Download Suppl. Material

Supplementary information 1
TABLE S1. Predicted Conjugative Transfer genes from ICEAfe1
ORF
Gene (Cluster)
Equivalence
Function
Motif (E-value)
AFE1047
AFE1065
AFE1066
AFE1067
AFE1068
AFE1078
AFE1079
AFE1080
AFE1081
AFE1082
AFE1084
AFE1084´
AFE1087
AFE1089
AFE1091
AFE1095
AFE1096
AFE1097
AFE1104
AFE1235
AFE1251
traAP
trbNP (I)
traFF(I)
traHF(I)
traGF(I)
traLF (II)
traEF (II)
traKF (II)
traBF (II)
traVF (II)
traA1F (II)
traA2F (II)
pilB (III)
traCF (III)
traFF(III)
traWF
traUF
traNF
traN-like
trbEP
traDF
VirD2
VirB1
VirB3
VirB5
VirB9
VirB10
VirB7
VirB2
VirB2
VirB11
VirB4
VirB4
VirD4
Relaxase
Soluble lytic murein transglycosylase
Pilus assembly protein
Relaxosome auxiliary protein
Mating-cell interactions stabilizer
Minor pilus subunit ?
Pilus tip ahdhesin ?
Secretin-like protein
Secretin-like protein
Lipoprotein
Mayor pilin subunit
Mayor pilin subunit
ATPase
ATPase
Pilin signal Peptidase
Conjugative transfer protein
Conjugative transfer protein
Mating-cell interactions stabilizer
Unknown
ATPase
Conjugative coupling protein
TIGR02768 (3.68E-28)
pfam01464 (1.77E-15)
pfam13728 (3.10E-58)
pfam06122 (2.48E-34)
pfam07916 (8.98E-22)
pfam07178 (1.69E-07)
TIGR02761 (2.65E-43)
pfam06586 (2.19E-15)
pfam03743 (3.02E-13)
pfam09676 (1.26E-12)
TIGR02538 (1.00E-93)
pfam11130 (5.37E-23)
TIGR02771 (1.60E-06)
TIGR02743 (2.61E-11)
pfam06834 (7.52E-54)
PRK12355 (9.46E-26)
COG0213 (0.062)
COG3451 (8.00E-07)
TIGR03743 (1.62E-17)
Sequence similarity and motif/domain conservation analysis indicates that ICEAfe1 encodes
the required set of proteins for proficient pilus formation, stabilization of mating pairs and
conjugative DNA metabolism (Table 1 supplementary information). These proteins include
two highly similar copies (identity 90 %) of the pilin TraA1/TraA2, the lipoprotein TraV F
(pfam09676), the secretin-like proteins TraBF (pfam03743) and TraKF (pfam06586), TraLF
(pfam07178) and TraEF (TIGR02761) which resemble the VirB3 and VirB5 components of
the P-type T4SS of IncP plasmids and the ATPases TraCF (pfam11130) and TrbEP
(COG3451) which provide energy for assembly and function of the T4SS. Auxiliary genes,
essential for pilus assembly, retraction and mating pair stabilization found in ICEAfe1
encode for the TraFF pilin signal peptidase (TIGR02771), TraGF (pfam07916), TraNF
(PRK12355), TraUF (pfam06834) and TraWF (TIGR02743), all hallmarks of F-like T4SS.
ICEAfe1 predicted relaxosome components are TraAP (TIGR02768) and TraHF
(pfam06122). The relaxosome is a protein–DNA complex that assembles at the origin of
transfer of a plasmid or other conjugative elements. The key enzyme of the relaxosome is
the conjugative DNA relaxase required to initiate DNA transfer during plasmid
conjugation, called TraI in the case of F-plasmid [Byrd et al., 2002; Matson and Ragonese,
2005] and TraA in P-type plasmids [Kopec et al., 2005; Lanka and Wilkins, 1995; Yang et
al., 2007]. ICEAfe1 encoded relaxase contains domains distinctive of the TraA P-type of
enzymes with an N-terminal single strand exonuclease (COG0507) and a C-terminal
helicase domain (pfam13538). This protein, which shares similarity to the central region of
TraI-type relaxasases likely fills the same role as TraIF, nicking ICEAfe1 at its oriT [Datta
et al., 2003] and unwinding the coiled circularized ICE prior to its conjugative transfer
[Matson et al., 2001]. One auxiliary protein present in ICEAfe1, TraHF, is predicted to form
part and stabilize the relaxosome. Based on sequence similarity analysis the product of
ICEAfe1 traD gene, TraDF (TIGR03743), is proposed to be the coupling protein (T4CP)
that recruits the relaxosome to the transferosome [Llosa et al., 2002], through which
ICEAfe1 DNA may pass on its way to the recipient cell.
2
Suplementary information 2.
Microarrays results for integration/excision and transfer mRNAs.
Supplementary figure 1. Microarray transcriptional profiles of phage and plasmid-related
conjugative and stabilization genes present in ICEAfe1. Color coded expression
corresponds to median log ratios of experiments (A) 9K-Iron 62 mM grown cells vs 9KSulfur, (B) 9K-Iron 200 mM vs 9K-Iron 62 mM and (C) self-self hybridization of Iron 62
mM grown cells labeled with either Cy3 or Cy5 fluorophores. Red represents expression
greater that reference, green is less than reference, yellow is equal and gray is missing or
excluded data. Open reading frames are numbered according to NCBI annotation
(NC_011761.1). Gene names and predicted functional assignments of each ORF are shown
to the right of the heat map.
3
REFERENCES SUPPLEMENTARY MATERIAL
Byrd DR, Sampson JK, Ragonese HM, Matson SW: Structure-function analysis of
Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase
domains. J Biol Chem 2002; 277:42645-42653.
Datta S, Larkin C, Schildbach JF: Structural insights into single-stranded DNA binding and
cleavage by F factor TraI. Structure 2003; 11:1369-1379.
Kopec J, Bergmann A, Fritz G, Grohmann E, Keller W: TraA and its N-terminal relaxase
domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as
dimers in solution. Biochem J 2005; 387:401-409.
Lanka E, Wilkins BM: DNA processing reactions in bacterial conjugation. Annu Rev
Biochem 1995; 64:141-169.
Llosa M, Gomis-Rüth FX, Coll M, de la Cruz Fd F: Bacterial conjugation: a two-step
mechanism for DNA transport. Mol Microbiol 2002; 45:1-8.
Matson SW, Ragonese H: The F-plasmid TraI protein contains three functional domains
required for conjugative DNA strand transfer. J Bacteriol 2005; 187:697-706.
Matson SW, Sampson JK, Byrd DR: F plasmid conjugative DNA transfer: the TraI helicase
activity is essential for DNA strand transfer. J Biol Chem 2001; 276:2372-2379.
Yang JC, Lessard PA, Sengupta N, Windsor SD, O'brien XM, Bramucci M, Tomb JF,
Nagarajan V, Sinskey AJ: TraA is required for megaplasmid conjugation in Rhodococcus
erythropolis AN12. Plasmid 2007; 57:55-70.
4