Differential regulation of two closely related integrative and conjugative elements from Streptococcus thermophilus - PubMed (original) (raw)

Nicolas Carraro et al. BMC Microbiol. 2011.

Abstract

Background: Two closely related ICEs, ICESt1 and ICESt3, have been identified in the lactic acid bacterium Streptococcus thermophilus. While their conjugation and recombination modules are almost identical (95% nucleotide identity) and their regulation modules related, previous work has demonstrated that transconjugants carrying ICESt3 were generated at rate exceeding by a 1000 factor that of ICESt1.

Results: The functional regulation of ICESt1 and ICESt3 transcription, excision and replication were investigated under different conditions (exponential growth or stationary phase, DNA damage by exposition to mitomycin C). Analysis revealed an identical transcriptional organization of their recombination and conjugation modules (long unique transcript) whereas the transcriptional organization of their regulation modules were found to be different (two operons in ICESt1 but only one in ICESt3) and to depend on the conditions (promoter specific of stationary phase in ICESt3). For both elements, stationary phase and DNA damage lead to the rise of transcript levels of the conjugation-recombination and regulation modules. Whatever the growth culture conditions, excision of ICESt1 was found to be lower than that of ICESt3, which is consistent with weaker transfer frequencies. Furthermore, for both elements, excision increases in stationary phase (8.9-fold for ICESt1 and 1.31-fold for ICESt3) and is strongly enhanced by DNA damage (38-fold for ICESt1 and 18-fold for ICESt3). Although ICEs are generally not described as replicative elements, the copy number of ICESt3 exhibited a sharp increase (9.6-fold) after mitomycin C exposure of its harboring strain CNRZ385. This result was not observed when ICESt3 was introduced in a strain deriving ICESt1 host strain CNRZ368, deleted for this element. This finding suggests an impact of the host cell on ICE behavior.

Conclusions: All together, these results suggest a novel mechanism of regulation shared by ICESt1, ICESt3 and closely related ICEs, which we identified by analysis of recently sequenced genomes of firmicutes. This is the first report of a partial shutdown of the activity of an ICE executed by a strain belonging to its primary host species. The sharp increase of ICESt3 copy number suggests an induction of replication; such conditional intracellular replication may be common among ICEs.

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Figures

Figure 1

Figure 1

Comparison of ICE_St1_ and ICE_St3_ regulation, conjugation and recombination modules. Location and orientation of ORFs and a truncated IS are indicated by arrowed boxes and a rectangle, respectively. ORF names beginning with "orf" are abbreviated with the corresponding letters or numbers. The pattern of the arrowed boxes depicts the relationships of each ORF deduced from functional analyses or from BLAST comparisons. White arrowed boxes correspond to unrelated ORFs of the two elements. Black arrowed box is the chromosomal fda gene. The grey areas indicate closely related sequences with the nucleotide identity percentage value. The angled arrows and the lollipops indicate the experimentally demonstrated promoters and rho-independent transcription terminators predicted from in silico analysis (black) or unpredicted (grey). The star corresponds to the putative transfer origin. Horizontal lines delimitate functional modules with their names above. Dashed lines indicate the A, B and C intergenic regions of both ICEs; their nucleotide sequence alignments are detailed below. (A) Region upstream from the orfQ gene, (B) Region upstream from the arp2 gene, (C) Parp2s region. The position of the ribosome binding sites (RBS), initiation and stop codons are annotated in bold. Coding regions are boxed. The -10 and -35 boxes of the promoters and transcriptional start sites (+1) determined by 5'RACE PCR are in boldface and underlined. Numbers indicate the nucleotide position on the ICE sequence [GenBank:AJ278471 for ICE_St1_ and GenBank:AJ586568 for ICE_St3_]. For region upstream from the orfQ gene (A), arrows indicate the rho-independent transcription terminator inverted repeats. For region upstream from the arp2 gene (B), horizontal lines below the sequences delimitate the putative stems regions and dashed lines indicate the loop part.

Figure 2

Figure 2

Transcriptional analysis of the arp2/orfM region of ICE_St3_. (A) Schematic representation of the arp2/orfM intergenic region of ICE_St3_. Primers used for PCR analysis are represented by triangles and promoters are represented by angled arrows. (B) RT-PCR mapping Pcr promoter of ICE_St3_. Amplicons are generated with primers mentioned above the gels on genomic DNA (gDNA) or cDNA synthesized from RNA extracted from cells in exponential growth phase (expo0.6). Amplicon size is given on the left. Results were identical for three independent biological replicates. (C) RT-PCR mapping Parp2 promoter of ICE_St3_. Amplicons are generated with primers mentioned on the left of the gels on genomic DNA (gDNA) or cDNA synthesized from RNA extracted from exponential growth phase (expo0.6) and stationary phase (stat) cells. The transcriptional activity upstream from the Parp2 promoter was detected during stationary phase. Results were identical for three independent biological replicates.

Figure 3

Figure 3

Quantification of the transcripts of the core regions of ICE_St1_ (A) and ICE_St3_ (B). Arrows correspond to transcripts. Primer pairs used for cDNA quantification are represented by convergent triangles below the corresponding transcript. Other symbols used in the map are identical to those used in Figure 1. cDNA quantities determined from cells grown in LM17 medium and harvested in exponential growth phase (expo0.6) or stationary phase (stat) or after 2.5 hours of exponential growth with mitomycin C (MMC) at MIC/2 are normalized to the quantity of cDNA of gyrA whose transcription is constitutive [39]. Lack of amplicon is mentioned as non-detected (ND). For each condition, data are average and standard deviation from three independent biological replicates.

Figure 4

Figure 4

Quantification of ICE excision. (A) Localization of amplicons used for quantitative PCR. The total ICE copy number is quantified by amplification of ICE internal fragments corresponding to orfJ/orfI and orfM/orfL junctions (J/I and M/L, respectively) whereas the total chromosome number is quantified by amplification of an internal fragment of fda. The two products of excision, i.e circular ICE and chromosome devoid of ICE, are quantified by amplification of the recombination sites resulting from excision, attI and attB respectively. The star represents the putative transfer origin. (B) Effect of growth phase on excision. qPCR amplifications were performed on total DNA extracted from cells harvested during exponential growth in LM17 medium at OD600 nm = 0.2 (expo0.2) or OD600 nm = 0.6 (expo0.6) or after 1.5 hours in stationary phase (stat). (C) Effect of MMC treatment on excision. qPCR amplifications were performed on total DNA extracted from cells grown in LM17 medium treated or not (expo0.6) during 2.5 hours with MMC at MIC/2 and harvested at OD600 nm = 0.6 (MMC). Excision percentage is calculated as (attB/fda)×100. Data are presented as average and standard deviation from three independent biological replicates.

Figure 5

Figure 5

Strain effect on ICE excision. qPCR amplification was performed on total DNA extracted from cells harvested during exponential growth in LM17 medium at OD600 nm = 0.6 (expo0.6) or treated for 2.5 hours by MMC at MIC/2 and harvested at OD600 nm = 0.6 (MMC). ICE and host strains studied are indicated below. ICE_St3_, in strains CNRZ368 and LMG18311, was tagged with the cat gene, conferring chloramphenicol resistance, for transconjugant selection. To avoid ICE interference, strain CNRZ368 was previously deleted of ICE_St1_ prior ICE_St3cat_ transfer. Excision percentage is calculated as (attB/fda)×100. Data are presented as average and standard deviation from three independent replicates.

Figure 6

Figure 6

Comparison of the conserved structure of streptococcal ICEs. ICE names or host strain names are mentioned on the right. ORFs location and orientation of each ICE are indicated by arrowed boxes. Above, ORF names are abbreviated with the corresponding letter or number. The pattern of the arrowed boxes depicts the related ORFs, homologs to ICE_St3_ regulation and conjugation genes deduced from functional analyses or from BLAST comparisons. The grey areas indicate closely related sequences between GIs (> 70% nucleotide identity); the identity percentage between pairs of GIs is given. Homologous ORFs of unknown function and unrelated ORFs are represented by black or white arrowed boxes, respectively. The identity percentage indicated on right goes until and includes the orfA conjugation gene. The angled arrows and the lollipops indicate the promoters and rho-independent transcription terminators experimentally demonstrated (black) or predicted from in silico analysis (white). Sequences used for this analysis are from the putative ICE ICE_Spn8140_ of S. pneumoniae [GenBank:FR671412[22] and from the partially or completely sequenced genomes of S. parasanguinis ATCC15912 [GeneBank:NZ_ADVN00000000] and F0405 [GenBank:NZ_AEKM00000000], S. infantis ATCC 700779 [GeneBank:NZ_AEVD00000000] and S. australis ATCC700641 [GeneBank:NZ_AEQR00000000].

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