Functional analysis, diversity, and distribution of the ean cluster responsible for 17ß-estradiol degradation in sphingomonads.

17ß-estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2.IMPORTANCESteroid estrogens have been detected in diverse environments, ranging from oceans and rivers to soils and groundwater, posing serious risks to both human health and ecological safety. The United States National Toxicology Program and the World Health Organization have both classified estrogens as Group 1 carcinogens. Several model organisms (proteobacteria) have established the 4,5-seco pathway for estrogen degradation. In this study, the newly isolated Sphingomonas colocasiae C3-2 could degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway. The novel gene cluster ean (including eanA, eanB1, eanC, and eanD) responsible for E2 degradation by the 4,5-seco pathway was identified; the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 through the 9,10-seco pathway. The TetR family transcriptional regulator EanR acts as a transcriptional repressor of the ean cluster. The cluster ean was also found to be present in other reported E2-degrading sphingomonads, indicating the ubiquity of the E2 metabolism in the environment.

Smartphone-assisted biosensor based on broom-like bacteria-specific magnetic enrichment platform for colorimetric detection of Listeria monocytogenes.

Pathogenic bacteria contamination poses a major threat to human health. The detection of low-abundance bacteria in complex samples has always been a knotty problem, and high-sensitivity bacterial detection remains challenging. In this work, a novel magnetic platform with high enrichment efficiency for L. monocytogenes was developed. The magnetic platform was designed by branched polyglutamic acid-mediated indirect coupling of cefepime on magnetic nanoparticles (Cefe-PGA-MNPs), and the specific enrichment of low-abundance L. monocytogenes in real samples was achieved by an external magnet, with a capture efficiency over 90%. A controllable and highly active platinum-palladium nanozyme was synthesized and further introduced in the magnetic nanoplatform for the construction of enzymatic colorimetric biosensor. The total detection time for L. monocytogenes was within 100 min. The colorimetric signals generated by labelled nanozyme were corresponding to different concentrations of L. monocytogenes, with a limit of detection (LOD) of 3.1 × 101 CFU/mL, and high reliability and accuracy (with a recovery rate ranging from 96.5% to 116.4%) in the test of real samples. The concept of the developed method is applicable to various fields of biosensing that rely on magnetic separation platforms.

Cloning and expression of the phenazine-1-carboxamide hydrolysis gene pzcH and the identification of the key amino acids necessary for its activity.

Phenazine-1-carboxamide (PCN), a phenazine derivative, can cause toxicity risks to non target organisms. In this study, the Gram-positive bacteria Rhodococcus equi WH99 was found to have the ability to degrade PCN. PzcH, a novel amidase belonging to amidase signature (AS) family, responsible for hydrolyzing PCN to PCA was identified from strain WH99. PzcH shared no similarity with amidase PcnH which can also hydrolyze PCN and belong to the isochorismatase superfamily from Gram-negative bacteria Sphingomonas histidinilytica DS-9. PzcH also showed low similarity (˂ 39%) with other reported amidases. The optimal catalysis temperature and pH of PzcH was 30 °C and 9.0, respectively. The Km and kcat values of PzcH for PCN were 43.52 ± 4.82 µM and 17.028 ± 0.57 s-1, respectively. The molecular docking and point mutation experiment demonstrated that catalytic triad Lys80-Ser155-Ser179 are essential for PzcH to hydrolyze PCN. Strain WH99 can degrade PCN and PCA to reduce their toxicity against the sensitive organisms. This study enhances our understanding of the molecular mechanism of PCN degradation, presents the first report on the key amino acids in PzcH from the Gram-positive bacteria and provides an effective strain in the bioremediation PCN and PCA contaminated environments.

MXene@Au based electrochemical biosensor with pretreatment by magnetic nanoparticles for determination of MRSA from clinical samples.

Pathogenic bacteria are associated with high morbidity rates and present significant diagnostic challenges in terms of rapid detection. This study introduces a magnetic separation-based electrochemical biosensor for the detection of Methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin (Van) was used to modify on the surface of polyethyleneimine (PEI) mediated MBs (MBs-PEI-Van) for separation and enrichment of MRSA. The MBs-PEI-Van shown a satisfactory stability and applicability with capture effective (CE) > 85% in both PBS and cerebrospinal fluid (CSF) samples. MXene@Au with controllable size of AuNPs was synthesized by a self-reduction method and employed to modify the glassy carbon electrode (GCE). Immunoglobulin G (IgG) was loaded onto the modified electrode to immobilize MRSA, and ferroceneboronic acid (Fc-BA) was used as a probe for quantitative determination. The differential pulse voltammetry (DPV) current was plotted against the concentration of MRSA from 3.8 × 101 to 3.8 × 107 CFU/mL with a limit of detection (LOD) of 3.8 × 101 CFU/mL. In addition, MRSA was successfully detected in spiked CSF samples with satisfactory recoveries (94.35-107.81 %) and validation results (RSD < 11 %). Overall, this study presents a promising method for the detection of MRSA, with the potential to be further developed into a universal pathogen detection method.

PcaR, a GntR/FadR Family Transcriptional Repressor Controls the Transcription of Phenazine-1-Carboxylic Acid 1,2-Dioxygenase Gene Cluster in Sphingomonas histidinilytica DS-9.

In our previous study, the phenazine-1-carboxylic acid (PCA) 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster) in Sphingomonas histidinilytica DS-9 was identified to be responsible for the conversion of PCA to 1,2-dihydroxyphenazine (Ren Y, Zhang M, Gao S, Zhu Q, et al. 2022. Appl Environ Microbiol 88:e00543-22). However, the regulatory mechanism of the pcaA1A2A3A4 cluster has not been elucidated yet. In this study, the pcaA1A2A3A4 cluster was found to be transcribed as two divergent operons: pcaA3-ORF5205 (named A3-5205 operon) and pcaA1A2-ORF5208-pcaA4-ORF5210 (named A1-5210 operon). The promoter regions of the two operons were overlapped. PcaR acts as a transcriptional repressor of the pcaA1A2A3A4 cluster, and it belongs to GntR/FadR family transcriptional regulator. Gene disruption of pcaR can shorten the lag phase of PCA degradation. The results of electrophoretic mobility shift assay and DNase I footprinting showed that PcaR binds to a 25-bp motif in the ORF5205-pcaA1 intergenic promoter region to regulate the expression of two operons. The 25-bp motif covers the -10 region of the promoter of A3-5205 operon and the -35 region and -10 region of the promoter of A1-5210 operon. The TNGT/ANCNA box within the motif was essential for PcaR binding to the two promoters. PCA acted as an effector of PcaR, preventing it from binding to the promoter region and repressing the transcription of the pcaA1A2A3A4 cluster. In addition, PcaR represses its own transcription, and this repression can be relieved by PCA. This study reveals the regulatory mechanism of PCA degradation in strain DS-9, and the identification of PcaR increases the variety of regulatory model of the GntR/FadR-type regulator. IMPORTANCE Sphingomonas histidinilytica DS-9 is a phenazine-1-carboxylic acid (PCA)-degrading strain. The 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster, encoding dioxygenase PcaA1A2, reductase PcaA3, and ferredoxin PcaA4) is responsible for the initial degradation step of PCA and widely distributed in Sphingomonads, but its regulatory mechanism has not been investigated yet. In this study, a GntR/FadR-type transcriptional regulator PcaR repressing the transcription of pcaA1A2A3A4 cluster and pcaR gene was identified and characterized. The binding site of PcaR in ORF5205-pcaA1 intergenic promoter region contains a TNGT/ANCNA box, which is important for the binding. These findings enhance our understanding of the molecular mechanism of PCA degradation.

Portable dual-mode biosensor based on smartphone and glucometer for on-site sensitive detection of Listeria monocytogenes.

Contamination of Listeria monocytogenes (L. monocytogenes) in the environment and food can pose a serious threat to human health, and there is an urgent need to establish sensitive on-situ detection methods to mitigate its hazards. In this study, we have developed a field assay that combines magnetic separation technology with antibody-labeled ZIF-8 encapsulating glucose oxidase (GOD@ZIF-8@Ab) to capture and specifically identify L. monocytogenes while GOD catalyzes glucose catabolism to produce signal changes in glucometers. On the other side, horseradish peroxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) were added to recombined with the H2O2 generated by the catalyst to form a colorimetric reaction system that changes from colorless to blue. The smartphone software was used for RGB analysis to complete the on-site colorimetric detection of L. monocytogenes. This dual-mode biosensor showed good detection performance for the on-site application of L. monocytogenes in lake water and juice samples, both with a limit of detection up to 101 CFU/mL and a good linear range of 101-106 CFU/mL. Therefore, this dual-mode on-site detection biosensor has a promising application for the early screening of L. monocytogenes in environmental and food samples.

Cefepime-modified magnetic nanoparticles and enzymatic colorimetry for the detection of Listeria monocytogenes in lettuces.

A novel sandwich assay for the detection of L. monocytogenes was designed based on antibiotic magnetic separation and enzymatic colorimetry. PEG-mediated cefepime functionalized magnetic nanoparticles (Cefe-PEG-MNPs) was reported for the first time to anchor L. monocytogenes cells with excellent bacterial capture capacity. The capture efficiency of L. monocytogenes in lettuce sample with high concentration (3.1 × 106 CFU/mL) was more than 73.8%. Anti-L. monocytogenes monoclonal antibody was adopted as the second anchoring agent to ensure the specificity for L. monocytogenes, which was co-modified with HRP on the surface of gold nanoparticles (AuNPs-HRP/mAb) to form AuNPs-HRP/mAb@L. monocytogenes@Cefe-PEG-MNPs sandwich complexes, and TMB was added to generate a colorimetric signal. The limit of detection in contaminated lettuce, watermelon juice, and fresh meat samples were both 3.1 × 102 CFU/mL, and the whole assay takes about 110 min. Based on the above facts, the proposed method has great potential for rapid separation and detection of pathogenic bacteria in food.

Portable sensor based on magnetic separation and enzyme-mediated immune nanomaterials for point-of-care testing of Listeria monocytogenes in food.

Listeria monocytogenes (L. monocytogenes), a typical foodborne pathogen, poses a serious threat to public health safety. This stimulates to develop a point-of-care testing (POCT) method to achieve rapid, sensitive detection of L. monocytogenes. In this study, polyethylene glycol (PEG) mediated ampicillin functionalized magnetic beads (Amp-PEG-MBs) was prepared successfully and it achieved high efficiency (>90%) and rapid (5 min) capture for L. monocytogenes at room temperature. The innovative combination of antibody (Ab), glucose oxidase (GOD) and graphene oxide (GO) prepared Ab@GO@GOD for the specific recognition of L. monocytogenes. Finally, Amp-PEG-MBs and Ab@GO@GOD were successfully assembled into Amp-PEG-MBs@L. monocytogenes-Ab@GO@GOD sandwich structure which could catalyze the glucose, and the final detection results were recorded by a blood glucose meter (BGM). Magnetic separation (MS) combined with enzyme-catalyzed sensor (MS-Ab@GO@GOD-BGM) was successfully established to achieve the detection of L. monocytogenes in artificially contaminated juice within 66 min with the limit of detection was 101 CFU/mL. This sensor has potential for other pathogens detection by modifying specific antibodies.

Functional analysis, diversity, and distribution of carbendazim hydrolases MheI and CbmA, responsible for the initial step in carbendazim degradation.

Strains Rhodococcus qingshengii djl-6 and Rhodococcus jialingiae djl-6-2 both harbour the typical carbendazim degradation pathway with the hydrolysis of carbendazim to 2-aminobenzimidazole (2-AB) as the initial step. However, the enzymes involved in this process are still unknown. In this study, the previous reported carbendazim hydrolase MheI was found in strain djl-6, but not in strain djl-6-2, then another carbendazim hydrolase CbmA was obtained by a four-step purification strategy from strain djl-6-2. CbmA was classified as a member of the amidase signature superfamily with conserved catalytic site residues Ser157, Ser181, and Lys82, while MheI was classified as a member of the Abhydrolase superfamily with conserved catalytic site residues Ser77 and His224. The catalytic efficiency (kcat /Km ) of MheI (24.0-27.9 µM-1  min-1 ) was 200 times more than that of CbmA (0.032-0.21 µM-1  min-1 ). The mheI gene (plasmid encoded) was highly conserved (>99% identity) in the strains from different bacterial genera and its plasmid encoded flanked by mobile genetic elements. The cmbA gene was highly conserved only in strains of the genus Rhodococcus and it was chromosomally encoded. Overall, the function, diversity, and distribution of carbendazim hydrolases MheI and CbmA will provide insights into the microbial degradation of carbendazim.

Passively Q-switched erbium-doped fiber laser using evanescent field interaction with gold-nanosphere based saturable absorber.

We demonstrate an all-fiber passively Q-switched erbium-doped fiber laser (EDFL) using a gold-nanosphere (GNS) based saturable absorber (SA) with evanescent field interaction. Using the interaction of evanescent field for fabricating SAs, long nonlinear interaction length of evanescent wave and GNSs can be achieved. The GNSs are synthesized from mixing solution of chloroauricacid (HAuCl4) and sodium citrate by the heating effects of the microfiber's evanescent field radiation. The proposed passively Q-switched EDFL could give output pulses at 1562 nm with pulse width of 1.78 µs, a repetition rate of 58.1 kHz, a pulse energy of 133 nJ and a output power of 7.7 mW when pumped by a 980 nm laser diode of 237 mW.

Broadband Kerr frequency combs and intracavity soliton dynamics influenced by high-order cavity dispersion.

We numerically investigate the influence of high-order dispersion (HOD) on temporal and spectral characteristics of microresonator-based optical frequency combs. Theoretical analysis based on the moment method associated with numerical simulations are utilized to study the comb evolution dynamics, showing that temporal shifts of steady-state intracavity solitons are induced by high-odd-order dispersion rather than high-even-order dispersion. The role of HOD on comb spectral envelopes is also elucidated through analyzing the intracavity dispersive wave generations. We further demonstrate that the spectral envelope of a broadband optical frequency comb can be engineered by using a cavity dispersion profile with multiple zero dispersion wavelengths.