Cloning and sequencing of the gene encoding the AmpC β-lactamase of Morganella morganii

FEMS Microbiology Letters 148 (1997) 15^20

Cloning and sequencing of the gene encoding the AmpC L-lactamase of Morganella morganii Guileéne Barnaud a , Guillaume Arlet a *, Claude Danglot b , Alain Philippon ;

a

a

è Paris VII Denis Diderot, Ho ê pital Saint-Louis, avenue Claude Vellefaux, 75475 Paris Cedex 10, France Service de Microbiologie, Universite

b

CRECEP, Paris, France

Received 24 December 1996; accepted 2 January 1997

Abstract

The chromosomal L-lactamase gene of a clinical isolate of Morganella morganii was cloned in Escherichia coli and sequenced. The L-lactamase had a pI of 7.4 and conferred a typical AmpC susceptibility pattern. The insert obtained was found to encode a protein of 379 amino acids. Its deduced amino acid sequence revealed it to be a class C L-lactamase: 39^56% identity with chromosomal AmpC L-lactamases of Serratia marcescens, Yersinia enterocolitica, Citrobacter freundii, Enterobacter cloacae and Escherichia coli ; and 37^56% identity with plasmid-mediated L-lactamases (MOX-1, CMY-1, FOX-1, ACT-1, LAT-1, BIL-1 and CMY-2). The ampC gene was linked to a gene only part of which (450 bp) was cloned homologous to the regulatory ampR genes of chromosomal class C L-lactamases. Keywords : Morganella morganii

;

ampC

gene; L-Lactamase

1. Introduction

Many members of the Enterobacteriaceae including Escherichia coli, Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, and Yersinia enterocolitica are naturally resistant to ampicillin or amoxicillin and ¢rst generation cephalosporins. The resistance phenotype to the second generation cephalosporins is variable. All these species produce a chromosomally encoded L-lactamase belonging to class C (ampC gene) [1,2], mainly inducible. The AmpC induction mechanisms in Citrobacter freundii and Enterobacter cloacae involve other genes, such as ampR, ampD. The ¢rst is located immediately up* Corresponding author. Tel.: +33 (1) 42 49 94 87; fax: +33 (1) 42 49 92 00; e-mail: [email protected]

stream from the ampC gene and is divergently transcribed [3^6]. It encodes a transcriptional regulator of the LysR family [7]. The product of the ampD gene interacts with AmpR protein and mutations in the ampD gene resulting in overproduction of the Llactamase [6] are responsible for the resistance to third generation cephalosporins observed among these species in the 1980s. The nucleotide sequences of the chromosomal L-lactamases of C. freundii, E. cloacae, Y. enterocolitica, S. marcescens, and E. coli have been determined [8^11]. More recently, seven plasmid-mediated AmpC type L-lactamases were described in clinical isolates of Klebsiella pneumoniae and E. coli [12]. Some have high DNA or amino acid sequence similarity with the chromosomal AmpC L-lactamases of C. freundii or E. cloacae, whereas the phylogeny of the others is unclear

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 0 0 6 - 2

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16

(review in [13], P. Bradford et al., 36th Interscience Congress on Antimicrobial Agents and Chemotherapy (ICAAC), New Orleans, 1996, Abstract C31). However, the nucleotide sequences of several chromosomally

encoded

L-lactamases

AmpC

are

un-

known. We therefore isolated the chromosomal

L-

Fig.

ampC gene from a clinical isolate of Morganella morganii and determined its nucleotide se-

1. Restriction map and

ORF map of the insert cloned

E. coli

pBGS 18+ conferring resistance to ampicillin in

lactamase

(B,

BamHI ;

E,

EcoRV ;

in

JM101

SmaI).

S,

quence.

2.3. L-Lactamase assays 2. Materials and methods Analytical

2.1. Bacterial strains and plasmids

isoelectric

polyacrylamide

gels

focusing

[16]

with

was

crude

performed cell-free

in

soni-

cated extracts. TEM-1 (pIP 1100, pI 5.4), TEM-3

M. morganii

(pCFF 04, pI 6.3), TEM-4 (pUD 16, pI 5.9) and

strain SLM01 was isolated at Saint-

Louis hospital (Paris) in 1996 from a clinical speci-

OXA-1

men (urinary tract infection) and identi¢ed with the

markers.

è rieux S.A., Marcy l'Etoile, API system 20 E (bioMe

classic

E. coli JM101 (supE thi v(lac-proAB) ‡ lacIq lacZ-vM15]) was used as FP[traD36 proAB host

for

transformation.

The

plasmid

238,

pI

L-Lactamase

chromogenic

7.4)

were

used

as

pI

activity was detected by the

nitroce¢n

method.

Inhibition

of benzylpenicillin hydrolysis by cloxacillin (1 mM)

France).

the

(RGN

and clavulanic acid (1 mM) was determined as de-

vector

scribed elsewhere [17].

pBGS18 [14] carrying a kanamycin resistance marker

2.4. Enzymes

was used for cloning and was prepared by the alkaline lysis method [15].

All

2.2. Determination of susceptibility pattern

used

enzymes according

supplier MICs were determined by an agar dilution method

for to

DNA the

manipulations

recommendations

(Boehringer-Mannheim

were of

France

the

S.A.,

France).

using Mueller-Hinton agar (Sano¢ Diagnostics Pas-

4

2.5. Cloning the ampC gene

teur, Marnes-la-Coquette, France). Inocula of 10 ^ 10

5

CFU per spot were delivered using a multipoint

inoculator.

Chromosomal DNA of

M. morganii

SLM01 was

Table 1 In vitro activities of nine

L-lactams

for

L-Lactam

M. morganii

SLM01,

M. morganii Amoxicillin Amoxicillin+clavulanic acid (2 Ticarcillin Cephalothin Cefuroxime

E. coli

MIC of strain (

Wg/ml)

v v v

transformant (pMOR) and

E. coli

JM101 recipient

Wg/ml)

SLM01

E. coli

JM101(pMOR)

E. coli

512

256

8

512

256

4

8

16

4

512

512

16

16

16

2

Cefoxitin

8

8

4

Cefotaxime

0.06

2

0.06

Cefepime

0.06

0.12

0.12

Moxalactam

0.25

0.25

0.12

Imipenem

1

0.25

0.25

FEMSLE 7434 24-3-97

JM101

G. Barnaud et al. / FEMS Microbiology Letters 148 (1997) 15^20

Fig. 2. Nucleotide sequence of

3

35 and

3

M. morganii

AmpC

L-lactamase

and partial nucleotide sequence of

BamHI

M. morganii

AmpR regulator. Putative

10 regions of the two genes are boxed.

prepared by the method of Grimont [18], partially digested

17

with site

of

Sau3A pBGS18.

and

ligated

Recombinant

were introduced into

E. coli

JM101 by transforma-

the

tion with CaCl2 [15] and transformants were selected

plasmids

on Mueller-Hinton agar supplemented with amoxi-

into

FEMSLE 7434 24-3-97

18

G. Barnaud et al. / FEMS Microbiology Letters 148 (1997) 15^20

Fig. 3. Multiple sequence alignment of the amino acid sequences of AmpC L-lactamases: 1, E. cloacae P99; 2, C. freundii OS60; 3, E. coli K12; 4, M. morganii SLM01; 5, Y. enterocolitica IP97; 6, S. marcescens SR50. Identical amino acids are boxed.

cillin (20 Wg/ml) and kanamycin (50 Wg/ml). The molecular size of the insert was estimated using restriction enzymes and electrophoresis on 1^3% agarose gels. 2.6. Sequencing

DNA sequences were determined with the procedure of Sanger et al. [19], using £uorescent dye-la-

beled dideoxynucleotides, thermal cycling with Taq polymerase and the ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA, USA). Three runs were performed. The BLASTN program at the NCBI was used for database searches. The Clustal V program was used for the alignment of multiple protein sequences. The GenBank accession number for the nucleotide sequence reported in this paper is 10283.

Table 2 Percent identity of amino acid sequences of the AmpC L-lactamase from M. morganii SLM01 with other chromosomal AmpC-type L-lactamases of Enterobacteriaceae Strain M. morganii E. coli C. freundii E. cloacae Y. enterocolitica S. marcescens SLM01 K12 OS60 P99 IP97 SR50 M. morganii SLM01 100 56 55 54 53 39 E. coli K12 100 74 68 55 37 C. freundii OS60 100 73 56 39 E. cloacae P99 100 54 41 Y. enterocolitica IP97 100 34 S. marcescens SR50 100

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G. Barnaud et al. / FEMS Microbiology Letters 148 (1997) 15^20

19

Fig. 4. Multiple sequence alignment of the partial amino acid sequences of AmpR genes: 1, S. marcescens SR50; 2, C. freundii OS60; 3, E. cloacae P99; 4, Y. enterocolitica IP97; 5, M. morganii SLM01. Identical amino acids are boxed. The predicted helix-turn-helix DNA-binding motif of LysR family proteins is shown (HTH). 3. Results and discussion

3.1. Cloning the ampC gene and expression in E. coli recipient

One E. coli transformant was obtained on selective medium supplemented with kanamycin and amoxicillin. It harbored a recombinant plasmid designated pMOR with an insert of about 3.4 kb. The MICs for the parental strain of M. morganii, the E. coli transformant and the E. coli JM101 recipient strain were determined (Table 1). The pattern of L-lactam susceptibility of the transformant was similar to that of the clinical isolate of M. morganii : resistance to amoxicillin, amoxicillin-clavulanate in combination, cephalothin, and cefuroxime. The susceptibility to cefotaxime of the transformant was moderately lower than that of the parental strain. Susceptibility to ticarcillin, cefoxitin, cefepime, moxalactam and imipenem was not a¡ected by the presence of the recombinant plasmid. These results are similar to those previously reported [20]. The antagonism observed between cefoxitin or imipenem and cefotaxime in the clinical strain by the di¡usion method was not observed in the E. coli transformant. By isoelectric focusing, the single band of L-lactamase activity in the E. coli transformant cofocused with the cephalosporinase of the wild-type strain of M. morganii SLM01 with an isoelectric point of 7.4 (results not shown). Yang and Livermore report that eight M. morganii strains produced L-lactamases focusing at pI 7.6 [20]. As assessed by the iodometric procedure, benzylpenicillin hydrolysis activity of this enzyme

was strongly inhibited by cloxacillin and not by clavulanic acid. Thus the susceptibility pattern and enzymatic properties of the E. coli recombinant suggest that it expressed a typical AmpC L-lactamase [21]. 3.2. Nucleotide sequence

The restriction map and the nucleotide sequence were determined. It was 3307 bp long and contained three open reading frames (ORFs) (Fig. 1). The ¢rst of 1425 bp designated ORF1 could not be identi¢ed after searches in the NCBI database with the BLASTN program. ORF2 (Fig. 2) was from an ATG start codon at position 546 to a stop codon at position 1683. GenBank database searches with ORF2 revealed similarities (less than 60%) with chromosomal and plasmidmediated L-lactamases of class C. This structural gene encoded a protein of 379 amino acids (aa) and its sequence was 39^56% identical to those of chromosomal AmpC L-lactamases from C. freundii, E. cloacae, Y. enterocolitica, S. marcescens and E. coli (Table 2) and 37^56% with those of seven plasmid-mediated AmpC L-lactamases, MOX-1, CMY-1, FOX-1, ACT-1, LAT-1, BIL-1 and CMY-2 (results not shown). The deduced peptide sequence contained conserved motifs found in serine L-lactamases [22]: the SXSK motif of the active site of AmpC, the class C typical motif YXN and the KTG domain (Fig. 3). The L-lactamase from M. morganii is thus a new member of the class C L-lactamases. A third ORF (ORF3) (Fig. 2) of 435 bp was found. Its direction of transcription was opposite

FEMSLE 7434 24-3-97

G. Barnaud et al. / FEMS Microbiology Letters 148 (1997) 15^20

20

to that of

ampC.

It was incomplete and started with

the ATG codon at position 435. By analogy with the

ampC-ampR

arrangement of the

genes in Enterobac-

L-lactamases,

teriaceae producing inducible AmpC

this ORF may correspond to the regulatory gene

ampR.

The predicted translation product of 145 res-

idues aligned with AmpR protein sequences from

freundii, E. cloacae

and

Y. enterocolitica

C.

(Fig. 4). It

possessed the helix-turn-helix motif involved in the DNA binding properties of these proteins [23]. The 110-bp region between the

ampR

and

ampC

start codons contained overlapping promoters (Fig.

ampC

beta-lactamase gene.

è, [9] Galleni, M. Lindberg, F. Normark, S., Cole, S.T., Honore é re, J.M. (1988) Sequence and comparative N., Joris, B. and Fre analysis of three

Enterobacter cloacae ampC

beta-lactamase

genes and their products. Biochem. J. 250, 753^760. [10] Nomura, K. and Yoshida, T. (1990) Nucleotide sequence of the

Serratia marcescens

SR50 chromosomal

ampC

beta-lacta-

mase gene. FEMS Microbiol. Lett. 58, 295^299. [11] Seoane, A., Francia, M.V. and Garcia, L.J. (1992) Nucleotide sequence of the ampC-ampR region from the chromosome of Yersinia enterocolitica. Antimicrob. Agents Chemother. 36, 1049^1052. [12] Philippon, A., Arlet, G. and Lagrange, P.H. (1994) Origin and impact of plasmid-mediated extended-spectrum

L-lactamases.

[13] Bauernfeind, A., Stemplinger, I., Jungwirth, R., Wilhelm, R.

strains although it was smaller (110 bp

and Chong, Y. (1996) Comparative characterization of the

was identical to that in

In conclusion we have cloned and determined the

ampC gene (and part of the ampR gene) of M. morganii.

entire sequence of the of

the

Although an AmpC-type

L-lactamase,

it was dissim-

ilar to the chromosomally encoded and plasmidmediated AmpC-type

Eur. J. Clin. Microbiol. Infect. Dis. 13, Suppl. 1, 17^29.

cephamycinase

versus 140 bp) [3^5,11].

sequence

OS60 chromosomal

M. morganii E. cloacae, C. freundii and Y.

2). The arrangement of this region in

enterocolitica

bacter freundii

Eur. J. Biochem. 156, 441^445.

L-lactamases.

L-lactamase

er

Lla CMY-1 gene and its relationship with othgenes. Antimicrob. Agents Chemother. 40,

1926^1930. [14] Spratt, B.G., Hedge, P.J., Heesen, T.S., Edelman, A. and Broome-Smith, J.K. (1986) Kanamycin-resistant vectors that are

analogues

of

plasmids

pUC8,

pUC9,

pEMBL8,

and

pEMBL9. Gene 41, 337^342. [15] Sambrook, J., Fritsch, E.F. and Maniatis, T.(1989) Molecular Cloning : A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [16] Matthew, M., Harris, A.M., Marshall, M.J. and Ross, G.W.

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