scholarly journals Inactivation of mrcA gene derepresses the basal-level expression of L1 and L2  -lactamases in Stenotrophomonas maltophilia

2011 ◽  
Vol 66 (9) ◽  
pp. 2033-2037 ◽  
Author(s):  
C.-W. Lin ◽  
H.-C. Lin ◽  
Y.-W. Huang ◽  
T.-C. Chung ◽  
T.-C. Yang
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
L1 And L2 ◽  
Basal Level Expression

scholarly journals AmpDI Is Involved in Expression of the Chromosomal L1 and L2 β-Lactamases of Stenotrophomonas maltophilia

2009 ◽  
Vol 53 (7) ◽  
pp. 2902-2907 ◽  
Author(s):  
Tsuey-Ching Yang ◽  
Yi-Wei Huang ◽  
Rouh-Mei Hu ◽  
Shao-Cheng Huang ◽  
Yu-Tzu Lin
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Stenotrophomonas Maltophilia ◽  
Genomic Analysis ◽  
Comparative Genomic Analysis ◽  
Comparative Genomic ◽  
Genomic Context ◽  
One Step ◽  
L1 And L2

ABSTRACT Two ampD homologues, ampD I and ampD II, of Stenotrophomonas maltophilia have been cloned and analyzed. Comparative genomic analysis revealed that the genomic context of the ampD II genes is quite different, whereas that of the ampD I genes is more conserved in S. maltophilia strains. The ampD system of S. maltophilia is distinct from that of the Enterobacteriaceae and Pseudomonas aeruginosa in three respects. (i) AmpDI of S. maltophilia is not encoded in an ampDE operon, in contrast to what happens in the Enterobacteriaceae and P. aeruginosa. (ii) The AmpD systems of the Enterobacteriaceae and P. aeruginosa are generally involved in the regulation of ampR-linked ampC gene expression, while AmpDI of S. maltophilia is responsible for the regulation of two intrinsic β-lactamase genes, of which the L2 gene, but not the L1 gene, is linked to ampR. (iii) S. maltophilia exhibits a one-step L1 and L2 gene derepression model involving ampD I, distinct from the two- or three-step derepression of the Enterobacteriaceae and P. aeruginosa. Moreover, the ampD I and ampD II genes are constitutively expressed and not regulated by the inducer and AmpR protein, and the expression of ampD II is weaker than that of ampD I. Finally, AmpDII is not associated with the derepression of β-lactamases, and its role in S. maltophilia remains unclear.

scholarly journals AmpN-AmpG Operon Is Essential for Expression of L1 and L2 β-Lactamases in Stenotrophomonas maltophilia

2010 ◽  
Vol 54 (6) ◽  
pp. 2583-2589 ◽  
Author(s):  
Yi-Wei Huang ◽  
Cheng-Wen Lin ◽  
Rouh-Mei Hu ◽  
Yu-Tzu Lin ◽  
Tung-Ching Chung ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Gene Dosage ◽  
Gram Negative Bacteria ◽  
Beta Lactamase ◽  
Gene Dosage Effect ◽  
Gram Negative ◽  
E Coli ◽  
Murein Sacculus ◽  
Lactamase Gene ◽  
L1 And L2

ABSTRACT AmpG is an inner membrane permease which transports products of murein sacculus degradation from the periplasm into the cytosol in Gram-negative bacteria. This process is linked to induction of the chromosomal ampC beta-lactamase gene in some members of the Enterobacteriaceae and in Pseudomonas aeruginosa. In this study, the ampG homologue of Stenotrophomonas maltophilia KJ was analyzed. The ampG homologue and its upstream ampN gene form an operon and are cotranscribed under the control of the promoter P ampN. Expression from P ampN was found to be independent of β-lactam exposure and ampN and ampG products. A ΔampN allele exerted a polar effect on the expression of ampG and resulted in a phenotype of null β-lactamase inducibility. Complementation assays elucidated that an intact ampN-ampG operon is essential for β-lactamase induction. Consistent with ampG of Escherichia coli, the ampN-ampG operon of S. maltophilia did not exhibit a gene dosage effect on β-lactamase expression. The AmpG permease of E. coli could complement the β-lactamase inducibility of ampN or ampG mutants of S. maltophilia, indicating that both species have the same precursor of activator ligand(s) for β-lactamase induction.

The role of AmpR in regulation of L1 and L2 β-lactamases in Stenotrophomonas maltophilia

2009 ◽  
Vol 160 (2) ◽  
pp. 152-158 ◽  
Author(s):  
Cheng-Wen Lin ◽  
Yi-Wei Huang ◽  
Rouh-Mei Hu ◽  
Kai-Hung Chiang ◽  
Tsuey-Ching Yang
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
L1 And L2

scholarly journals Plasmid Location and Molecular Heterogeneity of the L1 and L2 β-Lactamase Genes of Stenotrophomonas maltophilia

2001 ◽  
Vol 45 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Matthew B. Avison ◽  
Catherine S. Higgins ◽  
Charlotte J. von Heldreich ◽  
Peter M. Bennett ◽  
Timothy R. Walsh
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Kinetic Properties ◽  
Rrna Gene ◽  
Molecular Heterogeneity ◽  
Gram Negative Bacteria ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
L1 And L2 ◽  
Hydrolysis Of ◽  
Clinical Problems

ABSTRACT An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 β-lactamase genes. The location of L1 andL2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes,L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1(L1a), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lowerk cat/Km ratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (L2b, L2c, andL2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275L2 (L2a) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.

scholarly journals AmpI Functions as an Iron Exporter To Alleviate β-Lactam-Mediated Reactive Oxygen Species Stress in Stenotrophomonas maltophilia

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Yi-Wei Huang ◽  
Hsin-Hui Huang ◽  
Kai-Hung Huang ◽  
Wei-Chien Chen ◽  
Yi-Tsung Lin ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Stenotrophomonas Maltophilia ◽  
Iron Homeostasis ◽  
Reactive Oxygen ◽  
Iron Level ◽  
Oxygen Species ◽  
Intracellular Iron ◽  
Iron Storage ◽  
Content Type ◽  
L1 And L2

ABSTRACT Stenotrophomonas maltophilia is an organism with a remarkable capacity for drug resistance with several antibiotic resistance determinants in its genome. S. maltophilia genome codes for L1 and L2, responsible for intrinsic β-lactam resistance. The Smlt3721 gene (denoted ampI), located downstream of the L2 gene, encodes an inner membrane protein. The existence of an L2 gene-ampI operon was verified by reverse transcription-PCR (RT-PCR). For aerobically grown S. maltophilia KJ, inactivation of ampI downregulated siderophore synthesis and iron acquisition systems and upregulated the iron storage system, as demonstrated by a transcriptome assay, suggesting that AmpI is involved in iron homeostasis. Compared with the wild-type KJ, an ampI mutant had an elevated intracellular iron level, as revealed by inductively coupled plasma mass spectrometry (ICP-MS) analysis, and increased sensitivity to H2O2, verifying the role of AmpI as an iron exporter. The β-lactam stress increased the intracellular reactive oxygen species (ROS) level and induced the expression of the L1 gene and L2 gene-ampI operon. Compared to its own parental strain, the ampI mutant had reduced growth in β-lactam-containing medium, and the ampI mutant viability was improved after complementation with plasmid pAmpI in either a β-lactamase-positive or β-lactamase-negative genetic background. Collectively, upon challenge with β-lactam, the inducibly expressed L1 and L2 β-lactamases contribute to β-lactam resistance by hydrolyzing β-lactam. AmpI functions as an iron exporter participating in rapidly weakening β-lactam-mediated ROS toxicity. The L1 gene and L2 gene-ampI operon enable S. maltophilia to effectively cope with β-lactam-induced stress.

scholarly journals Induction of L1 and L2 β-Lactamase Production in Stenotrophomonas maltophilia Is Dependent on an AmpR-Type Regulator

2008 ◽  
Vol 52 (4) ◽  
pp. 1525-1528 ◽  
Author(s):  
Aki Okazaki ◽  
Matthew B. Avison
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
L2 Production ◽  
L1 And L2

ABSTRACT A divergently oriented ampR has been located upstream of bla L2 in Stenotrophomonas maltophilia. AmpR is necessary for L1 and L2 β-lactamase induction in response to β-lactam challenge, and activation of AmpR is sufficient to induce L1 and L2 production. L1 induction requires more activation of AmpR than does L2 induction.

scholarly journals 1437. Biochemical characterization of L1 and L2 β-lactamases from clinical isolates of Stenotrophomonas maltophilia

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S723-S723
Author(s):  
Maria F Mojica ◽  
Joseph Rutter ◽  
Magdalena A Taracila ◽  
Krisztina M Papp-Wallce ◽  
James Spencer ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Biochemical Characterization ◽  
Amino Acid Sequences ◽  
Structural Basis ◽  
Intrinsic Resistance ◽  
Research Support ◽  
Thermal Stabilities ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
L1 And L2

Abstract Background Stenotrophomonas maltophilia is a Gram-negative, non-fermenting opportunistic pathogen. Two β-lactamases provide intrinsic resistance to β-lactams: a class B Metallo- β-lactamase L1, and a class A serine β-lactamase (SβL) L2. Recently, we described novel variants of the L1 and L2 in a collection of clinical S. maltophilia isolates collected in the US, and showed through analyses of the amino acid sequences that L1 and L2 grouped into 4 (A-D, B, C, and E) and 2 (A and D) clades, respectively. We aimed to characterize the new L1 and L2 clinical variants biochemically. Methods Representative blaL1 and blaL2 genes from each of the identified clades were cloned into pBC-SK and pET24 vectors and transformed into E. coli DH10B and BL21 (DE3) cells, respectively. Minimal inhibitory concentrations (MICs) were determined using CLSI approved methods. Cell-based assays and biochemical characterization performed on purified enzymes, including circular dichroism (CD), thermal stability, and steady-state kinetics assays, were performed. Results Susceptibility testing results using DH10-B E. coli strains expressing the L1 and L2 variants are shown in Table 1. Remarkably, while all L1 variants confer the same level of resistance to carbapenems, L2B conferred higher MICs to 3rd gen cephalosporins and aztreonam than L2D. Kinetics assays confirmed differences in the kcat of both enzymes to ceftazidime (32s-1 for L2B vs. 7s-1 for L2D) and avibactam inhibition constant Ki (1.7 μM for L2B vs. 4.5 μM for L2D). Structurally, L2B and L2D present distinctive CD spectra and thermal stabilities (ΔTm 5°C). Table 1 Conclusion As opposed to the L2 variants, our results suggest that the L1 variants may not be functionally nor structurally different. Differences between L2B and L2D might have arisen due to the use of cephalosporins and SβL inhibitors. Further experiments are on the way to determine the structural basis of these observations and the implication of these for the design of novel β-lactamase inhibitors. Disclosures Krisztina M. Papp-Wallce, PhD, Entasis (Grant/Research Support)Merck (Grant/Research Support)Venatorx (Grant/Research Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

scholarly journals Induction of L1 and L2 β-Lactamases of Stenotrophomonas maltophilia

2007 ◽  
Vol 52 (3) ◽  
pp. 1198-1200 ◽  
Author(s):  
Rouh-Mei Hu ◽  
Kuang-Jay Huang ◽  
Lii-Tzu Wu ◽  
Ying-Ju Hsiao ◽  
Tsuey-Ching Yang
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Gene Knockout ◽  
Gene Replacement ◽  
Induction Kinetics ◽  
Knockout Mutants ◽  
L1 And L2 ◽  
Kinetics Of ◽  
Dioxygenase Activity

ABSTRACT Isogenic L1 and L2 gene knockout mutants of Stenotrophomonas maltophilia KJ (KJΔL1 and KJΔL2, respectively) were constructed by xylE gene replacement. Induction kinetics of the L1 and L2 genes were evaluated by testing catechol 2,3-dioxygenase activity in the mutants. The results suggested that the induction of the L1 and L2 genes was differentially regulated.

scholarly journals NagZ-Dependent and NagZ-Independent Mechanisms for β-Lactamase Expression in Stenotrophomonas maltophilia

2012 ◽  
Vol 56 (4) ◽  
pp. 1936-1941 ◽  
Author(s):  
Yi-Wei Huang ◽  
Rouh-Mei Hu ◽  
Cheng-Wen Lin ◽  
Tung-Ching Chung ◽  
Tsuey-Ching Yang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stenotrophomonas Maltophilia ◽  
Double Mutant ◽  
Content Type ◽  
Content System ◽  
L1 And L2

ABSTRACTβ-N-Acetylglucosaminidase (NagZ), encoded by thenagZgene, is a critical enzyme for basal-levelampCderepression (ampCexpression in the absence of β-lactam challenge) inampDanddacBmutants ofPseudomonas aeruginosa. Three mutants with a phenotype of basal-level L1 and L2 β-lactamase derepression inStenotrophomonas maltophiliahave been reported, including KJΔDI (ampDImutant), KJΔmrcA (mrcAmutant), and KJΔDIΔmrcA (ampDIandmrcAdouble mutant). In this study,nagZofS. maltophiliawas characterized, and its roles in basal-level β-lactamase derepression, induced β-lactamase activities, and β-lactam resistance of KJΔDI, KJΔmrcA, and KJΔDIΔmrcA were evaluated. Expression of thenagZgene was constitutive and not regulated by AmpR, AmpDI, AmpN, AmpG, PBP1a, and NagZ. Introduction of ΔnagZinto KJΔDI nearly abolished basal-level derepressed β-lactamase activity; conversely, introduction of ΔnagZinto KJΔmrcA did not affect it. At least two activator ligands (ALs) are thus considered responsible for β-lactamase expression in theS. maltophiliasystem, specifically, the NagZ-dependent (AL1) and NagZ-independent (AL2) ligands responsible for the basal-level derepressed β-lactamase activities of KJΔDI and KJΔmrcA, respectively. The contributions of AL1 and AL2 to the induced β-lactamase activities may vary with the types of β-lactams.nagZinactivation did not affect aztreonam-, cefoxitin-, and carbenicillin-induced β-lactamase activities, but it attenuated cefuroxime- and piperacillin-induced β-lactamase activities. Introduction of ΔnagZinto KJ, KJΔDI, KJΔmrcA, and KJΔDIΔmrcA did not significantly change the MICs of the β-lactams tested except that the MICs of cefuroxime and piperacillin moderately decreased in strains KJΔZ and KJΔDIΔZ (nagZmutants).

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