Biochemical characterization of the L1-like metallo-β-lactamase from Stenotrophomonas lactitubi

ABSTRACT

L1-like metallo-β-lactamases (MBLs) exhibit diversity and are highly conserved. Although the presence of the bla_L1-like gene is known, the biochemical characteristics are unclear. This study aimed to characterize an L1-like MBL from Stenotrophomonas lactitubi. It showed 70.9–99.7% similarity to 50 L1-like amino acid sequences. The characteristic kinetic parameter was its high hydrolyzing efficiency for ampicillin and nitrocefin. Furthermore, L1-like from S. lactitubi was distinctly more susceptible to inhibition by EDTA than that to inhibition by 2,6-pyridinedicarboxylic acid.

INTRODUCTION

Metallo-β-lactamases (MBLs) are highly diverse enzymes and are classified into B1, B2, and B3 subclasses (1, 2). B1 MBL genes, such as bla_IMP, bla_VIM, and bla_NDM, exist on mobile elements in several species (2). B3 MBL genes are frequently present in non-glucose-fermenting Gram-negative organisms, with a few exceptions, such as SMB-1 and AIM-1 (3–9). L1 MBL is a subclass of B3 MBL produced by Stenotrophomonas maltophilia; it exhibits high genetic diversity (10). The bla_L1-like diversity can be attributed to species diversity and rapid evolutionary changes (11).

Stenotrophomonas lactitubi was first reported by Weber et al. in 2018 (12). Stenotrophomonas species are distributed in various environments, such as soil; S. lactitubi was originally isolated from biofilms on milking equipment retainers (12). Infectious diseases caused by S. lactitubi have not been reported; however, we isolated S. lactitubi TUM20371 as a colonizer from the sputum of a hospitalized patient. The presence of the bla_L1-like gene in S. lactitubi was confirmed in our previous genome-wide study; however, the biochemical characteristics of this MBL encoded by bla_L1-like were not explored (11). Therefore, this study aimed to characterize the L1-like MBL derived from S. lactitubi.

We obtained a draft whole-genome sequence using MiSeq (Illumina, San Diego, CA, USA) (GenBank accession no. NZ_BRFG00000000). Molecular species identification using average nucleotide identity (13) showed high similarities of 95.9% and 94.0% to S. lactitubi M15^T and Stenotrophomonas indicatrix WS40^T, respectively. Therefore, we identified TUM20371 as S. lactitubi harboring the bla_L1-like gene.

The MICs of all tested β-lactams were determined using the broth microdilution method according to the guidelines stipulated by the Clinical and Laboratory Standards Institute (14). All tested β-lactams showed high MICs against TUM20371 (Table 1).

TABLE 1.

Minimum inhibitory concentrations (mg/L) for TUM20371 strain, BL21, and their transformants

Antibiotics	MIC (mg/L)
		Escherichia coli BL21a
	TUM20371	pET9a (vector only)	pET9a_blaL1b	pET9a_blaL1-like	pET9a_blaL1-like + EDTAc	pET9a_blaL1-like + DPAc
Ampicillin	>128	2	＞128	＞128	>128	>128
Cephalothin	>128	1	＞128	＞128	128	>128
Cefoxitin	128	1	＞128	＞128	128	>128
Cefotaxime	128	<0.125	64	64	16	64
Ceftazidime	8	0.25	64	64	32	64
Ceftazidime/clavulanate	8	0.25	64	64	32	64
Cefepime	64	<0.125	0.5	1	<0.125	1
Imipenem	128	0.25	64	64	4	64
Meropenem	32	<0.125	32	64	16	64
Aztreonam	128	<0.125	<0.125	<0.125	<0.125	<0.125

^a The MICs remained changed under induced (100 µM IPTG) and non-induced conditions.

^b bla_L1 derived from S. maltophilia NCTC 10257.

^c Contains EDTA and DPA at a final concentration of 100 µM.

The bla_L1-like gene from TUM20371 consisted of an 870-bp nucleotide sequence with 68.2% GC content and a predicted 289 amino acid sequences (GenBank accession no. NZ_BRFG01000003, 278911...279780). Phylogenetic analysis using 44 L1-like sequences published in the β-lactamase database (15) and six L1-like sequences identified from S. maltophilia NCTC 10257^T, Stenotrophomonas pavanii LMG 25348^T, Stenotrophomonas geniculata ATCC 19374^T, S. indicatrix WS40^T, and S. lactitubi M15^T is shown in Fig. 1. These 50 sequences were 70.9–99.7% similar, indicating high diversity. The L1-like from S. lactitubi and other L1-like enzymes possess conserved zinc-binding sites (H-X-H-X-D-H at positions 116–121, 196H, and 263H) (Fig. 2) (16).

Fig 1.

Phylogenetic tree for L1-like based on amino acid sequence, constructed using MEGA X with maximum likelihood and 10,00 replication bootstrapping using the Jones-Taylor-Thornton model.

Fig 2.

Amino acid alignment of L1-like. Red box indicates zinc-binding regions.

We compared the bla_L1-like genes of the five Stenotrophomonas species using EasyFig version 2.2.2 (Fig. 3) (17). The neighboring genomes were well conserved despite the partial insertion and deletion of open reading frames (Fig. 3). The bla_L1-like gene is highly conserved across multiple species, highlighting its role as a marker of species diversity. The S. maltophilia complex, which harbors the bla_L1-like gene, is subdivided into at least 20 distinct species (11).

Fig 3.

Comprehensive similarities in the genome sequences of neighboring bla_L1-like genes in L1-like producing Stenotrophomonas species. The block arrows indicate putative open reading frames and their orientation. Black and orange arrows indicate genes encoding L1-like and other protein coding sequences, respectively.

To characterize the kinetic properties of the L1-like from S. lactitubi, the recombinant protein was purified from Escherichia coli BL21 (DE3). Cloning was performed using the In-Fusion HD Cloning kit (TaKaRa Bio, Shiga, Japan) after the PCR amplification of bla_L1-like and the linear pET9a vector, using the following primers with a 15-bp overlapping sequence (underlined): pET9a_F_Infusion, 5′-TAACAAAGCCCGAAAGGAAGC-3′; pET9a_R_Infusion, 5′-ATGTATATCTCCTTCTTAAAGTTA-3′; F-L1-like_infusion, 5′-GAAGGAGATATACATATGCGCCTGCAGACCCTC-3′; and R-L1-like_infusion, 5′-TTTCGGGCTTTGTTATCAGCGGGCCCCGGCCGC-3′. The antimicrobial susceptibilities of the transformed recombinant and wild-type strains were determined according to the Clinical and Laboratory Standards Institute guidelines (Table 1). The cells were grown at 35°C in Luria-Bertani broth containing 50 g/mL kanamycin and induced using 1 mM isopropyl-thiogalactopyranoside for 4 h. The cell pellet was collected via centrifugation, resuspended in 20 mM MES buffer (pH 6.0), and sonicated, followed by ultracentrifugation at 50,000 rpm for 30 min. The supernatant was loaded onto a HiTrap SP column (Cytiva, MA, USA) and recombinant L1-like was eluted using a linear NaCl gradient (0–200 mM) in 20 mM MES buffer (pH 6.0). The purified enzyme was dialyzed using 50 mM HEPES buffer (pH 7.5) with 50 µM ZnSO₄ and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant L1-like was obtained with >95% purity at a concentration of 3.8 mg/mL, which complies with the consensus for β-lactamases (18). In addition, L1 from S. maltophilia NCTC 10257 was purified using ion exchange chromatography according to the protocol described earlier. The following cloning primers with a 15-bp overlapping sequence (underline) were used: F_L1_NCTC, 5ʹ-GAAGGAGATATACATATGCGTTTCACGCTGCTC-3ʹ; and R_L1_NCTC, 5ʹ-TTTCGGGCTTTGTTATTATCTTGTTCCTGCTGTTTC-3ʹ. The molecular mass (without the predicted signal peptide sequence) and the predicted isoelectric point of L1-like were 28,814 kDa and 6.11, respectively, as observed using ExPASy (https://web.expasy.org/compute_pi/).

The kinetic parameters of the purified L1-like and L1 from S. maltophilia were determined as described previously (19–21). The K_i values for ampicillin and the IC₅₀ values of EDTA (Nacalai Tesque, Kyoto, Japan) and 2,6-pyridinedicarboxylic acid (DPA; Sigma-Aldrich, St. Louis, MO, USA) were determined using nitrocefin as the reporter substrate. The mean ± standard deviation for K_m, k_cat, and K_i were calculated in triplicates (Table 2).

TABLE 2.

Kinetic parameters of L1-like form Stenotrophomonas lactitubi and other class B3 metallo-β-lactamases^e

Substrate	L1-like (S. lactitubi TUM 20371)				L1 (S. maltophilia NCTC 10257)				PAM-2 (Pseudomonas tohonis TUM18999)				PJM-1 (Pseudoxanthomonas japonensis TUM 20250)
	kcat (s−1)	Km (µM)	kcat/Km (M−1 s−1)	Relative hydrolyzing efficiency (%)	kcat (s−1)	Km (µM)	kcat/Km (M−1 s−1)	Relative hydrolyzing efficiency (%)	kcat (s−1)	Km (µM)	kcat/Km (M−1 s−1)	Relative hydrolyzing efficiency (%)	kcat (s−1)	Km (µM)	kcat/Km (M−1 s−1)	Relative hydrolyzing efficiency (%)
Ampicillin	110 ± 5.2	11 ± 0.58b	1.0 × 107	100	760 ± 3.2	78 ± 0.83b	1.0 × 107	100	170 ± 4.0	18 ± 0.91b	9.3 × 106	100	370 ± 3.1	51 ± 0.55b	7.2 × 106	100
Cephalothin	12 ± 0.36	11 ± 0.60	1.1 × 106	11	180 ± 9.5	3.3 ± 0.73	5.6 × 107	560	130 ± 2.1	15 ± 0.29	8.5 × 106	91	38 ± 0.36	13 ± 0.19	2.9 × 106	40
Cefuroxime	18 ± 0.17	38 ± 1.3	4.9 × 105	4.9	290 ± 6.0	42 ± 2.2	6.9 × 106	69	180 ± 2.1	59 ± 1.6	3.0 × 106	32	86 ± 1.8	120 ± 3.8	6.9 × 105	10
Cefoxitin	0.62 ± 0.014	3.2 ± 0.25	1.9 × 105	1.9	5.2 ± 0.62	0.47 ± 0.045b	1.1 × 107	110	11 ± 0.17	12 ± 0.34	9.6 × 105	10	31 ± 0.50	26 ± 0.98	1.2 × 106	17
Cefotaxime	22 ± 0.27	38 ± 0.31	5.7 × 105	5.7	180 ± 7.1	28 ± 2.7	6.6 × 106	66	87 ± 3.7	40 ± 1.0	2.2 × 106	24	90 ± 1.9	130 ± 7.0	7.1 × 105	10
Ceftazidime	1.2 ± 0.026	77 ± 4.0	1.5 × 104	0.15	31 ± 0.66	58 ± 2.2	5.4 × 105	5	5.6 ± 0.12	68 ± 3.3	8.3 × 104	0.89	0.091 ± 0.00036	150 ± 3.7	6.2 × 102	0.01
Cefepime	2.3 ± 0.065	190 ± 10	1.2 × 104	0.12	5.5 ± 0.11	90 ± 3.0	6.1 × 104	0.61	58 ± 2.6	200 ± 9.0	2.9 × 105	3.1	1.0 ± 0.027	230 ± 6.1	4.3 × 103	0.06
Imipenem	35 ± 1.7	24 ± 1.2	1.5 × 106	15	350 ± 11	19 ± 2.0	1.9 × 107	190	13 ± 0.14	95 ± 4.2b	1.4 × 105	1.5	66 ± 2.1	36 ± 1.4b	1.8 × 106	25
Meropenem	23 ± 0.099	11 ± 0.55	2.2 × 106	22	240 ± 6.5	6.6 ± 0.74	3.7 × 107	370	67 ± 0.89	11 ± 0.43	5.9 × 106	63	110 ± 3.8	34 ± 2.3	3.3 × 106	46
Aztreonama	NH	ND	ND	ND	NH	ND	ND	ND	NHc	ND	ND	NDd	NH	ND	ND	ND
Nitrocefin	7.3 ± 0.12	0.41 ± 0.034	1.8 × 107	180	160 ± 1.8	2.6 ± 0.28	6.0 × 107	600	29 ± 0.31	2.0 ± 0.12	1.5 × 107	150	79 ± 1.5	2.6 ± 0.11	3.1 × 107	280

^a The K_i value of aztreonam exceeded the measurable range.

^b K_i value.

^c NH, No hydrolysis.

^d ND, Not determined.

^e Relative hydrolytic efficiency was calculated with respect to hydrolysis efficiency against ampicillin, which was set at 100%. The reference values for PAM-2 and PJM-1 were 7 and 9, respectively.

The MICs of almost all β-lactams, except aztreonam, were higher in E. coli producing recombinant L1-like than that in L1-like non-producing E. coli (Table 1). No significant changes in the MIC value were observed compared with E. coli producing recombinant L1 from S. maltophilia NCTC 10257. This outcome may be attributed to the use of the pET vector, suggesting that the gene expression may not be effectively regulated in this context. The MICs remained unaltered in the presence of 100 µM DPA and marginally decreased in the presence of 100 µM EDTA. Therefore, EDTA had a stronger inhibitory effect on L1-like from S. lactitubi than DPA. However, EDTA can disrupt the lipopolysaccharides in the outer membrane of Gram-negative bacteria, which could lead to decreased MICs. Therefore, caution should be exercised during MIC measurements that involve the addition of EDTA. Consistently, the IC₅₀ for EDTA in 50 µM nitrocefin was lower than that for DPA (180 vs 410 µM). Thus, L1-like exhibited resistance to DPA inhibition, similar to that observed earlier in PJM-1 (7).

Kinetic parameters are influenced by various factors, such as enzyme stability, presence of tags, and assay conditions. These parameters were determined under the same conditions, and the relative hydrolysis efficiency (%) of different substrates was assessed based on a reference value of 100% for ampicillin. We compared the kinetic properties with our previous data for PAM-2 and PJM-1 (Table 2). The relative hydrolysis efficiencies of ceftazidime and cefepime were lower than that of ampicillin, which is a common characteristic of MBLs used in this study.

The L1-like from S. lactitubi showed high k_cat/K_m values for ampicillin and nitrocefin among the tested substrates (>10⁷ M⁻¹ s⁻¹), suggesting a preference for these substrates. The k_cat/K_m values were approximately 10⁶ M⁻¹ s⁻¹ for cephalothin and carbapenems, which were also preferred substrates. The k_cat/K_m values for cefuroxime, cefoxitin, and cefotaxime were approximately 10⁵ M⁻¹ s⁻¹, while those for ceftazidime and cefepime were 10⁴ M⁻¹ s⁻¹.

The k_cat/K_m value of L1-like for ampicillin was similar to that of L1 from NCTC 10257, whereas the k_cat/K_m value of L1-like for other substrates was lower than that of L1 from S. maltophilia (Table 2). However, the reason for this difference could not be determined based on the results of this study. L1-like enzymes constitute a large family with intricate taxonomic classifications. Therefore, future studies should include further evaluations, including enzymatic characterizations and enzyme nomenclature analyses.

The kinetic properties of L1-like from S. lactitubi were consistent with the antimicrobial susceptibility of L1-like-producing recombinant E. coli. The L1-like from S. lactitubi exhibited high hydrolyzing efficiency for ampicillin and nitrocefin and greater resistance to inhibition by DPA than that to inhibition by EDTA.