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Keywords:

  • Small cytoplasmic RNA;
  • Cloning strategy;
  • Listeria monocytogenes;
  • Phylogenetic analysis

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

A molecular cloning strategy has been designed to isolate the gene that encodes the small cytoplasmic RNA (scRNA) component of bacterial signal recognition particles. Using this strategy a putative Listeria monocytogenes scRNA λgt11 recombinant clone was isolated. A previously described complementation assay developed to genetically select functional homologues of 4.5S RNA and scRNA of bacteria confirmed that the λgt11 recombinant clone isolated encoded for the scRNA from L. monocytogenes. A secondary structure for this scRNA is proposed and a phylogenetic comparison of the 276 base L. monocytogenes scRNA with previously characterised Gram-positive bacterial scRNAs is also presented.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Signal recognition particles (SRP) are ribonucleoproteins that have been identified in Eukaryotae, Archaea and Eubacteria cells. In bacteria, SRP ribonucleoproteins may act as chaperones which are specific for signal sequences in nascent membrane bound pre-proteins and as such are employed in maintaining the pre-protein in a translocation-competent conformation prior to cellular secretion [1]. Analysis of bacterial SRP associated RNAs has demonstrated that they all share a highly homologous central structural motif, helix 8, there is, however, considerable sequence and structural heterogeneity associated throughout the remainder of these RNA species [2]. SRPs associated RNAs from the Gram-negative bacteria are termed 4.5S RNAs and have been characterised from Escherichia coli[3], Thermus thermophilus[4], Legionella pneumophilia and Pseudomonas aeruginosa[5]. Characterisation of SRP-associated RNAs from Gram-positive bacteria has been confined to three Mycoplasma species [6–8]Micrococcus luteus[5], Clostridium perfringens[9] and 13 Paenibacillus, Brevibacillus and Bacillus species [10]. The Mycoplasma species 4.5S RNAs, in common with the Gram-negative species examined, comprise of helix 8 and a partial helix 5, while the Micrococcus SRP associated RNA comprise of helix 8 and an extended helix 5. To date, the only bacterial SRP associated RNAs that contain, in common with the Archaea and Eukaryotae SRP associated RNAs, helices 1, 2, 3, 4, 5 and 8, are the scRNAs from C. perfringens and the endospore-forming genus Bacillus. We sought to expand the range of scRNAs characterised from Gram-positive bacteria, by developing a general molecular cloning based strategy for this purpose. The species of choice for this analysis was Listeria monocytogenes a non-spore forming Gram-positive rod shaped bacterium. It was selected primarily because of the phylogenetic relatedness to the Bacillus species [11]. Characterisation of the SRP associated RNA from L. monocytogenes could resolve whether the additional structural features of Bacillus species and C. perfringens scRNAs are wide spread amongst Gram-positive bacteria.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial strains, plasmids, DNA and RNA manipulations

L. monocytogenes serotype 1/2b was obtained from NCIMB. E. coli S1610 and plasmids pTUBE809 and pTUBE822 were used for scRNA complementation analysis [5, 12]. L. monocytogenes was cultured in Listeria broth (3% Tryptone soya broth, 0.6% yeast extract) at 30°C. Other E. coli cloning hosts were used as recommended by the manufacturers of the λgt11 cDNA cloning kit (Amersham). Chromosomal DNA and total RNA preparations were carried out as previously described [13, 14].

2.2Molecular cloning strategy

The initial aim of this cloning strategy was to isolate and characterise a partial nucleotide sequence of the L. monocytogenes scRNA gene for use as a specific oligonucleotide DNA probe to screen a L. monocytogenes EcoRI λgt11 genomic library constructed in E. coli Y1090 (data not presented). Alignment of known scRNA sequences using the CLUSTAL W alignment program [15], indicated that polymerase chain reaction (PCR) oligonucleotide primers could be designed which would amplify a short variable nucleotide sequence of scRNA genes, approximating to the nucleotide sequence spanning helices 5 and 8. Subsequently, sequence information derived from this short amplified variable region would then be used to design a specific oligonucleotide probe for use in screening the L. monocytogenesλgt11 library to identify putative scRNA gene recombinants.

A 17-mer 5′ degenerate oligonucleotide PCR primer A (5′-T/G G/T TTGG T/G TC T/C C/T C/G CGCAA-3′), corresponding to helix 5 of 4.5S/scRNA genes, and an 18-mer 3′ homologous oligonucleotide PCR primer B (5′-TCGAAGGAAGGCCTGGAC-3′), corresponding to the conserved sequence motif of helix 8 were synthesised and purchased from Genosys. PCR amplification was carried out using 250 ng of L. monocytogenes genomic DNA, with amplification conditions set at 94°C for 30 s, 50°C for 30 s and 72°C for 30 s in the presence of 2.5 mM MgCl2, 250 pmol of each PCR primer, 1 mM dNTPs, 1×PCR reaction buffer and 2.5 U of Taq DNA polymerase (Boehringer) to a final volume of 100 μl in a Perkin-Elmer thermocycler. The 75-bp amplified PCR product was purified from a 4% NuSieve (FMC) agarose gel and sequenced directly using the homologous 3′ PCR primer B (data not shown). Sequence data indicated that a specific DNA oligonucleotide probe could be generated for use in screening for scRNA recombinants from the L. monocytogenesλgt11 library and that the possibility of non-specific cross hybridisation with E. coli, (the library host used), 4.5S RNA sequences was eliminated. The 18-mer L. monocytogenes specific scRNA oligonucleotide C, corresponding to the short variable region spanning helices 5 and 8, 5′-TGGGAACCTGTGAACCAT-3′ and its complement D, were synthesised. Oligonucleotide C was then [γ-32P]ATP (Amersham) 5′-end-labelled with T4 polynucleotide kinase (Promega) and used as an oligonucleotide probe to screen 3000 recombinants of the L. monocytogenesλgt11 library. Hybridisation conditions for library screening was as follows. Nytran filters were prehybridised for 2 h in 100 ml of 6×SSC, 10×Denhardts and 0.1% SDS. The prehybridisation solution was removed and hybridisation was carried out in 5 ml of 6×SSPE, 0.1% SDS with 10 ng ml−1 of end labelled oligonucleotide probe (107 cpm) at 50°C for 2 h. Three washes (100 ml each) were then carried out in 6×SSC and 0.1% SDS at room temperature for 5 min, with a final wash (100 ml) in the same solution at the hybridisation temperature for 2 min. Autoradiography was carried out for 48 h at −70°C.

2.3Nucleotide sequence characterisation of the putative L. monocytogenes scRNA λgt11 recombinant clone

Small-scale λ preparations were carried out on isolated recombinant clones [13] and EcoRI restriction analysis determined the molecular weight of the cloned inserts from these recombinants. Recombinant inserts were subsequently gel purified from 2.5% NuSieve agarose gels and direct sequencing of the putative L. monocytogenes scRNA λgt11 clones were then carried out using oligonucleotide C for sequencing in the 5′–3′ direction and D in the 3′–5′ direction.

Mapping of the 5′-end of the mature scRNA was carried out using oligonucleotide B as a reverse transcriptase (BRL) extension primer [16]. The cDNA reaction products were analysed on a 6% DNA sequencing gel. The length of the extended DNA fragment was estimated by comparison with sequencing ladders from an M13 control transcript. 3′-end mapping was determined by comparative sequence analysis of CLUSTAL W multiple-sequence alignments of Bacillus, Brevibacillus and Paenibacillus species scRNAs.

2.4Complementation and genetic selection analysis

The putative L. monocytogenes scRNA nucleotide sequence was PCR amplified from the λgt11 recombinant clone using PCR primers sc276.1 (5′-GTTGATGAGCGTGAAGCC-3′), corresponding to the 5′-end of the gene and sc276.2, (5′-TTAGTGTCGCGCACCTCA-3′), corresponding to the 3′-end of the gene. The amplified product was then subcloned into the SmaI site of the E. coliB. subtilis shuttle vector, pTUBE809, and the resultant construct, pTUBE922 was subjected to the in vivo complementation assay system used to genetically select 4.5S/scRNA homologues of bacteria [5]. Essentially, this procedure consists of an in vivo complementation assay system dependent on the presence of a plasmid borne 4.5S/scRNA to complement a 4.5S chromosomal defect in the strain E. coli S1610. In this strain, the sole intact copy of the gene for 4.5S RNA is present on a thermoinducible prophage, rendering the bacterium temperature sensitive for growth. Since 4.5S RNA is essential for growth, the cured progeny of this strain are non-viable unless transformed with a plasmid encoding a 4.5S homologue.

2.5Computer analysis and secondary structure prediction

Secondary structure prediction of the L. monocytogenes scRNA was achieved by comparative analysis of multiple-sequence alignments with the secondary structure predictions of previously characterised 4.5S RNA and scRNA sequences. All multiple alignments were calculated by CLUSTAL W, the secondary structure for scRNA was drawn using the secondary structure drawing program Loop-D-loop (written by D. Gilbert, available from http://ftp.bio.indiana.edu/molbio/loopdloop). Phylogenetic trees were constructed using the Neighbor-Joining method [17], as implemented by CLUSTAL W, and Maximum parsimony as implemented by the GCG 9.1 PAUP program, the reliability of different phylogenetic trees was estimated using bootstrapping [18].

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

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).

image

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).

image

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 [19]. Analysis of protein coding sequences indicates that even the closely related species B. amyloliquefaciens and B. subtilis have 22% divergence in sequence identity [20]. 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[21], with the exception that the 16S rRNA sequence of L. monocytogenes is included and that since the original phylogenetic analysis of the genus Bacillus[21], 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).

image

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 [21]. 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 [10]. 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)
(a)
Bacillus species0–24%
B. brevis19–24%
L. monocytogenes14–20%
(b)
Bacillus species0–20%
B. brevis19–38%
L. monocytogenes38–41%

3.3Concluding remarks

We have demonstrated that the scRNA helices 1, 2, 3, and 4 which had only previously been identified within the spore-forming Bacillaceae and C. perfringens are also present in the non-spore forming L. monocytogenes. Genetic complementation and deletion experimentation of L. monocytogenes and other phylogenetic related scRNAs with the inducer dependent scRNA gene of B. subtilis strain SC200NA [12], will be useful in determining discrete functional biological sequence motifs of these scRNAs.

3.4Nucleotide sequence accession number

The L. monocytogenes scRNA has been submitted to the signal recognition particle database and GenBank (accession number U15684).

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

We thank P. Sharp for the use of his laboratory and helpful comments and C. Zwieb and N. Larsen for signal recognition particle associated RNA alignment analysis. We also thank S. Browne for E. coli S1610 and K. Nakamura for plasmids pTUBE809 and pTUBE822. J.P. is funded by a studentship from the University of Nottingham, B.G. and M.K. are funded by a studentship from Enterprise Ireland.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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