Assembling of RtTA1 chromosomal restriction fragments into a map
Chromosomal map backbone was constructed using three I-CeuI fragments - C1, C5
and C7 (Table 1, Fig. 2A). In the next step 16S rRNA (rrs) ribosomal probe was used against
RtTA1 genomic DNA digested with MssI, SmiI and PacI to anchor subsequent restriction
fragments. It hybridized to M2 and M9; S4, S7 and S8; P1 and P2 fragments, respectively
(Table 3, Fig. 2A). The 2D electrophoresis of MssI/I-CeuI digestion products (Table 2)
indicated that M2 is cleaved twice with I-CeuI giving three subfragments, C5, CM1 and CM3,
whereas M9 is cut only once with this restriction enzyme, resulting in CM2 and CM4 (Table
2). These results and the hybridization of rrs probe to the M2 and M9 fragments allowed us to
match M9 to the I-CeuI chromosomal map skeleton and, furthermore, to join P1 and P2
fragments (Fig. 2A).
2D PFGE experiments following M2 digestion with SmiI demonstrated that the
fragment contained three SmiI restriction sites (Table 2, Fig. 2A), which gave S7, SM4 and
S15 subfragments. Their total size was 1015 kb, in contrast to M2, which is 1026 kb. The
lacking SM14 subfragment (11 kb) was found after the conventional agarose electrophoresis
(data not shown). Furthermore, taking into consideration the results of rrs probing against
SmiI digestion of RtTA1 genomic DNA (Table 3), we were able to conclude that SM4 was a
part of larger S4 fragment. These data unequivocally linked the SmiI fragments in the order
S4-S15-S7 (Fig. 2A). In order to orientate the hitherto created map with respect to the putative
origin of replication, in the next step we used dnaK gene as a molecular probe, which,
similarly to rrs, might be located near the chromosomal replication origin (Marczyński and
Shapiro 1992, Ward-Rainey et al. 1996). The dnaK probe hybridized to the M9, S8 and P1, as
well as C1 fragments (Table 3), indicating the overlapping of S8 and M9, and consequently
permitting a linkage of S8 with S7 (Fig. 2A). Moreover, it defined the only possible (as
presented in Fig. 2A) orientation of P1 and P2 fragments. As rrs and dnaK probes both gave
hybridization signal with M9, we decided to orient the map with respect to this restriction
fragment because it is likely to contain the putative origin of replication. Subsequent
macrorestriction fragments were joined to this map in a similar manner. M5 was linked to M2
by employing Pss-I and Pss-II regions as molecular probes, which hybridized to the S4
fragment. 2D results of M5 digestion with SmiI showed that it contained only a single site for
this restriction enzyme, resulting in SM2 and SM7 subfragments (Table 2, Fig. 2A). The size
of SM2 (573 kb) suggested that it is a derivative of a larger SmiI fragment. This was further
confirmed with bioA and lpcAB molecular probes, which hybridized to SM2 as well as S2
1
(Table 3), locating this fragment next to S4 (Fig. 2A). Consequently, the hybridization of
orf11 probe to M10 and S2 revealed the location of M10 next to M5 (Fig. 2A). Completion of
the MssI circular map was done with the data from several Southern hybridizations with
defined molecular probes. The significant sequence similarity between the R. leguminosarum
biovars viciae and trifolii allowed us to design a series of PCR primer pairs that were further
used for amplification of DNA fragments containing potential RtTA1 MssI (PmeI) sites.
Using the six PCR primer pairs, we were able to obtain five amplification products, namely,
Pme1, Pme2, Pme4, Pme5 and Pme6; however, Pme1 and Pme5 lacked the MssI site (Table
3). Using them as molecular linking probes, we were able to pair the following MssI
restriction fragments: M11-M1 were coupled by Pme2 probe, Pme4 joined M10-M5
(confirming a previous hybridization result with orf11 probe), whereas M5-M2 were linked
by Pme6, also verifying their established order (Fig. 2A, Table 3). Despite the observation
that the amplified Pme1 fragment lacked MssI site, it appeared to be useful as probe. It
hybridized to M11, S3 and P1 fragments (Table 3). These data, Pme2 hybridization to S9 and
P3 fragments and 2D electrophoresis that revealed the presence of PacI and SmiI sites in M11
(Table 2) allowed us to put together S3-S9 and P1-P3, respectively (Fig. 2A).
The results of 2D PFGE of M1 SmiI or PacI digestion products were combined
afterwards to organize a physical map of the chromosome. SmiI cleaved M1 in four - SM5,
S6, S12 and SM16 (the last subfragment was barely visible even after the conventional
electrophoresis), whereas PacI in three - MP1, P8 and MP8, subfragments, respectively
(Table 2). The hybridization of Pme2 probe to SM5 and S9 revealed SM5 to be a part of S9,
further indicating that MP1 is a part of P3 fragment (Fig. 2A).
The presence of PacI site within S6 fragment and the results of acpXL probing showed
that fragment S12 is located next to S9. The hybridization of acpXL to S12 and P3 (Table 3)
unequivocally linked the fragments within M1 in the order S9-S12-S6 (Fig. 2A). Since it was
shown that M11 overlapped S9 and S3, we were able to extend the previously created
restriction fragment contig to S3-S9-S12-S6-SM16. We noticed that the size of S2 fragment
was almost identical with the sum of M10 and SM2. The absence of SmiI restriction site
within M10 suggested that it must be located marginally in the neighboring MssI fragment.
We deduced that SM16 residing inside M1 could indeed be the lacking part of S2 and on the
basis of this assumption we connected the S8-S7-S15-S4-S2 contig with S3-S9-S12-S6, and
furthermore, M9-M2-M5-M10 with M11-M1 (Fig. 2A). Assembling the remaining M3 and
M12 fragments was done by means of their SmiI digestion product 2D electrophoresis that
resulted in SM9 and SM11 for M12, and SM3, SM6, S14 subfragments for M3, respectively
2
(Table 2). The sizes of SM3 and SM6 showed that these two fragments must be a parts of
larger fragments S3 and S11, respectively, also indicating that M3 is located next to M11,
concomitantly orientating the M12 fragment with respect to the asymmetric SmiI site between
M9 and M3 (Fig. 2A). Finally, the last S14 fragment was placed between S11 and S3 closing
the circular map.
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