LETTER
In a recent paper, Nigam and colleagues analyzed the stress-related effects of the
endoribonuclease toxin MazF on the Escherichia coli proteome (1). The authors from
the lab of Hanna Engelberg-Kulka—the discoverer of the mazEF toxin-antitoxin system
(2)—claim that MazF creates a unique stress-induced translation machinery (STM). The
STM hypothesis states that the toxin cleaves selected mRNAs within 5′-leader sequences
to produce a pool of leaderless transcripts that are, in turn, translated by special
stress ribosomes (3, 4). The latter are formed when the toxin cleaves off an anti-Shine-Dalgarno
sequence-containing fragment from the 3′ end of 16S rRNA in mature ribosomes (3).
Thus, MazF is postulated to reshape translation in stressed E. coli similarly to how
the σS factor reshapes transcription.
Independent studies failed to support these findings. Transcriptome-wide mapping of
the cleavage sites indicated that MazF cleaves most transcripts within their coding
regions and produces very few full-length, leaderless mRNAs (5, 6). Contradicting
the STM model, MazF does not cleave rRNA in mature, fully assembled ribosomes but
instead targets rRNA precursors (5, 7). Finally, stable isotope labeling by amino
acids in cell culture (SILAC)-based proteomics revealed that MazF generally inhibits
protein synthesis and no proteins are selectively synthesized in response to the toxin
(6). This result is at odds with the paper of Nigam and coworkers (1), who also used
SILAC proteomics and report a group of 42 MazF-mediated, stress-induced E. coli proteins.
Here we reanalyze their data and highlight several technical issues.
The setup of the proteomics experiment and the lack of statistical analysis make it
impossible to determine whether the reported differences in proteomes were caused
by MazF or random fluctuations. The authors aimed to test which proteins are synthesized
in the ΔmazEF mutant and its wild-type (wt) parent strain upon treatment with the
quinolone antibiotic nalidixic acid (NA). To do that, Nigam et al. (1) grew bacteria
in the light medium, added NA to the culture, and after 10 min, added heavy lysine
and arginine in order to label the new proteins. The relative amounts of newly synthetized
proteins were estimated based on heavy and light isotope ratio (H/L ratio) after an
additional 5-min incubation. The experiment was repeated three times. The short length
of pulse labeling resulted in low H/L values, which could, possibly, account for the
high variability of results (see below). While the authors state that they “checked
several time points and deduced that 5 min is the optimal time point to figure out
which are the differential new proteins,” they do not present the relevant supporting
data. The authors state that the differences between the wt and ΔmazEF proteomes are
specifically induced by stress but do not provide an essential control, i.e., proteomic
analysis of these strains without NA treatment. Nigam and colleagues admit the lack
of statistical analysis and, instead, chose all the proteins “that were induced more
in the WT than in the mazEF mutants in all the repeats” as differentially expressed.
They further state that “as the purpose of the study was to identify the new proteins
rather than to calculate the turnover of the proteins, no complex statistical test
was used and no logarithmic transformation was done”. We statistically reanalyzed
the data to control for the false-positive rate of assignment into the group of differentially
expressed proteins. We found similar levels of covariation between the intrastrain
replicate experiments and interstrain comparisons (Fig. 1A), while no spike of small
P values appeared on the P value histogram obtained from Student’s t test of log2-transformed
data (Fig. 1B). This result is consistent with the null hypothesis of no differentially
expressed proteins, which results in a flat distribution of P values. A volcano plot
demonstrates an almost equal number of overexpressed and less-expressed heavy proteins
in the ΔmazEF mutant strain compared to the wild type, while no P values surpass the
Bonferroni-corrected significance level (Fig. 1C). We also could not detect any differentially
expressed proteins at a false-discovery rate (FDR) of 0.1 using a less conservative
Benjamini-Hochberg method. The lowest q-value for a particular protein was 0.88, which
means that we can accept this protein as differentially expressed only at a 0.88 false-discovery
rate level.
FIG 1
Statistically significant, differentially abundant proteins were not detected upon
reanalysis of the proteomics data of Nigam and colleagues (1). H/L ratios of the 192
proteins, which were measured in all three replicate experiments in both E. coli MC4100
relA
+ and ΔmazEF relA
+ strains, were taken from Table S1 in reference 1 and analyzed using the Perseus
computational platform (11). (A) R
2 coefficients of determination for individual experiments. (B) Histogram of the Student’s
t test P values. (C) Volcano plot showing differences between the median H/L ratios
of individual proteins and their statistical significance. The horizontal dotted line
denotes the Bonferroni-corrected (P = 0.0003) significance level.
The technical issues compromising the SILAC analysis are further confounded by the
lack of experimental validation of MazF activation and cutting of the mRNA leader
sequences at the listed sites (see Table 1 in reference 1) upon NA treatment. NA targets
type II topoisomerases but does not inhibit RNA or protein synthesis and is not expected
to stop production of the MazE antitoxin to activate the toxin. The authors refer
to a paper that reports NA-triggered, MazF-mediated programmed cell death (PCD) but
does not present evidence of RNA fragmentation (8). Other researchers could not reproduce
the mazEF-dependent PCD (9) and have found that the E. coli MC4100 relA
+ and ΔmazEF relA
+ strains used by Nigam et al. harbor a frameshift mutation in relA and are phenotypically
relA deficient (relaxed, relA mutant [9, 10]). Inactivation of the relA-mediated stringent
response, a central mechanism of stress adaptation, further complicates interpretation
of the results.
Finally, we note the absence of citations to papers critical of the STM hypothesis
(5
–
7).