Detailed sequence analysis of Tn55P7 Compared to that of TnPf

The following analysis o f the element will begin at bp 1, which has been designated the left end. Tn55P7 begins with a GA dinucleotide, the importance o f which will be discussed in chapter 4. Most o f the details o f the putative orfs are listed in Table 3.2 above and Table 3.3 below which shows the nearest homologue to the Tn55P7 putative orfs. The description below will

concentrate mainly on the intergenic regions and the important differences between Trv5397 and TnP7d.

Table 3.3. Nearest Homologue to Tn5397 orfs

Orf H om ologous orfs and/or proteins® Reference

orf23 an o(/23-like protein from B. subtilis Kasahara et al., 1997

orf22 an orp3-\]kQ protein from B. subtilis Kasahara et a l , 1997

o r f l l related to putative cell division proteins from Treponem a pallidum Tomb et a l , 1997; Aim et

and H elicobacter pylori. a l , 1999

orflO no hom ologues N one

o r f 19 no hom ologues None

o r f 18 hom ology to anti-restriction proteins from conjugative plasmids, Chilley and Wilkins, 1995;

Yersinia p e s t is and E. coli. Hu et a l , 1998; Belogurov

et a l , 1992

o r f 17 no hom ologues N one

o r f 16 no hom ologues N one

o r f 15 no hom ologues N one

o r f 14 Invasion associated Protein from Listeria m onocytogenes Kohler et a l , 1990

Intronic o r f Intron encoded protein from B. m egaterium Huang et a l , 1999

o r f 13 no hom ologues N one

o rfl5 no hom ologues N one

o r flô no hom ologues N one

tet(M) tet(M ) gene o f T n /5^ 5 Martin et a l , 1986

orfô no hom ologues N one

orf9 weak hom ology to repressor proteins C elli and Trieu-Cuot, 1998

o r f 10 no hom ologues N one

o r p RNA polymerase sigma factors Flannagan et al, 1994

o r P no hom ologues N one

tn dX tn pX from C. perfringens transposons Tn4451 Bannam et a l , 1995

The first 180 bp o ïT n 5 3 9 7 are unrelated to Tn916 (shown in Chapter 4) and replace a region of 201 bp in Tn916, that includes the 5' end (left end) and the first seven nucleotides o f orf24, the first o r f o f T n 9 16. Following this region, the two transposons are almost identical, however, a number o f insertions and deletions are present in the Tn5397 sequence with respect to Tn9I6. In

Tn9I6, there is an intergenic region o f 21 bp (31 4- 335) between orf24 and the next o r f orf23.

This region is almost identical in both conjugative transposons.

At the 3' end o f orf22 in Tn5397, 32 bp o f non-homologous sequence have replaced 25 bp present in Tn916. Interestingly this region o f non-homologous DNA contains a centrally located region (GAT A A AC AG), which has been inverted in one o f the elements, (Figure 3.4). The orf is also one amino acid longer in Jn5397.

T n 5 3 9 7 : CAC7\ACCTCAACCAAAGAAAGAACCTGTTATAGATAAACAGTA G CACTTCCGTAGA G A G A A A G G A G G I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I T n 9 1 6 : CTCAACCAC A A G C A A A G A A A G A A C C G A C A C A A G A C A A A T A G --- T C G CTAGGTAGAAAGGAGG F i g u r e 3.4. N u c l e o t i d e s e q u e n c e c o m p a r i s o n o f th e 3 ' e n d o f o r f 2 2 f r o m T n 5 3 9 7 ( i n d i c a t e d b y an a r r o w ) a n d o r / 2 2 f r o m T n 9 I 6 . T h e s t o p c o d o n s are u n d e r li n e d th e i n v e r t e d r e g io n ( G A T A A A C A G ) is s h o w n in b o l d , o n e c o p y o f w h i c h is p r e s e n t in e a c h t r a n s p o s o n .

Following or/27 in Tn5597 is a large intergenic region that extends from 2454-2846 (392 bp). This region contains a sequence homologous to the o r/T o f TnP7d (Jaworski et a l , 1995). The

actual nie site from ln916, F and Tn5397 is identical over an eight-nucleotide region TGGTGTGG (2464-2471 inT nJjP T ).

The region between the end o f orflO and the end o f o r f 18 is highly conserved in both elements. An intergenic region o f 87 bases follows o r f 18 in Tn5397. The start site listed for o rfI7 in

Tn916 does not have a cognate RBS, also if the same site is used in Tn5397 the peptide is

truncated at a site 75 bp downstream. There is also a single base pair deletion in Tn5397, which would change the frame o f the orf. There is, however, another ATG start codon present 17 bp downstream from this 1 bp deletion. This reading frame is in-frame with the original reading frame listed for Tn916. There is a RBS in both lw5397 and Tn916 and it is suspected that this site is used in both conjugative transposons (Figure 3.5).

Tn535 7: C A T T T C T A C C A A T C A T G G G A T TTTTGAAATC ACTCATTAAGTCTGTCGGTACATTACTACTG I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Tn 915: T A T C T C A A C C A A T C A T G G G A T TTTTGAAATC GTCTATTAAATCTGTCGGTACATTACTACTG Tn539 7: GCAGAGTTTCTTGTTTGCGGGGTAGCTTAAA CAGCTATCCCTATTTTT- I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I T n 9 I 5: GCAGATTTTCTATTTTACGGGGTGGCTCAAT CAGCTACCCCTATTTTTT Tn539 7: A T GA A A G G A TTGATTCTATGAAGAAA I I I I I I I I I I I I I I I I I I I I I I I I

T n 9 1 6 : ATGAAAGGAT TGAT TACATGAAGAAA

F ig u r e 3.5 C o m p a r i s o n o f th e i n te r g e n ic r e g io n b e t w e e n o r f l S a n d o r f l l a n d t h e i r h o m o l o g u e s in J n 9 1 6 . T h e first

A T G ( b o l d ) r e p r e s e n t s th e p u t a t i v e sta rt c o d o n t h a t h a s n o c o g n a t e R B S , th is is l o c a t e d w i t h in o r f l S . T h e sto p

c o d o n ( T A A ) fo r o r f l 8 in b o t h t r a n s p o s o n s is a ls o s h o w n in bo ld . T h e r e is a sto p c o d o n ( T A A ) l o c a t e d in the

T n 5 i P 7 s e q u e n c e w h i c h w o u l d t r u n c a te a n y p e p t i d e f o r m e d f r o m th e first sta rt c o d o n , th is is s h o w n in b o l d on th e

s e c o n d line. T h e s in g le b p d e le t io n is a ls o s h o w n on th e s e c o n d line. U n d e r l i n e d is th e R B S fo r th e s e c o n d p u ta tiv e

The regions o f the transposons that contain o r f 16 and o r f 15 are highly homologous and show no major rearrangements. The next orf in 1x15397, o r f 14 contains the group II intron and is

considered in detail in Chapter 6.

In l n 5 3 9 7 there are three possible start codons for o r f 13, all with a suitable RBS and all in the

same frame. These are located at 13433, 13452 and 13455. The first o f these incorporates the TG o f the o r f 14 stop codon (TGA). This area o f both elements is shown in Figure 3.6

T n 5 3 9 7 : G C A G G A C G A A T C A A A C A A T GAGAAAGGAAGA TTCAArGATGAAATTC

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

T n 9 1 6 : G C A G G A C G A A T C A A A C A A TGAG A A A G G A A G A T T T A A T G A T G A A A T T T

F i g u r e 3.6 . C o m p a r i s o n o f th e e n d o f o r f l 4 a n d th e sta rt o f o r f l S in T n 5 3 9 7 a n d th e h o m o l o g o u s r e g io n in T n 9 I 6 .

T h e l etters in b o l d r e p r e s e n t th e p o s s ib l e R B S s fo r th e o r f l S s tart c o d o n s . T h e u n d e r l i n e d r e g i o n r e p r e s e n t s th e sto p

c o d o n o f o r f 14 in b o t h e le m e n t s . O v e r l a p p i n g this r e g i o n in ita lic s is th e first p o t e n t i a l s ta rt c o d o n fo r o r f l S ,

f o l l o w i n g th is is t h e R B S ( b o l d ) fo r th e s e c o n d a n d t h ir d p o s s ib l e s ta rt c o d o n o f o r f l 3 (ita lic s).

In 1\\5397 orfl3 is followed by a region that has undergone major deletions and re-arrangements

when compared to the analogous region in lxx916. This sequence has been studied in detail and is thought to play a crucial role in detecting tetracycline in the medium, these results are

presented in Chapter 5. This region is followed by the gene. A phylogenetic tree showing the relationship o f the Tn53P7 Tet(M) polypeptide to other tetracycline resistance proteins is shown in Figure 3.7.

Tet(M ) M 74049 Streptom yces lividans

OtrA X 53401 Streptom yces rim osus

Tet(Q) L33696 P rev o te lla rum inicola

TetA(Q)3 Y 08615 B acteroides fra g ilis

Tet(Q) X 58717 B actero id es thetaiotaom icron

Tet(Q) U 73497 P rev o te lla interm idia

TetA(Q)2 Z21523 B acteroides fra g ilis

Tet(T) L 52544 S treptococcu s p yo g en es

Tet(O) Y 07780 S treptococcu s pneum oniae

Tet(O) M 20925 S treptococcu s mutans

Tet(O) P23835 C am pylobacter coli

Tet(O) M l 8896 C am pylobacter je ju n i

Tet(S) L 09756 L isteria m onocytogenes

Tet(S) X 92946 L actococcu s lactis

Tet(M ) M 85225 J n 9 1 6 Enterococcus fa ec a lis

Tet(M ) U 58985 G ardn erella vaginalis

Tet(M ) U 08812 U reaplasm a urealyticum

Tet(M ) X 75073 N eiserria m eningitidis

Tet(M ) X 92947 TnFO 1 E n terococcus fa ec a lis

— TetA(M ) M 21136 S taphylococcu s aureus

— Tet(M ) Y 90939 Tr\5251 S treptococcu s pneum oniae

— Tet(M ) X 04388 T n l5 4 5 Enterococcus fa e c a lis

— Tet(M) AF333235 Tn5597 Clostridium difficile

Tet(W ) A J2227699 B u tyrivibrio fibrisolven s

— TetB(P) L 20800 C lostridium perfringens

Figure 3.7 Average Distance Tree aligning all o f the tetracycline resistance proteins that are hom ologous to Tet(M) from Tn55P7 (as determined by a BLAST search). The labels give the name o f the protein, follow ed by the

accession number, the m obile element (where appropriate) and the host organism. The Tet(M ) protein from Tn55P7 (bold) is more closely related to the proteins from T n l5 4 5 and Tn5257 than it is to TnP7<5.

This tree shows that Tet(M) from Tn5397 is more related to the Tet(M) proteins from T n l5 4 5

(Martin et al., 1986) and Tn5251 (Ayoubi et a l , 1991) than to Tn916, both of which belong to

the T n 9 I6 family o f conjugative transposons. Each node in the tree (where two individual

branches meet) represents a hypothetical common ancestral gene (Page and Holmes, 1998). The number o f ancestral genes separating Tn916 and T n JJP ? are greater than the number separating

Tn5397 and Tn7J^5. The next orf, or/6 starts, in Tn9I6, with an ATT start codon. The only

published ATT start codon (AUU in the RNA strand) is for the E. coli InfC gene (Butler et al,

1987). In both Tn5397 and Tn9J6 there is an in-frame TTG codon which would allow an N- terminally truncated polypeptide to form. The RBS for the ATT start codons is more close to the consensus, GGAGGA than that for either o f the two TTG codons. However the requirement for the RBS can be as little as three bases which are complementary to nine bases at the 3' end o f the 16S rRNA strand, (ACCUCCUUA), (Kozak, 1999). This means that theoretically all of the four start codons in Figure 3.7 could be used to initiate translation. This region is shown in Figure 3.8

T n 9 1 6 : A A GGAG G A GATAGTTATTATTIAACTACAAGTGG A TATTGTGTCCTGTATGTGGAAAT