Article Figures & Data
Figures
- Fig. 1.
(A) Intracellular multiplication of strain LELA3896 within HL-60-derived macrophages. Differentiated HL-60 cells (1.5 × 106 per well) were infected with approximately 104 bacteria on day 0. The number of CFU of each strain per milliliter was determined daily for 5 days, in triplicate, on ABCYE plates. ▪, JR32; □, LELA3896; •, LELA3896(pMMB207); ○, LELA3896(pMW100). Error bars indicate the standard error of the mean. (B) Cytotoxicity of strain LELA3896 for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the absorbance at 570 nm (A570). ▪, JR32(pMMB207); □, JR32(pMW100); •, LELA3896; ○, LELA3896(pMMB207); ▴, LELA3896(pMW100). Values are the average of three determinations. Error bars indicate the standard error of the mean.
- Fig. 2.
(A) EcoRI restriction map of the genomic insert on plasmid pMW100. EcoRI restriction sites are labeled R. The approximate size of each EcoRI restriction fragment is shown in kilobases. Ptac of the pMMB207 vector directs transcription to the left into the 5.5-kbEcoRI fragment. The dotted line at the 3′-end of the 5.5-kbEcoRI fragment represents the region not sequenced. Mak− DNA hybridization groups represented on the genomic insert of pMW100 as shown by Southern analysis are shown above theEcoRI fragments. (B) ORFs present on the genomic insert of pMW100 and locations of Tn903dIIlacZ insertions (▹) and deletion substitutions (▪) in the Mak−mutants. Triangles on top of one another indicate that the LELA strains contain a Tn903dIIlacZ insertion at the same nucleotide. Vertical numbers indicate the strain number. ORFs shown were identified by nucleotide sequencing and MacDNAsis analysis. Solid arrows indicate the direction of transcription of each ORF. The location with respect to each EcoRI restriction site and size of each ORF is approximate. icmE is represented as a partial ORF, as indicated by a dotted 5′ end.
- Fig. 3.
Cytotoxicity of the icmF mutant strains for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 cells per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A570. (A) icmF mutant strain LELA1275. ▪, JR32(pMMB207); □, LELA1275(pMMB207); •, LELA1275(pMW100). (B) icmF mutant strain LELA1718. ▪, JR32(pMMB207); □, LELA1718(pMMB207); •, LELA1718(pMW100). Values are the average of three determinations. Error bars indicate the standard error of the mean.
- Fig. 4.
Representation of predicted transmembrane domains in theicm gene products. The hydropathic profiles of the predicted Icm proteins are shown according to the Kyte-Doolittle scale with a window size of 20 amino acids. The transmembrane domains as identified by the Psort program are represented as bars above the graphs. The vertical axis represents the hydrophobic value as measured on the Kyte-Doolittle scale, and the horizontal axis represents the number of amino acids in the icm gene products.
- Fig. 5.
Cytotoxicity of the icmB mutant strains containing different plasmids for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A570. (A) icmBmutant strain LELA3393. ▪, JR32(pMMB207); □, LELA3393(pBC SK+); •, LELA3393(pMW100); ○, LELA3393(pMW560). (B) icmBmutant strain LELA1012. ▪, JR32(pMMB207); □, LELA1012(pBC SK+); •, LELA1012(pMW100); ○, LELA1012(pMW560). (C) icmBmutant strain LELA1223. ▪, JR32(pMMB207); □, LELA1223(pBC SK+); •, LELA1223(pMW100); ○, LELA1223(pMW560). (D) icmBmutant strain LELA3896. ▪, JR32(pMMB207); □, LELA3896(pBC SK+); •, LELA3896(pMW100); ○, LELA3896(pMW560). Values are the average of three determinations. Error bars indicate the standard error of the mean.
- Fig. 6.
Cytotoxicity assay of L. pneumophila strains containing different plasmids for HL-60-derived macrophages. Differentiated HL-60 cells (4 × 105 cells per well) were infected with the indicated strains of bacteria at concentrations ranging from 10 to 106 CFU/ml. After 6 days, the remaining viable macrophages in the monolayer were quantitated by adding the dye MTT and measuring the A570. (A) icmJallelic exchange strain MW656. ▪, JR32(pMMB207); □, MW656(pMMB207αb); •, MW656(pMW100); ○, MW656(pMW680);
, MW656(pMW681); ▵, MW656(pMW560); ▾▴, MW656(pMW560, pMW790). (B) icmD mutant strain LELA1205. ▪, JR32(pMMB207); □, LELA1205(pMMB207αb); •, LELA1205(pMW100); ○, LELA1205(pMW734); , LELA1205(pMW736). (C) icmC allelic exchange strain MW645. ▪, JR32(pMMB207); □, MW645(pMMB207αb); •, MW645(pMW100); ○, MW645(pMW604); , MW645(pMW728); ▵, MW645(pMW730); ▾▴, MW645(pMW734). (D) icmG allelic exchange strain MW635. ▪, JR32(pMMB207); □, MW635(pMMB207αb); •, MW635(pMW100); ○, MW635(pMW741); , MW635(pMW743). Values are the average of three determinations. Error bars indicate the standard error of the mean.
Tables
- Table 1.
Bacterial strains used in this study
Strain Genotype and features Reference or source L. pneumophila JR32 NaCl-sensitive isolate of AM511 28 LELA1205 JR32icmD1205::Tn903dIIlacZ, DNA hybridization group II 28 LELA1506 JR32icmB1506::Tn903dIIlacZ, DNA hybridization group II 28 LELA1984 JR32icmE1984::Tn903dIIlacZ, DNA hybridization group II 28 LELA1996 JR32icmB1996::Tn903dIIlacZ, DNA hybridization group II 28 LELA2517 JR32icmC2517::Tn903dIIlacZ, DNA hybridization group II 28 LELA3244 JR32icmD3244::Tn903dIIlacZ, DNA hybridization group II 28 LELA3379 JR32icmB3379::Tn903dIIlacZ, DNA hybridization group II 28 LELA3393 JR32icmB3393::Tn903dIIlacZ, DNA hybridization group II 28 LELA3563 JR32icmB3563::Tn903dIIlacZ, DNA hybridization group II 28 LELA3896 JR32icmB3896::Tn903dIIlacZ, DNA hybridization group II 28 LELA1223 JR32icmB1223::Tn903dIIlacZ, DNA hybridization group IV 28 LELA1566 JR32icmB1566::Tn903dIIlacZ, DNA hybridization group IV 28 LELA3150 JR32icmB3150::Tn903dIIlacZ, DNA hybridization group IV 28 LELA3323 JR32icmB3323::Tn903dIIlacZ, DNA hybridization group IV 28 LELA1012 JR32icmB1012::Tn903dIIlacZ, DNA hybridization group VI 28 LELA2947 JR32icmB2947::Tn903dIIlacZ, DNA hybridization group VI 28 LELA1275 JR32icmF1275::Tn903dIIlacZ, DNA hybridization group XVII 28 LELA1718 JR32icmF1718::Tn903dIIlacZ, DNA hybridization group XVII 28 MW635 JR32icmG::Kmr This study MW645 JR32 icmC::Kmr This study MW656 JR32icmJ::Kmr This study MW627 JR32 tphA::Kmr This study Philadelphia-1 Virulent, serogroup 1 16 E. coli DH5α F−endA1 hsdRI7(r−k m+k) supE44 thi-1 λ− recA1 relA1 Δ(argF-lacZYA) U169 φ80dlacZΔM15 deoR gyrA96Nalr 33 LE392 F− e14− (McrA−)hsdR514 (r−km+k) supE44 supF58 lacY1 or Δ(lacIZY)6 galK2 galT22 metB1 trpR55 3 LW211 LE392 with an integrated RP4 (Δbla tetA::Mu) transferred by P1 transduction from SM10; Tra+ KmrApr Tcs 28 MW67 DH5α containing a L. pneumophilaPhiladelphia-1 chromosomal DNA library consisting of partialEcoRI fragments (10–20 kb) in pMMB207 This study - Table 2.
Plasmids used in this study
Plasmid Genotype and features Reference or source pAB13 Tn903dIIlacZ-containingEcoRI fragment from LELA1012 in pBSK KS+ 28 pAB14 Tn903dIIlacZ-containingEcoRI fragment from LELA1566 in pBSK KS+ 28 pBluescript II KS (+) oriR (f1) MCS oriR (ColE1), Apr Stratagene pBC SK (+) oriR (f1) MCS oriR (ColE1), Cmr Stratagene pBR322 oriR (ColE1), AprTcr New England Biolabs pCR2.1 oriR(f1) MCS oriR (ColE1), Apr Kmr Invitrogen pMMB207 oriR (RSF1010), Cmr 25 pMMB207αb pMMB207 MCS, Cmr 30 pMMB207αb::Gm pMMB207 MCS, Gmr This study pMW100 icmEGCDJB-, tphA-, andicmF-containing fragment in pMMB207 This study pMW150 Tn903dIIlacZ-containingEcoRI fragment from LELA3563 in pMMB207 This study pMW152 Tn903dIIlacZ-containingEcoRI fragment from LELA1223 in pMMB207 This study pMW318 Tn903dIIlacZ-containingEcoRI fragment from LELA1996 in pMMB207 This study pMW320 Tn903dIIlacZ-containingEcoRI fragment from LELA1506 in pMMB207 This study pMW420 1,864-bp icmJ-containing fragment in pBSK KS+ This study pMW424 2,892-bp icmGCD-containing fragment in pBSK KS+ This study pMW525 3,257-bpHindIII fragment from pMW100 in pBSK KS+ This study pMW528 icmB-containing fragment in pBSK KS+ This study pMW560 icmB-containing fragment in pBC SK+ This study pMW564 897-bp icmC-containing fragment in pCR2.1 This study pMW565 811-bpicmD-containing fragment in pCR2.1 This study pMW566 1,495-bp icmG-containing fragment in pCR2.1 This study pMW576 2,004-bp tphA-containing fragment in pUC18 This study pMW582 pMW424 with aKpnI site at bp 2993 This study pMW584 2,540-bpicmGCD-containing fragment in pUC18 This study pMW587 pMW420 containing ΔicmJ with aSalI site at bp 2993 This study pMW589 pMW576 containing ΔtphA with a SalI site at bp 6862 This study pMW591 pMW584 containing ΔicmGwith a SalI site at bp 995 This study pMW593 pMW584 containing ΔicmC with aSalI site at bp 1831 This study pMW596 pMW589 with Km cassette inserted in ΔtphA This study pMW598 pMW591 with Km cassette inserted in ΔicmG This study pMW600 pMW593 with Km cassette inserted in ΔicmC This study pMW602 1,331-bpHindIII-BamHI fragment from pMW587 in pUC18 This study pMW604 2,540-bp icmGCD-containing fragment in pMMB207 This study pMW606 pMW602 with Kmr cassette inserted in ΔicmJ This study pMW616 ΔtphA::Kmr in pLAW344 This study pMW618 ΔicmG::Kmr in pLAW344 This study pMW620 ΔicmC::Kmr in pLAW344 This study pMW622 ΔicmJ::Kmr in pLAW344 This study pMW680 1,864-bp icmJ-containing fragment in pBC, opposite orientation to PlacUV5 This study pMW681 1,864-bpicmJ-containing fragment in pBC, same orientation to PlacUV5 This study pMW728 897-bpicmC-containing fragment in pMMB207αb, same orientation to Ptac This study pMW730 897-bpicmC-containing fragment in pMMB207αb, opposite orientation to Ptac This study pMW734 811-bpicmD-containing fragment in pMMB207αb, same orientation to Ptac This study pMW736 811-bpicmD-containing fragment in pMMB207αb, opposite orientation to Ptac This study pMW741 1,495-bpicmG-containing fragment in pMMB207αb, same orientation to Ptac This study pMW743 1,495-bpicmG-containing fragment in pMMB207αb, opposite orientation to Ptac This study pMW790 1,894-icmJ-containing fragment in pMMB207αb::Gm, opposite orientation to Ptac This study pLB41 HindIII fragment containing Gmr in pBR322 L. Babiss and D. Figurski pUC18 oriR(ColE1) MCS, Apr New England Biolabs - Table 3.
Characteristics of predicted ORFs
Potential ORF identified No. of amino acidsa Mol mass (kDa)b Predicated cellular locationc Predicted motifsd Predicted homologye icmE icmG 269 29.7 IM (1 TM) None None icmC 194 20.3 IM (4 TM) None None icmD 132 13.5 IM (1 TM) None None icmJ 212 24.2 IM (1 TM) None None icmB 1,009 112.1 IM (1 TM) ATP/GTP binding site None tphA 418 46.4 IM (12 TM) Sugar transport protein ProP icmF 973 110.7 IM (2 TM) ATP/GTP binding site None ↵a The number of amino acids encoded by each complete ORF is reported. icmE is partial on pMW100; therefore the total number of amino acids is not reported.
↵b The predicted molecular mass of each complete ORF is shown. The molecular mass of the partial icmE cannot be determined.
↵c The location of each ORF was predicted by the Psort program (26). IM designates an inner membrane location. The number of transmembrane domains (TM) as determined by the Psort program is shown in parentheses.
↵d Motif searches were performed with the GCG motif search program.
↵e Homology searches were performed at the amino acid and nucleotide levels by using the TFASTA, BLASTP, and BLASTX (18) programs.
