Eve Vedler

The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D(+) phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D(+) phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazine-catabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 beta subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the alpha, beta, and gamma subgroups) that it belongs to a new IncP1subgroup, the delta subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids.

Pelagibacterium halotolerans gen. nov., sp. nov. and Pelagibacterium luteolum sp. nov., novel members of the family Hyphomicrobiaceae

Two Gram-negative, motile, aerobic bacterial strains, designated B2(T) and 1_C16_27(T), were respectively isolated from a seawater sample collected from the East China Sea and a semi-coke sample from north-eastern Estonia. Their genetic, phenotypic and chemotaxonomic properties were studied. The isolates were short rods with polar flagella and were positive for catalase and oxidase activities. Q-10 was the predominant respiratory ubiquinone. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids. The major fatty acids were nonadecanoic (C(19 : 0) cyclo), octadecanoic (C(18 : 0) and C(18 : 0) 3-OH), octadecenoic (C(18 : 1)) and hexadecanoic (C(16 : 0)) acids. The G+C content of the genomic DNA was 58.1-59.3 mol%. 16S rRNA gene sequence analysis revealed that the two isolates represent a distinct lineage within the family Hyphomicrobiaceae. The phylogenetically closest relatives were Cucumibacter (92.7-93.7 % 16S rRNA gene sequence similarity), Devosia (92.9-94.4 %) and Zhangella (91.7-92.1 %). Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strains B2(T) and 1_C16_27(T) could be differentiated from each other and from members of the genera Cucumibacter, Devosia and Zhangella. Therefore, it is proposed that strains B2(T) and 1_C16_27(T) represent two novel species in a new genus, for which the names Pelagibacterium halotolerans gen. nov., sp. nov. (the type species; type strain B2(T)  = CGMCC 1.7692(T)  = JCM 15775(T)) and Pelagibacterium luteolum sp. nov. (type strain 1_C16_27(T)  = CGMCC 1.10267(T)  = JCM 16552(T)  = CELMS EEUT 1C1627(T)) are proposed.

Analysis of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pEST4011 of Achromobacter xylosoxidans subsp. denitrificans strain EST4002.

The 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002, isolated in Estonia more than 10years ago, was found to contain the 70kb plasmid pEST4011 that is responsible for the bacterium having had obtained a stable 2,4-D(+) phenotype. The tfd-like genes for 2, 4-D degradation of the strain EST4002 were located on a 10.5kb region of pEST4011, but without functional genes coding for chloromuconate cycloisomerase and chlorodienelactone hydrolase. The latter two genes are probably encoded by homologous, tcb-like genes, located elsewhere on pEST4011. We also present evidence of two copies of insertion element IS1071-like sequences on pEST4011. IS1071 is a class II (Tn3 family) insertion element, associated with different catabolic genes and operons and globally distributed in the recent past. We speculate that this insertion element might have had a role in the formation of plasmid pEST4011. The 28kb plasmid pEST4012 is generated by deletion from pEST4011 when cells of A. xylosoxidans EST4002 are grown in the absence of 2,4-D in growth medium. We propose that this is the result of homologous recombination between the two putative copies of IS1071-like sequences on pEST4011.

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil, shale oil or diesel fuel

The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes.

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH(+) phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees.

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20.

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 ISPre1-like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 ISPre2-like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer - we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of ISPre2-like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.

TfdR, the LysR-type transcriptional activator, is responsible for the activation of the tfdCB operon of Pseudomonas putida 2, 4-dichlorophenoxyacetic acid degradative plasmid pEST4011.

In Pseudomonas putida EST4021, the tfdCB operon of plasmid pEST4011 encodes enzymes involved in 2,4-dichlorophenoxyacetic acid degradation. We have identified a gene, tfdR, important for the regulation of the tfdCB operon. Sequence analysis of the tfdR gene revealed an open reading frame with amino acid sequence similar to the LysR family of transcriptional activators. The tfdR gene is located upstream and transcribed divergently from the tfdCB operon. Utilizing primer extension analysis, the transcription initiation sites of the gene tfdR and the tfdCB operon were localized 85 (84)bp and 292bp upstream from the coding sequences of these genes, respectively. Multiple sequence analysis revealed that the genes tfdR, tfdC and tfdB of plasmid pEST4011 are most similar to the regulatory gene tfdR and the module 2 genes tfdC(II) and tfdB(II) of pJP4, respectively. The promoter-operator sequences of tfdR and its target tfdCB operon of pEST4011 have regions with highly conserved nucleotides characteristic for the catechol-subgroup LysR-type transcriptional activators. We showed that the pEST4011 tfdR gene product activates the expression of the tfdCB operon and the effector molecule for TfdR is 2,4-dichloro-cis,cis-muconate. Our data indicate that the structure and the mode of regulation of tfd genes are similar, despite the bacteria being isolated from different geographical regions.

Occurrence of plasmids in the aromatic degrading bacterioplankton of the baltic sea.

Plasmids are mobile genetic elements that provide their hosts with many beneficial traits including in some cases the ability to degrade different aromatic compounds. To fulfill the knowledge gap regarding catabolic plasmids of the Baltic Sea water, a total of 209 biodegrading bacterial strains were isolated and screened for the presence of these mobile genetic elements. We found that both large and small plasmids are common in the cultivable Baltic Sea bacterioplankton and are particularly prevalent among bacterial genera Pseudomonas and Acinetobacter. Out of 61 plasmid-containing strains (29% of all isolates), 34 strains were found to carry large plasmids, which could be associated with the biodegradative capabilities of the host bacterial strains. Focusing on the diversity of IncP-9 plasmids, self-transmissible m-toluate (TOL) and salicylate (SAL) plasmids were detected. Sequencing the repA gene of IncP-9 carrying isolates revealed a high diversity within IncP-9 plasmid family, as well as extended the assumed bacterial host species range of the IncP-9 representatives. This study is the first insight into the genetic pool of the IncP-9 catabolic plasmids in the Baltic Sea bacterioplankton.

Microbial population dynamics in response to Pectobacterium atrosepticum infection in potato tubers

Endophytes are microbes and fungi that live inside plant tissues without damaging the host. Herein we examine the dynamic changes in the endophytic bacterial community in potato (Solanum tuberosum) tuber in response to pathogenic infection by Pectobacterium atrosepticum, which causes soft rot in numerous economically important crops. We quantified community changes using both cultivation and next-generation sequencing of the 16S rRNA gene and found that, despite observing significant variability in both the mass of macerated tissue and structure of the endophytic community between individual potato tubers, P. atrosepticum is always taken over by the endophytes during maceration. 16S rDNA sequencing revealed bacteria from the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Fusobacteria, Verrucomicrobia, Acidobacteria, TM7, and Deinococcus-Thermus. Prior to infection, Propionibacterium acnes is frequently among the dominant taxa, yet is out competed by relatively few dominant taxa as the infection proceeds. Two days post-infection, the most abundant sequences in macerated potato tissue are Gammaproteobacteria. The most dominant genera are Enterobacter and Pseudomonas. Eight days post-infection, the number of anaerobic pectolytic Clostridia increases, probably due to oxygen depletion. These results demonstrate that the pathogenesis is strictly initiated by the pathogen (sensu stricto) and proceeds with a major contribution from the endophytic community.