Genome mining for methanobactins - BioMed Central | The Open

RESEARCH ARTICLE Open Access

Genome mining for methanobactinsGrace E Kenney and Amy C Rosenzweig*

Abstract

Background: Methanobactins (Mbns) are a family of copper-binding natural products involved in copper uptakeby methanotrophic bacteria. The few Mbns that have been structurally characterized feature copper coordinationby two nitrogen-containing heterocycles next to thioamide groups embedded in a peptidic backbone of varyingcomposition. Mbns are proposed to derive from post-translational modification of ribosomally synthesized peptides,but only a few genes encoding potential precursor peptides have been identified. Moreover, the relevance ofneighboring genes in these genomes has been unclear.

Results: The potential for Mbn production in a wider range of bacterial species was assessed by mining microbialgenomes. Operons encoding Mbn-like precursor peptides, MbnAs, were identified in 16 new species, includingboth methanotrophs and, surprisingly, non-methanotrophs. Along with MbnA, the core of the operon is formed bytwo putative biosynthetic genes denoted MbnB and MbnC. The species can be divided into five groups on thebasis of their MbnA and MbnB sequences and their operon compositions. Additional biosynthetic proteins,including aminotransferases, sulfotransferases and flavin adenine dinucleotide (FAD)-dependent oxidoreductaseswere also identified in some families. Beyond biosynthetic machinery, a conserved set of transporters wasidentified, including MATE multidrug exporters and TonB-dependent transporters. Additional proteins of interestinclude a di-heme cytochrome c peroxidase and a partner protein, the roles of which remain a mystery.

Conclusions: This study indicates that Mbn-like compounds may be more widespread than previously thought,but are not present in all methanotrophs. This distribution of species suggests a broader role in metal homeostasis.These data provide a link between precursor peptide sequence and Mbn structure, facilitating predictions of newMbn structures and supporting a post-translational modification biosynthetic pathway. In addition, testable modelsfor Mbn transport and for methanotrophic copper regulation have emerged. Given the unusual modificationsobserved in Mbns characterized thus far, understanding the roles of the putative biosynthetic proteins is likely toreveal novel pathways and chemistry.

Keywords: methanobactin, methanotroph, particulate methane monooxygenase, copper, chalkophore, TonB-dependent transporter, natural product, post-translational modification

BackgroundMethanotrophs are Gram-negative bacteria that usemethane, a potent greenhouse gas, as their sole source ofcarbon and energy [1]. As the only biological methanesink, methanotrophs have attracted much attention as ameans of mitigating methane emissions [2-4]. The firststep in their metabolic pathway, the oxidation of methaneto methanol, is catalyzed by methane monooxygenase(MMO) enzymes, which are of broad interest in the questto exploit abundant natural gas reserves as fuel and chemi-cal feedstocks. Most methanotrophs utilize particulate

methane monooxygenase (pMMO), a copper-dependentintegral membrane enzyme [5,6]. Under copper-limitinggrowth conditions, some methanotrophs can also expressan alternative, soluble form of MMO (sMMO) that utilizesiron [7]. In these methanotroph strains, the switchbetween pMMO and sMMO is controlled by copper: cop-per represses transcription of the sMMO genes and causesformation of intracytoplasmic membranes that housepMMO [8-10]. The details of this “copper switch” regula-tory mechanism are not understood and represent a majoroutstanding question in the field.An important part of the copper switch puzzle is the

discovery of methanobactins (Mbns), a family of copper-binding natural products initially detected in the

* Correspondence: [email protected] of Molecular Biosciences and of Chemistry, NorthwesternUniversity, Evanston, IL 60208, USA

Kenney and Rosenzweig BMC Biology 2013, 11:17http://www.biomedcentral.com/1741-7007/11/17

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methanotroph Methylosinus trichosporium OB3b[11-13], and potentially useful in applications rangingfrom wastewater copper removal in the semiconductorindustry [14] to treatment of Wilson disease, a humandisorder of copper metabolism [15]. Mbns are believedto be secreted under copper limiting conditions in acopper-free (apo) form to acquire copper from theenvironment and then internalized in a copper-loadedform to provide essential copper to the methanotroph[16,17]. In support of this model, methanobactin (Mbn)promotes the copper switch [18,19] and can mediaterelease of copper from insoluble mineral sources [19,20].In addition, direct uptake of copper-loaded Mbn(CuMbn) by Methylosinus trichosporium OB3b has beendemonstrated, and proceeds via an active transport pro-cess [21]. Because this model for Mbn function as wellas aspects of its structure (vide infra) are reminiscent of

iron siderophores, Mbn has also been referred to as achalkophore [13] (chalko- is derived from the Greekword for copper whereas sidero- is from the Greek wordfor iron).Mbn molecules from Methylosinus trichosporium

OB3b, Methylocystis strain SB2, Methylocystis hirsutaCSC-1, Methylocystis strain M and Methylocystis roseaSV97T have been characterized by mass spectrometry,nuclear magnetic resonance (NMR) and crystallography(Figure 1; Additional file 1, Figure S1). These data reveala peptidic backbone and copper coordination by twonitrogen-containing heterocycles next to thioamidegroups [13,22-25]. The Methylosinus trichosporiumOB3b Mbn backbone has the sequence 1-(N-(mercapto-(5-oxo-2-(3-methylbutanoyl)-oxazol-(Z)-4-ylidene)methyl)-Gly1-Ser2-Cys3-Tyr4)-pyrrolidin-2-yl-(mercapto-(5-oxo-oxazol-(Z)-4-ylidene)methyl)-Ser5-Cys6-Met7 and

HN

OH

NH

OO

HNO

HN

ON

ON

NO

NH

NH

OOH

NH

O

O

OHO

S

OHCu+

S

S

S-

S-O

O

HN

OH

NH

OO

HNO

HN

ON

ONH

HNO

NH

NH

OOH

NH

O

SH

OHO

S

OH

O

O

NH

HS SH

SH

A

B

N

HN

S ONH

OHO

HN

NO

O

NHS-

O

NHNH

O

Cu+

OSO

O O-

NOH

HO

NH

NH2+

H2N

NH

HN

O ONH

OHO

HN

HNO

NHOO

NHNH

O

OHO

SH

NH SH

HO

O

NH

NH2O

HO

O

HO

O

NH

NH2O

HO

O

NH+H2N

H2N

MTVKIAQKKVLPVIGRAAA

MTIRIAKRITLNVIGRASA

Leu1

Cys2Gly3 Ser4

Cys5

Tyr6

Cys8

Ser9

Cys10Met11

Pro7

Arg1

Cys2Ala3

Ser4

Thr5

Cys6

Ala7

Ala8

Thr9

Asn10

Gly11

Figure 1 Post-translational modifications required to produce methanobactins from Methylosinus trichosporium OB3b andMethylocystis rosea SV97T. (A) Mbn from Methylosinus trichosporium OB3b is generated from the precursor peptideMTVKIAQKKVLPVIGRAAALCGSCYPCSCM. Post-translational modifications needed to produce the final natural product include leader peptidecleavage and subsequent N-terminal transamination (orange), oxazolone formation (green), thioamide formation (blue), and disulfide bondformation (yellow). (B) Mbn from Methylocystis rosea SV97T is generated from the precursor peptide MTIRIAKRITLNVIGRASARCASTCAATNG. Post-translational modifications needed to produce the final natural product include leader peptide cleavage, pyrazinedione formation (purple)oxazolone formation (green), thioamide formation (blue), and threonine sulfonation (pink). Several residues that are present in the precursorpeptide are missing in the reported structure (gray); the loss of a C-terminal threonine and asparagine had been previously reported, butidentification of the precursor peptide indicates that a final glycine is also lost.


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is thought to derive from the peptide backboneLCGSCYPCSCM (Figure 1A).By comparison, Methylocystis Mbns are alanine-rich,

and the first nitrogen-containing heterocycle is not anoxazolone [24]. All the Methylocystis Mbns have a similarbackbone. The N-terminal residue is either arginine- ormethionine-derived (the latter only in Methylocystishirsuta CSC-1), and immediately precedes the first het-erocycle. The heterocycle/thioamide pair (pyrazinadionein all structures except the NMR structure of Methylocys-tis strain SB2) is followed by an alanine, a serine and asulfonated threonine (Additional file 1, Figure S1). Nextis an oxaozlone/thioamide pair and an alanine followedby a methionine (Methylocystis sp. M) or a second ala-nine (Additional file 1, Figure S1). Additional C-terminalresidues are present in some forms of the molecule. TheMethylocystis rosea SV97T Mbn contains a Thr-Asnsequence [24], and likely derives from a peptide backbonecontaining the sequence RCASTCAATN (Figure 1B).Despite these structural differences, these Mbns retaintheir strong and specific affinity for copper [24].The Mbn biosynthetic pathway has not been eluci-

dated, and was initially suggested to involve nonriboso-mal peptide synthetases [16,26], similar to production ofmany siderophores [27]. However, sequencing of theMethylosinus trichosporium OB3b genome [28] led tothe identification of a 30-amino acid open-readingframe (ORF) with similarities to the peptidic Mbn back-bone [23], supporting previous suggestions that Mbn isproduced via post-translational modification of a riboso-mally synthesized precursor peptide [22]. A similar pre-cursor peptide was identified in an unrelated species,Azospirillum sp. B510, along with several conservedneighboring genes [17,22], but analogous ORFs were notdetected in other available methanotroph genomes, andthe relevance of many of the neighboring genes sur-rounding the precursors was unclear.Genes encoding the precursors of small ribosomally-

produced natural products can be difficult to detect andannotate, and the underdetection of biologically relevantsmall ORFs is a known problem [29-31]. However, theever-increasing rate at which bacterial genomes arereleased has prompted the design of genome mining toolsfor widespread classes of ribosomally synthesized andpost-translationally modified peptide natural products(RiPPs), such as lantibiotics [32-37]. With the aim of iden-tifying the potential for Mbn production in a wider rangeof bacterial species, we mined the available microbialgenomes in the National Center for Biotechnology Infor-mation (NCBI) and Joint Genome Institute (JGI)/Inte-grated Microbial Genomes (IMG) databases, identifying 18new Mbn-like precursors and accompanying biosyntheticgenes from 16 species, including unknown or provisionallyidentified species present in metagenomic samples.

Surprisingly, many of these precursor peptides and theiroperons are from non-methanotrophic species and severalwell-studied methanotrophic species seem to lack Mbnoperons similar to that of Methylosinus trichosporiumOB3b. Beyond biosynthesis-related genes, we also identi-fied a widely-conserved set of transporters and sigma fac-tors, which has implications for Mbn export and import aswell as its involvement in cellular copper homeostasis.Finally, this bioinformatics study provides new tools tobetter detect Mbn-like gene clusters in novel genomes.

Results and discussionUsing a variety of bioinformatics techniques, we were ableto detect putative biosynthesis operons for Mbn-like nat-ural products in 14 new species, as well as several uniden-tified or tentatively identified species present inmetagenomic studies (Figure 2; Additional file 2, TableS1). While five of the identified species are Type II metha-notrophs like the first identified Mbn-producer, Methylosi-nus trichosporium OB3b, the remaining species are not.Operons were detected in b- and g-proteobacteria as wellas a-proteobacteria, to which the Type II methanotrophsbelong. Both the precursor peptides and the range ofnon-core biosynthesis genes present in the operon hint ata set of potential modifications that may define the Mbnfamily. Furthermore, genes likely to be related to export,import and copper regulation are found in almost everyoperon. Based on sequence analysis, the presence of speci-fic Mbn-related genes and the overall operon structure, wehave provisionally divided the operons into five groups(Figure 2).

Locating the precursor peptide MbnAAutomated detection of small peptide sequences in newly-sequenced genomes is problematic [29]. Short sequencesare poorly detected by Basic Local Alignment Search Tool(BLAST) and similar sequence analysis methods, anduncurated small ORF detection results in the annotationof many spurious small ORFs. For well-established classesof small ribosomally-produced natural products, such asbacteriocins, hidden Markov model (HMM)-based tools,such as BAGEL and BAGEL2, have been developed to bet-ter detect precursors in newly sequenced genomes [32,33].With only two published precursor peptide sequences(MbnAs), Mbn was not a good candidate for this detectionmethod [17,23]. A TIGRFAM group (TIGR04071) doesexist for the precursor, and is a member of the Gen-Prop0962 family (which also includes TIGRFAM groupsfor MbnC and half of MbnB) [38], but it is based on onlythe two previously published precursor peptide sequencesand a third suggested MbnA homologue from Gluconace-tobacter sp. SXCC-1 [38]. Four possible MbnAs detectedhere are also mentioned in the 2013 TIGRFAM update[38], but are not included in the available HMM.


Page 3 of 17

Because of the limitations of direct precursor peptidedetection, we pursued an alternate genome mining strat-egy focusing on the detection of biosynthetic proteins,followed by manual identification of unannotated precur-sors. This method has been used with some success for avariety of natural products, including radical S-adenosylmethionine (SAM)-modified peptides, bacteriocins in

cyanobacteria, and a new class of lantibiotic-like naturalproducts stemming from nitrile hydratase or Nif11 leaderpeptides [34-36,39]. We used the MbnB and MbnCsequences from Methylosinus trichosporium OB3b [38] asseeds in a tBLASTn search through the NCBI’s Non-redundant (NR) and Whole Genome Shotgun (WGS)databases, as well as the microbial genomes available at

Gluconacetobacteroboediens 174bp2

Gluconacetobactersp. SXCC-1

Pseudomonasextremaustralis 14-3, substr. 14-3b

Pseudomonasfluorescens NZI7

Azospirillumsp. B510

Methylocystisrosea SV97T

Methylocystis sp. SC2

Bioreactor metagenome PBDCA2†

Methylocystisparvus OBBP, contig03

Cupriavidusbasilensis B-8

Tistrella mobilis KA081020-065

Methylosinussp. LW3, v1

Methylosinussp. LW3, v2

Methylocystisparvus OBBPcontig41

Methylosinustrichosporium OB3b

Vibriocaribbenthicus BAA-2122

Photorhabdusluminescens substr. laumondii TT01

copC lyaseNAD-dep.ald. dehyd.

aldehydereductase lysR-like

cydB cydA

(multiple variants on fragmentary contigs)

phytanoylCoA synth. terC

lyase

dgoA dgoKmembr. flavidoxin

reductasearaC -like

tonB

Methylosinussp. LW4

tauD

moaA ß-hydrox.dehydratase

Zn, DNA-binding

moaA

3-hydroxy-isobutyrate

dehydratase

ATP/Znprotease

Type I rest./mod. enz.

acyltransferase

cystathioneß-synth.

cystathione-lyase

ECF 70 factor, FecI-like (MbnI)

MbnB (unknown Mbnbiosynthesis protein 1)

FecR-like membrane sensor (MbnR)

MbnC (unknown Mbnbiosynthesis protein 2)

TonB-dependent transporter (MbnT)

MATE effluxpump (MbnM)

Aminotransferase(MbnN)

Periplasmic bindingprotein

Partner of MbnH (MbnP)

FAD-dependent oxidoreductase (MbnF)

Di-heme cytochrome c peroxidase (MbnH)

Sulfotransferase(MbnS)

Mobile geneticelement

MbnA (Mbn precursor)

Annotated proteins ofunknown relevance

Putative/hypotheticalprotein

Annotated region

End of contig

yobA yebZIV

III

II

I

V

ECF factor

ECF factor

ECF factor

ECF factor

ECF factor

DUF46112kb

3kb

hisE hisI hisF

carbohydrateporin

11kb

25kb

cusR

ECF factor

fecR-like35kb

2kb

100kb

1kb

ß-lactamaseregulator 39kb

4kb

65kb

deoD deoB

cusR cusS

yobA17kb

epimerase DUF297 tetratricopeptide repeat

copC

tauD cusR cusS

cusS

copG?kdpF

kdpAkdpB

acrB

8kb

trbI trbG trbF20kb

fur-like

Azospirillumsp. B506†

cydB cydA

DUF4615kb

Methylobacteriumsp. B34†

Figure 2 The genetic organization of Mbn-producing operons. Genomic regions containing all identified Mbn operons are depicted.Operons are grouped into five families on the basis of operon content, MbnB conservation, and MbnA sequence. All operons contain MbnA(black) and MbnB (orange) and at least one transport protein, a MATE efflux pump (purple), a TonB-dependent transporter (blue) or both. Mostoperons contain additional biosynthesis-related genes, including MbnC (green), aminotransferases (MbnN, brown) or sulfotransferases (MbnS,dark purple). Additional genes may be related to regulation (MbnR, yellow and MbnI, red) or may play an unknown role in copper homeostasis(MbnP, gold and MbnH, dark blue) Genetic mobility elements of several varieties (light teal) are common in the vicinity of these operons.Metagenomic samples corresponding to unidentified or provisionally identified species are marked (†).


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JGI/IMG. For every MbnB homologue detected, a 2 kbregion preceding and following that gene was manuallyexamined for 45 to 150 bp ORFs coding for short peptideswith at least one cysteine in the last 10 amino acids and anN-terminal region containing multiple arginine or lysineresidues.A total of 18 novel MbnA-like ORFs were identified

using these methods, one preceding every close MbnBhomologue excluding truncated homologues from metage-nomic sequencing. Two Methylosinus species (Methylosi-nus sp. LW3 and Methylocystis parvus OBBP, which maybe misclassified as Methylocystis [40]) have two distinctMbnA genes encoding unique Mbns. While it is notuncommon for bacteria to produce multiple siderophoresto control iron acquisition in different environments [41],a similar phenomenon has not yet been observed forchalkophores. As shown in the Multiple Alignment withFast Fourier Transform (MAFFT) alignment (Figure 3A),both the leader and core sequences exhibit some conserva-tion over the 20 complete sequences. MbnA-likesequences range from 23 to 35 amino acids (aa), with

predicted core sequences ranging from 7 to 15 aa. Theleader peptides are better conserved than the core pep-tides, perhaps indicating the involvement of the leaderpeptide in interactions with biosynthesis proteins [42].The leader sequences are lysine/arginine rich, with at leasttwo such residues occurring near the beginning and onepresent in a conserved area immediately prior to the coresequence (Figure 3B). The core sequences are more vari-able, but all contain at least one C (G|A|S) (S|T) motif. Ofthe complete MbnA sequences, 18 have a second corecysteine and 11 contain one or two additional cysteines.One basis for the proposed five operon groups (Figure 3C)

is the nature of the MbnA sequences, including the structu-rally, but not genomically, characterized MethylocystisMbns. The Group I MbnA sequences, primarily fromMethylosinus genera are long (11 to 15 aa), with four non-adjacent core peptide cysteines, and contain core prolines. Itis unknown whether the presence of four cysteines allowsfor the formation of disulfide bonds as found in Methylosi-nus trichosporium OB3b Mbn or whether they lead to theproduction of additional oxazole/thioamide pairs, analogous

P. luminiscens sub. laumondii TT01V. caribbenthicus BAA-2122

G. oboediens 174bp2Gluconacetobacter sp. SXCC-1

Azospirillum sp. B510Methylobacterium sp. B34

Azospirillum sp. B506P. fluorescens NZI7

P. extremaustralis 14-3 sub.14-3bC. basilensis B-8

T. mobilis KA081020-065Methylocystis sp. SC2

M. rosea SV97TM. parvus OBBP, ctg3

Methylosinus sp. LW3, v2Bioreactor metagenome PBDCA2

M. trichosporium OB3bMethylosinus sp. LW3, v1

Methylosinus sp. LW4M. parvus OBBP, ctg41

M - - Q K K T S V E I E V E V E D K E - M T C G S Y N K - - - - - - - - - - - -M - - - - K N D K K V V V K V K D K E - M T C G A F N K - - - - - - - - - - - -M T I T I T I L K T K Q I S V P V R A G L Q C G S G - - - - - - V C G Y N A - -M A I T I T I L K T K Q I S V P V R A G L Q C G S G - - - - - - V C G Y N A - -M - - T I K I A K K Q T L S V A G R A G A C C G S C - - - - - - - C A P V G V NM - - T I K I A K K Q T L S V A G R A G A C C G S C - - - - - - - C A P V G V NM - - T I K I A K K Q T L S V A G R S G A C C G S C - - - - - - - C A P V G V NM - - S I K I A K K H T L H I A G R A G A C C A S C - - - - - - - C A P V G V NM - - S I K I A K K H T L Q I A G R A G A C C A S C - - - - - - - C A P L G V NM - - T I K I S K K E A I E V R G R S G A C C G S C - - - - - - - C A A I G A -M - - S I K I S A R K A L Q I A G R A G A R C A T I - - - - - - - C A V A G - -M - - T I R I A K R I T L N V I G R A G A M C A S T - - - - - - - C A A T N G -M - - T I R I A K R I T L N V I G R A S A R C A S T - - - - - - - C A A T N G -M - - T I K I V K R T A L A V N G R A G A D C G T A - - - - - - - C W A - - - -M - - A I N I V K R T T L V V N G R S G A D C G T A - - - - - - - C W G - - - -M - - A I K I V K R V A L R V I S R N G A C C G S A - - - - - - - C W - - - - -M - - T V K I A Q K K V L P V I G R A A A L C G S C Y P - - - - - C S C M - - -M - - A I K I A K K E V L P V V G R L G A M C S S C P M - - - - - C G P L C P -M - - T I K V V K K E I L P V I G R V Q A M C A C N P P - - - - W C G T C - - -M - - A I K I V K K E I L P V I G R V Q A F C S S C S G G G Q C G C G P A - - -

prob

abili

ty0

1

M - I T I K I A K K + T L S V + G R A G A C C G S C + + - - - - V C A P V G V N

leader sequence core sequence

leader sequence core sequence

CA

B

-10 +1 +10-1-20

-10 +1 +10-1-20

C. basilensis B-8

Methylobacterium sp. B34

Azospirillum sp. B51066Azospirillum sp. B506 57

P. fluorescens NZI7

P. extremaustralis 14-3 sub.14-3b

73

T. mobilisKA081020-065

50


M. rosea SV97T

95

21

15

Bioreactor metagenome PBDCA2M. parvus OBBP, ctg3

Methylosinus sp.LW3, v1

71725

M. trichosporium OB3bMethylosinus sp. LW3, v1

M. parvus OBBP, ctg41

Methylosinus sp. LW4

67

72

52

22

49

Gluconacetobactersp. SXCC-1

G. oboediens174bp2

100

P. luminiscens sub.laumondii TT01

V. caribbenthicusBAA-2122

99

I

IIIII

IV

V

Figure 3 Alignment and classification of the precursor peptide MbnA. (A) A MAFFT alignment of the 20 sequenced MbnA precursorsorganized by family. Likely leader and core peptides are indicated on the basis of the structurally characterized Mbns and their correspondingMbnA sequences. The alignment is visualized in Jalview and residues are colored following the Taylor color scheme [109], with intensitymodified by a conservation color increment of 30%. (B) Logo showing that both leader peptide and core peptide exhibit well-conserved motifs,but the leader peptide is less variant. (C) Phylogenetic tree (constructed in MEGA 5.0 as described in the Methods section) based on aClustalOmega alignment of the 20 full MbnA. Not pictured in this tree are the three species with structurally characterized Mbns for which nogenomic information exists; however, their core peptide sequences indicate that they would be grouped with Methylocystis sp. SC2 andMethylocystis rosea SV97T. Gamma distribution parameter 4.0462.


Page 5 of 17

to the multiple thiazoles and oxazoles present in manybacteriocins [42].The primarily Methylocystis Group II MbnA sequences

are shorter, contain only two or three cysteines, and manyhave a conserved threonine which, based on NMR andcrystal structures [23,24], is likely to be a sulfotransferasetarget. Interestingly, the sequences from Methylocystisstrain SC2 and Methylocystis rosea SV97T appear to bemerged with an extracytoplasmic function (ECF) sigmafactor, at least based on the annotation [43]. It is not clearwhether the precursor peptide is cleaved from these sigmafactors and whether sigma factor activity remains or isaltered. Although there is no structure for Mbn fromMethylocystis strain SC2, its MbnA sequence and the simi-larity of its operon structure to that of Methylocystis roseaSV97T suggest that its Mbn will resemble Methylocystisrosea SV97T Mbn and will be identical to Methylocysishirsuta CSC-1 Mbn [24]. Similarly, although there are nogenomes for Methylocystis strain SB2, Methylocystis strainM and Methylocystis hirsuta CSC-1, we can predict thatthe core peptides for their structurally characterized Mbnswill be RCASTCAA, RCASTCAMT and MCASTCAAT,respectively (likely followed by -TNG, -NG and -NG), andthat their leader sequences will resemble those fromMethylocystis rosea SV97T and Methylocystis strain SC2[24,43]. A subfamily of Group II MbnAs from Methylosi-nus or related species (Methylosinus sp. LW3, Methylocys-tis parvus OBBP and a bioreactor metagenome) do nothave the CASTCA(A) motif. Instead, the second cysteineis followed by a tryptophan. If the core peptide sequencedictates cysteine modification, these residues lack the C(G|A|S) motif associated with cyclization and thioamide for-mation in existing Mbn structures.The remaining families include MbnAs from a variety

of non-methanotrophic species. The species that haveGroup III MbnA sequences include two Pseudomonasspecies, two Azospirillum species and single species eachfrom the Cupriavidus, Tistrella and Methylobacteriumgenera. In this group, the two Pseudomonas sequences,the two Azospirillum sequences and the Methylobacter-ium sequence are most similar, with somewhat lengthyand near-identical MbnA core sequences containing twocysteine doublets. The less similar Cupriavidus basilen-sis B-8 MbnA preserves the cysteine doublets whereasthe Tistrella mobilis KA081020-065 sequence containsonly two non-adjacent cysteines.The Group IV MbnA sequences are currently only

found in the two Gluconacetobacter species. These twosequences are nearly identical, and feature only two corecysteines, with a leader sequence potentially extended bytwo amino acids. Finally, the Group V MbnA-likesequences are found in Vibrio caribbenthicus ATCCBAA-2122 and Phorhabdus luminescens subsp. laumon-dii TT01. These sequences are short and somewhat

divergent, containing only a single cysteine, which maysuggest that they represent a natural product with somestructural similarities to Mbn that either does not che-late copper or does not chelate copper in the same waythat other chalkophores do. This overall classificationscheme extends to the MbnB and MbnC sequences(vide infra), and will be subject to future modification asmore MbnA sequences are identified in new genomes.

The first unknown biosynthesis protein: MbnBMbnB is the core protein in the Mbn biosynthesis operon,and was detected in 19 operons, including truncatedforms in several metagenomic samples (Figures 2 and 4;Additional file 2, Table S1). However, the initial identifica-tion of this protein in Methylosinus trichosporium OB3bhas been problematic. In Methylosinus trichosporiumOB3b, and one other operon detected (Methylobacteriumsp. B34), MbnB is split into two ORFs, formerly annotatedas MettrDRAFT_3894 and MettrDRAFT_3895, but rean-notated as one entity (MettrDRAFT_3422) in a recentlyassembled genome build available on IMG [23]. ATIGRFAM HMM (TIGR04159) exists for the half of theprotein that resembles MettrDRAFT_3894, but does notcover MettrDRAFT_3895 [38]. Therefore, a conjugatewith a glycine replacing the stop codon between the twoORFs was used for BLAST detection and annotation.Despite the addition of new members to the MbnB

family, no motifs or domains of known function have beenidentified beyond occasional classification as TIM-barrelproteins [44]. MbnB homologues may be a subfamily inthe larger DUF692 family (PFAM class PF05114).However, when conducting a BLAST search or a HMM-based search for homologues, MbnB-like proteins repre-sent a distinct subgroup, with a sharp drop-off in expecta-tion value between the last MbnB-like protein (E <1E-50,except for sequences truncated by the end of a contig/scaf-fold) and other DUF692-like proteins. Notably, in theGroup V operons, the MbnB gene is separated from theMbnA-like precursor by a gene-sized ORF.A comparison of MbnB sequences (Figure 4A)

strongly supports the five operon families assigned onthe basis of the MbnA sequences (Figure 4B). There areabout six different regions that are strikingly well-pre-served, even in the Group V homologues. Withoutknowledge of the structure or function of MbnB, it isdifficult to interpret which of these conserved regionsare important. However, given that MbnB and MbnCare the only proteins with unassigned functions that arepreserved in both the Methylosinus trichosporium OB3band Methylocystis rosea SV97T operons, it is possiblethat one or both are responsible for the nitrogen-con-taining heterocycles and the neighboring thioamidesthat have been present in every Mbn structure obtainedthus far.


Page 6 of 17

The second unknown biosynthesis protein: MbnCMbnC is the second unknown Mbn biosynthesis protein,and as with MbnB, there is an existing, if limited, TIGR-FAM class (TIGR04061) [38]. We detected MbnC-likeproteins in 17 novel operons, a number that includes twofragmentary hits in a bioreactor metagenome (Figure 5A).As with MbnB, there is a broader class of distantly related

hits (with high Pseudomonas representation and a moredivergent C-terminal region), visible after a sharp declinein expectation value quality. This set of more distant rela-tives appears to correspond to the TIGRFAM familyTIGR04061.MbnC homologues are present in Groups I to IV Mbn

operons. For those operons, the phylogenetic tree

T. mobilis KA081020-065C. basilensis B-8

Azospirillum sp. B510Azospirillum sp. B506

P. extremaustralis 14-3 sub. 14-3bP. fluorescens NZI7

M. trichosporium OB3bM. parvus OBBP, ctg. 41

Methylosinus sp. LW4Methylosinus sp. LW2, v. 1

M. parvus OBBP, ctg. 3Methylosinus sp. LW2, v. 2

Methylocystis sp. SC2M. rosea SV97T

P. luminescens TT01V. caribbenthicus BAA-2122

Gluconacetobacter sp. SXCC-1G. oboediens 174bp2

10 20 30 40 50 60 70 80 90 100

MW G A A V R P L P I P D T I P D D M P D D G G A A L R I G F N F T L G A T A P M V R R L I A A G R I D Y V E L L V D N F L A V P P A Q I A A A F D A - P V G L H I M F S K F M E R P E A E L D E L A L R L T A I I- - - - - - - - - - - - - - - - - - - - - - - - - - M K I G F N F S L G T T Y P L V T Q L V R E R A I D F C E I L I D N F L Q V P A V E L A A A F D C - P V A F H I M F S K Y M E S D Q A A L E D L A A R L R E Y I- - - - - - - - - - - - - M V A A R S A E G E A V P M K I G F N F T L G E T L P L V R S L L A S G R I D Y V E L L I D N F L A V P E E E L A A G F D V - P V G F H I M F S K F I E A D E A A L D S L A S R L R S L I- - - - - - - - - - - - - - - - - - - - - - - - - - M K I G F N F T L G E T L P L V R S L L A S G R I D Y V E L L I D N F L A I P E E E L A A G F D V - P V G F H I M F S K F I E N D A V A L D S L A S R L R R L I- - - - - - - - - - - - - - - - - - - - - - - - - - M L I G F N Y T L G D T A P L V R K L L A G R H I D Y V E L L I D N F L A V P L E E L M D G F A A - P V G F H I M F S K F I E N D T P A L E R L A Y R L R E L I- - - - - - - - - - - - - - - - - - - - - - - - - - M R I G F N Y T L G D T A P L V R K L L A G G H I D Y V E L L I D N F L A V P I D E L E T G F A A - P V G F H I M F S K F I E N D T P A L E S L A R R L R E L I- - - - - - - - - - - - - - - - - - - - - - - - - - MQ I G F N F T L T G T L D M V Q Q M I K E R K I D Y V E M L I D N F V H L P P E Q I A D S F D C - P V A F H I M L S K Y L E R D R E E L E K L G K R L R R F I- - - - - - - - - - - - - - - - - - - - - - - - - - MQ I G F N F T L N G T L D M V R R M V R E R Q I D Y V E L L I D N F L H F P V E D L T D A F D C - P V A F H I M F S R H L E N D R E S L R K F A E R V R V F I- - - - - - - - - - - - - - - - - - - - - - - - - - MQ I G F N F T L N G T L D M V R R M L R E R Q I D Y C E L L I D N F L N V P V K E L A D A F E C - P V G F H I M L S K F L E N D R E T L E K L A K Q I R I L I- - - - - - - - - - - - - - - - - - - - - - - - - - - L I G F N F T L N A T L D M V Q R M I K A R Q I D Y C E L L I D N F L H V P P K E L E T T L D C - P I A F H I M F S K F L E S D D E V L G K L A K R I R I L I- - - - - - - - - - - - - - - - - - - - - - - - - - M R I G F N F T L T E T A D M V R R M L A E G H I D Y C E F L I D N F I G V P P A E L A R A F D C - P V G F H I M F S E F I D A D Q E T L E D F A K R I R V L I- - - - - - - - - - - - - - - - - - - - - - - - - - M R I G F N F T L T E T T E M V R R M I A E G H I D Y C E L L I D N F L C V P P A E L A R A F D C - P T G F H I M F S E F I D E D L D A L K D F A T R L R P F I- - - - - - - - - - - - - - - - - - - - - - - - - - M R L G F N F T L G D T Y D M V Q R L L A E G H I D Y C E F L I D N F F C V D P E E L S K A F D C - P I G L H I M Y S K F L E A D R P T L V D F A A R L R D Y I- - - - - - - - - - - - - - - - - - - - - - - - - - M R L G F N F T L G D T Y D M A Q R L I G E G H I D Y C E F L I D N F F C V D P D E L A K A F D C - P V G L H I M Y S K F L E A D R P T L V D F A A R L R D Y I- - - - - - - - - - - - - - - - - - - - - - - - - - M K I G T N W S G N R E L S V L K E I I S L R K V D F I E I L I D N F L Q C T P D S I L G I S K G L P I A F H I M N S K Y M H R S K Q D L E S I G K R I K L L Q- - - - - - - - - - - - - - - - - - - - - - - - - - M N V G I N W S G Q R E L P C I N Q L F L T R D I D F V E L L I D N F L T T D V D S I K A F L A G R P C A F H I M N S Q F L H K D E R E L L A M A K I I N K L I- - - - - - - - - - - - - - - - - - - - - - - M C N G S I G I N F T G G Q S Y S S I M S L I D E G H I D Y V E L L I D N F L H L N P T E I A K K F P C - P V A F H I MQ S R F L E R G E E E L K Y I S S R I T Q M S- - - - - - - - - - - - - - - - - - - - - - - M C N G S I G I N F T G G Q S Y S S I M S L I D E G H I D Y V E L L I D N F L H L N P T E I A K K F P C - P V A F H I MQ S R F L E R G E E E L K Y I S S R I T Q M S

110 120 130 140 150 160 170 180 190 200 210

R E T R P I Y V S D H I A R F T H Q G R H L L H L A E I D Y A G E - - - F E R V A D R V A R WQ D R L G C T I A F E N Y P S I M D G G R D - A P D F F R R L T A R T G C T V L F D V S N A V C A T H N C G L P F E AD I L R P L Y V S D H I A A F S H L G R H L Y H L A E V D Y A G A - - - Y R Q I R E R T L WWQ E R L G C R V H L E N Y P S I M D G G H D - A P A F F A R L T K E T G C G L L F D I S N A V C A Q R N C G L P L D AD R M N P L Y V S D H V A R F T H D G R Q L Y H L A E I D Y A A D - - - Y G R V R E R V D WWQ Q R L G R R L F L E N Y P S I L D G G H D - A P A F F E R L C R D T G C G V L F D A S N A V C A H L N C G V P L E AD R M S P L Y V S D H V A R F T H D G R Q L Y H L A E I D Y A A D - - - Y G R V R E R V D WWQ Q R L G R R L F L E N Y P S I L D G G H D - A P A F F E R L C R D T G C G V L F D A S N A V C A H L N C G V P L E AD R L R P L Y V S D H V A R F S H E G R Q L Y H L A E I D Y L N D - - - Y A R V R E R V D WWQ Q A L G C Q L L L E N Y P S I M D G G H D - A P A F H E R L Y R D T G A G V L F D A S N A V C A H L N C G V P L E AE R L R P L Y V S D H V A R F S H D G R Q L Y H L A E I D Y V G D - - - Y P Q V R E R V D WWQ Q Q L G C Q L L L E N Y P S I M D G G H D - A P A F H E R L Y R D T G A G V L F D A S N A V C A H L N C G V P L E AD V M R P V Y V S D H I L Y F T H N G R S L F H L G E I D Y - G E - - - Y D H V R S K V E Q WQ D M L G T R L Y L E N Y P S I M D G A W D - A P S F Y E R L S R E T G V G V L F D A S N A I C A Q N N T G A P V E LD A M K P L Y V S D H L L H F S H D G R R L F H L G E I D Y - A A - - - Y D S V R Q R V E Q WQ E M L G V R L Y L E N Y P S I M E G G W D - A P D F Y R R L S R E T G S G V L F D A S N A V V A M L N C G A P V D MD A L Q P A Y V S D H L L R F A P N G R N L F Y L G E I D Y - A Q - - - Y A P V R N R V E Q WQ D M L G V Q L H L E N Y P S I M D G G W E - Q P E F F R R L C R E T G A G I L F D A S N A V V A R H N C G A P L D AD A L N P I Y V S D H L L R F T H E G R C F Y N L G E I D Y E T Q - - - F D A V R R R V E K WQ D L L G R R L Y L E N F P S I M E G G W D - A P A F Y R R L S K E T G A G V L F D A S N A V C A K H N C G A P L D LR D MQ P L Y V S D H G A S F T H R G V N L F H L G E L D Y V K D - - - Y D R V R T R V D L WQ E M L G Q R L F I E N Y P S I L E G G W E - A P R F F E R L I A D T G A S V L F D A S N A V C A N L N C G A P L E AR D M K P L Y V S D H G T S F T H H G V N L F H L G E L D Y A R D - - - Y D R V R V R V D L WQ E M L G Q R I F I E N Y P S I M E G G W E - A P R F F E R L MQ D T G A G A L F D A S N S V C A N L N C G A P L E AD V L N P M Y V S D H I L R F T H E G R A F F H L G E I D Y S T E - - - Y E S V R D R V V A WQ E I V G Q R I H F E N Y P S I M D G G R E - A P A F F E R L M K E T G A G V L F D A S N A V C S H R N C G T P F E DD A L N P I Y V S D H I L R F S H E G R A F F H L G E I D Y S A E - - - Y E S V R D K V V Q WQ E M V G Q R I H F E N Y P S I M D G G L E - A P A F F E R L M K E T G A G V L F D A S N A V C A N R N C G T P F E AK E L N P I Y I S D H V G I F Y H N S Y P L P T M G E I D Y S S E - - - K D K Y F D S V S L WQ D I I G E K I Y L E N F P S I L D E N A K L Q P D F F K E M T K K C G N G L L F D I S N A V I A Q E N T G T P F E EH S L Q P I Y I S D H I G K F Y H R G Q A L P Q M L E V D Y G L Q - - - T H S T I K K V K A W S S L L D G K L L L E N Y P S I F P Q D M S - Q I D F F K R I L E E T Y C G L L F D I S N A F I A E V N I K Q S R T SE A F Q P L Y I S D H I A L F E H N G T N F Y Q L A E I N Y A I G S I Y F D K I R - - - - R WQ D M L G H T L Y L E N Y P S L L D G S Y E - A P T F F E H I S K K T E C N V L F D I S N A V C A S R N T G A D I D DE A F Q P L Y I S D H I A L F E H N G T N F Y Q L A E I N Y A I G S I Y F D K I R - - - - R WQ D M L G H T L Y L E N Y P S L L D G S Y E - A P T F F E H I S K K T E C N V L F D I S N A V C A S R N T G A D I D D

220 230 240 250 260 270 280 290 300 310

W T E V I R S A R Q F H V A G Y D H S V G Q P P I V L D T H D R A L A A D T R A A L A L C R A L V T D P D A T I T Y E R D D D L D E D R I I A D I D T L R A V M G R E P A H V - - - - - - - - - - - - - - - - - - -W S P L A A E A R H F H I A G F T G S I L S P R L A I D A H D T D L A P D T L A Y L E R I G P A L A L P G R T L T Y E R D G N I E Y E A I V R D L H R L R T A L A C P K P S H G H A A V N A G A S E D R N A N V T AW D G V I D A S R H F H T A G Y N L S I L Q P H L V L D T H D R A L S E A T M A F L E S R R G L F D K P G A T M T Y E R D D N F D E A D I A A D L D R L R A L F V R D P V A P A L E R A V P - - - - - - - - - - - -W D G I I D A A S H F H T A G Y N L S I L Q P H L V L D T H D R A L S D A T M A F L E S R R E R F D T P G A T M T Y E R D D N F D E L D I A S D L D R L R A L F A D D A V A P P V E R A A P - - - - - - - - - - - -W E G I I E L S S H F H T A G Y N L S I L Q P H L I L D T H D Q A L S D A T L D F L Q R Y R MQ F D T P H A T M T Y E R D D R F D E H E I V A D L Q R L R G V F M P P S Q A L Q S - - - - - - - - - - - - - - - - -W E G V I E H S S H F H T A G Y N L S I L Q P H L I L D T H D Q V L S E A T L G F L Q R Y R G L F D G P E A T L T Y E R D G R F D E A E I I G D L Q R L R G I F P S S P M E P R T - - - - - - - - - - - - - - - - -W K K I I E T T R H F H V A G Y G T A F I E P R V K A D T H D R E M A E D T L D F L S R M R T S F D K P G A T I T Y E R D F D I D Y E S I S V D L K R L R D I F P C - V E E E R H E P V A H C A G - - - - - - - - -W K D I V A D T K H F H V A G Y G P S F I E D D V I V D T H D R E M S T E T L D F L R R M R T D F D K P G A T I T Y E R D F E I D Y E S I S V D L R R L R E I F P R - T E E E A H G P L I A C A G - - - - - - - - -W N D V I S S T R H F H V A G Y G P S F L D P R V I V D S H D R E M A P D T L E F L R S M R A A F D K P G S T I T Y E R D F E I E Y D S I A I D L E R L R E I F P H - A E D T T N E S L V A C A G - - - - - - - - -W N D I V E T T Q H F H V A G Y A Q S F S A P H V I V D A H D R E M A P D T I E F L S S M R S S F D K P G A T I T Y E R D I N I D Y D S I A V D L Q R L R E I F P H - T Q E L E H A D L V A C A S - - - - - - - - -W D K I I A E T P H F H V A G Y S R A T T P P Y V I H D S H S E E L S E K T L D F L R G R R D L F D K P N A T M T Y E R D G N I D Y E S I V I D L K R L R D I F S T T S E D Q R H E S N L A C A H - - - - - - - - -W D K V I A S T N H F H V A G Y S R A V N P P H I V H D S H A E E L A E D T L A F L R G R R H I F D K P D A T I T Y E R D G N I E Y D S I I A D L R R L R E I F T S G T E E R Q D E R A I A C A N - - - - - - - - -W R N V I A E A K H F H V A G Y R H S L V E P F I S L D T H A E A L A P D T L A F L R D F R S V F D K P G A T M T Y E R D D R I E F D D V V A D L K L L R E L F G Q - P E E R R H D L A L S A - - - - - - - - - - -W R N V I A K A Q H F H V A G Y R Q S L I E P F I S L D T H A E A L A P D T L A F L Q N F R S V F D K S G A T M T Y E R D D Q I E F D D I V V D L K A L R D L F G Q - P E E T R H D L A L T A - - - - - - - - - - -W L D I - - E M N H L H I G G Y A E T S L R P S F L V D T H A D R I S N L S L K Y F N K L G T E S K D N L T S L S V E R D D N F V L G D W I N D I E L C R Q - - - - - - - - - - - - - - - - - - - - - - - - - - - -W F D L I K H C Q H F H I A G F E N A P - D N Q F L V D T H S Q C I E E P V L S F L Q E V N N A - - T S I A T I S V E R D E N F D V S D W A L D I D N V R N R V S N G R D T R - - - - - - - - - - - - - - - - - - -W K N V I S K T L H Y H V A G Y T P A P S D N N I L I D S H S E Q I S S E T E I F I D R Y A D L F F R E N T T I T Y E R D G N F D Y D L I V E D L N S L R L K S K N Q S V Y N E Q S - - - - - - - - - - - - - - - -W K N V I S K T L H Y H V A G Y T P A P S D N N I L I D S H S E Q I S S E T E I F I D R Y A D L F F R E N T T I T Y E R D G N F D Y D L I V E D L N S L R L K S K N Q S V Y N E Q S - - - - - - - - - - - - - - - -

58

35

13

100

M. rosea SV97T100

36

63

C. basilensis B-8

T. mobilisKA081020-065 Azospirillum

sp. B510

Azospirillumsp. B506

95

100

99

68

43

98

100

100

P. extremaustralis14-3, substr. 14-3b

P. fluorescens NZI7

Gluoconacetobactersp. SXCC-1G. oboediens 174bp2

V. caribbenthicusBAA-2122

P. luminescenssub. laumondii TT01

M. parvus OBBP, ctg. 3

Methylosinussp. LW3, v.2

Methylocystissp. SC2


Methylosinussp. LW4

M. parvus OBBP, ctg. 41M. trichosporium OB3b

B

A



















III

III IV

V

Figure 4 MbnB: the core Mbn biosynthesis gene. (A) MUSCLE alignment of MbnB, including both domains. Residues are colored followingthe Taylor color scheme [109], with intensity modified by a conservation color increment of 30%. Truncated metagenomic sequences are notshown in this alignment. (B) Phylogenetic tree (constructed in MEGA 5.0 as described in the Methods section) based on a MUSCLE alignment ofall MbnB sequences, not including truncated metagenomic sequences. Gamma distribution parameter 1.5823.


Page 7 of 17

constructed for MbnC resembles that for MbnB andMbnA, supporting the proposed classification scheme(Figure 5B). In families with a true MbnC homologue, thepredicted MbnC ORF frequently overlaps MbnB by a sig-nificant number of residues, but it is not in frame withMbnB. As with MbnB, multiple alignment of MbnChomologues confirms the broad conservation of severalregions of the gene, but the relationship between the con-served regions and MbnC’s potential role in biosynthesisof thioamide and nitrogen-containing heterocyclesremains unclear.

The Group V operons, which appear to be the mostdistantly related to the Methylosinus trichosporiumOB3b operon, diverge with MbnC. There do not appearto be clear Group V homologues for MbnC as there arefor MbnB. There is, however, an unidentified ORFimmediately neighboring the precursor, conserved pri-marily in these two species. This ORF could possiblyencode a core biosynthetic protein for the Group Voperons (Additional file 1, Figure S2). These sequencesappear to have no close homologues in other species,and have a weak N-terminal similarity to the DUF692-





P. extremaustralis sp. 14-3 substr. 14-3bP. fluorescens NZI7


C. basilensis B-8T. mobilis KA081020-065


10 20 30 40 50 60 70 80 90 100

- - - - - - - - - - M S L L P T A P V R I D A D L Y D D L A N P A R Q S L Y P R D S R G F I R I D I S L R A Y W H T L F D T C P R L L E L S G - - - - - - - - - - - - - - - P S G G A I F L P F M A W A R E N N L A- - - - - - - - - - M G R L S P A P V R I D A D L L E D F A D P A R Q D A Y P R D S R G Y V R V D I S I R A Y W H T L F D Q V P Q L L D L C G - - - - - - - - - - - - - - - P D G R T I F L P F MQ W A K E K K V S- - - - - - - - - - M S L L S P A P A R I D A D L Y Q D MM N P S R L D A F A R E S R S H V R L D I S Y R A Y W H V L F D A V P Q L L E L S G - - - - - - - - - - - - - - - P D G R A I F L P F MQ W A Q E K N L T- - - - - - - - - - M P I L S P A P A R I D A D L Y A D L I D P A R L A T Y P R E S R S Y I R I D I S L R A Y W H T L F D T C P L L L D L S G - - - - - - - - - - - - - - - P D G A S I F H P F M A W A R Q E K L S- - - - - - - - - - - - M S Q T S L A R T D P E L Y D V L T S P K L I L N Y P P E S R A Y A R I D V S L R A Y W H S T F D I C P E L L E L S G - - - - - - - - - - - - - - - P D G M S I F S P F M E W A R E R G V R- - - - - - - - - - - - M N E P S L A R T D R E L F D V L T S P S R I L D Y P P N S R A Y G R I D V S L R A Y W H S T F D I C P E L L E L S G - - - - - - - - - - - - - - - P D G M T I F A P F M E W A R E Q G I R- - - - - - - - - - - M I L L S L P S R T D R D L Y P A L M S P E L N A K Y P T D H K A F V R I D V S L R M Y W H T L F D I C P E L L E L S G - - - - - - - - - - - - - - - L D G M S I F R P F M A W A G E K K L A- - - - - - - - - - - M T L L S L P S R T D R D L Y P A L M S P Q L N A K Y P A D H K A F V R I D V S L R I Y W H T L F D I C P E L L E L S G - - - - - - - - - - - - - - - P D G M S I F R P F M A W A E E K K L A- - - - - - - - - - M S V R G L S P - R T D A D L L D A L S V P E R Q A D Y P R D S R A F V R I D S S L R V Y W H T L F D I C P G L L A L S G - - - - - - - - - - - - - - - P D G L A I F R P F M G W A A E Q R L S- - - - - - - - - - M K T E H P L P - R T D S Q L L H A L S V P Q R Q G D Y P R D S R A F V R I D S S L R V Y W H T L F D I C P G L L Q L T G - - - - - - - - - - - - - - - P D G L A I F R P F M S W A A Q Q R L S- - - - - - M T M T A M T P G W A K R L V D A D L L G A L T V P A R Q G D Y P R A S R A F V R I D T S L R I Y W H T L F D I C P G L L D L S G - - - - - - - - - - - - - - - P D G L S I F R P F M A W A A D E R L S- - - - - - - - V T V M T Q G R A E R L I D A N L L E A L T V P A R Q G D Y P R E S R A F V R I D T S L R I Y W H T L F D I C P G L L E L S S - - - - - - - - - - - - - - - P D G L S I F R P F M A W A A D E G L SM A M P P S MQ A P V K T A M P M S P L N D T D L L E A L A T P D L Q K R Y P R D S K A F V R I D T S L R A Y W H T L F D V C P A L L D L A G - - - - - - - - - - - - - - - P D G L A I F R P F M A W A A S R R L S- - - - - - - - - - - - - - - M S E R R I D S D L L D H L T V P A R H D E L G V G S R A W V R V D T S L R I Y W H T L F D I C P G L L R L D G E D G R P S D G Q P S D G Q P S D G M S I F R P F M A F A A E R G L S- - - - - - - - - - - - M N N H R K Y L R D I D V I D F M S R P D Q F S L F S D N R R A L V R L D I A W R G Y W H A L Y D P C P S L L D F S G D - - - - - - - - - - - - - - Q D G F N I F R P F M R W G S E R K I C- - - - - - - - - - - - M N N H R K Y L R D I D V I D F M S R P D Q F S L F S D N R R A L V R L D I A W R G Y W H A L Y D P C P S L L D F S G D - - - - - - - - - - - - - - Q D G F N I F R P F M R W G S E R K I C

110 120 130 140 150 160 170 180 190 200 210

F D W S F F L W V Y V W L Q Q S E F R E R L - - D E D Q L L P - V M T A S A T R W L M I D R D I D A C Q I V L G S R S L A - - - - G A - A V V G A K I D S I H C R L E Q V Q Q V E F E K P L P L P D G E F G Y F L TF N W A F Y L W V Y E W L L Q S E F R D R L - - D T D L L L K - MM S A A G G R W I G T D R D T D H C V I I M G C R L L E - - - - G S - V V V G W K L N S L A T T V I P V E R I E F E K P L P S P S G L F G C F Y TF D W A Y H I W A A E W L L Q S E F R E R V - - G A D L A L K - L M A A A A S R W S G R D R D L D Y C G I V I A S R L I G - - - - P T K A V A G W K L S S I E T R F I E V E Q V E F E C E L P A P E D N F G C F R TF D W T Y F L W V Y Q W L A Q S E F R D R L - - S E E L L L S - MM T A S A A R W L G Y D R N V D D L G V V I G T P L I D - - - - G - - V V V G W K M D S V Y C G L N Q V E R L E F E E P L P A P D G L F G Y F F TF T W S Y Y L W L Y V W L R Q S A F A D R L - - T K D L L Q S - L I G A S A A R W A V R D R S - A A M G L V I G C K E V P - - - - N - - L V V G W K C R S L D A G - R Q V E M I E L E E P L P L G D E F F G Y F T VF T W S Y Y L W L Y R W L R Q S V F R D R L - - G D E L L I S - L M G A S A A R W A I R D R G - A A H G L A I G C A A T P - - - - T - - F V V G W K C R S L S A G - R Q V E L V E L D Q P I A V G D A F F G F F T TF D W S Y Y L W V Y A W L R Q S P F K E R L L A D D K Y L R S - V M A A S A A R W A I L D R G - R D C G I V I G C A D T T - - - - N - - L V I G W K C R S V R L G - R E I E L I E P E E P L P A P P D I F G Y F T LF D W S Y Y L W V Y V W L R Q S P F K E R L L A D D K Y L R S - I M A A S A A R W A I L D R G - R S R G I V I G C A D T T - - - - D - - L V I G W K C R N V R L G - R E I E L I E P E E P L P A P P D L F G Y F T LL N W T Y Y L W V D V W L Q Q S Q F R Q Q L - - T P A L R Y S - L M G A S V A R W A T G D R S - E S N G I I V S A A D L P - - - - D - - L V C A W K T Q S L E G A - R E V E Q I E L S E P L P S P A A A F G F F T VL N W T Y Y L W L D V W L Q Q S S F A R H V - - T P A L R Y Q - L M G A S A A R W A T G D R S - E A N G I F L A A A D L P - - - - D - - L V C G W K T Q T L E G A - R D I E Q I E L A E P L P A P G A A F G Y F T VL N W T Y Y L W V D V W L R R S P F A G R V - - T P E L R L S - L I G A S A A R W A T G D R L - E A G G I V L S G L D A A - - - - D - - L V C G W K A R S I D A G - R Q V E R I G L E D P L P T A P E P F G F F T VL N W T Y Y L W V D V W L R R S P F A G R V - - T P E L R L S - L I G A S A A R W A T G D R S - E A G G I V L S G L D A A - - - - D - - L V C G W K A R S I D A G - R Q I E R I E L E D P L P A A P E P F G F F T VM N W A F Y I W A Y R W L D G S D F A D S L - - P D S L A E S - M L A A S A A R W A T V D R G - R A A G I V L G D A K S S - - - - G - - L I A G W K C R S V D G A - R S V E R I E L E A P L A P P C D R F G F F L VF D W T Y Y L W V D V W L Q G S G F R D R V - - D D D L R L T - L I G A A A A R W A V T D R S - P A V G L V I G T R S A D R G A L P - - L V V G W K P A D L E S G - R E I E T L E L E D P L P D P A T P F G W F T TF G W P L H L W V I I W L Q E N E M L N S S - - N S E I I L S E L A G A A A A R W A I S D R T - E A L G I A I Y R G I K Q - - - - N - - W T V G W K C R A V D L Q - R E I Q E I E I G A D L S P E D D I P S Y F L IF GWP L H L WV I I W L Q E N E M L N S S - - N S E I I L S E L A G A A A A R W A I S D R T - E A L G I A I Y R G I K Q - - - - N - - W T V G W K C R A V D L Q - R E I Q E I E I G A D L S P E D D I P S Y F L I

220

P G F E I D H - F P G W R P L P RP T F E L D H - F P G WQ P I P RP G Y E L D H - F P G W R P I P LP S L R L G H - F P G W R P I P RA A P E I T T - F P G W R S L P LP G S E I A E - F P G W R S L P LQ S F E L D I - F P G WQ S L A RP S F E L D I - F P G WQ S L A RA S R N L E E G F P G W T D V P RH S R H L Q D G F P G W T D V P RP G G E L D G G F P G W T P I P RP G G E L D G G F P G W T P I P RP G F A L E T - F P G W S P I P LT S R S L D H - F P G W R D L P LF D N K I S V - F P G W S R I L -F D N K I S V - F P G W S R I L -

95

4099

100

100

59

35

9998

5654

42100

M. rosea SV97T

C. basilensis B-8

T. mobilisKA081020-065


P. extremaustralis14-3, substr. 14-3b

P. fluorescens NZI7

Gluoconacetobactersp. SXCC-1

G. oboediens 174bp2





Methylosinus sp. LW4


M. trichosporium OB3b

B

A

















I

II

III

IV

Figure 5 MbnC: the secondary Mbn biosynthesis gene. (A) MUSCLE alignment of MbnC putative biosynthesis proteins. The Group Vsequences are divergent and not included. Residues are colored following the Taylor color scheme [110], with intensity modified by aconservation color increment of 30%. Truncated metagenomic sequences are not shown in this alignment. (B) Phylogenetic tree (constructed inMEGA 5.0 as described in the Methods section) based on a MUSCLE alignment of all MbnC sequences, not including truncated metagenomicsequences. Gamma distribution parameter 2.1637.


Page 8 of 17

like domain (PF05114), which is more like MbnB thanMbnC.

Other biosynthesis proteins: MbnN, MbnS and MbnFThe Mbns from Methylosinus and Methylocystis speciesexhibit post-translational modifications beyond the forma-tion of nitrogen-containing heterocycles and neighboringthioamides. Mbn biosynthesis in Methylosinus trichospor-ium OB3b requires a transamination reaction on theN-terminal amine group of the core peptide followingleader peptide removal, as well as the formation of a disul-fide bond, and all four Methylocystis Mbns contain a sulfo-nated threonine group [22,24]. Although specific proteasesand disulfide-forming proteins are not evident, we havediscovered proteins likely responsible for transaminationand threonine sulfonation in the Mbn biosynthesis oper-ons of several genomes. Transaminases are present inthree operons only: Methylosinus trichosporium OB3b(annotated as “histidinol phosphate transaminase/cobyricacid decarboxylase” and with a PFAM classification ofPF00155 or Class I/II aminotransferase), Methylosinus sp.LW4 (also PF00155 or class I/II aminotransferase), andGluconacetobacter sp. SXCC-1 (classified as PF00202 orClass III aminotransferase) (Figure 2). The transaminasehas tentatively been designated MbnN. The paucity oftransaminases in Mbn operons suggests that the N-term-inal transamination present in Methylosinus trichosporiumOB3b Mbn may not be a common modification.Like the N-terminal transamination, threonine sulfo-

nation may only be present in a subset of Mbns. Todate, it has only been observed in the four structures ofMbns produced by Methylocystis species [23,24]. Sulfo-transferases with domains corresponding to Pfam familyPF00685 were detected only in the two Group II Methy-locystis operons. Although no structure for Mbn fromMethylocystis strain SC2 is available, the similarity of itsMbnA to that of Methylocystis rosea SV97T combinedwith the presence of a sulfotransferase in its operonstrongly suggests that its Mbn will also be sulfonated,presumably at the same threonine. This sulfotransferasehas been designated MbnS.Finally, the gene encoding MbnF, generally annotated as

a flavin adenine dinucleotide (FAD)-dependent monooxy-genase or an FAD-dependent oxidoreductase (PfamPF01494), is also present in six Group I and II operons(including all known Methylocystis genomes and someMethylosinus genomes), always following MbnM(Figure 2). The function of MbnF is unclear, but given itspresence in the Methylocystis rosea SV97T operon andabsence in the Methylosinus trichosporium OB3b operon,it could play a role in pyrazinedione biosynthesis (Figure 1),possibly hydroxylating the heterocycle. Without structuresof Mbn-like products from non-methanotrophs, it is diffi-cult to connect other neighboring genes (annotated or

not) to potential biosynthetic modifications and todetermine the effective ending point of the operon andpotentially the end of any multicistronic mRNA tran-scripts. In both Methylocystis species, MbnS is followed bya gene resembling MoaA, a protein responsible for thefirst step in molybdenum cofactor biosynthesis [45] (whichinvolves the conversion of a guanosine derivative to pre-cursor Z) and a gene generally annotated as a 3-hydroxyi-sobutyrate dehydrogenase. Hypothetical unknownproteins (including the MbnC replacement in Group Voperons) are present in several operons, and a range ofproteins of unknown relevance, including several varietiesof known copper-related proteins, appear in a few operonsonly (Figure 2).

Exporting methanobactin via MbnMA proton/sodium-dependent multidrug export pump(MATE), belonging to the PFAM class PF01554, isfound in 13 of the identified operons (Figure 2). Of theremaining operons, several are on small contigs in morefragmented draft genomes making it difficult to rule outthe presence of a similar exporter. Excluding Vibriocaribbenthicus and Photorhabdus luminescens, whichappear to have dissimilar MATE transporters, perhapsreflecting a less similar final Mbn-like product, thisexporter is well-conserved, even in the non-methano-trophs Pseudomonas fluorescens NZI7 and Azospirillumsp. B510 and B506 (Additional file 1, Figure S3.) Inprokaryotes, MATE transporters primarily function asexporters of antibiotics and similar toxic compounds,simultaneously importing Na+ or H+ and exportingmostly cationic natural products [46-48]. Native naturalproducts are primarily exported by non-MATE effluxpumps, such as the resistance-nodulation-cell division(RND) or major facilitator superfamily (MFS) exportersthat are believed to transport some siderophores out ofthe cell [49-51]. However, many MATE transporters donot have known substrates, and MATE transporters areeven found in antibiotic hypersensitive strains [52].Thus, the ability of a MATE transporter to secreteMbn-like compounds is plausible, if unprecedented.

Importing copper-loaded methanobactin via MbnTA family of small molecule importers, known as TonB-dependent transporters (TBDTs), are also commonlyassociated with the Mbn biosynthesis operons. The onlygenomes for which nearby TBDTs are not observed areVibrio caribbenthicus and Photorhabdus luminescens, aswell as the second Mbn operon in Methylosinus sp. LW3,which is small and surrounded by transposon elements;contig truncation of several other operons may be hidingadditional potential transporters in other species. Wehave shown previously that CuMbn is imported via anactive process [17,21] and TBDTs are good candidates


Page 9 of 17

for importers since they play a similar role for sidero-phores [53-56]. TBDTs found in the vicinity of Mbnoperons are generally annotated as siderophore receptorsand classifiable under models including TIGR01783 (fullsiderophore-specific TBDT model), PF00593 (TBDTbarrel only), PF07715 (TBDT plug domain only) and insome cases PF07660 (an extended N-terminal region,which appears to approximate the published N-terminalextension (NExT) domain [57]); they have provisionallyhave been designated the MbnT family. Conservation ofthese TBDTs is weaker than that of MbnB, MbnC orMbnM; even the plug domain displays less homology(Additional file 1 Figure S4A, B). However, differences inthe core peptide backbone sequence may require mark-edly different binding approaches. While methanotrophMbn-related genes are generally relatively similar, theplug domain sequences of Methylocystis Group II TBDTsand Methylosinus Group I TBDTs diverge markedly,perhaps reflecting the structural differences of the finalcompounds (Additional file 1, Figure S4A, B.)

MbnT may have a FecIRA-like regulation system inMethylosinus speciesIn four operons from Methylosinus species, the TBDT hasan extra N-terminal domain (Additional file 1,Figure S4C.) These larger TBDTs are preceded by an ORFgenerally annotated as an “Fe(III) dicitrate membrane sen-sor” (PFAM PF04773) and an “ECF sigma factor” (withconserved s-70-like regions 2 (PFAM PF04542) and 4(PFAM PF08281)), designated MbnR and MbnI, respec-tively (Figure 2). This pairing is generally observed forFecIRA-like systems, in which the holo siderophore-boundTBDT interacts with the membrane sensor, which theninteracts with the ECF sigma factor to regulate expressionof siderophore biosynthesis and transport proteins [57-60].The earliest example of this system is the eponymousFecIRA system, which controls the transcription of ironcitrate transporters [57,59,61,62]. Similar systems exist forsiderophores, such as pseudobactins BN7 and BN8 (thePupBRI system) [63], pyoverdines (FpvARI/PvdS) [64] anda range of other siderophores. Not all of these systemshave identical regulatory pathways. The pyoverdine trans-port system has two ECF sigma factors (FpvI and PvdS)which regulate different operons [64], and the HasISR sys-tem, which transports heme, has an unusual regulatoryscheme in which the membrane-bound sigma factor HasSinhibits the activity of the ECF sigma factor HasI untilheme binding to the TBDT HasR [65].Strikingly, only the four Methylosinus MbnT TBDTs

have the N-terminal extensions necessary for FecIRAsignaling [57], suggesting a possible regulatory mechan-ism for Mbn production and transport in Group I oper-ons (Figure 6). In this model, when CuMbn binds toMbnT, a periplasmic TonB-mediated interaction with

MbnR results in an altered cytoplasm-side interactionwith MbnI. The MbnI ECF sigma factor may then inter-act with RNA polymerase to either upregulate or inhibitMbn biosynthesis and transport and may also regulateother operons that are highly expressed at low copper,such as the sMMO operon. If MbnIRT is a positive reg-ulation system, a negative regulator that binds copperand represses Mbn biosynthesis and transport, amongother systems, may also be present.The TBDTs in other operons beyond the Methylosinus

(Group I) species lack N-terminal extension domainsand are not adjacent to FecIR homologues. Although aFecIRA-like system could still be present in these spe-cies in a distant small operon, it is less likely. It may bethat models analogous to different siderophore regula-tory systems are more relevant to these Mbn operons.For example, iron-loaded pyochelin is taken up into thecell and binds to the transcription factor PchR, whichregulates its biosynthesis and transport [66-69]. If sucha system exists for chalkophores (Additional file 1,Figure S5), the regulators do not appear to be consis-tently encoded near the biosynthesis operon. However,genes encoding periplasmic binding proteins, commonlyassociated with natural product import via adenosinetriphosphate (ATP)-binding cassette (ABC) transporters,are located downstream of TBDTs in both completeMethylocystis and Azospirillum operons, and could berelevant to the need for cytoplasmic uptake in a PchR-like model (Figure 2).

MbnP and MbnH: mysterious partnersThe genes encoding MbnP and MbnH are conserved asa pair far beyond the group of Mbn producers analyzedhere and are defined by an existing set of TIGRFAMHMMs (TIGR04039 and TIGR04052) and an associatedgenome property (GenProp0940). The pair consists ofthe di-heme cytochrome c peroxidase MbnH, frequentlyannotated as resembling MauG, and its neighboringpartner protein, MbnP. In two non-Group V genomes(Methylosinus sp. LW3 and LW4), there are cases wherethis pair is not immediately proximal to an Mbn operon,but is present elsewhere in the genome. Methylosinustrichosporium OB3b has two such additional pairs.Interestingly, these isolated pairs are located nearMbnT-like TBDTs that also have adjacent MbnI andMbnR homologues.A somewhat similar pair of proteins are found in

some methanotroph species that lack Mbn operons. InMethylococcus capsulatus (Bath), the proteins are calledSACCP (the di-heme cytochrome c peroxidase) andMopE (the partner protein). MopE is known to be thesubject of a post-translational modification (possibly bySACCP, which is similar to MauG [70]) in which a tryp-tophan converted to kynurenine participates in a copper


Page 10 of 17

binding site [71]. Additionally, while the intact MopEprotein is surface-associated, a C-terminal region is fullysecreted [72]. In Methylomicrobium album BG8, theseproteins are called CorB (the di-heme cytochrome c per-oxidase) and CorA (the partner protein) [73,74]. Thegenes encoding these proteins are downregulated in thepresence of copper [75-77]. However, although there areseveral well-conserved tryptophans in the MbnP pro-teins, the sequence is not markedly similar to MopE orCorA (Additional file 1, Figure S6), and there are nodata linking any close MbnP homologues or their di-heme cytochrome c peroxidase partners to copper. Therelevance of this gene pair to Mbn biosynthesis, regula-tion or transport thus remains unclear.

Overall structure of the Mbn operonThe core of the Mbn operon (Figure 2) is the MbnBbiosynthesis gene, located directly downstream of MbnAin all operons except for the two Group V operons,which have an unknown gene between MbnA andMbnB. MbnC encodes a secondary core protein, present

immediately downstream of MbnB in all operons exceptGroup V operons. All components beyond that core aremore flexible. When present, MbnM follows the corebiosynthesis peptides. Other biosynthesis-related genes,such as MbnN and MbnS follow MbnM. In some cases,the MbnP/MbnH pair appears after the biosynthesisproteins. In others, it is present before them on thesame strand, or before them but on the complementarystrand. MbnT, downstream of MbnI/R in Group I oper-ons, primarily occurs prior to the biosynthesis clusteron the same strand and frequently neighbors the MbnP/MbnH pair as well.In many of the operons, factors related to genetic mobi-

lity, such as insertion sequences, transposases, integrases,insertion sites, shufflons and conjugation-related proteins,occur on one or both sides of the Mbn operon or withinseveral kilobases (Figure 2). These elements may suggestan explanation for the seemingly unrelated assortment ofspecies in which these operons have been detected, andfor the lack of operon detection in several well-studiedmethanotroph species, including Methylocystis str.

Figure 6 Proposed Mbn signal transduction pathway featuring the MbnIRT triad. Mbn is secreted from the cell via the MATE multi-drugexporter MbnM and an unknown outer membrane partner. CuMbn is readmitted to the cell via the TonB-dependent transducer MbnT. CuMbnbinding to MbnT induces a conformational change that results in contact with both the inner-membrane TonB-ExbD-ExbB complex and MbnRvia the unique N-terminal extension of MbnT. CuMbn may or may not enter the cytoplasm intact, but either way, MbnR activates MbnIanalogous to a standard FecIRA system. MbnI replaces s70 in the active RNA polymerase complex, activating transcription of Mbn biosynthesisand transport genes, and potentially other operons needed in low copper conditions. In siderophore systems, the negative regulator Fur bindsiron as intracellular iron levels rise, and the holo Fur binds to siderophore biosythesis and transport promoter regions, inhibiting transcription. Asimilar negative regulator might be needed to trigger the copper switch to pMMO production.


Page 11 of 17

Rockwell [78]. Siderophores are sometimes transportedbetween species on virulence or fitness cassettes [79].Similarly, it may be that chalkophores are transported inthis fashion and adapted by species that have a specialneed for copper-binding compounds.

ConclusionsWe have detected a total of 18 novel Mbn-like precursorslocated in full or partial biosynthesis/transport operons in16 species or metagenomic samples. Of the methanotrophspecies, operons are present in both strains that undergothe copper switch from sMMO to pMMO (for example,Methylosinus trichosporium OB3b [28], Methylocystis str.M [80,81], Methylocystis hirsuta CSC-1 [82]) and thosethat only express pMMO (for example, Methylocystisparvus OBBP [40], Methylocystis rosea SV97T [83]). The16 species are not limited to methanotrophic bacteria,providing compelling evidence that Mbn-like compoundsmay play a broader role in proteobacterial metal homeos-tasis. This analysis reveals the precursor peptide forMethylocystis rosea SV97T Mbn [24] and identifies in thesame operon genes encoding enzymes that would benecessary to produce the novel features of this Mbn,specifically the sulfonated threonine. Moreover, these dataallow us to predict that the Mbn produced by Methylocys-tis strain SC2 will be very similar to that of Methylocystisrosea SV97T and likely identical to that of Methylocystishirsuta CSC-1. Conversely, we can predict that the Mbnoperons of Methylocystis str. SB2, Methylocystis str. M andMethylocystis hirsuta CSC-1 will have the same corecomponents as the two Methylocystis operons presentedhere. Taken together, these findings provide strong newsupport for a post-translational modification biosyntheticpathway.Beyond the four Methylocystis Mbns, the only other

structurally characterized Mbn is the original compoundfrom Methylosinus trichosporium OB3b, which has aGroup I Mbn operon. As the related natural productsfrom Group I, III, IV and V familes are characterized,the extent of structural diversity in the Mbn familyshould become more clear. The roles of MbnB andMbnC as well as the less universal MbnN, MbnS andMbnF proteins in biosynthesis are unknown or uncon-firmed and need to be investigated biochemically. Thisis particularly important since Mbns contain uncommonpost-translational modifications, such as thioamidegroups, a modification rare enough that Mbns havedoubled the number of compounds known to contain it[84]. In addition, there are no other examples of RiPPscontaining pyrazinediones [85,86], and even oxazolonerings are uncommon, with oxazoles and thiazoles consti-tuting the more common products of serine, threonineand cysteine cyclization. The combination of thesemotifs with the possibility of more unknown post-

translational modifications in Mbns from Groups I andIII to V suggests that novel biochemical mechanismsmay be involved in Mbn biosynthesis.The two identified Group V operons may represent a

different natural product subfamily, albeit one that sharessome similar biosynthesis proteins and modificationswith the main Mbn family. Notably, their MbnAsequences contain only a single modifiable cysteine,suggesting that if the final products bind copper at all,they do not use the paired heterocycle/thioamide coordi-nation scheme. Instead of MbnC homologues, theseoperons include a third unidentified putative proteinwhich neighbors MbnA, and Vibrio caribbenthicus alsohas a second unknown protein following MbnB. Bothhave nearby exporters, but no TBDT-like importers.The identification of MbnM and MbnT as common

members of the Mbn operon provides candidate transpor-ters for both Mbn import and export. The possible invol-vement of MATE-type exporters is somewhat surprising,but the ability of TBDTs to import metal-loaded sidero-phores is well documented, and the association of suchtransporters with Mbn operons supports experimentalwork showing that Mbn uptake is an active process[21,53-55]. Furthermore, in the case of Group I operons,the N-terminal transduction element in MbnT combinedwith the presence of MbnI and MbnR is consistent withFecIRA-style regulation. This model, along with ahypothetical pyochelin-like route for non-Group I oper-ons, provides testable mechanisms for CuMbn involve-ment in methanotrophic copper regulation, and may helpunravel the mystery of the copper switch.A final point of interest lies in what was not found in

this analysis. There are a variety of methanotroph gen-omes, including but not limited to Methylococcus capsula-tus (Bath) [87], Methylocella sylvesteris BL2 [88],Methylocystis str. Rockwell (ATCC 49242) [78] andMethylomicrobium album BG8, in which we detect noMbn biosynthesis/transport operons. Based on theirgenomes, if these species produce a chalkophore as sug-gested [89], it is not similar to existing structurally charac-terized Mbns and its biosynthetic enzymes do not closelyresemble MbnB and MbnC. While one of these speciesonly produces sMMO, the rest produce pMMO andsome, including Methylocystis str. Rockwell, produce onlypMMO. If these methanotrophs do not produce their ownchalkophores, they might scavenge chalkophores fromother species, similar to what is observed for siderophores[90], and may still possess Mbn-transporting TBDTs.Alternatively, these strains may have other, yet to be uni-dentified, mechanisms of copper uptake. Taken together,these data provide new insight into Mbn and Mbn-likecompounds and their biosynthesis, provide new tools forinvestigating these processes, and have implications forthe broader question of bacterial heavy metal homeostasis.


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MethodsGenomesThe contigs, scaffolds or complete genomes containingMbn operons discussed in this paper include: Azospirillumsp. B506 (GenBank: BADK01001132), Azospirillum sp.B510 (GenBank: NC_013854) [91], Bioreactor metagenomePBDCA2 (GenBank: AGTN01410593, AGTN01295401and AGTN01527378), Cupriavidus basilensis B-8 (Gen-Bank: AKXR01001597), Gluconacetobacter sp. SXCC-1(GenBank: NZ_AFCH01000034) [92], Gluconacetobacteroboediens 174bp2 (GenBank: NZ_CADT01000094), Marinemetagenome (GenBank: JCVI_SCAF_1096627660232),Methylocystis sp. SC2 (GenBank: NC_018485) [43], Methy-lobacterium sp. B34 (GenBank: BADE01000957), Methylo-cystis parvus OBBP (GenBank: AJTV01000003 andAJTV01000041) [40], Methylocystis rosea SV97T (IMG:A3OODRAFT_scaffold1.1), Methylosinus sp. LW3 (IMG:MetLW3DRAFT_contig2.2), Methylosinus sp. LW4 (IMG:MetLW4DRAFT_scaffold2.2), Methylosinus trichosporiumOB3b (IMG: MettrDRAFT_Contig106; GenBank version isoutdated) [28], Photorhabdus luminescens subsp. laumondiiTT01 (GenBank: BX571860) [93], Pseudomonas extremaus-tralis 14-3 substr. 14-3b (GenBank: NZ_AHIP01000040)[94], Pseudomonas fluorescens NZI7 (GenBank:AJXF01000037) [95], Tistrella mobilis KA081020-065(GenBank: NC_017956) [96] and Vibrio caribbenthicusATCC BAA-2122 (GenBank: NZ_AEIU01000072).

Gene cluster identification and classificationGene sequences from Methylosinus trichosporium OB3band Azospirillum sp. B510 were used as seeds for searchesagainst the NCBI WGS and NR databases [97] (NationalCenter for Biotechnology Information, Bethseda,Maryland, USA), as well as the IMG database [98] (DOEJoint Genome Institute, Walnut Creek, California, USA),using the tBLASTn [99] (National Center for Biotechnol-ogy Information, Bethseda, Maryland, USA) algorithm toidentify genes even in unannotated regions. Hits of E <1E-20 were manually examined for the presence of otherrelated genes and potential precursors. In genes of interest,annotation was confirmed or potential roles for unanno-tated genes were identified via BLAST (National Centerfor Biotechnology Information, Bethseda, Maryland, USA)and via the Pfam (European Bioinformatics Institute, Hinx-ton, England, UK) and TIGRFAM (J. Craig Venter Insti-tute, Rockville, Maryland, USA) databases [38,100]. In thecases of Cupriavidus basilensis B-8, Methylocystis parvusOBBP, Pseudomonas fluorescens NZI7, Azospirillum sp.B506 and Methylobacterium sp. B34, the relevant contigswere manually examined for ORFs. ORFs of interest wereprovisionally identified using BLAST and PFAM. The IGSAnnotation Engine (http://ae.igs.umaryland.edu/cgi/index.cgi) [101] (Institute for Genome Sciences, University ofMaryland, Baltimore, Maryland, USA) was used for

structural and functional annotation of the first three ofthese sequences (Additional files 3, 4, 5, 6). Manatee wasused to view annotations (http://manatee.sourceforge.net/).

Alignment and phylogenyInitial multiple sequence alignments were generated usingClustalOmega [102] (University College Dublin, Dublin,Ireland), MAFFT [103] (Computational Biology ResearchCenter, Tokyo, Japan) or MUSCLE [104] (Drive5, Tiburon,California, USA), applying the default settings. Alignmentswere visualized using the Jalview 2 (University of Dundee,Dundee, Scotland, UK) package and were organizedaccording to a simple tree based on the Neighbor-Joinalgorithm using the BLOSUM62 model. Phylogenetic andevolutionary analyses were conducted using the MEGA5package [105] (Center for Evolutionary Medicine andInformatics, Arizona State University, Tempe, Arizona,USA). During phylogentic tree construction, the evolu-tionary history was inferred by using the Maximum Likeli-hood method based on the JTT matrix-based model [106].The bootstrap consensus tree inferred from 1,000replicates [107] was taken to represent the evolutionaryhistory of the taxa analyzed [106]. Branches correspondingto partitions reproduced in less than 50% bootstrap repli-cates were collapsed. Initial tree(s) for the heuristic searchwere obtained automatically by applying Neighbor-Joinand BioNJ algorithms to a matrix of pairwise distancesestimated using a JTT model, and then selecting thetopology with superior log likelihood value. A discreteGamma distribution was used to model evolutionary ratedifferences among sites (five categories (+G, parametervaried by alignment)).

Precursor gene identificationThe Methylosinus trichosporium OB3b and Azospirillumsp. B510 MbnB sequences were used as described aboveto identify potential Mbn biosynthesis operons. Whensuch a protein was identified, small ORFs (10 to 50 aa) ina 2 kb region up- and downstream of the gene wereanalyzed for the presence of cysteines in the last 10 resi-dues, a lysine or arginine preceded by a hydrophobicregion located a few residues prior to the cysteine, andmultiple lysines and/or arginines within the first 10 resi-dues. Motifs showing the amino acid frequency across theprecursor peptide were generated using Jalview 2 [108].

HMM generation and analysisHMMs were generated for MbnA (Additional file 7),MbnB (Additional file 8) and MbnC (Additional file 9)using HMMER3 [109] (Howard Hughes Medical InstituteJanelia Farm, Ashburn, Virginia, USA). Prior to HMMgeneration, curated seed alignments (featuring removalof truncated or otherwise unacceptable sequences andtrimming of extra domains) were manually generated.


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http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=BADK01001132

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NC_013854

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AGTN01410593



http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AKXR01001597

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NZ_AFCH01000034

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NZ_CADT01000094

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=JCVI_SCAF_1096627660232


http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=BADE01000957

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AJTV01000003

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AJTV01000041

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=BX571860

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NZ_AHIP01000040

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AJXF01000037


http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NZ_AEIU01000072

http://ae.igs.umaryland.edu/cgi/index.cgi

http://ae.igs.umaryland.edu/cgi/index.cgi

http://manatee.sourceforge.net/

HMMs were used to scan the existing protein databasesfor additional operons, but all extant Mbn operonsappear to have been located via tBLASTn, prior to HMMconstruction. A revised NExT HMM (NexTNew) wasused to confirm the identification of TBDTs possessingthe N-terminal extension required for TonB-mediatedsignal transduction [57].

Additional material

Additional file 1: Figures S1-S6. Figures of additional Mbn structures,alignment of potential biosynthesis protein in Group V operons,alignment of MbnM sequences, alignment of MbnT sequences andphylogenetic tree, figure of alternate Mbn regulation scheme, andalignment of MbnP sequences.

Additional file 2: Table S1. Table containing information regarding theGenBank or JGI contigs/scaffolds and locus IDs (where present) for thegenes discussed in the paper.

Additional file 3: Annotated C. basilensis B-8 scaffold 1267_1,GenBank format. A GenBank-formatted file consisting of C. basilensis B-8scaffold 1267_1 (containing an Mbn operon), as annotated by the IGS.

Additional file 4: Annotated M. parvus OBBP contig 003, GenBankformat. A GenBank-formatted file consisting of M. parvus OBBPcontig003 (containing an Mbn operon), as annotated by the IGS.

Additional file 5: Annotated M. parvus OBBP contig 041, GenBankformat. A GenBank-formatted file consisting of M. parvus OBBPcontig041 (containing an Mbn operon), as annotated by the IGS.

Additional file 6: Annotated P. fluorescens NZI7contig00040_contig01, GenBank format. A GenBank-formatted fileconsisting of P. fluorescens NZI7 contig00040_contig01 (containing anMbn operon), as annotated by the IGS.

Additional file 7: HMMER3 hidden Markov model for MbnA. AHMMER3-compatible hidden Markov model constructed using a curatedalignment of the MbnA sequences discussed in the paper.

Additional file 8: HMMER3 hidden Markov model for MbnB. AHMMER3-compatible hidden Markov model constructed using a curatedalignment of the complete MbnB sequences discussed in the paper.

Additional file 9: HMMER3 hidden Markov model for MbnC. AHMMER3-compatible hidden Markov model constructed using a curatedalignment of the MbnC sequences discussed in the paper.

AbbreviationsAa: amino acids; ABC: ATP-binding cassette; ATP: adenosine triphosphate;BLAST: Basic Local Alignment Search Tool; ECF: extracytoplasmic function;FAD: flavin adenine dinucleotide; HMM: hidden Markov model; MATE:multidrug and toxic compound extrusion; Mbn: methanobactin; MFS, majorfacilitator superfamily; MMO: methane monooxygenase; Mrna: messengerribonucleic acid; NExT: N-terminal extension; NMR: nuclear magneticresonance; ORF: open reading frame; pMMO: particulate methanemonooxygenase; RiPP: ribosomally synthesized and post-translationallymodified peptide natural product; RND: resistance-nodulation-cell division;SACCP: surface-associated cytochrome c peroxidase; SAM: S-adenosylmethionine; sMMO: soluble methane monooxygenase; TBDT: tonB-dependent transporter.

Authors’ contributionsGEK carried out the bioinformatics analyses. GEK and ACR conceived of thestudy and wrote the manuscript. Both authors read and approved the finalmanuscript.

Competing interestsThe authors declare that they have no competing interests.

AcknowledgementsWe thank Dr. Ralf Koebnik for providing us with the NExT HMM. We alsothank the Institute for Genome Sciences Annotation Engine service at theUniversity of Maryland School of Medicine for providing structural andfunctional annotation of the Cupriavidus basilensis B-8, Methylocystis parvusOBBP and Pseudomonas fluorescens NZI7 genomes. This work was supportedby NSF grant MCB0842366. GEK was supported in part by National Institutesof Health Training Grant GM08061.

Received: 21 December 2012 Accepted: 26 February 2013Published: 26 February 2013

References1. Semrau JD, Dispirito AA, Yoon S: Methanotrophs and copper. FEMS

Microbiol Lett 2010, 34:496-531.2. Scheutz C, Kjeldsen P, Bogner JE, De Visscher A, Gebert J, Hilger HA, Huber-

Humer M, Spokas K: Microbial methane oxidation processes andtechnologies for mitigation of landfill gas emissions. Waste Manag Res2009, 27:409-455.

3. Huber-Humer M, Gebert J, Hilger H: Biotic systems to mitigate landfillmethane emissions. Waste Manag Res 2008, 26:33-46.

4. Jiang H, Chen Y, Jiang PX, Zhang C, Smith TJ, Murrell JC, Xing XH:Methanotrophs: Multifunctional bacteria with promising applications inenvironmental bioengineering. Biochem Eng J 2010, 49:277-288.

5. Culpepper MA, Rosenzweig AC: Architecture and active site of particulatemethane monooxygenase. Crit Rev Biochem Mol Biol 2012, 47:483-492.

6. Balasubramanian R, Smith SM, Rawat S, Stemmler TL, Rosenzweig AC:Oxidation of methane by a biological dicopper centre. Nature 2010,465:115-119.

7. Rosenzweig AC, Frederick CA, Lippard SJ, Nordlund P: Crystal structure of abacterial non-haem iron hydroxylase that catalyses the biologicaloxidation of methane. Nature 1993, 366:537-543.

8. Prior SD, Dalton H: The effect of copper ions on membrane content andmethane monooxygenase activity in methanol-grown cells ofMethylococcus capsulatus (Bath). J Gen Microbiol 1985, 131:155-163.

9. Murrell JC, McDonald IR, Gilbert B: Regulation of expression of methanemonooxygenases by copper ions. Trends Microbiol 2000, 8:221-225.

10. Hakemian AS, Rosenzweig AC: The biochemistry of methane oxidation.Ann Rev Biochem 2007, 76:223-241.

11. Téllez CM, Gaus KP, Graham DW, Arnold RG, Guzman RZ: Isolation ofcopper biochelates from Methylosinus trichosporium OB3b and solublemethane monooxygenase mutants. Appl Environ Microbiol 1998,64:1115-1122.

12. DiSpirito AA, Zahn JA, Graham DW, Kim HJ, Larive CK, Derrick TS, Cox CD,Taylor AB: Copper-binding compounds from Methylosinus trichosporiumOB3b. J Bacteriol 1998, 180:3606-3613.

13. Kim HJ, Graham DW, DiSpirito AA, Alterman MA, Galeva N, Larive CK,Asunskis D, Sherwood PMA: Methanobactin, a copper-aquisitioncompound from methane oxidizing bacteria. Science 2004, 305:1612-1615.

14. Ruiz A, Ogden KL: Biotreatment of copper and isopropyl alcohol in wastefrom semiconductor manufacturing. IEEE Trans Semicond Manuf 2004,17:538-543.

15. Summer KH, Lichtmannegger J, Bandow N, Choi DW, DiSpirito AA,Michalke B: The biogenic methanobactin is an effective chelator forcopper in a rat model for Wilson disease. J Trace El Med Biol 2011,25:36-41.

16. Balasubramanian R, Rosenzweig AC: Copper methanobactin: a moleculewhose time has come. Curr Op Chem Biol 2008, 12:245-249.

17. Kenney GE, Rosenzweig AC: Chemistry and biology of the copperchelator methanobactin. ACS Chem Biol 2012, 7:260-268.

18. El Ghazouani A, Basle A, Firbank SJ, Knapp CW, Gray J, Graham DW,Dennison C: Copper-binding properties and structures ofmethanobactins from Methylosinus trichosporium OB3b. Inorg Chem 2011,50:1378-1391.

19. Knapp CW, Fowle DA, Kulczycki E, Roberts JA, Graham DW: Methanemonooxygenase gene expression mediated by methanobactin in thepresence of mineral copper sources. Proc Natl Acad Sci USA 2007,104:12040-12045.

20. Kulczycki E, Roberts JA: Methanobactin-promoted dissolution of Cu-substituted borosilicate glass. Geobiology 2007, 5:251-263.


Page 14 of 17

http://www.biomedcentral.com/content/supplementary/1741-7007-11-17-S1.PDF

http://www.biomedcentral.com/content/supplementary/1741-7007-11-17-S2.XLSX

http://www.biomedcentral.com/content/supplementary/1741-7007-11-17-S3.GBF




http://www.biomedcentral.com/content/supplementary/1741-7007-11-17-S7.HMM



http://www.ncbi.nlm.nih.gov/pubmed/19584243?dopt=Abstract



























21. Balasubramanian R, Kenney GE, Rosenzweig AC: Dual pathways for copperuptake by methanotrophic bacteria. J Biol Chem 2011, 286:37313-37319.

22. Behling LA, Hartsel SC, Lewis DE, Dispirito AA, Choi DW, Masterson LR,Veglia G, Gallagher WH: NMR, mass spectrometry and chemical evidencereveal a different chemical structure for methanobactin that containsoxazolone rings. J Am Chem Soc 2008, 130:12604-12605.

23. Krentz BD, Mulheron HJ, Semrau JD, DiSpirito AA, Bandow NL, Haft DH,Vuilleumier S, Murrell JC, McEllistrem MT, Hartsel SC, Gallagher WH: Acomparison of methanobactins from Methylosinus trichosporium OB3band Methylocystis strain SB2 predicts methanobactins are synthesizedfrom diverse peptide precursors modified to create a common core forbinding and reducing copper ions. Biochemistry 2010, 49:10117-10130.

24. El Ghazouani A, Basle A, Gray J, Graham DW, Firbank SJ, Dennison C:Variations in methanobactin structure influences copper utilization bymethane-oxidizing bacteria. Proc Natl Acad Sci USA 2012, 109:8400-8404.

25. Bandow N, Gilles VS, Freesmeier B, Semrau JD, Krentz B, Gallagher W,McEllistrem MT, Hartsel SC, Choi DW, Hargrove MS, Heard TM, Chesner LN,Braunreiter KM, Cao BV, Gavitt MM, Hoopes JZ, Johnson JM, Polster EM,Schoenick BD, Umlauf AM, DiSpirito AA: Spectral and copper bindingproperties of methanobactin from the facultative methanotrophMethylocystis strain SB2. J Inorg Biochem 2012, 110:72-82.

26. Kim HJ, Galeva N, Larive CK, Alterman M, Graham DW: Purification andphysical-chemical properties of methanobactin: a chalkophore fromMethylosinus trichosporium OB3b. Biochemistry 2005, 44:5140-5148.

27. Crosa JH, Walsh CT: Genetics and assembly line enzymology of siderophorebiosynthesis in bacteria. Microbiol Molec Biol Rev 2002, 66:223-249.

28. Stein LY, Yoon S, Semrau JD, DiSpirito AA, Crombie A, Murrell JC,Vuilleumier S, Kalyuzhnaya MG, Op den Camp HJ, Bringel F, Bruce D,Cheng JF, Copeland A, Goodwin L, Han S, Hauser L, Jetten MS, Lajus A,Land ML, Lapidus A, Lucas S, Médigue C, Pitluck S, Woyke T, Zeytun A,Klotz MG: Genome sequence of the obligate methanotroph Methylosinustrichosporium strain OB3b. J Bacteriol 2010, 192:6497-6498.

29. Boekhorst J, Wilson G, Siezen RJ: Searching in microbial genomes forencoded small proteins. Microb Biotechnol 2011, 4:308-313.

30. Harrison PM, Carriero N, Liu Y, Gerstein M: A ‘PolyORFomic’ analysis ofprokaryote genomes using disabled-homology filtering revealsconserved but undiscovered short ORFs. J Mol Biol 2003, 333:885-892.

31. Heo HS, Lee S, Kim JM, Choi YJ, Chung HY, Oh SJ: tsORFdb: Theoreticalsmall open reading frames (ORFs) database and massProphet: peptidemass fingerprinting (PMF) tool for unknown small functional ORFs.Biochem Biophys Res Commun 2010, 397:120-126.

32. de Jong A, van Hijum S, Bijlsma JJ, Kok J, Kuipers OP: BAGEL: a web-basedbacteriocin genome mining tool. Nucleic Acids Res 2006, 34:W273-W279.

33. de Jong A, van Heel AJ, Kok J, Kuipers OP: BAGEL2: mining forbacteriocins in genomic data. Nucleic Acids Res 2010, 38:W647-W651.

34. Wang H, Fewer DP, Sivonen K: Genome mining demonstrates thewidespread occurrence of gene clusters encoding bacteriocins incyanobacteria. PLoS ONE 2011, 6:e22384.

35. Velásquez JE, van der Donk WA: Genome mining for ribosomallysynthesized natural products. Curr Opin Chem Biol 2011, 15:11-21.

36. Murphy K, O’Sullivan O, Rea MC, Cotter PD, Ross RP, Hill C: Genome miningfor radical SAM protein determinants reveals multiple sactibiotic-likegene clusters. PLoS One 2011, 6:e20852.

37. Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA,Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M,Dittmann E, Donadio S, Dorrestein PC, Entian K-D, Fischbach MA, Garavelli JS,Goransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C,Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, et al: Ribosomallysynthesized and post-translationally modified peptide natural products:overview and recommendations for a universal nomenclature. Nat ProdRep 2013, 30:108-160.

38. Haft DH, Selengut JD, Richter RA, Harkins D, Basu MK, Beck E: TIGRFAMsand Genome Properties in 2013. Nucleic Acids Res 2013, 41:D387-D395.

39. Haft DH, Basu MK, Mitchell DA: Expansion of ribosomally producednatural products: a nitrile hydratase-and Nif11-related precursor family.BMC Biol 2010, 8.

40. del Cerro C, García JM, Rojas A, Tortajada M, Ramón D, Galán B, Prieto MA,García JL: Genome sequence of the methanotrophic poly-β-hydroxybutyrate producer Methylocystis parvus OBBP. J Bacteriol 2012,194:5709-5710.

41. Grass G: Iron transport in Escherichia coli: All has not been said anddone. Biometals 2006, 19:159-172.

42. Oman TJ, van der Donk WA: Follow the leader: the use of leader peptidesto guide natural product biosynthesis. Nat Chem Biol 2010, 6:9-18.

43. Dam B, Dam S, Kube M, Reinhardt R, Liesack W: Complete genomesequence of Methylocystis sp strain SC2, an aerobic methanotroph withhigh-affinity methane oxidation potential. J Bacteriol 2012, 194:6008-6009.

44. Copley RR, Bork P: Homology among (βα)8 barrels: implications for theevolution of metabolic pathways. J Mol Biol 2000, 303:627-640.

45. Hanzelmann P, Schindelin H: Crystal structure of the S-adenosylmethionine-dependent enzyme MoaA and its implications formolybdenum cofactor deficiency in humans. Proc Natl Acad Sci USA 2004,101:12870-12875.

46. Omote H, Hiasa M, Matsumoto T, Otsuka M, Moriyama Y: The MATEproteins as fundamental transporters of metabolic and xenobioticorganic cations. Trends Pharmacol Sci 2006, 27:587-593.

47. Kuroda T, Tsuchiya T: Multidrug efflux transporters in the MATE family.Biochim Biophys Acta 2009, 1794:763-768.

48. He XA, Szewczyk P, Karyakin A, Evin M, Hong WX, Zhang QH, Chang G:Structure of a cation-bound multidrug and toxic compound extrusiontransporter. Nature 2010, 467:991-994.

49. Brickman TJ, Armstrong SK: Bordetella AlcS transporter functions inalcaligin siderophore export and is central to inducer sensing in positiveregulation of alcaligin system gene expression. J Bacteriol 2005,187:3650-3661.

50. Allard KA, Viswanathan VK, Cianciotto NP: lbtA and lbtB are required forproduction of the Legionella pneumophila siderophore legiobactin. JBacteriol 2006, 188:1351-1363.

51. Hannauer M, Yeterian E, Martin LW, Lamont IL, Schalk IJ: An efflux pump isinvolved in secretion of newly synthesized siderophore by Pseudomonasaeruginosa. FEBS Lett 2010, 584:4751-4755.

52. Li XZ, Nikaido H: Efflux-mediated drug resistance in bacteria: an update.Drugs 2009, 69:1555-1623.

53. Noinaj N, Guillier M, Barnard TJ, Buchanan SK: TonB-dependent transporters:regulation, structure, and function. Ann Rev Microbiol 2010, 64:43-60.

54. Wiener MC: TonB-dependent outer membrane transport: going forBaroque? Curr Op Struct Biol 2005, 15:394-400.

55. Krewulak KD, Vogel HJ: TonB or not TonB: is that the question? BiochemCell Biol 2011, 89:87-97.

56. Schauer K, Rodionov DA, de Reuse H: New substrates for TonB-dependenttransport: do we only see the ‘tip of the iceberg’? Trends Biochem Sci2008, 33:330-338.

57. Koebnik R: TonB-dependent trans-envelope signalling: the exception orthe rule? Trends Microbiol 2005, 13:343-347.

58. Postle K, Larsen RA: TonB-dependent energy transduction between outerand cytoplasmic membranes. Biometals 2007, 20:453-465.

59. Ferguson AD, Amezcua CA, Halabi NM, Chelliah Y, Rosen MK,Ranganathan R, Deisenhofer J: Signal transduction pathway of TonB-dependent transporters. Proc Natl Acad Sci USA 2007, 104:513-518.

60. Brooks BE, Buchanan SK: Signaling mechanisms for activation ofextracytoplasmic function (ECF) sigma factors. Biochim Biophys Acta 2008,1778:1930-1945.

61. Braun V, Mahren S, Ogierman M: Regulation of the Fecl-type ECF sigmafactor by transmembrane signalling. Curr Opin Microbiol 2003, 6:173-180.

62. Braun V, Endriss F: Energy-coupled outer membrane transport proteinsand regulatory proteins. Biometals 2007, 20:219-231.

63. Koster M, Vanklompenburg W, Bitter W, Leong J, Weisbeek P: Role for theouter membrane ferric siderophore receptor PupB in signal transductionacross the bacterial cell envelope. EMBO J 1994, 13:2805-2813.

64. Beare PA, For RJ, Martin LW, Lamont IL: Siderophore-mediated cellsignalling in Pseudomonas aeruginosa: divergent pathways regulatevirulence factor production and siderophore receptor synthesis. MolMicrobiol 2003, 47:195-207.

65. Lefevre J, Delepelaire P, Delepierre M, Izadi-Pruneyre N: Modulation bysubstrates of the interaction between the HasR outer membranereceptor and its specific TonB-like protein, HasB. J Mol Biol 2008,378:840-851.

66. Michel L, Bachelard A, Reimmann C: Ferripyochelin uptake genes areinvolved in pyochelin-mediated signalling in Pseudomonas aeruginosa.Microbiology 2007, 153:1508-1518.


Page 15 of 17






































































































67. Heinrichs DE, Poole K: PchR, a regulator of ferripyochelin receptor gene(fptA) expression in Pseudomonas aeruginosa, functions both as anactivator and as a repressor. J Bacteriol 1996, 178:2586-2592.

68. Michel L, Gonzalez N, Jagdeep S, Nguyen-Ngoc T, Reimmann C: PchR-boxrecognition by the AraC-type regulator PchR of Pseudomonas aeruginosarequires the siderophore pyochelin as an effector. Mol Microbiol 2005,58:495-509.

69. Youard ZA, Wenner N, Reimmann C: Iron acquisition with the naturalsiderophore enantiomers pyochelin and enantio-pyochelin inPseudomonas species. Biometals 2011, 24:513-522.

70. Wilmot CM, Yukl ET: MauG: a di-heme enzyme required for methylaminedehydrogenase maturation. Dalton Trans 2013, 42:3127-3135.

71. Helland R, Fjellbirkeland A, Karlsen OA, Ve T, Lillehaug JR, Jensen HB: Anoxidized tryptophan facilitates copper binding in Methylococcuscapsulatus-secreted protein MopE. J Biol Chem 2008, 283:13897-13904.

72. Karlsen OA, Berven FS, Stafford GP, Larsen Ø, Murrell JC, Jensen HB,Fjellbirkeland A: The surface-associated and secreted MopE protein ofMethylococcus capsulatus (Bath) responds to changes in theconcentration of copper in the growth medium. Appl Environ Microbiol2003, 69:2386-2388.

73. Berson O, Lidstrom ME: Cloning and characterization of corA, a geneencoding a copper-repressible polypeptide in the type I methanotroph,Methylomicrobium albus BG8. FEMS Microbiol Lett 1997, 148:169-174.

74. Karlsen OA, Larsen O, Jensen HB: Identification of a bacterial di-haemcytochrome c peroxidase from Methylomicrobium album BG8.Microbiology 2010, 156:2682-2690.

75. Karlsen OA, Lillehaug JR, Jensen HB: The presence of multiple c-typecytochromes at the surface of the methanotrophic bacteriumMethylococcus capsulatus (Bath) is regulated by copper. Mol Microbiol 2008,70:15-26.

76. Karlsen OA, Kindingstad L, Angelskår SM, Bruseth LJ, Straume D,Puntervoll P, Fjellbirkeland A, Lillehaug JR, Jensen HB: Identification of acopper-repressible C-type heme protein of Methylococcus capsulatus(Bath): a member of a novel group of the bacterial di-heme cytochromec peroxidase family of proteins. FEBS J 2005, 272:6324-6335.

77. Karlsen OA, Larsen O, Jensen HB: The copper responding surfaceome ofMethylococccus capsulatus Bath. FEMS Microbiol Lett 2011, 323:97-104.

78. Stein LY, Bringel F, DiSpirito AA, Han S, Jetten MS, Kalyuzhnaya MG, Kits KD,Klotz MG, Op den Camp HJ, Semrau JD, Vuilleumier S, Bruce DC, Cheng JF,Davenport KW, Goodwin L, Han S, Hauser L, Lajus A, Land ML, Lapidus A,Lucas S, Médigue C, Pitluck S, Woyke T: Genome sequence of themethanotrophic alphaproteobacterium Methylocystis sp strain Rockwell(ATCC 49242). J Bacteriol 2011, 193:2668-2669.

79. Carniel E: The Yersinia high-pathogenicity island: an iron-uptake island.Microbes Infect 2001, 3:561-569.

80. Gilbert B, McDonald IR, Finch R, Stafford GP, Nielsen AK, Murrell JC: Molecularanalysis of pmo (particulate methane monooxygenase) operons from twotype II methanotrophs. Appl Environ Microbiol 2000, 66:966-975.

81. McDonald IR, Uchiyama H, Kambe S, Yagi O, Murrell JC: The solublemethane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp. strain M. Appl EnvironMicrobiol 1997, 63:1898-1904.

82. Lindner AS, Pacheco A, Aldrich HC, Staniec AC, Uz I, Hodson DJ:Methylocystis hirsuta sp nov., a novel methanotroph isolated from agroundwater aquifer. Int J Syst Evol Microbiol 2007, 57:1891-1900.

83. Wartiainen I, Hestnes AG, McDonald IR, Svenning MM: Methylocystis roseasp nov., a novel methanotrophic bacterium from Arctic wetland soil,Svalbard, Norway (78°N). Int J Syst Evol Microbiol 2006, 56:541-547.

84. Banala S, Sussmuth RD: Thioamides in nature: in search of secondarymetabolites in anaerobic microorganisms. Chembiochem 2010,11:1335-1337.

85. Chinworrungsee M, Kittakoop P, Saenboonrueng J, Kongsaeree P,Thebtaranonth Y: Bioactive compounds from the seed fungusMenisporopsis theobromae BCC 3975. J Nat Prod 2006, 69:1404-1410.

86. Savard ME, Melzer MS, Boland GJ, Bensimon C, Blackwell BA: A new 1-hydroxy-2,6-pyrazinedione associated with hypovirulent isolates ofSclerotinia minor. J Nat Prod 2003, 66:306-309.

87. Ward N, Larsen O, Sakwa J, Bruseth L, Khouri H, Durkin AS, Dimitrov G,Jiang L, Scanlan D, Kang KH, Lewis M, Nelson KE, Metheacute B, Wu M,Heidelberg JF, Paulsen IT, Fouts D, Ravel J, Tettelin H, Ren Q, Read T,DeBoy RT, Seshadri R, Salzberg SL, Jensen HB, Birkeland NK, Nelson WC,

Dodson RJ, Grindhaug SH, Holt I, et al: Genomic insights intomethanotrophy: the complete genome sequence of Methylococcuscapsulatus (Bath). PLoS Biol 2004, 2:e303.

88. Chen Y, Crombie A, Rahman MT, Dedysh SN, Liesack W, Stott MB, Alam M,Theisen AR, Murrell JC, Dunfield PF: Complete genome sequence of theaerobic facultative methanotroph Methylocella silvestris BL2. J Bacteriol2010, 192:3840-3841.

89. Choi DW, Bandow NL, McEllistrem MT, Semrau JD, Antholine WE,Hartsel SC, Gallagher W, Zea CJ, Pohl NL, Zahn JA, DiSpirito AA: Spectraland thermodynamic properties of methanobactin from gamma-proteobacterial methane oxidizing bacteria: A case for coppercompetition on a molecular level. J Inorg Biochem 2010, 104:1240-1247.

90. Greenwald J, Nader M, Celia H, Gruffaz C, Geoffroy V, Meyer JM, Schalk IJ,Pattus F: FpvA bound to non-cognate pyoverdines: molecular basis ofsiderophore recognition by an iron transporter. Mol Microbiol 2009,72:1246-1259.

91. Kaneko T, Minamisawa K, Isawa T, Nakatsukasa H, Mitsui H, Kawaharada Y,Nakamura Y, Watanabe A, Kawashima K, Ono A, Shimizu Y, Takahashi C,Minami C, Fujishiro T, Kohara M, Katoh M, Nakazaki N, Nakayama S,Yamada M, Tabata S, Sato S: Complete genomic structure of thecultivated rice endophyte Azospirillum sp B510. DNA Res 2010, 17:37-50.

92. Du XJ, Jia SR, Yang Y, Wang S: Genome sequence of Gluconacetobacter spstrain SXCC-1, isolated from Chinese vinegar fermentation starter. JBacteriol 2011, 193:3395-3396.

93. Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, Taourit S,Bocs S, Boursaux-Eude C, Chandler M, Charles JF, Dassa E, Derose R,Derzelle S, Freyssinet G, Gaudriault S, Medigue C, Lanois A, Powell K,Siguier P, Vincent R, Wingate V, Zouine M, Glaser P, Boemare N, Danchin A,Kunst F: The genome sequence of the entomopathogenic bacteriumPhotorhabdus luminescens. Nat Biotechnol 2003, 21:1307-1313.

94. Tribelli PM, Iustman LJR, Catone MV, Di Martino C, Revale S, Mendéz BS,López NI: Genome sequence of the polyhydroxybutyrate producerPseudomonas extremaustralis, a highly stress-resistant Antarcticbacterium. J Bacteriol 2012, 194:2381-2382.

95. Godfrey SA, Marshall JW, Klena JD: Genetic characterization ofPseudomonas ’NZ17-a novel pathogen that results in a brown blotchdisease of Agaricus bisporus. J Appl Microbiol 2001, 91:412-420.

96. Xu Y, Kersten RD, Nam SJ, Lu L, Al-Suwailem AM, Zheng HJ, Fenical W,Dorrestein PC, Moore BS, Qian PY: Bacterial biosynthesis and maturationof the didemnin anti-cancer agents. J Am Chem Soc 2012, 134:8625-8632.

97. Benson DA, Karsch-Mizrachi I, Clark K, Lipman DJ, Ostell J, Sayers EW:GenBank. Nucleic Acids Res 2012, 40:D48-D53.

98. Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E, Grechkin Y,Ratner A, Jacob B, Huang JH, Williams P, Huntemann M, Anderson I,Mavromatis K, Ivanova NN, Kyrpides NC: IMG: the Integrated MicrobialGenomes database and comparative analysis system. Nucleic Acids Res2012, 40:D115-D122.

99. Gertz EM, Yu Y-K, Agarwala R, Schaffer AA, Altschul SF: Composition-basedstatistics and translated nucleotide searches: improving the TBLASTNmodule of BLAST. BMC Biol 2006, 4:41.

100. Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL,Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR,Bateman A: The Pfam protein families database. Nucleic Acids Res 2010,38:D211-D222.

101. Galens K, Orvis J, Daugherty S, Creasy HH, Angiuoli S, White O, Wortman J,Mahurkar A, Giglio MG: The IGS standard operating procedure forautomated prokaryotic annotation. Stand Genomic Sci 2011, 4:244-251.

102. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li WZ, Lopez R,McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG: Fast,scalable generation of high-quality protein multiple sequencealignments using Clustal Omega. Mol Syst Biol 2011, 7:539.

103. Edgar RC: MUSCLE: multiple sequence alignment with high accuracy andhigh throughput. Nucleic Acids Res 2004, 32:1792-1797.

104. Katoh K, Standley DM: MAFFT multiple sequence alignment softwareversion 7: improvements in performance and usability. Mol Biol Evol 2013.

105. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance, and maximum parsimony methods. Mol Biol Evol2011, 28:2731-2739.

106. Jones DT, Taylor WR, Thornton JM: The rapid generation of mutation datamatrices from protein sequences. Comput Appl Biosci 1992, 8:275-282.


Page 16 of 17


































































































107. Felsenstein J: Confidence limits on phylogenies-an approach using thebootstrap. Evolution 1985, 39:783-791.

108. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ: JalviewVersion 2-a multiple sequence alignment editor and analysis workbench.Bioinformatics 2009, 25:1189-1191.

109. Finn RD, Clements J, Eddy SR: HMMER web server: interactive sequencesimilarity searching. Nucleic Acids Res 2011, 39:W29-W37.

110. Taylor WR: Residual colours: a proposal for aminochromography. ProteinEng 1997, 10:743-746.

doi:10.1186/1741-7007-11-17Cite this article as: Kenney and Rosenzweig: Genome mining formethanobactins. BMC Biology 2013 11:17.

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