Enterobacter asburiae L1 is a quorum sensing bacterium isolated from lettuce leaves. In this study, for the first time, the complete genome of E. asburiae L1 was sequenced using the single molecule real time sequencer (PacBio RSII) and the whole genome sequence was verified by using optical genome mapping (OpGen) technology. In our previous study, E. asburiae L1 has been reported to produce AHLs, suggesting the possibility of virulence factor regulation which is quorum sensing dependent. This evoked our interest to study the genome of this bacterium and here we present the complete genome of E. asburiae L1, which carries the virulence factor gene virK, the N-acyl homoserine lactone-based QS transcriptional regulator gene luxR and the N-acyl homoserine lactone synthase gene which we firstly named easI. The availability of the whole genome sequence of E. asburiae L1 will pave the way for the study of the QS-mediated gene expression in this bacterium. Hence, the importance and functions of these signaling molecules can be further studied in the hope of elucidating the mechanisms of QS-regulation in E. asburiae. To the best of our knowledge, this is the first documentation of both a complete genome sequence and the establishment of the molecular basis of QS properties of E. asburiae.
Over the past decades, Enterobacter spp. have been identified as challenging and important pathogens. The emergence of multidrug-resistant Enterobacteria especially those that produce Klebsiella pneumoniae carbapenemase has been a very worrying health crisis. Although efforts have been made to unravel the complex mechanisms that contribute to the pathogenicity of different Enterobacter spp., there is very little information associated with AHL-type QS mechanism in Enterobacter spp. Signaling via N-acyl homoserine lactone (AHL) is the most common quorum sensing (QS) mechanism utilized by Proteobacteria. A typical AHL-based QS system involves two key players: a luxI gene homolog to synthesize AHLs and a luxR gene homolog, an AHL-dependent transcriptional regulator. These signaling molecules enable inter-species and intra-species interaction in response to external stimuli according to population density. In our recent study, we reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. Whole genome sequencing and in silico analysis revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easI/R, in strain L1. In a QS system, a LuxR transcriptional protein detects and responds to the concentration of a specific AHL controlling gene expression. In E. asburiae strain L1, EasR protein binds to its cognate AHLs, N-butanoyl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL), modulating the expression of targeted genes. In this current work, we have cloned the 693 bp luxR homolog of strain L1 for further characterization. The functionality and specificity of EasR protein in response to different AHL signaling molecules to activate gene transcription were tested and validated with β-galactosidase assays. Higher β-galactosidase activities were detected for cells harboring EasR, indicating EasR is a functional transcriptional regulator. This is the first report documenting the cloning and characterization of transcriptional regulator, luxR homolog of E. asburiae.
In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.
Pectobacterium carotovorum M022T has been isolated from a waterfall source in Selangor district (Malaysia). Using genomic and phenotypic tests, we re-examined the taxonomical position of this strain. Based on 14 concatenated housekeeping genes (fusA, rpoD, rpoS, acnA, purA, gyrB, recA, mdh, mtlD, groEL, secY, glyA, gapA and rplB), multi-locus sequence analysis revealed that strain M022T falls into a novel clade separated from the other Pectobacterium species. The in silico DNA-DNA hybridization and average nucleotide identity values were lower than the 70 and 95 % threshold values, respectively. In addition, by combining genomic and phenotypic tests, strain M022T may be distinguished from the other Pectobacterium isolates by its incapacity to grow on d(+)-xylose, l-rhamnose, cellobiose and lactose. Strain M022T (=CFBP 8629T=LMG 30744T) is proposed as the type strain of the Pectobacteriumfontis sp. nov.
Strains 2B12T, FVG1-MFV-O17 and FVG10-MFV-A16 were isolated from fresh water samples collected in Asia and Europe. The nucleotide sequences of the gapA barcodes revealed that all three strains belonged to the same cluster within the genus Dickeya. Using 13 housekeeping genes (fusA, rpoD, rpoS, glyA, purA, groEL, gapA, rplB, leuS, recA, gyrB, infB and secY), multilocus sequence analysis confirmed the existence of a new clade. When the genome sequences of these three isolates and other Dickeya species were compared, the in silico DNA-DNA hybridization and average nucleotide identity values were found to be no more than 45.50 and 91.22 %, respectively. The closest relative species was Dickeya fangzhongdai. Genome comparisons also highlighted genetic traits differentiating the new strains from D. fangzhongdai strains DSM 101947T (=CFBP 8607T) and B16. Phenotypical tests were performed to distinguish the three strains from D. fangzhongdai and other Dickeya species. The name Dickeya undicola sp. nov. is proposed with strain 2B12T (=CFBP 8650T=LMG 30903T) as the type strain.
The under-treated wastewater, especially remaining carcinogenic aromatic compounds in wastewater discharge has been expansively reported, wherein the efficiency of conventional wastewater treatment is identified as the primary contributor source. Herein, the advancement of wastewater treatments has drawn much attention in recent years. In the current study, combined sequential and hybridized treatment of thermolysis and coagulation-flocculation provides a novel advancement for environmental emerging pollutant (EP) prescription. This research is mainly demonstrating the mitigation efficiency and degradation pathway of pararosaniline (PRA) hybridized and combined sequential wastewater treatment. Notably, PRA degradation dominantly via a linkage of reaction: thermal cleavage, deamination, silication and diazene reduction. Thermolysis acts as an initiator for the PRA decomposition through thermally induced bond dissociation energy (BDE) for molecular fragmentation whilst coagulation-flocculation facilitates the formation of organo-bridged silsesquioxane as the final degradation product. Different from conventional treatment, the hybridized treatment showed excellent synergistic degradability by removing 99% PRA and its EPs, followed by combined sequential treatment method with 86% reduction. Comprehensive degradation pathway breakdown of carcinogenic and hardly degradable aromatic compounds provides a new insight for wastewater treatment whereby aniline and benzene are entirely undetectable in effluent. The degradation intermediates, reaction derivatives and end products were affirmed by gas chromatography-mass spectrometry, Fourier transform infrared spectroscopy and ultraviolet-visible spectrophotometry (GC-MS, FTIR and UV-Vis). This finding provides valuable guidance in establishing efficient integrated multiple-step wastewater treatments.
The theme of present research demonstrates performance of copper (II) sulfate (CuSO4) as catalyst in thermolysis process to treat reactive black 5 (RB 5) dye. During thermolysis without presence of catalyst, heat was converted to thermal energy to break the enthalpy of chemical structure bonding and only 31.62% of color removal. With CuSO4 support as auxiliary agent, the thermally cleaved molecular structure was further destabilized and reacted with CuSO4. Copper ions functioned to delocalize the coordination of π of the lone paired electron in azo bond, C=C bond of the sp2 carbon to form C-C of the sp3 amorphous carbon in benzene and naphthalene. Further, the radicals of unpaired electrons were stabilized and RB 5 was thermally decomposed to methyl group. Zeta potential measurement was carried out to analyze the mechanism of RB 5 degradation and measurement at 0 mV verified the critical chemical concentration (CCC) (0.7 g/L copper (II) sulfate), as the maximum 92.30% color removal. The presence of copper (II) sulfate catalyst has remarkably increase the RB 5 dye degradation as the degradation rate constant without catalyst, k1 is 6.5224 whereas the degradation rate constant with catalyst, k2 is 25.6810. This revealed the correlation of conversion of thermal energy from heat to break the chemical bond strength, subsequent fragmentation of RB 5 dye molecular mediated by copper (II) sulfate catalyst. The novel framework on thermolysis degradation of molecular structure of RB 5 with respect to the bond enthalpy and interfacial intermediates decomposition with catalyst reaction were determined.
Pseudomonas aeruginosa is a rod-shaped Gram-negative bacterium which is notably known as a pathogen in humans, animals, and plants. Infections caused by P. aeruginosa especially in hospitalized patients are often life-threatening and rapidly increasing worldwide throughout the years. Recently, multidrug-resistant P. aeruginosa has taken a toll on humans' health due to the inefficiency of antimicrobial agents. Therefore, the rapid and advanced diagnostic techniques to accurately detect this bacterium particularly in clinical samples are indeed necessary to ensure timely and effective treatments and to prevent outbreaks. This review aims to discuss most recent of state-of-the-art molecular diagnostic techniques enabling fast and accurate detection and identification of P. aeruginosa based on well-developed genotyping techniques, e.g., polymerase chain reaction, pulse-field gel electrophoresis, and next generation sequencing. The advantages and limitations of each of the methods are also reviewed.
Bacterial communication or quorum sensing (QS) is achieved via sensing of QS signaling molecules consisting of oligopeptides in Gram-positive bacteria and N-acyl homoserine lactones (AHL) in most Gram-negative bacteria. In this study, Enterobacteriaceae isolates from Batavia lettuce were screened for AHL production. Enterobacter asburiae, identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was found to produce short chain AHLs. High resolution triple quadrupole liquid chromatography mass spectrometry (LC/MS) analysis of the E. asburiae spent supernatant confirmed the production of N-butanoyl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). To the best of our knowledge, this is the first report of AHL production by E. asburiae.