Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Effects of Naphthalene Degradative Plasmids on the Physiological Characteristics of Rhizosphere Bacteria of the Genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

We studied the specific growth rate, duration of the lag phase, stability of plasmids, and activities of the key enzymes involved in naphthalene biodegradation in rhizosphere pseudomonades carrying the structurally similar plasmids pOV17 and pBS216. It was demonstrated that these plasmids determined various levels of catechol-2,3-dioxygenase activities. The structural rearrangements in the plasmid pBS216 could “switch off” the genes of the catechol oxidation meta-pathway. It was shown that certain combinations of degradative plasmids and bacterial hosts, such as Pseudomonas chlororaphis PCL1391(pBS216), P. chlororaphis PCL1391(pOV17), and P. putida 53a(pOV17), were considerably more efficient than natural variants in their growth characteristics and the stability of the biodegradation activity, having a potential for bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs).     

Salicylate degradation by a cold-adapted <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Pseudomonas sp. strain MC1 was characterized as a cold-adapted, naphthalene-degrading bacterium that is able to grow in a broad temperature range of 5–30°C. MC1 harbors a catabolic plasmid, designated pYIC1, which is almost identical to the archetypal NAH7 plasmid from the mesophilic bacterium Pseudomonas putida G7. On pYIC1, the catabolic genes for naphthalene degradation are clustered in two operons: nahAa-Ab-Ac-Ad-B-F-C-Q-E-D encoding the conversion of naphthalene to salicylate, and nahG-T-H-I-N-L-O-M-K-J encoding the conversion of salicylate through meta-cleavage pathway to pyruvate and acetyl CoA. NahH, the bona fide extradiol dioxygenase in MC1 salicylate metabolism, is thermolabile and is a cold-adapted enzyme. The thermal profiles of the NahH enzyme activities expressed in different hosts indicate the presence of a factor(s) or mechanism(s) to protect the thermolabile NahH enzyme (100% aa identity with MC1 counterpart) in G7. Overall, the results reported in the present work suggest that the thermolabile NahH might be a product of the cold-adaptation process of MC1 and thus contribute to the survival and growth ability of MC1 on salicylate and naphthalene in cold environments.     

Salicylate degradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains not involving the “Classical” <Emphasis Type="Italic">nah2</Emphasis> operon

Genetic systems for salicylate catabolism were analyzed in 12 strains of Pseudomonas putida, isolated from polluted soil samples collected in the Murmansk and Tula oblasts. All of the studied P. putida strains utilize salicylate in the ortho-pathway of catechol cleavage without employing the enzymes of the “classical” nah2 operon. The data demonstrates that salicylate degradation in the studied strains is performed with the involvement of the salicylate hydroxylase gene analogous to the nahU gene of strain P. putida ND6. New variants of salicylate hydroxylase genes nahG1 and nahU were found.     

Aerobic degradation of 2,4-dichlorophenoxyacetic acid and other chlorophenols by <Emphasis Type="Italic">Pseudomonas</Emphasis> strains indigenous to contaminated soil in South Africa: Growth kinetics and degradation pathway

Three indigenous pseudomonads, Pseudomonas putida DLL-E4, Pseudomonas reactans and Pseudomonas fluorescens, were isolated from chlorophenol-contaminated soil samples collected from a sawmill located in Durban (South Africa). The obtained isolates were tested for their ability to degrade chlorophenolic compounds: 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP) in batch cultures. The isolates were found to effectively degrade up to 99.5, 98.4 and 94.0% with a degradation rate in the range of 0.67–0.99 (2,4-D), 0.57–0.93 (2,4-DCP) and 0.30–0.39 (2,4,6-TCP) mgL^–1 day^–1 for 2,4-D; 2,4-DCP and 2,4,6-TCP, respectively. The degradation kinetics model revealed that these organisms could tolerate up to 600 mg/L of 2,4-DCP. Catechol 2,3-dioxygenase activity detected in the crude cell lysates of P. putida DLL-E4 and P. reactans was 21.9- and 37.6-fold higher than catechol 1,2-dioxygenase activity assayed, suggesting a meta-pathway for chlorophenol degradation by these organisms. This is also supported by the generally high expression of C23O gene (involved in meta-pathway) relative to tfdC gene (involved in ortho-pathway) expression. Results of this study will be helpful in the exploitation of these organisms and/or their enzymes in bioremediation strategies for chlorophenol-polluted environment.     

Development and relations of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence; the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed the development of the F. culmorum mycelium in soil, but stimulated chlamydospore formation. Decreased mycelial density in the presence of P. fluorescens was more pronounced in soil without additions and less pronounced in the case of introduction of glucose or cellulose. F. culmorum had no effect on P. fluorescens growth in soil.     

Diversity of Oil-Degrading Microorganisms in the Gulf of Finland (Baltic Sea) in Spring and in Summer

Diversity of the oil-degrading microbial strains isolated from the water and sediments of the Gulf of Finland (Baltic Sea) in winter and in summer was studied. Substrate specificity of the isolates for aliphatic and aromatic hydrocarbons was studied. The isolates belonged to 32 genera of the types Proteobacteria (alpha-, beta-, and gammaproteobacteria), Actinobacteria,Firmicutes, and Bacteroidetes. Seasonal variations of the oil-degrading microbial communities was revealed. The presence of the known genes responsible for the degradation of oil aliphatic and aromatic hydrocarbons was determined. The alkB sequence of the alkane hydroxylase gene was found in ~16% of the studied strains. The sequence of the phnAc phenanthrene 3,4- dioxygenase was found in Sphingobacterium sp. and Arthrobacter sp. isolates retrieved in winter and summer. In five Pseudomonas sp. strains from winter samples, the classical operons of naphthalene degradation (nah) were localized in catabolic plasmids, of which three belonged to IncР-9, one, to IncР-7, and two to an unidentified incompatibility group. Burkholderia and Delftia strains contained the operons for naphthalene degradation via salicylate and gentisate (nag). The presence of nag genes has not been previously reported for Delftia spp. strains. The sequences of the nagG salicylate 5-hydroxylase gene were also found in Achromobacter, Sphingobacterium, and Stenotrophomonas strains.     

Phenanthrene degradation by bacteria of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> in model soil systems

Degradation of phenanthrene by strains Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown. Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS1143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. Putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Catabolism of naphthalene and salicylate byPseudomonas fluorescens

Analysis of spent naphthalene growth media ofPseudomonas fluorescens by GC-MS revealed the presence of salicylate. Gentisate 1,2-dioxygenase and pyrocatechol 1,2-dioxygenase were induced by growth on naphthalene, whereas only pyrocatechol 1,2-dioxygenase was induced during growth on salicylate. These results suggest the existence of alternative degradative routes of salicylate,via gentisate and pyrocatechol, which are involved in the catabolism of naphthalene.     

Epiphytic microorganisms degrading aromatic hydrocarbons from the phyllosphere of urban woody plants

From the leaves of three urban trees (Tilia sp., Acer sp., and Fraxinus sp.), 180 strains degrading phenanthrene, naphthalene, and salicylate were isolated by direct plating and enrichment cultures. The leaves of each tree species were characterized by a specific profile of aromatic hydrocarbon-degrading microflora. Members of the type Actinobacteria were predominant in the case of direct plating on media with phenanthrene and naphthalene. Enrichment cultures with phenanthrene and salicylate were shown to yield microbial consortia, the composition of which changed with time. Members of the type Proteobacteria were predominant in these consortia. No plasmids of polycyclic aromatic hydrocarbon degradation of the P-7 and P-9 incompatibility groups were revealed in the studied strains.     

Modulation of microbial consortia enriched from different polluted environments during petroleum biodegradation

Environmental microbial communities are key players in the bioremediation of hydrocarbon pollutants. Here we assessed changes in bacterial abundance and diversity during the degradation of Tunisian Zarzatine oil by four indigenous bacterial consortia enriched from a petroleum station soil, a refinery reservoir soil, a harbor sediment and seawater. The four consortia were found to efficiently degrade up to 92.0% of total petroleum hydrocarbons after 2 months of incubation. Illumina 16S rRNA gene sequencing revealed that the consortia enriched from soil and sediments were dominated by species belonging to Pseudomonas and Acinetobacter genera, while in the seawater-derived consortia Dietzia, Fusobacterium and Mycoplana emerged as dominant genera. We identified a number of species whose relative abundances bloomed from small to high percentages: Dietzia daqingensis in the seawater microcosms, and three OTUs classified as Acinetobacter venetianus in all two soils and sediment derived microcosms. Functional analyses on degrading genes were conducted by comparing PCR results of the degrading genes alkB, ndoB, cat23, xylA and nidA1 with inferences obtained by PICRUSt analysis of 16S amplicon data: the two data sets were partly in agreement and suggest a relationship between the catabolic genes detected and the rate of biodegradation obtained. The work provides detailed insights about the modulation of bacterial communities involved in petroleum biodegradation and can provide useful information for in situ bioremediation of oil-related pollution.     

Isolation and characterization of heavy polycyclic aromatic hydrocarbon-degrading bacteria adapted to electrokinetic conditions

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria capable of growing under electrokinetic conditions were isolated using an adjusted acclimation and enrichment procedure based on soil contaminated with heavy PAHs in the presence of an electric field. Their ability to degrade heavy PAHs under an electric field was individually investigated in artificially contaminated soils. The results showed that strains PB4 (Pseudomonas fluorescens) and FB6 (Kocuria sp.) were the most efficient heavy PAH degraders under electrokinetic conditions. They were re-inoculated into a polluted soil from an industrial site with a PAH concentration of 184.95 mg kg^?1. Compared to the experiments without an electric field, the degradation capability of Pseudomonas fluorescens and Kocuria sp. was enhanced in the industrially polluted soil under electrokinetic conditions. The degradation extents of total PAHs were increased by 15.4 and 14.0 % in the electrokinetic PB4 and FB6 experiments (PB4 + EK and FB6 + EK) relative to the PB4 and FB6 experiments without electrokinetic conditions (PB4 and FB6), respectively. These results indicated that P. fluorescens and Kocuria sp. could efficiently degrade heavy PAHs under electrokinetic conditions and have the potential to be used for the electro-bioremediation of PAH-contaminated soil, especially if the soil is contaminated with heavy PAHs.     

Novel bacteria capable of degrading phenanthrene in activated sludge revealed by stable-isotope probing coupled with high-throughput sequencing

The indigenous microorganisms responsible for degrading phenanthrene (PHE) in activated biosludge were identified using DNA-based stable isotope probing. Besides the well-known PHE degraders Burkholderia, Ralstonia, Sinobacteraceae and Arthrobacter, we for the first time linked the taxa Paraburkholderia and Kaistobacter with in situ PHE biodegradation. Analysis of PAH-RHD_α gene detected in the heavy DNA fraction of ¹³C-PHE treatment suggested the mechanisms of horizontal gene transfer or inter-species hybridisation in PAH-RHD gene spread within the microbial community. Additionally, three cultivable PHE degraders, Microbacterium sp. PHE-1, Rhodanobacter sp. PHE-2 and Rhodococcus sp. PHE-3, were isolated from the same activated biosludge. Among them, Rhodanobacter sp. PHE-2 is the first identified strain in its genus with PHE-degrading ability. However, the involvement of these strains in PHE degradation in situ was questionable, due to their limited enrichment in the heavy DNA fraction of ¹³C-PHE treatment and lack of PAH-RHD_α gene found in these isolates. Collectively, our findings provide a deeper understanding of the diversity and functions of indigenous microbes in PHE degradation.     

Molecular biological taxonomy of some free-living nitrogen-fixing bacteria

A novel approach to the taxonomy of several free-living nitrogen-fixing bacteria is proposed on the basis of two DNA parameters. 1) DNA base composition, expressed as average molar (guanine + cytosine) content, determined by thermal denaturation and 2) DNA homology, determined by DNA hybridization with bothPseudomonas fluorescens andPseudomonas putida. The following taxonomic conclusions emerged:

1. 1.
   The existence ofBeijerinckia andDerxia as individual genera seems justified.     
   
   

Genetic tools for reliable gene expression and recombineering in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we determined the copy numbers of several broad host range origins and found that plasmid copy numbers are significantly higher in P. putida KT2440 than in the synthetic biology workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.     

Bacteria of the sulfur cycle in the sediments of gold mine tailings,Kuznetsk Basin,Russia

The number and diversity of culturable microorganisms involved in sulfur oxidation and sulfate reduction were investigated in the oxidized sediments of gold mine tailings, Kuznetsk Basin, Russia. The sediments had a low pH (2.4–2.8), high SO ₄ ^2? content (up to 22 g/l), and high concentrations of dissolved metals. The arsenic content was as high as 1.9 g/l. Bacterial phylogeny in microcosms was investigated by amplification of 16S rRNA gene fragments with subsequent denaturing gradient gel electrophoresis (DGGE). Spore-forming bacteria Desulfosporosinus were the only bacteria revealed for which the capacity for dissimilatory sulfate reduction is known. Strain Desulfosporosinus sp. DB was obtained in pure culture, and it was phylogenetically remote from other cultured and uncultured members of the genus. No sulfate-reducing members of the Deltaproteobacteria were detected. The Firmicutes members were the most numerous phylotypes in the microcosms, including a separate cluster with the similarity to Pelotomaculum not exceeding 94%. Acidithiobacillus ferrooxidans and A. caldus were found in anaerobic and microaerophilic microcosms. The number of sulfate reducers did not exceed 9.5 × 10² cells/ml.     

Characterization of bacterial composition and diversity in a long-term petroleum contaminated soil and isolation of high-efficiency alkane-degrading strains using an improved medium

Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9–C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

Mobilization of genes for simazine degradation by the pSa plasmid

The plasmid pSa was found to mobilize the genes for simazine degradation (smz) of the rhizosphere bacterium Herbaspirillum sp. B601 by forming hybrid pSa-Smz plasmids. The independent migration of smz genes into various loci of genomes during the transfer and elimination of the hybrid plasmids indicated that the genes are parts of a catabolic island, which could be unstable under certain conditions.     

Insights into the degradation capacities of <Emphasis Type="Italic">Amycolatopsis tucumanensis</Emphasis> DSM 45259 guided by microarray data

The analysis of catabolic capacities of microorganisms is currently often achieved by cultivation approaches and by the analysis of genomic or metagenomic datasets. Recently, a microarray system designed from curated key aromatic catabolic gene families and key alkane degradation genes was designed. The collection of genes in the microarray can be exploited to indicate whether a given microbe or microbial community is likely to be functionally connected with certain degradative phenotypes, without previous knowledge of genome data. Herein, this microarray was applied to capture new insights into the catabolic capacities of copper-resistant actinomycete Amycolatopsis tucumanensis DSM 45259. The array data support the presumptive ability of the DSM 45259 strain to utilize single alkanes (n-decane and n-tetradecane) and aromatics such as benzoate, phthalate and phenol as sole carbon sources, which was experimentally validated by cultivation and mass spectrometry. Interestingly, while in strain DSM 45259 alkB gene encoding an alkane hydroxylase is most likely highly similar to that found in other actinomycetes, the genes encoding benzoate 1,2-dioxygenase, phthalate 4,5-dioxygenase and phenol hydroxylase were homologous to proteobacterial genes. This suggests that strain DSM 45259 contains catabolic genes distantly related to those found in other actinomycetes. Together, this study not only provided new insight into the catabolic abilities of strain DSM 45259, but also suggests that this strain contains genes uncommon within actinomycetes.