Apr 6, 1993 - 100. L. E. M A C. E L Q. K. M. N Y N S. L. F. D H. T. L L. R. A. D A. S. 101 ... 300. N. S S V. K I C T V V G. F. P L G Q T. S T. K. Q. K. V. Y. E. T. K.
Vol. 175, No. 16
JOURNAL OF BACrERIOLOGY, Aug. 1993, p. 5281-5285
0021-9193/93/165281-05$02.00/0
Copyright X 1993, American Society for Microbiology
Identification of Mycoplasma pirum Genes Involved in the Salvage Pathways for Nucleosides T. N. THAM, S. FERRIS, R. KOVACIC, L. MONTAGNIER, AND A. BLANCHARD* Departement du SIDA et des retrovirus, Oncologie Virale, Institut Pasteur, 28, Rue du Dr. Roux, 75724 Paris Cedex 15, France Received 6 April 1993/Accepted 3 June 1993 Genes encoding enzymes involved in the salvage pathway for nucleosides have been cloned and sequenced from the mollicute Mycoplasma pirum. One of them, encoding deoxyriboaldolase, was functionally identified by complementation of an Escherichia coli mutant. These genes are clustered, suggesting an operon organization, and they are immediately followed by the putative gene for the triose phosphate isomerase, an enzyme used during glycolysis.
Mycoplasmas (class Mollicutes) are the smallest selfreplicating bacteria, with a genome which can be as small as 600 kbp (4). It has been hypothesized that during evolution from the ancestral gram-positive walled bacterium (30), mycoplasmas have lost many metabolic activities found in most other bacteria, including the ability to synthesize a cell wall. Mycoplasmas obtain many nutrients from the host cells that they parasitize. In vitro, they have complex nutritional requirements (20). All mycoplasmas tested lack the orotic pathway for pyrimidine synthesis and the enzymatic pathways for de novo synthesis of purine bases (19). Uracil, thymine, and guanine are required for growth. Lacking these capacities for de novo synthesis, mycoplasmas form nucleotides by salvage pathways from more complex precursors. Consequently, they must possess a range of enzymatic activities to facilitate the use of the more complex precursors. One of the salvage pathways that is possibly active in mycoplasmas involves enzymes which in Escherichia coli are encoded by the deo operon. These enzymes form a pathway by which nucleosides from the medium provide bases that serve as precursors for nucleic acid synthesis or that are used to form pentose or deoxypentose, which can be used as carbon and energy sources. Enzymes from this pathway include thymidine phosphorylase and purine nucleoside phosphorylase, which have been detected in several mycoplasma species (for a recent review, see reference 6). However, unlike for E. coli, there is no genetic documentation for these metabolic pathways in mycoplasmas, the only exception being the published sequence of the gene for the deoxyriboaldolase in Mycoplasma pneumoniae (13). We report here the cloning and sequencing of a DNA fragment from Mycoplasma pirum with deduced polypeptides homologous to enzymes both of the deo operon and of side reactions to glycolysis. M. pirum BER was previously isolated from a culture of peripheral blood mononuclear cells purified from the blood of a patient infected with human immunodeficiency virus type 1 (17). This mycoplasma was cultivated in SP-4 medium (25), and genomic DNA was purified by conventional procedures (14). Genomic DNA was digested with the restriction enzyme HindIII, and Southern blot hybridization under low-stringency conditions was performed with an oligonucleotide probe. This oligonucleotide (5'GGCGTAGTATC *
Corresponding author.
TACTCCCCTTGTTAACC'1TATCAATGGCCAG3')
was initially chosen from a region showing a high degree of homology between the genes for adhesins in M. pneumoniae and Mfycoplasma genitalium (9) and was thought likely to hybridize to the adhesin-like gene in M. pirum. Three DNA fragments hybridized with this probe, and one of them (4.6 kbp) was partially purified from the agarose gel by electroelution (21), ligated to the plasmid vector pBluescript SK(Stratagene, La Jolla, Calif.), and used to transform E. coli XL1-Blue competent cells. The DNA insert in the recombinant plasmid (pMPDEO) was sequenced by using Sanger dideoxy reactions (22) with modified T7 DNA polymerase (Sequenase; U.S. Biochemical Corp., Cleveland, Ohio). Assuming that UGA codons would code for tryptophan (Trp), as is the case in all other species from the genus Mycoplasma analyzed so far (5), analysis of the nucleotidic sequence revealed four open reading frames (ORFs) corresponding to the following nucleotide positions (shown in Fig. 1): ORF2, from 150 to 810; ORF3, from 829 to 2086; ORF4, from 2106 to 2505; and ORF5, from 2526 to 4158. In addition, one ORF without an initiation codon was found at the 5' end of the DNA fragment (ORF1, from 1 to 129) and another ORF without a termination codon was found at the 3' end (ORF6, from 4171 to 4621). The deduced protein sequences from these ORFs were compared with the protein sequences available from the SwissProt data base (version 23.0) by using the GCG sequence analysis software package (Genetics Computer Group, Inc., Madison, Wis.). The highest levels of homology found are shown in Table 1. Taking into account that mycoplasmas are phylogenetically distant from E. coli and Bacillus stearothermophilus (a fortiori from Homo sapiens), these levels of homology are significantly high and indicate that the proteins share the same enzymatic activity. Therefore, the putative polypeptides encoded by the DNA fragment cloned from M. pirum are purine phosphorylase (ORF1), deoxyriboaldolase (ORF2), thymidine phosphorylase (ORF3), cytidine deaminase (ORF4), phosphomannomutase (ORF5), and triose phosphate isomerase (ORF6). In order to demonstrate that ORF2 encodes deoxyriboaldolase, the plasmid pMPDEO was tested for complementation of strain S0063 of E. coli, deficient for the gene deoC, which encodes deoxyriboaldolase (26). This strain was kindly provided by P. Valentin-Hansen (Odense Universitet, Odense, Denmark). Growth was obtained on selective medium containing thymidine as a carbon and energy 5281
5282
J. BACTERIOL.
NOTES
A
1
L
101
D
S
L
L
S
E
K
T
I
Q
R
E
L
S
S
V
M
T
N
F
T
T
L
K
A
E
C
Q
L
N
M
K
N
Y
S
H
D
F
L
L
L
T
100
S
A
D
A
R
200
TTTAGAAATGGCTTGTGAATTACAAAAATAAATTAAGGTGTTAACAATTATGAATTACAATAGCTTATTTGATCACACATTATTGCGTGCTGATGCTTCT E
E
Q
K
I
L
E
D
C
A
K
V
N
F
C
V
S
F
F
V
Y
S
P
N
V
H
L
Q
E
K
V
Y
P
GTAGAAGAAATTAAACAACTTTGTGATGAAGCAGTAAAGTTTAATTTTTTTTCAGTTTGTGTTAATCCATCATATGTACCTTATGTAAAAGAACAATTAC K
V
S
S
N
301
S
V
T
L
A
M
E
V
201
L
T
L
AAGCTTTAACATTACTAACTGTTAGTGATTCTTTAATCACAAAAGAATCTTTATCTTCTTTAGAAAGACAAACAACATTTAACACTATGGTTAAACTTGC
I
T
C
V
V
F
G
P
L
Q
G
S
T
T
V
K
Q
K
I
A
I
K
T
E
Y
300
G
E
K
400
ATAATTCAAGTGTCAAAATTTGCACAGTAGTTGGTTTTCCATTAGGACAAACATCTACAAAACAAAAAGTATATGAAACCAAAATAGCTATCAAGGAAGG D
A
M
D
I
E
I
N
L
V
F
E
S
K
E
N
N
V
V
C
A
C
I
E
K
Y
K
R
K
K
C
V
K
500
401
CGCGGATGAAATAGATATGGTTTTAAATATATCTGAATTTAAAGAAAATTGCGCCTGTGTAGTTAACGAAATTAGAAAATATAAAAAAGTATGCAAGAAA
501
AAAATATTAAAAGTTATTGTTGAAACAGCATTATTAAGTGAGAATGAAATTGAAAAAGCAACTTTAGTTGTTATTGATGGAGGAGCGGATTTTATAAAAA
601
CATCAACAGGTTTTTCTTCTCGAGGCGCTTCAATCAAAGATATTGAAATAATGAAAAACGTTATTGAAAAAAATAATAGCAAATTAAAAATTAAAGCATC
701
TGGTGGAATAAAAACATTAACGTTTGTTGAGGAATTAATAAAAGCTGGTGCCGAAAGAATTGGTTCTTCAAAAAGTGTAGAAATAATAAAAGAAACATTA
801
AATAAAAATTAAATTTAAAACAAAATTTATGAATATTATAGAAATTATTGAATTAAAAAAAATAAAAAAATTGTCTCAAGATCAAATTAATTTTTGT
901
ATTTCAGGATTAGTAAACAAATCAATTCCAGATTATCAAATTAGTGCATTGTTAATGGCTATATGATTTAACGGATTAGATGATAATGMTTATATTTTT
1001
TAACAAAAGCAATGATTGATTCGGGAAAAATTTATAAATTTCATCCCGAGTATAAAAAAATTTTAATAGACAAACATTCTACAGGTGGCATAGGCGATAA
1101
AGTAAGTATTGCTTTACGGCCAATATTAGTTTCTTTTGATTTAGGTGTTGCAAAATTATCTGGCCGAGGATTAGGTTTTACTGGCGGGACTATAGATAAG
1201
TTAGAATCTATTAATGTAAATACTGATATTGATTTAAAAAATTCTAAAAAAATATTAAATATAGCAAACATGTTTATTGTGGGACAAACAAATGATATTG
K
L
I
S
I
G
G
N
T
V
A
P
N
I
V
K
D
P T
L
L
I
D
A
Y
F
S
L
D
R
L
L
K
D
T
T
G
V
S
L
S
D
I
N
L
G
A
L
P
I
F
F
M
N
A
A
I
S
G
G
I
D
K
K
S
1300
L
1400
1301
TTCCTGCTGATAAATTATTATATGCGTTACGTGATGTAACAGGAACAGTTGATTCTTTGCCTTTAATAGCAGCATCAATTTTATCTAAAAAATTTGCTTT
1401
AGAGTCGGATTACATTTTTATTGATATTAAATATGGTCAAGGAGCATTTTGTCATGATATAGAAACAGCAAAAAAAATAAGTAATATCATGAAAAATTTA
E
S
A
K
K
I
Y
D
K
F
I
F K
R
I
D V
K
Y
F
Y
Q
G
V
L
G
D
S
F
A
M
C V
E
N
D
H
I G
L
T
E
A
T
N
K
K
V
G
I
K
M
I
V
E
I
A
N
N
S
L
N
E
K
I
A
1600
GCTAAAAAATTTAAACGAAAAGTTTATTTTGTTTTAAGTGATATGAATGAAGTATTAGGGAATACTGTGGGTAATGCAATTGAAGTAAAAGAGGCCATAG
1601
ATTTTTTAAAAAATAATTCTGATGTTGGAACATATTTTAAAAAATTAATGTTTGATTTAGTAACTTTAATTCTTTTGAAAACTAAAAAATGCAAAACAAA
L
K
1701
E K
D
V
D
Y
F
F
K
L
G
I
K
K
G
S
L E
G
D
F
I
D
N
N
F
A
V
L
L
T
F
N
C
L
L
I I
W
K
E
K
T Q
L
K G
N
K
C
I
N
K
T
T
W
N
A
I
A
K
W
I
K
G
S
S
I
I
S
K
Y
A E
A
L
E
K
K
E
K
D
D
I
F
Q
I
G
A
Y
A
H
L
K
E
N
S
K
S
Y
S
K
P
Q
K
I
D
L
D
I
K
K
K
A
K
I
I
I
D
Y
I
F
K
Q
E
N
L
L
I
N
S
A
V
Y
P
S
Y
N
K
I
V
F
W
A
V
G
N
S
N
E
I
Y
A
P
S
T
E
A
C
I
V
A
S
R
F
I
Q
V
K
Y
I
T
L
D
I
T
V
D
K
G
I
P
T
P
E
T
I
P
T
I
Y
N
L
G
K
K
E
F
T
Y
F
P
L
L
Q
E
L
M
S
Q
E
N
N
M
K
K
V
I
D
S
S
L
W
N
P
V
I
D
K
2300
K
E
E
N
L
E
L
A
S
F
L
P
A
N
I
G
A
T
G
F
S
P
A
M
K
A
R
2500
K
ATGAAAAATGAAGAGTTAGAATTAGCTTTCTCTAATGCGCCGTTATCGTTTGGCACAGCTGGTATAAGAGCTAAAATGGCTCCTGGCACTCAATTTTTAA T
I
Q
Y
Y
M
A
T
Y
G
G
K
L
F
K
K
N
F
S
I
N
Q
N
S
A
V
I
V
N
D
H
I
G
D
F
S
I
T
V
D
L
S
T
L
I
N
L
C
I
F
E
L
E
L
L
N
I
I
K
R
L
S
Y
A
I
R
K
L
N
A
Q
G
A
V
I
V
T
A
S
H
N
P
K
E
D
N
G
F
K
I
Y
2800
Q
TAATAATGGTATTGATTTTTCTATTGATGTAACTAATATCTTAACTTCGCTAGAATTAGAGTTTATTTGTTTGAAAATAATCAACTTACTTCTACGCCAA F
2700
R
ATAAAATTACATACTATCAAATGGCAACAGGATATGGAAAATTTTTAAAAAATAAATTTTCTAATCAGAATATTAGCGTGATTGTTGCTCATGATAATCG N
2600
N
L
F
Q
T
G
2400
L
S
I
2200
A
F
A
D
K
2100
S
E
S
N
F
D
T
Q
2000
L
T
I
L
V
A
F
1900
I
L
L
C
S
S
R
C
V
G
C
1800
I
S
R
F
1700
K
AAAATAAAAAGGACACACACATATTATGAATAATGAAATTGTTAAAAAATGATTATCTAGTGATAATGTTCCACAAACAGATAAAGATATTATTTCTAAA
L
2901
K
N
F
M
CAAAACCCGAAACACCTATAATTACTTATAATCTTAAAGGTGAAAAATTTTTTTATACTCTCGAGCAATTATTACCGTTTGCTTTTAATAAAGATGCATT
N
2801
Y
R
R
L
T
K
G
K
K
2701
L
GGTTTTAAACAAATTTTTAAAGTATATATTTTGACTGACACGATTGTTAAAGATATTGGAACTCCTTGTGGGGTTTGTCGTCAAGTTTTAAGTGAATTTG
M
2601
K
K
E
L
K
P
K
2501
V
F
AACTGATGGAGGATGATTCGCTGGAGTTAATATAGAAAATTCTGCATATTCACCAACTATATGTGCCGAGCGTAGTGCTGTTTCATCTATGATTACATCG
K
2401
N
I
Y
T
CGATAATGAAAGAAAAAGATATTTATTTTCAAAAACTTAATGAATTAATAAGTAACGCATATGTTCCATATTCGAATTTTAGAGTTAGTTGTTTATTATT
G
2301
G
V
AAAAGATAAAATATTAACATTATATTCTTCTAAACCAATAAAGCAAGACTTAATTGATAAAGCTAAAAAAATAATAAAAATTTCTTAATTTATAAGGTAA
T
2201
K
E T
D
N
M
2101
D
TAGGAGTTGATTTGGGTTCTGGTAGAAGAAAAAAAGAAGATAAAATAGATTTTCAAGCCGGAATTTATTTACATGCTAAATCAAATGAAAAAATTAAAAT K
2001
K
A
S
ATTAAAAATGATACTTTTTTCAAACCTAAATATTGAACTAATATAGCAGCATGGAAATCAGGAAAAATTTCATACAAAAGTATCATAGAATTAGCTGAAA G
1901
N
N
AAAAGAAGCTAAAGAAAAAATAAATTATGTTTTAGAAAACAAAATAGCATTTAATAATTTTTGTAACTGAATTGAATTGCAAAATGGAAATATTGCAAAA I
1801
K
1500
D
1501
F
1200
V
A
F
1100
K
D
N
T
1000
K
D
I
T
Q
L
G
900 L
F
Y
I
800
C
F
L
G
G
V
E
G
T
I
N
T
S
G
L
D
D
H
K
D
R
L
G
700
L
T
N
600
S
A
E
I
T
K
K
K
Q
D
Q
S
I
I
E
V
L
N
I
G
K
F
L
S
I
K
L
K
S
I
F
D
A
G
G
N
K
K
W
I
L
I
K
I
K
K
A
K
S
K
Y
V
A
S
K
D
N
K
S
N
I
V
E
G
K
M
L
E
G
N
V
P
H
D
L
I
K
V
I
V
R
L
L
T
N
E
E
A
A
K
A
I
S
F
K
G
I
I
Q
Y
V
E
M
I
A
K
E
I
E
K
I
Y
I
L
I
N
D
K
I
I
E
I
D
L
N
P
G
S
R
L
A
S
D
I
K
E
N
E
S
I
E
I
S
L
S
A
F
K
N
M
A I
E
V
L
K S
L
G
T
L
L
A
M
G
S
V
T
K
R
S
S
T
E
V
N
K
I
F
G
T
I
V
K
N
2900
E
TTGTTTTCGTATGCTATAAGAAAATTAAATGCACAAGGAGCAGTTATTGTTACTGCTAGTCATAATCCTAAAGAAGATAATGGCTTTAAAATCTATAATG
3000
FIG. 1. Nucleotide sequence of the M. pirum 4.6-kbp DNA fragment. Deduced polypeptides are indicated above the nucleotidic sequence.
NOTES
VoL. 175, 1993 T
3001
G
I
N
G
D
E
Q
N
G
Q
I
G
K
L
V
V
L
E
M
P
V
N
F
E
I
M
D
L
K
A
V
N
D
S
D
I
F
Q
R
Y
Y
D
E
C
K
Q
L
A
I
K
N
T
I
N
S
E
K
E
G
H
T
A
K
C
R
L
P
F
E
L
K
L
L
G
Y
N
K
I
V
L
I
E
E
F
S
T
N
P
T
N
P
P
E
R
N
A
A
D
W
L
S
E
I
Y
A
K
D
N
C
Q
N
A
D
V
P
A
D
D
R
A
F
L
G
R
V
Y
N
K
S
R
W
F
S
L
G
N
M
Q
G
D
Y
L
I
K
N
K
T
F
K
T
K
P
Y
V
I
S
S
V
Y
S
T
L
N
I
D
I
R
I
Y
E
V
Y
R
V
T
G
G
F
V
W
K
G
K
D
I
N
K
K
I
D
E
S
E
F
V
G
V
F
E
N
L
A
T
S
I
N
R
K
D
D
A
Q
Y
A
A
L
A
A
L
E
Y
I
N
E
L
C
K
N
D
I
L
H
K
E
N
I
Y
G
K
Y
G
I
I
N
H
T
D
S
I
T
F
F
V
N
E
N
E
K
W
K
K
S
L
D
K
I
L
K
Y
S
E
K
I
T
G
N
T
R
I
T
S
K
I
Y
E
N
V
G
G
3700 3800
L
AATATTATTGATCATTTAGAAAAAAATATTTATGGAAAATATGGAATTATTCATAATGACACTATTTCATTTACTTTTGTGGAAAATAATTGAAAAGAAT V
3600
I
N
TGTTGGCGCTTTAAATTCAACAATAAACAGAGACAAAGATGCATATCAAGCAGCTGCATTGGCATTAGAAATATACAATGAATGTTTAAAAAACAACATT I
3500
A
E
ATGGAGAAGTTTATCGAGTTGGAACTGGTTTTAAATGAGTTGGAGATAAAATTAATAAAATCAAAGATTCAGAAGAATTTGTTGTTGGATTTGAAGAAGC G
3400
H
ACAGATTACATACTTAAAAACAAAACATTTACCAAACCATATATTGTTTCTTCATACGTATCAACAAATTTAATTGATCGTATTATCAAAGAATATC E
3300
Y
I
K
3200
I
V
N
I
3100
S
F
GATTATTCAAGTTGATCCTGATGCTGATAGATTTGCCCTTGGTGTTCGCTATAAAAATTCTTGACGTTTTTTAAGTGGAAATCAAATGGGCATTATTTAC
N
3801
D
TTTTTGATGGGAATTTTTCTAATACTCCAACACCAAATCCAGAAAATCGTGCAGCGTGAGATTTGTCAATA¢AGTATGCAGATAAAAACAATGCAAATGT
V
3701
L
S
D
G
3601
D
P
ATAGTTTTTAGTGGTCAACATGGTACTGCATGTAAAAGATTACCTGAATTTCTTAAACTACTAGGTTATAAAAACATAATTTTAGTTGAAGAACAATGTA
T
3501
Y
F
I
3401
T
V
F
3301
L
ATTAATTACATATTTAAATGAAGATATTTTTAGACAATATTATGAAGATTGTAAACAAGCATTAATTAAAACTAATATCAATGAATCTAAAGAATTTTCA I
3201
V
AAACAGGTGCTCAAGTTTTGCCTGATGATGGTTTAAAAGTTGTTGAATTAATGCCTAATGTATTTGAAATGATAGATTTAAAAGTTGCTAATGATGATTC L
3101
Q
A
5283
C
3900
Y
3901
TAGTAAAAAAAAGTTTAGATAAAATTTTAAAATATAGTGAAAAAACAATAGGAAATAGAACTATTACTTCTATAAAATATAATGAAGTTGGTGGTTGTTA
4000
4001
W I L D G D S W L R F R M S G T E P K F K V Y Y N L Y G E N L N TGATTGAATATTAGATGGAGATTCTTGACTACGTTTTAGAATGTCTGGAACAGAACCTAAATTTAAAGTTTATTATAATTTATATGGCGAAAACTTAAAT
4100
4101
I I G N W M K K R I Q E A K T I N D Q I K T L L N L GCACTTAGTCAAGAAGCTAAAACAATAAATGATCAAATTAAAACATTATTAAATTTATAAGAGTTATTGTATGAAAAAAAGAATTATTATTGGTAATTGG
4200
4201
Q K E V K E F F K I L N A S L K N K N I C C T F G V A P V AAAACAAATAAAACTCAAAAAGAAGTTAAAGAATTTTTTAAAATTTTAAATGCTTCACTTAAAAACAAAAATATTTGTTGCACATTTGGGGTAGCTCCTG
4300
4301
H L E L A K S L A P K Q M I I A A Q D A N Y I S S G A F T G T TAGCAATTCATTTAGAATTAGCAAAATCATTAGCACCAAAACAAATGATTATTGCTGCACAAGATGCTAATTATATTTCTTCAGGAGCATTCACTGGCAC
4400
4401
Q L K D I K I K Y V I V G H S E R R M Y Y N E T D D I V N TATAAGTTGATCACAATTAAAAGATATTAAAATTAAGTATGTAATTGTTGGACATTCAGAACGAAGAATGTACTATAATGAAACAGACGATATAGTAAAT
4500
4501
Q V C A AAAAAAGTTAAAAATTTACTTGAAAATAAAATGATTCCAATATTATGTATTGGGGGAAATATAGAAGAATTTAATAACAAAAAAACATATCAAGTTTGTG
4600
4601
L K K A CAACTCAATTAAAAAAAGCTT
D
A
L
K
S
T
A
K
S
W
S
T
T
I
I
K
K
N
V
K
N
L
L
E
N
K
M
I
P
I
L
C
I
G
G
N
I
E
E
F
N
N
K
K
T
Y
Q
4621
FIG. 1-Continued.
source when this strain was transformed with the plasmid pMPDEO, whereas no growth was obtained with the same strain transformed with only the plasmid vector pBluescript SK-. Complementation of this gene was feasible as this gene does not contain any TGA triplet encoding the amino acid Trp. This is not the case for the other genes, for which the deduced polypeptides have 12 of the 14 Trp residues encoded by TGA, showing the preferential use for this codon compared with TGG. The use of TGA in these coding regions would therefore prevent the functional expression of these genes in the study of complementation in E. coli mutants. The first three genes of the sequenced DNA fragment from M. pirum are part of the deo operon described for E. coli (for a review, see reference 12), but in this bacterium the order of the genes, from 5' to 3', is deoC (deoxyriboaldolase), deoA (thymidine phosphorylase), deoB (deoxyribomutase), and deoD (purine phosphorylase). The three-dimensional structure of the thymidine phosphorylase from E. coli has been determined (29), and three residues most likely to be involved in binding thymine have been detected. These residues, Arg-168, Ser-183, and Lys-187 (numbering from the E. coli sequence), are conserved in the M. pirum sequence. In humans, this enzyme and the platelet-derived endothelial cell growth factor have been characterized as being identical (7). Purine phosphorylase and thymidine phosphorylases from mycoplasmas have been implicated in various cytogenetic effects of mycoplasmal infection of cell cultures, interfering, for example, with the
incorporation of radiolabelled thymidine or of bromodeoxyuridine (16). In addition, tests based on the detection of these enzymes have been developed for the detection of mycoplasmal contamination in cell cultures (3, 15). Although these two enzymes have been detected in several mycoplasmas, the plasmid pMPDEO did not hybridize (under high-stringency conditions) to genomic DNAs from other related mycoplasmas (M. genitalium, M. pneumoniae, and Mycoplasma penetrans [21) or from Mycoplasma fermentans when used as a probe in Southern blotting experiments (data not shown). When the same experiment was repeated under low-stringency conditions, weak bands appeared in the profiles from these four mycoplasmas; however, it is not possible without sequence information to state that these DNA bands are encoding related genes. This apparent lack of genetic homology may be partially explained by the unusually low percent G+C in the M. pirum genome (25.5%), compared with 40, 30.5, and 32.4% for M. pneumoniae, M. penetrans, and M. genitalium, respectively. For the only deoC gene, the difference in the G+C content is striking: 27.7% for M. pirum compared with 41.2% for M. pneumoniae. In addition, as two distinct EcoRI bands (10 and 11 kbp) were found to be hybridized with pMPDEO in the M. pirum profile and since there are no EcoRI sites in the sequenced DNA fragment, at least some of these genes may exist as two copies in the genome. In E. coli, triose phosphate isomerase is an enzyme used in the glycolytic pathway. In the same organism, phosphomannomutase is in-
5284
J. BACTERIOL.
NOTES
TABLE 1. Highest levels of homology found between the polypeptides deduced from the ORFs of the M. pirum 4.6-kbp DNA fragment and proteins in the SwissProt data base
ORFP
Molecular mass (kDa) of
deduced
%
%
Similarity Identity
Origin (molecular mass [kDa])
Gene (enzyme)
of protein compared
Accession
no.
in SwissProt
Reference(s)
polypeptideb
ORFiC
NA
64
52
deoD (purine phosphorylase)
E. coli (26.0)
p09743
8, 11
ORF2
24.4
53 74
32 61
deoC (deoxyriboaldolase)
E. coli (27.7) M. pneumoniae (24.8)
p00882 p09924
27 13
ORF3
46.9
53 56
34 31
deoA (thymidine phosphorylase) E. coli (47.3) H. sapiens (49.9)
p07650 p19971
28, 29 7, 10
p19079
23
p24175 p26341
None 24
p04790 p00943
18 1
ORF4
15.0
55
38
cdd (cytidine deaminase)
Bacillus subtilis (14.8)
ORF5
61.4
50 48
21 22
cpsG (phosphomannomutase)
E. coli (50.4) Salmonella typhimurium
57
39
tpi (triose phosphate isomerase)
ORF6d
NA
(49.9)
E. coli (26.9) B. stearothermophilus (26.8)
a The putative polypeptides were deduced from the different ORFs, and homologous sequences were searched in SwissProt. NA, not applicable (partial sequences). ' Partial sequence from M. pirum; deoD, 42 amino acids of the COOH end (239 for E. coli). d Partial sequence from M. pirum; tpi, 149 amino acids of the NH2 end (255 for E. coli). b
volved in a side reaction to the glycolytic pathway used for the introduction of mannose in polysaccharide synthesis. Although the homology between the deduced polypeptide from ORF5 and phosphomannomutase is relatively high (Table 1), we cannot exclude the possibility that ORF5 is encoding another phosphomutase, the deoxyribomutase. As described above, deoxyribomutase in E. coli is the product of deoB, a gene from the deo operon. However, the sequence for this gene has not yet been published and therefore comparison with ORF5 was not possible. In addition, there is a significant difference of molecular weights between the putative product of ORF5 and phosphomannomutase
7.
8.
9.
(Table 1). The close spacing between these genes (10 to 21 nucleotides) suggests their organization in a putative operon and also suggests the use of the salvage pathway for nucleosides as common energy and carbon sources in this microorgan-
10.
ism. Nucleotide sequence accession number. The nucleotide sequence reported here will appear in the EMBL/GenBank/ DDBJ Nucleotide Sequence Data Libraries under the accession number L13289.
11.
We thank Lloyd Finch and Kendall King for carefully reading the
12.
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