Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains.

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.

Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids.

In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+C content of 53.75%. A total of 135 open reading frames (ORFs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. ORFs encoding proteins with putative functions in stress response, transposition and site-specific recombination were also predicted. Analysis of the organization and nucleotide sequence of pD2RT backbone region revealed high degree of similarity to the draft genome sequence data of the plant-pathogenic pseudomonad Pseudomonas syringae pv. glycinea strain B076, exhibiting relatedness to pPT23A plasmid family. The pD2RT backbone is also closely related to that of pGRT1 of Pseudomonas putida strain DOT-T1E and pBVIE04 of Burkholderia vietnamiensis strain G4, both plasmids are associated with resistance to toluene. The ability of pD2RT to self-transfer by conjugation to P. putida recipient strain PaW340 was experimentally determined. Genetic organization of toluene-degrading (xyl) genes and flanking DNA segments resembles the structure of Tn1721-related class II transposon Tn4656 of TOL plasmid pWW53 of P. putida strain MT53. The complete sequence of the plasmid pD2RT extends the known range of xyl genes carriers, being the first completely sequenced TOL plasmid, which is not related to well-studied IncP plasmid groups. We also verified the functionality of the catabolic route encoded by pD2RT by monitoring the expression of the xylE gene in pD2RT bearing hosts along with bacterial strains containing TOL plasmid of IncP-9 group. The growth kinetics of plasmid-bearing strains was found to be affected by particular TOL plasmid.

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.

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.

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.

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.

Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-cresol-degradative strains of Pseudomonas fluorescens.

p-Cresol methylhydroxylase (PCMH), a key enzyme responsible for the catabolism of p-cresol via the protocatechuate ortho pathway, was used as a tool to characterize catabolic differences between phenol- and p-cresol-degrading Pseudomonas fluore-scens strains PC18 and PC24. Although both strains catabolize p-cresol using PCMH, different whole-cell kinetic parameters for this compound were revealed. Affinity for the substrate and the specific growth rate were higher in PC18, whereas maximum p-cresol tolerance was higher in PC24. In addition, PCMH of strain PC18 was induced during growth on phenol. In both strains, the pchACXF operon, which encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, was sequenced. Transcriptional regulation of these operons by PchR, a putative sigma(54)-dependent regulator, was shown. Although the promoters of these operons resembled sigma(54)-controlled promoters, they differed from the consensus sequence by having T instead of C at position -12. Complementation assays confirmed that the amino acid sequence differences of the PchR regulators between the two strains studied led to different effector-binding capabilities of these proteins: (1) phenol was a more efficient effector for PchR of PC18 than p-cresol, (2) phenol did not activate the regulator of PC24, and (3) both regulators responded similarly to p-cresol.

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.

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.

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.