Displaying publications 1 - 20 of 26 in total

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  1. Fulltext Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production
    Remali J, Sarmin N'M, Ng CL, Tiong JJL, Aizat WM, Keong LK, et al.
    PeerJ, 2017;5:e3738.
    PMID: 29201559 DOI: 10.7717/peerj.3738
    Background: Streptomyces are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy) carbonyl) phenazine-1-carboxylic acid (HCPCA) extracted from Streptomyces kebangsaanensis, an endophyte isolated from the ethnomedicinal Portulaca oleracea.

    Methods: The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s) believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of S. kebangsaanensis in producing other useful secondary metabolites.

    Results: The S. kebangsaanensis genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35%) consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis.

    Discussion: The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of S. kebangsaanensis for producing various antibiotics and secondary metabolites.

    Matched MeSH terms: rRNA Operon
  2. Fulltext Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems
    Chan WT, Espinosa M, Yeo CC
    Front Mol Biosci, 2016;3:9.
    PMID: 27047942 DOI: 10.3389/fmolb.2016.00009
    In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I-VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.
    Matched MeSH terms: Operon
  3. Fulltext The Importance of the Expendable: Toxin-Antitoxin Genes in Plasmids and Chromosomes
    Díaz-Orejas R, Espinosa M, Yeo CC
    Front Microbiol, 2017;8:1479.
    PMID: 28824602 DOI: 10.3389/fmicb.2017.01479
    Toxin-antitoxin (TA) genes were first reported in plasmids and were considered expendable genetic cassettes involved in the stable maintenance of the plasmid replicon by interfering with growth and/or viability of bacteria in which the plasmid was lost. TAs were later found in bacterial chromosomes and also in integrated mobile genetic elements; they were proposed to be involved in the bacterial response to stressful situations. At present, 100s of TAs have been identified and classified in up to six families (I to VI), with those belonging to the type II (constituted by two protein components) being the most studied. Based on well-characterized examples of several type II TAs, we discuss in this review that irrespective of their locations in plasmids or chromosomes, TAs functionally overlap as indicated by: (i) in both locations they can mediate the maintenance of genetic elements to which they are physical linked, and (ii) they can induce persistence or virulence in response to stress situations. Examples of functional confluences in homologous TA systems with different locations are also given. We also consider whether the physiological role of TAs is due to their genetic organization as operons or to their inherent properties, like the short lifespan of the antitoxin component.
    Matched MeSH terms: Operon
  4. Fulltext Genetic regulation of the yefM-yoeB toxin-antitoxin locus of Streptococcus pneumoniae
    Chan WT, Nieto C, Harikrishna JA, Khoo SK, Othman RY, Espinosa M, et al.
    J Bacteriol, 2011 Sep;193(18):4612-25.
    PMID: 21764929 DOI: 10.1128/JB.05187-11
    Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae, there are at least eight putative TAS, one of them being the yefM-yoeB(Spn) operon studied here. Through footprinting analyses, we showed that purified YefM(Spn) antitoxin and the YefM-YoeB(Spn) TA protein complex bind to a palindrome sequence encompassing the -35 region of the main promoter (P(yefM2)) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to P(yefM2), since YefM(Spn) behaved as a weak repressor with YoeB(Spn) as a corepressor. Interestingly, a BOX element, composed of a single copy (each) of the boxA and boxC subelements, was found upstream of promoter P(yefM2). BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation, and genetic competence. In the yefM-yoeB(Spn) locus, the boxAC element provided an additional weak promoter, P(yefM1), upstream of P(yefM2) which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that P(yefM1) was constitutive albeit weaker than P(yefM2). Intriguingly, the coupling of the boxAC element to P(yefM1) and yefM(Spn) in cis (but not in trans) led to transcriptional activation, indicating that the regulation of the yefM-yoeB(Spn) locus differs somewhat from that of other TA loci and may involve as yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeB(Spn) locus suggested that its presence may provide a selective advantage to the bacterium.
    Matched MeSH terms: Operon
  5. Fulltext The dnd operon for DNA phosphorothioation modification system in Escherichia coli is located in diverse genomic islands
    Ho WS, Ou HY, Yeo CC, Thong KL
    BMC Genomics, 2015;16:199.
    PMID: 25879448 DOI: 10.1186/s12864-015-1421-8
    Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands.
    Matched MeSH terms: Operon/genetics*
  6. Genomic characterization of endemic Salmonella enterica serovar Typhimurium and Salmonella enterica serovar I 4,[5],12:i:- isolated in Malaysia
    Ngoi ST, Yap KP, Thong KL
    Infect Genet Evol, 2018 08;62:109-121.
    PMID: 29684710 DOI: 10.1016/j.meegid.2018.04.027
    Salmonella enterica serovar Typhimurium (S. Typhimurium) and the monophasic variant Salmonella I 4,[5],12:i:- are two clinically-important non-typhoidal Salmonella serovars worldwide. However, the genomic information of these two organisms, especially the monophasic variant, is still lacking in Malaysia. The objective of the study was to compare the genomic features of a monophasic variant and two endemic S. Typhimurium strains isolated from humans. All three strains were subjected to whole genome sequencing followed by comparative genomic and phylogenetic analyses. Extensive genomic deletion in the fljAB operon (from STM2757 to iroB) is responsible for the monophasic phenotype of STM032/04. The two S. Typhimurium genomes (STM001/70 and STM057/05) were essentially identical, despite being isolated 35 years apart. All three strains were of sequence type ST19. Both S. Typhimurium genomes shared unique prophage regions not identified in the monophasic STM032/04 genome. Core genome phylogenetic analyses showed that the monophasic STM032/04 was closely-related to the S. Typhimurium LT2, forming a distinctive clade separated from the two endemic S. Typhimurium strains in Malaysia. The presence of serovar Typhimurium-specific mdh gene, conserved Gifsy and Fels-1 prophages, and the close genomic resemblance with S. Typhimurium LT2 suggested that the monophasic STM032/04 was originated from an LT2-like S. Typhimurium ancestor in Malaysia, following an evolutionary path different from the S. Typhimurium strains. In conclusion, the monophasic Salmonella I 4,[5],12:i:- and the S. Typhimurium strains isolated in Malaysia descended from different phylogenetic lineages. The high genomic resemblance between the two S. Typhimurium strains isolated for at least 35 years apart indicated their successful evolutionary lineage. The identification of multiple virulence and antimicrobial resistance determinants in the Salmonella I 4,[5],12:i:- and S. Typhimurium genomes explained the pathogenic nature of the organisms.
    Matched MeSH terms: Operon
  7. Fulltext Draft Genome Sequence of Arsenic-Resistant Microbacterium sp. Strain SZ1 Isolated from Arsenic-Bearing Gold Ores
    Mohd Bahari Z, Ibrahim Z, Jaafar J, Shahir S
    Genome Announc, 2017 Oct 26;5(43).
    PMID: 29074663 DOI: 10.1128/genomeA.01183-17
    Microbacterium sp. strain SZ1 isolated from gold ores of a Malaysia gold mine was found to be highly resistant to arsenic. Here, we report the draft genome sequence of SZ1, which may provide further insights into understanding its arsenic resistance mechanism. In this draft genome, a complete set of ars operons and two additional scattered ars genes were encoded.
    Matched MeSH terms: Operon
  8. Cloning and characterization of poly(3-hydroxybutyrate) biosynthesis genes from Pseudomonas sp. USM 4-55
    Tan Y, Neo PC, Najimudin N, Sudesh K, Muhammad TS, Othman AS, et al.
    J Basic Microbiol, 2010 Apr;50(2):179-89.
    PMID: 20082371 DOI: 10.1002/jobm.200900138
    Pseudomonas sp. USM 4-55 is a locally isolated bacterium that possesses the ability to produce polyhydroxyalkanoates (PHA) consisting of both poly(3-hydroxybutyrate) [P(3HB)] homopolymer and medium-chain length (mcl) monomers (6 to 14 carbon atoms) when sugars or fatty acids are utilized as the sole carbon source. In this study, the P(3HB) biosynthesis operon carrying the phbC(Ps) P(3HB) synthase was successfully cloned and sequenced using a homologous probe. Three open reading frames encoding NADPH-dependent acetoacetyl-coenzyme A reductase (PhbB(Ps)), beta-ketothiolase (PhbA(Ps)) and P(3HB) synthase (PhbC(Ps)) were found in the phb operon. The genetic organization of phb operon showed a putative promoter region, followed by phbB(Ps)-phbA(Ps)-phbC(Ps). phbR(Ps)which encoded a putative transcriptional activator was located in the opposite orientation, upstream of phbBAC(Ps). Heterologous expression of pGEM''ABex harboring phbC(Ps) in Escherichia coli JM109 resulted in P(3HB) accumulation of up to 40% of dry cell weight (DCW).
    Matched MeSH terms: Operon
  9. The FloR master regulator controls flotation, virulence and antibiotic production in Serratia sp. ATCC 39006
    Quintero-Yanes A, Lee CM, Monson R, Salmond G
    Environ Microbiol, 2020 07;22(7):2921-2938.
    PMID: 32352190 DOI: 10.1111/1462-2920.15048
    Serratia sp. ATCC 39006 produces intracellular gas vesicles to enable upward flotation in water columns. It also uses flagellar rotation to swim through liquid and swarm across semi-solid surfaces. Flotation and motility can be co-regulated with production of a β-lactam antibiotic (carbapenem carboxylate) and a linear tripyrrole red antibiotic, prodigiosin. Production of gas vesicles, carbapenem and prodigiosin antibiotics, and motility are controlled by master transcriptional and post-transcriptional regulators, including the SmaI/SmaR-based quorum sensing system and the mRNA binding protein, RsmA. Recently, the ribose operon repressor, RbsR, was also defined as a pleiotropic regulator of flotation and virulence factor elaboration in this strain. Here, we report the discovery of a new global regulator (FloR; a DeoR family transcription factor) that modulates flotation through control of gas vesicle morphogenesis. The floR mutation is highly pleiotropic, down-regulating production of gas vesicles, carbapenem and prodigiosin antibiotics, and infection in Caenorhabditis elegans, but up-regulating flagellar motility. Detailed proteomic analysis using TMT peptide labelling and LC-MS/MS revealed that FloR is a physiological master regulator that operates through subordinate pleiotropic regulators including Rap, RpoS, RsmA, PigU, PstS and PigT.
    Matched MeSH terms: Operon
  10. Fulltext Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa
    Wong CF, Rahman RNZRA, Basri M, Salleh AB
    Iran J Biotechnol, 2017;15(3):194-200.
    PMID: 29845069 DOI: 10.15171/ijb.1524
    Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
    Matched MeSH terms: Lac Operon
  11. Fulltext CsoR metalloregulatory protein: function, mechanism and relevance
    Mangavelu, Ashwaani, Yahaya M. Normi, Leow, Adam Thean Chor, Mohd Shukuri Mohammad Ali, Raja Noor Zaliha Raja Abd. Rahman
    The Pertanika Journal of Scholarly Research Reviews, 2017;3(1):24-31.
    MyJurnal
    Transition metals are required constituent in bacterial metabolism to assist in some enzymatic reactions. However, intracellular accumulations of these metal ions are harmful to the bacteria as it can trigger unnecessary redox reactions. To overcome this condition, metalloregulatory proteins assist organisms to adapt to sudden elevated and deprived metal ion concentration in the environment via metal homeostasis. CsoR protein is a copper(I) [Cu(I)] sensing operon repressor that is found to be present in all major classes of eubacteria. This metalloregulatory protein binds to the operator region in its apo state under Cu(I) limiting condition and detaches off from the regulatory region when it binds to the excess cytosolic Cu(I) ion, thus derepressing the expression of genes involved in Cu(I) homeostasis. CsoR proteins exist in dimeric and tetrameric states and form certain coordination geometries upon attachment with Cu(I). Certain CsoR proteins have also been found to possess the ability to bind to other types of metals with various binding affinities in some Gram positive bacteria. The role of this metalloregulatory protein in host pathogen interaction and its relation to bacterial virulence are also discussed.
    Matched MeSH terms: Operon
  12. Fulltext Multiphasic strain differentiation of atypical mycobacteria from elephant trunk wash
    Chan KG, Loke MF, Ong BL, Wong YL, Hong KW, Tan KH, et al.
    PeerJ, 2015;3:e1367.
    PMID: 26587340 DOI: 10.7717/peerj.1367
    Background. Two non-tuberculous mycobacterial strains, UM_3 and UM_11, were isolated from the trunk wash of captive elephants in Malaysia. As they appeared to be identical phenotypes, they were investigated further by conventional and whole genome sequence-based methods of strain differentiation. Methods. Multiphasic investigations on the isolates included species identification with hsp65 PCR-sequencing, conventional biochemical tests, rapid biochemical profiling using API strips and the Biolog Phenotype Microarray analysis, protein profiling with liquid chromatography-mass spectrometry, repetitive sequence-based PCR typing and whole genome sequencing followed by phylogenomic analyses. Results. The isolates were shown to be possibly novel slow-growing schotochromogens with highly similar biological and genotypic characteristics. Both strains have a genome size of 5.2 Mbp, G+C content of 68.8%, one rRNA operon and 52 tRNAs each. They qualified for classification into the same species with their average nucleotide identity of 99.98% and tetranucleotide correlation coefficient of 0.99999. At the subspecies level, both strains showed 98.8% band similarity in the Diversilab automated repetitive sequence-based PCR typing system, 96.2% similarity in protein profiles obtained by liquid chromatography mass spectrometry, and a genomic distance that is close to zero in the phylogenomic tree constructed with conserved orthologs. Detailed epidemiological tracking revealed that the elephants shared a common habitat eight years apart, thus, strengthening the possibility of a clonal relationship between the two strains.
    Matched MeSH terms: rRNA Operon
  13. Fulltext The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella
    Kushwaha SK, Bhavesh NLS, Abdella B, Lahiri C, Marathe SA
    Sci Rep, 2020 12 03;10(1):21156.
    PMID: 33273523 DOI: 10.1038/s41598-020-77890-6
    Salmonellae display intricate evolutionary patterns comprising over 2500 serovars having diverse pathogenic profiles. The acquisition and/or exchange of various virulence factors influences the evolutionary framework. To gain insights into evolution of Salmonella in association with the CRISPR-Cas genes we performed phylogenetic surveillance across strains of 22 Salmonella serovars. The strains differed in their CRISPR1-leader and cas operon features assorting into two main clades, CRISPR1-STY/cas-STY and CRISPR1-STM/cas-STM, comprising majorly typhoidal and non-typhoidal Salmonella serovars respectively. Serovars of these two clades displayed better relatedness, concerning CRISPR1-leader and cas operon, across genera than between themselves. This signifies the acquisition of CRISPR1/Cas region could be through a horizontal gene transfer event owing to the presence of mobile genetic elements flanking CRISPR1 array. Comparison of CRISPR and cas phenograms with that of multilocus sequence typing (MLST) suggests differential evolution of CRISPR/Cas system. As opposed to broad-host-range, the host-specific serovars harbor fewer spacers. Mapping of protospacer sources suggested a partial correlation of spacer content with habitat diversity of the serovars. Some serovars like serovar Enteritidis and Typhimurium that inhabit similar environment/infect similar hosts hardly shared their protospacer sources.
    Matched MeSH terms: Operon/genetics
  14. Transcriptional responses of Streptococcus gordonii and Fusobacterium nucleatum to coaggregation
    Mutha NVR, Mohammed WK, Krasnogor N, Tan GYA, Choo SW, Jakubovics NS
    Mol Oral Microbiol, 2018 12;33(6):450-464.
    PMID: 30329223 DOI: 10.1111/omi.12248
    Cell-cell interactions between genetically distinct bacteria, known as coaggregation, are important for the formation of mixed-species biofilms such as dental plaque. Interactions lead to gene regulation in the partner organisms that may be critical for adaptation and survival in mixed-species biofilms. Here, gene regulation responses to coaggregation between Streptococcus gordonii and Fusobacterium nucleatum were studied using dual RNA-Seq. Initially, S. gordonii was shown to coaggregate strongly with F. nucleatum in buffer or human saliva. Using confocal laser scanning microscopy and transmission electron microscopy, cells of different species were shown to be evenly distributed throughout the coaggregate and were closely associated with one another. This distribution was confirmed by serial block face sectioning scanning electron microscopy, which provided high resolution three-dimensional images of coaggregates. Cell-cell sensing responses were analysed 30 minutes after inducing coaggregation in human saliva. By comparison with monocultures, 16 genes were regulated following coaggregation in F. nucleatum whereas 119 genes were regulated in S. gordonii. In both species, genes involved in amino acid and carbohydrate metabolism were strongly affected by coaggregation. In particular, one 8-gene operon in F. nucleatum encoding sialic acid uptake and catabolism was up-regulated 2- to 5-fold following coaggregation. In S. gordonii, a gene cluster encoding functions for phosphotransferase system-mediated uptake of lactose and galactose was down-regulated up to 3-fold in response to coaggregation. The genes identified in this study may play key roles in the development of mixed-species communities and represent potential targets for approaches to control dental plaque accumulation.
    Matched MeSH terms: Operon
  15. Fulltext First report on the molecular epidemiology of Malaysian Staphylococcus epidermidis isolated from a university teaching hospital
    Sani NA, Sapri HF, Neoh HM, Hussin S
    BMC Res Notes, 2014;7:597.
    PMID: 25186825 DOI: 10.1186/1756-0500-7-597
    Staphylococcus epidermidis is a pathogen associated with nosocomial infections whose medical importance has increased due to progressively invasive medical procedures. In this study, we characterized the molecular epidemiology of S. epidermidis strains circulating in our university hospital situated in Kuala Lumpur, Malaysia.
    Matched MeSH terms: Operon
  16. Fulltext Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109
    Yee LN, Chuah JA, Chong ML, Phang LY, Raha AR, Sudesh K, et al.
    Microbiol Res, 2012 Oct 12;167(9):550-7.
    PMID: 22281521 DOI: 10.1016/j.micres.2011.12.006
    In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA(Co) gene, 1182 bp), acetoacetyl-CoA reductase (phaB(Co) gene, 738 bp) and PHA synthase, class I (phaC(Co) gene, 1694 bp) were identified. Sequence analysis of the phaA(Co), phaB(Co) and phaC(Co) genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaC(Co) and phaAB(Co)) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaC(Co)AB(Re) and pGEM'-phaC(Re)AB(Co) were shown to be functionally active synthesising 33 wt.% and 17 wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCAB(Co)) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCAB(Co) could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively.
    Matched MeSH terms: Operon
  17. Fulltext The cry toxin operon of Clostridium bifermentans subsp. malaysia is highly toxic to Aedes Larval Mosquitoes
    Qureshi N, Chawla S, Likitvivatanavong S, Lee HL, Gill SS
    Appl Environ Microbiol, 2014 Sep;80(18):5689-97.
    PMID: 25002432 DOI: 10.1128/AEM.01139-14
    The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic bacteria Bacillus thuringiensis and Lysinibacillus sphaericus have been used for vector control for a number of decades. But a more cost-effective use would be an anaerobic bacterium because of the ease with which these can be cultured. More recently, the anaerobic bacterium Clostridium bifermentans subsp. malaysia has been reported to have high mosquitocidal activity, and a number of proteins were identified as potentially mosquitocidal. However, the cloned proteins showed no mosquitocidal activity. We show here that four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, are all required for toxicity, and these toxins collectively show remarkable selectivity for Aedes rather than Anopheles mosquitoes, even though C. bifermentans subsp. malaysia is more toxic to Anopheles. Hence, toxins that target Anopheles are different from those expressed by the Cry operon.
    Matched MeSH terms: Operon*
  18. Functional identification of the prnABCD operon and its regulation in Serratia plymuthica
    Liu X, Yu X, Yang Y, Heeb S, Gao S, Chan KG, et al.
    Appl Microbiol Biotechnol, 2018 Apr;102(8):3711-3721.
    PMID: 29511844 DOI: 10.1007/s00253-018-8857-0
    The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum of antimicrobial activities. The PRN biosynthetic gene cluster remains to be characterised in Serratia plymuthica, though it is highly conserved in PRN-producing bacteria. To better understand PRN biosynthesis and its regulation in Serratia, the prnABCD operon from S. plymuthica G3 was cloned, sequenced and expressed in Escherichia coli DH5α. Furthermore, an engineered strain prnind which is a conditional mutant of G3 prnABCD under the control of the Ptac promoter was constructed. This mutant was able to overproduce PRN with isopropylthiogalactoside (IPTG) induction by overexpressing prnABCD, whilst behaving as a conditional mutant of G3 prnABCD in the absence of IPTG. These results confirmed that prnABCD is responsible for PRN biosynthesis in strain G3. Further experiments involving lux-/dsRed-based promoter fusions, combined with site-directed mutagenesis of the putative σS extended -10 region in the prnA promoter, and liquid chromatography-mass spectrometry (LC-MS) analysis extended our previous knowledge about G3, revealing that quorum sensing (QS) regulates PRN biosynthesis through cross talk with RpoS, which may directly activated prnABCD transcription. These findings suggest that PRN in S. plymuthica G3 is produced in a tightly controlled manner, and has diverse functions, such as modulation of cell motility, in addition to antimicrobial activities. Meanwhile, the construction of inducible mutants could be a powerful tool to improve PRN production, beyond its potential use for the investigation of the biological function of PRN.
    Matched MeSH terms: Operon/genetics*
  19. Fulltext Functional validation of putative toxin-antitoxin genes from the Gram-positive pathogen Streptococcus pneumoniae: phd-doc is the fourth bona-fide operon
    Chan WT, Yeo CC, Sadowy E, Espinosa M
    Front Microbiol, 2014;5:677.
    PMID: 25538695 DOI: 10.3389/fmicb.2014.00677
    Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between 4 and 10 putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary(19A)-6 which contains all possible 10 TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.
    Matched MeSH terms: Operon
  20. Fulltext The Streptococcus pneumoniaeyefM-yoeB and relBE Toxin-Antitoxin Operons Participate in Oxidative Stress and Biofilm Formation
    Chan WT, Domenech M, Moreno-Córdoba I, Navarro-Martínez V, Nieto C, Moscoso M, et al.
    Toxins (Basel), 2018 09 18;10(9).
    PMID: 30231554 DOI: 10.3390/toxins10090378
    Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein⁻protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.
    Matched MeSH terms: Operon
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