Oxidative Degradation of Squalene by Arthrobacter Species

An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K₂HPO₄ to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, β,β′-dimethylacrylic, geranic, and (+)-(R)-citronellic acids. Among these acids, α,β-saturated carboxylic acids were found to be predominant in quantity.     

Catching speciation in the act: Metschnikowia bowlesiae sp. nov., a yeast species found in nitidulid beetles of Hawaii and Belize

We describe the species Metschnikowia bowlesiae sp. nov. based on the recovery of six isolates from Hawaii and Belize. The species belongs to the Metschnikowia arizonensis subclade of the large-spored Metschnikowia clade. The isolates are haploid and heterothallic. Both Hawaiian strains had the mating type h ⁺ and the Belizean strains were h ^?. Paraphyletic species structures observed in some ribosomal DNA sequence analyses suggest that M. bowlesiae sp. nov. might represent an intermediate stage in a succession of peripatric speciation events from Metschnikowia dekortorum to Metschnikowia similis and might even hybridize with these species. The type of M. bowlesiae sp. nov. is strain UWOPS 04-243x5 (CBS 12940^T, NRRL Y-63671) and the allotype is strain UWOPS 12-619.1 (CBS 12939^A, NRRL Y-63670).     

Metabolic ability and gene characteristics of Arthrobacter sp. strain DNS10, the sole atrazine-degrading strain in a consortium isolated from black soil

Strain DNS10 was the only member that could utilize atrazine as the sole nitrogen source for growth in an atrazine-degrading consortium which was isolated from black soil previously in our laboratory. It belongs to the genus Arthrobacter according to the sequence of 16S rRNA gene and is designated as Arthrobacter sp. DNS10. 16S rRNA gene phylogenetic analysis showed that strain DNS10 was located in a different evolutionary branch comparing with other Arthrobacter sp. atrazine-degrading strains. The degrading genes such as trzN, atzB and atzC harbored in strain DNS10 revealed high sequence similarity with those in Arthrobacter aurescens TC1 and Pseudomonas sp. ADP. These genes enabled the strain DNS10 to decompose atrazine to cyanuric acid. This was further proved by the results that the strain DNS10 (10⁸ CFU mL⁻¹) could degrade the whole atrazine (100 mg L⁻¹) in the medium within 24 h at 30 °C and there was 66.13 ± 2.11 mg L⁻¹ cyanuric acid accumulated at 24 h. These results imply that the strain DNS10 seems to be an excellent atrazine-degrading strain. Furthermore, this paper helps us in the better understanding of the strain evolution by comparing the metabolic ability and gene characteristics of strain DNS10 with other geographically distinct atrazine-degrading strains.     

A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level.     

Pathway for Degradation of 2-Chloro-4-Nitrophenol in <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. SJCon

Degradation of 2-Chloro-4-nitrophenol (2C4NP) was studied by Arthrobacter sp. SJCon, isolated from the soil of a pesticide contaminated site. This strain utilized 2C4NP as sole source of carbon and energy and degraded 2C4NP with stoichiometric release of nitrite and chloride ions. A metabolite was detected during the study of 2C4NP degradation and identified as chlorohydroquinone (CHQ) by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC–MS). Inhibition study using 2,2′-dipyridyl showed that CHQ is a terminal aromatic compound in degradation pathway of 2C4NP. CHQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain SJCon that suggested the cleavage of the CHQ to maleylacetate (MA). Our study clearly showed that Arthrobacter sp. SJCon degraded 2C4NP via formation of CHQ that further cleaved to MA by CHQ dioxygenase. This mechanism of degradation of 2C4NP differs from previously reported degradation pathways of 2C4NP.     

Production of a Polyunsaturated Isoprenoid Wax Ester during Aerobic Metabolism of Squalene by Marinobacter squalenivorans sp. nov.

This paper describes the production of 5,9,13-trimethyltetradeca-4E,8E,12-trienyl-5,9,13-trimethyltetradeca-4E,8E,12-trienoate during the aerobic degradation of squalene by a Marinobacter strain, 2Asq64, isolated from the marine environment. A pathway involving initial cleavage of the C₁₀-C₁₁ or C₁₄-C₁₅ double bonds of the squalene molecule is proposed to explain the formation of this polyunsaturated isoprenoid wax ester. The isoprenoid wax ester content reached 1.1% of the degraded squalene at the mid-exponential growth phase and then decreased during the stationary phase. The wax ester content increased by approximately threefold in N-limited cultures, in which the ammonium concentration corresponds to conditions often found in marine sediments. This suggests that the bacterial formation of isoprenoid wax esters might be favored in such environments. The bacterial strain is then characterized as a member of a new species, for which we propose the name Marinobacter squalenivorans sp. nov.     

mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.     

Microbial production of squalene by a nicotinic acid-resistant mutant derived from Fusarium sp. no. 5-128B

A nicotinic acid-resistant mutant, designated NA201, was obtained from Fusarium sp. no. 5-128B by treatment with ultraviolet light. This mutant strain could grow in the presence of up to 500 mM nicotinic acid in the culture medium, although the parent strain could not grow at concentrations of nicotinic acid above 200 mM. The NA201 strain exhibited morphological mutations, neither forming aerial hyphae nor secreting a red-brown pigment. However, it retained the resistance to kabicidin at 25 mg l^−t of the parent strain. The mutant NA201 cells contained high levels of squalene and low levels of ergosterol, about 53 times higher and five to six times lower, respectively, than those of the parent strain under standard culture conditions. The volumetric oxygen transfer coefficient (Kd) affected the level of squalene in the mutant cells. The Kd for the maximum production of squalene by the mutant was 24 mmol O₂I⁻¹h⁻¹atm⁻¹ and the level of squalene in the mutant cells was 26 mg (g cell)⁻¹ on a dry weight basis. The greatest accumulation of squalene by the NA201 strain, corresponding to 323 mg per liter of culture medium and 35 mg (g cell)⁻¹ on a dry weight basis, was achieved in a culture in which the Kd was changed from a high to a low value on the third day, with the simultaneous addition of 3% glucose (w/v).     

Efficiency of natural and engineered bacterial strains in the degradation of 4-chlorobenzoic acid in soil slurry

Two bacterial strains, the natural isolate Arthrobacter sp. FG1 and the engineered strain Pseudomonas putida PaW340/pDH5, were compared for their efficiency in the degradation of 4-chlorobenzoic acid in a slurry phase system. The recombinant strain was obtained by cloning the Arthrobacter sp. FG1 dehalogenase encoding genes in P. putida PaW340. In the slurry inoculated with pre-adapted cultures of Arthrobacter sp. FG1, the 4-chlorobenzoic acid degradation was found to be slower than that observed in the slurry inoculated with the recombinant strain P. putida PaW340/pDH5, regardless of the presence or absence of soil indigenous bacteria. Slurry inoculated with mixed cultures of Arthrobacter sp. FG1 and the 4-hyroxybenzoic acid degrader P. putida PaW340 did not show any improvement in 4-chlorobenzoic acid degradation.     

trans-Stilbene degradation by Arthrobacter sp. SL3 cells

trans-Stilbene degradation was examined by the reaction using resting cells of microorganisms isolated through the enrichment culture using trans-stilbene. The strain SL3, showing the highest trans-stilbene-degrading activity, was identified as Arthrobacter sp. One of the reaction products was identified to be cis,cis-muconic acid. Arthrobacter sp. SL3 cells also transformed benzaldehyde, benzoic acid and catechol into cis,cis-muconic acid, suggesting that one benzene ring of trans-stilbene was converted into cis,cis-muconic acid via benzaldehyde formed by its C_α=C_β bond cleavage.     

Relationship between Transport of Bacteria and Their Clogging Efficiency in Sand Columns

In an earlier article, we reported that, under conditions in which neither exopolymers nor bacterial mats were produced, Arthrobacter sp. strain AK19 was an effective plugging agent in sand columns, whereas the bacterial strain SLI^- had no significant effect on the permeability of the medium. A laboratory experiment with sand columns was carried out to elucidate the causes of this difference in behavior. Measured values of the saturated hydraulic conductivity of the sand were explained in terms of biomass accumulation, which was estimated by solving a mass balance equation. The relationship between the saturated hydraulic conductivity and the biomass density within the sand was exponential, although two different exponential coefficients were needed to fit the data for biomass densities above or below 13 mg (wet weight) per cm³, suggesting that two different clogging mechanisms may be involved in different ranges of biomass densities. The experimental results suggest that the SLI^- strain was a poor clogging agent partly because of its lower yield coefficient relative to the limiting nutrient (oxygen) and partly because 60% of the biomass produced in situ was washed out from the column, compared with only 1.2% in the case of Arthrobacter sp. strain AK19.     

Evidence of atrazine mineralization in a soil from the Nile Delta: Isolation of Arthrobacter sp. TES6, an atrazine-degrading strain

The s-triazine herbicide atrazine was rapidly mineralized (i.e., about 60% of ¹⁴C-ring-labelled atrazine released as ¹⁴CO₂ within 21 days) by an agricultural soil from the Nile Delta (Egypt) that had been cropped with corn and periodically treated with this herbicide. Seven strains able to degrade atrazine were isolated by enrichment cultures of this soil. DNA fingerprint and phylogenetic studies based on 16S rRNA analysis showed that the seven strains were identical and belonged to the phylogeny of the genus Arthrobacter (99% similarity with Arthrobacter sp. AD38, EU710554). One strain, designated Arthrobacter sp. strain TES6, degraded atrazine and mineralized the ¹⁴C-chain-labelled atrazine. However, it was unable to mineralize the ¹⁴C-ring-labelled atrazine. Atrazine biodegradation ended in a metabolite that co-eluted with cyanuric acid in HPLC. This was consistent with its atrazine-degrading genetic potential, shown to be dependent on the trzN, atzB, and atzC gene combination. Southern blot analysis revealed that the three genes were located on a large plasmid of about 175 kb and clustered on a 22-kb SmaI fragment. These results reveal for the first time the adaptation of a North African agricultural soil to atrazine mineralization and raise interesting questions about the pandemic dispersion of the trzN, atzBC genes among atrazine-degrading bacteria worldwide.     

Draft Genome Sequence of the Volatile Organic Compound-Producing Antarctic Bacterium Arthrobacter sp. Strain TB23, Able To Inhibit Cystic Fibrosis Pathogens Belonging to the Burkholderia cepacia Complex

Arthrobacter sp. strain TB23 was isolated from the Antarctic sponge Lissodendoryx nobilis. This bacterium is able to produce antimicrobial compounds and volatile organic compounds (VOCs) that inhibit the growth of other Antarctic bacteria and of cystic fibrosis opportunistic pathogens, respectively. Here we report the draft genome sequence of Arthrobacter sp. TB23.     

TLC screening of thraustochytrid strains for squalene production

Screenings of thraustochytrids (Labyrinthulomycetes) have been conducted for 176 strains isolated from various sites in the Asian region to investigate what type of species and strains accumulate high levels of squalene. Thin layer chromatography (TLC) screening for squalene production revealed that 38 strains were rated as “+” (high), 29 as “±” (medium), and 109 as “?” (low). Further, high performance liquid chromatography analysis strongly supported the TLC screening results. Besides the 18W-13a strain of Aurantiochytrium sp., which was previously recognized as a squalene-rich strain, several strains produced squalene at approximately 1 g L^?1 of culture volume. Squalene production was strongly related to locality, colony color, and phylogenetic clade. Most strains with “+” squalene spots were isolated from Okinawa, a subtropical region of Japan, while the strains with “±” and “?” squalene spots were isolated from wide geographical regions from tropical to subarctic. Approximately half the strains with orange colonies on GTY medium plates produced a high amount of squalene, whereas the other strains with different colors showed less or no squalene spots on TLC. All the squalene-rich strains were assigned to the Aurantiochytrium clade. Overall, our results suggest that (1) the thraustochytrids show tendentious locality in terms of squalene production, (2) a relationship exists between the metabolic synthesis of carotenoid pigments and squalene production, and (3) the Aurantiochytrium clade may have evolved to accumulate squalene.     

Destruction of mustard gas hydrolysis products by marine and soil bacteria

Destruction of mustard gas hydrolysis products by bacterial cultures isolated from soils and bottom waters at the sites of chemical weapons disposal has been studied. Among the tested microorganisms, the soil bacteria Pseudomonas putida Y-21 and Rhodococcus erythropolis 8D and the marine bacteria Achromobacter sp. 75-1, Arthrobacter sp. 23-3, and Pseudomonas sp. 93-2 show the highest activity. Thiodiglycol is utilized by two pathways; one of them, with formation of [(2-hydroxyethyl)thio]acetic and thiodiglycolic and thioglycolic acids, is a common pathway for all bacteria under study. The results demonstrate both the possibility of self-purification of natural objects by natural communities of microorganisms and the prospects for application of microorganisms-destructors in bioremediation of polluted territories.     

Identification and evaluation of a potential biocontrol agent,Bacillus subtilis,against Fusarium sp. in apple seedlings

To find a potential biocontrol agent against Fusarium sp. in apple seedlings, an endophytic bacterium strain was isolated from apple tree tissues. The inhibitive efficiency of the isolated strain against the hyphal growth of Fusarium sp. and Rhizoctonia solani was tested. Strain Y-1 showed significant inhibitory effects against Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani and R. solani. Its antifungal activity against F. oxysporum was the highest, reaching up to 64.90 %. In vivo tests indicated that strain Y-1 effectively protects apple from F. oxysporum infections. The control effect reached 92.26 % when bacterial inoculation was performed 3 days prior to pathogen inoculation. Strain Y-1 could colonize the rhizosphere and tissues within 30 days. It was also able to induce systemic resistance in apple seedlings as shown by the activities of SOD and POD. Strain Y-1 significantly increased the root length, root wet and dry weights, and plant height of the apple seedlings compared with the control group. The homology analysis of the 16S rRNA sequence, together with morphological, physical, and biochemical analyses, revealed that strain Y-1 is Bacillus subtilis.     

Interconversion of trans, trans and cis, trans farnesol by enzymes from Andrographis

When trans, trans-farnesol [4,8,12-¹⁴C₃,1-³H₂] is isomerized to cis, trans-farnesol by soluble enzymes from Andrographis paniculata tissue cultures, 50% of the tritium label is lost. The same loss is observed when isomerization occurs in the opposite direction. This is in accordance with the proposed mechanism for isomerization via aldehydes.     

Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent.

Methodology/Principal Findings

The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens.

Conclusions/Significance

The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.     

Catabolism of Arylboronic Acids by Arthrobacter nicotinovorans Strain PBA

Arthrobacter sp. strain PBA metabolized phenylboronic acid to phenol. The oxygen atom in phenol was shown to be derived from the atmosphere using ¹⁸O₂. 1-Naphthalene-, 2-naphthalene-, 3-cyanophenyl-, 2,5-fluorophenyl-, and 3-thiophene-boronic acids were also transformed to monooxygenated products. The oxygen atom in the product was bonded to the ring carbon atom originally bearing the boronic acid substituent with all the substrates tested.