Biosynthesis of the acetyl-CoA carboxylase-inhibiting
antibiotic, andrimid in Serratia is regulated by Hfq and
the LysR-type transcriptional regulator, AdmX
Miguel A. Matilla,1,2* Veronika Nogellova,1
Bertrand Morel,2 Tino Krell2 and
George P. C. Salmond1**
1Department of Biochemistry, University of Cambridge,
Tennis Court Road, Cambridge, CB2 1QW, UK.
2Department of Environmental Protection, Estacion
Experimental del Zaidın, Consejo Superior de
Investigaciones Cientıficas, Prof. Albareda 1, Granada
18008, Spain.
Summary
Infections due to multidrug-resistant bacteria repre-
sent a major global health challenge. To combat this
problem, new antibiotics are urgently needed and
some plant-associated bacteria are a promising
source. The rhizobacterium Serratia plymuthica A153
produces several bioactive secondary metabolites,
including the anti-oomycete and antifungal hateruma-
lide, oocydin A and the broad spectrum polyamine
antibiotic, zeamine. In this study, we show that A153
produces a second broad spectrum antibiotic, andri-
mid. Using genome sequencing, comparative
genomics and mutagenesis, we defined new genes
involved in andrimid (adm) biosynthesis. Both the
expression of the adm gene cluster and regulation of
andrimid synthesis were investigated. The biosyn-
thetic cluster is operonic and its expression is
modulated by various environmental cues, including
temperature and carbon source. Analysis of the
genome context of the adm operon revealed a gene
encoding a predicted LysR-type regulator, AdmX,
apparently unique to Serratia strains. Mutagenesis
and gene expression assays demonstrated that
AdmX is a transcriptional activator of the adm gene
cluster. At the post-transcriptional level, the expres-
sion of the adm cluster is positively regulated by the
RNA chaperone, Hfq, in an RpoS-independent man-
ner. Our results highlight the complexity of andrimid
biosynthesis – an antibiotic with potential clinical
and agricultural utility.
Introduction
The discovery of antibiotics is one of the main milestones
in the history of medicine. However, excessive overuse of
antibiotics has encouraged the emergence of multidrug-
resistant bacteria, leading to a global increase in the spec-
trum of untreatable infections, which are currently
responsible for around 50,000 annual deaths in Europe
and the United States (Woodford et al., 2011; Blair et al.,
2015). There is therefore an urgent need to identify new
antibiotics, but efforts focussed on discovery and develop-
ment of new antibiotics have met with only limited success
(Lewis, 2013; Pidot et al., 2014). New platforms for antibi-
otic discovery include the generation of synthetic
antimicrobials and development of species-specific antibi-
otics (Fischbach and Walsh, 2009; Lewis, 2013; Liu et al.,
2013; Pidot et al., 2014). It has been estimated that 90%
of microbial natural products, and more than 99% of the
total number of secondary metabolites, remain to be dis-
covered. Consequently, recent approaches to antibiotic
discovery include screening of microbes from new ecologi-
cal niches and attempts at exploitation of previously
uncultured microbes (Fischbach and Walsh, 2009).
Natural products (and their synthetic derivatives) com-
prise most of the antibiotics used clinically (Newman and
Cragg, 2007), many of which are based on non-ribosomal
peptides and/or polyketides. Both families of secondary
metabolites are synthesised by multifunctional enzymes,
known as non-ribosomal peptide synthetases (NRPSs)
and polyketide synthases (PKSs), through sequential
rounds of condensation of amino acids and acyl-CoA build-
ing units, respectively (Sattely et al., 2008; Hertweck,
2009). The great structural diversity of non-ribosomal pep-
tides and polyketides results from the number of
condensed building units and a range of pre- and post-
assembly processing reactions (Sattely et al., 2008; Hert-
weck, 2009). This chemical diversity is consequently
reflected in a broad spectrum of biological activities
Received 27 November, 2015; accepted 20 January, 2016. *For
correspondence. E-mail miguel.matilla@eez.csic.es; Tel. 134 958
181600; Fax 134 958 135740. **E-mail gpcs2@cam.ac.uk; Tel.
144 (0)1223 333650; Fax 144 (0)1223 766108.
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
Environmental Microbiology (2016) 18(11), 3635–3650 doi:10.1111/1462-2920.13241
(Sattely et al., 2008; Fischbach and Walsh, 2009; Hertweck,
2009; Pidot et al., 2014; Mousa and Raizada, 2015).
Some bacteria can devote up to 10% of their genomes
to secondary metabolism (Udwary et al., 2007; Nett et al.,
2009; Chowdhury et al., 2015). The biological synthesis of
such metabolites can be energetically costly and so pro-
duction is generally highly regulated (Coulthurst et al.,
2005; Williamson et al., 2006; Liu et al., 2013). Biosyn-
thetic gene clusters are often linked to their own regulatory
genes (Chen et al., 2006; Zhao et al., 2010; Gurney and
Thomas, 2011; Liu et al., 2013), the products of which are
involved in sensing factors such as physiological state,
population density and diverse environmental cues. As a
result, the synthesis of the cognate secondary metabolite
can be modulated appropriately. Quorum sensing regula-
tory circuits (Coulthurst et al., 2005; Williamson et al.,
2006; M€uller et al., 2009; Matilla et al., 2015), two-
component systems (Sola-Landa et al., 2003; Haas and
Defago, 2005; Williamson et al., 2006), orphan transcrip-
tional regulators (Williamson et al., 2006; Lu et al., 2011;
Klaponski et al., 2014) and post-transcriptional regulators
(Vogel and Luisi, 2011; Romeo et al., 2013) can all be
involved in the regulatory complexity of bacterial secondary
metabolite control.
The rhizosphere is one of the most complex environ-
ments on earth, with many organisms interacting and
competing for nutrients and space (Lugtenberg and
Kamilova, 2009; Mendes et al., 2013). Many rhizosphere
microbes have evolved the capacity to synthesize bioactive
secondary metabolites that allow them to efficiently antag-
onize diverse niche competitors (Berg et al., 2002;
De Vleesschauwer and H€ofte, 2007; Raaijmakers et al.,
2009; Pidot et al., 2014; Mousa and Raizada, 2015). Con-
sequently, this defines the rhizosphere as a habitat with
great potential for exploitation as a source of new natural
products with pharmacological, chemotherapeutic and
agricultural applications.
Serratia plymuthica strains are near-ubiquitous in nature
but have been commonly isolated from soil and the
rhizosphere of many economically important crops
(De Vleesschauwer and H€ofte, 2007). Serratia plymuthica
strains possess great potential as biocontrol agents by
antagonizing the growth of plant-pathogens through the
production of diverse bioactive secondary metabolites,
siderophores and lytic enzymes (Alstr€om, 2001;
De Vleesschauwer and H€ofte, 2007; Matilla et al.,
2015). The strain used in this study, Serratia plymuthica
A153, was isolated from the rhizosphere of wheat
(A˚strom and Gerhardson, 1988) and it has been shown
to possess bioactivity against fungi, oomycetes, bacteria
and nematodes (Thaning et al., 2001; Matilla et al.,
2012; Hellberg et al., 2015). These activities are mainly
due to the synthesis of NRPS- and PKS-based second-
ary metabolites, such as the haterumalide, oocydin
A (Thaning et al., 2001), and the polyamine antibiotic,
zeamine (Hellberg et al., 2015).
Our previous work showed that the strain A153, in addi-
tion to zeamine, produces an unidentified second
antibacterial compound (Hellberg et al., 2015). In this
study, we employed genome sequencing, comparative
genomics and mutagenesis approaches to identify the
genes involved in the biosynthesis of the unknown second-
ary metabolite. The regulation of the production of the
antibacterial compound was also investigated and the
results showed that the expression of the biosynthetic
genes is tightly regulated at transcriptional and post-
transcriptional levels. Different environmental cues control-
ling the transcription of the biosynthetic genes were also
identified.
Results
Serratia plymuthica A153 produces the hybrid
non-ribosomal peptide-polyketide antibiotic, andrimid
Characterization of the biocontrol rhizobacterium, S. ply-
muthica A153, showed that this strain possesses a strong
bioactivity against Bacillus subtilis (Fig. 1A). The observed
antibacterial activity was not associated with the produc-
tion of other known bioactive secondary metabolites
produced by A153, namely oocydin A (Matilla et al., 2012)
or zeamine (Hellberg et al., 2015).
During the in silico analysis of the A153 genome
sequence (Matilla et al., 2016) we identified at least five
candidate biosynthetic PKS and NRPS gene clusters
which could be responsible for the synthesis of the
unknown antibacterial compound. To identify the genes
responsible for this bioactivity, a random transposon inser-
tion strain library was constructed and screened for
mutants defective in antibacterial activity against Bacillus
subtilis. Several transposon insertion mutants showing
loss of antibacterial properties were isolated and all the
insertions were transduced back into the wild type genetic
background using the transducing phage /MAM1 (Matilla
and Salmond, 2014) to confirm the link between transpo-
son insertions and mutant phenotype. Random primed
PCR confirmed that most of the transposons were located
in a hybrid PKS/NRPS gene cluster described previously
as responsible for the biosynthesis of the broad-spectrum
antibiotic, andrimid (Figs. 1 and Supporting information
Fig. S1) (Jin et al., 2006).
We had access to another plant-associated oocydin A
producing strain, Serratia marcescens MSU97. This strain
also showed strong antibacterial activity towards B. subtilis
(Supporting information Fig. S2) and sequencing of its
genome (Matilla and Salmond, unpubl. data) revealed that
the andrimid (adm) gene cluster is also present in this plant
epiphytic bacterium (Supporting information Fig. S3). We
reported previously that MSU97 is recalcitrant to various
3636 M. A. Matilla et al.
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Environmental Microbiology, 18, 3635–3650
genetic tools (Matilla et al., 2012) and attempts at isolating
mutants defective in the adm gene cluster of this strain
were unsuccessful.
Comparative analyses of sequenced andrimid gene
clusters
The biosynthesis of andrimid has been demonstrated in a
broad range of bacteria (Fredenhagen et al., 1987; Long
et al., 2005; Jin et al., 2006; Wietz et al., 2011; Sanchez
et al., 2013). However, few adm gene clusters have been
sequenced, including those of the marine bacteria, Vibrio-
nales SWAT-3 (PATRIC Genome ID 391574.12) and Vibrio
coralliilyticus S2052 (Machado et al, 2015), and the plant-
associated enterobacterium Pantoea agglomerans Eh335
(Jin et al., 2006; GenBankTM Accession No. AY192157.1).
Additionally, our genomic analyses revealed that the adm
gene cluster is also present in the recently sequenced rhi-
zobacterium Serratia marcescens 90-166 (Supporting
information Figs. S3 and S4) (Jeong et al., 2015).
Comparative analyses showed that the genomic con-
text of the sequenced adm gene clusters in A153,
MSU97, Eh335, S2052 and SWAT-3 is completely differ-
ent and, consequently, the upstream and downstream
predicted ends of the biosynthetic clusters were
assigned based only on their homologies (Supporting
information Figs. S3 and S4). These analyses allowed
the identification of a gene, designated admV, located
immediately upstream of admA. AdmV was not previ-
ously associated with andrimid biosynthesis (Jin et al.,
2006) and in silico analyses did not shed light on its
putative function. However, we found that the gene
admV is conserved in all adm gene clusters (Supporting
information Figs. S3 and S4). To further investigate its
role in the synthesis of andrimid, we constructed an in
frame deletion mutant defective in admV, thereby avoid-
ing polar effects in the expression of the downstream
adm genes. The resulting mutant strain no longer exhib-
ited antibacterial activity and the bioactivity could
be complemented by the in-trans expression of AdmV
(Fig. 2).
The adm gene clusters of A153, MSU97, 90-166,
Eh335, SWAT-3 and S2052 are 25.1, 24.9, 24.8, 24.7, 25.6
and 25.6 kb, respectively, and they are between 70.1%
and 99.0% identical at the DNA level (Supporting informa-
tion Fig. S4 and Table S1) suggesting that adm
biosynthetic clusters may have been moved horizontally
between the producing strains. In accordance with this
hypothesis, the overall genomic G1C content of A153
(56.0%), MSU97 (58.9%), 90-166 (59.1%), SWAT-3
(44.5%) and S2052 (45.7%) is considerably different from
the G1C content of their respective adm biosynthetic
clusters, which are 45.8%, 47.6%, 46.27, 51.1% and
51.2%, respectively. Furthermore, remnant sequences of
Fig. 1. Identification and characterization of the andrimid gene cluster in Serratia plymuthica A153.
A. Antibacterial activities against Bacillus subtilis of Serratia plymuthica A153, and derivative strains with mutations in the zeamine (zmn) and
andrimid (adm) biosynthetic gene clusters.
B. Genetic organization of the adm gene cluster in S. plymuthica A153. The same genetic organization was found in S. marcescens MSU97
and S. marcescens 90-166 (Fig. S3). Location of the Tn-KRCPN1 transposon insertions and in-frame deletion mutants are indicated by black
and red arrows, respectively. Colour code representing the functional category of each gene of the gene cluster is given where possible, based
on the biosynthetic pathway for andrimid proposed by Jin et al. (2006). Genes admV, admW and admX were not previously associated with
the regulation or biosynthesis of andrimid.
Regulation of andrimid biosynthesis 3637
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
transposable genetic elements were found flanking the gene
clusters of all the producing strains, although located in dif-
ferent regions in these loci (Supporting information Fig. S3).
In contrast, some remarkable differences were found
between the six biosynthetic clusters. First, the inter-
genic regions of several adm contiguous genes are not
conserved between the adm gene clusters (Supporting
information Fig. S4) which could suggest differential reg-
ulation in the expression of the biosynthetic clusters.
Second, the intergenic region between admS and admT
is considerably larger in A153, MSU97 and 90-166, and
we identified a putative ORF, admW, in these three Ser-
ratia strains (Supporting information Figs. S3 and S4).
However, the in frame deletion of admW did not alter
the antibacterial activity of A153 (Fig. 1A). Finally, a
gene encoding a LysR-type transcriptional regulator was
found upstream of admT in the adm gene clusters of
Vibrionales SWAT-3 and Vibrio coralliilyticus S2052
(Supporting information Fig. S3).
The andrimid gene cluster consists of a large
polycistronic unit
The adm gene cluster in Serratia consists of 22 predicted
ORFs and its genetic organization, together with the small
intergenic distances between contiguous genes, suggests
the presence of a single transcriptional unit (Figs. 3A and
Supporting information Fig. S3). To further understand the
biosynthesis of andrimid, we investigated its transcriptional
regulation. With primers spanning the 30 end of the
upstream gene and the 50 end of the contiguous down-
stream gene (Fig. 3A), we used reverse transcription-PCR
(RT-PCR) to assess co-transcription of the adm genes in
S. plymuthica A153. PCR products were obtained across
all intergenic regions covering the complete adm
Fig. 3. The andrimid gene cluster is organized as a polycistronic transcriptional unit.
A. Schematic representation of the adm gene cluster in Serratia plymuthica A153. Lines labelled 1-21 above the gene cluster represent the
regions amplified by RT-PCR and shown in B. Numbers below the arrows represent the intergenic distance in base pairs, and negative
numbers indicate overlapping genes.
B. Transcript analysis by RT-PCR using primers designed to span the intergenic region between two adjacent genes. For each region, three
PCR analyses were carried out: 1, RT-PCR on cDNA; 2, negative control with no reverse transcriptase; c, positive control with genomic DNA
as template. Culture samples for RNA isolation were taken at early stationary phase (Fig. 4).
Fig. 2. Role of the hypothetical protein AdmV in the biosynthesis of
andrimid. Bioactivities against Bacillus subtilis of an in-frame admV
deletion mutant of Serratia plymuthica A153. Induction of the
expression of the wild type proteins was done by addition of 1 mM
of IPTG. The bioassays were repeated at least three times, and
representative results are shown. Pictures were taken after 48 h of
incubation at 25 8C.
3638 M. A. Matilla et al.
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Environmental Microbiology, 18, 3635–3650
biosynthetic cluster, indicating the presence of one long
polycistronic transcript (Fig. 3B) – although the possibility
of internal promoters cannot be discarded.
The transcription of the adm biosynthetic cluster
is growth phase dependent
We identified several mutants in which the Tn-KRCPN1
transposon generated b-galactosidase transcriptional
fusions (Table 1). Because our RT-PCR analyses demon-
strated the presence of an operonic adm biosynthetic
cluster, we investigated transcription throughout growth in
a Lac- derivative of A153. Using a chromosomal fusion
located in the first multidomain PKS/NRPS-encoding gene
of the biosynthetic cluster (admK) our b-galactosidase
assays showed that the transcription of the adm operon
started in late-logarithmic phase of growth (Figs. 4A and
5B). Expression of the biosynthetic cluster correlated per-
fectly with the presence of andrimid in cell-free
supernatants (Fig. 4B).
Temperature and carbon source regulate the
transcription of the adm operon
The biosynthesis of secondary metabolites can be ener-
getically costly and other secondary metabolites produced
by Serratia strains have been shown to be highly regulated
by various environmental cues (Williamson et al., 2006;
Coulthurst et al., 2005). To shed light on the regulation of
andrimid production in A153, we investigated the impact of
different environmental parameters in the transcription of
the adm operon.
At the optimal growth temperature (30 8C) for A153,
andrimid production was abolished (Fig. 5A). However, as
the temperature decreased, a gradual increase in the pro-
duction of andrimid was observed, with higher production
levels at 15 8C than at 25 8C (Fig. 5A). To examine whether
the increase in andrimid production was reflected in the
expression of the adm gene cluster, we evaluated the
impact of temperature on the transcription of the biosyn-
thetic cluster. b-galactosidase assays showed that the
transcription was also thermoregulated (Fig. 5B). In
accordance with this, no adm expression was observed at
temperatures above 30 8C, confirming the tight correlation
between andrimid production and transcription of the bio-
synthetic operon (Fig. 5B).
Serratia plymuthica A153 was originally isolated from
the rhizosphere (Astrom and Gerhardson, 1988), a soil
environment which is under the direct influence of plant-
root exudates. The composition of root exudates is chemi-
cally complex but quantitatively it consists mainly of
carbon-based compounds, primarily organic acids and
sugars (Uren, 2007; Badri and Vivanco, 2009; Suzuki
et al., 2009). Thus, we investigated the production of andri-
mid in response to different carbon sources found in plant
root exudates. Our results showed that andrimid is differ-
entially produced depending on carbon source. Maximal
antibacterial activity was observed in the presence of
citrate, gluconate or glycerol whereas no activity was
detected in the presence of arabinose or succinate as sole
carbon sources (Fig. 6A). Although the growth rates of
A153 varied between the tested carbon sources (Support-
ing information Fig. S5), the observed antibacterial activity
was not correlated with the growth rate per se or the final
optical density reached in each of the culture media. Con-
sistent with these observations, higher transcriptional
levels of the adm operon were observed in media contain-
ing citrate or gluconate, whereas no transcription was seen
in arabinose media (Fig. 6B), confirming that both tran-
scriptional activity and andrimid production, were carbon
source-dependent.
Finally, we also evaluated the production of andrimid at
different pH, aeration conditions and NaCl concentrations,
but no impacts on antibacterial synthesis were observed
(not shown).
Fig. 4. AdmX and Hfq regulate andrimid production by activating
the expression of the adm biosynthetic gene cluster.
A. b-galactosidase activity (filled symbols) throughout growth
measured from a chromosomal fusion admK::lacZ in Serratia
plymuthica A153 LacZ (circles), and its DadmX (squares) and Dhfq
(triangles) derivative strains in LB medium at 25 8C. Open symbols
represent bacterial growth. Data are the mean and standard
deviation of three biological replicates. Arrow, time point when
samples for RT-PCR and qPCR were taken (Figs. 3 and S6).
B. Andrimid production by S. plymuthica A153 strain JH6 (zeamine
negative) throughout growth in LB medium at 25 8C. For the
assays, a Bacillus subtilis top agar lawn was prepared and 300 ml
of filter-sterilized supernatants were added to holes punched in the
Bacillus bioassay plates.
Regulation of andrimid biosynthesis 3639
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
The LysR-type regulator, AdmX, activates andrimid
production
Analysis of the genomic context of the adm gene clusters
in A153, MSU97, 90-166, Eh335, S2052 and SWAT-3
revealed a 2 kb region upstream of admV which was highly
conserved in the Serratia strains (Supporting information
Figs. S3 and S4). This region contained a gene encoding a
putative LysR-type transcriptional regulator (LTTR; Sup-
porting information Figs. S3 and S4). The family of
LTTRs comprises one of the largest classes of transcrip-
tional regulators in bacteria, functioning either as
repressors or activators of the transcription of their tar-
get genes (Maddocks and Oyston, 2008). To investigate
the potential role of the identified LTTR in andrimid
Table 1. Bacteria and phages used in this study.
Bacteria/phage Genotype or relevant characteristica Reference or source
Escherichia coli DH5a supE44 lacU169(Ø80lacZD M15) hsdR17 (r2K m
2
K )
recA1 endA1 gyrA96 thi-1 relA1
Woodcock et al. (1989)
E. coli CC118kpir araD, D(ara, leu), DlacZ74, phoA20, galK, thi-1,
rspE, rpoB, argE, recA1, kpir
Herrero et al. (1990)
E. coli HH26 Mobilizing strain for conjugal transfer Kaniga et al. (1991)
E. coli b2163 F- RP4-2-Tc::Mu DdapA::(erm-pir); KmR EmR Demarre et al. (2005)
Serratia plymuthica A153 Wild type, rhizosphere isolate H€okeberg et al. (1997)
LacZ A153 DlacZ (1470 bp D) Matilla et al. (2015)
VN1 A153 transposon mutant admH::Tn-KRCPN1lacZ; KmR This study
VN2 A153 transposon mutant admK::Tn-KRCPN1lacZ; KmR This study
VN3 A153 transposon mutant admO::Tn-KRCPN1lacZ; KmR This study
VN7 A153 transposon mutant admM::Tn-KRCPN1; KmR This study
VN9 A153 transposon mutant admJ::Tn-KRCPN1; KmR This study
VN11 A153 transposon mutant admC::Tn-KRCPN1lacZ; KmR This study
VN12 A153 transposon mutant admP::Tn-KRCPN1; KmR This study
VN17 A153 transposon mutant admO::Tn-KRCPN1; KmR This study
VN21 A153 transposon mutant admK::Tn-KRCPN1; KmR This study
VN22 A153 transposon mutant admP::Tn-KRCPN1lacZ; KmR This study
VN23 A153 transposon mutant admF::Tn-KRCPN1; KmR This study
VN24 A153 transposon mutant admE::Tn-KRCPN1lacZ; KmR This study
VN26 A153 transposon mutant admS::Tn-KRCPN1lacZ; KmR This study
A153JH6 A153 DlacZ, zmn13::Tn-KRCPN1; Zeamine-; KmR Hellberg et al. (2015)
ANDV A153 DadmV (336 bp D) This study
ANDW A153 DadmW (150 bp D) This study
ANDX A153 DadmX (789 bp D) This study
XJH6 A153 DadmX, zmn13::Tn-KRCPN1; andrimid-, zeamine-; KmR This study
ARpoS A153 rpoS::Km; KmR Matilla et al. (2015)
AHfq A153 Dhfq::Km (252 bp D); KmR Matilla et al. (2015)
A153H A153 Dhfq (252 bp D) This study
A153HL A153 DlacZ, Dhfq This study
LVN2 A153 DlacZ, admK::Tn-KRCPN1lacZ; generated by
transduction using /MAM1; KmR
This study
HLVN2 A153 DlacZ, Dhfq, admK::Tn-KRCPN1lacZ; generated
by transduction using /MAM1; KmR
This study
XLVN2 A153 DlacZ, DadmX, admK::Tn-KRCPN1lacZ; generated
by transduction using /MAM1; KmR
This study
ASptI A153 in-frame sptI (483 bp D) Matilla and Salmond (unpubl. data)
ASptR A153 sptR::Km; KmR Matilla and Salmond (unpubl. data)
ASplR A153 splR::Km; KmR Matilla and Salmond (unpubl. data)
ASpsR A153 spsR::Km; KmR Matilla and Salmond (unpubl. data)
A153C A153 in-frame csrB (267 bp D) Matilla and Salmond (unpubl. data)
Serratia marcescens MSU97 Wild type, plant epiphyte, pigmented Strobel et al. (1999)
Bacillus subtilis JH642 pheA1 trpC2 J.A. Hoch
Dickeya solani MK10 Wild type, plant pathogen Pritchard et al. (2013)
Xanthomonas campestris
pv. campestris
Wild type, plant pathogen R. Penyalver
Phages
/MAM1 Generalized transducing phage for S. plymuthica A153 Matilla and Salmond (2014)
a. The following abbreviations are used: Km, kanamycin; Tc, tetracycline; Em, erythromycin.
3640 M. A. Matilla et al.
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Environmental Microbiology, 18, 3635–3650
biosynthesis, we engineered an in frame deletion mutant in
S. plymuthica A153. The resulting mutant did not inhibit
growth of B. subtilis but antibacterial activity was fully
restored by in trans expression of the LTTR encoding gene
(Fig. 7A). These results suggested that the regulator, desig-
nated AdmX, is responsible for activating transcription of
the adm gene cluster and b-galactosidase assays con-
firmed that expression of the biosynthetic cluster was
abolished in an admX-deficient strain (Fig. 4A). These
results were supported by qPCR analyses showing
decreased adm transcript levels in the A153 DadmX strain
(Supporting information Fig. S6).
To assess the transcription of admX throughout growth,
we constructed a transcriptional fusion of the admX pro-
moter to the reporter gene lacZ. b-galactosidase assays
showed that the transcription of admX started in late-
logarithmic phase of growth, correlating with transcription
of the adm biosynthetic cluster (Supporting information
Fig. S7). We explored the effect of AdmX on expression of
its cognate gene. However, the transcription of admX
Fig. 6. Effect of carbon source on the production of andrimid and expression of the adm gene cluster in Serratia plymuthica A153.
A. Halos of antibiosis against Bacillus subtilis of filter-sterilized supernatants of A153 strain JH6 (zeamine negative) grown in minimal medium
with different carbon sources. The bioassays were repeated at least three times, and representative results are shown. All the carbon sources
were used at a final concentration of 15 mM. With the exception of lactose, all carbon sources used are frequently found in plant root
exudates. Bars, 5 mm.
B. b-galactosidase activity (filled symbols) throughout growth measured from the chromosomal fusion admK::lacZ in Serratia plymuthica A153
LacZ in minimal medium with different carbon sources. Open symbols represent bacterial growth. Data are the mean and standard deviation of
three biological replicates. Growth and doubling times of A153 in all the carbon sources used are shown in Fig. S5.
Fig. 5. The production of andrimid in Serratia plymuthica A153 is temperature-dependent and correlates with the expression of the adm gene
cluster.
A. Halos of antibiosis against Bacillus subtilis of filter-sterilized supernatants of A153 strain JH6 (zeamine negative) grown in LB at different
temperatures. The bioassays were repeated at least three times, and representative results are shown.
B. b-Galactosidase activity (filled symbols) throughout growth measured from the chromosomal fusion admK::lacZ in Serratia plymuthica A153
LacZ. Open symbols represent bacterial growth. Data are the mean and standard deviation of three biological replicates. Doubling times at 20,
25, 30 and 35 8C were 66.86 0.4, 47.360.7, 40.760.4 and 40.560.2 minutes respectively.
Regulation of andrimid biosynthesis 3641
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
remained unaltered in an admX mutant strain (Supporting
information Fig. S7). Finally, we also investigated whether
andrimid thermoregulation is mediated by AdmX. Previous
studies showed that the expression of another LTTR
(PecT), and consequently the expression of its target
genes, is thermoregulated (Herault et al., 2014). However,
no differences in transcripts levels of admX were observed
(using qPCR analysis) at 25, 30 and 37 8C (data not
shown).
The chaperone Hfq regulates the expression of the
adm gene cluster
The biosynthesis of secondary metabolites is frequently
regulated at post-transcriptional level and, in gammapro-
teobacteria, this is mainly mediated by the Csr/Rsm
(Romeo et al., 2013) and Hfq (Vogel and Luisi, 2011)
systems. To assess a potential role of these post-
transcriptional regulatory systems, we analysed the pro-
duction of andrimid in different genetic backgrounds in
S. plymuthica A153. A mutant with a deletion in the
non-coding sRNA, csrB, showed the same antibacterial
activity as the wild type strain (not shown). However, in
frame deletion of hfq led to complete loss of andrimid
production (Fig. 7B) and this phenotype was partially
complemented by the in trans expression of hfq (Sup-
porting information Fig. S8).
b-galactosidase assays were performed to assess the
expression of the adm gene cluster throughout growth in
A153 Dhfq. The results showed that transcription of the
andrimid operon was abolished in a hfq-deficient strain,
indicating that the chaperone Hfq positively regulates
expression of the adm biosynthetic cluster (Fig. 4A). qPCR
analyses confirmed that the transcriptional levels of the
adm gene cluster were reduced by 99.9% in an hfq-defi-
cient background (Supporting information Fig. S6). To
investigate whether Hfq regulates admX expression,
qPCR experiments were also performed. However, admX
transcript levels were unaltered in the Dhfq mutant (Sup-
porting information Fig. S6).
The expression of the adm gene cluster starts in late-
logarithmic phase of growth (Fig. 4) and so we investigated
whether quorum sensing (QS) could be regulating andri-
mid production. A153 carries sptI, sptR, splR and spsR
genes encoding LuxI- and LuxR-type proteins very similar
to those of the three QS systems identified in the taxo-
nomically related rhizobacterium, S. plymuthica G3 (Liu
et al., 2011; Duan et al., 2012). However mutations in each
of these four genes in A153 had no observable impacts on
antibacterial activity (not shown). Similarly, the role of the
stationary phase sigma factor, RpoS, in the production of
andrimid was also investigated, but the antibacterial activ-
ity of an rpoS mutant was indistinguishable from that of the
wild type strain (Fig. 7B).
Discussion
Since the golden age of antimicrobial discovery in the
1940–1960s, there has been a diminution in the rate of dis-
covery of new antibiotics and this problem has been
exacerbated by the progressive emergence worldwide of
antibiotic-resistant bacteria (Lewis, 2013; Blair et al.,
2015). However, there are many antibiotics described in
the literature that, although not currently exploited, could
prove to be lead molecules for drug discovery programmes
Fig. 7. The LysR-type regulator AdmX, and the RNA chaperone
Hfq positively regulate the biosynthesis of andrimid. Bioactivities
against Bacillus subtillis of S. plymuthica A153 and derivative
strains are shown (A, B). In frame deletion of admX was
functionally complemented by the in trans expression of AdmX
using the pQE80L-based vector, pMAMV185 (A). Induction of the
expression of the AdmX was done by addition of 0.1 mM of IPTG.
Complementation of the Dhfq strain was done using a pQE-80L-
based vector (Fig. S8). The bioassays were repeated at least three
times, and representative results are shown. Pictures were taken
after 48 h of incubation at 25 8C.
3642 M. A. Matilla et al.
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
leading to clinical or agricultural utility. The hybrid NRPS/
PKS antibacterial compound, andrimid, was first isolated in
the late 1980s from a brown planthopper intracellular sym-
biont belonging to the Enterobacter genus (Fredenhagen
et al., 1987). After its discovery, three related antibacterial
compounds, moiramides A-C (Supporting information Fig.
S1), were also isolated from Pseudomonas fluorescens
(Needham et al., 1994).
Andrimid is a nanomolar inhibitor of the bacterial acetyl-
CoA carboxylase (Freiberg et al., 2004), an enzyme
responsible for the conversion of acetyl-CoA to malonyl-
CoA in the first committed step for the synthesis of fatty
acids (Harwood, 2007). The discovery of the andrimid tar-
get, together with its unusual chemistry, has stimulated
interest in the biochemistry and genetics of the antibiotic.
The andrimid gene cluster was first described almost a
decade ago and a model for its biosynthesis proposed (Jin
et al., 2006). In this model, the function for most of the
Adm proteins was predicted and, in some cases, later
demonstrated biochemically (Fortin et al., 2007; Magarvey
et al., 2008; Ratnayake et al., 2011). However, the roles of
the hypothetical proteins AdmB, AdmL and AdmN in the
synthesis of andrimid remain unknown. In this study, we
redefined the length of the adm gene cluster by identifying
a new hypothetical protein encoding gene, admV, located
immediately upstream of admA and forming part of the
adm operon (Figs. 3 and Supporting information Fig. S3).
Using mutagenesis and complementation analyses we
demonstrated that AdmV is required for the biosynthesis of
andrimid. Our bioinformatic analyses did not categorically
clarify the function of AdmV. However, in silico studies pre-
dicted three alpha helices with a high content of positively
charged amino acids at the C-terminal end, suggesting
that AdmV may be a DNA binding protein.
Andrimid has been shown to be a broad-spectrum anti-
bacterial compound that acts on Gram-positive and Gram-
negative bacteria. It was first isolated after it was shown to
have potent activity against the different pathovars of the
phytopathogen, Xanthomonas campestris (Fredenhagen
et al., 1987) but further research showed that it is also
active against bacterial pathogens belonging to Bacillus,
Enterococcus, Escherichia, Salmonella, Staphylococcus,
Vibrio and Yersinia genera (Needham et al., 1994; Singh
et al. 1997; Long et al., 2005; Wietz et al., 2010; Sanchez
et al., 2013). Resistance to andrimid is dependent on multi-
drug efflux pumps (Freiberg et al., 2004; Jin et al., 2006) or
the presence of andrimid-resistant acetyl-CoA carboxyl-
transferases (ACC) hypothesized to show decreased
affinity for the inhibitor (Liu et al., 2008). Interestingly, we
showed that the plant-pathogen Agrobacterium tumefa-
ciens and multiple rhizobacteria strains in the family
Enterobacteriaceae (i.e., Kluyvera, Pantoea, Serratia,
Weeksella, Xenorhabdus, and the emerging phytopatho-
gen, Dickeya solani) (Supporting information Figs. S9 and
S10) are sensitive to andrimid. However, the differential
sensitivities to the antibiotic of these plant-associated
strains could also indicate the presence of weaker intrinsic
resistance mechanism(s) of different efficiencies.
NRPS and PKS constitute the main enzymatic source of
secondary metabolites in bacteria; 36% of which are gen-
erated by hybrid NRPS/PKS gene clusters (Wang et al.,
2014). Secondary metabolites have been associated with
important roles in the physiology and development of the
bacterial host, but are also implicated in overcoming com-
petitors in the same nutritional niche (Liu et al., 2013; Pidot
et al., 2014; Wang et al., 2014; Mousa and Raizada,
2015). The synthesis in situ of andrimid has been demon-
strated in a sponge-associated bacterium (Oclarit et al.,
1994; Long et al., 2005) and its production is favoured over
that of other metabolites in conditions mimicking natural
environments - suggesting an important ecophysiological
role of andrimid (Wietz et al., 2011). Consistent with this
view, even sub-lethal concentrations of andrimid can pro-
mote a negative chemotactic response (in sensitive
competitors) away from the antibiotic, thereby enhancing
competitiveness of the producers (Graff et al., 2013). In S.
plymuthica A153, the synthesis of andrimid is carbon
source-dependent and induced in the presence of sugars
and organic acids commonly found in plant root exudates
(Fig. 6). Carbon source has been shown to regulate antibi-
otic synthesis at both molecular and physiological levels
and, in general, rapidly metabolized carbon sources are
involved in repression of bacterial secondary metabolites
(Sanchez et al., 2010). However, our results did not show
a correlation between preferred carbon sources and andri-
mid production (Figs. 6 and S5). Importantly, in A153, the
production of the antibiotic zeamine is high in the presence
of carbon sources which show low andrimid production
(i.e., arabinose, sorbitol and succinate) whereas the syn-
thesis of zeamine is repressed by carbon sources that
stimulate andrimid production (i.e., citrate, gluconic acid
and glycerol) (Fig. 6; Hellberg et al., 2015). This “mirror
image” metabolic regulation of andrimid and zeamine pro-
duction when presented with different environmental
carbon sources could be beneficial in maintaining the
capacity of A153 to overcome bacterial competitors in its
natural niche, the rhizosphere, where specific carbon
source availability is likely to fluctuate.
Soil bacteria are subjected to daily and seasonal abiotic
variations and temperature is considered a key abiotic fac-
tor influencing bacterial metabolic activity – which may
therefore modulate inter- and intra-specific interactions
between microbes. The results reported here show that
andrimid synthesis in A153 is thermoregulated, with
enhanced antibiotic production at lower temperature. Pre-
vious studies also showed that andrimid production in
Vibrio (Long et al., 2005; Wietz et al., 2011) and Serratia
strains (Sanchez et al., 2013) is also thermosensitive. In
Regulation of andrimid biosynthesis 3643
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
this study, we demonstrate that the observed thermoregu-
lation is exerted at the transcriptional level (Fig. 5) and in a
AdmX-independent fashion. During the analysis of the bio-
active properties of A153, we showed that the production
of the haterumalide, oocydin A, is also thermosensitive
(Matilla and Salmond, unpubl. data) and this may indicate
that a common control pathway is modulating the thermo-
regulation of the biosynthesis of secondary metabolites in
A153. The ecological role of this thermosensitivity is
unknown, although it could be related to competitiveness
and efficient colonization of specific niches by the produc-
ing rhizobacterium, S. plymuthica A153.
To date, most of the published research on andrimid has
focussed on its biochemistry, but the regulation of its bio-
synthesis has attracted little attention. The first adm gene
cluster was identified in Pantoea agglomerans but no regu-
latory proteins were found in this biosynthetic cluster (Jin
et al., 2006). However, our analysis of the genomic context
of the andrimid gene cluster in A153, MSU97 and 90-166,
revealed a LysR-type regulator-encoding gene, admX,
which was highly conserved in these producing strains
(Supporting information Figs. S3 and S4). Mutagenesis,
complementation and gene expression analyses demon-
strated that AdmX is a positive regulator of andrimid
biosynthesis (Figs. 4 and 7A). AdmX is a 305 amino acid
protein composed of a helix-turn-helix motif-containing
DNA-binding domain and a probable effector binding
domain at the N-terminal and C-terminal regions, respec-
tively (Supporting information Fig. S11). By analogy with
other LTTR structures (Ezezika et al., 2007; Monferrer
et al., 2010; Devesse et al., 2011), AdmX is proposed to
possess a potential effector binding site located in between
the two lobes of the effector binding domain (Supporting
information Fig. S11). Inspection of a homology model
shows that this site is primarily composed of amino acids
with hydrophobic side chains (Supporting information Fig.
S11) and further investigations are necessary to identify
candidate effector molecules.
In general, LysR transcriptional regulators activate the
expression of their target genes while negatively autoregu-
lating their own transcription (Maddocks and Oyston,
2008). LTTRs have been shown to regulate genes involved
in metabolism, virulence, motility, chemotaxis, quorum
sensing and biofilm formation (Maddocks and Oyston,
2008). However, only a limited number of LTTRs have
been found to regulate the biosynthesis of secondary
metabolites – such as actinorhodin (Mao et al., 2013), phe-
nazines (Klaponski et al., 2014), pyoluteorin (Li et al.,
2012), ralfuranones (Kai et al., 2014) and undecylprodigio-
sin (Mao et al., 2013). LTTRs are broadly distributed within
the prokaryotic kingdom, suggesting dissemination and
acquisition by horizontal gene transfer (HGT; Maddocks
and Oyston, 2008). In accordance with this notion, sequen-
ces reminiscent of transposases were found flanking
admX (Supporting information Figs. S3 and S4) and the
G1C content of the admX genes in A153, MSU97 and
90-166 is considerably higher than that of their respective
adm gene clusters. Although AdmX seems to be restricted
to the adm biosynthetic clusters present in Serratia strains,
BLAST analyses showed that AdmX is up to 84% identical
(90% similar) to an orphan LysR-type regulator highly con-
served within Enterobacter and Klebsiella genera, again
suggesting that this LTTR-encoding gene may have been
acquired by HGT. Interestingly, we found an LTTR-
encoding gene immediately upstream of admT in the adm
gene clusters of Vibrio coralliilyticus S2052 and Vibrionales
SWAT-3 (Supporting information Fig. S3), perhaps indicat-
ing an important regulatory role in the expression of the
adm gene clusters in these strains. It has been proposed
that the fragmented andrimid biosynthetic pathway reflects
a recent evolutionary origin (Magarvey et al., 2008) and so
the insertion of the LTTR-encoding genes in the adm gene
clusters of Vibrio and Serratia may represent a step for-
ward in the evolution of these biosynthetic clusters.
The post-transcriptional regulation of the adm gene clus-
ter in A153 was also investigated and our results showed
that the RNA binding protein, Hfq, positively regulates the
expression of the andrimid operon. Previous studies of
Serratia strains showed that the synthesis of secondary
metabolites such as a carbapenem antibiotic (Wilf et al.,
2011), prodigiosin (Wilf et al., 2011) and pyrrolnitrin (Zhou
et al., 2012) is regulated by Hfq, and we recently showed
that Hfq positively regulates the expression of the oocydin
A (Matilla et al., 2015) and zeamine (Hellberg et al., 2015)
gene clusters in S. plymuthica A153. It is known that Hfq
also stimulates rpoS translation (Vogel and Luisi, 2011)
and the deletion of hfq results in reduced rpoS transcripts
levels in A153 (Matilla et al., 2015). However, contrary to
our observations on regulation of oocydin A in A153 (Mat-
illa et al., 2015), Hfq-mediated regulation of andrimid is
independent of RpoS (Fig. 7B), as observed in other Ser-
ratia strains for the biosynthesis of zeamine (Hellberg
et al., 2015), carbapenem (Wilf and Salmond, 2012) and
prodigiosin (Wilf and Salmond, 2012) antibiotics.
In summary, we have shown that the plant-associated
bacterium, Serratia plymuthica A153, produces the broad
spectrum antibacterial compound, andrimid. Comparative
genomics, molecular genetics and transcriptional
approaches led us to re-define the borders of the adm
gene clusters and identify new genes involved in the bio-
synthesis and regulation of andrimid. Further, in vivo
assays expanded the spectrum of bacterial strains which
are sensitive to andrimid. For the first time, the regulation
of the biosynthesis of andrimid was investigated at tran-
scriptional and post-transcriptional levels. Future research
will provide information about the effectors recognized by
AdmX and such knowledge, coupled with the work
described in this study, may encourage the use of plant-
3644 M. A. Matilla et al.
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
associated andrimid-producing strains as biocontrol agents
in sustainable agriculture strategies.
Experimental procedures
Bacterial strains, culture media and growth conditions
Bacterial strains used in this study are listed in Table 1 and
Supporting Information Table S2. Serratia, Bacillus, Agrobac-
terium, Dickeya, Kluyvera, Pantoea, Weeksella,
Xanthomonas, Xenorhabdus, Yersinia and their derivative
strains were grown routinely at 30 8C, unless otherwise indi-
cated, in Luria Broth (LB; 5 g yeast extract l21,10 g Bacto
tryptone l21 and 5 g NaCl l21) or minimal medium (0.1%, w/v,
(NH4)2SO4, 0.41 mM MgSO4, 40 mM K2HPO4, 14.7 mM
KH2PO4, pH 6.9–7.1) with glucose (0.2%; w/v) as carbon
source, unless otherwise indicated. Escherichia coli strains
were grown at 37 8C in LB. Escherichia coli DH5a was used
as a host for gene cloning. Media for propagation of E. coli
b2163 were supplemented with 300 mM 2,6-diaminopimelic
acid. When appropriate, antibiotics were used at the following
final concentrations (in mg ml21): ampicillin, 100; chloram-
phenicol, 25; kanamycin, 25 (E. coli strains) and 50 (Serratia
strains); streptomycin, 50; tetracycline, 10. Sucrose was
added to a final concentration of 10% (w/v) when required to
select derivatives that had undergone a second crossover
event during marker-exchange mutagenesis. Bacterial growth
(OD600 nm) was measured on a Unicam Hekios spectropho-
tometer at 600 nm, 1 cm path length.
In vitro nucleic acid techniques and bioinformatic
analyses
Plasmid DNA was isolated using the Anachem Keyprep plas-
mid kit. For DNA digestion, the manufacturer’s instructions
were followed (New England Biolabs, Roche and Fermentas).
Separated DNA fragments were recovered from agarose gels
using the Anachem gel recovery kit. Ligation reactions and
total DNA extraction were performed as previously described
(Sambrook et al., 1989). Competent cells were prepared using
calcium chloride and transformations were performed by
standard protocols (Sambrook et al., 1989). PhusionVR high
fidelity DNA polymerase (New England Biolabs) was used in
the amplification of PCR fragments for cloning. PCR reactions
were purified using the Anachem PCR Clean-up kit. PCR frag-
ments were verified by DNA sequencing that was carried out
at the University of Cambridge DNA Sequencing Facility
(Cambridge, UK) or at the Institute of Parasitology and Biome-
dicine Lopez-Neyra (CSIC; Granada, Spain). Sequence
comparison analyses were performed employing the wgVISTA
online tool (Frazer et al., 2004). Open reading frames (ORFs)
in the andrimid gene clusters were predicted using Glimmer
3.0 (Delcher et al., 1999). Blast analyses were used for the
functional gene assignment. Protein domain organization was
identified using the NCBI conserved domains database. Multi-
ple sequence alignments were carried out with ClustalW2
(European Bioinformatics Institute). Artemis software (Well-
come Trust Sanger Institute) was used to visualize genomic
sequences.
Random transposon mutagenesis
Random transposon mutagenesis of S. plymuthica A153
using Tn-KRCPN1 was performed by biparental conjugation
mating using Escherichia coli b2163, as described previously
(Matilla et al., 2012). In total, three thousand kanamycin-
resistant insertion mutants were screened for their antibacte-
rial activity against Bacillus subtilis using dual drop culture
bioassays. Auxotrophic mutants were discarded and insertion
mutations were transduced into the wild type strain A153
using phage /MAM1 (Matilla and Salmond, 2014). The inser-
tion site of transposon Tn-KRCPN1 in mutants of interest was
determined using random primed PCR following the method
described previously (Matilla et al., 2012) and using primers
described in Supporting Information Table S3.
Antibacterial assays
Antibiotic activity was tested using agar lawn assays. Briefly,
indicator plates for andrimid production contained a 0.8% LB
agar (LBA) top lawn containing 200 ml of an overnight culture
of the bacterial strain to test. Five microliters of overnight cul-
tures of the andrimid-producing strains were spotted on the
surface of the indicator agar lawn and incubated for 48 h at
25 8C, unless otherwise indicated. To determine andrimid lev-
els in bacterial supernatants, culture samples were taken,
bacterial cells were pelleted by centrifugation (10,0003g, 10
min), and the supernatant was filtered (0.2 mm). Three hun-
dred microliters of the filter-sterilized supernatant were added
to wells cut into the LBA plate and incubated at 25 8C for 24 h.
All experiments were repeated at least three times.
Construction of strains and plasmids
Chromosomal mutants of Serratia plymuthica strains were
constructed by homologous recombination using derivative
plasmids of the suicide vector pKNG101. These plasmids,
which are listed in Supporting Information Table S4, were con-
firmed by DNA sequencing and they carried mutant in-frame
deletions for the replacement of wild type genes in the chro-
mosome. Primers used in this study are listed in Supporting
Information Table S3. In all cases, plasmids for mutagenesis
were transferred to S. plymuthica strains by triparental conju-
gation using E. coli CC118kpir and E. coli HH26 (pNJ500) as
helper. The plasmids for the construction of the in-frame dele-
tion mutants were generated by amplifying the up- and
downstream flanking regions of the gene, or domain to be
deleted. The resulting PCR products were digested with the
enzymes specified in Supporting Information Table S4 and
ligated in a three-way ligation into pUC18Not, previously
cloned into the marker exchange vector pKNG101. The in-
frame deletion mutant strains ANDX, ANDV and ANDW were
generated using plasmids pMAMV175, pMAMV191 and
pMAMV192, respectively. Mutant strains defective in hfq,
A153H and A153HL, were constructed using plasmid
pMAMV193. All relevant mutations were confirmed by PCR
and sequencing.
For the construction of the complementing plasmids, the
genes were amplified using primers described in Supporting
Information Table S3 and cloned into pTRB30. All the inserts
Regulation of andrimid biosynthesis 3645
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
were confirmed by PCR and sequencing. Complementing
plasmids were used to transform A153 by electroporation.
Genetic complementation assays
Complementation of mutations was carried out by the intro-
duction of a wild type copy of the corresponding mutated gene
in trans on plasmid pTRB30. For the complementation assays,
LBA containing the appropriate antibiotic (to maintain the plas-
mid) and isopropyl-b-D-thiogalactopyranoside (IPTG) at 0.1 or
1 mM were added to holes punched in Bacillus subtilis bioas-
say plates. Then, 5 ll of overnight cultures of the selected
strains were spotted on the surface of the LBA containing the
antibiotic and IPTG and were incubated at 25 8C for 2 days.
Generalized transduction
The generalized transducing viunalikevirus, /MAM1, was
used for transduction of chromosomal mutations, as described
previously (Matilla and Salmond, 2014).
b-Galactosidase assays
Expression of the lacZ reporter gene was performed using
the fluorogenic substrate 4-methylumbelliferyl b-D-galacto-
side (Melford Cat No. M1095) at a final concentration of
0.125 mg ml21, as described previously (Ramsay, 2013).
Samples were measured in a SpectraMax Gemini XPS fluo-
rescence microplate reader (Molecular Devices) using the
following settings: excitation 360 nm, emission 450 nm, cut-
off 435 nm, reading every 30 s for 20 min at 37 8C.
b-Galactosidase activity was expressed as relative fluores-
cent units s21 and normalize to the OD600 nm of the
corresponding sample. Alternatively, b-galactosidase activ-
ity was measured as described previously (Miller, 1972)
using 2-Nitrophenyl b-D-galactopyranoside (ONPG; Sigma–
Aldrich Cat No. N1127) as substrate. All the transcriptional
fusion assays were carried out using S. plymuthica A153
LacZ (control) or derived mutants.
RNA extraction, cDNA synthesis, reverse
transcription-PCR (RT-PCR) and quantitative
real time PCR analyses
RNA was extracted from early stationary phase cultures
grown in LB medium using an RNeasy mini kit (Qiagen)
according to the manufacturer’s instructions. RNA concentra-
tion was determined spectrophotometrically and RNA integrity
was assessed by agarose gel electrophoresis. Genomic DNA
contamination was eliminated by treating total RNA with Turbo
DNA-free (Ambion). The synthesis of cDNA was performed
using random hexamers (GE Healthcare) and SuperScript II
reverse transcriptase (Invitrogen) in a 30 ml reaction with 2 mg
of total RNA and incubation at 42 8C for 2 h. As negative con-
trol the reaction was performed omitting the reverse
transcriptase. For the RT-PCR analysis, the equivalent of 50
ng of total RNA was subjected to PCR amplification using pri-
mers to amplify across the junctions (Supporting Information
Table S3). Positive and negative control PCR reactions were
performed using genomic DNA and no-RT cDNA samples,
respectively, as templates. PCR conditions consisted of 30
cycles of denaturation for 1 min at 94 8C, annealing for 1 min
at 62 8C, and extension for 40 s at 72 8C. qPCRs were per-
formed as described previously (Burr et al., 2006) using
primers specific for admX and admV (Supporting Information
Table S3). qPCR amplifications were performed using an
MyiQTM2 Two-Color Real-Time PCR Detection System (Bio-
Rad). To confirm the absence of contaminating genomic DNA,
control PCRs were carried out using no RT cDNA samples as
templates. Melting curve analyses were conducted to ensure
amplification of a single product. The relative gene expression
was calculated using the critical threshold (DDCt) method
(Pfaffl, 2001) and using 16S rRNA as the internal control to
normalize the data.
Acknowledgements
We thank Kornelia Smalla and Ian Toth for the generous dona-
tion of bacterial strains. Work in the Salmond laboratory is
supported by funding through the Biotechnology and Biologi-
cal Sciences Research Council (UK). M.A.M. was supported
by the EU Marie-Curie Intra-European Fellowship for Career
Development (FP7-PEOPLE-2011-IEF) Grant No. 298003
and the Spanish Ministry of Economy and Competitiveness
Postdoctoral Research Program, Juan de la Cierva (JCI-
2012-11815). The Krell laboratory is supported by FEDER
funds and Fondo Social Europeo through grants from the
Junta de Andalucıa (Grant CVI-7335) and the Spanish Minis-
try for Economy and Competitiveness (Grants BIO2013-
42297 and RTC-2014-1777-3).
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Supporting information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Fig. S1. Structures of andrimid and moiramide B. The
structure of andrimid consists of an unsaturated fatty acid
chain, a pyrrolidinedione ring, a valine and glycine derived
b-ketoamide and the amino acid b-phenylalanine. Chemical
synthesis studies showed that the fatty acid chain and
b-phenylalanine are involved in bacterial cell penetration of
the antibiotic, whereas the pyrrolidinedione head and the b-
ketoamide moiety are responsible for the antibacterial activ-
ity (Pohlmann et al., 2005; Freiberg et al., 2006).
Fig. S2. Antibacterial activity of Serratia marcescens
MSU97 against Bacillus subtilis.
Fig. S3. Schematic representation of the andrimid gene
clusters of Serratia marcescens MSU97, Serratia marces-
cens 90-166, Pantoea agglomerans Eh335, Vibrio coralliily-
ticus S2052 and Vibrionales bacterium SWAT-3. Numbers
below the arrows represent the intergenic distance between
contiguous genes, with negative numbers indicate overlap-
ping genes
Fig. S4. DNA homology between the andrimid gene cluster
of Serratia plymuthica A153 and the andrimid gene clusters
of the other producing strains. A, Schematic representation
of the adm gene cluster in Serratia strains. B-F, Alignments
representing the percentage of DNA homology between the
adm gene cluster of A153 and those of S. marcescens
MSU97 (B), S. marcescens 90-166 (C), Pantoea agglomer-
ans Eh335 (D) Vibrio coralliilyticus S2052 (E) and Vibrio-
nales SWAT-3 (F). Alignments were performed using
wgVISTA (Frazer et al., 2004).
Fig. S5. Growth of Serratia plymuthica A153 in minimal
medium with different carbon sources. Growth curves show-
ing the doubling time in sorbitol (181.261 min), mannitol
(1536 1 min), fructose (145.86 2 min), galactose
(115.26 1 min), mannose (181.662 min), lactose
(413.46 6 min), xylose (208.26 2 min), succinic acid
(142.86 1 min), maltose (145.26 1 min), sucrose
(106.26 1 min), glucose (115.26 1 min), glycerol (1216 1
min), gluconic acid (96.66 1 min), arabinose (235.26 2
min) and citrate (158.76 3 min) as sole carbon source.
Data are the mean and standard deviation of three biologi-
cal replicates. The assays were done at 25 C with shaking
at 200 rpm.
Fig S6. Impact of Hfq (A) and AdmX (B) on the expression
of admV and admX. Quantitative real-time PCR was used
to measure transcript levels of admV (grey bars) and admX
(white bars) in Serratia plymuthica A153, and derivative
strains. The values showed the average expression relative
to wild type expression. The arrow in Fig. 4A indicates the
time point when the samples for qPCR were taken. The
Regulation of andrimid biosynthesis 3649
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650
data are the mean and standard deviation of three biologi-
cal replicates.
Fig. S7. AdmX transcription correlates with the expression
of the andrimid gene cluster. Transcription of the admX
(PadmX::lacZ; pMAMV244) promoter fusion throughout
growth in Serratia plymuthica A153 strains. b-Galactosidase
activity (filled symbols) and growth curves (open symbols)
were determined in LacZ (red) and DadmX (blue) in LB
medium at 25 8C. A153 wt harbouring the empty reporter
plasmid (black) was used as negative control in the assays.
Data are the mean and standard deviation of three biologi-
cal replicates.
Fig. S8. Genetic complementation of Serratia plymuthica A153
strain A153H. Expression of hfq in trans in A153 Dhfq restored
andrimid production and therefore the antibacterial activity
against Bacillus subtilis. Induction of Hfq expression was done
by addition of 0.1 mM of IPTG. The bioassays were repeated at
least three times, and a representative figure is shown. Pictures
were taken after 48 h of incubation at 25 8C.
Fig. S9. Sensitivities of different bacterial strains to the antibi-
otic andrimid. Bioactivities of Serratia plymuthica A153 and the
non-andrimid producing mutant of A153, VN2, against ecologi-
cally different bacterial strains. For the assays, an indicator top
agar lawn was prepared as described in “Experimental
procedures,” and 5 ll overnight cultures of the A153 strains
were spotted on the surface of the bacterial indicator agar
lawns. The bioassays were repeated three times, and represen-
tative results are shown. Pictures were taken after 48 h of incu-
bation at 25 8C. The strains used are described in Table 1 and
supplementary Table S2.
Fig. S10. Andrimid shows antibacterial activity against Bacil-
lus subtilis, Dickeya solani and Xanthomonas campestris pv.
campestris. Recovery of viable Bacillus, Dickeya and Xantho-
monas cells grown in the presence of A153 JH6 (andrimid
positive, zeamine negative) and A153 XJH6 (andrimid and
zeamine negative) supernatants. The values showed the per-
centage of viable cells in the presence of JH6 supernatants
relative to the number of viable cells in the presence of XJH6
supernatants. For the assays, overnight bacterial cultures of
Bacillus, Dickeya and Xanthomonas were adjusted to an opti-
cal density at 600 nm (OD600) of 0.1 and grown at 30 C
with orbital shaking (225 rpm). At an OD600 of 0.4, 10 mL of
the bacterial culture was removed and pelleted by centrifuga-
tion at 4,000 x g for 10 min at room temperature. The pellet
was resuspended in 5 ml of 2X LB and 5 ml supernatants of
an overnight culture of A153 JH6 or A153 XJH6 were added
to the bacterial culture. Samples were taken after 5 and 10 h
of incubation and the number of colony forming units (CFU)
were determined. Data are the mean and standard deviation
of three biological replicates.
Fig. S11. Homology model of AdmX. The model was gen-
erated by the Geno3D modeling algorithm (Combet et al.,
2000) and the structure of the BenM transcriptional regula-
tor (PDB ID 3K1N) as template. The site for the binding of
potential effector molecules is indicated.
Table S1. Identity at DNA level of the andrimid gene clus-
ters between producing strains.
Table S2. Additional bacterial strains used in this study.
Table S3. Oligonucleotides used in this study.
Table S4. Plasmids used in this study.
3650 M. A. Matilla et al.
VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.,
Environmental Microbiology, 18, 3635–3650