Inhibition of Pseudomonas aeruginosa swarming motility by 1-naphthol and other bicyclic compounds bearing hydroxyl groups.

Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants, and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyperswarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. We showed that this 1-naphthol-dependent swarming inhibition is independent of changes of rhamnolipid production and the intracellular level of signaling molecule cyclic-di-GMP (c-di-GMP). Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and with pyochelin, flagellar, and pilus synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa.

Microbial degradation of aliphatic and aliphatic-aromatic co-polyesters.

Biodegradable plastics (BPs) have attracted much attention since more than a decade because they can easily be degraded by microorganisms in the environment. The development of aliphatic-aromatic co-polyesters has combined excellent mechanical properties with biodegradability and an ideal replacement for the conventional nondegradable thermoplastics. The microorganisms degrading these polyesters are widely distributed in various environments. Although various aliphatic, aromatic, and aliphatic-aromatic co-polyester-degrading microorganisms and their enzymes have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. In this review, we have reported some new microorganisms and their enzymes which could degrade various aliphatic, aromatic, as well as aliphatic-aromatic co-polyesters like poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), poly(L-lactic acid) (PLA), poly(3-hydroxybutyrate) and poly(3-hydoxybutyrate-co-3-hydroxyvalterate) (PHB/PHBV), poly(ethylene terephthalate) (PET), poly(butylene terephthalate) (PBT), poly(butylene adipate-co-terephthalate (PBAT), poly(butylene succinate-co-terephthalate) (PBST), and poly(butylene succinate/terephthalate/isophthalate)-co-(lactate) (PBSTIL). The mechanism of degradation of aliphatic as well as aliphatic-aromatic co-polyesters has also been discussed. The degradation ability of microorganisms against various polyesters might be useful for the treatment and recycling of biodegradable wastes or bioremediation of the polyester-contaminated environments.

Analysis of genes for succinoyl trehalose lipid production and increasing production in Rhodococcus sp. strain SD-74.

Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species.

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica: surface plasmon resonance and atomic force microscopy study.

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica JCM10317 (PaE) was analyzed by surface plasmon resonance (SPR). The adsorption of PaE and the degradation rate for polyester films were quantitatively monitored by a positive and negative SPR signal shifts, respectively. The decrease in SPR signal and the erosion depth of amorphous poly(L-lactide) (a-PLLA) film measured by atomic force microscopy (AFM) had a linear relationship, and the weight loss was estimated from the AFM data combined with a density of a-PLLA film. Furthermore, SPR sensorgrams for various polyester films showed that degradation rate of poly(ε-caprolactone) and poly(butylene succinate-co-adipate) which contain C6 units was higher than that of other polyesters such as poly(butylene succinate) and a-PLLA. These results suggest that C6 is the preferred chain length as substrates for PaE.

Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization.

Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable plastics (BPs) such as agricultural mulch films composed of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA). An enzyme named PaE was isolated and the gene encoding PaE was cloned from the strain by functional complementation in Saccharomyces cerevisiae. The deduced amino acid sequence of PaE contains 198 amino acids with a predicted molecular weight of 20,362.41. High identity was observed between this sequence and that of cutinase-like enzymes (CLEs) (61-68%); therefore, the gene encoding PaE was named PaCLE1. The specific activity of PaE against emulsified PBSA was 54.8±6.3 U/mg. In addition to emulsified BPs, PaE degraded solid films of PBS, PBSA, poly(ε-caprolactone), and poly(lactic acid).

Characterization of phospholipids in membrane vesicles derived from Pseudomonas aeruginosa.

Many Gram-negative bacteria release membrane vesicles (MVs), but their phospholipid properties are poorly understood. Phosphatidylglycerol was present at high levels in MVs derived from Pseudomonas aeruginosa, but not in the cellular outer membrane. The ratio of stearic acid in MVs was high compared to that in the cellular outer membrane. These findings suggest that membrane rigidity is associated with MV biogenesis.

Variation of physiochemical properties and cell association activity of membrane vesicles with growth phase in Pseudomonas aeruginosa.

Pseudomonas aeruginosa and other Gram-negative bacteria release membrane vesicles (MVs) from their surfaces, and MVs have an ability to interact with bacterial cells. Although it has been known that many bacteria have mechanisms that control their phenotypes with the transition from exponential phase to stationary phase, changes of properties in released MVs have been poorly understood. Here, we demonstrate that MVs released by P. aeruginosa during the exponential and stationary phases possess different physiochemical properties. MVs purified from the stationary phase had higher buoyant densities than did those purified from the exponential phase. Surface charge, characterized by zeta potential, of MVs tended to be more negative as the growth shifted to the stationary phase, although the charges of PAO1 cells were not altered. Pseudomonas quinolone signal (PQS), one of the regulators related to MV production in P. aeruginosa, was lower in MVs purified from the exponential phase than in those from the stationary phase. MVs from the stationary phase more strongly associated with P. aeruginosa cells than did those from the exponential phase. Our findings suggest that properties of MVs are altered to readily interact with bacterial cells along with the growth transition in P. aeruginosa.

Isolation of a novel Co2+-resistant bacterium and the application of its siderophore in Co2+ recovery from an aqueous solution.

Siderophores are considered to have a good potential as decontamination agents owing to their metal-chelating abilities. In order to confirm whether siderophores can be used in the recovery of metal ions, a siderophore (or metallophore) exhibiting Co2+-chelating activity was screened to demonstrate its ability to recover Co2+ from an aqueous solution. A siderophore-producing bacterium, Pandoraea sp. HCo-4B, was identified from a screen of Co2+-resistant bacteria grown in an aerobic enrichment culture with a Co2+-supplemented medium. After incubation of the crude extracted siderophore in a Co2+-containing solution, the Co2+-siderophore complex was adsorbed on to a C18 column. The bound Co2+ was eluted from the column by the addition of 10 mM H2SO4. The recovered amount of Co2+ was proportional to the amount of the added siderophore. We observed that the siderophore identified in this study binds to Co2+ at a 1:1 ratio.

Outer membrane machinery and alginate synthesis regulators control membrane vesicle production in Pseudomonas aeruginosa.

The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.

Ecological impact evaluation by constructing in situ microcosm with porous ceramic arrowhead.

In recent years, bioremediation has been used as an effective technique for the cleaning of polluted sites. However, bioremediation treatment efficacy varies considerably; thus, characterization of indigenous pollutant-degrading soil microorganisms and assessment of the changes in microbial composition by pollutants are essential for designing efficient bioremediation methods. In this study, an ecological impact evaluation method that is cost-efficient and has low contamination risk was developed to assess the indigenous microbial composition. An "in situ microcosm" was constructed using a porous ceramic arrowhead. Phenol, a common environmental pollutant, was used to assess the evaluation efficacy of this method. Our data showed that phenol gradually percolated into the soil adjacent to the arrowhead and stimulated unique indigenous microorganisms (Bacillus sp., Streptomyces sp., and Cupriavidus sp.). Furthermore, the arrowhead approach enabled efficient evaluation of the ecological impact of phenol on soil microorganisms. Thus, the arrowhead method will contribute to the development of bioremediation methods.

Potential use of methane fermentation digested slurry as a low-cost, environmentally-friendly nutrient for bioethanol production from crude glycerol by Klebsiella variicola TB-83D.

A methane fermentation digested slurry (MFDS) was evaluated as a substitute for the commercial nutrient, yeast extract (YE), in ethanol production from glycerol by Klebsiella variicola strain TB-83D. In pH-controlled fed-batch cultures, partial replacement of YE by MFDS did not reduce ethanol productivity significantly. However, non-sterilized MFDS had negative effects on glycerol fermentation by this strain. Although ethanol production decreased when YE was completely replaced by sterilized MFDS, the use of crude glycerol and sterilized MFDS achieved a yield of 14.6 g/L ethanol. This is the first study to report the use of MFDS as the sole nutrient for ethanol production from glycerol, which contributes to the development of a low-cost glycerol biorefinery derived from the biodiesel fuel industry.

Identification and functional analysis of genes required for desulfurization of alkyl dibenzothiophenes of Mycobacterium sp. G3.

Mycobacterium sp. G3 was reported as a dibenzothiophene (DBT)-degrading microorganism and the first strain to have the ability to degrade high-molecular-weight alkyl DBTs, such as 4,6-dibutyl DBT and 4,6-dipentyl DBT, by the C-S bond cleavage pathway. Three genes (mdsA, mdsB, and mdsC) for desulfurization, which form a cluster, were cloned from Mycobacterium sp. G3. The expression of each gene in Escherichia coli JM109 showed that MdsC oxidized DBT to DBT sulfone, MdsA transformed DBT sulfone into 2-(2'-hydroxyphenyl)benzene sulfinate (HPBS), and MdsB desulfinated HPBS into 2-hydroxybiphenyl (HBP), indicating that the gene products of mdsABC are functional in the recombinant. MdsC oxidized 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT and 4,6-dibutyl DBT to each sulfone form, suggesting that MdsC covers a broad specificity for alkyl DBTs.

Screening of novel cellulose-degrading bacterium and its application to denitrification of groundwater.

To establish an environmentally friendly groundwater bioremediation process using a cellulose carrier combined with cellulose-utilizing, denitrifying microorganisms, a novel psychrophilic bacterium, designated CL-5, which can degrade a commercial-based cellulose carrier as the sole carbon source, was screened. Since the denitrification capability of CL-5 is low, complex microbial systems were constructed together with other denitrifying bacteria designated NR-1 and NR-2 that were also isolated from soil. The nitrate-reducing activities of mixed cultures were much higher than those of the pure cultures of CL-5, NR-1 and NR-2. The highest N(2)O and N(2) formation activities were observed in the mixed culture of CL-5+NR-2.

Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils.

The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with the unplanted system. Among the representative alkane hydroxylase genes alkB, CYP153, almA and ladA, the first two were detected in this study, and the genotypes of both genes were apparently different among the systems studied. Their diversity was also higher on the rhizoplanes of the grasses than in unplanted oil-contaminated soils. Actinobacteria-related genes in particular were among the most diverse alkane hydroxylase genes on the rhizoplane in this study, indicating that they are one of the main contributors to degrading alkanes in oil-contaminated soils during phytoremediation. Actinobacteria-related alkB genes and CYP153 genes close to the genera Parvibaculum and Aeromicrobium were found in significant numbers on the rhizoplanes of grasses. These results suggest that the increase in diversity and genotype differences of the alkB and CYP153 genes are important factors affecting petroleum hydrocarbon-degrading ability during phytoremediation.

Improved ethanol tolerance and ethanol production from glycerol in a streptomycin-resistant Klebsiella variicola mutant obtained by ribosome engineering.

To improve the ethanol tolerance of the Klebsiella variicola strain TB-83, we obtained the streptomycin-resistant, ethanol-tolerant mutant strain TB-83D by a ribosome engineering approach. Strain TB-83D was able to grow in the presence of 7% (v/v) ethanol and it showed higher ethanol production than strain TB-83. Examination of various culture conditions revealed that yeast extract was essential for ethanol production and bacterial growth. In addition, ethanol production was elevated to 32g/L by the addition of yeast extract; however, ethanol production was inhibited by formate accumulation. With regard to cost reduction, the use of corn steep liquor (CSL) markedly decreased the formate concentration, and 34g/L ethanol was produced by combining yeast extract with CSL. Our study is the first to improve ethanol tolerance and productivity by a ribosome engineering approach, and we found that strain TB-83D is effective for ethanol production from glycerol.

A simple screening procedure for heterotrophic nitrifying bacteria with oxygen-tolerant denitrification activity.

Various naturally occurring strains of heterotrophic nitrifying bacteria were isolated by enrichment culture using acetamide as the C and N source, and 21 strains were identified as heterotrophic nitrifiers. Using a new simple procedure, these 21 strains were also investigated for the ability to carry out denitrifcation in the presence of oxygen. Several of the nitrifying strains were found to exhibit a distinct activity that allows for denitrifcation via nitrite (NO2-) in the presence of oxygen, indicating that they have an oxygen-tolerant denitrifcation system. A wide variety of bacteria possessing both nitrification and denitrifcation capabilities in the presence of oxygen were isolated and partially characterized by using the simple screening combinatorial procedure described in this paper.

Matrix metalloproteinase-2 inhibition and Zn2+-chelating activities of pyoverdine-type siderophores.

Pyoverdine-type siderophores from fluorescent Pseudomonas species were purified by Zn2+-chelate chromatography, and their matrix metalloproteinase-2 (MMP-2) inhibition and metal-ion-chelating activities were studied. Structurally different pyoverdines showed different MMP-2 inhibition activities, and the inhibition activity was correlated with Zn2+-chelating activity. The IC50 value of a pyoverdine ((P113A1)-2, MW 1187) for MMP-2 was 0.27 microg/ml (0.23 microM).

Cloning and sequence analysis of poly(tetramethylene succinate) depolymerase from Acidovorax delafieldii strain BS-3.

A gene encoding poly(tetramethylene succinate), PBS, depolymerase, pbsA, has been cloned from Acidovorax delafieldii strain BS-3 chromosomal DNA. The clone expressed in Escherichia coli showed the ability to degrade both PBS and poly[(tetramethylene succinate)-co-adipate] that are kinds of biodegradable plastics. PBS depolymerase was considered to be a kind of lipase, since it also degrades olive oil. It had no apparent hydrophobic-amino-acid-rich region which exists in other known plastic-degrading enzymes. From the result of amino acid homology search, PbsA was found to have some similarities with lipases of Streptomyces sp. and Mollaxella sp. In the motif surrounding the active site Ser residue (Gly-X1-Ser-X2-Gly), PbsA was revealed to have a Trp residue in the X1 position instead of His which is most likely found in other bacterial lipases.

Ethanol production from glycerol-containing biodiesel waste by Klebsiella variicola shows maximum productivity under alkaline conditions.

Biodiesel fuel (BDF) waste contains large amounts of crude glycerol as a by-product, and has a high alkaline pH. With regard to microbial conversion of ethanol from BDF-derived glycerol, bacteria that can produce ethanol at alkaline pH have not been reported to date. Isolation of bacteria that shows maximum productivity under alkaline conditions is essential to effective production of ethanol from BDF-derived glycerol. In this study, we isolated the Klebsiella variicola TB-83 strain, which demonstrated maximum ethanol productivity at alkaline pH. Strain TB-83 showed effective usage of crude glycerol with maximum ethanol production at pH 8.0-9.0, and the culture pH was finally neutralized by formate, a by-product. In addition, the ethanol productivity of strain TB-83 under various culture conditions was investigated. Ethanol production was more efficient with the addition of yeast extract. Strain TB-83 produced 9.8 g/L ethanol (0.86 mol/mol glycerol) from cooking oil-derived BDF waste. Ethanol production from cooking oil-derived BDF waste was higher than that of new frying oil-derived BDF and pure-glycerol. This is the first report to demonstrate that the K. variicola strain TB-83 has the ability to produce ethanol from glycerol at alkaline pH.

Rapid and simple colorimetric assay for detecting the enzymatic degradation of biodegradable plastic films.

We developed a rapid and simple method for evaluating the degradation of solid biodegradable plastics (BPs). Dye-containing BP films were used as substrates and the release of dye caused by the degradation of BPs was confirmed by a color change in the enzyme solution after a reaction time of 24 h.