El virreinato de las Indias Occidentales 1493-

The sequencing analysis revealed an 84 bp deletion upstream o f tet{M) in Tn5397 compared to

Tn916 (Figure 5.6). Despite these differences it appears that Tn55P7 can still detect tetracycline

in the medium and respond by increasing transcription o f the tet(M) gene. For this reason the area upstream o f was analysed in detail. There is an 84 bp deletion in T n5iP7 which deletes the 5' region o f o r fl2 and results in the formation o f another o r f This rearrangement generates orf26 as shown in Figure 5.7. orf25 is present in both Tn916 and Tn55P7. The orf26

start codon and RJBS is present in TnP7d but the peptide would only extend 6 amino acids, as an in-frame stop codon is present.

There are also three inverted repeats in this area, in the RNA strand they are predicted to form stable secondary structures (Figure 5.7). One of the stem-loop structures (3L:4L) forms a typical terminator structure. This is believed to play an important role in the regulation o f Tn55P7.

T n 5 3 9 7 Tn916 T n 5 J 9 7 T n 9 1 6 T n 5 J 9 7 T n 9 1 6 T n 5 3 9 7 T n 9 1 6 T n 5 J 9 7 Tn9]6 T n 5 3 9 7 Tn916 T n 5 3 9 7 T n 9 1 6 T n 5 3 9 7 T A T A G A A T A A C A A A T A T TG G T A C A T T A T TA C A G C T A T TT T G T A A T C A C T A T A A A ATAACTTTTAGTACATGGTTAT AGCTATTTTGTAACCAC IR GTACTCTCTTTGATAAAAAATTGGAGATTCC TTTACAAATATGCTCTTAC 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 GTTCTC-CTTTGACAAAAAATCGGATATATC TTTACAAATATACTCTTAT -35 G TGC T A T TATTTAAGTATCTATTTAAAAGGAG T T A A T A A A T A T GCGGCAA 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 G T G C T A A T G TTAATATAAGTATATAAAAGGAGTTAATAAA TATGCGGCAA - 1 0 GGTATTATTAAATAAACTGTCAATTTGATAG CGGGAACAAATAATTGGAT 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 GGTATTCTTAAATAAACTGTCAATTTGATAG TGGGAACAAATAATTAGAT RBS0J-/25 G T C C T T T T TTAGGAGGGCTTAGTTTTTTGTAC C C A G T TTAAGAATACCTT 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 DR GTCCCTTTTTAGGA G G G G T T A G T T T T T T G T A --- R B Soj.^26 T A T C A T G T G A T T C T A A A G T A T C C G G A G A A T A T C T G T A T G C T T T G T A T G C 84 bp deletion in Tn5397 C T A T G G T T ATGCATAAAAATCCCA(ÿGATAAGAGTATTTATCACTGGGA --- CCCAGTGATAAGAGTATCTGTCACTGGGA DR -TTTTTATGCCTTTTGGGCTTTTGAATGGAG GAAAATCACATGAAAATTA 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

TTTTTTATGCCTTTTTGGCTTTTGAATGGAG GAAAATCACA T G A AAATTA RBS let (M) F i g u r e 5.6. C o m p a r i s o n o f th e o r fJ 2 r e g io n o f T n 9 / 6 a n d th e c o r r e s p o n d i n g r e g io n in T n 5 3 9 7 . T h e first line s h o w s th e s t o p c o d o n ( T A A ) ( u n d e r l i n e d ) f o r o r f l 3 in b o th c o n ju g a t iv e t r a n s p o s o n s . A l s o s h o w n is an i m p e r f e c t i n v e r t e d r e p e a t s e q u e n c e ( i n d i c a t e d w i t h a r r o w s a n d l a b e l e d IR ), o n e c o p y o f w h i c h is p r e s e n t in T n P / d a n d o n e c o p y in T n 5 5 9 7 . T h e s e c o n d line s h o w s th e p o s i t i o n o f th e - 3 5 r e g io n o f a p r o m o t e r in T n P / d ( J a w o r s k i a n d C l e w e l l , 1 9 9 5 ), th e t h i r d line s h o w s th e p o s i t io n o f th e - 1 0 r e g io n . T n 5 3 9 7 h a s a slig h t m i s m a t c h in th e - 1 0 r e g io n b u t t h e - 3 5 r e g io n is i d en tica l.

T h e n e x t line s h o w s th e R B S ( i ta lic s) a n d sta rt c o d o n ( b o l d ) fo r o r /2 5 in T n 5 i 9 7 . T h e fifth line s h o w s th e s t o p c o d o n ( u n d e r l i n e d ) fo r o r /2 5 a n d th e R B S (ita lic s) a n d start c o d o n ( T T G ) ( b o l d ) fo r o r/2 6 . T h is is f o l l o w e d b y a n 84 b p

d e l e t i o n in T n 5 3 9 7 c o m p a r e d to T n 9 1 6 ( s h o w n in b o l d in T n 9 / 6 ) . I n t e r e s t in g l y a 6 b p d i r e c t r e p e a t is p r e s e n t

( u n d e r l i n e d w i t h an a r r o w a n d l a b e l le d D R ) , o n e c o p y is p r e s e n t in T n 9 / d (this h a s b e e n d e le t e d in T n 5 J 9 7 a n d o n e c o p y i m m e d i a t e l y f la n k s th e d e le t io n ( p r e s e n t in T n 9 I 6 a n d T n 5 3 9 7 ) . T h e last line s h o w s th e sto p c o d o n ( u n d e r l i n e d )

A . orf25 orf26 5:6 UU u

^iL

GG GAACAAAUAAUUAGAUGUCC CU UUUUAGGAGGCmUC fGGAUUUUUUAUGCUU UUGAAUGGAGGAAAAU CA CA UGA GC 3L: 4L GU A A B . / A C \ G U A C AU UG GC UA 3 S : 4 S GC 5 : 6 AU UUU CG U U CG CG CG CG

GG G A A C A A A U AA UU A GAU GUCCCU UUUUAG GAG GGGUUAGUUUUUUGUAAUUUUUUAUGCU UUUGAAUGGA GGAAAAUCACA U G A

Figure 5.7. A variety o f stem loop structures can be formed from RNA derived from the region between o r f l3 and rer(M). The bases in bold show the position o f

the start codons for orf25 (A U G ) and orf26 (U U G ) and for tet{M ) (AU G ). R BSs are shown in italics and correspond to the nearest start codon, stop codons are

underlined. The arrows represent the orfs with which they are labelled. The direction o f the arrows indicates the probable direction o f transcription. Structures 1:2 and 3L;4L can not exist at the same time as they incorporate the same bases into their structure.

5.4 Discussion

In Tn916 circularisation is required to obtain high levels o f transcription o f the transfer

genes from promoters in the left end o f the element. Our results indicate that this is also the case with Tn5397 as transcription has been detected from to the end o f Tn5397

and into the left end when the element is in the circular form (the probable transposition intermediate). When the cells were grown in tetracycline containing medium a marked increase in the amount o f transcript was detected from the end o f tet(M) to orf9. Similar results were obtained in T nP id (Celli and Triou-Cuot 1998). Also, transcription can be detected in samples from both tetracycline positive and negative cultures, this indicates that there is also a basal level o f transcription throughout this region. Again, similar results to those observed for Tn916 (Su et al, 1992). That up-regulation occurs is not surprising as it would be metabolically expensive and wasteful to continue to produce the Tet(M) protein and downstream gene products in large quantities when they were not needed.

There are two potential orfs upstream o f tetfWf) in Tn5397. These may play a crucial role in sensing tetracycline. Based on the RT-PCR results and on the analysis o f the sequence data the following models for the up regulation o f transcription through tet(M) and downstream genes is proposed. The first model will assume that the stem-loop 1:2 does form (Figure 5.7). In the absence o f tetracycline, transcription continues along the DNA from the tet{M) promoter, orf25 will be translated first as RBSory25 will be freely available

to the ribosome. Also the stem loop structure 1:2 and 3S:4S will form (Figure 5.7). Stem loop structure 1:2 will occlude RBSor/26 and the TTG start codon. Therefore orf26 will not

be translated. As the ribosome continues along orf25, it will disrupt stem loops 1:2 and 3S:4S. This ribosome will also continue to occlude the RBSqæô The stem loop 3L:4L can then form. As can be seen stem loop 3L:4L is a typical transcriptional terminator and under normal conditions the transcription will terminate (Figure 5.8). In the presence of tetracycline, initially most ribosomes in the cell will not be functional. Only those ribosomes that are protected by the basal level o f Tet(M) will be able to initiate transcription at RBSorf25- Therefore it is likely that there will be a delay in ribosome

binding at RBSorf25 enabling the stem-loop structures 1:2 and 3S:4S to remain intact. This

will prevent the formation o f the terminator (3L:4L) and allow transcription o f the /e/(M) and downstream genes. The second model explaining the up-regulation o f transcription o f

tet(}A) assumes that the stem-loop structure 1:2 does not form. When tetracycline is

absent the terminator can form freely which would result in a low level o f te t( ^ )

expression. In this situation the continual presence o f ribosomes on the RNA strand would occlude RBSorfzô- However, when tetracycline is present, again the majority o f the ribosomes will be inactive, this delay in attachment to RBSorf25 would allow other,

Tet(M) protected ribosomes to bind to RBSorf26- This RBS more closely matches the

consensus than the RBSorfzs- Ribosomes translating this region would disrupt the

terminator, leading to higher levels o f expression o f tet(M) and downstream genes. These hypotheses allow for the formation or non-formation o f stem-loop 1:2 and are not

mutually exclusive. To strengthen these hypotheses Northern blots need to be carried out to determine the length o f the transcripts that cover Tn5397, this would show us if the transcription o f the transfer genes is dependent upon the circularisation o f the element.

The above hypothesis differs from the situation thought to occur in TnP7d in that regulation in T n5iP7 is on two distinct levels, the transcriptional terminators (mediated by stem-loops 3L:4L and 5:6) and by RBS occlusion (mediated by stem-loop 1:2 and by the ribosome). Recently, IS200 has been shown to possess both transcriptional and translational control mechanisms for the transcription o f its transposase gene. These mechanisms involve both transcriptional terminations at a rho independent terminator and RBS occlusion by the formation o f a second stem-loop structure (Beuzon et a l, 1999). Interestingly, one o f the closest related IS element to \S200 is the C. perfringens IS1470

(Brynestad et a l, 1997) which also has the potential to form a stem loop structure, which would occlude the RBS o f the transposase gene.

5.5 Conclusions

Tetracycline upregulates transcription in Tn5397. The presence o f tetracycline in the medium is thought to be detected upstream o f tet(M), by a mechanism that involves transcriptional termination and translational attenuation via RBS occlusion.

Chapter 6