1 Catabolic pathway of arginine in Anabaena involves a novel bifunctional
2 enzyme that produces proline from arginine
3
4 Mireia Burnat, Silvia Picossi, Ana Valladares, Antonia Herrero, and Enrique
5 Flores*
6 Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Américo
7 Vespucio 49, E-41092 Sevilla, Spain.
8
9 *Correspondence to: Enrique Flores ([email protected])
10
11
12
13 Key words: Archaea; Arginine catabolism; Arginine dihydrolase; Cyanobacteria; Ornithine
14 cyclodeaminase
1 15 Summary
16 Arginine participates widely in metabolic processes. The heterocyst-forming cyanobacterium
17 Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of
18 ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic
19 pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA
20 protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In
21 contrast, AgrE is a hitherto unrecognized enzyme that contains both an N-terminal propeller
22 domain and a unique C-terminal domain of previously unidentified function. In vitro analysis of
23 the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of
24 the N-terminal domain and ornithine cyclodeaminase activity of the C-terminal domain, overall
25 producing proline from arginine. In the diazotrophic filaments of Anabaena, -aspartyl-arginine
26 dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing
27 aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in
28 vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE-PutA
29 pathway mainly in the vegetative cells. Expression in Anabaena of a homologue of the C-
30 terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an
31 archaeal ornithine cyclodeaminase.
32
33
2 34 Introduction
35 Arginine (2-amino-5-guanidinopentanoic acid) is the amino acid with highest N:C ratio. This is
36 due to the presence of a guanidine group capping its 3-carbon aliphatic side chain. In addition to
37 being a proteinaceous amino acid, arginine has multiple metabolic uses in the living cell (Morris,
38 2016). For example, it is the substrate for biosynthesis of nitric oxide, and its metabolism gives
39 rise to polyamines and metabolites of the urea cycle. In various bacteria, arginine can serve as a
40 source of carbon, nitrogen and energy. In cyanobacteria, oxygenic phototrophs, arginine
41 accumulates in a cell inclusion called cyanophycin (multi-L-arginyl-poly[L-aspartic acid]) that
42 can eventually be mobilized. Consistent with such diverse metabolic uses, arginine can be
43 metabolized by numerous enzymes, including those of catabolic pathways that make its nitrogen
44 atoms available for metabolism. Some of these pathways produce glutamate, which is a major
45 distributor of nitrogen in the cell (Lu, 2006). Commonly, first steps in catabolism of arginine
46 involve its conversion to ornithine, either directly by arginase (with concomitant release of urea)
47 or arginine amidinotransferase (with transfer of urea to an acceptor metabolite) or indirectly via
48 citrulline produced by arginine deiminase and further metabolized by ornithine
49 carbamoyltransferase (Lu, 2006). Ornithine is then converted to glutamate either via glutamate-
50 semialdehyde or via proline. In the latter case, ornithine cyclodeaminase, an enzyme discovered
51 in Clostridia (Costilow and Laycock, 1971; Goodman et al., 2004), produces the proline that is
52 further catabolized to glutamate by PutA, which is a bifunctional enzyme with proline
53 dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase activities (Liu et al., 2017).
54 Some cyanobacteria grow as chains of cells (filaments) that, under nitrogen deprivation,
55 contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and
56 heterocysts that fix N2 (Flores and Herrero, 2010). In rapidly growing filaments, heterocysts and
3 57 vegetative cells are found in a ratio of about 1 to 10. The heterocysts provide the vegetative cells
58 with fixed nitrogen, and the vegetative cells provide the heterocysts with reduced carbon, mainly
59 in the form of sucrose, through intercellular molecular transfer (Nürnberg et al., 2015).
60 Cyanophycin accumulates conspicuously in the polar regions of the heterocysts, and degradation
61 of cyanophycin takes place in two steps: cyanophycinase produces a dipeptide, -aspartyl-
62 arginine, that is transferred to the adjacent vegetative cells where it is hydrolyzed by isoaspartyl
63 dipeptidase producing aspartate and arginine (Burnat et al., 2014). Those amino acids, together
64 with glutamine (Thomas et al., 1977), serve as nitrogen sources for the vegetative cells. Whereas
65 aspartate and glutamine can make their nitrogen atoms available for metabolism by glutamine-
66 oxoglutarate transamidase and aspartate transaminase, respectively (Thomas et al., 1977; Xu et
67 al., 2015), the pathway for arginine utilization was elusive.
68
69 Results
70 Physiological-genetic identification of the agrE-putA pathway
71 When filaments of the model heterocyst-forming cyanobacterium, Anabaena sp. strain PCC
72 7120 (hereafter Anabaena), were incubated in the presence of [14C]arginine, labelled proline and
73 glutamate were produced at substantial amounts, and ornithine was occasionally detected at low
74 levels (Fig. 1A; see also Burnat and Flores, 2014). We previously showed that a putative
75 ureohydrolase encoded in the Anabaena genome is an agmatinase rather than an arginase and is
76 not required for the production of proline and glutamate from arginine (Burnat and Flores, 2014).
77 Open reading frame (ORF) alr4995 of the Anabaena genome has been discussed as a possible
78 arginine deiminase or arginine amidinotransferase (Schriek et al., 2007). A more detailed
79 analysis of Alr4995 showed however that it has two distinct domains (Fig. 2). The N-terminal
4 80 part of the protein is predicted to have the structure of an / propeller domain characteristic of
81 arginine deiminases and arginine amidinotransferases (Shirai et al., 2006), and the C-terminal
82 part is homologous to a Methanococcus protein annotated as lysine-oxoglutarate
83 reductase/saccharopine dehydrogenase (LOR/SDH) protein 2 whose function was unknown (we
84 shall refer this Methanococcus protein as Mls2). We denoted ORF alr4995 gene agrE (from
85 arginine-guanidine removing enzyme) and constructed an Anabaena strain (agrE) with deletion
86 of an internal fragment of this gene (see Experimental procedures and Fig. S1). Neither proline
87 nor glutamate was observed in filaments of strain agrE when incubated in the presence of
88 [14C]arginine (Fig. 1A), indicating that protein AgrE is needed to catabolize arginine to proline
89 and glutamate. Anabaena filaments incubated with [14C]ornithine also produced labelled proline
90 and glutamate, and this production was also arrested in strain agrE (Fig. 1B), indicating that
91 AgrE also mediates catabolism of ornithine (Fig. 1C), which could be an intermediate in the
92 reaction that produces proline from arginine.
93 Because arginine deiminases produce citrulline from arginine, it was also possible that
94 citrulline rather than ornithine is an intermediate in the reaction catalyzed by AgrE, and that in
95 our in vivo assays with [14C]ornithine, [14C]citrulline was produced from [14C]ornithine by
96 ornithine carbamoyltransferase. We thereupon constructed an Anabaena strain (∆argF::C.K3)
97 with an insertion of gene-cassette C.K3 in the argF gene that encodes ornithine
98 carbamoyltransferase (Fig. S2). In this mutant, labelled proline and glutamate were produced
99 from [14C]ornithine as in the wild type (Fig. 3), indicating that conversion to citrulline was
100 unnecessary for ornithine catabolism. These observations suggested that AgrE can indeed use
101 ornithine as a substrate as well as produce it as an intermediate in the conversion of arginine into
102 proline. We hypothesize that (i) the N-terminal domain of AgrE catalyzes the formation of
5 103 ornithine from arginine, and (ii) the C-terminal domain of AgrE catalyzes the production of
104 proline from ornithine.
105 We then constructed derivatives of the Anabaena agrE mutant expressing Strep-tagged
106 Anabaena AgrE or Methanococcus Mls2 (Fig. S3 and S4, respectively). The Anabaena agrE
107 mutant complemented with the Strep-tagged AgrE produced [14C]proline and [14C]glutamate
108 from both [14C]arginine and [14C]ornithine, as the wild type (Fig. 4). The Anabaena agrE
109 mutant complemented with the Strep-tagged Mls2 produced [14C]proline and [14C]glutamate
110 from [14C]ornithine but not from [14C]arginine (Fig. 4). These results corroborate that AgrE is
111 involved in the production of proline from arginine in Anabaena and show that the
112 Methanococcus Mls2 protein provides Anabaena with the capability to synthesize proline from
113 ornithine. Therefore, Mls2 is an ornithine cyclodeaminase.
114 ORF alr0540 of the Anabaena genome encodes a putative PutA protein (bifunctional
115 proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase). To investigate whether
116 this enzyme is responsible for the second part of the arginine catabolism pathway, production of
117 glutamate from proline, we created an alr0540::C.S3 insertional mutant of Anabaena (Fig. S5).
118 This mutant did not produce [14C]glutamate from [14C]arginine or [14C]ornithine, but instead
119 accumulated [14C]proline (Fig. 1A, B). To test directly the role of alr0540 in proline catabolism,
120 we additionally tested the fate of [14C]proline in the wild-type and mutant cells. Whereas the
121 wild type produced [14C]glutamate and, further, [14C]glutamine –by amination of [14C]glutamate
122 by glutamine synthetase, the alr0540::C.S3 mutant did not produce [14C]glutamate or
123 [14C]glutamine when supplied extracellularly with [14C]proline (Fig. 1D). Hence, alr0540
124 encodes a PutA protein of Anabaena.
125
6 126 In vitro activity of AgrE
127 The complete AgrE protein and its N-terminal AT/ADI domain were produced, separately, in
128 Escherichia coli as His-tagged proteins, and the products of in vitro assays were analyzed by
129 HPLC after derivatization with phenylisothiocyanate (Heinrikson and Meredith, 1984). When
130 incubated with arginine in a buffer, each of these proteins produced ornithine (Fig. 5A, B;
131 enzymatic reactions tested are summarized in Table 1.1 to 1.3), but no simultaneous production
132 of urea (determined colorimetrically) was observed as it would have been expected from an
133 arginase-like reaction. Instead, in addition to ornithine, production of ammonium was found. The
134 whole protein produced 1.71 ± 0.16 mol of NH4+ per mol of ornithine (mean and SD, n = 4
135 independent enzyme preparations), and the N-terminal domain produced 1.84 mol of NH4+ per
136 mol of ornithine. These results indicate that the N-terminal domain of AgrE is an arginine
137 dihydrolase, reminiscent of the succinylarginine dihydrolase activity of some / propeller
138 proteins (Shirau et al., 2006). The same conclusion has been recently reported for a homologous
139 protein from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Zhang et al., 2018).
140 No production of proline from ornithine was, however, observed with the whole His-tagged
141 AgrE protein assayed under different incubation conditions.
142 We next produced and isolated a Strep-tagged AgrE protein from an Anabaena strain
143 carrying the corresponding gene construct on a plasmid (Fig. S3). Using arginine as substrate,
144 this protein was observed to produce ammonium and ornithine with a stoichiometry of 1.91 mol
145 NH4+ per mol of ornithine (Fig. 5C and Table 1.4). Using ornithine as substrate, this protein,
146 incubated in the presence of a reducing agent and 1 mM NAD+, was observed to produce
147 ammonium and proline with a stoichiometry of 1.16 ± 0.12 mol NH4+ per mol of proline (n = 4)
148 (Fig. 5D and Table 1.4). The reaction was shown to require NAD+ that could be partially
7 149 substituted by NADP+ (Table 1.4), but did not require ATP which instead was inhibitory (Table
150 1.5). Importantly, ornithine cyclodeaminases are known to use NAD+ as a catalytic cofactor
151 (Goodman et al., 2004). These results indicate that AgrE can catalyze the cyclodeamination of
152 ornithine. Because the N-terminal, / propeller domain is an arginine dihydrolase, and because
153 the C-terminal, LOR/SDH-like domain is homologous to the Methanococcus Mls2 protein that
154 provides Anabaena with the capability to produce proline from ornithine (Fig. 4D), we ascribe
155 the ornithine cyclodeaminase activity of AgrE to the C-terminal domain of the protein (Fig. 2).
156 Using arginine as substrate some preparations of Strep-tagged AgrE were observed to
157 produce more than 2 mol NH4+ per mol of ornithine (see Table 1.6). This result suggested that
158 the Strep-tagged protein isolated from Anabaena is able to carry out the two consecutive
159 reactions of AgrE, also producing ammonium from ornithine, although the concomitant
160 production of proline could not be determined because of coincidence of proline and arginine in
161 the chromatography. We then carried out assays analyzing the products of the reaction with a
162 different amino acid derivatization and HPLC method (Fabiani et al., 2002). For these assays, we
163 used Strep-tagged AgrE isolated from Anabaena in the presence of a reducing agent and NAD+,
164 which were the conditions that permitted to observe consistently the ornithine cyclodeaminase
165 activity. Using arginine as substrate, the production of ammonium, ornithine and proline could
166 be detected (Fig. 6 and Table 1.7). The amount of ornithine produced was 1.65-fold higher than
167 the amount of proline, and the amount of ammonium produced (29.2 nmol min-1) corresponded
168 to that expected from the production of 2 mol of NH4+ per mol of ornithine (2 x 7.62 nmol min-1)
169 plus 3 mol of NH4+ per mol of proline (3 x 4.62 nmol min-1), according to the reaction depicted
170 in Fig. 2. Hence, the studies carried out in vitro with different versions of the AgrE protein
8 171 corroborate that this enzyme can catalyze the conversion of arginine into proline with
172 concomitant release of ammonium.
173
174 Physiology and cell specificity of the AgrE-PutA pathway
175 We have recently described a pathway producing sym-homospermidine from arginine in
176 Anabaena (Burnat et al., 2018). The first enzyme in this pathway is arginine decarboxylase
177 encoded by speA (all3401), and an speA mutant is drastically hampered in diazotrophic growth
178 apparently because of a requirement for polyamines during heterocyst differentiation (Burnat et
179 al., 2018). Here we studied the growth rate constants of the agrE and putA mutants in liquid
180 medium. As shown in Fig. 7A, both mutants showed substantial diazotrophic growth.
181 Nonetheless, strain agrE was frequently observed to have increased granulation in the
182 cytoplasm that can correspond to accumulation of cyanophycin (Fig. 7B), which may reflect a
183 restricted catabolism of arginine in this mutant. To investigate whether further metabolic
184 pathways can use arginine in Anabaena, we created an agrE speA double mutant by transferring
185 an speA inactivating construct (speA::C.S3) to the agrE strain (Fig. S6). Filaments of strain
186 agrE speA::C.S3 did not generate any significant 14C-labeled product when incubated in the
187 presence of [14C]arginine (Fig. 8A), indicating that no other major pathway degrading arginine is
188 expressed in Anabaena under the growth conditions tested. Like the speA mutant, the double
189 agrE speA::C.S3 mutant was unable to grow diazotrophically (Fig. 8B).
190 In diazotrophically-grown filaments of Anabaena, hydrolysis of -aspartyl-arginine takes
191 place mainly in vegetative cells rather than in heterocysts (Burnat et al., 2014). To investigate the
192 cell type in which the AgrE-PutA pathway is active, we prepared an Anabaena strain producing
9 193 AgrE fused to superfolder green fluorescent protein (sf-GFP) (Fig. S7). In filaments incubated
194 under diazotrophic conditions, the AgrE-sf-GFP fusion protein accumulated in vegetative cells at
195 substantially higher levels than in heterocysts (Fig. 9A,B). Expression of the putA gene was
196 studied by northern blot analysis with RNA isolated from whole filaments incubated with
197 different nitrogen sources and from purified heterocysts (Fig. 9C). The heterocyst RNA was
198 validated by hybridization with probes of the nifH gene (encoding a nitrogenase component,
199 expressed only in heterocysts) and the rbcL gene (encoding a subunit of ribulose-1,5-
200 bisphosphate carboxylase/oxygenase, expressed only in vegetative cells) as described previously
201 (Picossi et al., 2005). The putA gene is 2,991-bp long, and a transcript of about 3.6 kb was
202 observed with RNA from whole filaments but not from heterocysts (Fig. 9C), showing that that
203 putA is expressed specifically in vegetative cells. These results together suggest that arginine is
204 degraded by the AgrE-PutA pathway mainly in vegetative cells.
205
206 Discussion
207 Our work has identified in the heterocyst-forming cyanobacterium Anabaena an arginine
208 catabolism pathway that produces glutamate mediated by the products of only two genes, agrE
209 (alr4995 in the Anabaena genome) and putA (alr0540). Figure 10 depicts this pathway along
210 with the homospermidine biosynthesis pathway that we have recently described in Anabaena
211 (Burnat et al., 2018). Among the pathways that catabolize arginine producing glutamate and
212 ammonium, the use of ornithine as an intermediate is common. However, ornithine is normally
213 produced by an arginase that also releases urea or by a dihydrolase if arginine is activated with
214 succinyl-CoA (Lu, 2006; Vander Wauven and Stalon, 1985). AgrE is unique in having arginine
215 dihydrolase activity, which can be ascribed to the N-terminal domain of the protein (Fig. 2; see
10 216 also Zhang et al., 2018). In many bacteria, ornithine (or succinyl ornithine) is further catabolized
217 through glutamate semialdehyde (or succinyl glutamate semialdehyde) to glutamate (Lu, 2006),
218 but in a few bacteria including some clostridia and Agrobacterium a specific ornithine
219 cyclodeaminase produces proline (Costilow and Laycock, 1971; Goodman et al., 2004), which is
220 then oxidized to glutamate by the bifunctional proline oxidase PutA (Lu, 2006). As we have
221 shown in this work, AgrE from Anabaena bears a C-terminal domain that carries out an ornithine
222 cyclodeaminase-like reaction producing proline, although AgrE is not homologous to
223 conventional ornithine cyclodeaminases and therefore represents a new type of protein with this
224 activity. Proline produced by AgrE in Anabaena is further oxidized to glutamate by PutA.
225 Overall, AgrE and PutA represent a straightforward pathway to make available to metabolism
226 the four nitrogen atoms of arginine, three as ammonium ions and one as glutamate (Fig. 10).
227 As observed repeatedly in this work, when using arginine as substrate, AgrE can release
228 ornithine in vitro. It is likely that ornithine release from AgrE can also take place in vivo, since
229 [14C]ornithine has been occasionally observed in our experiments of [14C]arginine catabolism by
230 Anabaena (see, e.g., Fig. 4A). Nonetheless, in vitro, AgrE can produce proline from arginine as
231 shown in Fig. 6, implying that the C-terminal domain of AgrE can directly use ornithine
232 produced by the N-terminal domain. This observation raises the possibility that AgrE carries out
233 substrate channeling, a process by which an intermediate is transferred from one enzyme active
234 site to another without diffusing out of the enzyme (Liu et al., 2017). If this were the case, the Km
235 of the enzyme for ornithine would be expected to be higher than for arginine. Consistently,
236 available information permits to estimate a Km of AgrE for arginine of about 6 mM and for
237 ornithine of about 50 mM (Table 1). Although further work will be necessary to increase our
238 knowledge of the biochemistry of AgrE, we speculate that channeling in AgrE may prevent the
11 239 use of ornithine in arginine biosynthesis under physiological conditions that favor arginine
240 catabolism, thus avoiding the operation of a futile cycle. A similar situation, channeling of the
241 intermediate P5C/GSA, may occur in the second enzyme of this pathway, PutA (see Fig. 10),
242 thus avoiding the use of the intermediate in proline biosynthesis. Substrate channeling has been
243 investigated comprehensively in other bacterial PutA proteins (Liu et al., 2017).
244 In Anabaena, arginine can be taken up from the outer medium by a high-affinity ABC
245 transporter (Pernil et al., 2008) or can be produced from cyanophycin (Burnat et al., 2014).
246 Under diazotrophic conditions, -aspartyl-arginine released from cyanophycin in the heterocysts
247 is transferred to the vegetative cells, in which it is split producing aspartate and arginine (Burnat
248 et al., 2014). The agrE and putA genes are expressed mainly in the vegetative cells of
249 diazotrophic filaments, which is consistent with the idea that the AgrE-PutA pathway will
250 contribute to the utilization by the vegetative cells of the nitrogen fixed in the heterocysts. This
251 pathway is however not essential for diazotrophic growth. The use of arginine as substrate for the
252 biosynthesis of homospermidine may also contribute to support growth of the vegetative cells
253 under diazotrophic conditions, since, in addition to producing polyamines, the homospermidine
254 biosynthesis pathway releases urea and ammonia that can be used in metabolism (Fig. 10). For
255 this, urea is further degraded to two molecules of ammonia and CO2 by urease, which is present
256 in Anabaena (Valladares et al., 2002).
257 The agrE and putA genes are present in a large number of sequenced cyanobacterial
258 genomes (tested at https://img.jgi.doe.gov/cgi-bin/m/main.cgi) indicating that the agrE-putA
259 pathway is widely distributed in these organisms. However, these genes are not present in
260 cyanobacteria such as Synechococcus elongatus or the marine Prochlorococcus spp. and
261 Synechococcus spp., which do not produce cyanophycin. These observations suggest a role of the
12 262 agrE-putA pathway in catabolism of arginine made available by mobilization of cyanophycin.
263 Previous work done with Synechocystis (which also accumulates cyanophycin) suggested a role
264 of the proline biosynthesis protein ProC in production of glutamate during arginine and ornithine
265 catabolism, but the proC mutant still showed substantial production of proline implying the
266 existence of another enzyme synthesizing proline during arginine catabolism (Quintero et al.,
267 2000). The Synechocystis ORF Sll1336 encodes a 705-amino acid residue protein that is 74 %
268 identical to Anabaena AgrE, and could therefore be responsible for the observed production of
269 proline in the Synechocystis proC mutant.
270 Whereas the putA gene is of wide distribution in the living world, agrE is restricted to
271 cyanobacteria and a few other bacteria. Notably, a protein homologous to the C-terminal domain
272 of AgrE is found in Methanococcus maripaludis and related archaea. These organisms have been
273 known to express ornithine cyclodeaminase activity (Graupner and White, 2001), but no gene
274 encoding a conventional ornithine cyclodeaminase is found in their sequenced genomes leading
275 to the idea that they express a different type of ornithine cyclodeaminase. Hence, our work
276 showing that the M. maripaludis protein homologous to the C-terminal part of AgrE provides
277 Anabaena with the capability of producing proline from ornithine has also identified the gene
278 encoding archaeal ornithine cyclodeaminase. The M. maripaludis gene encoding this protein,
279 MMP1218, appears to be not essential in either minimal medium or a complex rich medium
280 (Sarmiento et al., 2013). Our results identifying this gene’s product as an ornithine
281 cyclodeaminase could open the possibility of performing a specific test of its role in the
282 physiology of this type of archaea.
283
284
13 285 Experimental procedures
286 Strains and growth conditions
287 Anabaena sp. strain PCC 7120 was grown axenically in BG11 medium (containing NaNO3),
288 BG110 medium (free of combined nitrogen) or BG110NH4+ medium (BG110 containing 4 mM
289 NH4Cl and 8 mM TES-NaOH buffer, pH 7.5). In every case, ferric citrate replaced the ferric
290 ammonium citrate used in the original recipe (Rippka et al., 1979). For plates, medium was
291 solidified with 1% separately autoclaved Difco agar. Cultures were grown at 30 ºC in the light
292 (25 μmol photons m-2 s-1), with shaking (80-90 rpm) for liquid cultures. Alternatively, cultures
293 (referred to as bubbled cultures) were supplemented with 10 mM of NaHCO3 and bubbled with a
294 mixture of CO2 and air (1 % v/v) in the light (50 to 75 mol photons m-2 s-1). For mutants
295 described below, antibiotics were used at the following concentrations: erythromycin (Em), 2 μg
296 mL-1 for liquid cultures and 5 μg mL-1 for solid media; neomycin (Nm) at 20 μg mL-1 for both
297 liquid and solid media; streptomycin sulfate (Sm) and spectinomycin dihydrochloride
298 pentahydrate (Sp), 5 μg mL-1 each for both liquid and solid media. DNA was isolated from
299 Anabaena sp. by the method of Cai and Wolk (1990). The ΔargF::C.K3 mutant is an arginine
300 auxotroph and was supplemented with 0.5 mM L-arginine.
301 Escherichia coli strain DH5α was used for plasmid constructions, strains HB101 and
302 ED8654 for conjugations with Anabaena sp., and strain BL21(DE3) for overexpression of
303 proteins. They were grown in Luria-Bertani medium supplemented when appropriate with
304 antibiotics at standard concentrations.
305
306 Plasmid constructions and genetic procedures
14 307 Open reading frame (ORF) alr4995 of the Anabaena chromosome (Kaneko et al., 2001) was
308 inactivated by removing an internal fragment of 1,755 bp. DNA fragments upstream (490 bp)
309 and downstream (500 bp) from the central region of the gene were amplified by PCR using DNA
310 from Anabaena as template and primers alr4995-3/alr4995-4 and alr4995-5/alr4995-6 (all
311 oligodeoxynucleotide primers are listed in Table S1). The external primers, alr4995-3 and
312 alr4995-6, included a PstI-site in their 5’ ends, and primers alr4995-4 and alr4995-5 were
313 complementary in their 5’ ends and joined together by the megaprimer PCR protocol. The DNA
314 fragment from this gene was cloned in pSPARK (Canvax Biotech S.L., Spain) and its sequence
315 was corroborated by sequencing. The cloned fragment was then transferred as a PstI-ended
316 fragment to PstI-digested pCSRO (Merino-Puerto et al., 2013), producing pCSDH8.
317 Conjugation of Anabaena with E. coli HB101 carrying the cargo plasmid (pCSDH8) with
318 helper and methylation plasmid pRL623 was effected by the conjugative plasmid pRL443,
319 carried in E. coli ED8654, and performed as described (Elhai et al., 1997) with selection for
320 resistance to Sm and Sp. Exconjugants were spread on BG110NH4+ medium supplemented with
321 5% sucrose (Cai and Wolk, 1990), and individual SucR colonies were checked by PCR looking
322 for clones that had replaced the wild-type locus by a locus bearing the deletion. The genetic
323 structure of selected clones was studied by PCR with DNA from those clones and primers
324 alr4995-6/alr4995-7 and alr4995-7/alr4995-8. A clone homozygous for the mutant chromosomes
325 was named strain CSMI43 (Δalr4995).
326 The plasmid carrying fusion gene alr4995-sf-gfp was prepared as follows. A 603-bp
327 fragment from the 3’-terminal part of alr4995 was amplified by PCR using Anabaena DNA as
328 template and primers alr4995-9 (which contains a HindIII site) and alr4995-10 (which lacks the
329 stop codon of the gene and contains a BsaI site in its 5’ end), and the resulting fragment was
15 330 cloned as a HindIII/BsaI-ended fragment in HindIII/BsaI-digested pCSAL39 producing plasmid
331 pCSMI72 that carries the fusion of the sf-gfp gene to the 3’ end of alr4995 (pCSAL39 is a
332 pMBL-T- derived vector that contains the sf-gfp gene, a sequence encoding a 4-Gly linker and a
333 BsaI site in its 5’ end). The insert of pCSMI72 was corroborated by sequencing and the resulting
334 fusion was transferred as a KpnI-ended fragment to KpnI-digested pCSV3, which provides
335 resistance to Sm and Sp (Valladares et al., 2011), producing pCSMI73. This plasmid, which
336 bears the alr4995-sf-gfp fusion gene, was transferred to Anabaena by triparental mating as
337 described above, with selection for SmR SpR. Insertion into alr4995 and segregation of
338 chromosomes carrying the fusion was confirmed by PCR using template DNA from exconjugant
339 clones and primers alr4995-11 and gfp-6 for testing insertion of sf-gfp, and alr4995-11 and
340 alr4995-6 for testing segregation of the mutated chromosomes. A homozygous clone bearing the
341 alr4995-sf-gfp construct was named strain CSMI32.
342 ORF alr4907 (argF) encodes ornithine carbamoyltransferase (Kaneko et al., 2001). To
343 inactivate alr4907, two DNA fragments, one encompassing 522 bp from sequences upstream
344 from the central region of the gene and the other including 646 bp from sequences downstream
345 from the central region of the gene, were amplified by PCR using DNA from Anabaena as
346 template and primer pairs alr4907-1/alr4907-2 and alr4907-3/alr4907-4, respectively (alr4907-2
347 and alr4907-3 bear SmaI sites at their 5’ ends). The two DNA fragments, joined together by the
348 megaprimer PCR protocol, were cloned into pSPARK producing plasmid pCSMI67 and, after
349 corroboration by sequencing and digestion with SmaI, were ligated to SmaI-ended gene cassette
350 C.K3 encoding Kmr/Nmr, producing plasmid pCSMI68. The insert of the resulting plasmid,
351 excised with SacI, was transferred to SacI-digested pCSRO (Merino-Puerto et al., 2013),
352 producing pCSMI69. Conjugation of Anabaena with E. coli HB101 carrying pCSMI69 and
16 353 helper and methylation plasmid pRL623 was effected by the conjugative plasmid pRL443,
354 carried in E. coli ED8654, and performed as described (Elhai et al., 1997) with selection for
355 resistance to Nm. Exconjugants were isolated, and double recombinants were identified as clones
356 resistant to Nm, resistant to sucrose (Cai and Wolk, 1990), and sensitive to Sm and Sp, for which
357 the resistance determinant was present in the vector portion of the transferred plasmid. The
358 genetic structure of selected clones was studied by PCR with DNA from those clones and
359 primers alr4907-5/alr4907-6 and alr5045-5/alr4907-4. Clones homozygous for the mutant
360 chromosomes were only obtained for the indirect orientation of the C.K3 cassette, and were
361 named strain CSMI30b (Δalr4907::C.K3).
362 ORF alr0540 (putA) encodes proline oxidase. To inactivate alr0540, a 2.3-kb fragment
363 from the central part of alr0540 was amplified by PCR using Anabaena DNA as template and
364 primers PA-1 and PA-2, and the resulting fragment was cloned into pGEM-T producing plasmid
365 pCSS6. Gene cassette C.S3 encoding Smr Spr was introduced in the HindIII site of pCSS6,
366 producing plasmid pCSS9. The insert of the resulting plasmid, excised with PvuII, was
367 transferred to NruI-digested pRL278, resulting in plasmid pCSS11. Conjugation of Anabaena
368 and identification of exconjugants was performed as for the CSMI30b mutant (but using Smr Spr
369 for positive selection). A clone homozygous for the mutant chromosomes was named CSS5.
370 ORF all3401 (speA) encodes arginine decarboxylase, and the arginine decarboxylase
371 deletion mutant (∆speA::C.S3) has been described previously (Burnat et al., 2018). To create a
372 double ∆agrE ∆speA::C.S3 mutant, plasmid constructs used to create the speA mutant were
373 transferred to Anabaena ∆agrE by triparental mating, as described above, with selection for SmR
374 SpR. Exconjugants were isolated, and double recombinants were identified as clones resistant to
375 Sm and Sp, resistant to sucrose, and sensitive to Nm, for which the resistance determinant was
17 376 present in the vector portion of the transferred plasmid. The genetic structure of selected clones
377 was studied by PCR with DNA from those clones and appropriate primer pairs (Fig. S6). A clone
378 homozygous for both mutant chromosomes was obtained and named strain CSMI43-MI35
379 (∆agrE ΔspeA::C.S3).
380
381 Construction of strains producing Alr4995 (AgrE)
382 For overexpression of protein Alr4995 (AgrE), two strategies were used. For the first strategy
383 (expression in E. coli), the 6xHis tag was added in frame to alr4995 and the N-terminal AT/ADI
384 domain of alr4995. Using DNA from Anabaena as template and primers alr4995-11 and
385 alr4995-12 (which includes a PstI site close to its 5’ end), and alr4995-11 and alr4995-13 (which
386 also includes a PstI site close to its 5’ end), the alr4995 gene and the AT/ADI domain of alr4995
387 were amplified by PCR. The PCR products were PstI-digested and cloned into SfoI/PstI-digested
388 pPROEX-HTb expression vector, producing plasmids pCSMI81 and pCSMI82, respectively,
389 whose inserts were corroborated by sequencing. These plasmids were introduced into E. coli
390 BL21(DE3) by electroporation.
391 The second strategy, used for overexpression of Alr4995 in Anabaena and for
392 complementation of the Anabaena alr4995 deletion mutant, was performed using Strep tag II
393 and a plasmid replicating in Anabaena. Using DNA from Anabaena as template and primers
394 alr4995-19 (which includes an NdeI site close to its 5’end) and alr4995-17 (which includes a
395 BamHI site close to its 5’ end), ORF alr4995 was amplified by PCR and ligated into pSPARK
396 (Canvax, Biotech SL) producing plasmid pCSMI93, whose insert was corroborated by
397 sequencing. This insert was excised from pCSMI93 by digestion with NdeI/SalI, and transferred
398 to NdeI/XhoI-digested pCMN28b (which is a vector derived from pET28b in which the His tag
18 399 has been replaced by a Strep-tag linker, as described previously [Napolitano et al., 2013]),
400 producing plasmid pCSMI94. Plasmid pCSMI94 was BglII-digested, receded 3’ends were filled
401 with Klenow fragment, and BamHI-digested. The resulting excised fragment was transferred to
402 SmaI/BamHI-digested pRL3845 replacing all1711 by alr4995, producing pCSMI95. (pRL3845
403 is a CmR EmR-plasmid that contains a PglnA-all1711 construct and can replicate in Anabaena
404 [López-Igual et al., 2012].) As is the case for all1711 in pRL3845, alr4995 in pCSMI95 is
405 expressed from the Anabaena glnA promoter. This plasmid was conjugated into Anabaena wild
406 type and strain CSMI43 (Δalr4995) as described above, with selection for EmR, producing
407 strains CSMI40 and CSMI43-C, respectively.
408
409 Construction of strains producing LOR/SDH protein 2 of Methanococcus
410 Cloning of the LOR/SDH protein 2 of Methanococcus (hereafter protein Mls2) was performed as
411 above for alr4995, with some modifications. Using genomic DNA from Methanococcus
412 maripaludis strain S0001 (which is derived from the wild-type M. maripaludis S2; William B.
413 Whitman laboratory, University of Georgia) as template and primers LOR/SDH-4 (which
414 includes a BamHI site close to its 5’end) and LOR/SDH-5 (which includes an NdeI site close to
415 its 5’ end), the Mls2-encoding gene was amplified by PCR and ligated into pSPARK producing
416 plasmid pCSMI99, whose insert was corroborated by sequencing. This insert was excised from
417 pCSMI99 by digestion with NdeI/SalI and transferred to NdeI/XhoI-digested pCMN28b
418 producing plasmid pCSMI100. This plasmid was XbaI-digested, filled in with Klenow fragment
419 and BamHI-digested. The resulting excised fragment was transferred to SmaI/BamHI-digested
420 pRL3845 replacing all1711 by Mls2, producing pCSMI101. This plasmid was conjugated into
19 421 Anabaena strain CSMI43 (Δalr4995) as described above, with selection for EmR, producing
422 strain CSMI43-CMls2.
423
424 Growth tests, microscopy and northern blot analysis
425 Protein concentration and chlorophyll a (Chl) content of the cultures were determined by a
426 modified Lowry procedure (Markwell et al., 1978) and by the method of Mackinney (1941),
427 respectively. The growth rate constant ( = ln2/td, where td is the doubling time) was calculated
428 from the increase of protein content, determined in 0.2-mL samples, of shaken liquid cultures.
429 Cultures were inoculated with cells containing about 5 µg of protein mL-1 and grew
430 logarithmically until reaching about 40 µg of protein mL-1.
431 For growth tests on solid media, cultures grown in BG11 medium were harvested and
432 washed three times with BG110 medium, and dilutions were prepared in BG110 medium.
433 Samples of 10-μl of the resulting suspensions were spotted on agar plates with different nitrogen
434 sources and incubated at 30 °C in the light (25 μmol photons m-2 s-1).
435 GFP fluorescence was analyzed by confocal microscopy. Samples from cultures of strain
436 CSMI32 or Anabaena wild type (used as a control) grown in bubbled cultures with BG11 or
437 BG110 medium were visualized using a Leica HCX PLAN-APO 63X 1.4 NA oil immersion
438 objective attached to a Leica TCS SP2 confocal laser-scanning microscope. GFP was excited
439 using 488-nm irradiation from an argon ion laser. Fluorescence emission was monitored by
440 collection across windows of 500-520 nm (GFP imaging) and 630-700 nm (cyanobacterial
441 autofluorescence). Under the conditions used, optical section thickness was about 0.4 µm. GFP
442 fluorescence intensity was analyzed using ImageJ 1.43m software.
20 443 Northern analysis was performed using a DNA probe of putA obtained with primers PA-1
444 and PA-2 (Table S1) and control probes of the nifH, rbcL and rnpB genes as described
445 previously (Picossi et al., 2005).
446
447 Amino acid catabolism
448 Cells grown in BG11 medium were harvested by centrifugation at 4,000 rpm at room
449 temperature, washed twice with 25 mM N-tris(hydroxymethyl)-methylglycine (Tricine)-NaOH
450 buffer (pH 8.1), and resuspended in the same buffer. The uptake assays were carried out at 30 °C
451 in the light (white light from fluorescent lamps, about 175 μmol photons m-2 s-1) and were started
452 by mixing a suspension of cells (2.1 mL) containing 5 to 10 μg of Chl· mL-1 with a solution (0.1
453 mL) of L-[U-14C]arginine (274 mCi·mmol-1), L-[1-14C]ornithine (57.1 mCi·mmol-1), both
454 purchased from Perkin Elmer (Massachusetts, USA), or L-[U-14C]proline (257 mCi·mmol-1)
455 from Amersham Life Science (UK). The final concentration of both arginine and ornithine in the
456 experiments was 1 μM, and the final concentration of proline was 1.9 µM. The amount of
457 metabolite taken up in the assays was estimated in 0.5-mL samples of the cell suspensions. The
458 samples were filtered (0.45 μm-pore-size Millipore HA filters were used) and the cells on the
459 filters were washed with 5 to 10 mL of 5 mM Tricine–NaOH buffer (pH 8.1). The filters
460 carrying the cells were then immersed in 5 mL of scintillation cocktail, and their radioactivity
461 was measured. Retention of radioactivity by boiled cells was used as a blank.
462 To determine metabolites produced from the provided labeled amino acid, samples of 0.5
463 mL of the cell suspension were immediately (<15 s) mixed, without filtering the cells, with 1.5
464 mL of water at 100 °C and further incubated for 5 min in a bath of boiling water. The resulting
465 suspensions were centrifuged, and samples from the supernatants were analyzed by thin-layer
21 466 chromatography (TLC) and electronic autoradiography as described previously (Burnat and
467 Flores, 2014).
468
469 Expression and purification of Anabaena Alr4995
470 Plasmids pCSMI81 and pCSMI82 contain the alr4995 gene and the 5’ terminal part of this gene
471 encoding the AT/ADI domain, respectively, fused in frame to a sequence encoding a 6xHis tag
472 under an IPTG inducible promoter. These plasmids were transferred to E. coli BL21-lacIq. A
473 pre-inoculum of this strain grown overnight in LB medium supplemented with 50 μg of
474 ampicillin (Ap) mL−1 (50 μg of Km mL−1 for pCSMI94) and 1 % glucose was washed with LB
475 medium and used to inoculate 0.5 L of LB medium + Ap or Km. The culture was incubated at 37
476 °C up to an OD600 of 0.6, and protein expression was induced by addition of 1 mM isopropyl-β-
477 D-1-thiogalactopyranoside (IPTG). After 2-3 h at 37 °C, cells were collected by centrifugation at
478 4,000 x g (10 min, 4º C), washed with a buffer containing 100 mM Tris-HCl (pH 8.0) and 150
479 mM NaCl and centrifuged at 7,000 g (10 min, 4 ºC). Cells were resuspended with lysis buffer
480 (200 mM Tris-HCl [pH 8.0], 500 mM NaCl, 5% glycerol, and 5 mM imidazole) at 5 mL g−1 of
481 cells. DNase I and protease inhibitor cocktail complete Mini EDTA-free (Roche) were added just
482 before breakage of the cells by passage twice through a French pressure cell at 20,000 psi. After
483 centrifugation at 27,216 g (30 min, 4 °C), the 6xHis-Alr4995 or 6xHis-AT/ADI Alr4995 protein
484 was purified from the supernatant by chromatography through a 5-mL His-Select column from
485 Sigma, using imidazole in the same buffer described above to elute the retained proteins.
486 Samples obtained after purification were subjected to SDS-PAGE to confirm the presence of the
487 proteins (see Fig. S8A, B).
488 Anabaena strain CSMI40 carries pCSMI95 in which alr4995 is cloned fused to a
22 489 sequence encoding a Strep-Tag II as described above. Cyanobacterial cells were incubated in 50
490 mL BG11 medium supplemented with 5 µg Em mL-1 for 5-7 days, and then, transferred to 800
491 mL of bubbled BG11 medium supplemented with 2 µg Em mL-1, and incubated for 5-7 days.
492 After harvesting by filtration, cells were resuspended in 50 mM Tris-HCl (pH 8), 200 mM NaCl
493 and 20 % glycerol. Protease inhibitor cocktail complete Mini EDTA-free (Roche) was added just
494 before breakage of the cells by passage twice through a French pressure cell at 20,000 psi. After
495 centrifugation at 27,216 g (30 min, 4 °C), soluble fractions were subjected to purification using
496 StrepTrap HP columns and 0.53 mg mL-1 desthiobiotin in the same buffer as above as eluent (GE
497 Healthcare), and the protein was desalted using PD-10 columns (see Fig. S8C). After initial
498 checking of different conditions, 1 mM NAD+ and 10 mM dithiothreitol (DTT) were added to
499 lysis and purification buffers.
500
501 In vitro enzymatic assays of Alr4995
502 To study the enzymatic activity in vitro, L-arginine and L-ornithine supplied at different
503 concentrations were used as substrates. Different buffers and co-factors were tried: 100 mM Tris-
504 HCl (pH 9) buffer, 100 mM phosphate buffer (pH 7.5), 10 mM dithiothreitol (DTT), 10 mM β-
505 mercaptoethanol, and NAD+ at different concentrations. Anoxic conditions obtained by flushing
506 argon through solutions were also tried. Substrates and products were analyzed by HPLC, except
507 urea that was determined colorimetrically (Boyde and Rahmatullah, 1980). The HPLC method
508 used by default involved a derivatization of amino acids with phenylisothiocyanate (PITC)
509 (Heinrikson and Meredith, 1984) and was performed as described (Burnat et al., 2014). An
510 alternative HPLC method involved derivatization with 9-fluorenylmethyl-chloroformate and was
511 performed as described by Fabiani et al. (2002).
23 512
513 Acknowledgements
514 We thank Dorothee Heinemann for her participation in the construction of the agrE mutant,
515 Ignacio Luque (CSIC, Sevilla, Spain) for useful suggestions, William B. Whitman (University of
516 Georgia, Athens, GA) for a sample of DNA from Methanococcus maripaludis, and Anthony J.
517 Michael (University of Texas Southwestern Medical Center, Dallas, TX), Vicente Rubio (CSIC,
518 Valencia, Spain) and C. Peter Wolk (Michigan State University, East Lansing, MI) for critical
519 reading of the manuscript. We also thank the editor, James A. Imlay, for excellent suggestions.
520 Work was supported by grant nos. BFU2014-56757-P and BFU2017-88202-P (EF) and
521 BFU2016-77097 (AH) from the Spanish Government, co-financed by the European Regional
522 Development Fund. The authors have no conflict of interest to declare.
523
524 Author contributions
525 MB designed and performed work related to AgrE and drafted parts of the manuscript; SP
526 designed and performed work related to PutA; AV performed biochemical work related to AgrE;
527 AH designed and supervised research; EF conceived the study, designed and supervised
528 research, and wrote the manuscript; all authors analyzed results and made manuscript revisions.
529
24 530 References
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538 Burnat M, Li B, Kim SH, Michael AJ, Flores E (2018) Homospermidine biosynthesis in the 539 cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal 540 diazotrophic growth. Mol Microbiol 109(6):763-780. 541 Cai YP, Wolk CP (1990) Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double 542 recombinants and to entrap insertion sequences. J Bacteriol 172(6):3138-3145. 543 Costilow RN, Laycock L (1971) Ornithine cyclase (deaminating). Purification of a protein that converts ornithine to 544 proline and definition of the optimal assay conditions. J Biol Chem 246:6655-6660. 545 Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP (1997) Reduction of conjugal transfer efficiency by 546 three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179(6):1998-2005. 547 Fabiani A, Versari A, Parpinello GP, Castellari M, Galassi S (2002) High-performance liquid chromatography 548 analysis of free amino acids in fruit juices using derivatization with 9-fluorenylmethyl-chloroformate. J 549 Chromatogr Sci 40(1):14-18. 550 Flores E, Herrero A (2010) Compartmentalized function through cell differentiation in filamentous cyanobacteria. 551 Nat Rev Microbiol 8(1):39-50. 552 Goodman JL, Wang S, Alam S, Ruzicka FJ, Frey PA, Wedekind JE (2004) Ornithine cyclodeaminase: structure, 553 mechanism of action, and implications for the mu-crystallin family. Biochemistry 43:13883-13891. 554 Graupner M, White RH (2001) Methanococcus jannaschii generates L-proline by cyclization of L-ornithine. J 555 Bacteriol 183(17):5203-5. 556 Heinrikson RL, Meredith SC (1984) Amino acid analysis by reverse-phase high-performance liquid 557 chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem 136(1):65-74. 558 Kaneko T, Nakamura Y, Wolk CP, Kuritz T, Sasamoto S, Watanabe A, Iriguchi M, Ishikawa A, Kawashima 559 K, Kimura T, Kishida Y, Kohara M, Matsumoto M, Matsuno A, Muraki A, Nakazaki N, Shimpo S, Sugimoto 560 M, Takazawa M, Yamada M, Yasuda M, Tabata S (2001) Complete genomic sequence of the filamentous 561 nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8(5): 227-253. 562 Liu LK, Becker DF, Tanner JJ (2017) Structure, function, and mechanism of proline utilization A (PutA). Arch 563 Biochem Biophys 632:142-157.
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27 611 Figure legends
612
613 Fig. 1. Catabolism of [14C]arginine, [14C]ornithine and [14C]proline in Anabaena. (A, B)
614 Visualization of catabolic products. Filaments of Anabaena grown in BG11 medium were
615 incubated with 1 µM L-[U-14C]arginine (A) or L-[1-14C]ornithine (B) for 10 min and extracted.
616 Samples were subject to thin layer chromatography (TLC) and visualized by electronic
617 autoradiography as described in Experimental procedures.. Empty ovals indicate the approximate
618 locations of the spots of Pro and Glu (not produced in the corresponding mutant). Note that
619 citrulline and arginine are synthesized from ornithine in the agrE mutant. (C) Schematic
620 showing the putative metabolic path of the labeled amino acids. (D) Catabolism of 1.9 µM L-[U-
621 14C]proline in Anabaena wild type and the putA::C.S3 mutant (incubation time, 15 min).
622 Arrowheads point to the origin of the chromatography.
623
624 Fig. 2. AgrE of Anabaena (Alr4995) is a 703-amino acid residue protein containing two well
625 differentiated domains: an N-terminal / propeller domain (amino acid residue 10-269) and a
626 C-terminal domain homologous to Methanococcus LOR/SDH protein 2 (amino acid residue 285-
627 694), here identified as an ornithine cyclodeaminase and in which two further homology domains
628 are detected. The proposed reactions of the AgrE protein and their stoichiometry are shown.
629
630 Fig. 3. Production of 14C-labeled metabolites from L-[14C]arginine and L-[1-14C]ornithine in
631 filaments of strain CSMI30b (argF::C.K3). Suspensions of filaments grown in BG11 medium
632 and containing 5 to 10 μg of Chl·ml-1 were incubated for 10 min with 1 μM L-[14C-(U)]arginine
633 (A) or 1 μM L-[1-14C-(U)]ornithine (B). Metabolites in the cell suspensions were extracted and
28 634 analyzed by TLC and autoradiography as described in Experimental procedures. The amino
635 acids identified were: Arg, Cit, Pro, Glu, Gln and Orn. Note that the glutamine and citrulline
636 spots overlap. White triangles point to the origin of the chromatography. (C) Alternative pathway
637 via citrulline that is ruled out by analysis of the argF mutant (i.e., if this pathway were operative
638 in Anabaena, [14C]proline and [14C]glutamate would not be produced from [14C]ornithine in the
639 argF mutant).
640
641 Fig. 4. Production of 14C-labeled metabolites from [14C]arginine or [14C]ornithine in filaments of
642 the Anabaena agrE mutant complemented with the Anabaena ArgE protein (A, B) or with the
643 Methanococcus Mls2 protein (C, D). Filaments grown in BG11 medium supplemented with 2 µg
644 Em/mL were incubated for 10 min with 1 μM L-[U-14C]arginine (A, C) or L-[1-14C]ornithine (B,
645 D). Metabolites in the cell suspensions were extracted and analyzed by TLC and autoradiography
646 as described in Experimental procedures. The amino acids identified were: Arg, Cit, Pro, Glu,
647 Gln and Orn. Note that the glutamine and citrulline spots overlap. Arrowheads point to the origin
648 of the chromatography. Empty ovals indicate the approximate locations of the spots of Pro or
649 Glu (not produced in the indicated mutant).
650
651 Fig. 5. In vitro activity of the Anabaena AgrE protein and of its N-terminal domain. The
652 indicated proteins were produced and isolated from E. coli or Anabaena as described in
653 Experimental procedures and Fig. S8. The reactions were carried out in 100 mM phosphate
654 buffer (pH 7.5) supplemented with the indicated substrate and, when indicated, 10 mM -
655 mercaptoethanol and 1 mM NAD+, and the reaction products were analyzed by HPLC after
29 656 derivatization with phenylisothiocyanate (Heinrikson and Meredith, 1984). The molar ratios of
657 products detected are indicated.
658
659 Fig. 6. Whole activity of the Strep-tagged AgrE protein isolated from Anabaena. The protein
660 (10.75 µg mL-1) was assayed in 100 mM phosphate buffer (pH 7.5) supplemented with 1 mM
661 NAD+, 1 mM -mercaptoethanol and 40 mM L-arginine. (A) HPLC analysis of the 60-min
662 reaction products after derivatization with 9-fluorenylmethyl-chloroformate (Fabiani et al.,
663 2002). Arg, substrate; FMOC, 9-fluorenylmethyl-chloroformate; React., unknown reactant;
664 Proline (Pro*) and ornithine (Orn*) are noted to partially overlap reactants. (B) Quantification of
665 the reaction products after subtraction of time zero values. The reaction rates were calculated
666 from the slope of the lines: 29.20 nmol ammonium min-1, 42.51 nmol ornithine min-1 and 25.77
667 nmol proline min-1.
668
669 Fig. 7. Physiology of Anabaena (WT) and mutant strains CSMI43 (agrE) and CSS5
670 (putA::C.S3). (A) Growth rate constants determined in liquid media. Mean and SD (n = 3 to 6).
671 nd, not determined. (B) Filaments from cultures incubated for 6 days in BG11 medium and
672 visualized by light microscopy. Note the presence of increased granulation in strain CSMI43.
673 Magnification, 100x.
674
675 Fig. 8. Study of an Anabaena agrE ΔspeA::C.S3 double mutant. (A) Catabolism of 1 µM
676 [14C]arginine for 10 min in wild-type Anabaena and the agrE ΔspeA::C.S3 double mutant, and
677 analysis by TLC of labeled products. Incubation with wild-type filaments that had been subjected
678 to boiling is shown as a negative control. (B) Growth tests of the indicated strains on solid
30 679 medium with ammonium (BG110NH4+), nitrate (BG11) or N2 (BG110) as the nitrogen source.
680 Each spot was inoculated with an amount of cells containing the indicated amount of chlorophyll
681 a (Chl), and the plates were incubated under culture conditions for 10 days and photographed.
682
683 Fig. 9. Expression of agrE and putA. (A) Cellular localization of AgrE in the filaments of
684 Anabaena. Filaments of strain CSMI32 (alr4995-sf-gfp) grown in bubbled BG110 medium for 24
685 h or 48 h, were visualized by confocal microscopy and their GFP fluorescence was quantified.
686 Average background fluorescence from wild-type cells (lacking sf-GFP) was subtracted. VC,
687 vegetative cell; H, heterocyst. (B) Quantification of the sf-GFP fluorescence in cells of strain
688 CSMI32. Average background fluorescence from wild type cells was subtracted. Figures are the
689 mean and standard deviation of the mean of the fluorescence recorded in cells grown in bubbled
690 BG110 medium for 24 h (365 vegetative cells and 39 heterocysts counted; Student’s t test p = 5.1
691 x 10-37) or 48 h (367 vegetative cells and 37 heterocysts counted; Student’s t test p = 8.3 x 10-58).
692 (C) Northern analysis of the expression of the alr0540 gene in Anabaena cells grown with
693 ammonium (NH4+), nitrate (NO3-) or N2 as the nitrogen source, as well as in isolated heterocysts
694 (Het). The probe used is shown in the top scheme. The arrowhead points to a transcript of about
695 3.6 kb. As loading control, the membrane was hybridized with a rnpB probe. As controls for the
696 quality of the RNA isolated from heterocysts, the same membrane was hybridized with a nifH
697 and a rbcL probe. (Data of the heterocyst controls are from Picossi et al., 2005.)
698
699 Fig. 10. The arginine metabolism pathways of Anabaena. The homospermidine biosynthesis
700 pathway is represented to the left and the arginine-glutamate catabolic pathway to the right.
701 SpeA, arginine decarboxylase (all3401 gene product); SpeB, agmatinase (alr2310); SpeY,
31 702 deoxyhypusine synthase-like protein (alr3804); AgrE, arginine-guanidine removing enzyme
703 (alr4995); PutA, bifunctional proline oxidase (alr0540) that carries out two sequential reactions:
704 proline dehydrogenase and glutamate--semialdehyde dehydrogenase; [H], reducing power,
705 predictably in the form of FADH2 (product of the proline dehydrogenase reaction) and NADPH
706 (product of the glutamate--semialdehyde dehydrogenase reaction). The positions of ornithine
707 (Orn) as an intermediate in AgrE and of 1-pyrroline-5-carboxylate/glutamate--semialdehyde
708 (P5C/GSA) as intermediates in PutA are indicated.
709
710
32 711 Table 1. Activities of the AgrE proteina.
Assays Enzyme Substrate Additions/conditions Products Ratio set prep (nmol/µg protein)
NH4+ Orn Pro NH4+/ NH4+/ Orn Pro
1 AgrEb 40 mM Arg - 323.5 200.2 1.62 0.5 mM NAD+ 390.1 203.6 1.92
2 AgrEb 40 mM Arg - 304.3 173.4 1.75 4 mM Arg - 139.4 80.2 1.74
3 AT/ADI 40 mM Arg - 90.12 48.85 1.84 domainc
4 AgrEd 40 mM Arg - 214.8 112.6 1.91
40 mM Orn 10 mM β-m-etOH + 1 mM NAD+ 17.31 13.05 1.33 1 mM FAD+ 0 0.33 - 1 mM NADP+ 4.26 2.72 1.57 1 mM PLP 0 0 -
5 AgrEd 10 mM β-m-etOH + 0.1 mM DTT + 1 mM NAD+ + 1 mM Orn - 0 0.96 - 1 mM Orn 5 mM ATP 0 0 - 10 mM Orn - 27.87 26.56 1.05 10 mM Orn 5 mM ATP 0 4.14 - 50 mM Orn - 86.29 74.33 1.16 50 mM Orn 5 mM ATP 41.31 37.71 1.10
6 AgrEd 40 mM Arg 10 mM β-m-etOH + 188.0 77.15 2.44f 1 mM NAD+, 37 ºC
7e AgrEd 40 mM Arg 1 mM β-m-etOH + 162.98 42.51 25.77 2.0g 3.0g 1 mM NAD+
712 a Assays were performed with 100 mM phosphate buffer (pH 7.5) at 30 ºC. Exceptions to these standard conditions 713 are described in the table. No activity was found at pH 9. Figures for NH4+, ornithine (Orn) and proline (Pro) 714 correspond to products detected by HPLC 60 min after the addition of the substrate. DTT, dithiothreitol; β-m-etOH, 715 β-mercaptoethanol; PLP, pyridoxal 5-phosphate. 716 b His-tagged AgrE protein isolated from E. coli (see Fig. S8A). 717 c His-tagged AgrE N-terminal (AT/ADI) domain isolated from E. coli (see Fig. S8B). 718 d Strep-tagged AgrE protein isolated from Anabaena (see Fig. S8C). The protein used in Assays set 5, 6 and 7 was 719 isolated in the presence of 10 mM DTT and 1 mM NAD+. 720 e The products of this assay were analyzed by the derivatization and HPLC methods described by Fabiani et al. 721 (2002). 722 f This figure exceeds the stoichiometry of 2 mol NH4+/mol of ornithine expected for the arginine dihydrolase 723 reaction, which may imply that the reaction proceeded further to proline releasing extra ammonium. 724 g The amount of NH4+ observed in this assay (162.98 nmol/µg protein) matched that expected from the detected 725 amounts of ornithine (42.51) and proline (25.77), which together make 162.33 nmol/µg protein according to the 726 equation: nmol NH4+ = 2 [nmol ornithine] + 3 [nmol proline]. 727
33 A [14C]Arginine B [14C]Ornithine C WT WT Pro Pro [14C]Arginine Orn Arg
Glu Glu [14C]Ornithine
ΔagrE ΔagrE AgrE Orn
Arg Cit Arg
[14C]Proline putA::C.S3 putA::C.S3 Pro Pro Orn PutA Arg
[14C]Glutamate
D [14C]Proline
WT putA::C.S3
Pro Pro
Gln
Glu a/b propeller LOR/SDH-like protein domain N C LOR/SDH NAD-dependent n-domain DHS
Arg dihydrolase Ornithine cyclodeaminase Arg Orn Pro
+ + 2 NH4 + CO2 NH4
+ mol NH4 produced = 2 x (mol ornithine) + 3 x (mol proline) A [14C]Arginine C
[14C]Arginine [14C]Ornithine Pro Orn ArgF Gln/Cit Arg [14C]Citrulline Glu AgrE
B [14C]Ornithine
[14C]Proline Pro Orn PutA
Glu [14C]Glutamate 14 A [ C]Arginine B [14C]Ornithine
ΔagrE + AgrE ΔagrE + AgrE
Pro Pro
Orn Orn Gln/Cit Gln/Cit Arg Arg
Glu Glu
14 C [ C]Arginine D [14C]Ornithine
ΔagrE + Mls2 ΔagrE + Mls2
Pro
Arg Orn
Glu A His-tagged a/b propeller domain B His-tagged whole AgrE protein
6xHis a/b propeller 6xHis a/b propeller LOR/SDH-like protein tag domain tag domain N C N C
40 mM L-arginine substrate; 40 mM L-arginine substrate; protein isolated from E.coli. protein isolated from E.coli. 0 min 0 min Arg Arg ) ) units units arbitrary ( arbitrary ( 60 min 60 min Arg Arg Orn Orn
+ 1.84 NH /Orn Fluorescence + Fluorescence 4 1.75 NH4 /Orn NH + + 4 NH4
0 10 20 30 (min) 0 10 20 30 (min)
Strep-tagged whole AgrE protein Strep II a/b propeller LOR/SDH-like protein tag domain N C Muestra: 5 0 Vial: 6 Vol iny. 10 µL C Secuencia: C:\EZChrom Elite\Enterprise\Projects\Aminoacidos D PICT\Sequence\parada 1'0.seq Usuario: Carlos 40 mM L-arginine substrate; 40 mM L-ornithine substrate + 1 mM NAD+ + Fecha: 06/07/2017 9:55:11 protein isolated from Anabaena 10 mM b-m-etOH + protein from Anabaena
2000 2000 0 min 0 min Arg Orn 1500 1500 ) ) 1000 1000 mAU mAU units Muestra: 5 60 units Vial:500 16 500 Vol iny. 10 µL Secuencia: C:\EZChrom Elite\Enterprise\Projects\Aminoacidos PICT\Sequence\parada 1'0.seq Usuario:0 Carlos 0 Fecha: 06/07/2017 9:54:03 arbitrary arbitrary ( ( 0 5 10 15 20 25 30 Minutes 2000 60 min 2000 60 min Arg Orn Orn
1500 1500
+
Fluorescence 1000 1000 1.33 NH /Pro Fluorescence mAU mAU 4 1.91 NH +/Orn NH + 4 500 4 500 + Pro NH4
0 0
0 5 10 15 20 25 30 0 10 Minutes 20 30 (min) 0 10 20 30 (min) Muestra: arg s9 60 Vial: 3 Vol iny. 10 µL Secuencia: C:\EZChrom Elite\Enterprise\Projects\Aminoacidos PICT\Sequence\parada 1'0.seq Usuario: Carlos Fecha: 12/11/2018 12:49:01 A 500 500 Arg FMOC ) 400 Orn* 400
units 300 300 mAU mAU
200 200 React. Fluorescence arbitrary 100 100 ( + Pro* NH4
0 0 0 5 10 15 20 25 30 0 10 Minutes 20 30 (min)
B 2000 + NH4
1500 (µM)
1000 product
Reaction Orn 500
Pro
0 0 20 40 60 80 Reaction time (min)
-500 A Growth rate, µ (day-1)
+ BG110NH4 BG11 (Nitrate) BG110 (N2) Set 1 PCC 7120 (WT) 0.53 ± 0.16 0.86 ± 0.19 0.87 ± 0.56 CSMI43 (ΔagrE) 0.56 ± 0.08 0.91 ± 0.42 0.83 ± 0.51 Set 2 PCC 7120 (WT) nd 0.86 ± 0.10 0.50 ± 0.17 CSS5 (putA::C.S3) nd 0.82 ± 0.04 0.36 ± 0.05
B PCC 7120 CSMI43 (WT) (DagrE) A Anabaena WT ΔagrE ΔspeA::C.S3 Boiled filaments
Agm Pro
Orn Arg Gln/Cit Arg Arg Glu
B + BG110NH4 BG11 BG110 PCC 7120 (WT)
ΔagrE ΔspeA::C.S3
ΔagrE
ΔspeA::C.S3
(ng Chl) 10 5 2.5 1.25 0.6 10 5 2.5 1.25 0.6 10 5 2.5 1.25 0.6 A 70 C 60 50 alr0540 alr0541 40
fluorescence 30 PA-1 PA-2 20 2.3-kb probe 10 Relative Het N NO - NH + 0 2 3 4 putA VC VC VC VC H VC VC VC VC
B 120 vegetative cells heterocysts 100
80 rnpB
60 fluorescence nifH 40
Relative rbcL 20
0 24 h -N 48 h -N Arginine SpeA
+ 2 NH4 + CO2 Agmatine CO 2 AgrE Orn NH + SpeB 4 Urea Proline
Putrescine [H] Putrescine SpeY PutA P5C/GSA
+ [H] NH4 Sym-homospermidine Glutamate