1 or 3 restriction enzymes, as suggested in some works [10,15], may either not detect polymorphism during identification of closely related microorganisms or, on the contrary, lead to too much variance due to one or more random mutations. On the other hand, the use of 10 different restriction endonucleases does not result in identification of additional fragment length polymorphism in DNA cleavage products and is also vastly excessive [9].

 Fig. 1. Theoretically calculated electrophoretic separation pictures of amplified DNA 16S RNA genes, after cleavage by restriction endonucleases Sse9I (1), Tru9I (2), BsuRI (3), MspI (4), BstMBI(5) and RsaI (6). Run M - molecular weight markers.

 

 

Fig. 1. Theoretically calculated electrophoretic separation pictures of amplified DNA 16S RNA genes, after cleavage by restriction endonucleases Sse9I (1), Tru9I (2), BsuRI (3), MspI (4), BstMBI(5) and RsaI (6). Run M - molecular weight markers.



 

 

  Streptomyces albus Micrococcus luteus Bacillus subtilis Staphylococcus aureus Hemophilus influenzae Escherichia coli Pseudomonas aerugenosa Neisseria denitrificans
Escherichia coli 22 20 27 24 25 _ 27 20
Streptomyces albus _ 37 28 20 12 22 16 12
Hemophilus influenzae 20 20 24 16 _ 24 24 20
Neisseria denitrificans 22 26 22 22 18 22 26 _

Table 1. Table 1 DNA fragments with identical lengths of 16S RNA genes, from representatives of various microorganisms' genera



Figure1 represents computer simulated electrophoretic pictures of gene 16S RNA cleavage by the above suggested set of six restriction endonucleases (Sse9I, Tru9I, BsuRI, MspI, BstMBI and RsaI). Genes of 16S RNA were selected from the genetic sequences databank (GEN BANK). Selection of microorganisms was completely random. All bacteria belong to different genera, and are both grampositive and gramnegative. Results of 16S RNA gene cleavage by the six restriction endonucleases, revealed a unique mix of DNA fragments for each microorganism. The number of DNA fragments is about 25-30 (fragments with lengths less than 100 bp of nucleotides were disregarded).
Percentages of DNA fragments with identical lengths were calculated for all different pairs of microorganisms and are presented in Table 1. We believed DNA fragments length to be identitical, when fragments length varied by no more than 5%. This table provides only a portion of all possible microorganism pairs, presented in Fig. 1. However, the results are quite typical, and reveal that in various microorganisms, 12-28% of fragments have identical lengths. Thus, the data show that the pictures of DNA fragments, produced as a result of 16S RNA genes cleavage by six restriction endonucleases, can serve as basis for identification of tribal bacterial cells.

Fig. 2. Electrophoretic separation of cleavage of amplified DNA 16S RNA genes from the strains-producers of thermolabile alkaline phosphatase, by restriction endonucleases Sse9I (2), Tru9I (3), BsuRI (4), MspI (5), BstMBI (6) and RsaI (7). Run 1 - molecular weight markers.

 

 

Fig. 2.Electrophoretic separation of cleavage of amplified DNA 16S RNA genes from the strains-producers of thermolabile alkaline phosphatase, by restriction endonucleases Sse9I (2), Tru9I (3), BsuRI (4), MspI (5), BstMBI (6) and RsaI (7). Run 1 - molecular weight markers.

 




The Fig. 2 presents hydrolysis of the amplified 16S RNA genes, which are identified as strains-producers of thermolabile alkaline phosphatase. From the simple visual comparison of DNA fragments patterns for strains-producers 20 and 48, we can see that DNA cleavage products are identical in lengths for all six restriction enzymes. This demonstrates that these particular strains are either identical or closely related microorganisms. It should be noted that the existence of weak minor bands on the electrophoretic pictures may be a result of either incomplete amplified DNA cleavage, or to the presence of several copies of the gene 16S RNA in the microorganism genome, some of which may have variations in the sequence [10]. These bands do not significantly affect the analysis of the experimental results, since these minor bands were not considered.

 

Sse9I Tru9I BsuRI
27 48 20 A.und 27 48 20 A.und 27 48 20 A.und
537 548 550 548   469 471   470     478
470             409   313 319 330
  439 442   349 356 360 357 281 283 285 287
385               262      
249 245 250 252 310     314   205 207  
168       269 279 282   187 184 188 185
128 129 134   143 146 145 140 140 136 141 137
  121 125 123 129       109 110 114  
96                 90 95  
        100 96 95 97       83
MspI BstMBI RsaI
27 48 20 A.und 27 48 20 A.und 27 48 20 A.und
  552 550 539 665 659 641   900     886
524 504 508         539   485 500  
      488       500   445 442  
310         403 400   360     359
246       321         241 243  
176       276 286 287 277   231 232  
143 140 146 140 169 170 171 172 155     155
122 117 120 116 102       136 134 136 133
  79 83 79                

Table 2 Length of DNA fragments, obtained from amplified DNA 16S RNA genes of the strains-producers 27, 48, 20 and Alteromonas undina, after cleavage by the 6 restriction endonucleases


As can be seen from the results in Table 2, after cleavage of amplified genes 27, 48, 20 and Alteromonas undina with the six restriction endonucleases, there is a high percentage of bands, which are equal in length. Bands were accepted as equal in length (as in the case for comparison of computer simulated electrophoretic pictures) when their length differed by no more than 5%. This percentage corresponds to the experimental error value in determining DNA fragments length.

Fig. 3. Percentages of 16S RNA gene DNA fragments with identical lengths in strains-producers of thermolabile alkaline phosphatase

 

 

Fig. 3. Percentages of 16S RNA gene DNA fragments with identical lengths in strains-producers of thermolabile alkaline phosphatase



 

 

 

  Streptomyces albus Micrococcus luteus Bacillus subtilis Staphylococcus aureus Hemophilus influenzae Escherichia coli Pseudomonas aerugenosa Neisseria denitrificans
Alteromonas undina 11 22 18 11 22 15 22 18

Table 3 Percentages of DNA fragments with identical lengths in Alteromonas undina and different microorganisms from various genera


Results presented in Fig. 3 show that for all strains-producers of thermolabile alkaline phosphatase, more than 50% of DNA fragments are identical in length. This value indicates that these microorganisms are related. Comparison of the fragments lengths, produced by cleavage of amplified DNA 16S RNA gene from Alteromonas undina, to that of the same gene from the control microorganisms (Streptomyces albus, Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, Hemophilus influenzae, Escherichia coli, Pseudomonas aerugenosa and Neisseria) revealed a 11-22% overlap. This percent overlap represents random coincidence value. We used this data as control to confirm that the microorganisms are not related and belong to different genera.

Fig. 4. Theoretically calculated electrophoretic separation pictures of amplified DNA 16S RNA genes from the Alteromonas genus, after cleavage by restriction endonucleases Sse9I (1), Tru9I (2), BsuRI (3), MspI (4), BstMBI(5) and RsaI (6). Run M - molecular weight markers.

 

 

Fig. 4. Theoretically calculated electrophoretic separation pictures of amplified DNA 16S RNA genes from the Alteromonas genus, after cleavage by restriction endonucleases Sse9I (1), Tru9I (2), BsuRI (3), MspI (4), BstMBI(5) and RsaI (6). Run M - molecular weight markers.

 




We suggested that the 50% overlap in number of DNA fragments with identical length characterizes microorganisms as one genus. This premise was confirmed through the following analysis of the existing primary structures database of different DNA types in the same bacteria genus. For comparison of both methods of microorganisms relationship identification we analyzed computer reconstructed images of 16S RNA gene DNA cleavage of a number representatives from the Alteromonas genus. We constructed calculated patterns of DNA cleavage with the same set of restriction endonucleases, which were used in the amplified DNA hydrolysis of the16S RNA gene from the newly discovered microorganisms, described earlier (Fig. 4). In the genetic sequences databank (GEN BANK), we selected the 16S RNA gene sequences for the following microorganisms: A(P). atlantica, A. litorea, A. marina SW47, A. marina SW49 and A. stellaepolaris. As shown in Fig. 4, 16S RNA genes A. marina SW47 and A. marina SW49 have identical DNA cleavage patterns for all six restriction endonucleases. Therefore, we compared our data to only one of these microorganisms. We compared cleavage patterns of the strain-producer 27, 20 and 48 with those obtained from the databank for Alteromonas genus microorganisms, and also with patterns for A. undina. The comparison of the strain-producer 27 patterns, revealed that identical DNA fragment lengths were present in 45% to 58% of all cases (as presented in Table 4), with average value of (49±4)%. For the strains-producers 20 and 48, which produce identical patterns, 53-60% of all fragment lengths were identical, with the average value of (58±3)%. For comparison of Alteromonas undina patterns with those from the databank, revealed 49-57% of identically long fragments; average a (54±3)% average. We have compared data on the percentages of identical fragments lengths, obtained after DNA cleavage of 16S RNA gene, between a number of microorganisms, specifically between the recently discovered strains-producers and the representatives of the Alteromonas genus, as well as between various microorganisms of unrelated genera. As a result, we have found a statistically reliable difference in this data, when comparing newly discovered strains and the Alteromonas genus, average of 50% or more, and the data on lengths similarities between all other unrelated microorganisms, average of only 11-22% (Table 3).

 

  A(P). atlantica A. litorea A. marina SW47, A. marina SW49 A.stellaepolaris
A(P). atlantica
-
62
63
65
A. litorea
62
-
80
77
A. marina SW47, A. marina SW49
63
80
-
87
A.stellaepolaris
65
77
87
-

 Table 4. Percentages of DNA fragments with identical lengths in 16S RNA genes from the Alteromonas genus


As shown in Table 5, within the Alteromonas genus, the percentage of DNA fragments with identical lengths, ranges from 62 to 87%. It should be noted, that a proposed division of the Alteromonas genus into Alteromonas and Psevdoalteromonas genera[8], is, in our opinion an isolation of a more closely related group of microorganisms within the Alteromonas genus. Proof of this fact is in the percentage of identically long DNA fragments of the 16S RNA genes of A(P). atlantica and Alteromonas undina. This value is 53%. According to the new classification, they belong to the Pseudoalteromonas genus. Similarly, the percentage of length similarity of 16S RNA genes DNA fragments of these microorganisms and A. litorea, A. marina SW47, A. marina SW49 and A. stellaepolaris ranged from 49 to 65%. According to the new classification, the latter 4 microorganisms, belong to the Alteromonas genus. Fragment lengths similarity of A. litorea, A. marina SW47 and A. stellaepolaris ranges from 77 to 87%, and is, perhaps, a sign of a closer relationship.

 

  A(P). atlantica A. litorea A. marina SW47, A. marina SW49 A.stellaepolaris
Alteromonas undina
53
49
57
57
27
46
48
50
45
20, 48
63
53
58
59

Table 5. Percentages of DNA fragments with identical lengths in strain-producers of thermolabile alkaline phosphatase and the Alteromonas genus.


Thus, the data suggest that the coincidence of (50±5)% or more in the DNA fragments lengths, obtained through hydrolosys of 16S RNA genes of various microorganisms with a set of 6 specific restriction endonucleases (Sse9I, Tru9I, BsuRI, MspI, and RsaI BstMBI) characterized these microorganisms to be of one genus. Consequently, we can conclude that strains-producers of thermolabile alkaline phosphatase (20, 27 and 48, respectively) can be classified as Alteromonas and marked accordingly: Alteromonas species 20, Alteromonas species 27 and Alteromonas species 48.
Experimental results show that using the proposed combination of restriction endonucleases for cleavage of 16S RNA gene, along with data in the genetic sequences databank, can be a simple, affordable and universal method for identification of microorganisms. In addition, amplified ribosomal DNA restriction analysis (ARDRA) is much less sensitive to the presence of impurities in DNA compared to the polymerase chain reaction (PCR), which is used in determining sequences of the 16S RNA gene. This fact also simplifies the use of this method for identifying microorganisms.
We thank N. Sanamyan for technical assistance in acquiring the under-water samples of microorganisms.

 

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