The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii - PubMed (original) (raw)

The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii

L M Hales et al. J Bacteriol. 1999 Aug.

Abstract

To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.

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Figures

FIG. 1

FIG. 1

Schematic diagram of the ORFs (indicated by arrows) encoded on pLM507. At the top is a restriction map of pLM507. Restriction sites: R, _Eco_RI; S, _Sal_I; H, _Hin_dIII; P, _Pst_I; V, _Eco_RV. Plasmid pLM507 contains two _Eco_RI fragments 7.2 and 0.8 kb in size. It is not known if these fragments are contiguous on the L. pneumophila chromosome, and the 0.8-kb fragment was not studied. A _Pst_I digest of pLM507 produced two fragments 3.2 and 2.5 kb in size. One of the _Pst_I sites of the 2.5-kb fragment is from the vector pMMB207 and is 39 bp from the _Eco_RI site of L. pneumophila DNA. The 5,658-bp L. pneumophila contiguous _Pst_I-_Eco_RI fragment is represented below pLM507. An enlarged view of the ORFs (represented as arrows) is shown. The gene names given the respective ORFs are based on homology from searches of the GenBank database (Table 2).

FIG. 2

FIG. 2

Western blot analysis of crude cell extracts. Six times more L. pneumophila than E. coli crude extract was loaded. (A) Relative amounts of E. coli RpoS from log-phase (lanes 1, 3, and 5) and stationary-phase (lanes 2, 4, and 6) cultures of LM5000 (lanes 1 and 2), LM5005 (lanes 3 and 4), and LM5005 containing pLM806 (lanes 5 and 6). (B) Relative amounts of L. pneumophila RpoS from log-phase (lanes 1, 3, and 5) and stationary-phase (lanes 2, 4, and 6) cultures of JR32 (lanes 1 and 2), LM1376 (lanes 3 and 4) and LM1376 containing pLM806 (lanes 5 and 6).

FIG. 3

FIG. 3

Growth curves of wild-type JR32 (squares) and rpoS null strain LM1376 (diamonds) in AYE medium. (A) Absorbance at 600 nm, measured every 2 to 4 h during a 64-h time course, plotted as a function of time; (B) CFU, measured by plating dilutions of bacterial strains on ABCYE plates during the same time course, plotted as a function of time.

FIG. 4

FIG. 4

Ability of L. pneumophila strains to survive under stress. The log of the percent survival of each culture is plotted as a function of time. Individual experiments were performed two to four times, and the results of one representative experiment are shown. (A) Survival of log-phase (filled squares) and stationary-phase (open squares) JR32 at pH 3. (B) Survival of log-phase (filled squares) and stationary-phase (open squares) JR32 in the presence of 10 mM hydrogen peroxide. (C) Survival of log-phase (filled squares) and stationary-phase (open squares) JR32 in the presence of 5 M sodium chloride. (D) Survival of log-phase (filled squares) and stationary-phase (open squares) LM1376 at pH 3. (E) Survival of log-phase (filled squares) and stationary-phase (open squares) LM1376 in the presence of 10 mM hydrogen peroxide. (F) Survival of log-phase (filled squares) and stationary-phase (open squares) LM1376 in the presence of 5 M sodium chloride.

FIG. 5

FIG. 5

Growth of L. pneumophila strains in eukaryotic hosts. Assays were performed in triplicate; the standard error bars indicate standard errors of the means and may not be visible. (A) Replication of wild-type JR32 (open squares), mutant 25D (circles), and rpoS null strain LM1376 (filled squares) in HL-60-derived macrophages. A monolayer of HL-60 cells was differentiated and infected with L. pneumophila bacteria at an MOI of 0.01. Infection wells were sacrificed, and the net log number of CFU was plotted as a function of time. (B and C) Cytotoxicity of HL-60 (B)- and THP-1 (C)-derived macrophages by wild-type JR32 (open squares), mutant 25D (circles), and rpoS null strain LM1376 (filled squares). An MTT assay was performed, the number of viable macrophages 5 days after infection with L. pneumophila was measured, and the absorbance at 570 nm was plotted as a function of the log of the number of bacteria in the wells. (D) Replication of wild-type JR32 (open squares), mutant 25D (circles), rpoS null strain LM1376 (filled squares), rpoS null strain LM1376 containing pLM806 (triangles), and JR32 containing pLM806 (hatched squares) within A. castellanii. CFU were measured from infection supernatants, and net log growth was plotted as a function of time.

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