Sequence of the IktD gene from Actinobacillus actinomycetemcomitans


Members of the RTX family of protein toxins are secreted without the concomitant cleavage of a signal peptidel 2. In Pasteurella haemolytica and E. coli., the structural gene for the RTX toxin is upstream of two genes, lktB/hlyB and lktD/hlyD, required for toxin secretionl 3. In the preceding paper, we reported the sequence of the lktB gene from the leukotoxin operon of Actinobacillus actinomycetemcomitans; it shared a high degree of sequence similarity with the equivalent proteins from P. haemolytica and E. coli. This was unexpected since the RTX toxins from these bacteria are secreted while the leukotoxin from A. actinomycetemcomitans is reportedly retained in the periplasm4. Thus, to determine whether a sequence analogous to lktD/hlyD was present in A. actinomycetemcomitans, a 6 kb Barn HI fragment containing 2.5 kb of DNA 3' to lktB was cloned from strain JP-2. The region immediately downstream of the lktB gene was sequenced and an open reading frame was identified. The deduced protein, LktD, demonstrates significant homology to the equivalent polypeptides from related bacteria: it shares 61% identity with the LktD protein from P. haemolytical and 68% identity with the HlyD protein from E. coli3. Identities are found in all three proteins at 52 % of the residues. Moreover, the lktD gene is expressed in A. actinomycetemcomitans as shown by Northern blot analysis using an IktD-specific probe (Spitznagel, Kraig, and Kolodrubetz, in preparation). The fact that the LktB and LktD proteins of A. actinomycetemcomitans are so similar to the leukotoxin/hemolysin secretion proteins from other organisms suggests that further investigations into the possible secretion of LktA from A. actinomycetemcomitans are necessary. 
E K D E N E F L P A N L E L I E T P V S N A P R F V S Y S I M L F L T L A I 217 ATTGTTTCTATCTTTAGCAATGTGGAATTATCGCACGGCTTCAGGAAATTTGCACTAAGCGGGAGAAGCAAGAMTTAACCGATTGAATTCATTGGTTAAACATATT I V S I F S N V E I I A T A S G K F A L S G R S K E I K P I E N S L V K H I 331 TTTGTGAAAGAGGGCGAATACGTGAAAGGGTGAACTATTGTTAAAACTGACCGCACTTGGTGCCGAAGCGGATACGTTAAAACGAGACATCGCTTTCTCAAGCTAACTG F V K E G E Y V K K G E L L L K L T A L G A E A D T L K T K T S L S Q A K L 445 GAGGATTTCGTTATAATCTTTATTGGAGCTGTTGAAGATCAATTACCAATATTAGATTTTTCTAAATTGATTTACCTTTTATGACGGAACGATCAAAAAAGAGTG E E F R Y K S L L E A V E K D Q L P I L D F S K I D L P F M T E N D Q K R V 559 ACTTTACTGATTGAAGAACATTCTCTACTTGGCAAACAACGCCATCAGAACCTTGAATCTTAATAAAAAGAAGCGGAAAACTCAGTTATTTGGCACGAATAAA T L L I E E O F S T W Q K Q R H Q K T L N L N K K E A E K L S Y L A R I K K 673 TATGAAGGTCTGATTAATACAGAGCAAGTCCGATTGGATGATTTTAGAGCCTTATATAAGGAACATGCTATTGCAACATACAGTTTTAGATGAAGAGAATAAATATCAGGAT Y E G L I N T E Q V R L D D F R A L Y K E H A I A K H T V L D E E N K Y Q D 787 GCAATCAATGAGCTTGAGGTATATAGGCGAGTTTAATGCAAGTTGAAACGAAGTTTTATTGGCCAAAGAGAGCAGGATTAGTCACACAATTGTTCAAAAATGATATTTTA A I N E L E V Y K A S L M Q V E N E V L L A K E E Q E L V T Q L F K N D I L 901 GACAAGCTAAAACAGGCGACGGATAATGTGAATTTATTGACATTCGAACTGGACAAACAATCAACGCCAGCAAGTCTCTGAATTCGAGCTCCTGTATCAGGTACTGTACAA D K L K Q A T D N V N L L T F E L D K N N Q R Q Q V S E I R A P V S G T V Q 1015 CAATTAAAGTTCACACAATAGATGGCGTTGTTACTACGGCTGAACATTAATGGTAGTTGTGCCGGAAGAAGATTCCCTTGAAGTCACTGCACTTATTCAAATAAGATATT Q L K V H T I D G V V T T A E T L M V V V P E E D S L E V T A L I Q N K D I 1129 GGTTTTGTGAAAGAAGGGCAAGAGGTTGTAATTAAGGTCGAAGCCTTCCCCTATACCCGTTATGGTTATTTMCGGGAMAGTGATATTACTTTAGATGCGATTGMCAT G F V K E G Q E V V I K V E



  103 EK. II1 FIPAN.LL.E. L I .... I AA TP.,NPFVSY ML. I A . I . I A . FLII ATA I 




  A F P Y T R Y G Y L T G K V K N I T L D A I E N 1243 CCAAAGCTTGGACTTGTATTTA TACGDATTTAAAAACACTTTCTACC TCCCACTTTCCGCAGGATGG TTACTGCAATG P K L G L V F N T I I E L D K K T L S T E E K E I P L S A G M E I T A E I K 


ACKNOWLEDGEMENTS

We wish to thank Terry Dailey for valuable technical assistance. This work was supported by Public Health Service grants DE08521 and DE00152. J.M.G. is a Dentist-Scientist Awardee.
Janet M Guthmiller, Ellen Kraig, Marianna P Cagle, David Kolodrubetzl
Sequence of the IktD gene from Actinobacillus actinomycetemcomitans