Ben Genes Research Articles

Biodegradation of phthalate isomers by newly isolated Klebsiella variico SY1 and its functional genomic analysis

Phthalate isomers (ortho-, meta-, and para-) are important industrial aromatic dicarboxylic acids and key intermediates of pollutants degradation such as waste polyethylene terephthalate (PET) and plasticizers. Recently, microbial treatment of such pollutants has been received much attention. In this study, Klebsiella variico SY1, which could simultaneously degrade three phthalate isomers, was isolated from river sludge. The degradation priority order of the three phthalate isomers was phthalate (PA), terephthalate (TPA) and isophthalate (IPA), and the tolerant concentration could reach 5500 mg/L. The degradation results showed that SY1 degraded 3000 mg/L of three isomers at the maximum rates of 186.38 mg/L∙h for TPA and 195.90 mg/L∙h for IPA, respectively. The analysis of comparative genome, key enzyme activity, gene expression and degradation intermediates, showed that SY1 initially degraded these three phthalate isomers to a common intermediate protocatechuic acid (PCA), and then accomplished a subsequent degradation through a bifurcated pathway. In addition, it was surmised that the ben gene cluster might also be involved in the degradation of phthalate isomers. In summary, Klebsiella variico SY1 was demonstrated as a chassis cell able to biodegrade phthalates and their derived contaminants due to its high degradation efficiency and tolerance against the three phthalate isomers.

Cloning of the ben gene and its functional identification in Cordyceps militaris

Selectable marker genes are very important for the screening of target mutants. At least two selectable marker genes are usually required to identify the function of a gene by gene knockout, gene complementation, and gene overexpression. However, there are few selectable marker genes that can be applied to Cordyceps militaris. In order to obtain the selectable marker gene which can be used to C. militaris, the sensitivity of C. militaris to benomyl as a fungicide was studied by the concentration gradient method. Then the benomyl resistance gene (the ben gene) was cloned from Neurospora crassa. The binary plasmid of the ben gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the ben gene in C. militaris was identified by the Agrobacterium tumefaciens-mediated transformation (ATMT) method. The results showed that 3 μg/mL benomyl could completely inhibit the growth of C. militaris. The ben gene with a length of 1344 bp was successfully cloned from N. crassa. The binary plasmid pCAMBIA0390-Ben containing the ben gene expression cassette was efficiently constructed by the DJ-PCR method and the homologous recombination method. The ben gene of the plasmid pCAMBIA0390-Ben was successfully integrated into the genome of C. militaris by the ATMT method, and the C. militaris transformants with resistance to benomyl were obtained. Moreover, the ben gene could be highly expressed in C. militaris. It was confirmed that the ben gene derived from N. crassa could be used as a selectable marker gene in C. militaris. These results provide technical support for the identification of C. militaris gene function and the elucidation of the biosynthetic pathways of bioactive components in C. militaris.

Synthetic control of plasmid replication enables target- and self-curing of vectors and expedites genome engineering of Pseudomonas putida

Genome engineering of non-conventional microorganisms calls for the development of dedicated synthetic biology tools. Pseudomonas putida is a Gram-negative, non-pathogenic soil bacterium widely used for metabolic engineering owing to its versatile metabolism and high levels of tolerance to different types of stress. Genome editing of P. putida largely relies on homologous recombination events, assisted by helper plasmid-based expression of genes encoding DNA modifying enzymes. Plasmid curing from selected isolates is the most tedious and time-consuming step of this procedure, and implementing commonly used methods to this end in P. putida (e.g. temperature-sensitive replicons) is often impractical. To tackle this issue, we have developed a toolbox for both target- and self-curing of plasmid DNA in Pseudomonas species. Our method enables plasmid-curing in a simple cultivation step by combining in vivo digestion of vectors by the I-SceI homing nuclease with synthetic control of plasmid replication, triggered by the addition of a cheap chemical inducer (3-methylbenzoate) to the medium. The system displays an efficiency of vector curing >90% and the screening of plasmid-free clones is greatly facilitated by the use of fluorescent markers that can be selected according to the application intended. Furthermore, quick genome engineering of P. putida using self-curing plasmids is demonstrated through genome reduction of the platform strain EM42 by eliminating all genes encoding β-lactamases, the catabolic ben gene cluster, and the pyoverdine synthesis machinery. Physiological characterization of the resulting streamlined strain, P. putida SEM10, revealed advantageous features that could be exploited for metabolic engineering.

Antiviral activity of the inducible humoral immunity and its suppression by eleven BEN family members encoded in Cotesia plutellae bracovirus

Upon parasitization by some endoparasitoids, polydnaviruses (PDVs) play a crucial role in inducing host immunosuppression. This study reports a novel immunosuppressive activity against humoral immune responses by BEN family genes encoded in Cotesia plutellae bracovirus (CpBV). A total of 11 BEN family members are encoded in 10 different CpBV DNA segments. When the CpBV segments were individually injected, specific BEN genes were expressed and suppressed the expression of antimicrobial peptide (AMP) and prophenoloxidase genes following bacterial challenge. The suppressive activities of the BEN genes were reversed by injection of the double-stranded RNA (dsRNA) specific to each BEN gene. The suppression of the AMP gene expressions by the BEN genes was also confirmed using an inhibition zone assay against Gram-positive and Gram-negative bacterial growth. The significance of the suppressive activity of BEN genes against humoral immune responses was analyzed in terms of suppression of antiviral activity by the host humoral immunity. When CpBV was incubated with the plasma obtained from the larvae challenged with bacteria, the immunized plasma severely impaired the expression activity of the viral genes. However, an expression of BEN gene significantly rescued the viral gene expression by suppressing humoral immune response. These results suggest that BEN family genes of CpBV play a crucial role in defending the antiviral response of the parasitized Plutella xylostella by inhibiting humoral immune responses.

The pWW0 plasmid imposes a stochastic expression regime to the chromosomalorthopathway for benzoate metabolism inPseudomonas putida

Environmental plasmids often expand the metabolic repertoire of bacteria that carry them, but they also interfere with the biochemical and genetic network of the host. The pWW0 plasmid born by Pseudomonas putida mt-2 encodes the TOL pathway for degradation of toluene/m-xylene through production of intermediate compounds benzoate/3-methylbenzoate. These can be also recognized as substrates by the chromosomally encoded ben and cat gene products, thereby creating a manifest regulatory and biochemical conflict. In this context, we have investigated how the introduction of the pWW0 plasmid into P. putida affects behaviour of the promoter of the ben pathway (Pb) in single cells. Using a series of standardized transcriptional fusions to green fluorescent protein, we found that acquisition of the TOL pathway switches the activation course of the Pb promoter from unimodal/graded to bimodal/stochastic when cells were exposed to benzoate. This behaviour was propagated downstream into the Pc promoter of the cat gene cluster, which responds to the benzoate-degradation intermediate cis,cis-muconate. The TOL plasmid thus imposes expression of the chromosomal Pb with a stochastic behaviour likely to result in biochemical heterogeneity of the otherwise genetically clonal population when exposed to benzoate as a growth substrate.

Cloning, Characterization and Analysis of cat and ben Genes from the Phenol Degrading Halophilic Bacterium Halomonas organivorans

BackgroundExtensive use of phenolic compounds in industry has resulted in the generation of saline wastewaters that produce significant environmental contamination; however, little information is available on the degradation of phenolic compounds in saline conditions. Halomonas organivorans G-16.1 (CECT 5995T) is a moderately halophilic bacterium that we isolated in a previous work from saline environments of South Spain by enrichment for growth in different pollutants, including phenolic compounds. PCR amplification with degenerate primers revealed the presence of genes encoding ring-cleaving enzymes of the β-ketoadipate pathway for aromatic catabolism in H. organivorans.FindingsThe gene cluster catRBCA, involved in catechol degradation, was isolated from H. organivorans. The genes catA, catB, catC and the divergently transcribed catR code for catechol 1,2-dioxygenase (1,2-CTD), cis,cis-muconate cycloisomerase, muconolactone delta-isomerase and a LysR-type transcriptional regulator, respectively. The benzoate catabolic genes (benA and benB) are located flanking the cat genes. The expression of cat and ben genes by phenol and benzoic acid was shown by RT-PCR analysis. The induction of catA gene by phenol and benzoic acid was also probed by the measurement of 1,2-CTD activity in H. organivorans growth in presence of these inducers. 16S rRNA and catA gene-based phylogenies were established among different degrading bacteria showing no phylogenetic correlation between both genes.Conclusions/SignificanceIn this work, we isolated and determined the sequence of a gene cluster from a moderately halophilic bacterium encoding ortho-pathway genes involved in the catabolic metabolism of phenol and analyzed the gene organization, constituting the first report characterizing catabolic genes involved in the degradation of phenol in moderate halophiles, providing an ideal model system to investigate the potential use of this group of extremophiles in the decontamination of saline environments.

Regulation of benzoate degradation in Acinetobacter sp. strain ADP1 by BenM, a LysR-type transcriptional activator.

In Acinetobacter sp. strain ADP1, benzoate degradation requires the ben genes for converting benzoate to catechol and the cat genes for degrading catechol. Here we describe a novel transcriptional activator, BenM, that regulates the chromosomal ben and cat genes. BenM is homologous to CatM, a LysR-type transcriptional activator of the cat genes. Unusual regulatory features of this system include the abilities of both BenM and CatM to recognize the same inducer, cis,cis-muconate, and to regulate some of the same genes, such as catA and catB. Unlike CatM, BenM responded to benzoate. Benzoate together with cis,cis-muconate increased the BenM-dependent expression of the benABCDE operon synergistically. CatM was not required for this synergism, nor did CatM regulate the expression of a chromosomal benA::lacZ transcriptional fusion. BenM-mediated regulation differs significantly from that of the TOL plasmid-encoded conversion of benzoate to catechol in pseudomonads. The benM gene is immediately upstream of, and divergently transcribed from, benA, and a possible DNA binding site for BenM was identified between the two coding regions. Two mutations in the predicted operator/promoter region rendered ben gene expression either constitutive or inducible by cis,cis-muconate but not benzoate. Mutants lacking BenM, CatM, or both of these regulators degraded aromatic compounds at different rates, and the levels of intermediary metabolites that accumulated depended on the genetic background. These studies indicated that BenM is necessary for ben gene expression but not for expression of the cat genes, which can be regulated by CatM. In a catM-disrupted strain, BenM was able to induce higher levels of catA expression than catB expression.

Bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog

The Drosophila bendless (ben) gene was originally isolated as a mutation affecting the escape jump response. This behavioral defect was ascribed to a single lesion affecting the connectivity between the giant fiber and the tergotrochanter motor neuron. A closer examination of the ben phenotype suggests that ben activity is broader and affects a variety of other neurons including photoreceptor cells and their axons. Mosaic analysis indicates that the focus of ben activity is presynaptic. We have cloned the ben gene through a chromosomal walk and show that it is homologous to a class of ubiquitin-conjugating enzymes. The major role of ubiquitination in the protein degradative pathway suggests that ben regulates neural developmental processes such as growth cone guidance by targeting specific proteins for degradation.

Mapping ofbengenes ofPseudomonas aeruginosa

Four ben genes responsible for the conversion of benzoate to catechol in Pseudomonas aeruginosa PAO have been mapped to a 4.6 kb KpnI fragment, ben-1 and ben-4 were known to be separate genes but now ben-1508 has been found to be different from ben-2. The two genes were distinguished by Tn5 mutagenesis of a cosmid clone and deletion mapping. It is likely that the four genes mapped (ben-4, ben-2, ben-1508 and ben-1) correspond to the previously characterized benR (regulatory gene) and benABC (benzoate dioxygenase) respectively.

The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes

The bendless (ben) mutation of Drosophila alters synaptic connectivity between a subset of CNS neurons. Here, we show that ben also causes morphological abnormalities within the visual system, suggesting that ben functions in a number of different developmental processes. We show that the ben gene encodes a protein which is closely related to ubiquitin-conjugating enzymes and that a missense mutation in the highly conserved active site region is associated with the ben mutation. High levels of ben expression are restricted to the nervous system during development. These results suggest a role for ubiquitin-mediated protein modification in nervous system development, including, but not exclusive to, the regulation of synaptic connectivity.

Effect of benomyl and benomyl resistance on cellulase formation by Trichoderma reesei and Trichoderma harzianum

To investigate the possible relationship between resistance to benomyl and the production of cellulases by Trichoderma spp., we investigated the effect of benomyl on growth and cellulase formation in Trichoderma reesei, Trichoderma harzianum, and the benomyl-resistant mutant T. harzianum T95. While T. reesei produced the highest and T. harzianum the lowest cellulase amounts, growth of both strains was equally inhibited by 2 μg/mL benomyl. However, sublethal doses of benomyl (0.2–0.5 μg/mL) promoted growth, stimulated cellulase production, and produced a highly branched, crippled morphology. The same phenomenon was observed with T. harzianum T-95, albeit at higher (5–10 μg/mL) benomyl concentrations. Introduction of the Neurospora crassa ben gene, coding for a benomyl-resistant β-tubulin, into a T. reesei by transformation yielded a series of transformants, which exhibited increased growth, increased cellulase formation, and highly branched, crippled morphology. Key words: Trichoderma harzianum, Trichoderma reesei, benomyl, rhizosphere competence, cellulase.

Cloning and expression in Escherichia coli of Acinetobacter calcoaceticus genes for benzoate degradation.

The catabolic genes necessary for the conversion of benzoate to catechol have been cloned from Acinetobacter calcoaceticus into Escherichia coli. The cloned genes, benABCD, encoded both a benzoate 1,2-dioxygenase system, composed of NADH-cytochrome c reductase and terminal oxygenase components, and a cis-diol dehydrogenase. The dioxygenase system appears to be encoded by three genes, benABC, whose products, 53-, 19-, and 38-kilodalton proteins, correspond in size to those of components in other bacterial dioxygenases. The cloned dioxygenase system is expressed at high level in E. coli, enabling the conversion of benzoate to a cis-diol, 2-hydro-1,2-dihydroxybenzoate, at a rate comparable to that of fully induced A. calcoaceticus cultures. A cis-diol dehydrogenase, the product of the A. calcoaceticus benD gene, when present in E. coli enables this organism to convert the cis-diol intermediate to catechol. The dehydrogenase has been partially purified and is a dimer with two identical 31-kilodalton subunits. The ben genes are clustered on the A. calcoaceticus chromosome with independently regulated genes needed for the dissimilation of catechol. In a 16-kilobase-pair region of the chromosome there are 10 genes for benzoate catabolism, organized in no fewer than three transcriptional units. This kind of arrangement, termed supraoperonic clustering, has been observed previously in pseudomonads.

Location on the Escherichia coli genome of a gene specifying O-acetylserine (thiol)-lyase.

The plasmid pAB65, derived from a specialized transducing phage carrying DNA from about 52 min on the Escherichia coli genome, coded for two polypeptides of Mr approx. 34 000. The expression of one was regulated by cyst(e)ine and the cysB gene product and the other by the cysB gene product only. One of these polypeptides was a subunit of O-acetylserine (thiol)-lyase (EC 4.2.99.8); the other, associated with the E. coli membrane, was the N-terminus of the product of the lambda ben gene. The pattern of peptide synthesis directed by plasmids carrying smaller DNA fragments indicated that the gene for O-acetylserine (thiol)-lyase was transcribed clockwise. The spectrum, amino acid composition and subunit number of the enzyme were determined. The enzyme appears homologous with the Salmonella typhimurium cysK gene product. This provides further evidence for the inversion of this region of the genome.

The ben gene of bacteriophage λ : Mapping, identification and control of synthesis

The bacteriophage λ ben gene is responsible for the induction of a novel endonuclease following infection of Escherichia coli. We have mapped ben to the right side of the b-region of the λ genome. Genetic and biochemical methods have been used to show that the product of the ben gene corresponds to gpbEa59, one of several previously identified lambda b-region proteins. Expression of ben is shown to depend on N-gene function and to require p L-initiated transcription. Some strains of λ carry a previously undetected, putative missense mutation in ben, which is shown to have arisen during selection for the Sam 7 mutation.