Potential of Indigenous Strains Isolated from the Wastewater Treatment Plant of a Crude Oil Refinery.

Contamination of the environment with crude oil or other fuels is an enormous disaster for all organisms. The microbial communities for bioremediation have been an effective tool for eliminating pollution. This study aimed to determine individual cultures' and a strain mixture's ability to utilize alkanes (single alkanes and crude oil). The proper study of pure cultures is necessary to design synergistically working consortia. The Acinetobacter venetianus ICP1 and Pseudomonas oleovorans ICTN13 strains isolated from a wastewater treatment plant of a crude oil refinery can grow in media containing various aromatic and aliphatic hydrocarbons. The genome of the strain ICP1 contains four genes encoding alkane hydroxylases, whose transcription depended on the length of the alkane in the media. We observed that the hydrophobic cells of the strain ICP1 adhered to hydrophobic substrates, and their biofilm formation increased the bioavailability and biodegradation of the hydrocarbons. Although strain ICTN13 also has one alkane hydroxylase-encoding gene, the growth of the strain in a minimal medium containing alkanes was weak. Importantly, the growth of the mixture of strains in the crude oil-containing medium was enhanced compared with that of the single strains, probably due to the specialization in the degradation of different hydrocarbon classes and co-production of biosurfactants.

Variance in translational fidelity of different bacterial species is affected by pseudouridines in the tRNA anticodon stem-loop.

Delicate variances in the translational machinery affect how efficiently different organisms approach protein synthesis. Determining the scale of this effect, however, requires knowledge on the differences of mistranslation levels. Here, we used a dual-luciferase reporter assay cloned into a broad host range plasmid to reveal the translational fidelity profiles of Pseudomonas putida, Pseudomonas aeruginosa and Escherichia coli. We observed that these profiles are surprisingly different, whereas species more prone to translational frameshifting are not necessarily more prone to stop codon readthrough. As tRNA modifications are among the factors that have been implicated to affect translation accuracy, we also show that translational fidelity is context-specifically influenced by pseudouridines in the anticodon stem-loop of tRNA, but the effect is not uniform between species.

Pseudomonas putida Biofilm Depends on the vWFa-Domain of LapA in Peptides-Containing Growth Medium.

The biofilm of Pseudomonas putida is complexly regulated by several intercellular and extracellular factors. The cell surface adhesin LapA of this bacterium is a central factor for the biofilm and, consequently, the regulation of lapA expression, for example, by Fis. It has been recently shown that peptides in growth media enhance the formation of P. putida biofilm, but not as a source of carbon and nitrogen. Moreover, the peptide-dependent biofilm appeared especially clearly in the fis-overexpression strain, which also has increased LapA. Therefore, we investigate here whether there is a relationship between LapA and peptide-dependent biofilm. The P. putida strains with inducible lapA expression and LapA without the vWFa domain, which is described as a domain similar to von Willebrand factor domain A, were constructed. Thereafter, the biofilm of these strains was assessed in growth media containing extracellular peptides in the shape of tryptone and without it. We show that the vWFa domain in LapA is necessary for biofilm enhancement by the extracellular peptides in the growth medium. The importance of vWFa in LapA was particularly evident for the fis-overexpression strain F15. The absence of the vWFa domain diminished the positive effect of Fis on the F15 biofilm.

Tryptone in Growth Media Enhances Pseudomonas putida Biofilm.

Extracellular factors and growth conditions can affect the formation and development of bacterial biofilms. The biofilm of Pseudomonas putida has been studied for decades, but so far, little attention has been paid to the components of the medium that may affect the biofilm development in a closed system. It is known that Fis strongly enhances biofilm in complete LB medium. However, this is not the case in the defined M9 medium, which led us to question why the bacterium behaves differently in these two media. Detailed analysis of the individual medium components revealed that tryptone as the LB proteinaceous component maintains biofilm in its older stages. Although the growth parameters of planktonic cells were similar in the media containing tryptone or an equivalent concentration of amino acids, only the tryptone had a positive effect on the mature biofilm of the wild type strain of P. putida. Thus, the peptides in the environment may influence mature biofilm as a structural factor and not only as an energy source. Testing the effect of other biopolymers on biofilm formation showed variable results even for polymers with a similar charge, indicating that biopolymers can affect P. putida biofilm through a number of bacterial factors.

Monitoring the growth, survival and phenol utilization of the fluorescent-tagged Pseudomonas oleovorans immobilized and free cells.

Bioaugmentation in wastewater treatment plants (WWTPs) is challenging due to low survival and persistence of applied microbes. This study aimed to track the capacity and survival of fluorescent-tagged Pseudomonas oleovoransICTN13 as a model organism applicable in bioaugmentation of phenol-containing wastewater. The isolate was immobilized in alginate biopolymer, and enhanced efficacy and survival for biodegradation of phenol against free cells were studied. Encapsulated cells resulted in enhanced phenol removal efficiency (~94%) compared to free cells (~72%). Encapsulation of cells facilitated an extended storage time of 30 days. Remarkably, phenol and COD removal efficacy of encapsulated cells was sustained up to ~ 92-93% in a reactor after 45 days, while free cells could produce ~ 80-84% removal efficiency. Fluorescence microscopy showed high survival of the encapsulated cells, whereas gradual deterioration of free cells was observed. Thus, the findings highlight the importance of bio augmented strain in WWTPs where encapsulation is a crucial factor.

Pseudouridines of tRNA Anticodon Stem-Loop Have Unexpected Role in Mutagenesis in Pseudomonas sp.

Pseudouridines are known to be important for optimal translation. In this study we demonstrate an unexpected link between pseudouridylation of tRNA and mutation frequency in Pseudomonas species. We observed that the lack of pseudouridylation activity of pseudouridine synthases TruA or RluA elevates the mutation frequency in Pseudomonas putida 3 to 5-fold. The absence of TruA but not RluA elevates mutation frequency also in Pseudomonas aeruginosa. Based on the results of genetic studies and analysis of proteome data, the mutagenic effect of the pseudouridylation deficiency cannot be ascribed to the involvement of error-prone DNA polymerases or malfunctioning of DNA repair pathways. In addition, although the deficiency in TruA-dependent pseudouridylation made P. putida cells more sensitive to antimicrobial compounds that may cause oxidative stress and DNA damage, cultivation of bacteria in the presence of reactive oxygen species (ROS)-scavenging compounds did not eliminate the mutator phenotype. Thus, the elevated mutation frequency in the absence of tRNA pseudouridylation could be the result of a more specific response or, alternatively, of a cumulative effect of several small effects disturbing distinct cellular functions, which remain undetected when studied independently. This work suggests that pseudouridines link the translation machinery to mutation frequency.

Microbial Metabolic Potential of Phenol Degradation in Wastewater Treatment Plant of Crude Oil Refinery: Analysis of Metagenomes and Characterization of Isolates.

The drilling, processing and transportation of oil are the main sources of pollution in water and soil. The current work analyzes the microbial diversity and aromatic compounds degradation potential in the metagenomes of communities in the wastewater treatment plant (WWTP) of a crude oil refinery. By focusing on the degradation of phenol, we observed the involvement of diverse indigenous microbial communities at different steps of the WWTP. The anaerobic bacterial and archaeal genera were replaced by aerobic and facultative anaerobic bacteria through the biological treatment processes. The phyla Proteobacteria, Bacteroidetes and Planctomycetes were dominating at different stages of the treatment. Most of the established protein sequences of the phenol degradation key enzymes belonged to bacteria from the class Alphaproteobacteria. From 35 isolated strains, 14 were able to grow on aromatic compounds, whereas several phenolic compound-degrading strains also degraded aliphatic hydrocarbons. Two strains, Acinetobacter venetianus ICP1 and Pseudomonas oleovorans ICTN13, were able to degrade various aromatic and aliphatic pollutants and were further characterized by whole genome sequencing and cultivation experiments in the presence of phenol to ascertain their metabolic capacity in phenol degradation. When grown alone, the intermediates of catechol degradation, the meta or ortho pathways, accumulated into the growth environment of these strains. In the mixed cultures of the strains ICP1 and ICTN13, phenol was degraded via cooperation, in which the strain ICP1 was responsible for the adherence of cells and ICTN13 diminished the accumulation of toxic intermediates.

Narrative of a versatile and adept species Pseudomonas putida.

Pseudomonas putida is a fast-growing bacterium found mostly in temperate soil and water habitats. The metabolic versatility of P. putida makes this organism attractive for biotechnological applications such as biodegradation of environmental pollutants and synthesis of added-value chemicals (biocatalysis). This organism has been extensively studied in respect to various stress responses, mechanisms of genetic plasticity and transcriptional regulation of catabolic genes. P. putida is able to colonize the surface of living organisms, but is generally considered to be of low virulence. A number of P. putida strains are able to promote plant growth. The aim of this review is to give historical overview of the discovery of the species P. putida and isolation and characterization of P. putida strains displaying potential for biotechnological applications. This review also discusses some major findings in P. putida research encompassing regulation of catabolic operons, stress-tolerance mechanisms and mechanisms affecting evolvability of bacteria under conditions of environmental stress.

Integration Host Factor IHF facilitates homologous recombination and mutagenic processes in Pseudomonas putida.

Nucleoid-associated proteins (NAPs) such as IHF, HU, Fis, and H-NS alter the topology of bound DNA and may thereby affect accessibility of DNA to repair and recombination processes. To examine this possibility, we investigated the effect of IHF on the frequency of homologous recombination (HR) and point mutations in soil bacterium Pseudomonas putida by using plasmidial and chromosomal assays. We observed positive effect of IHF on the frequency of HR, whereas this effect varied depending both on the chromosomal location of the HR target and the type of plasmid used in the assay. The occurrence of point mutations in plasmid was also facilitated by IHF, whereas in the chromosome the positive effect of IHF appeared only at certain DNA sequences and/or chromosomal positions. We did not observe any significant effects of IHF on the spectrum of mutations. However, despite of the presence or absence of IHF, different mutational hot spots appeared both in plasmid and in chromosome. Additionally, the frequency of frameshift mutations in the chromosome was also strongly affected by the location of the mutational target sequence. Taking together, our results indicate that IHF facilitates the occurrence of genetic changes in P. putida, whereas the location of the target sequence affects both the IHF-dependent and IHF-independent mechanisms.

Mutation and Recombination Rates Vary Across Bacterial Chromosome.

Bacteria evolve as a result of mutations and acquisition of foreign DNA by recombination processes. A growing body of evidence suggests that mutation and recombination rates are not constant across the bacterial chromosome. Bacterial chromosomal DNA is organized into a compact nucleoid structure which is established by binding of the nucleoid-associated proteins (NAPs) and other proteins. This review gives an overview of recent findings indicating that the mutagenic and recombination processes in bacteria vary at different chromosomal positions. Involvement of NAPs and other possible mechanisms in these regional differences are discussed. Variations in mutation and recombination rates across the bacterial chromosome may have implications in the evolution of bacteria.

Seasonal bacterial community dynamics in a crude oil refinery wastewater treatment plant.

The biological treatment of oil refinery effluents in wastewater treatment plants (WWTPs) relies on specialized bacteria contributing to remove organic load, nitrogen, sulfur, and phosphorus compounds. Knowledge about bacterial dynamics in WWTPs and how they affect the performance of the wastewater treatment is limited, particularly in tropical countries. The bacterial communities from three compartments of an oil refinery WWTP in Uran, India, were assessed using 16S-metabarcoding, in winter and monsoon seasons, upstream (from the surge pond) and downstream the biotower (clarifier and guard pond), to understand the effects of seasonal variations in WWTP's efficiency. The organic load and ammonia levels of the treated wastewater increased by 3- and 9-fold in the monsoon time-point. A decreased abundance and diversity of 47 genera (325 OTUs) comprising ammonia and nitrite oxidizing bacteria (AOB, NOB, denitrifiers) was observed in the monsoon season downstream the biotower, whereas 23 OTUs of Sulfurospirillum, Desulfovibrio, and Bacillus, putatively performing dissimilatory nitrate reduction to ammonia (DNRA), were 3-fold more abundant in the same compartments (DNRA/denitrifiers winter ratio < 0.5 vs. monsoon ratio around 3). The total abundance of reported sulfate- and sulfite-reducing bacteria also increased 250- and 500-fold downstream the biotower, in the monsoon time-point. Bacteria performing DNRA may thus outcompete denitrification in this WWTP, limiting the biodegradation process. The alterations detected in bacterial populations involved in the removal of nitrogen and sulfur species evidenced a reduced quality of the released wastewater and may be good candidates for the following monitoring strategies and optimization of the wastewater treatment.

Involvement of transcription-coupled repair factor Mfd and DNA helicase UvrD in mutational processes in Pseudomonas putida.

Stalled RNA polymerases (RNAPs) pose an obstacle for the replicating complexes, which could lead to transcription-replication conflicts and result in genetic instability. Stalled RNAPs and DNA lesions blocking RNAP elongation are removed by transcription-coupled repair (TCR), the process which in bacteria is mediated by TCR factor Mfd and helicase UvrD. Although the mechanism of TCR has been extensively studied, its role in mutagenesis is still obscure. In the current study we have investigated the role of Mfd and UvrD in mutational processes in soil bacterium Pseudomonas putida. Our results revealed that UvrD helicase is essential to prevent the emergence of mutations, as the loss of uvrD resulted in elevated mutant frequency both in exponential- and stationary-phase bacterial cultures. UvrD was also found to be necessary to survive DNA damage, but NER or MMR pathways are not completely abolished in UvrD-deficient P. putida. Mfd-deficiency had a moderate impact on surviving DNA damage and did not influence the frequency of mutations occurred in exponentially growing bacteria. However, the absence of Mfd caused approximately a two-fold decline in stationary-phase mutant frequency compared to the P. putida wild-type strain and suppressed the elevated mutant frequency observed in the ΔuvrD strain. Remarkably, the Mfd-deficient strain also formed less UV-induced mutants. These results suggest that in P. putida the Mfd-mediated TCR could be associated with UV- and stationary-phase mutagenesis.

The Effect of Cellular Redox Status on the Evolvability of New Catabolic Pathways.

Oxidation of aromatic compounds can be mutagenic due to the accumulation of reactive oxygen species (ROS) in bacterial cells and thereby facilitate evolution of corresponding catabolic pathways. To examine the effect of the background biochemical network on the evolvability of environmental bacteria hosting a new catabolic pathway, Akkaya and colleagues (mBio 9:e01512-18, 2018, https://doi.org/10.1128/mBio.01512-18) introduced the still-evolving 2,4-dinitrotoluene (2,4-DNT) pathway genes from the original environmental Burkholderia sp. isolate into the genome of Pseudomonas putida KT2440. They show that the mutagenic effect of 2,4-DNT oxidation, which is associated with the accumulation of ROS and oxidative damage on DNA, can be avoided by preserving high NADPH levels in P. putida The observations of this study highlight the impact of the cellular redox status of bacteria on the evolvability of new metabolic pathways.

Colonization efficiency of Pseudomonas putida is influenced by Fis-controlled transcription of nuoA-N operon.

Root colonization of plant growth-promoting bacteria is a complex multistep process that is influenced by several factors. For example, during adherence to plant roots, bacteria have to endure reactive oxygen species (ROS) produced by plants. In this study, we report that the global transcriptional regulator Fis is involved in the regulation of ROS-tolerance of Pseudomonas putida and thereby affects barley root colonization. Fis overexpression reduced both ROS-tolerance and adherence to barley roots and activated the transcription of the nuoA-N operon encoding NADH dehydrogenase I, the first enzyme of a membrane-bound electron-transport chain. The nuoA-N knockout mutation in the fis-overexpression background increased the ROS-tolerance and adherence to barley roots. We show that nuoA has two transcriptional start sites located 104 and 377 nucleotides upstream of the coding sequence, indicating the presence of two promoters. The DNase I footprint analysis revealed four Fis binding sites: Fis-nuo1 to Fis-nuo4, situated between these two promoters. Site-directed mutagenesis in a promoter-lacZ reporter and ß-galactosidase assay further confirmed direct binding of Fis to Fis-nuo2 and probably to Fis-nuo4 but not to Fis-nuo1 and Fis-nuo3. Additionally, the results implied that Fis binding to Fis-nuo4 could affect transcription of the nuoA-N operon by modification of upstream DNA topology. Moreover, our transposon mutagenesis results indicated that Fis might be involved in the regulation of several alternative ROS detoxification processes utilizing NADH.

Correction: The promoter region of lapA and its transcriptional regulation by Fis in Pseudomonas putida.

[This corrects the article DOI: 10.1371/journal.pone.0185482.].

The promoter region of lapA and its transcriptional regulation by Fis in Pseudomonas putida.

LapA is the biggest protein in Pseudomonas putida and a key factor for biofilm formation. Its importance and posttranslational regulation is rather thoroughly studied but less is known about the transcriptional regulation. Here we give evidence that transcription of lapA in LB-grown bacteria is initiated from six promoters, three of which display moderate RpoS-dependence. The global transcription regulator Fis binds to the lapA promoter area at six positions in vitro, and Fis activates the transcription of lapA while overexpressed in cells. Two of the six Fis binding sites, Fis-A7 and Fis-A5, are necessary for the positive effect of Fis on the transcription of lapA in vivo. Our results indicate that Fis binding to the Fis-A7 site increases the level of transcription from the most distal promoter of lapA, whereas Fis binding to the Fis-A5 site could be important for modifying the promoter area topology.

Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria.

Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2) and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source.

Ongoing evolution of Pseudomonas aeruginosa PAO1 sublines complicates studies of DNA damage repair and tolerance.

Sublines of the major P. aeruginosa reference strain PAO1 are derivatives of the original PAO1 isolate, which are maintained in laboratories worldwide. These sublines display substantial genomic and phenotypic variation due to ongoing microevolution. Here, we examined four sublines, MPAO1, PAO1-L, PAO1-DSM and PAO1-UT, originated from different laboratories, and six DNA polymerase-deficient mutants from the P. aeruginosa MPAO1 transposon library for their employment in elucidation of DNA damage repair and tolerance mechanisms in P. aeruginosa. We found that PAO1 subline PAO1-UT carries a large deletion encompassing the DNA damage inducible imuA-imuB-imuC cassette (PA0669-PA0671), which is implied in mutagenesis in several species. Furthermore, the genetic changes leading to variation in the functionality of the MexEF-OprN efflux system contributed largely to the phenotypic discordance between P. aeruginosa PAO1 sublines. Specifically, we identified multiple mutations in the mexT gene, which encodes a transcriptional regulator of the mexEF-oprN genes, mutations in the mexF, and complete absence of these genes. Of the four tested sublines, MPAO1 was the only subline with the functional MexEF-OprN multidrug efflux system. Active efflux through MexEF-OprN rendered MPAO1 highly resistant to chloramphenicol and ciprofloxacin. Moreover, the functions of specialized DNA polymerase IV and nucleotide excision repair (NER) in 4-NQO-induced DNA damage tolerance appeared to be masked in MPAO1, while were easily detectable in other sublines. Finally, the frequencies of spontaneous and MMS-induced Rifr mutations were also significantly lower in MPAO1 in comparison to the PAO1 sublines with impaired MexEF-OprN efflux system. The MexEF-OprN-attributed differences were also observed between MPAO1 and MPAO1-derived transposon mutants from the two-allele transposon mutant collection. Thus, the accumulating mutations and discordant phenotypes of the PAO1 derivatives challenge the reproducibility and comparability of the results obtained with different PAO1 sublines and also limit the usage of the MPAO1 transposon library in DNA damage tolerance and mutagenesis studies.

DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida.

Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.