Enrichment and characterization of a bacterial mixture capable of utilizing C-mannosyl tryptophan as a carbon source.

C-Mannosylation, a protein-modification found in various eukaryotes, involves the attachment of a single mannose molecule to selected tryptophan residues of proteins. Since C-mannosyl tryptophan (CMW) was detected in human urine, it is generally thought that CMW is not catabolized inside our body and instead is excreted via the urine. This paper reports enrichment of a bacterial consortium from soil that degrades CMW. The bacteria grew in minimal medium supplemented with CMW as the carbon source. Interestingly, even after successive clonal picks of individual colonies, several species were still present in each colony as revealed by 16S rRNA gene sequence analysis, indicating that a single species may not be responsible for this activity. A next generation sequencing (NGS) analysis was therefore carried out in order to determine which bacteria were responsible for the catabolism of CMW. It was found that a species of Sphingomonadaceae family, but not others, increased with simultaneous decrease of CMW in the media, suggesting that this species is most likely the one that is actively involved in the degradation of CMW.

Acetogenesis from H2 plus CO2 and nitrogen fixation by an endosymbiotic spirochete of a termite-gut cellulolytic protist.

Symbiotic associations of cellulolytic eukaryotic protists and diverse bacteria are common in the gut microbial communities of termites. Besides cellulose degradation by the gut protists, reductive acetogenesis from H2 plus CO2 and nitrogen fixation by gut bacteria play crucial roles in the host termites' nutrition by contributing to the energy demand of termites and supplying nitrogen poor in their diet, respectively. Fractionation of these activities and the identification of key genes from the gut community of the wood-feeding termite Hodotermopsis sjoestedti revealed that substantial activities in the gut--nearly 60% of reductive acetogenesis and almost exclusively for nitrogen fixation--were uniquely attributed to the endosymbiotic bacteria of the cellulolytic protist in the genus Eucomonympha. The rod-shaped endosymbionts were surprisingly identified as a spirochete species in the genus Treponema, which usually exhibits a characteristic spiral morphology. The endosymbionts likely use H2 produced by the protist for these dual functions. Although H2 is known to inhibit nitrogen fixation in some bacteria, it seemed to rather stimulate this important mutualistic process. In addition, the single-cell genome analyses revealed the endosymbiont's potentials of the utilization of sugars for its energy requirement, and of the biosynthesis of valuable nutrients such as amino acids from the fixed nitrogen. These metabolic interactions are suitable for the dual functions of the endosymbiont and reconcile its substantial contributions in the gut.

Rice Bran Amendment Suppresses Potato Common Scab by Increasing Antagonistic Bacterial Community Levels in the Rhizosphere.

Potato common scab (PCS), caused by pathogenic Streptomyces spp., is a serious disease in potato production worldwide. Cultural practices, such as optimizing the soil pH and irrigation, are recommended but it is often difficult to establish stable disease reductions using these methods. Traditionally, local farmers in southwest Japan have amended soils with rice bran (RB) to suppress PCS. However, the scientific mechanism underlying disease suppression by RB has not been elucidated. The present study showed that RB amendment reduced PCS by repressing the pathogenic Streptomyces population in young tubers. Amplicon sequencing analyses of 16S ribosomal RNA genes from the rhizosphere microbiome revealed that RB amendment dramatically changed bacterial composition and led to an increase in the relative abundance of gram-positive bacteria such as Streptomyces spp., and this was negatively correlated with PCS disease severity. Most actinomycete isolates derived from the RB-amended soil showed antagonistic activity against pathogenic Streptomyces scabiei and S. turgidiscabies on R2A medium. Some of the Streptomyces isolates suppressed PCS when they were inoculated onto potato plants in a field experiment. These results suggest that RB amendment increases the levels of antagonistic bacteria against PCS pathogens in the potato rhizosphere.

A heavy-metal tolerant novel bacterium, Alcaligenes pakistanensis sp. nov., isolated from industrial effluent in Pakistan.

Two strains, NCCP-650(T) and NCCP-667, were isolated from industrial effluent and their taxonomic positions were investigated using a polyphasic taxonomic approach. The strains were found to be Gram-stain negative, strictly aerobic, motile short rods, which are tolerant to heavy-metals (Cr(+2), As(+2), Pb(+2) and Cu(+2)). Cells were observed to grow at a temperature range of 10-37 °C (optimal 25-33 °C), pH range of 5.5-10.0 (optimal 6.5-7.5) and can tolerate 0-7 % NaCl (w/v) (optimum 0-1 %) in tryptic soya agar medium. Sequencing of the 16S rRNA gene and two housekeeping genes, gyrB and nirK, of the isolated strains revealed that both strains belong to the Betaproteobacteria showing highest sequence similarities with members of the genus Alcaligenes. The chemotaxonomic data [major quinones as Q-8; predominant cellular fatty acids as summed features 3 (C16 :1 ω7c/iso-C15 :0 2OH) and C16:0 followed by Summed features 2 (iso-C16 :1 I/C14 :0 3OH), C17:0 Cyclo and C18:1 ω7c; major polar lipids as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminolipid] also supported the affiliation of the isolated strains with the genus Alcaligenes. DNA-DNA hybridizations between the two strains and with closely related type strains of species of the genus Alcaligenes confirmed that both isolates belong to a single novel species within the genus Alcaligenes. On the basis of phylogenetic analyses, physiological, biochemical characteristics and DNA-DNA hybridization, the isolated strains can be differentiated from established Alcaligenes species and thus represent a novel species, for which the name Alcaligenes pakistanensis sp. nov. is proposed with the type strain NCCP-650(T) (=LMG 28368(T) = KCTC42083(T) = JCM 30216(T)).

Aniseikonia and retinal morphological changes in eyes undergoing macular hole surgery.

Even after idiopathic macular hole (MH) surgery and with successful closure of MH, aniseikonia is a common postoperative symptom. We investigated the correlation of MH diameter, retinal displacement and retinal layer thicknesses with aniseikonia in 41 eyes of 41 patients undergoing MH surgery with internal limiting membrane peeling. Aniseikonia was measured with the New Aniseikonia Test. Retinal displacement (RD%) was defined as change of retinal distance between the temporal margin of the optic papilla and the intersection of the retinal vessels. Changes of thicknesses of the inner nuclear layer (INL%) and the outer retinal layer (OR%) were calculated. Aniseikonia improved postoperatively. Preoperative aniseikonia and their improvement at 6 months correlated with MH diameters (P = 0.004-0.046). Improvement of aniseikonia correlated with temporal RD% (P = 0.002-0.012). Improvement of vertical aniseikonia correlated with INL% at 2 weeks and with the nasal OR% at 1, 3, and 6 months (P = < 0.001-0.028). MH diameter and age were significant predictors for improvement of aniseikonia. The greater the temporal retina displacement, and the thinner the postoperative INL and OR, the greater the improvement of aniseikonia. MH diameter and age are strong predictors for improvement of aniseikonia after MH surgery.

Predictors for metamorphopsia in eyes undergoing macular hole surgery.

Metamorphopsia is an important visual symptom of macular disease. We determined predictors for metamorphopsia investigating the relationships of macular hole (MH) diameter and retinal layer thicknesses with metamorphopsia after MH surgery. Forty-two eyes of 42 consecutive patients undergoing MH surgery were retrospectively studied. Metamorphopsia was measured with M-CHARTS. Inner nuclear layer (INL) and outer retinal layer (OR) thicknesses were measured 1000 µm away from central fovea at using Spectralis. Preoperative M-CHARTS scores correlated with MH diameters (P = 0.007-0.031) and changes of temporal OR thickness (P = 0.008-0.010). Postoperative M-CHARTS score at 3 months correlated with preoperative nasal and inferior OR thicknesses (P = 0.003 and 0.016) and with changes of superior INL at 3 and 6 months (P = 0.011 and 0.025), and score at 1 month with change of temporal OR at 6 months (P = 0.033). Postoperative improvement of M-CHARTS scores correlated with changes of temporal INL and superior OR (P = 0.026 and 0.002). Multiple regression analysis revealed that MH diameter was a significant predictor for metamorphopsia. Photoreceptor displacement and inner retinal change may generate metamorphopsia in MH undergoing surgery, however MH diameter is the most powerful predictor.

Retinal displacement and intraretinal structural changes after idiopathic macular hole surgery.

PURPOSE: To investigate the correlations of thickness of three retinal layers with retinal displacement after idiopathic macular hole surgery. STUDY DESIGN: Retrospective, consecutive, case series. METHODS: 42 eyes of 42 patients undergoing macular hole surgery with internal limiting membrane peeling were studied. Retinal distance was measured with near-infrared images between the optic nerve and the intersection of retinal vessels at four quadrants. Retinal thicknesses of inner retinal layer, inner nuclear layer and outer retinal layer were measured 1000 µm away from the central fovea using Spectralis. RESULTS: Retinal distances other than the nasal quadrant decreased postoperatively (p < 0.001). Retinal displacement (%) correlated significantly with the change in inner nuclear layer thickness in the temporal sector at 1, 3, and 6 months, in the superior sector at 2 weeks, 1, and 6 months, and in the inferior sector at 3 and 6 months postoperatively (r = 0.319-0.570, p < 0.001-0.040), but not in the inner or outer retinal layers. CONCLUSION: Internal limiting membrane peeling for macular hole enhances retinal displacement toward the optic disc, whose distances correlate with the changes in inner nuclear layer thickness.

The GAF-like-domain-containing transcriptional regulator DfdR is a sensor protein for dibenzofuran and several hydrophobic aromatic compounds.

Dibenzofuran (DF) is one of the dioxin carbon skeletal compounds used as a model to study the microbial degradation of dioxins. This study analyzed the transcriptional regulation of the DF dioxygenase genes dfdA1 to dfdA4 in the DF-utilizing actinomycetes Rhodococcus sp. strain YK2 and Terrabacter sp. strain YK3. An open reading frame designated dfdR was detected downstream of the dfdC genes. The C-terminal part of the DfdR amino acid sequence has high levels of similarity to several LuxR-type DNA binding helix-turn-helix domains, and a GAF domain sequence in the central part was detected by a domain search analysis. A derivative of YK2 with dfdR disrupted was not able to utilize DF and did not exhibit DF-dependent dfdA1 transcriptional induction ability, and these dysfunctions were compensated for by introduction of dfdR. Promoter analysis of dfdA1 in Rhodococcus strains indicated that activation of the dfdA1 promoter (P(dfdA1)) was dependent on dfdR and DF and not on a metabolite of the DF pathway. The cell extract of a Rhodococcus strain that heterologously expressed DfdR showed electrophoretic mobility shift (EMS) activity for the P(dfdA1) DNA fragment in a DF-dependent manner. In addition, P(dfdA1) activation and EMS activity were observed with hydrophobic aromatic compounds comprising two or more aromatic rings, suggesting that DfdR has broad effector molecule specificity for several hydrophobic aromatic compounds.

Analysis of two gene clusters involved in the degradation of 4-fluorophenol by Arthrobacter sp. strain IF1.

Arthrobacter sp. strain IF1 is able to grow on 4-fluorophenol (4-FP) as a sole source of carbon and energy. To clone the 4-FP degradation genes, DNA libraries were constructed and screened with a probe obtained by PCR using primers designed on the basis of conserved regions of aromatic two-component monooxygenases. Sequencing of positive clones yielded two gene clusters, each harboring a gene encoding a monooxygenase with high sequence similarity to the oxygenase component of 4-nitrophenol and 4-chlorophenol monooxygenase systems. Both these monooxygenase genes were differentially expressed during growth on 4-FP, as revealed by Northern blotting and reverse transcription-PCR. One cluster also contained a gene for a flavin reductase. The monooxygenase and reductase were purified from Escherichia coli cells expressing the corresponding genes, and together they catalyzed NADH-dependent hydroxylation and dehalogenation of 4-halophenols. The results indicate that strain IF1 transforms 4-FP to hydroquinone by a two-component monooxygenase system of which one component provides reduced flavin adenine dinucleotide at the expense of NADH and the other catalyzes para-hydroxylation of 4-FP and other 4-substituted phenols.

Functional expression of three Rieske non-heme iron oxygenases derived from actinomycetes in Rhodococcus species for investigation of their degradation capabilities of dibenzofuran and chlorinated dioxins.

The activity of Rieske non-heme iron oxygenases (aromatic hydrocarbon dioxygenases, AhDOs) is important for the bacterial degradation of aromatic pollutants such as polycyclic aromatic hydrocarbons and dioxins. During our analysis of the role of AhDOs in dioxin bioremediation, some enzymes derived from high G + C Gram-positive actinomycetes were difficult to produce in active form in the Escherichia coli protein expression system. In this study, we constructed a heterologous expression system for AhDOs in Rhodococcus species using a constitutive expression promoter, P(dfdB), and a shuttle vector, pRK401, and analyzed the ability of these enzymes to degrade dibenzofuran and deplete several chlorinated dioxins. Three active AhDOs expressed in Rhodococcus strains that were difficult to obtain by the E. coli system showed different regiospecificities for dibenzofuran bioconversion as well as different substrate depletion specificities for chlorinated dioxins. Moreover, AhDO derived from R. erythropolis TA421 showed relatively diverse depletion-substrate specificity for chlorinated dioxins.

Draft Genome Sequence of Tepidibacter mesophilus Strain JCM 16806T Isolated from Soil Polluted by Crude Oil in China.

Here, we report the draft genome sequence of Tepidibacter mesophilus strain JCM 16806T, which was isolated from an oil field. It is composed of 3,310,272 bp and contains 3,160 protein-coding genes, 8 5S rRNAs, 3 16S rRNAs, and 69 tRNAs.

Prokaryotic Communities at Different Depths between Soils with and without Tomato Bacterial Wilt but Pathogen-Present in a Single Greenhouse.

The characterization of microbial communities that promote or suppress soil-borne pathogens is important for controlling plant diseases. We compared prokaryotic communities in soil with or without the signs of tomato bacterial wilt caused by Ralstonia solanacearum. Soil samples were collected from a greenhouse at two different depths because this pathogen is present in deep soil. We used samples from sites in which we detected phcA, a key gene regulating R. solanacearum pathogenicity. The pyrosequencing of prokaryotic 16S rRNA sequences in four soil samples without disease symptoms but with phcA and in two soil samples with disease symptoms indicated that community richness was not significantly different between these two soils; however, microbial diversity in the lower soil layer was higher in soil samples without disease symptoms but with phcA. A difference in prokaryotic community structures between soil samples with and without bacterial wilt was only observed in the upper soil layer despite apparent similarities in the communities at the phylum level. Proteobacteria, Acidobacteria, Chloroflexi, Verrucomicrobia, and several Archaea were more abundant in soil samples without disease symptoms, whereas taxa in another eight phyla were more abundant in soil samples with disease symptoms. Furthermore, some prokaryotic taxa were abundant specifically in the lower layer of soil, regardless of whether disease was present. These prokaryotic taxa may suppress or accelerate the pathogenesis of bacterial wilt and are good targets for future studies on disease control.

Comparison of Prokaryotic and Eukaryotic Communities in Soil Samples with and without Tomato Bacterial Wilt Collected from Different Fields.

Biocontrol agents (BCA) effectively suppress soil-borne disease symptoms using natural antagonistic prokaryotes or eukaryotes. The main issue associated with the application of BCA is that disease reduction effects are unstable under different field conditions. In order to identify potentially effective BCA among several fields, we compared prokaryotic and eukaryotic communities in soil with and without tomato bacterial wilt from three different fields, each of which had the same field management and similar soil characteristics. Soil samples were collected from three fields and two depths because bacterial wilt pathogens were present in soil at a depth greater than 40 cm. We classified soil samples based on the presence or absence of the bacterial phcA gene, a key gene for bacterial wilt pathogenicity and tomato disease symptoms. Pyrosequencing of the prokaryotic 16S rRNA gene and eukaryotic internal transcribed spacer region sequences showed that the diversity and richness of the communities mostly did not correlate with disease symptoms. Prokaryotic and eukaryotic community structures were affected more by regional differences than the appearance of disease. Several prokaryotes and eukaryotes were more abundant in soil that lacked disease symptoms, and eight prokaryotes and one eukaryote of this group were commonly detected among the three fields. Some of these taxa were not previously found in disease-suppressive soil. Our results suggest that several prokaryotes and eukaryotes control plant disease symptoms.

Peroxisome deficiency represses the expression of n-alkane-inducible YlALK1 encoding cytochrome P450ALK1 in Yarrowia lipolytica.

Among the eight genes (YlALK1-YlALK8) encoding P450 cytochromes of the CYP52 family of the n-alkane-assimilating yeast Yarrowia lipolytica, Y1ALK1 is most highly induced by n-alkanes with short hydrocarbon chains, such as n-decane, and involved in the initial hydroxylation of n-alkane. To determine the factors regulating YlALK1 expression, we isolated an n-decane assimilation-deficient mutant, B0-6-1, whose YlALK1 expression level was lower than that of the wild-type. By complementation of the mutation of B0-6-1, we cloned a gene having an open reading frame of 1062 bp. The putative gene product is a protein of 354 amino acids and has significant homology to Pex10ps of other organisms. We named this gene YlPEX10. YlPex10p has a C(3)HC(4) ring finger motif common among Pex10ps in its C-terminal region. This motif was also essential for the function of YlPex10p. Both B0-6-1 and a null mutant of YlPEX10 failed to form peroxisome and showed low-level transcription of YlALK1 after the change of carbon source to n-decane. Furthermore, YlPEX5 and YlPEX6 disruptants also showed low-level transcription of YlALK1 like the YlPEX10 disruptant and B0-6-1 mutant. We propose that in this organism peroxisome deficiency represses the expression of n-alkane-inducible YlALK1 encoding cytochrome P450ALK1.

Chloroplast-type ferredoxin involved in reactivation of catechol 2,3-dioxygenase from Pseudomonas sp. S 47.

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XylTE products catalyze the dioxygenation of the aromatics. The xylT of the strain S-47 is located just upstream of the xylE gene. XylT is a typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2 (TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XylT for the reactivation of the catechol 2,3-dioxygenase (XylE) that is oxidized with hydrogen peroxide. The pRES1 that was treated with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 minutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XylT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.

Draft Genome Sequence of Clostridium straminisolvens Strain JCM 21531T, Isolated from a Cellulose-Degrading Bacterial Community.

Here, we report the draft genome sequence of a fibrolytic bacterium, Clostridium straminisolvens JCM 21531(T), isolated from a cellulose-degrading bacterial community. The genome information of this strain will be useful for studies on the degradation enzymes and functional interactions with other members in the community.

Draft Genome Sequence of Paenibacillus pini JCM 16418T, Isolated from the Rhizosphere of Pine Tree.

Paenibacillus pini strain JCM 16418(T) is a cellulolytic bacterium isolated from the rhizosphere of pine trees. Here, we report the draft genome sequence of this strain. This genome information will be useful for studies of rhizosphere bacteria.

Draft Genome Sequence of Bacteroides reticulotermitis Strain JCM 10512T, Isolated from the Gut of a Termite.

Here we report the draft genome sequence of Bacteroides reticulotermitis strain JCM 10512(T), a xylanolytic and cellulolytic bacterium isolated from the gut of a wood-feeding termite. The genome information will facilitate the study of this strain for biomass degradation and adaptation to the gut environment.

Draft Genome Sequences of Two Lactobacillus Strains, L. farraginis JCM 14108T and L. composti JCM 14202T, Isolated from Compost of Distilled Shochu Residue.

Here, we report the draft genome sequences of two type strains of Lactobacillus, Lactobacillus farraginis JCM 14108(T) and Lactobacillus composti JCM 14202(T), isolated from the compost of distilled shochu residue. Their genome information will be useful for studies of ecological and physiological functions of these Lactobacillus species.

Draft Genome Sequences of Three Strains of Bacteroides pyogenes Isolated from a Cat and Swine.

Here, we report the draft genome sequences of Bacteroides pyogenes JCM 6294(T), JCM 6292, and JCM 10003, which were isolated from a cat and swine and were recently classified into a single species, B. pyogenes. Comparative analyses of these genomes revealed the diversification of B. pyogenes strains isolated from different animals.