Bacterial
persisters, a dormant and multidrug tolerant subpopulation
that are able to resuscitate after antibiotic treatment, have recently
received considerable attention as a major cause of relapse of various
infectious diseases in the clinic. However, because of their low abundance
and inherent transience, it is extremely difficult to study them by
proteomics. Here we developed a magnetic-beads-based separation approach
to enrich Escherichia coli persisters and then subjected
them to shotgun proteomics. Rifampin pretreatment was employed to
increase persister formation, and the resulting cells were exposed
to a high concentration of ampicillin (10× MIC) to remove nonpersisters.
The survivors were analyzed by spectral counting-based quantitative
proteomics. On average, 710 proteins were identified at a false discovery
rate of 0.01 for enriched E. coli persisters. By
spectral counting-based quantification, 105 proteins (70 down-regulated,
35 up-regulated) were shown to be differentially expressed compared
with normal cells. A comparison of the differentially expressed proteins
between the magnetic beads-enriched persisters and nonenriched persisters
(a mixture of persisters and intact dead cells) shows only around
half (∼58%) overlap and different protein–protein interaction
networks. This suggest that persister enrichment is important to eliminate
the cumulative effect of dead cells that will obscure the proteome
of persisters. As expected, proteins involved in carbohydrate metabolism,
fatty acid and amino acid biosynthesis, and bacterial chemotaxis were
found to be down-regulated in the persisters. Interestingly, membrane
proteins including some transport proteins were up-regulated, indicating
that they might be important for the drug tolerance of persisters.
Knockout of the pal gene expressing peptidoglycan-associated
lipoprotein, one of the most up-regulated proteins detected in persisters,
led to 10-fold reduced persister formation under ampicillin treatment.
In biosynthesis of natural products, potential intermediates or analogs of a particular compound in the crude extracts are commonly overlooked in routine assays due to their low concentration, limited structural information, or because of their insignificant bio-activities. This may lead into an incomplete and even an incorrect biosynthetic pathway for the target molecule. Here we applied multiple compound mining approaches, including genome scanning and precursor ion scan-directed mass spectrometry, to identify potential pyrrolamide compounds in the fermentation culture of Streptomyces netropsis. Several novel congocidine and distamycin analogs were thus detected and characterized. A more reasonable route for the biosynthesis of pyrrolamides was proposed based on the structures of these newly discovered compounds, as well as the functional characterization of several key biosynthetic genes of pyrrolamides. Collectively, our results implied an unusual “iterative strategy” underlying the pyrrole polymerization in the biosynthesis of pyrrolamide antibiotics.
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