A New ICEclc Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain Pseudomonas stutzeri KF716

Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation o-f aromatic compounds, information on their genetic features is limited. We identified a new member of the ICEclc family carrying biphenyl catabolic bph genes and salicylic acid catabolic sal genes from the PCB-degrading strain Pseudomonas stutzeri KF716. The 117-kb ICEbph-salKF716 contains common core regions exhibiting homology with those of degradative ICEclc from P. knackmussii B13 and ICEXTD from Azoarcus sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from P. putida B6-2, P, alcaliphila JAB1, P. stutzeri AN10, and P. stutzeri 2A20 had highly conserved core regions with those of ICEbph-salKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICEbph-salKF716 was transferred from P. stutzeri KF716 to P. aeruginosa PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICEbph-salKF716 compared to other catabolic ICEs.

In silico modeling and interactive profiling of BPH resistant R genes with elicitor molecules of rice planthoppers

Brown planthopper resistant NBS-LRR specific R genes (Bph9, Bph14, Bph18, Bph26) have been reported in rice. BPH specific R genes were clustered with other R genes of rice on chromosome 12 (Bph9, Bph18, Bph26) and 3 (Bph14). Motif analysis of BPH specific R genes showed the predominant motifs as CC, NBS and LRR regions. Bph9, Bph18 and Bph26 R genes exhibited high degree of sequence similarity in their CC and NBS region and are considered as functional alleles of BPH resistance at chromosome 12. LRR region of BPH genes were interacting with the elicitor molecules of planthoppers and are the potential lignad binding site. Bph14 exhibited more number of LRR repeats and were interacting efficiently with all the tested salivary elictor molecules of planthoppers. Bph18 with no LRR region exhibited reduced interaction efficiency with the tested elicitor molecules of planthoppers. Our in silico studies confirms that Bph14 R gene resistance protein to be a promising candidate for providing broad spectrum resistance against planthoppers of rice. The study further provides new avenues to investigate the mechanism of receptor-ligand recognition and signaling mechanism against rice planthoppers.

Biphenyl/PCB Degrading bph Genes of Ten Bacterial Strains Isolated from Biphenyl-Contaminated Soil in Kitakyushu, Japan: Comparative and Dynamic Features as Integrative Conjugative Elements (ICEs)

We sequenced the entire genomes of ten biphenyl/PCB degrading bacterial strains (KF strains) isolated from biphenyl-contaminated soil in Kitakyushu, Japan. All the strains were Gram-negative bacteria belonging to β- and γ-proteobacteria. Out of the ten strains, nine strains carried a biphenyl catabolic bph gene cluster as integrative conjugative elements (ICEs), and they were classified into four groups based on the structural features of the bph genes. Group I (five strains) possessed bph genes that were very similar to the ones in Pseudomonas furukawaii KF707 (formerly Pseudomonas pseudoalcaligenes KF707), which is one of the best characterized biphenyl-utilizing strains. This group of strains carried salicylate catabolic sal genes that were approximately 6-kb downstream of the bph genes. Group II (two strains) possessed bph and sal genes similar to the ones in KF707, but these strains lacked the bphX region between bphC and bphD, which is involved in the downstream catabolism of biphenyl. These bph-sal clusters in groups I and II were located on an integrative conjugative element that was larger than 110 kb, and they were named ICEbph-sal. Our previous study demonstrated that the ICEbph-sal of Pseudomonas putida KF715 in group II existed both in an integrated form in the chromosome (referred to as ICEbph-salKF715 (integrated)) and in a extrachromosomal circular form (referred to as ICEbph-sal (circular)) (previously called pKF715A, 483 kb) in the stationary culture. The ICEbph-sal was transferred from KF715 into P. putida AC30 and P. putida KT2440 with high frequency, and it was maintained stably as an extrachromosomal circular form. The ICEbph-salKF715 (circular) in these transconjugants was further transferred to P. putida F39/D and then integrated into the chromosome in one or two copies. Meanwhile, group III (one strain) possessed bph genes, but not sal genes. The nucleotide sequences of the bph genes in this group were less conserved compared to the genes of the strains belonging to groups I and II. Currently, there is no evidence to indicate that the bph genes in group III are carried by a mobile element. Group IV (two strains) carried bph genes as ICEs (59–61 kb) that were similar to the genes found in Tn4371 from Cupriavidus oxalacticus A5 and ICEKKS1024677 from the Acidovorax sp. strain KKS102. Our study found that bph gene islands have integrative functions, are transferred among soil bacteria, and are diversified through modification.

IDENTIFICATION OF POLYMORPHISM ON SIMPLE SEQUENCE REPEATS MARKERS ASSOCIATED WITH BROWN PLANTHOPPER RESISTANCE GENES IN TWENTY RICE GENOTYPES AND THEIR GENETIC RELATIONSHIP

<p>Brown planthopper is one of the most destructive insect pest of rice in Indonesia and other Asian countries. Pyramiding some brown planthopper resistance genes is a valuable approach to create more durable resistance against the pest. The objective of this study was to identify polymorphisms of Brown Planthopper Resistance genes (Bph) on 20 genotypes of rice, and to obtain genetic relationship among genotypes tested. The experiment was conducted from June to September 2012 at Green House and Laboratory of Plant Analysis and Biotechnology, Faculty of Agriculture, Universitas Padjadjaran, Jatinangor. Twenty genotypes were analyzed, and two of them were used as check varieties. Simple Sequence Repeat (SSR) markers were applied to detect Bph3, Bph4, Qbph3, and Qbph4 genes. Polymorphic levels were analyzed by calculating PIC (Polymorphic Information Content). The grouping of rice genotypes were done based on principal components analysis (PCA) of SSR data, and the genetic relationship based on the presence of Bph genes was estimated using UPGMA (Unweighted Pair Group With Arithmetic mean). Results showed that RM313, RM8072, RM8213, RM5953, RM586, and RM589 markers were polymorphic. Rice genotypes PTB 33, Diah Suci, Cibogo, Cisantana, Digul, Ciherang, Inpari 13, Inpari 10, and Memberamo had Bph3, Qbph3, Bph4, and Qbph4. Meanwhile Bph3, Qbph3, and Bph4 were supposed to be belonged by IR 64, Aek Sibundong, Batang Gadis, IR 66, and Mekongga. Kalimas and Tukat Penatu had Bph3, Qbph3, dan Qbph4. IR 74 had Bph3 and Qbph3, and Fatmawati had Bph3 and Bph4. UPGMA clustering resulted in two main clusters, in which the first cluster consisted of 2 subclusters. PTB-33 was closely related with Memberamo, Tukat Penatu, Digul, Diah Suci, and Kalimas. The SSR markers used in this study were proven to be valuable in molecular detection of Bph genes and in estimating genetic relationsips of rice genotypes. PTB-33 was a good donor of resistance genes, as well as Memberamo, Tukat Penatu, Digul, Diah Suci, and Kalimas which were identified as promising donors in rice breeding resistance to brown planthopper. </p><p><strong>Keywords :</strong> Bph gene, Brown Planthopper, Genetic relationship, SSR markers.</p>

bph genes of the thermophilic PCB degrader, Bacillus sp. JF8: characterization of the divergent ring-hydroxylating dioxygenase and hydrolase genes upstream of the Mn-dependent BphC

Bacillus sp. JF8 is a thermophilic polychlorinated biphenyl (PCB) degrader, which utilizes biphenyl and naphthalene. A thermostable, Mn-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase, BphC_JF8, has been characterized previously. Upstream of bphC are five ORFs exhibiting low homology with, and a different gene order from, previously characterized bph genes. From the 5′ to 3′ direction the genes are: a putative regulatory gene (bphR), a hydrolase (bphD), the large and small subunits of a ring-hydroxylating dioxygenase (bphA1A2), and a cis-diol dehydrogenase (bphB). Hybridization studies indicate that the genes are located on a plasmid. Ring-hydroxylating activity of recombinant BphA1A2_JF8 towards biphenyl, PCB, naphthalene and benzene was observed in Escherichia coli cells, with complementation of non-specific ferredoxin and ferredoxin reductase by host cell proteins. PCB degradation by recombinant BphA1A2_JF8 showed that the congener specificity of the recombinant enzyme was similar to Bacillus sp. JF8. BphD_JF8, with an optimum temperature of 85 °C, exhibited a narrow substrate preference for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. The Arrhenius plot of BphD_JF8 was biphasic, with two characteristic energies of activation and a break point at 47 °C.