Pseudocitrobacter gen. nov., a novel genus of the Enterobacteriaceae with two new species Pseudocitrobacter faecalis sp. nov., and Pseudocitrobacter anthropi sp. nov,isolated from fecal samples from hospitalized patients in Pakistan

Four isolates of Gram-negative facultatively anaerobic bacteria, three of them producing NDM-1 carbapenemase, were isolated from hospitalized patients and outpatients attending two military hospitals in Rawalpindi, Pakistan, and studied for their taxonomic position. Initially the strains were phenotypically identified as Citrobacter species. Comparative analysis of 16S rRNA gene sequences then showed that the four strains shared >97%, but in no case >98.3%, 16S rRNA gene sequence similarities to members of the genera Citrobacter, Kluyvera, Pantoea, Enterobacter and Raoultella, but always formed a separate cluster in respective phylogenetic trees. Based on multilocus sequence analysis (MLSA) including partial recN, rpoA, thdF and rpoB gene sequence and respective amino acid sequence analysis it turned out that the strains also here always formed separate clusters. Based on further comparative analyses including DNA–DNA hybridizations, genomic fingerprint analysis using rep- and RAPD-PCRs and physiological tests, it is proposed to classify these four strains into the novel genus Pseudocitrobacter gen. nov. with a new species Pseudocitrobacter faecalis sp. nov. with strain 25 CIT^T (= CCM 8479^T = LMG 27751^T) and Pseudocitrobacter anthropi sp. nov. with strain C138^T (= CCM 8478^T = LMG 27750^T), as the type strains, respectively.     

Application of a novel bacterial consortium for mineralization of sulphonated aromatic amines

A novel bacterial consortium (TJ-2) for mineralization of aromatic amines resulting from decolorization of azo dyes was developed. Three bacterial strains were identified as Pseudomonas pseudoalcaligenes (TJ-21,EU072476), Pseudomonas citronellolis (TJ-22,EU072477) and Pseudomonas testosterone (TJ-23,EU072477) by 16S rRNA gene sequence analysis. Aromatic amine mineralization under aerobic conditions was observed to be significantly higher with the consortium as compared to pure strains indicating complementary interactions among these strains. It was observed that more than 90% mineralization of aromatic amines was achieved within 18 h for different initial aromatic amines concentrations. It was also observed that aromatic amine mineralization depends upon the structure of aromatic amine. Para- and meta-hydroxy substituted aromatic amine were easily mineralized as compared to ortho-substituted which undergoes autoxidation when exposed to oxygen. The consortium was capable of mineralizing other aromatic amines, thus, conferring the possibility of application of TJ-2 for the treatment of industrial wastewaters containing aromatic amines.     

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice—natural pond; Jordan's Park—artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies.     

Isolation and characterization of a fungus able to degrade pyrethroids and 3-phenoxybenzaldehyde

Fungal strain HU, isolated from activated sludge and identified as a member of the genus Cladosporium based on morphology and sequencing of 28S rRNA, was shown to degrade 90% of fenvalerate, fenpropathrin, β-cypermethrin, deltamethrin, bifenthrin, and permethrin (100 mg L⁻¹) within 5 days. Fenvalerate was utilized as sole carbon and energy source and co-metabolized in the presence of sucrose. Degradation of fenvalerate occurred at pH 5-10 at 18-38 °C. The fungus first hydrolyzed the carboxylester linkage to produce α-hydroxy-3-phenoxy-benzeneacetonitrile and 3-phenoxybenzaldehyde, and subsequently degraded these two compounds with a q_max, K_s and K_i of 1.73 d⁻¹, 99.20 mg L⁻¹ and 449.75 mg L⁻¹, respectively. Degradation followed first-order kinetics. These results show that the fungal strain may possess potential to be used in bioremediation of pyrethroid-contaminated environments.     

Osmoregulated trehalose-derived oligosaccharides in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium accumulating glutamate, N-acetylglutaminyl glutamine amide and trehalose in hyperosmolarity. Besides these compatible solutes, we highlighted several compounds in S. meliloti Rm1021 wild-type strain. The purification and the structural characterization based on liquid chromatography evaporative light scattering detector, electrospray ionization high resolution mass spectrometry and nuclear magnetic resonance techniques showed they were four linear oligosaccharides composed of 3, 4, 5 and 6 glucose units all linked by α-(1 → 2) linkages except a terminal α-(1 ↔ 1) linkage. These oligosaccharides were cytoplasmic and were observed in several wild-type strains suggesting they were common features in S. meliloti strains grown in hyperosmolarity.     

Metabolic engineering of Arabidopsis for remediation of different polycyclic aromatic hydrocarbons using a hybrid bacterial dioxygenase complex

The widespread presence of polycyclic aromatic hydrocarbons (PAHs) and their potential harm to various organisms has generated interest in efficiently eliminating these compounds from the environment. Phytoremediation is an efficient technology for cleaning up pollutants. However, unlike microorganisms, plants lack the catabolic pathway for complete degradation of these dangerous groups of compounds. One way to enhance the potential of plants for remediation of these compounds is by transferring genes involved in xenobiotic degradation from microbes to plants. In this paper, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as NahAa and NahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were transferred and ectopically expressed in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing the heterozygous naphthalene dioxygenase system exhibited enhanced tolerance toward 2–4 rings PAHs. Transgenic plants assimilated PAHs from the culture media faster and accumulated less in vivo than wild-type plants. Furthermore, examination of metabolic intermediates by gas chromatography–mass spectrometry revealed that the naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Taken together, our findings suggest that grafting the naphthalene dioxygenase complex into plants is a possible strategy to breed PAH-tolerant plants to efficiently degrade PAHs in the environment.     

Enhanced resistance of Saccharomyces cerevisiae to vanillin by expression of lacA from Trametes sp. AH28-2

Saccharomyces cerevisiae is affected by the presence of certain phenolic compounds such as vanillin during fermentation of pretreated lignocellulosic hydrolysates. Since vanillin can be polymerized in the presence of laccase into compounds with lower toxicity, the laccase gene, lacA, from Trametes sp. AH28-2 was fused to the α-factor signal sequence and transferred into S. cerevisiae CEN.PK strains for secretory expression. Furthermore, the chaperone gene, KAR2, was overexpressed to promote the translocation of laccase. In the presence of 8 mmol/L vanillin, a shorter lag phase was observed in the lacA gene expressing strains. The vanillin-specific conversion rate of the lacA-expressing strain BSJX0A2 was 0.069 g g⁻¹ biomass h⁻¹, while it was 0.065 g g⁻¹ biomass h⁻¹ in the reference strain.     

Isolation of Enterobacteria able to degrade simple aromatic compounds from the wastewater from olive oil extraction

Summary Enterobacteria growing on wastewater from olive oil extraction were selected. Among this microflora, strains of Klebsiella oxytoca and Citrobacter diversus able to degrade simple monomeric aromatic compounds were isolated by enrichment culture of the effluent lacking simple sugars. In this preliminary investigation, the phenolic acids tested on solid and liquid media were gentisic, protocatechuic, p-hydroxybenzoic, benzoic, vanillic and ferulic. It was shown that the biodegradation of an aromatic acid is tightly dependent on both the type and the position of the radical substituted on the aromatic ring. Citrobacter was the most efficient strain in metabolizing ferulic acid in liquid medium at a concentration of 1.5 g/l. The substrate biodegradation yield achieved exceeded 86%.     

Key aromatic residues at subsites + 2 and + 3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k_cat/K_m for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k_cat/K_m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k_cat/K_m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k_cat/K_m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.     

Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Two Types of Bacteria Having Angular Dioxygenases with Different Features

Two kinds of bacteria having different-structured angular dioxygenases—a dibenzofuran (DF)-utilizing bacterium, Terrabacter sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp. strain CA10—were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.     

Harvesting biohydrogen from cellobiose from sulfide or nitrite-containing wastewaters using Clostridium sp. R1

Harvesting biohydrogen from inhibiting wastewaters is of practical interest since the toxicity of compounds in a wastewater stream commonly prevents the bioenergy content being recovered. The isolated Clostridium sp. R1 is utilized to degrade cellobiose in sulfide or nitrite-containing medium for biohydrogen production. The strain can effectively degrade cellobiose free of severe inhibitory effects at up to 200 mg l⁻¹ sulfide or to 5 mg l⁻¹ nitrite, yielding hydrogen at >2.0 mol H₂ mol⁻¹ cellobiose. Principal metabolites of cellobiose fermentation are acetate and butyrate, with the concentration of the former increases with increasing sulfide and nitrite concentrations. The isolated strain can yield hydrogen from cellobiose in sulfide-laden wastewaters. However, the present of nitrite significantly limit the efficiency of the biohydrogen harvesting process.     

Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase

We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamines < o-aminophenols < o-diphenols, which agrees with the view that the transfer of the proton to the peroxide group of the oxy-tyrosinase form is the slowest step in the catalytic cycle. As regards the apparent inactivation constant, it remains within the same order of magnitude, although slightly lower in the case of the aromatic o-diamines and o-aminophenols than o-diphenols: o-diamines < o-aminophenols < o-diphenols. The efficiency of the second nucleophilic attack of substrate on CuA seems to be the determining factor in the bifurcation of the inactivation and catalytic pathways. This attack is more efficient in o-diamines (where it attacks a nitrogen atom) than in o-aminophenols and o-diphenols (where it attacks an oxygen atom), favouring the catalytic pathway and slowing down the inactivation pathway. The inactivation step is the slowest of the whole process. The values of r, the number of turnovers that 1 mol of enzyme carries out before being inactivated, follows the order aromatic o-diamines < o-aminophenols < o-diphenols. As regards the Michaelis constants, that of the o-diamines is slightly lower than that of the o-diphenols, while that of the o-aminophenols is slightly greater than that observed for the o-diphenols. As a consequence of the above, the inactivation efficiency, λ_max/K_m^S, follows this order: o-diphenols > o-aminophenols > aromatic o-diamines.     

Mating system and population genetic structure of Bruguiera gymnorrhiza (Rhizophoraceae), a viviparous mangrove species in China

In order to explain the diversity patterns and develop the conservation strategies, the population genetic structures and the mating systems of Bruguiera gymnorrhiza from the coastlines of south China were investigated in this study. The mating system parameters were analyzed using progeny arrays for allozyme markers. The multilocus outcrossing rates (tm) ranged from 0.845 (Fugong) to 0.267 (Dongzhai harbor). High allozyme variations within the five collected populations were determined and compared with the published data of other plant species with the mixed mating systems. At species level, the percentage of polymorphic loci (P) was 80%, the average number of alleles per locus (A) was 2.440, and the heterozygosity (He) was 0.293. The total gene diversity within each population (H_S = 0.2782) and the coefficient of genetic differentiation (G_ST = 0.0579) among the populations were estimated. On the basis of this population genetic structure, it is suggested that the gene flow (Nm = 3.85) is quite high, which is possibly related to its water-dispersed hypocotyls. It is also suggested that the mating system of this species is of mixed mating.     

Comparative study of biological hydrogen production by pure strains and consortia of facultative and strict anaerobic bacteria

In this paper, a simple and rapid method was developed in order to assess in comparative tests the production of binary biogas mixtures containing CO₂ and another gaseous compound such as hydrogen or methane. This method was validated and experimented for the characterisation of the biochemical hydrogen potential of different pure strains and mixed cultures of hydrogen-producing bacteria (HPB) growing on glucose.The experimental results compared the hydrogen production yield of 19 different pure strains and sludges: facultative and strict anaerobic HPB strains along with anaerobic digester sludges thermally pre-treated or not. Significant yields variations were recorded even between different strains of the same species by i.e. about 20% for three Clostridium butyricum strains. The pure Clostridium butyricum and pasteurianum strains achieved the highest yields i.e. up to 1.36 mol H₂/mol glucose compared to the yields achieved by the sludges and the tested Escherichia and Citrobacter strains.     

Complete modification maps for the cytosolic small and large subunit rRNAs of Euglena gracilis: functional and evolutionary implications of contrasting patterns between the two rRNA components

In the protist Euglena gracilis, the cytosolic small subunit (SSU) rRNA is a single, covalently continuous species typical of most eukaryotes; in contrast, the large subunit (LSU) rRNA is naturally fragmented, comprising 14 separate RNA molecules instead of the bipartite (28S + 5.8S) eukaryotic LSU rRNA typically seen. We present extensively revised secondary structure models of the E. gracilis SSU and LSU rRNAs and have mapped the positions of all of the modified nucleosides in these rRNAs (88 in SSU rRNA and 262 in LSU rRNA, with only 3 LSU rRNA modifications incompletely characterized). The relative proportions of ribose-methylated nucleosides and pseudouridine (∼ 60% and ∼ 35%, respectively) are closely similar in the two rRNAs; however, whereas the Euglena SSU rRNA has about the same absolute number of modifications as its human counterpart, the Euglena LSU rRNA has twice as many modifications as the corresponding human LSU rRNA. The increased levels of rRNA fragmentation and modification in E. gracilis LSU rRNA are correlated with a 3-fold increase in the level of mispairing in helical regions compared to the human LSU rRNA. In contrast, no comparable increase in mispairing is seen in helical regions of the SSU rRNA compared to its homologs in other eukaryotes. In view of the reported effects of both ribose-methylated nucleoside and pseudouridine residues on RNA structure, these correlations lead us to suggest that increased modification in the LSU rRNA may play a role in stabilizing a ‘looser’ structure promoted by elevated helical mispairing and a high degree of fragmentation.     

Stereospecific degradation of the phenoxypropionate herbicide dichlorprop

2,4-Dichlorophenoxyacetic acid (2,4-D)/α-ketoglutarate (α-KG) dioxygenase, TfdA, from Ralstonia eutropha JMP134, was purified from recombinant cells and shown by gas chromatographic and colorimetric methods to degrade only the S enantiomer of dichlorprop, a phenoxypropionate herbicide. Similarly, cell extracts of Burkholderia cepacia RASC, containing a biochemically and genetically related α-KG-dependent dioxygenase, also were shown to oxidize (S)-dichlorprop using chiral HPLC and colorimetric methods. In contrast, cell extracts of a mecoprop-degrading strain of Alcaligenes denitrificans were shown to catabolize (R)-dichlorprop. Although the A. denitrificans activity exhibited stereospecificity opposite to that of the JMP134 and RASC strains, its cofactor requirements were found to be characteristic of an α-KG-dependent dioxygenase. A PCR amplification product from the DNA of this strain was shown to encode an amino acid sequence that was 95% and 86% identical to the corresponding region of TfdA in RASC and JMP134, respectively. Thus, closely related herbicide-degrading gene products appear to be capable of exhibiting opposite stereochemical degradative capabilities.     

Gammaproteobacteria occurrence and microdiversity in Tyrrhenian Sea sediments as revealed by cultivation-dependent and -independent approaches

Bacterial diversity in Tyrrhenian Sea sediments was assessed using cultivation-dependent and -independent approaches. Samples collected from the different sediment layers (up to 30 cm) relative to four seamount and non-seamount stations, at depths from 3425 to 3580 m, were subjected to DNA extraction and 16S rRNA amplification targeting the V3 region. Denaturing gradient gel electrophoresis (DGGE) showed several heterogeneous profiles and 27 single bands were excised and sequenced. Sequence analysis revealed the presence of Firmicutes, Actinobacteria and Chloroflexi in 26% of the DGGE bands and a predominance of sequences affiliated to cultivable and uncultivable clones of Gammaproteobacteria (55%). To corroborate these findings, cultivation attempts were performed that allowed the isolation of 87 strains assigned to the proteobacterial classes. Identification was achieved by means of automated ribosomal intergenic spacer analysis (ARISA) and by 16S rDNA sequencing. The isolates were related to the gamma, alpha and beta subclasses of Proteobacteria with respective percentages of 77, 17 and 6%. The most predominant Gammaproteobacteria isolates, assigned to the Psychrobacter marincola and P. submarinus clade (n = 53) and to Halomonas aquamarina (n = 14), showed a huge intraspecific diversity with 29 distinct ARISA haplotypes. The detection by both approaches of these psychrophilic and moderately halophilic species and their extensive microdiversity indicated their predominance in Tyrrhenian Sea sediments where they constituted the indigenous microflora.     

Microbial treatment of high-strength perchlorate wastewater

To treat wastewater containing high concentrations of perchlorate, a perchlorate reducing-bacterial consortium was obtained by enrichment culture grown on high-strength perchlorate (1200 mg L⁻¹) feed medium, and was characterized in a sequence batch reactor (SBR) over a long-time operation. The consortium removed perchlorate in the SBR with high reduction rates (35-90 mg L⁻¹ h⁻¹) and stable removal efficiency over 200-day operations. The maximum specific perchlorate reduction rate (q_max), half saturation constant (K_s), and optimal pH range were 0.67 mg-perchlorate mg-dry cell weight⁻¹ h⁻¹, 193.8 mg-perchlorate L⁻¹, and pH 7-9, respectively. The perchlorate reduction yield was 0.48 mol-perchlorate mol-acetate⁻¹. A clone library prepared using the amplicons of cld gene encoding chlorate dismutase showed that the dominant (per)chlorate reducing bacteria in the consortium were Dechlorosoma sp. (53%), Ideonella sp. (28%), and Dechloromonas sp. (19%).     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.