Angewandte
Chemie
DOI: 10.1002/anie.201108214
Natural Products
Antiterminator-Mediated Unveiling of Cryptic Polythioamides in an
Anaerobic Bacterium**
Swantje Behnken, Thorger Lincke, Florian Kloss, Keishi Ishida, and Christian Hertweck*
The genus Clostridium comprises a highly heterogeneous
array of obligate anaerobic organisms that inhabit diverse
ecological niches, ranging from soil to human intestines.
These microorganisms have been extensively studied, not
only because of their ability to produce useful solvents, but
also because some species produce harmful protein toxins
such as botulinum toxin.[1,2] However, despite the large body
of knowledge on clostridia, until recently no secondary
metabolites have been isolated from these or any other
strict anaerobes. Yet, mining the sequenced genomes of
Clostridium species has revealed a widespread occurrence of
secondary metabolism genes, in particular polyketide syn-
thase and non-ribosomal peptide synthetase genes.[3,4] We
have shown that the encoded pathways remain silent under
standard laboratory conditions.[4] Obviously, such cryptic
biosynthesis genes are only activated in the presence of
particular stimuli.[5,6] This is plausible, because secondary
metabolite pathways require much ATP-bound energy, a rare
commodity in the anaerobic world. We recently discovered
that the addition of aqueous soil extract to a C. cellulolyticum
culture activates an otherwise silent metabolic pathway,
leading to a wholly unprecedented type of polythioamide
named closthioamide (1; Scheme 1).[7] Apart from its excep-
tional structural features, closthioamide is highly active
against a variety of bacteria, such as vancomycin-resistant
enterococci (VRE) and methicillin-resistant Staphylococcus
aureus (MRSA), and is the first antibiotic from a strictly
anaerobic bacterium.[7] However, the variable nature of soil
constrains constant production rates and hampers the com-
plicated fermentation of these organisms. As a result, various
minor closthioamide congeners evaded isolation and full
characterization, and biosynthetic studies were unapproach-
able. Herein we present a new strategy using an antitermi-
nator gene to trigger a cryptic biosynthetic pathway and
disclose the structures and antibacterial activities of seven
novel polythioamide congeners. Besides revealing structure–
activity relationships, we used synthetic closthioamide ana-
logues as probes to gain an initial insight into the biogenetic
relationships of the natural polythioamides.
An established approach for the targeted induction of
secondary metabolite biosynthesis is the manipulation of
pathway-specific regulatory genes.[5,8–10] However, as the
closthioamide biosynthesis gene locus still remains obscure,
this approach was out of reach. As an alternative, we focused
on regulatory elements that could be involved in a more
global activation of secondary metabolism.[6–11] When analyz-
ing the C. cellulolyticum genome, we noted the presence of
a putative antiterminator gene, nusG. N-utilizing factor G
(NusG) is an essential protein in E. coli that can increase the
overall rate of transcription. NusG has the ability to decrease
the occupancy of some of the paused RNA-polymerase
(RNAP) complexes by promoting the forward translocation
of RNAP.[12] NusG also enables RNAP to read through
transcription-terminating 1-dependent sites (Figure 1A),
especially in rrn operons, and plays a role in translation
owing to the presence of the Kyprides–Onzonis–Woese
(KOW) motif it shares with ribosomal protein families.[13]
Increasing the processivity of RNA polymerase aids in the
efficient synthesis of the corresponding transcripts and
produces polycistronic mRNA, which results in elevated
protein production. Thus, increasing the nusG expression rate
could potentially activate secondary metabolism in C. cellu-
lolyticum.
Scheme 1. Structure of closthioamide (1), the first antibiotic from
a strictly anaerobic bacterium, Clostridium cellulolyticum.[7]
[*] Dipl.-Biochem. S. Behnken, Dr. T. Lincke, Dipl.-Chem. F. Kloss,
Dr. K. Ishida, Prof. Dr. C. Hertweck
Leibniz Institute for Natural Product Research and Infection
Biology, HKI, Dept. of Biomolecular Chemistry, and Bio Pilot Plant
Beutenbergstrasse 11a, 07745 Jena (Germany)
E-mail: christian.hertweck@hki-jena.de
To generate a C. cellulolyticum mutant overexpressing the
antiterminator gene, nusG was PCR-amplified from genomic
DNA, subcloned and sequenced. It was then cloned down-
stream of the strong and constitutive promoter Pthl into the
high-copy vector pSOS95,[14] yielding E. coli–Clostridium
shuttle plasmid pSB050, which was introduced into C. cellu-
lolyticum. The successful transformation was verified by PCR
and plasmid re-isolation and restriction analysis.[15] To test the
ability of pSB050 to promote overexpression of the nusG
gene, reverse-transcription-quantitative polymerase chain
reaction (RT-qPCR) experiments were performed using
total RNA isolated from logarithmically growing cultures of
Prof. Dr. C. Hertweck
Friedrich Schiller University, Jena (Germany)
[**] We thank A. Perner for MS measurements and E. T. Papoutsakis for
providing plasmid pSOS95. We are grateful to U. Knꢀpfer for
support in fermentations. This work was supported by the “Pakt fꢀr
Forschung und Innovation” of the Free State of Thuringia and the
BMBF, and the International Leibniz Research School for Microbial
and Biomolecular Interactions (ILRS), as part of the excellence
graduate school “Jena School for Microbial Communication”
(JSMC).
Supporting information for this article, including full experimental
Angew. Chem. Int. Ed. 2012, 51, 2425 –2428
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2425