Table 1.
Bacterial strains used in this study.
Figure 1.
Major steps of the polyamine metabolism in E. coli.
The diagram depicts the pathway of polyamine biosynthesis in E. coli. Data are drawn from Ecocyc database [39]. Dotted boxes: precursor aminoacids. AdoMet dec: S-Adenosyl-L-Methioninamine. MTA: S-Methyl-5′-Thioadenosine.
Table 2.
Analysis of polyamine content in different EIEC and S. flexneri strains.
Figure 2.
Molecular and functional analysis of the ynfB-speG operon in EIEC.
(A) Genetic organization of the ynfB-speG operon in EIEC strains. The operon at the top is based on the E. coli K-12 MG1655 sequence as reported in (National Center of Biotechnology Information, Bethesda, Md.). The right-pointing arrow marks the transcription start site (+1). The asterisk (*) indicates genes carrying non-synonymous mutations. The nucleotide sequence at the bottom refers to the IS2 insertion into the ynfB-speG promoter of EIEC HN280 (IRL: left-end inverted repeat of IS2. Underlined: direct repeat at the IS2 insertion site). (B) Relative speG transcription in EIEC strains as monitored by in vivo Real-Time PCR using MG1655 as a control. Strains were grown at 37°C in M9 complete medium. At least three wells were run for each sample and the error bars display the calculated maximum (RQMax) and minimum (RQMin) expression levels that represent standard error of the mean expression level (RQ value). * denotes 0.05>p≥0.01; ** denotes p<0.01. (C) Primer extension analysis of the transcripts generated under the control of the ynfB-speG promoter of EIEC strains HN280, 13.80, and 4608. The autoradiograph is the result of a typical experiment performed with the peg primer on RNA extracted from strain 4608 (lane 1), 13.80 (lane 2), and HN280 (lane 3). Lanes G, A, T, and C show the sequencing ladder generated with the same primer. The asterisk marks the transcription start site (+1).
Figure 3.
Effect of speG on the sensitivity to oxidative stress.
The sensitivity to H2O2 was assayed on EIEC strain HN280 (wild type), on its speG-deleted derivative HN280DG, on HN280 complemented with a plasmid carrying speG under the control of its own promoter (pULS37) or of the Ptac promoter (pULS13), on strain 53638 (wild type), and on its speG defective derivative 53638 DG. Survival in the presence of H2O2 was measured after exposure of strains, grown in M9 complete medium, to H2O2 for 1 h at 37°C. The survival percentage is relative to growth without exposure to H2O2. Error bars indicate the standard deviations relative to at least three independent experiments. ** denotes p<0.05.
Figure 4.
Expression of speC in EIEC strains.
The in vivo speC transcription was monitored by Real-Time PCR and referred to the level observed in the MG1655 control. Strains were grown at 37°C in M9 complete medium. At least three wells were run for each sample and the error bars display the calculated maximum (RQMax) and minimum (RQMin) levels that represent standard error of the mean expression level (RQ value); * denotes 0.05>p≥0.01; ** denotes p<0.01.
Table 3.
Analysis of the polyamines levels in EIEC strains complemented or not with a functional copy cadC gene.
Figure 5.
Cadaverine interferes with speC expression.
Experiments were performed using strains lacking only the cadC gene (EIEC HN280, 4608 and 53638 and E. coli K-12 ULS655) or the entire cad operon (EIEC 13.80 and 6.81). Strains were transformed with pCC55 (a pACYC184 derivative containing a functional cadC gene) or pACYC184. (A) Transcription of speC in the presence or absence of cadaverine as monitored by Real-Time PCR. At least three wells were run for each sample and the error bars display the calculated maximum (RQMax) and minimum (RQMin) levels that represent standard error of the mean expression level (RQ value). * denotes 0.05>p≥0.01; ** denotes p<0.01 (B) Ornithine decarboxylase (ODC) activity in the presence or absence of cadaverine. ODC activity was measured in total cell extracts by assaying putrescine production. The synthesized putrescine was made fluorescent through chemical modification and separated by TLC. The fluorimetric data were normalized against the total protein content of each sample. Results are shown as variation (percentage) in strains carrying pCC55 (cadC) vs the corresponding wt strain carrying the backbone construct. * denotes 0.05>p≥0.01; ** denotes p<0.01. (C) Transcription of speC in E. coli K-12 (MG1655); ** denotes p<0.01. Transcription in strains producing or lacking cadaverine was monitored by Real-Time PCR as in panel A.
Figure 6.
A model of the pathoadaptive evolution of the polyamine profile in EIEC and Shigella spp.
In this view the loss of cad genes marks the transition from the polyamine profile of the commensal E. coli ancestor to that of enteroinvasive E. coli (EIEC), characterized by lack of cadaverine and increased putrescine content. The loss of the speG gene, occurring in a successive evolutionary step and inducing spermidine accumulation (and, consequently, higher resistance to oxidative stress), gives rise to the Shigella-type polyamine profile.