JB Accepted Manuscript Posted Online 1 June 2015 J. Bacteriol. doi:10.1128/JB.00302-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor
2 3
Corey L. Compton, Michael S. Fernandopulle, Rohith T. Nagari, and Jason K. Sello
4 5
Department of Chemistry, Brown University, 324 Brook St. Providence, RI 02912
6
[email protected]
7
Abstract
8
Pupylation is a post-translational modification peculiar to actinobacteria wherein proteins are
9
covalently modified with a small protein called the prokaryotic ubiquitin-like protein (Pup). Like
10
ubiquitination in eukaryotes, this phenomenon has been associated with proteasome-mediated
11
protein degradation in mycobacteria. Here, we report studies of pupylation in streptomycetes,
12
another large genus of actinobacteria. We constructed mutants of Streptomyces coelicolor
13
lacking PafA (Pup ligase), the proteasome, and the Pup-proteasome system. We found that these
14
mutants share a high susceptibility to oxidative stress, as compared to that of the wild-type strain.
15
Remarkably, we found that the pafA null mutant has a sporulation defect not seen in strains
16
lacking the Pup-proteasome system. In proteomics experiments facilitated by an affinity-tagged
17
variant of Pup, we identified 110 pupylated proteins in S. coelicolor strains having and lacking
18
genes encoding the 20S proteasome. Our findings shed new light on this unusual post-
19
translational modification and its role in Streptomyces physiology.
20 21
Importance
22
The presence of 20S proteasomes reminiscent of those in eukaryotes and a functional equivalent
23
of ubiquitin, known as the prokaryotic ubiquitin-like protein (Pup), in actinobacteria have
1
24
motivated re-evaluations of protein homeostasis in prokaryotes. Though the Pup-proteasome
25
system has been studied extensively in mycobacteria, it is much less understood in
26
streptomycetes, a large genus of actinobacteria known for highly choreographed life cycles in
27
which phases of morphological differentiation, sporulation, and secondary metabolism are often
28
regulated by protein metabolism. Here, we define constituents of the pupylome in Streptomyces
29
coelicolor for the first time and present new evidence that link pupylation and the oxidative
30
stress response in this bacterium. Surprisingly, we find that the Pup ligase has a Pup-independent
31
role in sporulation.
32 33
Key Words
34
Streptomyces, 20S proteasome, pupylation, proteomics, sporulation, post-translational
35
modification
36 37
Introduction
38
Protein homeostasis is a tightly regulated and critically important phenomenon in
39
bacterial physiology [1]. It is thought to be particularly complex in actinobacteria, which have
40
both the canonical proteolytic machinery of prokaryotes and 20S proteasomes analogous to those
41
of eukaryotes that are accompanied by a functional equivalent of ubiquitin known as the
42
prokaryotic ubiquitin-like protein (Pup). [2-7] Pupylation, the covalent attachment of Pup to
43
proteins, was the first protein-protein modification discovered in prokaryotes. This post-
44
translational modification has been extensively studied in mycobacteria wherein its mechanism
45
and physiological role are analogous to ubiquitination in eukaryotes.[4-7] In a common
46
mechanism, both ubiquitin and Pup are covalently attached to lysine residues of proteins via the
2
47
ATP-dependent activation of a constituent carboxylate moiety [3]. While ubiquitin is attached
48
via a C-terminal glycine residue [2], Pup is linked via the side chain of a glutamate residue at its
49
carboxy terminus [4-8]. In mycobacteria, this residue is the product of hydrolytic deamidation of
50
a glutamine side chain which is catalyzed by an enzyme called Dop (deamidase of Pup).[5-11]
51
The enzyme that catalyzes the ATP-dependent activation of the deamidated side chain of Pup
52
and its subsequent ligation to lysine residues of selected proteins is called either Pup ligase or
53
PafA (proteasomal accessory factor A). [5, 8-11] As is the case for the major physiological role
54
of ubiquitination, experimental evidence acquired in mycobacteria suggests that Pup-tagged
55
proteins are unfolded by the proteasomal ATPase MPa (also known as ARC) and translocated
56
into the barrel-shaped core of the 20S proteasome wherein hydrolytic degradation occurs. [4, 12]
57
Though pupylation has been closely tied to degradation of proteins by the 20S proteasome, it is
58
not yet clear if this phenomenon has other roles like controlling protein sub-cellular localizations,
59
associations, and activities as is the case for ubiquitination.
60
In Mycobacterium tuberculosis, genes encoding Pup, mediators of pupylation (Dop and
61
PafA), and components of the proteasome are dispensable for viability, yet essential for survival
62
under conditions of nitrosative stress. [13-15] The latter observations have been invoked as an
63
explanation for the capacity of M. tuberculosis to evade the innate immune response and persist
64
in the host. [13, 14] The genetic analyses of the Pup-proteasome system in mycobacteria have
65
been complemented by biochemical analyses wherein pupylated proteins (i.e., the pupylome)
66
have been identified in M. tuberculosis and Mycobacterium smegmatis.[16-19] In these
67
mycobacterial species, as many as 55 pupylated proteins have been identified in cell lysates. The
68
Pup modification was observed on proteins of nearly all structural and functional classes. [16]
69
While the relevance of the Pup-proteasome system to the pathogenicity of M.
3
70
tuberculosis has made it the focus of much attention, we and others [24, 25] have been intrigued
71
by the homologous system in Streptomyces bacteria. These actinobacteria bacteria are best
72
known as producers of antibiotics and for their complex life cycle during which both
73
morphological differentiation and sporulation occur [20-22]. Because protein metabolism plays
74
key regulatory roles in several aspects of Streptomyces physiology [21], we predicted that the
75
Pup-proteasome system would be a compelling subject for research. For our studies, we chose
76
Streptomyces coelicolor, which is the model organism of the genus. It has genes whose products
77
are homologous to proteins of the Pup-proteasome system in M. tuberculosis (Figure 1). The
78
genes encoding the 20S proteasome components (PrcA and PrcB) and Pup are clustered in a
79
genetic locus referred to as the Pup-proteasome system, pps. Though the cognate proteins share
80
at least 60% identity with those in M. tuberculosis, a remarkable and perhaps meaningful
81
difference is that Pup in S. coelicolor has a glutamate residue rather than a glutamine at its
82
carboxy terminus (Figure 1b). [23] This substitution obviates the need for the Dop-catalyzed
83
deamidation of Pup (which precedes pupylation in most other bacteria) and thus leaves open
84
questions about the function of the Dop in S. coelicolor. As was the case in M. tuberculosis, it
85
has been reported that the prc genes are not essential for viability. [24, 25] However, proteomic
86
analyses of the S. coelicolor prc null strain revealed a marked over-production of proteins that
87
mediate stress responses. [25] While the physiological significance of the prc locus has been
88
assessed in S. coelicolor, [25] the genes encoding Pup and the enzymes that catalyze pupylation
89
have not been characterized. To broaden our understanding of the Pup-proteasome system in S.
90
coelicolor, we constructed and characterized S. coelicolor strains lacking genes encoding the 20S
91
proteasome, the Pup-proteasome system, and Pup ligase. In addition to phenotypic analyses of
92
these pupylation deficient strains, we performed proteomic experiments to identify pupylated
4
93
proteins in S. coelicolor by expressing a gene encoding an affinity-tagged allele of Pup in both
94
proteasome (prc) null and wild type strains of S. coelicolor.
95 96
Methods and Materials
97
Detailed procedures regarding cloning, strain construction, and the proteomic analyses can be
98
found in the supporting information file that is available on-line.
99 100
Strains, Media and Culture Conditions. Streptomyces coelicolor strains were grown at 30°C on
101
mannitol soy flour medium (SFM), Difco nutrient agar medium (DNA), yeast extract-malt
102
extract medium (YEME), or Tryptone Soya Broth (TSB). [26] SFM was used for conjugations
103
between S. coelicolor and Escherichia coli and for generating spore stocks. Escherichia coli
104
strains DH5α and ET12567/pUZ8002 were grown on Luria-Bertani medium at 37°C for routine
105
subcloning. For the selection of E. coli, ampicillin, apramycin, chloramphenicol, hygromycin,
106
and kanamycin were employed at 100, 50, 25, 80, and 50 μg/ml, respectively. Nalidixic acid was
107
used at 20 μg/ml to counterselect E. coli in conjugations with S. coelicolor. Apramycin and
108
hygromycin were used at 50 μg/ml for the selection of S. coelicolor exconjugants.
109 110
Construction of pps (SCO1643-46), prc (SCO1634-44), and pafA (SCO1640) null mutants.
111
Strains of S. coelicolor M145 in which either the pps locus (SCO1643-46), the prc locus
112
(SCO1643-44) or the pafA gene (SCO1640) were replaced with an apramycin (apr) resistance
113
cassette were constructed via polymerase chain reaction (PCR)-targeted mutagenesis.[27] Gene
114
replacements were confirmed by genomic DNA isolation and amplification of the region by PCR.
115
Complementation of the pafA null strain was performed in two ways. In one strategy, the null
5
116
strain was conjugated with derivatives of the integrative plasmid pMS81 harboring the respective
117
genetic loci including their promoters. In the other strategy, the null strain was conjugated with
118
the integrative plasmid pIJ10257 harboring the genes of interest under the control of the
119
constitutive, ermE* promoter.
120 121
Scanning Electron Microscopy. Wild-type, pafA null strains, and pafA overexpression strains of
122
S. coelicolor were grown on SFM for four days. Biomass (i.e., spores and aerial hyphae) on the
123
surfaces of the confluent lawns was harvested by adherence to double-sided tape. In turn, the
124
tape was affixed to microscopy stubs for imaging. After desiccation for 48 hours in the presence
125
of Drierite, the samples were coated with Au by low vacuum sputter coating. The samples were
126
then imaged on a Hitachi 2700 Scanning Electron Microscope equipped with a lanthanum
127
hexaboride gun. Images were collected and analyzed with a Quartz PCI digital imaging system.
128
The microscope was set to a BC of 8, a WD of 15 mm, and a KV of 8. The images were
129
collected for 80 seconds with 1.5 K mag, 6 K mag, and 15 K mag, for collection at 20 nm, 5 nm,
130
and 2 nm respectively.
131 132
Oxidative Stress Assay. In assessments of the strains’ sensitivities to oxidative stress, equal
133
numbers of spores (1x108) of the wild-type, Δprc::apr, Δpps::apr, ΔpafA::apr, and ΔpafA::apr
134
with wild-type pafA (i.e., the complemented null strain) were plated on either SFM or DNA. To
135
the center of each freshly inoculated plate was added a sterilized and dry paper disc (6mm
136
diameter) that had been submerged in a 40% solution of cumene hydroperoxide in dH2O. After
137
growth at 30°C for 48 hours on DNA or 72 hours SFM, the size of the cleared zone was
6
138
measured. These experiments were replicated 4 times and the average sizes of the zones of
139
inhibition are reported.
140 141
Construction of His-tagged Pup. To facilitate the isolation and identification of pupylated
142
proteins in cell lysates from the S. coelicolor strains of interest, an affinity-tagged version of Pup
143
was constructed. Using a sub-cloning strategy described in the supporting information, the wild-
144
type pup gene in the pps locus was replaced with an allele encoding Pup with a hexa-histidine
145
affinity tag fused to its N-terminus. Using the aforementioned plasmid as template in a PCR, we
146
amplified a fragment containing the engineered pup gene and the downstream open reading
147
frame (encoding a hypothetical protein, SCO1645) in an effort to generate a plasmid from which
148
the engineered Pup and not the prc genes could be expressed. These strategies were employed to
149
ensure that the engineered pup gene would be transcribed from the native pup promoter.
150
Plasmids harboring either the entire engineered pup gene in its native context within the pps
151
locus (pJS873) or the engineered pup gene alone (pJS875) were cloned into the integrative
152
plasmid pMS81 and introduced into the S. coelicolor pps null strain by conjugation. The former
153
strain was employed for studies of pupylation in a mimic of wild-type S. coelicolor, whereas the
154
latter was used to characterize pupylation in the absence of a functional proteasome.
155 156
Isolation of Pupylated Proteins. S. coelicolor pps null strains harboring either pJS873 (pps locus
157
with an embedded pup allele encoding His-tagged Pup) or pJS875 (only pup allele encoding His-
158
tagged Pup) were grown to stationary phase in Tryptone Soya Broth (TSB). In replicate
159
experiments, growth of the cultures was assessed via measurements of dry cell weights at various
160
time points over a period of 36 hours after spore germination. Cultures of wild-type S. coelicolor
7
161
and the pafA null strain (both lacking the His-tagged Pup) were grown in parallel for control
162
experiments. In duplicate experiments, mycelia were harvested at single time points in early
163
exponential (16 hours post-spore germination), late exponential (20 hours post-spore
164
germination), and stationary phase (26 hours post-spore germination) by centrifugation at 4000
165
rpm for 10 minutes. (The growth rates were measured by quantification of dry cell weights.) The
166
mycelial pellets were resuspended in 1.4 mL lysis buffer (50mM NaH2PO4, 300mM NaCl,
167
10mM Imidazole, pH 8.0) and 4.5 μL 1:9 benzonase buffer (50mM Tris-HCl, 20mM NaCl,
168
20mM MgCl2, pH 8.0). For lysis, lysozyme was added to a final concentration of 1 mg/mL.
169
After incubation on ice for 1 hour, mycelia were lysed by sonication at 60% power three times at
170
30-second intervals with 2 minutes of rest between each sonication. The lysates were clarified at
171
4o C by centrifugation at 13,000 rcf for 15 minutes and applied to a Qiagen Ni-NTA column
172
under non-denaturing conditions. Following the manufacturer’s protocol, the column was
173
washed and the bound proteins were eluted. The eluted proteins were finally digested with
174
trypsin. Lysates from two different cultures were subjected to the affinity purification method. In
175
duplicate experiments, wild-type and the ΔpafA::apr strains grown to stationary phase were also
176
subjected to identical proteomic analyses without the Ni-NTA purification.
177 178
Protein Identification via Mass Spectrometry and Bioinformatics. To identify proteins isolated
179
from the lysates via Ni-NTA chromatography, the eluates were digested with trypsin and the
180
resulting peptides were loaded onto a reverse phase column and separated via an Agilent 1200
181
HPLC. The peptides were identified by tandem, high-resolution MS/MS analyses using a
182
coupled LTQ Orbitrap Velos mass spectrometer housed at the Brown University Center for
183
Genomics and Proteomics. The mass spectral data were searched using the MASCOT software
8
184
algorithm against the S. coelicolor protein database. In the bioinformatic identifications of the
185
peptide fragments, trypsin specificity with two missed cleavage sites was allowed and the MS
186
mass tolerance was 7 ppm while the MS/MS tolerance was 0.5 Da. A total of 857 proteins were
187
identified among all the biological samples. Using the target decoy method, the false discovery
188
rates were calculated for each sample. For nine of the twelve samples, they ranged between 0
189
and 6%. The remaining samples had false discovery rates of 8.3, 11.5, and 12.5%. Nevertheless,
190
the iterative nature of data analysis required for identification of pupylated proteins negated the
191
significance of the false discovery rates. Specifically, our criterion for the assignment of any of
192
the 857 proteins as a pupylation target was the identification of a constituent peptide having a
193
lysine reside and a 243 Dalton mass difference due to the presence of a characteristic tryptic
194
fragment of Pup. This Gly-Gly-Glu (GGE) fragment corresponds to last three residues at the C-
195
terminus of Pup and and is linked to lysine residues via an isopeptide linkage (Figure 1c).
196
Identifications were contingent on at least one unique peptide spectrum match (PSM) in the
197
molecular weight search (MOWSE) with a protein score cutoff of 1.
198 199
Results and Discussion
200
Phenotypic analyses of S. coelicolor pps, prc and pafA null mutants. To visually assess the
201
significance
202
differentiation and secondary metabolism, the prc (Δprc::apr), pafA (ΔpafA::apr), and pps
203
(Δpps:apr) null strains were grown on both soya flour media (SFM) and Difco nutrient agar
204
(DNA).[26] The former is a minimal medium optimized for spore formation in S. coelicolor,
205
while the latter is a nutrient-rich medium that promotes fast growth without sporulation.[26] The
206
production of the pigmented secondary metabolites by S. coelicolor can be easily visualized on
of
pupylation
and
proteasome-mediated
9
degradation
in
morphological
207
both media [26]. The prc and the pps null strains were virtually indistinguishable from the wild-
208
type strain on both SFM and DNA (see supporting information). These observations indicate that
209
pupylation and proteasome-mediated degradation of proteins are dispensable for secondary
210
metabolism and sporulation in S. coelicolor. In stark contrast, we found that the pafA null strain
211
exhibited defects in both secondary metabolism and morphological differentiation. On both SFM
212
and DNA, the pafA null strain overproduced the blue-colored secondary metabolite actinorhodin
213
(Figure S1). Another phenotype of the pafA null strain was that its confluent lawns lacked the
214
grey-colored sporulation pigment after four days of growth on SFM; whereas lawns of the wild-
215
type strain were completely grey at the same time point. Curiously, we found that the pafA null
216
strain’s sporulation defect was only apparent when the spores were plated at low densities (
1
Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor
2 3
Corey L. Compton, Michael S. Fernandopulle, Rohith T. Nagari, and Jason K. Sello
4 5
Department of Chemistry, Brown University, 324 Brook St. Providence, RI 02912
6
[email protected]
7
Abstract
8
Pupylation is a post-translational modification peculiar to actinobacteria wherein proteins are
9
covalently modified with a small protein called the prokaryotic ubiquitin-like protein (Pup). Like
10
ubiquitination in eukaryotes, this phenomenon has been associated with proteasome-mediated
11
protein degradation in mycobacteria. Here, we report studies of pupylation in streptomycetes,
12
another large genus of actinobacteria. We constructed mutants of Streptomyces coelicolor
13
lacking PafA (Pup ligase), the proteasome, and the Pup-proteasome system. We found that these
14
mutants share a high susceptibility to oxidative stress, as compared to that of the wild-type strain.
15
Remarkably, we found that the pafA null mutant has a sporulation defect not seen in strains
16
lacking the Pup-proteasome system. In proteomics experiments facilitated by an affinity-tagged
17
variant of Pup, we identified 110 pupylated proteins in S. coelicolor strains having and lacking
18
genes encoding the 20S proteasome. Our findings shed new light on this unusual post-
19
translational modification and its role in Streptomyces physiology.
20 21
Importance
22
The presence of 20S proteasomes reminiscent of those in eukaryotes and a functional equivalent
23
of ubiquitin, known as the prokaryotic ubiquitin-like protein (Pup), in actinobacteria have
1
24
motivated re-evaluations of protein homeostasis in prokaryotes. Though the Pup-proteasome
25
system has been studied extensively in mycobacteria, it is much less understood in
26
streptomycetes, a large genus of actinobacteria known for highly choreographed life cycles in
27
which phases of morphological differentiation, sporulation, and secondary metabolism are often
28
regulated by protein metabolism. Here, we define constituents of the pupylome in Streptomyces
29
coelicolor for the first time and present new evidence that link pupylation and the oxidative
30
stress response in this bacterium. Surprisingly, we find that the Pup ligase has a Pup-independent
31
role in sporulation.
32 33
Key Words
34
Streptomyces, 20S proteasome, pupylation, proteomics, sporulation, post-translational
35
modification
36 37
Introduction
38
Protein homeostasis is a tightly regulated and critically important phenomenon in
39
bacterial physiology [1]. It is thought to be particularly complex in actinobacteria, which have
40
both the canonical proteolytic machinery of prokaryotes and 20S proteasomes analogous to those
41
of eukaryotes that are accompanied by a functional equivalent of ubiquitin known as the
42
prokaryotic ubiquitin-like protein (Pup). [2-7] Pupylation, the covalent attachment of Pup to
43
proteins, was the first protein-protein modification discovered in prokaryotes. This post-
44
translational modification has been extensively studied in mycobacteria wherein its mechanism
45
and physiological role are analogous to ubiquitination in eukaryotes.[4-7] In a common
46
mechanism, both ubiquitin and Pup are covalently attached to lysine residues of proteins via the
2
47
ATP-dependent activation of a constituent carboxylate moiety [3]. While ubiquitin is attached
48
via a C-terminal glycine residue [2], Pup is linked via the side chain of a glutamate residue at its
49
carboxy terminus [4-8]. In mycobacteria, this residue is the product of hydrolytic deamidation of
50
a glutamine side chain which is catalyzed by an enzyme called Dop (deamidase of Pup).[5-11]
51
The enzyme that catalyzes the ATP-dependent activation of the deamidated side chain of Pup
52
and its subsequent ligation to lysine residues of selected proteins is called either Pup ligase or
53
PafA (proteasomal accessory factor A). [5, 8-11] As is the case for the major physiological role
54
of ubiquitination, experimental evidence acquired in mycobacteria suggests that Pup-tagged
55
proteins are unfolded by the proteasomal ATPase MPa (also known as ARC) and translocated
56
into the barrel-shaped core of the 20S proteasome wherein hydrolytic degradation occurs. [4, 12]
57
Though pupylation has been closely tied to degradation of proteins by the 20S proteasome, it is
58
not yet clear if this phenomenon has other roles like controlling protein sub-cellular localizations,
59
associations, and activities as is the case for ubiquitination.
60
In Mycobacterium tuberculosis, genes encoding Pup, mediators of pupylation (Dop and
61
PafA), and components of the proteasome are dispensable for viability, yet essential for survival
62
under conditions of nitrosative stress. [13-15] The latter observations have been invoked as an
63
explanation for the capacity of M. tuberculosis to evade the innate immune response and persist
64
in the host. [13, 14] The genetic analyses of the Pup-proteasome system in mycobacteria have
65
been complemented by biochemical analyses wherein pupylated proteins (i.e., the pupylome)
66
have been identified in M. tuberculosis and Mycobacterium smegmatis.[16-19] In these
67
mycobacterial species, as many as 55 pupylated proteins have been identified in cell lysates. The
68
Pup modification was observed on proteins of nearly all structural and functional classes. [16]
69
While the relevance of the Pup-proteasome system to the pathogenicity of M.
3
70
tuberculosis has made it the focus of much attention, we and others [24, 25] have been intrigued
71
by the homologous system in Streptomyces bacteria. These actinobacteria bacteria are best
72
known as producers of antibiotics and for their complex life cycle during which both
73
morphological differentiation and sporulation occur [20-22]. Because protein metabolism plays
74
key regulatory roles in several aspects of Streptomyces physiology [21], we predicted that the
75
Pup-proteasome system would be a compelling subject for research. For our studies, we chose
76
Streptomyces coelicolor, which is the model organism of the genus. It has genes whose products
77
are homologous to proteins of the Pup-proteasome system in M. tuberculosis (Figure 1). The
78
genes encoding the 20S proteasome components (PrcA and PrcB) and Pup are clustered in a
79
genetic locus referred to as the Pup-proteasome system, pps. Though the cognate proteins share
80
at least 60% identity with those in M. tuberculosis, a remarkable and perhaps meaningful
81
difference is that Pup in S. coelicolor has a glutamate residue rather than a glutamine at its
82
carboxy terminus (Figure 1b). [23] This substitution obviates the need for the Dop-catalyzed
83
deamidation of Pup (which precedes pupylation in most other bacteria) and thus leaves open
84
questions about the function of the Dop in S. coelicolor. As was the case in M. tuberculosis, it
85
has been reported that the prc genes are not essential for viability. [24, 25] However, proteomic
86
analyses of the S. coelicolor prc null strain revealed a marked over-production of proteins that
87
mediate stress responses. [25] While the physiological significance of the prc locus has been
88
assessed in S. coelicolor, [25] the genes encoding Pup and the enzymes that catalyze pupylation
89
have not been characterized. To broaden our understanding of the Pup-proteasome system in S.
90
coelicolor, we constructed and characterized S. coelicolor strains lacking genes encoding the 20S
91
proteasome, the Pup-proteasome system, and Pup ligase. In addition to phenotypic analyses of
92
these pupylation deficient strains, we performed proteomic experiments to identify pupylated
4
93
proteins in S. coelicolor by expressing a gene encoding an affinity-tagged allele of Pup in both
94
proteasome (prc) null and wild type strains of S. coelicolor.
95 96
Methods and Materials
97
Detailed procedures regarding cloning, strain construction, and the proteomic analyses can be
98
found in the supporting information file that is available on-line.
99 100
Strains, Media and Culture Conditions. Streptomyces coelicolor strains were grown at 30°C on
101
mannitol soy flour medium (SFM), Difco nutrient agar medium (DNA), yeast extract-malt
102
extract medium (YEME), or Tryptone Soya Broth (TSB). [26] SFM was used for conjugations
103
between S. coelicolor and Escherichia coli and for generating spore stocks. Escherichia coli
104
strains DH5α and ET12567/pUZ8002 were grown on Luria-Bertani medium at 37°C for routine
105
subcloning. For the selection of E. coli, ampicillin, apramycin, chloramphenicol, hygromycin,
106
and kanamycin were employed at 100, 50, 25, 80, and 50 μg/ml, respectively. Nalidixic acid was
107
used at 20 μg/ml to counterselect E. coli in conjugations with S. coelicolor. Apramycin and
108
hygromycin were used at 50 μg/ml for the selection of S. coelicolor exconjugants.
109 110
Construction of pps (SCO1643-46), prc (SCO1634-44), and pafA (SCO1640) null mutants.
111
Strains of S. coelicolor M145 in which either the pps locus (SCO1643-46), the prc locus
112
(SCO1643-44) or the pafA gene (SCO1640) were replaced with an apramycin (apr) resistance
113
cassette were constructed via polymerase chain reaction (PCR)-targeted mutagenesis.[27] Gene
114
replacements were confirmed by genomic DNA isolation and amplification of the region by PCR.
115
Complementation of the pafA null strain was performed in two ways. In one strategy, the null
5
116
strain was conjugated with derivatives of the integrative plasmid pMS81 harboring the respective
117
genetic loci including their promoters. In the other strategy, the null strain was conjugated with
118
the integrative plasmid pIJ10257 harboring the genes of interest under the control of the
119
constitutive, ermE* promoter.
120 121
Scanning Electron Microscopy. Wild-type, pafA null strains, and pafA overexpression strains of
122
S. coelicolor were grown on SFM for four days. Biomass (i.e., spores and aerial hyphae) on the
123
surfaces of the confluent lawns was harvested by adherence to double-sided tape. In turn, the
124
tape was affixed to microscopy stubs for imaging. After desiccation for 48 hours in the presence
125
of Drierite, the samples were coated with Au by low vacuum sputter coating. The samples were
126
then imaged on a Hitachi 2700 Scanning Electron Microscope equipped with a lanthanum
127
hexaboride gun. Images were collected and analyzed with a Quartz PCI digital imaging system.
128
The microscope was set to a BC of 8, a WD of 15 mm, and a KV of 8. The images were
129
collected for 80 seconds with 1.5 K mag, 6 K mag, and 15 K mag, for collection at 20 nm, 5 nm,
130
and 2 nm respectively.
131 132
Oxidative Stress Assay. In assessments of the strains’ sensitivities to oxidative stress, equal
133
numbers of spores (1x108) of the wild-type, Δprc::apr, Δpps::apr, ΔpafA::apr, and ΔpafA::apr
134
with wild-type pafA (i.e., the complemented null strain) were plated on either SFM or DNA. To
135
the center of each freshly inoculated plate was added a sterilized and dry paper disc (6mm
136
diameter) that had been submerged in a 40% solution of cumene hydroperoxide in dH2O. After
137
growth at 30°C for 48 hours on DNA or 72 hours SFM, the size of the cleared zone was
6
138
measured. These experiments were replicated 4 times and the average sizes of the zones of
139
inhibition are reported.
140 141
Construction of His-tagged Pup. To facilitate the isolation and identification of pupylated
142
proteins in cell lysates from the S. coelicolor strains of interest, an affinity-tagged version of Pup
143
was constructed. Using a sub-cloning strategy described in the supporting information, the wild-
144
type pup gene in the pps locus was replaced with an allele encoding Pup with a hexa-histidine
145
affinity tag fused to its N-terminus. Using the aforementioned plasmid as template in a PCR, we
146
amplified a fragment containing the engineered pup gene and the downstream open reading
147
frame (encoding a hypothetical protein, SCO1645) in an effort to generate a plasmid from which
148
the engineered Pup and not the prc genes could be expressed. These strategies were employed to
149
ensure that the engineered pup gene would be transcribed from the native pup promoter.
150
Plasmids harboring either the entire engineered pup gene in its native context within the pps
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locus (pJS873) or the engineered pup gene alone (pJS875) were cloned into the integrative
152
plasmid pMS81 and introduced into the S. coelicolor pps null strain by conjugation. The former
153
strain was employed for studies of pupylation in a mimic of wild-type S. coelicolor, whereas the
154
latter was used to characterize pupylation in the absence of a functional proteasome.
155 156
Isolation of Pupylated Proteins. S. coelicolor pps null strains harboring either pJS873 (pps locus
157
with an embedded pup allele encoding His-tagged Pup) or pJS875 (only pup allele encoding His-
158
tagged Pup) were grown to stationary phase in Tryptone Soya Broth (TSB). In replicate
159
experiments, growth of the cultures was assessed via measurements of dry cell weights at various
160
time points over a period of 36 hours after spore germination. Cultures of wild-type S. coelicolor
7
161
and the pafA null strain (both lacking the His-tagged Pup) were grown in parallel for control
162
experiments. In duplicate experiments, mycelia were harvested at single time points in early
163
exponential (16 hours post-spore germination), late exponential (20 hours post-spore
164
germination), and stationary phase (26 hours post-spore germination) by centrifugation at 4000
165
rpm for 10 minutes. (The growth rates were measured by quantification of dry cell weights.) The
166
mycelial pellets were resuspended in 1.4 mL lysis buffer (50mM NaH2PO4, 300mM NaCl,
167
10mM Imidazole, pH 8.0) and 4.5 μL 1:9 benzonase buffer (50mM Tris-HCl, 20mM NaCl,
168
20mM MgCl2, pH 8.0). For lysis, lysozyme was added to a final concentration of 1 mg/mL.
169
After incubation on ice for 1 hour, mycelia were lysed by sonication at 60% power three times at
170
30-second intervals with 2 minutes of rest between each sonication. The lysates were clarified at
171
4o C by centrifugation at 13,000 rcf for 15 minutes and applied to a Qiagen Ni-NTA column
172
under non-denaturing conditions. Following the manufacturer’s protocol, the column was
173
washed and the bound proteins were eluted. The eluted proteins were finally digested with
174
trypsin. Lysates from two different cultures were subjected to the affinity purification method. In
175
duplicate experiments, wild-type and the ΔpafA::apr strains grown to stationary phase were also
176
subjected to identical proteomic analyses without the Ni-NTA purification.
177 178
Protein Identification via Mass Spectrometry and Bioinformatics. To identify proteins isolated
179
from the lysates via Ni-NTA chromatography, the eluates were digested with trypsin and the
180
resulting peptides were loaded onto a reverse phase column and separated via an Agilent 1200
181
HPLC. The peptides were identified by tandem, high-resolution MS/MS analyses using a
182
coupled LTQ Orbitrap Velos mass spectrometer housed at the Brown University Center for
183
Genomics and Proteomics. The mass spectral data were searched using the MASCOT software
8
184
algorithm against the S. coelicolor protein database. In the bioinformatic identifications of the
185
peptide fragments, trypsin specificity with two missed cleavage sites was allowed and the MS
186
mass tolerance was 7 ppm while the MS/MS tolerance was 0.5 Da. A total of 857 proteins were
187
identified among all the biological samples. Using the target decoy method, the false discovery
188
rates were calculated for each sample. For nine of the twelve samples, they ranged between 0
189
and 6%. The remaining samples had false discovery rates of 8.3, 11.5, and 12.5%. Nevertheless,
190
the iterative nature of data analysis required for identification of pupylated proteins negated the
191
significance of the false discovery rates. Specifically, our criterion for the assignment of any of
192
the 857 proteins as a pupylation target was the identification of a constituent peptide having a
193
lysine reside and a 243 Dalton mass difference due to the presence of a characteristic tryptic
194
fragment of Pup. This Gly-Gly-Glu (GGE) fragment corresponds to last three residues at the C-
195
terminus of Pup and and is linked to lysine residues via an isopeptide linkage (Figure 1c).
196
Identifications were contingent on at least one unique peptide spectrum match (PSM) in the
197
molecular weight search (MOWSE) with a protein score cutoff of 1.
198 199
Results and Discussion
200
Phenotypic analyses of S. coelicolor pps, prc and pafA null mutants. To visually assess the
201
significance
202
differentiation and secondary metabolism, the prc (Δprc::apr), pafA (ΔpafA::apr), and pps
203
(Δpps:apr) null strains were grown on both soya flour media (SFM) and Difco nutrient agar
204
(DNA).[26] The former is a minimal medium optimized for spore formation in S. coelicolor,
205
while the latter is a nutrient-rich medium that promotes fast growth without sporulation.[26] The
206
production of the pigmented secondary metabolites by S. coelicolor can be easily visualized on
of
pupylation
and
proteasome-mediated
9
degradation
in
morphological
207
both media [26]. The prc and the pps null strains were virtually indistinguishable from the wild-
208
type strain on both SFM and DNA (see supporting information). These observations indicate that
209
pupylation and proteasome-mediated degradation of proteins are dispensable for secondary
210
metabolism and sporulation in S. coelicolor. In stark contrast, we found that the pafA null strain
211
exhibited defects in both secondary metabolism and morphological differentiation. On both SFM
212
and DNA, the pafA null strain overproduced the blue-colored secondary metabolite actinorhodin
213
(Figure S1). Another phenotype of the pafA null strain was that its confluent lawns lacked the
214
grey-colored sporulation pigment after four days of growth on SFM; whereas lawns of the wild-
215
type strain were completely grey at the same time point. Curiously, we found that the pafA null
216
strain’s sporulation defect was only apparent when the spores were plated at low densities (
