3.1Characterisation of the L. monocytogenes scRNA gene
A putative L. monocytogenes scRNA λgt11 clone was identified and isolated after three rounds of selective hybridisation screenings with oligonucleotide C. Small-scale λ preparation and restriction analysis revealed a recombinant insert of 2.2 kb. Direct sequencing with oligonucleotide C generated 200 bp of nucleotide sequence data, while direct sequencing with oligonucleotide D generated nucleotide sequence data of 175 bp (data not shown). 5′-Primer extension analysis identified the initial 5′-nucleotide of the mature putative scRNA, with a major single cDNA product observed from RNA isolated from two points on the L. monocytogenes growth curve (Fig. 1). This partial sequence data was aligned using CLUSTAL W with scRNAs previously characterised from 13 Bacillus, Brevibacillus and Paenibacillus species. Comparative sequence analysis of this alignment revealed that the putative L. monocytogenes scRNA was probably 276 bases in length, with an overall sequence identity with the other scRNAs sequences that ranged between 54 and 65%. This increased to 86% over the region homologous to 4.5S RNAs (i.e. bases 110–230).
Figure 1. Determination of the 5′-end of the L. monocytogenes scRNA by primer extension. Total RNA was prepared from mid-exponential phase, 4.5 h after inoculation (lane 1), and pre-stationary phase, 7 h after inoculation (lane 2) and subjected to primer extension analysis. The length of the extended cDNA fragments were estimated by comparison with sequencing ladders from an M13 control transcript. The 177 base cDNA extension products are arrowed.
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The secondary structure of the putative L. monocytogenes scRNA demonstrates the presence of helices 1–4. Compared with the scRNA of B. subtilis, helices 1, 2, 4 have fewer base pairing and the secondary interaction between 3 and 4 is weaker (Fig. 2).
Figure 2. Proposed secondary structure of the L. monocytogenes scRNA as deduced from comparative CLUSTAL W alignments. Uppercase bases are those conserved with B. subtilis scRNA, lower case bases are those unique to the L. monocytogenes scRNA. Helices are labelled with Arabic numerals (1, 2, 3, 4, 5 and 8), and dashed lines infer possible tertiary interactions between helices 3 and 4.
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Confirmation that recombinant gene isolated encoded for the scRNA of L. monocytogenes was carried out by subjecting the putative scRNA gene sequence to the genetic selection procedure. Plasmids pTUBE809 a negative control, pTUBE822 a positive control, a derivative of pTUBE809 containing the scRNA gene of B. subtilis and pTUBE922 containing the putative L. monocytogenes scRNA gene were transformed into E. coli S1610 and subjected to genetic selection. No survivors were detected upon temperature pulse curing at 42°C of E. coli S1610 transformed with pTUBE809, while approximately 106 CFU ml−1 heat-resistant survivors were obtained when E. coli S1610 was transformed, and subsequently pulsed cured at 42°C, with either pTUBE822 or pTUBE922.
3.2Phylogenetic analysis of the L. monocytogenes scRNA
The heterogeneous phylogenetic nature of the genus Bacillus has been well documented . Analysis of protein coding sequences indicates that even the closely related species B. amyloliquefaciens and B. subtilis have 22% divergence in sequence identity . A phylogenetic tree estimated from the 16S rRNA sequences of those Bacillus species whose scRNAs sequences have also been determined is presented in (Fig. 3a). The topology is essentially the same as that reported in a much more extensive phylogenetic analysis of the genus Bacillus, with the exception that the 16S rRNA sequence of L. monocytogenes is included and that since the original phylogenetic analysis of the genus Bacillus, the species Bacillus polymyxa, Bacillus macerans and Bacillus brevis have been reclassified as Paenibacillus polymyxa, Paenibacillus macerans and Brevibacillus brevis, respectively. Analysis of available 23S rRNA sequences data supports this clustering profile (data not shown).
Figure 3. Phylogenetic trees based on (a) 16S rRNA and (b) scRNA of L. monocytogenes and selected Bacillus species. Phylogenetic trees were constructed using the Neighbor-Joining method as implemented by the CLUSTAL W program, gaps in alignments were excluded because of the high divergences, corrections were not made for multiple substitutions and confidence values for individual branches were obtained by bootstrap analysis, in which 1000 bootstrap trees were generated from resampled data. The trees are unrooted and the distance between two species is obtained by summing connecting branch lengths, using the relevant distance scale. The percentage of the number of bootstraps out of 1000 replications, that support a phylogenetic group of more than 85%, is placed beside the relevant branch.
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It has been reported that the genus Bacillus can be subdivided into groups on the basis of comparative phylogenetic 16S rRNA sequence analysis . B. subtilis, B. amyloliquefaciens and B. pumilus are placed in group 1, with B. sphaericus in group 2, and B. stearothermophilus in group 5. The L. monocytogenes 16S rRNA sequence is shown to represent a separate branch equidistant from the three Bacillus groups, Brevibacillus brevis and Paenibacillus species. Surprisingly, a similar phylogenetic analysis of scRNA sequences produced a very different topology (Fig. 3b), with species, P. polymyxa, P. macerans, B. sphaericus, B. subtilis, B. amyloliquefaciens and B. pumilus clustering together. This topology was supported by a boot-strap analysis.
To determine whether the scRNA or 16S rRNA phylogenetic trees best represented the true species tree, phylogenetic trees of sequences with homologues in at least four species that included either P. polymyxa, P. macerans, or B. sphaericus were constructed. The sequences that met these conditions were the hyper variable region of the 23S rRNA, spoIIA, endo-β-1,3-1,4 glucanase, serine proteases, cytosine specific methylases, RNA polymerase sigma factors, spoA, bsuRI and cdgT. None of these phylogenetic trees supported the scRNA topology in preference to the 16S rRNA topology.
The P. polymyxa, P. macerans, B. sphaericus and B. subtilis scRNA sequences are obviously clustering together because of sequence identity. Why the scRNA from these otherwise quite distantly related species have such a high sequence identity is not clear. This sequence identity is unlikely to be the result of convergent evolution it is far more likely that is the result the horizontal transfer of these scRNAs between these species.
The most divergent scRNAs are L. monocytogenes and B. brevis (Fig. 3b). As the branch lengths have not been corrected for superimposed substitutions, this represents a conservative estimation of evolutionary distance. The scRNA of L. monocytogenes and these other Gram-positive species can be considered as having a region which is homologous to the E. coli 4.5S RNA (HEc4.5) and those regions which are not (nHEc4.5). Divergence values between these regions are outlined in Table 1. These divergences indicate that it is the nHEc4.5 regions of L. monocytogenes and B. brevis scRNA that account for their high sequence divergence. The high sequence divergence observed in the nHEc4.5 regions is indicative that the L. monocytogenes and B. brevis helices 1–4 are under a lower evolutionary constraint on sequence conservation than the other scRNAs. The combination of a low evolutionary constraint and the less frequent base-pairing in the secondary structure of L. monocytogenes for helices 1–4 would seem to imply that these helices may have a biologically diminished functional role in L. monocytogenes relative to the corresponding helices of the other scRNAs. The base-pairing in the secondary structure of the equivalent B. brevis scRNA helices is much more like the other Bacillus species . When we consider that L. monocytogenes is the only one of these species that is non-sporulating, it would support a hypothesis that the scRNA helices 1, 2, 3, 4 and 5 have a possible biological role in the ability of the other species and C. perfringens to sporulate [9, 12], but it does not adequately explain the high evolutionary rate of the equivalent helices of the B. brevis scRNA.
Table 1. Comparison of the divergence percentage values for scRNAs of Bacillus species, B. brevis and L. monocytogenes, (a) between the region that is homologous to E. coli 4.5S RNA (bases 110–230 L. monocytogenes– HEc4.5) and (b) the remaining part of their scRNA (bases 1–109, 231–276 L. monocytogenes– nHEc4.5)