Draft genomes and initial characteriaztion of siderophore producing pseudomonads isolated from mine dump and mine drainage.

Siderophores are of high interest for biotechnological, pharmaceutical, agricultural and industrial applications. Although they are synthesized by various organisms, the yield is usually low which hindrances their suitability for broad range uses. Thus, it is necessary to identify novel producers and to increase the understanding of the biosynthesis pathways. Herein we report the isolation of two novel Pseudomonas strains and the identification of the gene clusters for the biosynthesis of pseudomonine as well as pyochelin and pyoverdine.

Iron is an essential element for living organisms and involved in numerous cellular processes. Thus, the maintenance of a proper intercellular level is critical [1]. However, iron is not easily bioavailable due to the formation of insoluble forms at aerobic conditions and neutral pH, although it is one of the most abundant elements. One strategy to circumvent this issue is the employment of small carriers called siderophores [2]. These are produced as secondary metabolites by various organisms including prokaryotes (bacteria [3,4], cyanobacteria [5] and archaea [6]) as well as eukaryotes (plants [7], fungi [8] and mammals [9]). Siderophores differ in structure and chemical properties but can be classified according to their metal chelating functional groups into hydroxamate, catecholate, carboxylate and mixed type [10]. The chemical structure also defines their affinity for iron but also other metals. Besides their physiological role, they have been shown to be promising candidates for various industrial, agricultural and medical applications [4,11]. However, the biosynthesis pathways of siderophores are usually complex and the yield is modest. Thus, it is currently laborious and expensive to produce siderophores in sufficient amounts. Therefore, the isolation, identification and characterization of siderophore producers is of high importance to facilitate an application of these compounds. Especially pseudomonads are known to be versatile siderophore producers [12]. In this study, we isolated two novel strains that offer a steady siderophore production rate and especially showed a good affinity for other metals like Al and Cu.

The herein described strains have been isolated from a heap in “Neuhilbersdorf” (strain H3, 50°55’07.1“N 13°22’19.2“E, 2016/12/13) and wet soil next to the mine drainage region “Roter Graben” in Tuttendorf (strain RGB, 50°56’24.1“N 13°22’19.6“E, 2017/01/18). They were selected as potent siderophore producers by evaluation on Chrome azurol S (CAS)-agar plates [13,14] (Fig. 1). Further, they showed to be selective for other metals than iron, making them interesting candidates for further investigations.

Fig. 1

Isolates H3 (left) and RGB (right) plated on CAS-agar selection plates. Production and secretion of siderophores by the bacteria is indicated by formation of yellowish halos around the colonies.

Genome sequencing was done to reveal the genus and to gain information about siderophores types that are supposed to be produced. Therefore, both strains were cultivated in 50 ml LB media, harvested after 3 days and DNA was isolated as described previously [15].

The DNA library preparation, genome sequencing, assembly, annotation and analysis were done as described previously [16]. Therewith a genome coverage of 247x for strain H3 and 249x for strain RGB was obtained, respectively. The results of the genome sequencing are summarized in Table 1. For isolate H3, 49 contigs (49 > = 1000 bp) were identified that cover about 5.8 Mbp with an average G + C content of 58.8 % (N50 = 175970; N75 = 108569; L50 = 11; L75 = 21). For isolate RGB, the assembly resulted in 43 contigs (41 > = 1000 bp) covering about 6.3 Mbp with an average G + C content of 60.5 % (N50 = 359427; N75 = 161348; L50 = 6; L75 = 12). A 16S rDNA-based phylogenetic analysis was done for both isolates revealing that both are classified as Pseudomonas species (Fig. 2). Isolate H3 clusters together with P. rhodesiae (99.3 % identity to strain DSM 14020) and P. grimontii (99.2 % identity to strain DSM 17515). However, strain H3 forms a separate branch and might be related to a novel species, which has to be proven. Isolate RGB is situated close to P. auricularis NBRC 10220 (100 % identity to strain NBRC 10220) and is therefore probably associated to this species.

Table 1

Genome statistics of the isolated pseudomonads.

Genome Feature	Pseudomonas sp. H3	Pseudomonas sp. RGB
Sum of contig length (bp)	5,865,301	6,336,192
Contigs	49 (> 3000 bp)	43 (> 950 bp)
G + C content (%)	58.8	60.5
Protein coding genes	5361	5723
Average gene length (bp)	950	960
Coding percentage (%)	87	87
tRNAs	42	44
rRNAs	3	3

Fig. 2

Separated subtrees with focus on related strains of isolate H3 (A) and isolate RGB (B). Phylogenetic analysis of the isolates H3 and RGB was done based on a 16S rDNA sequences. The complete multiple sequence alignment was prepared with 167 16S rDNAs by applying the ClustalW algorithm. The maximum likelihood tree was constructed by using the MEGAX software and bootstraps of 5,000 replicates [39]. Bootstrap values above 50 % are indicated.

Biochemical typing of both strains was done by Api20 NE (Table 2). Most of the metabolic properties of the isolates resemble those of the reference strains. Both strains are Gram-negative as well as catalase and oxidase positive. Notable differences can be found for isolate H3 in comparison to the nearest relatives on 16S rDNA base. It does not show growth on L-Arabinose and N-acetylglucosamine, while species rhodesiae and grimontii are able to use these compounds as carbon source. Further, strain H3 did not show fluorescence on Kings B or CSGA medium [17] what indicates that this strain is producing a non-fluorescent siderophore. Strain RGB showed fluorescence only on CSGA media which indicates that this strain might produce fluorescent siderophores. Both strains are able to grow on beech wood hydrolysate (BWH) as cheap alternative to glucose as carbon source for the production of siderophores [18] (Fig. 3). Therefore, both strains were cultivated on minimal media [19] containing 5 mM BWH for 25 h at 30 °C and 160 rpm. The doubling time is with 1.75–2.25 h in a comparable order of magnitude to glucose-grown pseudomonads (Table 3).

Table 2

Growth of Pseudomonas isolates in comparison to Pseudomonas type strains.

	Pseudomonas sp. H3	Pseudomonas sp. RGB	P. fluorescens DSM 50090	P. fulvaDSM 17717	P. marginalis DSM 13124	P. poaeDSM 14396	P. simiaeDSM 18861	P. rhodesiae DSM 14020	P. grimontiiDSM 17515*
Growth at 4 °C	+	+	+	+	+	+	+	+	+
Growth at 20 °C	+	+	+	+	+	+	+	+	n.d.
Growth at 37 °C	–	–	–	+	–	–	–	–	n.d.
Fluorescence on Kings B media	–	–	+	–	+	+	+	n.d.	+
Fluorescence on CSGA media	–	+	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
Oxidase test	+	+	+	–	+	+	+	+	+
D-Glucose	+	+	+	+	+	+	+	+	+
L-Arabinose	–	+	+	+	+	+	+	+	+
D-Mannose	+-	+	+	+	+	+	+	+	+
Rhamnose	–	–	–	–	–	n.d.	–	–	+
Trehalose	+	+	+	–	+	+	n.d.	+	+
Succinate	+	+	+	+	n.d.	n.d.	n.d.	n.d.	+
D-Mannitol	+	+	+	–	+	+	+	+	+
N-Acetylglucosamine	–	+-	+	–	+	+	–	+	+
D-Maltose	–	–	–	–	–	–	–	–	–
Potassium gluconate	+	+	+	–	+	+	+	+	+
Decanoic acid	+-	+	+	+	+	+	+	+	n.d.
Adipic acid	–	–	–	–	–	–	–	–	n.d.
Malic acid	+	+	+	–	+	+	+	+	n.d.
Trisodium citrate	+	+	+	+	+	+	+	+	+
Phenylacetate	–	–	–	+	–	–	–	–	–
Benzoate	+	–	+	–	n.d.	n.d.	n.d.	n.d.	+-
Serine	+	+	+	+	n.d.	n.d.	n.d.	n.d.	+
Decane	+	–	–	+	n.d.	n.d.	n.d.	n.d.	n.d.

(+) growth; (−) no growth; (+−) slow growth; (n.d.) not determined; *determined for at least 90 % of the isolated strains.

Fig. 3

Growth of (A) Pseudomonas sp. H3 and (B) Pseudomonas sp. RGB on 5 mM beech wood hydrolysate at 30 °C and 160 rpm.

Table 3

Doubling times of Pseudomonas isolates and other Pseudomonas strains.

Strain	Doubling time in h	Substrate	References
Pseudomonas sp. H3	2.25	Beech wood hydrolysate	This study
Pseudomonas sp. RGB	1.75	Beech wood hydrolysate	This study
P. fluorescens	1.42	Glucose	[20]
P. putida	1.67	Glucose	[21]
P. fragi	0.83	Glucose	[22]
P. chlororaphis	1.25	Glucose	[23]

The biological subsystem distribution of the annotated genes based on RAST can be found in Table 4 [24]. A subsystem coverage of about 50 % was achieved for both isolates. Herein, 1.5 % of the genes of strain H3 and 2.4 % of the genes of strain RGB are supposed to be related to iron acquisition and metabolism, respectively. An additional genome analysis was executed on the antismash 5.0 platform to estimate the production of secondary metabolites and especially siderophores [25]. For Pseudomonas sp. H3, twelve secondary metabolite gene clusters were identified whereas four are annotated as putative siderophore gene clusters (cluster 1, 4, 9 and 10; Table 5). Cluster 1 contains several genes that can be found in the pyoverdine biosynthesis cluster, but no relevant peptide synthetase. Thus, it is unlikely that pyoverdines can be produced, especially as strain H3 does not show fluorescence. Cluster 2 and 9 contain IucA/IucC-like synthetases, which indicate the production of hydroxamate siderophores like aerobactin and desferrioxamine. This remains to be proven as these clusters also do not contain related biosynthesis components like an N-hydroxylase or a decarboxylase. At least, manual annotation showed that only cluster 10 contains the gene set for the production of the isoxazolidone siderophore pseudomonine (Table 6) [[26], [27], [28], [29]]. This siderophore was found in Pseudomonas entomophila and Pseudomonas fluorescens strains [30]. However, not much is known about the biochemical and metal binding properties of this siderophore.

Table 4

Biological subsystem distribution of annotated genes in the isolates H3 and RGB.

		Pseudomonas sp. H3		Pseudomonas sp. RGB
	Subsystem coverage	52 %		51 %
Code	Description	Value	Percent	Value	Percent
A	Cofactors, vitamins, prosthetic groups, pigments	317	7.8	348	8.1
B	Cell wall and capsule	214	5.2	180	4.2
C	Virulence, disease and defense	166	4.1	153	3.5
D	Potassium metabolism	30	0.7	27	0.6
E	Miscellaneous	100	2.5	84	1.9
F	Phages, prophages, transposable elements, plasmids	14	0.3	26	0.6
G	Membrane transport	231	5.7	251	5.8
H	Iron acquisition and metabolism	60	1.5	102	2.4
I	RNA metabolism	224	5.5	207	4.8
J	Nucleosides and nucleotides	115	2.8	128	3.0
K	Protein metabolism	279	6.8	289	6.7
L	Cell division and cell cycle	33	0.8	33	0.8
M	Motility and chemotaxis	120	2.9	137	3.2
N	Regulation and cell signaling	126	3.1	139	3.2
O	Secondary metabolism	6	0.1	4	0.1
P	DNA metabolism	110	2.7	120	2.8
Q	Fatty acids, lipids, and isoprenoids	199	4.9	190	4.4
R	Nitrogen metabolism	50	1.2	50	1.2
S	Dormancy and sporulation	5	0.1	3	0.1
T	Respiration	148	3.6	128	3.0
U	Stress response	195	4.8	199	4.6
V	Metabolism of aromatic compounds	101	2.5	130	3.0
W	Amino acids and derivatives	707	17.3	728	16.9
X	Sulfur metabolism	59	1.4	103	2.4
Y	Phosphorus metabolism	45	1.1	79	1.8
Z	Carbohydrates	423	10.4	480	11.1

Table 5

Secondary metabolite clusters identified in Pseudomonas sp. H3 with antiSMASH5.0.

Cluster	Type	From	To	Most similar known cluster	Similarity	MIBiG BGC-ID
1	Resorcinol	1	32,672	Pyoverdine	12 %	BGC0000413
2	NRPS-like	1	20,451	Mangotoxin	71 %	BGC0000387
3	Hserlactone	60,840	81,502	–	–	–
4	Siderophore	117,806	129,707	–	–	–
5	Arylpolyene	2,428	46,051	APE Vf	40 %	BGC0000837
6	Bacteriocin	16,482	28,126	–	–	–
7	Bacteriocin	50,089	60,982	–	–	–
8	Betalactone	12,457	35,644	Fengycin	13 %	BGC0001095
9	Siderophore	260,231	272,153	–	–	–
10	NRPS	131,179	182,321	Pseudomonine	100 %	BGC0000410
11	Ectoine	225,551	235,937	–	–	–
12	transAT-PKS,NRPS	245,969	338,222	Leinamycin	8 %	BGC0001101

Table 6

Pseudomonine related gene cluster in Pseudomonas sp. H3.

Putative siderophore: Pseudomonine
Pseudomonas sp. H3		Pseudomonas fluorescens WCS374 (GenBank: EF457930 and Y09356)
Locus	Gene	Description	Accession	% ID	% Coverage
4876	–	Ferrichrome-iron receptor	–	–	–
4877	orf10	Iron compound ABC uptake transporter substrate-binding protein	ABS50193	84	76
4878	orf9	Ferric siderophore ABC transporter, ATP-binding protein	ABS50192	89	100
4879	orf8	Iron compound ABC uptake transporter permease protein PiuC	ABS50191	85	99
4880	orf7	Iron compound ABC transporter, permease protein	ABS50190	95	100
4881	orf6	Putative ABC transporter permease/ATP-binding protein	ABS50189	87	100
4882	orf5	Putative ABC transporter permease/ATP-binding protein	ABS50188	87	100
4883	orf4	Sigma factor, ECF-superfamily	ABS50187	81	98
4884	pmsG	NRPS	ABS50186	79	100
4885	pmsF	N-hydroxylase	ABS50185	87	73
4886	hyp	Hypothetical protein	–	–	–
4887	pmsD	NRPS	ABS50184	82	100
4888	pmsC	Isochorismate synthase (EC 5.4.4.2)	CAA70528	86	100
4889	pmsE	NRPS	CAA70529	90	100
4890	pmsA	Pyridoxal-dependent histidine decarboxylase	CAA70530	86	97
4891	pmsB	Isochorismate pyruvate-lyase (EC 4.-.-.-)	CAA70531	76	100

For strain RGB, twelve secondary metabolite gene clusters were identified whereas three are annotated as putative siderophore gene cluster for the production siderophores (cluster 1, 2 and 8; Table 7). A manual analysis on genome level allowed for the identification of siderophore gene clusters contain the relevant genes for production of pyochelin (cluster 2) and pyoverdine (cluster 1 and 8). The annotation of the respective genes can be found in Table 8, Table 9. The pyochelin cluster is complete [31,32]. However, the cluster organization of pyoverdine is different in strain RGB compared to the reference in Pseudomonas aeruginosa PA01. Some regulatory genes and the cluster pvcABCD (relevant for the biosynthesis of the pyoverdine chromophore) are missing [33]. The fragmentation of the clusters might be a result of the draft genome sequence. However, all required genes that are needed for the biosynthesis and transport of pyoverdine are present in strain RGB [34]. Pyochelin as well as pyoverdines are known to be fluorescent, which harmonizes the observation of strain RGB on CSGA medium.

Table 7

Secondary metabolite clusters identified in Pseudomonas sp. RGB with antiSMASH5.0.

Cluster	Type	From	To	Most similar known cluster	Similarity	MIBiG BGC-ID
1	NRPS	33,192	100,383	Pyoverdine	10 %	BGC0000413
2	NRPS	48,274	100,804	Pyochelin	100 %	BGC0000412
3	Phenazine,hserlactone	196,591	219,395	Pyocyanine	100 %	BGC0000936
4	Thiopeptide	433,489	463,427	Lipopolysaccharide	5 %	BGC0000774
5	Arylpolyene	304,851	348,426	APE Vf	45 %	BGC0000837
6	NRPS	135,021	186,674	Viscosin	75 %	BGC0001312
7	NAGGN	85,373	100,323	–	–	–
8	NRPS	133,328	186,224	Pyoverdine	10 %	BGC0000413
9	Bacteriocin	593,687	604,532	–	–	–
10	Bacteriocin	417,217	428,095	–	–	–
11	Betalactone	179,482	202,682	Fengycin	13 %	BGC0001095
12	NRPS-like	1	21,957	Mangotoxin	71 %	BGC0000387

Table 8

Pyochelin related gene cluster in Pseudomonas sp. RGB.

Putative siderophore: Pyochelin
Pseudomonas sp. RGB		Pseudomonas aeruginosa PAO1 (GenBank: X82644 and AF074705 and U03161)
Locus	Gene	Description	Accession	% ID	% Coverage
3845	pchA	Menaquinone-specific isochorismate synthase (EC 5.4.4.2)	CAA57969	44	99
3846	pchB	Isochorismate pyruvate-lyase (EC 4.2.99.21)	CAA57968	61	100
3847	pchC	Pyochelin biosynthetic protein PchC, predicted thioesterase	CAA57967	62	92
3848	pchD	2,3-Dihydroxybenzoate-AMP ligase (EC 2.7.7.58)	CAA57966	71	95
3849	pchR	Transcriptional regulator PchR	NP_252917	71	100
3850	pchE	NRPS	AAC83656	59	100
3851	pchF	NRPS	AAC83657	62	100
3852	pchG	Thiazolinyl imide reductase	AAK01463	61	99
3853	pchH	Putative ABC iron siderophore transporter	AAK01464	54	99
3854	pchI	Putative ABC iron siderophore transporter	AAK01462	55	100
3855	fptA	Fe(III)-pyochelin outer membrane receptor	AAC43213	74	97
3856	fptB	Hypothetical protein in pyochelin cluster	AAC43214	58	21
3857	fptC	Putative iron-regulated membrane protein	AAC43215	51	72
3858	fptX	inner-membrane permease	NP_252908	67	93

Table 9

Pyoverdine related gene clusters in Pseudomonas sp. RGB.

Putative siderophore: Pyoverdine
Pseudomonas sp. RGB		Pseudomonas aeruginosa PA01 (GenBank: AY765259 and AE004091)
Locus	Gene	Description	Accession	% ID	% Coverage
1153	pvdP	Tyrosinase	AAX16288	65	96
1154	pvdM	Periplasmic enzyme	AAX16289	76	99
1155	pvdN	Periplasmic enzyme	AAX16290	62	100
1156	pvdO	Periplasmic enzyme	AAX16291	75	93
1157	pvdF	N5-hydroxyornithine transformylase	AAX16292	80	99
1158	pvdE	ABC transporter (secretion)	AAX16293	78	100
1159	fpvA	Ferripyoverdine receptor protein	AAX16294	65	98
1160	pvdD	NRPS	AAX16295	54	99
1161	pvdJ	NRPS	AAX16296	48	97
1162	pvdI	NRPS	AAX16297	56	100
2550	mbtH	MbtH-like NRPS chaperone	AAG05800	85	95
2551	pvdH	Aminotransferase	AAG05801	85	97
2560	pvdL	NRPS	AAG05812	74	100
2561	pvdG	Thioesterase	AAG05813	53	98
2562	pvdS	ECF sigma factor	AAG05814	92	93
4143	pvdQ	Ntn-type hydrolase	AAG05773	54	98
5639	opmQ	Outer membrane pyoverdine eflux protein	AAG05779	63	97
5640	pvdT	Pyoverdine efflux carrier and ATP binding protein	AAG05778	81	98
5641	pvdR	Pyoverdine specific efflux protein	AAG05777	72	99
5642	fpvI	ECF sigma factor	AAG05775	72	99
5643	pvdA	L-ornithine N5-oxygenase	AAG05774	75	99

The siderophore production was tested in 5 and 100 ml scale with the glucose as sole carbon source. Therefore, precultures were grown for three days in LB medium (30 °C, 160 rpm), harvested by centrifugation (10,000 × g), washed twice with sterile saline, and re-suspended in 10 % of the initial volume with sterile saline. The main culture was inoculated 1:50 with cell suspension. To produce siderophores, a M9 minimal medium with low phosphate content was chosen containing 1.28 g l−1 Na2HPO4, 0.3 g l−1 KH2PO4, 0.5 g l−1 NaCl, 1 g l−1 NH4Cl and 10 ml l−1 goodiemix. Goodiemix solution consists of 385 mM MgSO4, 10 mM CaCl2, 0.1 mM thiamine and 125 ml l−1 trace element solution (49 g l−1 MgCl2, 2 g l−1 CaCl2, 1.44 g l−1 ZnSO4 · 7 H2O, 0.85 g l−1 MnSO4 ∙ H2O, 0.24 g l−1 CuSO4 · 5 H2O, 0.06 g l−1 H3BO3, 51 ml l−1 HCl). 20 mM glucose was added as substrate. All glassware used for siderophore production was washed with 6 M HCl in order to remove iron [35]).

The highest siderophore production was determined after 3 days with about 130 μM* (* (desferriooxamine B) equivalent [14]) in 5 ml scale. This is in a similar range compared to P. aeruginosa strains, although it has to be mentioned that the cultivation conditions are slightly different and the concentration was determined already after 1 day [36,37] (Table 10). Further, it can be seen that the siderophore concentration is lower in a bigger cultivation volume. Binding of other metal ions (Al3+, Ga3+ and Cu2+) was determined in the culture supernatant by adapted CAS-assays (Fig. 4) [14]. All of the tested metal ions can be chelated by the siderophores. This is in accordance with previous findings on pyoverdin and pyochelin [38]. For pseudomonine no other ligands than iron have been tested so far.

Table 10

Siderophore production of strain H3 and RGB on glucose in 5 ml and 100 ml scale.

Strain	Scale in ml	Carbon source	Siderophore production in μM	After x days	References
Pseudomonas sp. H3	5	20 mM Glucose	135*	3	This study
	100	20 mM Glucose	80*	3
Pseudomonas sp. RGB	5	20 mM Glucose	130*	3	This study
	100	20 mM Glucose	70*	3
P. aeruginosa FP6	100	56 mM Glucose	20	n.s.	[37]
		34 mM Succinate	125	1.5
P. aeruginosa PSS	3500	56 mM Glucose	180	1.25	[36]
		25 mM Succinate	60	1
		7 mM Glutamic acid	140	1

n.s. – not specified.

Calculated in desferrioxamine B equivalents according to [14].

Fig. 4

Binding of the metal ions Al3+, Ga3+ and Cu2+ by culture supernatants of Pseudomonas sp. H3 and RGB. DFOBeq. - desferrioxamine B equivalents.

Nucleotide sequence accession numbers

The genome sequences were deposited at the DDBJ/ENA/GenBank under the accession numbers (Pseudomonas sp. H3: VMSG00000000; BioProject: ) and (Pseudomonas sp. RGB: VMSH00000000; BioProject: ).

Author contribution

MH isolated the strains. MH, VS and SH performed the strain characterization including growth experiments and siderophore production. MH and TH performed the DNA isolation, genome sequencing, annotation and analyzes. TH, MH and DT wrote the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.