The phototrophic bacterium Rhodopseudomonas capsulata sp108 encodes an indigenous class A beta-lactamase.
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ABSTRACT:
SUBMITTER: Campbell JI
PROVIDER: S-EPMC1138748 | biostudies-other | 1989 Jun
REPOSITORIES: biostudies-other
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Project description:Extracellular electron uptake (EEU) is the ability of microbes to take up electrons from solid-phase conductive substances such as metal oxides. EEU is performed by prevalent phototrophic bacterial genera, but the electron transfer pathways and the physiological electron sinks are poorly understood. Here we show that electrons enter the photosynthetic electron transport chain during EEU in the phototrophic bacterium Rhodopseudomonas palustris TIE-1. Cathodic electron flow is also correlated with a highly reducing intracellular redox environment. We show that reducing equivalents are used for carbon dioxide (CO2) fixation, which is the primary electron sink. Deletion of the genes encoding ruBisCO (the CO2-fixing enzyme of the Calvin-Benson-Bassham cycle) leads to a 90% reduction in EEU. This work shows that phototrophs can directly use solid-phase conductive substances for electron transfer, energy transduction, and CO2 fixation.
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Project description:The amino acid sequence of the principal soluble cytochrome c from the phototrophic acidophilic bacterium Rhodopseudomonas (or Rhodopila) globiformis was determined. By the criteria of percentage sequence identity and fewness of internal insertions and deletions it is more similar in sequence to some mitochondrial cytochromes c than to any known bacterial cytochrome. The organism does not have any properties that commend it as being particularly similar to postulated prokaryotic precursors of the mitochondrion. We consider that the relatively high degree of sequence similarity is an instance of convergence, and is an example of the limitations that are imposed on attempts to deduce distant evolutionary relationships from sequence information. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50136 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].
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Project description:Recent years have witnessed an increased prevalence of intrinsic and acquired beta-lactamase-producing bacteria, severely limiting human and veterinary medicine therapeutic options. The present study aimed to design specific oligonucleotides for rapid PCR detection of the cephalosporinase-encoding gene blaEC (BlaEC family class C beta-lactamase). A total of three primers were designed to detect 2281 variants of the blaEC gene and two sets of primer pairs were also tested against DNA from 11 strains. The study indicates that the proposed primers should be able to detect 100% of all described blaEC genes in different bacterial strains and monitor their spread. After comparing the amino acid sequences, a phylogenetic tree was created based on the presence of conserved amino acids and homologous motifs. More than 24 760 mutations in BlaEC enzymes have been identified. The mutations involving 371 amino acid positions and these hotspots can change the structure and activity of the monitored enzymes. We predicted several BlaEC enzymes with a broadened substrate activity against higher-generation cephalosporins.
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Project description:As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.
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Project description:The biogeography of the purple nonsulfur bacterium Rhodopseudomonas palustris on a local scale was investigated. Thirty clones of phototrophic bacteria were isolated from each of five unevenly spaced sampling locations in freshwater marsh sediments along a linear 10-m transect, and a total of 150 clones were characterized by BOX-PCR genomic DNA fingerprinting. Cluster analysis of 150 genomic fingerprints yielded 26 distinct genotypes, and 106 clones constituted four major genotypes that were repeatedly isolated. Representatives of these four major genotypes were tentatively identified as R. palustris based on phylogentic analyses of 16S rRNA gene sequences. The differences in the genomic fingerprint patterns among the four major genotypes were accompanied by differences in phenotypic characteristics. These phenotypic differences included differences in the kinetics of carbon source use, suggesting that there may be functional differences with possible ecological significance among these clonal linages. Morisita-Horn similarity coefficients (C(MH)), which were used to compare the numbers of common genotypes found at pairs of sampling locations, showed that there was substantial similarity between locations that were 1 cm apart (C(MH), >/=0.95) but there was almost no similarity between locations that were >/=9 m apart (C(MH), </=0.25). These calculations showed there was a gradual decrease in similarity among the five locations as a function of distance and that clones of R. palustris were lognormally distributed along the linear 10-m transect. These data indicate that natural populations of R. palustris are assemblages of genetically distinct ecotypes and that the distribution of each ecotype is patchy.
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Project description:The discovery that the mechanism of beta-lactam hydrolysis catalyzed by the class A (active site serine-dependent) beta-lactamases proceeds via an acyl-enzyme intermediate was made thirty years ago. Since this discovery, the active site circumstance that enables acylation of the active site serine and further enables hydrolytic deacylation of the acyl-serine intermediate, has received extraordinary scrutiny. The justification for this scrutiny is the direct relevance of the beta-lactamases to the manifestation of bacterial resistance to the beta-lactam antibiotics, and the subsequent (to the discovery of the beta-lactamase acyl-enzyme) recognition of the direct evolutionary relationship between the serine beta-lactamase acyl-enzyme, and the penicillin binding protein acyl-enzyme that is key to beta-lactam antibiotic activity. This short review describes the early events leading to the recognition that serine beta-lactamase catalysis proceeds via an acyl-enzyme intermediate, and summarizes several of the key mechanistic studies--including infrared spectroscopy, cryoenzymology, beta-lactam design, and x-ray crystallography--that have been exploited to understand this pivotal catalytic intermediate.
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Project description:β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-β-lactam combinations.
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Project description:Phototrophic Fe(II)-oxidizing bacteria couple the oxidation of ferrous iron [Fe(II)] to reductive CO(2) fixation by using light energy, but until recently, little has been understood about the molecular basis for this process. Here we report the discovery, with Rhodopseudomonas palustris TIE-1 as a model organism, of a three-gene operon, designated the pio operon (for phototrophic iron oxidation), that is necessary for phototrophic Fe(II) oxidation. The first gene in the operon, pioA, encodes a c-type cytochrome that is upregulated under Fe(II)-grown conditions. PioA contains a signal sequence and shares homology with MtrA, a decaheme c-type cytochrome from Shewanella oneidensis MR-1. The second gene, pioB, encodes a putative outer membrane beta-barrel protein. PioB is a homologue of MtrB from S. oneidensis MR-1. The third gene, pioC, encodes a putative high potential iron sulfur protein (HiPIP) with a twin-arginine translocation (Tat) signal sequence and is similar to the putative Fe(II) oxidoreductase (Iro) from Acidithiobacillus ferrooxidans. Like PioA, PioB and PioC appear to be secreted proteins. Deletion of the pio operon results in loss of Fe(II) oxidation activity and growth on Fe(II). Complementation studies confirm that the phenotype of this mutant is due to loss of the pio genes. Deletion of pioA alone results in loss of almost all Fe(II) oxidation activity; however, deletion of either pioB or pioC alone results in only partial loss of Fe(II) oxidation activity. Together, these results suggest that proteins encoded by the pio operon are essential and specific for phototrophic Fe(II) oxidation in R. palustris TIE-1.
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