Table 1.
NicK and HelP are the only ICE–encoded factors required for autonomous replication.
Figure 1.
Map of ICEBs1 and derivatives.
A–B. Schematic of ICEBs1 (A) and the helP mutant (B) integrated at the normal attachment site, attB. The antibiotic resistance marker (kan) that is inserted in rapI-phrI is not shown. A. Genetic map of ICEBs1. The linear integrated form of ICEBs1 is shown. Open arrows indicate open reading frames and the direction of transcription. The black rectangles at the ends of ICEBs1 represent the 60 bp direct repeats that contain the site-specific recombination sites in the left and right attachment sites, attL and attR. Some gene names are indicated. The genes encoding the ICEBs1 repressor immR and anti-repressor immA are indicated by R and A, respectively. The origin of transfer (oriT) is indicated by a black line in the 5′ end of nicK. oriT is needed for ICEBs1 transfer [15] and replication [6]. Primers used in ChIP-PCR experiments hybridize to nicK (oriT), and conE. B. Schematic of the ΔhelP155 mutation in ICEBs1. Thin horizontal lines represent the regions of ICEBs1 that are present in the mutant. The gap in the line represents the in-frame markerless deletion of helP. C–D. Diagram of the truncated ICEBs1 derivatives that were used to test complementation of ΔhelP donors. Both constructs are integrated at thrC and contain ICEBs1 genes represented by the horizontal lines, from attL to ydcO (C) or helP (D). Both derivatives contain cat (chloramphenicol resistance) in place of the part of ICEBs1 that is deleted, and neither can excise from the chromosome due to the absence of attR.
Table 2.
HelP is required for ICEBs1 transfer and replication.
Figure 2.
HelP, PcrA, and Ssb-GFP association with ICEBs1 DNA.
ICEBs1 gene expression was induced by overproduction of RapI from amyE::{Pspank(hy)-rapI spc} for one hour followed by DNA-protein crosslinking with formaldehyde. Association of HelP (A, D), PcrA (B, E), and Ssb-GFP (C, F) with DNA sequences near oriT (A–C) and conE (D–F) were measured by immunoprecipitation of lysates with polyclonal anti-HelP, anti-PcrA and anti-GFP antibodies respectively followed by qPCR (ChIP-PCR), relative to ydbT, adjacent to ICEBs1. When Ssb-GFP association was measured, strains contained the lacA::{PrpsF-ssb-GFPm tet} allele expressing GFP-tagged Ssb [6]. Strains included: wild type (JMA168 for HelP and PcrA; MMB834 for Ssb-GFP), white bars; ΔnicK (CAL306), horizontally striped bars; nicKY195F (JT340), dotted bars; ΔhelP (JT155 for HelP and PcrA; JT252 for Ssb-GFP), light grey bars; and ΔhelP, complemented with helP+ (JT335 for HelP and PcrA; JT398 for Ssb-GFP), dark grey bars. The c-Myc tag in the nicKY195F allele did not affect HelP association with conE which was unchanged in the NicK-Myc (JT308) control strain (421±96) compared to wild type (551±100) in three independent experiments. Error bars represent standard deviation.
Table 3.
UvrD permits ICEBs1 replication in a pcrA-defective strain.
Figure 3.
DNA unwinding by UvrD is stimulated by HelP.
Substrates for unwinding were M13mp18 ssDNA annealed to either a 22 (A) or 81 (B) complementary fluorophore-labeled oligonucleotide. Data presented are the averages of three independent experiments ± standard deviation. Substrate unwinding was undetectable in the presence of HelP alone (<0.5% substrate unwound after 45 minutes) for both substrates. These controls were done with only the 45 minute time point.
Figure 4.
Comparison of HelP to Orf22 and Orf23 of Tn916.
HelP (middle sequence), Orf22 of Tn916 (top sequence) and Orf23 of Tn916 (bottom sequence) were aligned using Clustal X. Markings above and below the HelP sequence indicate which amino acids are identical (asterisk), highly similar (colon) or weakly similar (period) to Tn916 Orf22 (marks above) and Tn916 Orf23 (marks below). Markings for the pairwise comparison of Orf22 to Orf23 are shown below the Tn916 Orf23 sequence. Needleman-Wunsch alignment scores indicate that HelP is more similar to Orf22 and Orf23 (N-W scores = 142 and 104, respectively) than Orf22 is to Orf23 (N-W score = 31). ICEBs1 HelP is 126 amino acids, Tn916 Orf22 is 128 amino acids, and Tn916 Orf23 is 104 amino acids.
Figure 5.
Organization of genes for HelP, ConQ (coupling protein) and NicK homologues in ICEBs1 and Tn916.
Schematic diagram showing the organization of genes encoding HelP, ConQ and NicK in ICEBs1 and their homologues Orf23, Orf22, Orf21, and Orf20 in Tn916. Most (>40) of the 72 ICEs in the ICEberg database that have helP homologues have this consecutive (helP)-helP-conQ-nicK configuration. Some ICEs have the same gene order but have an additional one or two genes located between the helP and conQ and/or between conQ and nicK. Not shown is a different gene organization found in ICEs related to ICE6013 in which the conQ is separate from helP and nicK.
Figure 6.
Phylogenic tree of HelP homologues.
Using the ICEberg database and search tools (HMMER3 and WU-BLAST2) (http://db-mml.sjtu.edu.cn/ICEberg/), we identified 128 HelP homologues in 72 ICEs. The homologues were analyzed using CLUSTAL X and grouped into clades if they were within a distance of <0.22. The diagram is of an unrooted tree with the number of homologues in each clade shown in parentheses. Branch lengths indicate relative phylogenic distances. The width and length of branch tips indicate the number of homologues and the relative phylogenic distance between homologues in the clade, respectively. Of the seven clades, six are named for a representative ICE and its HelP homologue and one is named “Orf23-like” to reflect its close relationship to the Tn916 Orf23 clade. Twelve homologues in the ICE6013 Orf14 clade have the identical 106 amino acid sequence. The two homologues in the ICEBs1 HelP clade only differ by 5 of 126 amino acids. The two homologues in the Orf23-like clade only differ by 7 of 108 amino acids. Sixteen ICEs encode a single homologue of HelP. These sixteen homologues are from three clades - ICEBs1 HelP (2), ICE6013 Orf14 (12) and Tn916 Orf23 (2). The remaining 56 ICEs encode pairs of HelP homologues. The members of each pair of homologues fall into separate clades as exemplified by Tn916 Orf22 (128 aa) and Tn916 Orf23 (104 aa), and ICESa2 SAPIG1865 (141 aa) and ICESa2 SAPIG1866 (107 aa).
Figure 7.
Model for association of the relaxase, helicase, HelP, and Ssb with ICEBs1 DNA.
Following excision from the chromosome (not shown), the double-stranded circular ICEBs1 DNA is nicked at oriT by the relaxase NicK (filled circle). By analogy to related relaxases, NicK likely becomes covalently attached to the 5′ end of the nicked DNA on the strand that is transferred during conjugation. HelP (open circles) and the host-encoded helicase PcrA (gray packman) associate with the nicked ICEBs1 at oriT. HelP facilitates processive unwinding of ICEBs1 by PcrA and subsequent association of the host-encoded Ssb (open triangles). Unwinding of ICEBs1 by PcrA and HelP is required for replication and conjugation of ICEBs1. HelP is depicted as binding to both single strands of ICEBs1 DNA adjacent to PcrA, although it is possible that HelP associates only with one of the two single strands.
Table 4.
Strains and plasmids.