［明治薬科大学研究紀要 42，1-18（2013）］

Molecular phylogenetics of β-lactamasesin Actinobacteria

Hiroshi Ogawara

総Review

説[明治薬科大学研究紀要 42, 1-18 (2013)]

Molecular phylogenetics of β-lactamases in Actinobacteria
Hiroshi Ogawara
HO Bio Institute, 33-9, Yushima-2, Bunkyo-ku, Tokyo 113-0034, Japan andMeiji Pharmaceutical University, 522-1, Noshio-2, Kiyose, Tokyo 204-8588, Japan
E-mail address: hogawara@sc5.so-net.ne.jp
tel/fax number: +813-3832-3474
Mailing address: HO Bio Institute, 33-9, Yushima-2, Bunkyo-ku, Tokyo 113-0034, Japan
Key words: β-lactamase; molecular phylogenetics; actinobacteria; Streptomyces; transposon; drug resistance.

ABSTRACT
β-Lactamases are the main cause of β-lactam resistance in many pathogenic bacteria. To elucidate the relationshipsof β-lactamases in soil-dwelling Gram-positive β-lactam-producing Actinobacteria and those in other bacteria, themolecular phylogenetics among β-lactamases in 19 Actinobacteria, whose genome sequences were completely clarified, were analyzed. The precise phylogenetic and amino acid alignment analyses revealed that 10 class A, 5 class B
and 1 class D probable β-lactamases were present. A class D sequence detected in Thermomonospora curvata belonged to OXA-5-type β-lactamase, an enzyme detected in Gram-negative bacteria. Another interesting finding is thatas many as 5 class B

β-lactamases were detected in this study and these class B β-lactamases were limited to sub-8)class B3 like Alaskan soil3). β-Lactamase candidates picked up through the primary and secondary screenings were
distributed randomly in Actinobacteria irrespective of their taxonomic position. In some species over 5 putative
β-lactamases were detected, but in some others no β-lactamase was detectable. Thirty five putative class C
β-lactamase candidates were identified by the preliminary phylogenetic analysis. Although these presumed
β-lactamases were found to be probableβ-lactamases or acyltransferases, it should be taken care that a few nucleotide
substitutions can convert them into active β-lactamases in the next generation.

It is intriguing in this connection thatabout 11kb transposon-like sequences including β-lactamases were encountered in Nocardia farcinica and Saccharopolyspora erythraea. This is the first report describing a transposon-like sequence including β-lactamase genein Actinobacteria. Although this paper does not represent a comprehensive survey of β-lactamases in Actinobacteria,
it will provide distinct characteristics of β-lactamases in Actinobacteria.
1. Introduction
β-Lactamases are the enzymes which hydrolyze the β-lactam antibiotics to produce antibacterially inactive compounds. Because of this, β-lactamases are responsible for β-lactam resistance in many pathogenic bacteria57). These
enzymes, however, are also produced by nonpathogenic bacteria such as Streptomyces45, 48), Bacillus2) and the
cyanobacteria36, 73). In addition, recent whole genome analyses expand the β-lactamase world to β-lactamase superfamily proteins55), β-lactamase domain-containing proteins15) and RNA-metabolising metallo-β-lactamases18,74).

­− 1 −

Consequently, β-lactamase -producing organisms were found to include a thermobacterium67) (GenBank accession
number: ADD27888), a green sulfur bacterium (GenBank accession number: ACF43212), a green non-sulfur bacterium (GenBank accession number: ABX07048), a methylotrophic bacterium37) (GenBank accession number:
ACT50532), a fungus78) (GenBank accession number: CAA93219.1) and even a plant38) (GenBank accession number: AAC49866) and mammals22, 61) (GenBank accession numbers: EDL84248 and NP_116246). Furthermore, analkalophilic and halotolerant Gram-positive bacterium Oceanobacillus iheyensis isolated from the bottom of the Pacific Ocean at a depth of 1050m71) and Pseudomonas fluorescens isolated from a remote Antarctic coastal area also
produced β-lactamases43). Moreover, screening of metagenomic libraries from soil bacterial communities for clones
that confer antibiotic resistance gave new types of β-lactamases such as oligomeric33) and bifunctional β-lactamases3,
19). A bifunctional β-lactamase was also isolated from a pathogen Mycobacterium tuberculosis H37Rv, an actinomy-

cete64). Owing to its diverse microbial community, the soil is a large environmental reservoir of antibiotic resistance16) and is called the antibiotic resistome17, 58). The human microflora is also the antibiotic resistance reservoir62).
Streptomyces species are filamentous, soil-dwelling, Gram-positive bacteria and are characterized by their ability
to undergo complex cellular differentiation like filamentous fungi12). In addition, Streptomyces species produce awide variety of secondary metabolites including β-lactam antibiotics, the substrate of β-lactamases39). Streptomyces
species are prokaryotic microorganisms and hence they must protect themselves from the attack of β-lactam antibiotics. The β-lactam antibiotic biosynthetic gene cluster contains a β-lactamase coding sequence in Streptomyces clavuligerus, a β-lactam producing Streptomyces39, 54). This is also the case with Lysobacter lactamgenus, another prokaryotic microorganism producing a β-lactam antibiotic34). Therefore, it is possible that β-lactamases in these bacteria are
involved in self-resistance against β-lactam antibiotics. However, β-lactamases are produced abundantly in Streptomyces irrespective of their resistance against β-lactam antibiotics45, 48, 50). The structural change of their targets, penicillin-binding proteins is supposed to be the major mechanism of resistance against β-lactam antibiotics in Streptomyces49, 52).
β-Lactamases are grouped into four classes on the basis of their amino acid sequences4, 8, 31, 56). This classificationreflects more or less the substrate specificity: class A (penicillin-hydrolyzing), class C (cephalosporin-hydrolyzing)
and class D (oxacillin-hydrolyzing) β-lactamases are active site serine enzymes and class B β-lactamases require one
or two zinc ions for their activity and are called as metallo-β-lactamases9, 23). A previous phylogenetic study indicatedthat class A β-lactamases could be divided into three subgroups47). β-Lactamases are not essential bacterial proteins inthemselves, but are speculated to have evolved from the essential penicillin-binding proteins in someβ-lactam-producing bacteria such as Streptomyces or related bacteria, because these bacteria have to have some
self-protective strategies against β-lactam antibiotics46, 73). Penicillin-binding proteins are involved in peptidoglycan
biosynthesis in prokaryotic microorganisms. Therefore, it is interesting to know the relationships of β-lactam antibiotics, β-lactamases and penicillin-binding proteins in Streptomyces, β-lactam-producing microorganisms. In addition,
the relationship of β-lactamases in Streptomyces and those in Actinobacteria should be clarified. In this paper, thephylogenetic relationships were analyzed among β-lactamases in Streptomyces and some of closely related Actinobacteria, whose genome sequences were wholly clarified.
2. Materials and methods
2. 1. Selection of candidate β-lactamases
Four Streptomyces and closely related 15 Actinobacteria listed in Table 1 were used for mining of β-lactamase
genes in Genome Information Broker24). β-Lactamase genes were selected at three steps. From the database, putative
β-lactamase genes in these bacteria were screened with keywords “lactamase” and “penicillin-binding protein” at the­− 2 −

first step. For each species, the screened sequences were analyzed at the second step by aligning and constructing phylogenetic trees by using ClustalW269) as implemented by MEGA466)
together with the representative sequences of β-lactamases(Table 1 of Supplement) with the default parameters. On thebasis of the phylogenetic trees and the alignments thus obtained,candidate sequences were tentatively chosen and classified intoclass A, class B, class C and class D β-lactamases. Thereafter,
the statistical significance of the classification was verified bypair-wise aligning a selected candidate and the representative
β-lactamases in either class with the default parameters (Pearson). When the E-value was over 1.0, the candidate sequence
was excluded from further analysis.
2. 2. Phylogenetic analyses
For the final selection or at the third step, phylogenetic
analyses were used. For phylogenetic analyses, the amino acidsequences were aligned using ClustalX 2.068) and MUSCLE
version 3.820) with default parameters. Ambiguous regions wereautomatically trimmed according to Gblocks11, 65) and concatenated alignments were constructed. Phylogenetic
analyses were performed by the neighbor-joining method with the bootstrap tree option using the MEGA4 programand allowing for 1000 bootstrap iterations66), the maximum likelihood method of the PhyML program package with
100 bootstrap iterations28) and Baysian analyses in MrBayes 3.159). As an outgroup, a class C β-lactamase (an Escherichia coli K-12 chromosomal enzyme, U14003) was used for class A and D trees and a class A β-lactamase (aStreptomyces cacaoi enzyme, D90201) was used for class C tree. A glyoxylase II (GLO2_ECOLI), a member ofmetallo-β-lactamase superfamily, was used as an outgroup for class B tree. For maximum likelihood (PhyML) andBayesian analyses, the best fit amino acid substitution models for each gene were selected based on Akaike Information Criteria (AIC) using ProtTest1).
3. Results
3. 1. General feature
As a result of secondary screening, 59 sequences in total were tentatively selected as candidates for further analysis among 529 putative β-lactamase and penicillin-binding protein sequences in 19 species (Table 2). They were 18
class A, 5 class B, 35 class C and 1 class D β-lactamase candidates. For example, 19 putative β-lactamase and 24
putative penicillin-binding proteins were picked up at the first selection in Streptomyces coelicolor A3(2). However
only 2 (Sco0088 and Sco3774) were screened as class A and 2 (Sco0830 and Sco3912) were as class C β-lactamase
candidates by pair-wise alignment. There were some characteristic features in the 59 selected candidate sequences.
First, β-lactamase candidates were not distributed uniformly at least in the strains analyzed in this study: while
Saccharomonospora viridis, Streptomyces avermitilis, Streptomyces scabiei and Streptosporangium roseum possessed as many as 5, 12, 5 and 5 candidate genes, respectively, Mycobacterium leprae, Nocardioides sp. JS614 andRhodococcus erythropolis had no candidate gene. In general, Streptomyces species produced β-lactamase candidates
at higher rates. However, the numbers of candidates were not necessarily related to the taxonomic classification. For­− 3 −

example, although Streptosporangium and Nocardioides were closely related taxonomically63), the numbers of
β-lactamase candidates in Streptosporangium were 5 but those in Nocardioides were zero. Even in the same genus,
Mycobacterium tuberculosis produced 4 but M. leprae produced none. β-Lactamases detected by their enzymatic
activity also distributed independently of actinobacterial taxonomy50). These results indicate strongly thatβ-lactamases evolve and are produced independent of actinobacterial taxonomy.
Second, most of the β-lactamases detected so far by their enzymatic activity in Gram-positive bacteria belong toclass A and a few belong to class B. However, this was not the case with whole genomic level. In this study, twice as
many as class C β-lactamase candidates than class A were detected. The similar results were obtained with Bacillus
subtilis and Bacillus cereus (unpublished results).
Third, the nucleotide sequence and hence the amino acid sequence of Mycobacterium tuberculosis Rv2068c werecompletely identical with that of Mycobacterium bovis JTY2081, indicating that a horizontal gene transfer occurred
between these two species very recently. Thus, Mycobacterium bovis JTY2081 β-lactamase was precluded from further analyses.
Fourth, it is intriguing that Thermomonospora curvata, a high G+C Gram-positive soil thermophilic bacterium,
produced two class B and one class D β-lactamase candidates but no class A and class C enzyme. Usually,
Gram-positive bacteria produce class A and class B β-lactamases, but not class C and class D. However, this was notdue to the fact that T.curvata is thermophile because another thermophilic Actinomycete, Thermobispora bispora
DSM43833 produced two class C but no class D β-lactamase candidate. Hereafter, 59 putative β-lactamases in thesegroups are analyzed in more detail at the third step.
Table 2. The tentative classification of putative β-lactamase genes.
㻮㼍㼏㼠㼑㼞㼕㼍
Actinosynnema mirum DSM 43827
Frankia alni ACN14a
Frankia sp. CcI3
Mycobacterium bovis BCG str. Tokyo 172
Mycobacterium leprae TN
Mycobacterium tuberculosis H37Rv
Nocardia farcinica IFM 10152
Nocardioides sp. JS614
Propionibacterium acnes KPA171202
Rhodococcus erythropolis PR4
Saccharomonospora viridis DSM 43017

㻼㼞㼑㼒㼕㼤
Amir
Fraal
Francci3
JTY
ML
Rv
NFA
Noca
Ppa
Rer
Svir

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Saccharopolyspora erythraea NRRL 2338
Streptomyces avermitilis MA-4680

Sace
SAV

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Streptomyces coelicolor A3(2)Sco
Streptomyces griseus subsp. griseus NBRC 13350 SGR
Streptomyces scabiei 87.22
SCAB
Streptosporangium roseum DSM 43021
Sros
Thermobispora bispora DSM 43833
Tbis
Thermomonospora curvata DSM 43183
Tcur

The numbers in the table are the names of the genes in each bacterium. The prefixes for A. mirum, F. alni, Frankia sp. , M. bovis, M.
leprae, M. tuberculosis, N. farcinica, Nocardioides sp., P. acnes, R. erythropolis, S. viridis, S. erythraea, S. avermitilis, S. coelicolor, S.
griseus, S. scabiei, S. roseum and T. curvata are Amir, Fraal, Francci3, JTY, ML, Rv, NFA, Noca, Ppa, Rer, Svir, Sace, SAV, Sco, SGR,
SCAB, Sros and Tcur, respectively.
㻖㻌㻖㻖㻌Not detectable.

3. 2. Class A + class D group
A phylogenetic tree was constructed with 18 selected sequences (17 class A excluding M. bovis JTY2081 and 1class D) together with 6 class A(Streptomyces cacaoi D90201, Streptomyces cellulosae D12653, Streptomyces­− 4 −

albus M28303, Streptomyces clavurigerus Z54190, Staphylococcus aureus M15526 and Mycobacterium fortuitus
L25634) and one class D enzymes (Pseudomonas aeruginosa Z22590) and the result is shown in Fig. 1. These sequences were divided roughly into three groups (A1, A2 and A3 in Fig. 1). Tcur2040, Sace0046 and NFA0820 madeone group together with Z22590 (OXA-10). The amino acid alignment of these sequences together with Z22590(OXA-10) and M25261 (OXA-2) showed that although Sace0046 and NFA0820 carried some of the signature aminoacid residues of Class D β-lactamases75) (S67TFK70 and K205TG207 in Table 3 and Fig. 1 of supplement), they had no
Table 3.

The list of active site residues discussed in this paper.

other conserved residues such as Y142G, W154,
W221XXG corresponding to box IV, box A and box VI

Class A: S70XXK70, S130DN132, R164W/YE166, D179, K234T/SG236

of Joris et al.32), respectively. Furthermore, neither ofClass B: H116XHXD120, G123, H196, H263

them carried conserved residues of class A β-lactamases

Class C: S64XXK67, Y150XN152, K315TG317S/A318

such as S130DN, W165EP and D179 and many extra amino

Class D: S67TFK70, S115XV, Y142G, W154, K205TG, W221XXG

acid sequences were inserted into the sequences. Molecular distance analysis by using FASTA Sequence

Comparison at the University of Virginia (Pearson) confirmed that neither NFA0820 nor Sace0046 belonged to anyof the β-lactamase classes. Moreover, Blast analysis indicates that these two sequences were closely related to
3LO7_A (penicillin binding protein A of Mycobacterium tuberculosis21)) in Molecular Modeling Database(MMDB77)) of the National Center for Biotechnology Information not only in their amino acid sequence but also intheir three dimensional levels. Thus, Sace0046 and NFA0820 were probable acyltransferases. On the other hand,
Tcur2040 carried two other signature sequences (S115 XV117 and Y142GX) of class D β-lactamases. In addition, statistical significance scores from shuffles (Pearson) of an alignment by aligning Tcur2040 and class D β-lactamases of
OXA-10 (2WGW_B, 1E4D_C or 2X01_A) were less than 4.9e-42, substantiating that Tcur2040 is closely relatedwith 2WGW_B, 1E4D_C or 2X01_A not only in their amino acid sequence but also in their three dimensional levels.
These sequences were retrieved in MMDB77) with Tcur2040 as a query. It is concluded, therefore, that only Tcur2040
belonged to the class D β-lactamase group among three candidates. At the present time, most of the class D
β-lactamases are detected in Gram-negative bacteria and only a few have been reported in Gram-positive bacteria6).
Thus, it is intriguing to know to which group Tcur2040 enzyme belongs, Gram-positive or Gram-negative class D
β-lactamases. Phylogenetic analysis (Fig. 2) clearly showed that Tcur2040 was more closely related to Gram-negativeclass D β-lactamases, especially to OXA-5 and OXA-27 type β-lactamases. Molecular distance analysis by MEGA4
and FASTA (Pearson) again confirmed this suggestion.
Sco0088, Sros5713, Sco3774 and Sros1443 formed one group (A2 in Fig. 1) and Sros5574 was an isolated gene.
The amino acid alignment showed that these sequences possessed signature amino acid residues of class A
β-lactamases (S70XXK73, S130DN132 and K234T/SG236 in Fig. 2 of supplement). However, all the molecular distances
analyzed by MEGA4 and FASTA Sequence Comparison at the University of Virginia (Pearson) were above 0.8 and0.001 between a class A β-lactamase such as D90201 and the one of the above sequences (Sco0088, Sros5713,
Sco3774, Sros1443 or Sros5574), respectively. In addition, except for Sros5574 they had E166, another conserved
residue, and a conserved basic residue (arginine in this case) in the omega loop region of residue 164 to 179 of class
A β-lactamases41). Thus they may be probable β-lactamases. In fact, Blast analysis by using MMDB indicated that
Sco0088, Sros5713, Sco3774 and Sros1443 were more related to BlaB protein (2WUQ_B), a regulatory protein from
Streptomyces cacaoi72) in their three dimensional level, and Sros5574 was only related to β-lactamase-like proteinsfrom Actinobacteria such as Catenulispora acidiphila (YP_03117446.1), Mycobacterium smegmatis (YP_890415.1)
and Leifsonia xyli (YP_061292.1).

­− 5 −

The third of class A + class D group consisted of 11 members including Mycobacterium bovis JTY2081 (A3 in Fig.
1). The phylogenetic (Fig. 3) and molecular distance analyses indicate that all of them and the representative sequences were closely related each other and belonged to typical class A β-lactamases. The amino acid alignmentshown in Fig. 3 of supplement indicates that these sequences conserved most of the amino acid residues (S70XXK73,
S130DN132, R164W/YE166, D179, K234T/SG236) involved in catalytic reactions with the exception of Amir2178 and abasic residue in the omega loop region of residue 164 to 179 of class A β-lactamases41). In Amir2178, lysine at 73,
serine at 130, asparagine at 132 and threonine at 235 were replaced by serine, aspartic acid, serine, and proline, respectively. Therefore, Amir2178 may be a probable acyltransferase, even though its amino acid sequence andthree-dimensional structure closely resembles 1YLT_A, Ctx-M β-lactamase13). More careful scrutiny of the phylogenetic and molecular distance analyses indicated that Sros5706 was closely related to Gram-negative carbapenem-hydrolyzing β-lactamases such as Serratia marcescens SME-1 (U60295) and Klebsiella pneumoniae KPC-2(AAK70220) β-lactamases, and cephalosporin-hydrolyzing β-lactamases such as Serratia marcescens GES-16(ADJ94120) and Bacteroides fragilis CepA (AAA21538) than to Gram-positive enzymes (Fig. 3). Other 8 sequences
formed a cluster with those from Streptomyces such as S. cellulosae (D12653), S. albus (M28303), S. clavuligerus(Z54190) and S. cacaoi (D90201). It is concluded therefore that the class A β-lactamases in Actinobacteria consistedof a mixture of Gram-negative carbapenem-hydrolyzing and Gram-positive type enzymes.
72
97

Z54190Rv2068c
Sace4283
SGR457
Amir3962+

NFA23080
Amir2178
L25634

A3

Sace1374
D12653100

M28303
SAV4452
SCAB38731

86

Sros5706
D90201
M1552666

Sros5574
Sco0088
Sros5713
100

Sco3774+

A2+

Sros1443+

Z22590100

Tcur2040
99

Sace0046

A1

NFA820Fig. 2. Bayesian evolutionary tree of aligned amino acid sequences of Tcur2040

U14003(square boxed) and 14 representative class D β-lactamases. Bayesian posterior㻜㻚㻝

probabilities were shown at the nodes. The representative β-lactamases from
Fig. 1. Phylogenetic tree based on amino acid sequences of 18 putative and 6

Gram-negative bacteria (accession numbers are indicated in parentheses) arerepresentative class A and 1 class D (Z22590) β-lactamases (square boxed)

OXA-1 (AJ238349), OXA-2 (M25261), OXA-3 (L07945), OXA-5 (X58272),

constructed by MEGA466) and the neighbor-joining program with the default

OXA-10parameters. Branch lengths were drawn to scale and were proportional to the(U37105),

OXA-17(AF060206),

OXA-18(U85514),OXA-25(AF201826), OXA-27 (AF201828) and OXA-29 (AJ400619), and those fromnumbers of amino acid changes. Bootstrap values (%) calculated from 1,000
replicates were shown at the nodes. A class C β-lactamase (U14003) was used as

Gram-positive bacteria (bacterial names were indicated in the parentheses) werean outgroup. The representative β-lactamases are as shown in Table 1 of

AP003358 (Staphylococcus aureus), AE016749 (Staphylococcus epidermis),
CAB13772 (Bacillus subtilis) and AE016750 (Staphylococcus epidermis). ș

supplement.

-Lactamases from Gram-positive bacteria were marked with “+”. A class C
β-lactamase (U14003) was used as an outgroup.

­− 6 −

1.00
1.00

1.00

1.00

0.72

1.00
0.58
1.00

0.98

0.73

1.00

1.00
0.97

1.00
1.00

0.84
0.99
1.00

0.98
1.00

0.81

1.00

0.81

0.74

0.93

Fig. 3. Bayesian evolutionary tree of aligned amino acid sequences of 10 putative(square boxed) and 18 representative class A β-lactamases. Bayesian posterior
probabilities were shown at the nodes. The representative β-lactamases from
Gram-negative bacteria (accession numbers were indicated in parentheses) wereTEM-1 (EU491958), TEM-3 (CAA45828), SHV (M59181), RTG-4 (ACJ61335),
PSE-1(ACL31204),

PER-1(AAW62294),

CepA (AAA21538),

GES-16(ADJ94120), SME-1 (U60295) and KPC-2 (AAK70220) and those from
Gram-positive bacteria (bacterial names are indicated in parentheses) are D12653(Streptomyces cellulosae), M28303 (Streptomyces albus), Z54190 (Streptomyces
clavuligerus), D90201 (Streptomyces cacaoi), X06599 (Bacillus cereus), V00093(Bacillus licheniformis) and M15526 (Staphylococcus aureus). A class C
β-lactamase (U14003) was used as an outgroup.

3. 3. Class B metallo-β-lactamase groupClass B β-lactamases were composed of five members: Rv1913, Ppa0914, SGR4992, Tcur2765 and Tcur4662 (Table
2). Amino acid alignment of these sequences together with three representative sequences disclosed that these sequences contained the essential sequence of H116XHXD120, G123 and two other histidine residues (His196 and His263)
proposed to be involved in the coordination of zinc atom26, 76) (Fig. 4 of supplement). Molecular distance analysis
confirmed that these proteins belonged to Class B metallo-β-lactamases. A phylogenetic tree constructed by using
MrBayes method showed that SGR4992，Rv1913, Tcur4662 and Tcur2765 formed a distinctive cluster different fromthat of Aeromonas CAA40386, Pseudomonas AJ492820, Serratia P52699, Bacillus M15350, Bacillus AAA22276,
Bacteroides AAA22904 and Bacteroides M63556 (Fig. 4). However, Ppa0914 was an isolated gene. A phylogenetic
tree constructed by using PhyML program gave an essentially similar topology. BLAST search with SGR4992 andRv1913 as query sequences revealed that these were more nearly related to metallo-β-lactamases from Actinobacteriasuch as Streptomyces, Mycobacterium, Nocardioides, Amycolatopsis, Stackebrandtia, Propionibacterium, Streptosporangium and Saccharopolyspora, but not to those from Bacillus, Bacteroides, Pseudomonas, Serratia and Aeromonas. Tcur4662 and Tcur2765 were highly related to metallo-β-lactamases from Actinobacteria such as Frankia,
Catenulispora, Streptosporangium, Amycolatopsis and Nocardiopsis as well as polyketide cyclases from Streptomyces7) and Sulfolobus (NP_343860). On the other hand, Ppa0914 was closely related to metallo-β-lactamases from
Mycobacterium, Streptomyces, Saccharomonospora and Saccharopolyspora.

­− 7 −

1.00
0.96

1.00

0.92

0.93
1.00
0.96
1.00

0.90
0.61

1.00

Fig. 4. Bayesian evolutionary tree of aligned amino acid sequences of 5 putative(square boxed) and 8 representative class B β-lactamases. Bayesian posterior
probabilities were shown at the nodes. A glyoxylase II (GLO2_ECOLI) was used as
an outgroup. The representative β-lactamases are as shown in Table 1 of
supplement.

3. 4. Class C group
Comparative amino acid alignment and phylogenetic analyses of 35 sequences tentatively classified to Class C
β-lactamases (Table 2) and 8 representative enzymes (Escherichia coli U14003, Citrobacter freundii AAM93471,
Enterobacter cloacae P05364, Morganella morganii AAC68582, Serratia marcescens X52964, Lysobacter lactamgenus CAA39987, Mycobacterium smegmatis AY442183 and Laribacter hongkongensis AAT46346) indicated thatthese sequences were grouped roughly into 3 clusters (C1, C2 and C3 in Fig. 5 of supplement). The first cluster included fifteen sequences: Sco3912, SAV4283, SCAB46171, SGR3667, Svir33850, Fraal6499, SAV2663, SAV4455,
SAV5004, Svir23940, Tbis0343, SCAV26931, Amir4262, JTY17443 and SCAB55861 (C1 in Fig. 5 of supplement).
The amino acid alignment in Fig. 6 of supplement shows that these sequences carried active site Ser64, Lys67 andTyr150 (References30, 40)). However, other residues involved in the catalytic reaction like Lys315, Thr316, Gly317 and
Ser/Ala318 were missing, suggesting that these are probable β-lactamases. However, a close inspection of the aminoacid sequence alignment in Fig. 6 of supplement revealed that in these sequences there was a conserved H/KxG motif
about 25 residues before KTG motif of AAM93471, which was detected in acyltransferases of the SxxK superfamilyof diverse functions like endopeptidase of Bacillus cereus and D-aminopeptidase of Ochrobactrum anthropi27). So
these may be probable D-aminopeptidases but not β-lactamases. The second cluster contained 5 sequences: SAV5764,
SCAB52151, Sros2550, Sco0830 and Sace4808 (C2 in Fig. 5 of supplement). In these sequences the active siteamino acid residues Lys315, Thr316, Gly317 and Ser/Ala318 and the conserved H/KxG motif were also missing. The
third cluster consisted of 10 members: SAV1304, SAV3611, Svir16190, Rv1923, Rv0907, SAV5791, Fracci3_0661,
Svir15330, SGR3563, SAV1783, Tbis1664, Svir35970, SAV1261 and SAV5918 (C3 in Fig. 5 of supplement). These
sequences again contained active site Ser64, Lys67, Tyr150 and Asn152, but the active site residues Lys315, Thr316 and
Gly317 and the conserved H/KxG motif were missing. In addition to these residues, Lys67 was also absent in
Amir3080. The important defect of these sequences tentatively classified as Class C was that all of them lacked
commonly the conserved amino acids of Lys315, Thr316 and Gly317, which are involved in the catalytic reaction30, 40).

­− 8 −

Instead of this, C1 group in Fig. 5 of supplement had a conserved H/KxG motif detected in D-aminopeptidases. In
accord with this fact, 8 representative class C β-lactamases formed a distinct cluster from that of the sequences fromActinobacteria (Fig.5 of supplement), although these representative class C enzymes were selected from various subgroups9, 30). Therefore, these sequences tentatively classified as class C β-lactamases are considered to be probable orreserved β-lactamases or acyltransferases for the next generation.
3. 5. Genomic organization
A comparative analysis of Nocardia farcinica and Saccharopolyspora erythraea disclosed that the genomic organizations of the β-lactamase genes (NFA0820/Sace0046) and the nearby regions were arranged in very similar orders
even though these bacteria were not so closely related taxonomically63): two protein kinases, NFA0820 or Sace0046,
a cell division protein, a protein phosphatase, two hypothetical proteins and tRNA (Fig. 5). In addition, amino acid
identities and similarities of these corresponding proteins from Nocardia farcinica29) and Saccharopolyspora erythraea51) were over 50 and 75%, respectively.

However, the genomic organization upstream of a reductase(NFA0780) and a transposase (Sace0042) and a hypothetical protein (NFA0790 or Sace0043) and that downstream ofa transcriptional regulator (NFA0870) and a phosphatase (Sace0051) differed completely, indicating that about 11kb
DNA fragment was inserted in this region by transposition or other mechanisms. A reductase (NFA0780) and atransposase (Sace0042), and a transcriptional regulator (NFA0870) and a phosphatase (Sace0051) were located about
4,000bp upstream and about 7,000bp downstream of the β-lactamase (NFA0820/Sace0046) gene in each bacterium,
respectively (Fig. 5). It is interesting that two transposases were present in Saccharopolyspora but not in Nocardia,
indicating that these transposases may function in gene transfer between these Actinobacteria.
Depending
(A)

on

the

bindingtoblue-dextran,theβ-lactamases of Streptomyces can be divided into twogroups35, 48). S. cacaoi (blue-dextran nonbinding) group and
S. cellulosae/S. fradiae (blue-dextran-binding) group. This
reflects somehow the genomic organization of the(B)
Function

Saccharopolyspora erythreae
Nocarida farcinica
Gene name Gene size (bp) Gene name Gene size (bp)

Homologyβ-lactamase gene and the adjacent region. The genomic

pabA/transposase

NFA770645

SACE041807

overarrangement of Actinosynnema mirum Amir3962 was very

reductase/transposase

NFA7801222

SACE042

1380

over

similar to that of Streptomyces cacaoi β-lactamase72):

hypothetical protein

NFA790

471

SACE043

357

overAmir3960/BlaB,

protein kinase

NFA800

1908

SACE0441962

2.00E-85

protein kinase

NFA8101524

SACE0451310

4.50E-40

䃑-lactamase

NFA8201467

SACE0461452

4.30E-66

cell division protein

NFA8301479

SACE0471437

1.20E-68

protein phosphatase

NFA8401530

SACE0481419

5.80E-54

hypothetical protein

NFA850

468

SACE049465

1.90E-38

hypothetical protein

NFA860

1431

SACE0501314

2.80E-19

tRNA-Leu

NFA80030

86 SACE8003

83

1.60E-04

regulator/phosphatase

NFA870

633

SACE051

1002

over

hypothetical protein

NFA880

2163

SACE052

1542

over

Amir3961/BlaA

and

Amir

3962/β-lactamase. In addition, the percentage identity andsimilarity at their amino acid sequence level were 62.8 and82.6%, 57.9 and 81.4%, and 47.1 and 68.1%, respectively.
BlaA and BlaB are transcriptional regulator or activator
proteins of S. cacaoi β-lactamase. This may be also a caseof the gene transfer between these species. No otherβ-lactamase detected in this study belonged to this class.
However, near a β-lactamase in N. farcinica (NFA23080)

Fig. 5. Genomic organizations of the ș-lactamase genes and nearby regions.

and that of S. avermitilis (Sace1374), there was a transcrip-(A) Gene arrangements of 14 genes nearby ș-lactamase genes in Nocardia

tional regulator whose sequence had some similarity withfarcinica and Saccharopolyspora erythreae, and (B) function, gene name, gene size

BlaA. Moreover, a MerR family transcriptional regulator

and homology between the corresponding genes. Homology was calculated
according to the method of Pearson. “over” means that the homology was notdetected between the two genes with the default parameters.

and a putative β-lactamase was present near a S. avermitilis
β-lactamase­− 9 −(SAV4452).

InS.

roseum,three

β-lactamase-like sequences (Sros5713, Sros5714 and Sros5715) existed in tandem together with an upstream transcriptional regulator (Sros5712). Sros5713 was shown to be very similar to BlaB in the three dimensional level, as
described before.
4. Discussion
In the Genome Information Broker24), 529 sequences are deposited as putative β-lactamases and penicillin-binding
proteins in 4 Streptomyces and 15 closely related Actinobacteria. On the basis of primary screening using the phylogenetic trees and the amino acid alignments, 59 sequences were selected as β-lactamase candidates for further
analysis. However, through more dense and cautious examination the numbers of β-lactamases were restricted to 10class A, 5 class B and one class D enzymes. No candidate remained as class C β-lactamase. The fact that most of the
β-lactamases detected in this study belonged to class A is in accord with the result of enzymatic study50). However, incontrast to the result on the basis of their enzymatic activity, the preliminary phylogenetic analysis and/or the secondary screening noticed twice as many as class C than class A β-lactamase candidates in whole genomic level (Table2). This result may be accounted for by the fact that most of the presumed class C β-lactamase candidates are considered to be enzymatically inactive probable β-lactamases or acyltransferases. However, because only a few nucleotide
substitution can convert them into active β-lactamases, strategies must be prepared for protecting us against thesenext generation β-lactamases. In addition, considering that β-lactam-interacting proteins including β-lactamases arevery flexible to adapt to carry out their catalytic reactions27, 43, 62, 70), it is possible that some of these class C
β-lactamase candidates catalyze the hydrolysis of β-lactams.
It is interesting that the amino acid sequence and the three-dimensional structure of Amir2178 in class A was very
closely related to 1YLT_A, Ctx-M (TEM-3-type) β-lactamase, although active site lysine73, serine130, asparagine132 and threonine235 were replaced by serine, aspartic acid, serine and proline, respectively. So Amir2178
maybe a probable β-lactamase or acyltransferase. Nine other class A sequences formed a cluster with β-lactamases
from Gram-positive bacteria except Sros5706. Sros5706 falled into the cluster of carbapenem-hydrolyzing
β-lactamases from Gram-negative bacteria in the phylogenetic tree (Fig. 3). Similar topology was obtained by PhyML
program. Blast analysis indicates that another sequence SAV4452 in class A was narrowly related to 2OV5_A(KPC-2 carbapenemase).
It should be pointed out here that a Gram-negative OXA-5 and OXA-27 type class D sequence of Tcur2040 was
found in a thermophilic soil bacterium, Thermomonospora curvata (Fig.2). T. curvata produced two class B and oneclass D β-lactamases but no class A and class C enzymes. This is very peculiar in Gram-positive bacteria, because
most of β-lactamases produced in Gram-positive bacteria belong to class A and a few class B β-lactamases. Anotherintriguing thing is that as many as 5 class B sequences were detected in this study and, in addition, all of them belonged to one (B3) of the three subgroups8) of known metallo-β-lactamases (Fig. 4). A similar situation, that is, thepresence of a relatively large number of subgroup B3 metallo-β-lactamases, was reported in remote Alaskan soil bacteria3).
Other interesting features emerged in this study are: First, the nucleotide sequence and hence the amino acid sequence of Mycobacterium tuberculosis Rv2068c10) are completely identical with that of Mycobacterium bovis
JTY208161). Comparison of these two bacteria disclosed that the nucleotide sequences of at least 50kb segment including the β-lactamase and the adjacent vitamin B12 biosynthetic gene cluster were almost completely identical witheach other, implying that a horizontal gene transfer occurred very recently. Probably, these two bacteria belong to thesame species, because the nucleotide sequences of 16S rRNAs were completely identical with each other. In thisconnection, it is intriguing that comparison of the nucleotide and the amino acid sequences of putative Bacillus cer-­− 10 −

eusβ-lactamases BCA2625 and BCA3308 revealed that 97% of the amino acid sequences were identical with eachother, suggesting that the gene duplication occurred very recently in this bacterium (unpublished data). These twogenes were separated by about 650kb. Thus, it is supposed that a horizontal gene transfer and a gene duplication are
not rare events in natural environment, especially in the soil.
Blast analysis in the National Center for Biotechnology Information indicated that NFA0820 and Sace0046 in A1of Fig. 1 were narrowly related to penicillin-binding protein A of M. tuberculosis, and Sros5706, SAV4452 andAmir2178 in A3 of Fig. 1 were to extended type β-lactamases of Gram-negative bacteria like NMC-A, KPC-2, andCTX-M. It is concluded therefore that Actinobacteria, members of soil bacteria, have a potential to produce a various
kind of β-lactamases including canonical class A, extended class A, class B, class C and class D β-lactamases andform a reservoir of these β-lactamases in the next generation.
No β-lactam biosynthesis and/or related gene was found out near the β-lactamase sequences analyzed so far, although thioesterases (Sace4285 and Sace4286) and a LD-carboxypeptidase (SGR456) involved in non-ribosomal
peptide biosynthesis and peptidoglycan recycling existed immediately downstream of a β-lactamase (Sace7283) and
upstream of a β-lactamase (SGR457), respectively. Therefore, the present analysis could not clarify the relationshipbetween the biosynthesis of β-lactam antibiotics and that of β-lactamases in Actinobacteria.
Acknowledgment
I am grateful to the National Institute of Genetics, Mishima (Japan), for permission to use the Unix system for the
genetic analysis.
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­− 13 −

Supplement information
Table 1 of Supplement. The list of β-lactamases used for the classification
Accession number
OriginClass
AF297554
A
Klebsiella pneumoniae
AF395881
A
Klebsiella pneumoniae
D12653
A
Streptomyces cellulosae
D90201A
Streptomyces cacaoi
L25634A
Mycobacterium fortuitus
M15526A
Staphylococcus aureus
M28303
A
Streptomyces albus
U60295
A
Serratia marcescens
V00093A
Bacillus licheniformis
X06599A
Bacillus cereus
Z54190A
Streptomyces clavuligerus
AAA22276
B1
Bacillus cereus
AAA22904
B1
Bacteroides fragilis
AJ492820
B2
Pseudomonas aeruginosa
CAA40386
B2
Aeromonas hydrophila
CAB96921
B3
Fluoribacter gormanii
M15350
B1
Bacillus sp.
M63556
B1
Bacteroides fragilis
P52699B1
Serratia marcescens
AAC68582
C
Morganella morganii
AAM93471
C
Citrobacter freundii
AAT46346
C
Laribacter hongkongensis
AY442183
C
Mycobacterium smegmatis
CAA39987
C
Lysobacter lactamgenus
P05364CEnterobacter cloacae
U14003C
Escherichia coli
X52964
C
Serratia marcescens
Z22590D
Pseudomonas aeruginosa
AP003358
D
Staphylococcus aureus
AE016749
D
Staphylococcus epidermidis
AE016750
D
Staphylococcus epidermidis
CAB13772
D
Bacillus subtilis­− 14 −

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
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1)$9$$3,$5$9/'$*/4**
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7FXU/*55//+5/*9/3<3$                                             Fig. 1 of Supplement. Amino acid alignment of 3 putative and 2 representative (marked with bold letters) class D ȕ-lactamases. The alignment was generated using the MUSCLE
version 3.820). The amino acid numbering was in accord with Ambler et al.5).
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'05,53755///*$9$3/$/93/9$&*4$6*6(6*443*/**&*76$+*6$'$+(.()5$/(..)'$+3*9<$,'75'*4(,7+5$'(5)$<*67).$/4$*$,/$49/5'*5(955*$($'*0'.99+<*4'$,/316397(.+9$''05.3766/75569/*$*/*/**$/$/*677$6$$6$*7736(13$$955/5$/(5(+4$5,*9)$/1/$7*$6//+5$+(/)30&69).7/$$$$9/5'/'+'*64/$59,5<7($'97.6*+$397.'+,'7
6FR//6/5'$$)/00696'17$$'///559*/'$9157$$5/*/7575$9<*)*(0/*705('$*3'*$5$/7'3+9,75/5$/'3$571+6735'075//*$9:5'($&33(<*$$055,0*/49:3+5/$6*)3
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'.':.9('.6*49.<*751',$99533*5$3,996906+*'74'$(3+'(/9$($*/99$'*/.
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Fig. 2 of Supplement. Amino acid alignment of 5 putative and 2 representative (marked with bold letters) class A ȕ-lactamases. The alignment was generated using the MUSCLE version 3.820). The amino acidnumbering was in accord with Ambler et al. 5).­− 15 −

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6&$%0/*/**/*9$*9/*6/*$*6$<$$$3*'$*/:55/*(/(5(<$$5/*9)$+'7$7*579$<5$+(5)3/&69).$/$$*$9/5'/'5'*$</*55
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6*5065/313+$5569/5//**$//6$3/7$&*7(6'$3$363$*.3$*733$5$'*5$)($/(.(<$$5/*9<$9'7*7*779$+5'*(5)$<$67).$/$$*$9/55<*/**/(5,5YF0515*)*55(//9$0$0/9697*&$5+$6*$53$677/3$*$'/$'5)$(/(55<'$5/*9<93$7*77$$,(<5$'(5)$)&67).$3/9$$9/+413/7+/'./
6DFH07$653$9755$//*0$$/93/$*&$5*($3$$333$99$3'+''+/$*/(5.)*$5/*9<$/$7*7*$79$+5$'(5)$)&67).$/$7$$9/+51306+/'$/
$PLU0/775$+5$*0$$//)$/9$*&*4$$33$4$*9$47$777$7657$$3/*63$)$$/(45)'$5/*9<$/'7*6*599$+5$'(5)$<$67).$/$)*$//$(569$'/'(5
6DFH0+41(.59*55$9/$*//$/39$*$&$$3$$3573$3$34$3'/$$5)*3$/$(/(55<'$5/*/7$91957*5(/.+5$'(5)$0&67).7<$$*$//$$+3/$**<)'.9
1)$0$$$$9,$//**&*$'$*6(3$77$$677$36$$7'$$7$()$$/(45)*$5/*9<$9'776*$99$<5$'(5)*0$67).*/$&*$//5(+3/66*<)'49

6URV9+:77$(/4(+6397*.+9.'*079$5/&($$,756'17$$10//.4,**3$*/7$<)+7/.'3965/'5:(7(/11:63.(.5'7773$60*5'/5$977*'$/'$5'5(5/1$:/7$1.7*'$5,
',5<7($'97.6*+$397.'+,'7*07,5'/&'$7,5<6'1&$$1///5(/**37$975)&56/*'3975/'5:(3(/16*(3'557'7763<$,$57<45/9/*1$/153'5$//7'://51777/77)
0,/<74''9(4$'*$*3(7*.341/$1$4/79((/&(96,7$6'1&$$1/0/5(/**3$$975)956/*'5975/'5:(3(/16$(3*597'77635$,757<*5/9/*'$/135'55//76://$1776*'5)
6$9,5<7(4<9.'6*<*397*5$(1/$$*079$(/&$$$96'6'1$$*1///5(/**37$,7*)&56,*'*575/'5:(3'/16$(3:5'7'77635$,*.7<$5/,/*'9/334'575/71:/,$17717$5)
6&$%,)<7.(<$6$$*<63,767'(19$$*079*(/&$$796+6'1*$*1///5(/**37$,75)&56/*'5$75/'5:(3(/17$(3:5,7'77735$,**7<$5///*5$/3'$'5(//7*:/,$16719(5)
'9+<*4'$,/316397(.+9$'*06/5(/&'$99$<6'17$$1//)'4/**55*6759/.4/*'+7760'5<(4(/*6$93*'35'76735$)$('/5$)$9('*(.$$/$31'5(4/1':06*657*'$/,
$PLU,7<(5$(/,*16397*659'**079$(/&'$$,$+''67$/1(//6/9**3($,7$)$)$/*'(475/'5:(3'/1$$(3*'(5'7673*$/$$*<51/9/*7$/(*$5(5$(/5*:/(***79/
6*597<55('/9'+6397(.+9$7*06/*$/&'$$95)6'17$*1//)'$9**35./4$9/$*/*'(9750955(7(/1(:73*$75'76735$/$('/5$)9/*'$/**3(5$5/74:/77177**(/,5YF,7<76'',56,639$44+947*07,*4/&'$$,5<6'*7$$1///$'/**3***7$$)7*</56/*'7965/'$((3(/15'33*'(5'7773+$,$/9/44/9/*1$/33'.5$//7':0$5177*$.5,
6DFH97<*($'/0.669,754+967*07,54/&'$$95<6'*7$*1///5'/**3$(/7$<$5*/*'(9750'59(3/,9($73*'35'76635$)*7')'(/9/.'$/3('.5$)/5'//45*77*$55,
$PLU,7)7*((/97<63,7(*.9'7*07/5(9$'$$95+6'17$*1///5(/**'$*/$**3$$)(5$/5$,*'77763$59(7(/1($93*'35'7673($/$$'/55<$9'$'9/$''55(//96705$177*'($,
6DFH,5<75$'/5316397(759'7*079$(/&($$,756'1$$*1///$(/**3$$,$3)$56,*'3775/'5:(9(/17$,3*'(5'7773$*,$$*<5$099*'$/*$3(5(4/..:/,$177**(5,1)$95<65((996<6397(759'7*079$(/&+$7,796'17$*14,/.//**3$*)7$)/56/*'(965/'5:(7(/1(933*((5'7773$$9$$1<5$/9/*'9/$(3(5$4/5':/9$177*'45,
6URV5$*/3.7:79*'.7*716.<*$*1',$99:3*.6$$3,,06,<715*$$'$$9''.9,$'7$$,/$5$/*./
'57*/3.*:79$'.6***'7<*751($$,$:73'*$39//7$/7+.36/37$3*'73/,,./$79/6($9$3$
05$*/3'':7/*'.7*$*5<*711'$*97:33*5$3,9/79/7$.7(4'$$5''*/9$'$$59/$(7/*
6$95$*/3$':,/$'.7***16<*9$1'9*99:331563,9079/77.+$$$*36'63/9$57$(//$$(/7
6&$%5$*/3$*:7/$'.7***6$<*9$1'9*99:33*533/9/69/67.<'3$*37'1$/9$5$$*/9$*$/7$
'5$*93.':.9('.6*49.<*751',$99533*5$3,996906+*'74'$(3+'(/9$($*/99$'*/.$PLU569935*:99**.3*$$(<*75&'$$,$:33'5'3/999/06753(5'$5$'*7//*'$$59$/*$0*9*
6*55$*93.*:79*'.7*$*67<*751',$99:33'$$3/9/$9/6157$$'$'+'17/,$.$$6$$97$/75
5YF5$*)3$':.9,'.7*7*'<*5$1',$99:637*93<99$906'5$***<'$(35($//$($$7&9$*9/$
6DFH.$$933*:79$1.7*7*'<*7/1',$99:33'6$3/9,$,066.3$.'$(<'($//$($$$+9,$7/7
$PLU5$*935*:99$'.7*6$5<**51',$9/:33'5$3,9/$9/6*.$7$'$(+''$//$($75$99(//'
6DFH5$*/331:97*'.7*6*'<*7$1'9$97:7(6*'$,9,$,/76.6$3'5.<'15/+$'7$5,99($/5
1)$5$*93$*:79*'.7***6+**11'9$9$:7(7*'3,9,$//6+57'3$$.$'1$//$($75$997$/5


Fig. 3 of Supplement. Amino acid alignment of 10 putative and 3 representative (marked with bold letters) class A ȕ-lactamases. The alignment was generated using the MUSCLE version 3.820). The aminoacid numbering was in accord with Ambler et al.5).


7FXU07$$5(53+0/(9$369+$<,43'**:&/11$*/,9*('&79/,'7$$7(5575$/'$$955/$34*3'//917+)+*'+7)*1$<).35
7FXU0$76*$'7$5(39/+(/*$*9)*:943'*7::911$*$,673(*7/,9'7&$7($5755)/$$95($7*'93/5<$917+/+*'+7+*16//3(63SD0+,3/:+65936$7/656+69//3/6/7$+63.5$5/9$06713(,34:)$/7359:7769(3(697&*/9$*('+9//,'7*673(4*+7/$06$$50/*539'5,997+++<'+6**/3*,7
6*50'9$:(')*:(5/*5*9*555/3*:'$79$/9$*$(*9//<'7*67/5(*9(/554$($//*5597+,$/6+3+)'+9/*7$$)$
5Y0+)':(5/7'69+5&5/3)&'979*/95*57*,//9'7*77/*($7$,$$'9.4,$*&497+99/7+.+)'+9/*669)'$-0163.65$//*)0*$)&///9$*$3/6$.66'+9'/3<1/7$7.,'6'9)997'5')<6619/9$.0/'*799,9663)(1/*747/0':9$.70.3..99$,17+)+/'*7**1(,<..0
00.79),/,60/)39$90$4.69.,6'',6,74/6'.9<7<96/$(,(*:*09361*0,9,11+4$$//'73,1'$47(0/91:97'6/+$.977),31+:+*'&,**/*</45.
00..17//.9*/&96//*774)967,6694$64.9(4,9,.1(7*7,6,64/1.19:9+7(/*<)1*($9361*/9/176.*/9/9'66:'1./7.(/,(09(..)4.597'9,,7+$+$'5,**,7$/.(5
7FXU$7,,$+(1&$5'4/$$*3$/5(/:3(9':*573,7$3'97<5*($9/+3**+59(/)+3*3$+7$*'799:/3'6*9/)7*'99:6$773)&/0*6,7*6/(9,*5/5(/5359993*+*39**7FXU79/,*+((75.*,/$'),,'*&33/:(3/3':*$975533(97/76(/$97/*'55,(/5+3*<$$+73*'999:/3((59/)7*'//)+*/730/*0*697*$/56/':,$6)(3'5993*+*3,&'*$
3SD'$(9:0+($$/$+&3'/+9'+396/0$<91/**/*$(9,+3*3$+7'*'/,9,95'(.,7)9*'/9(7$*(346'*77'95*:35$,'69,7*7'*$*/<93*+*43,'
6*5*959+*6$*/7$//5'*(4$/+*'$95+*93('($$56$'7/993+((95*(57/'/***559//$'/*3$+66+'/$9/93*6'*(3399/&*'/9((6*(34$*3'$93$5:3$$/*5//(/**('$/<93*+*$99'
5Y4$(9)&$3(99(</56$7'5/5('$/6<*$'7$(9'5$,$$/.334+*,<'$$9'/*'5797,7+3*6*+77$'/999$3$7*+$'*3799)7*'/9((6$'3','$'6'/$$:3$7/'59/$,**3'$6<93*+*.99'$-*$(7:66'/7.4/5/((1..'5,.$$()<.1('/.55,/66+393$'19)'/.4*.9)6)61(/9(96)3*3$+63'1999<)3...//)**&0,.3.(/*</*'$19.$:3'6$55/..)'$.,9,3*+*(:**0*946<$1407,'/$.(.*/393(+*)7'6/796/'*03/4&<</***+$7'1,99:/37(1,/)**&0/.'14$76,*1,6'$'97$:3.7/'.9.$.)36$5<993*+*'<**0*,.$+67$/7$(/$..6*<((3/*'/47971/.)*17.9(7)<3*.*+7('1,99:/34<4,/$**&/9.6$($.1/*19$'$<91(:676,(10/.5<51,1/993*+*.9*'
7FXU3(/)'47(6</7://(9$((*95$*53$/(7$5&$'/*5)$$//'6(5/9*1/+5$<$(/$*/33*$3,'9*6$)$(09()+*$/3$&+$
7FXU$/3'9/$$+(5<<5)//(9*7'*0.'*/63/($$45$'/*()$$:$'$(5/9)1/+.)<$'77*(3)'/$4$$/'797)1**306769
3SD$7'90145$4/$(5,371,3/7'5+,3/3
6*5$$)95$45$$/$(5)$$
5Y$4)95545$:/575$65435(73$7/3&.5$-3(091.7,.9$(.$9*(05/
07(/,(+7.4,914<,(676.3
0.*///+7/'//.


Fig. 4 of Supplement. Amino acid alignment of 5 putative and 3 representative (marked with bold letters) class B ȕ-lactamases. The alignment is generated using the MUSCLE version 3.820) The amino acidnumbering is in accord with Galleni et al., 25).
­− 16 −

Sco3912
SAV4283
SCAB46171
SGR3667
Svir33850
Fraal6499
SAV2663
SAV4455

C1

SAV5004
Svir23940
Tbis0343
SCAB26931
Amir4262
JTY17443
SCAB55861
SAV5764
SCAB52151
Sros2550

C2

Sco0830
Sace4808
U14003
AAM93471
P05364
AAC68582
X52964

Class CCAA39987
AY442183
AAT46346
SAV1304
SAV3611
Svir16190
Rv1923
Rv0907
SAV5791
Francci3
Svir15330

C3

SGR3563
SAV1783
Tbis1664
Svir35970
SAV1261
SAV5918
Amir3080
D90201㻜㻚㻝Fig. 5 of Supplement. Phylogenetic tree based on amino acid sequences of 35 putative and 8 representative class Cȕ-lactamases (square boxed). The tree was constructed by MEGA version 466) and the neighbor-joining programwith the default values. A class A ȕ-lactamase (D90201) was used as an outgroup.

­− 17 −


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-7<0&33,,/66$737*75&*75+*5$997(<95$/'5/3+(,$7$99(791&$'3*$$)'(/'$.,1$*0.$<$,3*9
$PLU063+'$33536$375$$56559*$/9$9$$$$//$*&*$$3*4*3$3+9773$$3(7$5/73$'91$:/'*9,3$*/(1**99*$
6&$%0555/++353$,*,73373(*3$07453$/'/$+:445/'7/5$$+*93*$7/$)/$+*(
6$90:9/6/)*:64307+6795/55*$97$,$9*7/093)$**$$6*$3$6$7$$$7$6$7$9(33736'6$6$67*')57/73$97.49'($955905($1
7ELV03.&3.,9*//0/*7$/66*7039*73$$$67**$$97*/*6$+5$$5$**+''35'*4:4695$5(54441//5'97/$/730446(7'65$/3<154.7'$07(6<6'45*/6',765$6*'4.163
6&$%0/$3/+$3/39**3$$+$*33**'76,$*)9557/35'***79/$$5*'(
6*5076+'$'9/3*755$//+5,$9$45'*5$36/9$$9554*5
6&$%07734((//3$755$//+5,$9$4$(*5736/9$$995**(
6FR07764*(//37755$/7+5,$9$4$(*5736/9$$995**5
6$90.764((//3$755$//+5,$9$4$(*5$36/$$$995**4
6YLU0/377()$//559$7(4$6*5$36/6$$9951*(
)UDDO0$3$653*3***//3$7(5///459$$(4$**5$36/9$$9$5'*5
6YLU053/5,39/*$9/6/$$7$),*3$9$9$6(7(7$$(,/5(*$4/*9'1*<3*9,*/95'*7'
6$9016675,$/&$$/$)$997*3$$6$)$'$*63$$*76$5$74*$*6/167$/54$/$*/3'''$7$$9959$*4
6$903.357$57357//$,396/$//*)$3**)4$3773$7'$//3//975**$3$$
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$$03<<)7:*.$',$11+397447/)(/*696.7)1*9/**'$,$5*(,./6'397.<:3(/7*.4:5*,6//+/$7<7$**/3/4,3''97
-7<$9$9:$**4(<9.*<*9719'+3039'*'79)5,*677.7)7*79005/9(5*.9'/'63955<,3')$9$'(6$6$797954//1+7$*:'*51*4')*5*''
$PLU79699+'*49/7$5*<*/$'7*(1*17*($3739'3(.+/)53*696./97$7$904494$*.,'/'$',.4</')'/37()'73,775+//7+73*)((59$1//5/'*
6&$%,+(/$6*9/67$7*9($77'69)4/*6,$.9<7$$/904/9(6*(/'/'9399.9/3()$79'3$$7(9,7350//6+76*/7&')+,'7*5*''
6$993*979*/:76'4*4+956)*9$'.6**(5063'/<05,*6(7.7)797$//(/9'(+.9*/''3,*.<9'*931*'.,7/5(/$*056*/)1<7(''*)).$/76'3553)
7ELV'713397$:$36'1$$*$$0/$</5'/$$515)7*6$/9955*59/$5)$$*:$'((.5,3153'79<5906,7.4)760/9/./5'5*//(/''3,&3</93.</.7&3$$:53,7,507/7+76*,31,(7/3*</$197537
6&$%/$<&$*)*7$'5$$*,$$6&579<'90607.4)7$$$,/./(90*5/597'5,*5+/*3935'.5*,79(+//7+76*/9(*/*''<'39
6*57$:6*$57&9(*+$3'$'74<5,*6,7.7)7$9/9/5/5'(*/,'/''3/(.+/3*7*6**979)4//*+6$*/*$($3$3::(573*6
6&$%79:1*$5769(*+$3'(194<5,*6,7.7)7$9/9/5/5'(*//'/*'3/(.+/3*7*$*($7,$(//$+7**/$$(6363::(573*$
6FR$9:+*$5769'*+*3'(194<5,*6,7.7)7$9/9/5/5'(*9/'/*'3/(.+/3*7*$*($7,$(//$+6**/$$(63$3::(573*6
6$979:<*65769'*+$3'(194<5,*6,7.7)7$9/9/5/5'(*9/'/*'3/(.+/3*7*$*($7,$(//$+7**/$$(63$3::(573*6
6YLU/9:6$$5*(9'*$73'157/<5,*6,7.6/9$$9905/5'(*5/'/1'3/'.+93*7()**/79$4//$+76*/76(63*$::(573**
)UDDO39:$9*5*5$9*(*&$9*(*&$9*(*5*6553'$'74<5,*6,7.7)7$9/905/5'(*5/$/*'3/'5+/3*7$9*'579$4//6+/**,$$(7333::(59$*4
6YLU7(<94$*/*'5)7593$'34$5)5,*617.$),$79//4/($('5/6/''7/'7:/3'9)$56*+'*67,7954//1+76*/3(</1'359/$6<$$1/13<5
6$9'*7:5*9**9+'7(7*5($,$'*5)5$*697.9)7$$99/4/9$(+59*/'5394'</.*/)3$'533,79$4//1+76*,43$'*$*'6)$'4<$+5)'67
6$9$//$5((6*,5)$5$*5*,'5$'+)5$*6,7.7),$799/4/$$(+5/6/6'69('+/3*/95*$*+'*5$97/5$//7+76*/$')7$'7$*7939

$$0'.$(//5)<41:434:73*$.5/<$166,*/)*$/99.+6*06<(($07559/43/./$+7:,79346(4.'<$:*<5(*.39+963*4/'$($<*9.669,'0$5:94$10'$6/
-7<$9$/<9.$075/34/733*7$)$<116*/99$*5,,(/9$*77<(67945///'3/4/$+75<)6'4,,*/19$$6+699'*.3,$97'):7)356&137**/067$5'4/5<$4)+/*'*5$31*(4,$PLU93$1/5($/97'33$49<$3*773$<61<*16/$*</9(597*75)((<9(5(9)'5,*07666)(43/3(5/.'506**<1'6$139$43)(,97733$*$/7$6$7'0$5)0/$+/*$47**73/
6&$%$,$5<9($$.*9$0'&33*7$96<6*,*<99/*5,9(9/7*/7:'4$/5(59)$3/*/$+707/3(($/3)59$0*+0$*('*7473$3$:'/035$$*3*$59/$7$*'995<$.$+/'**$*,
6$97344//'<$).+39/)33*(.)6<&171/,//*/99(.$**53/*'),5419/(3$*01+76/3$'$$)313+$4*<7'47$7*(9(1$7':136:*:$$*$0,6'/''/57:$579$7*,/3'*'6/
7ELV795./,54)91/3/').3*76:.<716*<9/$*$,,4$97+.3<*99/5./,7.3/./5+7*<657<335*<$.*<9.3*63$3//1*6(/)6$7*/<779'',95:'5$)*6<79
6&$%65((/95*$/$6./56$3*$()+<617*<6//$$,9(.$6*(*<($)/$5+/)$3$*0557*<9/35:355/9$9(<''5*565*53)'+3:$$'*3<:1/5*1**0/67$('9)5:+5$//*((9
6*5/53(/$'9/*'5345+3$*+5++<613*<7//*6/9($95*96:(($/57(,/(3/*0+5776*39$3+$$*:$9+3:$'90/6(3$('/*,0$3$*4/:67$$'//5)$$)/7(*''59
6&$%/53(/$'9/*(43+5+39*55)+<613*)7//*$/9(495*$$:('9/55(9/(3/*/5576:+3($3+$**:$9+3:$'90/3(.9('/*50*3$*4/:67$$'/$5)$9)/$5*''59
6FR/53(/$'9/*.43//+39*55++<613*<7//*$/9(./5*$3:((9/45(9/'3/(/557*7536$3+$**:$9+3:$'90/$(39('/*50$3$*4/:677*'/$5)$9)/7/*''59
6$9/53(/$'9/*(/3/5+39*55++<613*<7//*$/9((/5*$3:((9/55(9/(3/*05576*'35$3+$**:$9+3:$'90/3(3$('/*50$3$*4/:677*'/$5)$9)/$4*'(59
6YLU7:7(/166/*31(/.+53*55)+<619*<*9/*(/9$5/5*77:/4$95((,/53/*06+77'53(*5+$4*<$9+3)$'99/3(3(+'$*$0$3$*4/:6&,''/$5:7$)9**'7*(9)UDDO$+37/$'$/*3'3'5)53*55++<619*)$9/*(/9$5/5*76:$4$/+5(//$3/*09577$/39$3+$+*)$9+3+$'9//3(3$+'$*9/$3$*4/:67$$'/95:6$9/$*+7'$9
6YLU.:(35./9(,$/54*$7*$3*(5+6<6171<//$*09,($97*133+9(9545,,63/*/+'76)37'3534*(:/5*<)5,**)/5'979619',7*6$*$0967/''/$7)(5$//6*(/
6$97354/9$6$/6.3/()73*744'</1,1<79/*//,(.97*76<(+49455,/'3/+/5'7<)3*'',5,5*$+1+*<4$7$'*/9'97(:1$6$*:$$*1,,667$'/(5)77$/)**.,
6$973/4$95,$/7+33$'5*5)$<6171<9//*0994497*+6<$9($(55,973/5/7*76)3*$57$/3'3+*5$<$$'*7'97(/'359$*$$*(/977/$'/'5)<$$//**(/
$$094(.7/44*,(/$465<:5,*'0<4*/*:(0/1:3/.$'6,,1*6'6.9$/$$/3$9(9133$3$9.$6:9+.7*67**)*6<9$)93(.1/*,90/$1.6<3-7</6546/.$05613*$**7/:9(/7*0*97:0/536$(197,9(+**7:.*456*)9093'51)$079/716'**)+0,1'/)$6':$/45)$*/61/3$7345/*$9
$PLU/'($70./0+(*$/'$(4/**/$'*3507/*))'(651*+5,,*+**'71))+6+/4/<3*$*$*.*7*/)96)167*91**94*951$9/1*)$'5<)3.*$67**767(*$6$0(<'+'$$3/$5'($9(6&$%/53(796$04$5(7'93'.:796$'*:*/*:7/<':'*93*+*+'*$$7*4<$)/59$37.*9$9$//71**$65//<$'//5(/0$(3

6$9,63974.45/173377,3*$*<*/*,)194*:,*+1*6/3*<(6/7,</36$4$7/99//17',1+$*7(367/)*($,7.,97
7ELV$33$79.4$)734$.&331*&/1/366$<$)*:/9'5/4*+5/5<+3*/)0*<7$010</3''',4999/6194'7'7$*,$5+/$7/$/'
6&$%/3$5$5'./)(3595(3*6766<*<*:697'73'*3)$:+'**1':6/*//*563$'*9/9):,6
6*5/6$6695(0573$$3$(7*$6'<*/*/4,9**(*59/)*+6*6/3*)9$*/:,&(*('9$$99/$1976*/3$$09$$(//*,99'
6&$%/&$$695(0573$$3*($$'96$*9$<*/*04/4+5**5)/9*+**693*)/$4/7/69(''9$$99/71&76*3996(9$$'/95,9$(
6FR/*$$795(05$3$$3$(6$'99'*$$<*/*/4,4+5'*5//9*+6*6/3*)/$1/7,*9(''9$$99/71&76*3//$*9*$'/95,9$(
6$9/6$'695(0573$$3$($$'906*6$<*/*04/4<5'*5//9*+6*6/3*)/$1/7969(''/$$99/91&76*3036$9$$'/95,9$(
6YLU/+3'79$(05($$9,''6*3:7**<*/*/4/$49+*.5/$*+7*603*)/$&90,'3$757*$9$/$1776*,*,/6/7$'/,$0$''
)UDDO/6$'7/$(05(3$$9(*'6*(:5/*$*/*94//5(3**9/9*+**603*)9$$/:7535*7$$9$0$1$76*9$99'/$9*/,'$9'7
6YLU/3(34/$$/.'793/7('*6$*$*/*9)/7373&*39:4+6*$//*<77/:/76''*'.4999$$1(<+/9***3*(4+,$5
6$9935$4)5+0/(93$*,3*$9470*06509/3'*797<*.7*(5<*<$7*,*$,/''+*5,557/9<69167'$.6.1*1$59
6$9/3$+*/5(0/'757$+*6<*0*/<39./3&*779:*+1*5,6*6<957$$79'*559/7)5917'*96(3*/(

$$013$59($$:5,/(./4
-7<'/$3<(*5<,$.49$41*'/(779,')5$5'*4/$*6067''$13'*416$1/*/$)<53'<*/'/*3'1.37*6561)95*3'*1,$:)&64+*5/)554
$PLU/$$594*(<'66550'67//6$,'/7657797$5('*7//)43*3$6$63$9<($9*(+7:5(9**459/$75,('*597*,$<('9)7//373$456$*/9/39//*6/99/9/$996:3,$$9$556$*9$35'5$*
6&$%$*9703$3)$3$$(33$9',73:7*7<.5(*99,7,*(51*73+/7<$)9'*09*<63'/(0'/93/6(79)$*$***66)$(':0399)$7/6'*
6$93*+9)1/3$43$$5
7ELV/
6&$%14$65$*(:1/('9$4(/7/*/9(595$(*$
6*5$(3/,3(3:53/3(9''(//$/7*9:+:*7+397/5/7*'5*/4/73/**4*5*$$)7<*3'*7:7*41'<<$*(7/59955''*79'+/'/*6)9)
6&$%$(35,3(3:53/3$9'$69/(/$*3:<:*7<6)9/5/$*'**/6/'3/6$7/5*$5)5$1''*6:7*/1*<)$*(//5$/553'*796+/'/*6)9)
6FR$(35,3(3:53/5(9'6$9/(/$*4:<:*7+$)$/5/73'**96/*3/6*4*5565)5$1*'*6:7
6$9$(35,3(3:53/3'9'(*9/(/$*4:<:*74$)9/5/79'**96/(3/6*9*55$5/5$1*'*7:7*/'*<<$*(//.$9553'*696+/'/*6)9)
6YLU+(33/3$(:53$39'3$//(/7*/:+:*3$3<+/5,/31*:/'/63$9*7*5$65)53/*''5:/*/'*<<$*(7/79*5'6'*53+</'/$7)9)
)UDDO$(33/$$(:93$39$*5(3/'97*9:<:*3$3<$/6/53'**///$3/*56*5565)53*3**6*7)/*9'*<)$*(7/,34556'*$/6+/(/$6)9)
6YLU$$9($<&$/
6$9/3,94$:06
6$93$9/$$()&3576
$$0
-7<
$PLU5/$59/$.9*7/6*9/$$$*:$$697$/04/4(,3$$4,56/4//4//$$/$993$*95/<''$5557*:7599*6$/9//$/,*/$:)$9()1)/$3',6<
6&$%75&$<,*05$$3.9$
6$9
7ELV
6&$%
6*575(3<($*7$93**9'$(*:5*/$'
6&$%7543<((*$393**9'3(*:5*,3
6FR
6$97543<'(*$393**9'3(*:5*,3
6YLU7573<'37$393**9'$$*:4303(5
)UDDO75$3<$3$*393**9'$$*:5$63$3$3$3$3$$***4*
6YLU
6$9
6$9


Fig. 6 of Supplement. Amino acid alignment of 15 putative and one representative (marked with bold letters) class C ȕ-lactamases. The alignment was generated using the MUSCLE version 3.820).
The amino acid numbering was in accord with Lobkovsky et al, 40).­− 18 −