A New ICEclc Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain Pseudomonas stutzeri KF716.

Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation of aromatic compounds, information on their genetic features is limited. We identified a new member of the ICEclc family carrying biphenyl catabolic bph genes and salicylic acid catabolic sal genes from the PCB-degrading strain Pseudomonas stutzeri KF716. The 117-kb ICEbph-salKF716 contains common core regions exhibiting homology with those of degradative ICEclc from P. knackmussii B13 and ICEXTD from Azoarcus sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from P. putida B6-2, P, alcaliphila JAB1, P. stutzeri AN10, and P. stutzeri 2A20 had highly conserved core regions with those of ICEbph-salKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICEbph-salKF716 was transferred from P. stutzeri KF716 to P. aeruginosa PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICEbph-salKF716 compared to other catabolic ICEs.

N-terminal deletion of Swi3 created by the deletion of a dubious ORF YJL175W mitigates protein burden effect in S. cerevisiae.

Extreme overproduction of gratuitous proteins can overload cellular protein production resources, leading to growth defects, a phenomenon known as the protein burden/cost effect. Genetic screening in the budding yeast Saccharomyces cerevisiae has isolated several dubious ORFs whose deletions mitigated the protein burden effect, but individual characterization thereof has yet to be delineated. We found that deletion of the YJL175W ORF yielded an N-terminal deletion of Swi3, a subunit of the SWI/SNF chromatin remodeling complex, and partial loss of function of Swi3. The deletion mutant showed a reduction in transcription of genes encoding highly expressed, secreted proteins and an overall reduction in translation. Mutations in the chromatin remodeling complex could thus mitigate the protein burden effect, likely by reallocating residual cellular resources used to overproduce proteins. This cellular state might also be related to cancer cells, as they frequently harbor mutations in the SWI/SNF complex.

Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida.

Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

Biphenyl/PCB Degrading bph Genes of Ten Bacterial Strains Isolated from Biphenyl-Contaminated Soil in Kitakyushu, Japan: Comparative and Dynamic Features as Integrative Conjugative Elements (ICEs).

We sequenced the entire genomes of ten biphenyl/PCB degrading bacterial strains (KF strains) isolated from biphenyl-contaminated soil in Kitakyushu, Japan. All the strains were Gram-negative bacteria belonging to ß- and γ-proteobacteria. Out of the ten strains, nine strains carried a biphenyl catabolic bph gene cluster as integrative conjugative elements (ICEs), and they were classified into four groups based on the structural features of the bph genes. Group I (five strains) possessed bph genes that were very similar to the ones in Pseudomonasfurukawaii KF707 (formerly Pseudomonas pseudoalcaligenes KF707), which is one of the best characterized biphenyl-utilizing strains. This group of strains carried salicylate catabolic sal genes that were approximately 6-kb downstream of the bph genes. Group II (two strains) possessed bph and sal genes similar to the ones in KF707, but these strains lacked the bphX region between bphC and bphD, which is involved in the downstream catabolism of biphenyl. These bph-sal clusters in groups I and II were located on an integrative conjugative element that was larger than 110 kb, and they were named ICEbph-sal. Our previous study demonstrated that the ICEbph-sal of Pseudomonas putida KF715 in group II existed both in an integrated form in the chromosome (referred to as ICEbph-salKF715 (integrated)) and in a extrachromosomal circular form (referred to as ICEbph-sal (circular)) (previously called pKF715A, 483 kb) in the stationary culture. The ICEbph-sal was transferred from KF715 into P. putida AC30 and P. putida KT2440 with high frequency, and it was maintained stably as an extrachromosomal circular form. The ICEbph-salKF715 (circular) in these transconjugants was further transferred to P. putida F39/D and then integrated into the chromosome in one or two copies. Meanwhile, group III (one strain) possessed bph genes, but not sal genes. The nucleotide sequences of the bph genes in this group were less conserved compared to the genes of the strains belonging to groups I and II. Currently, there is no evidence to indicate that the bph genes in group III are carried by a mobile element. Group IV (two strains) carried bph genes as ICEs (59-61 kb) that were similar to the genes found in Tn4371 from Cupriavidus oxalacticus A5 and ICEKKS1024677 from the Acidovorax sp. strain KKS102. Our study found that bph gene islands have integrative functions, are transferred among soil bacteria, and are diversified through modification.

Novel N-Acyl Homoserine Lactone-Degrading Bacteria Isolated From Penicillin-Contaminated Environments and Their Quorum-Quenching Activities.

N-Acyl homoserine lactones (AHLs) are signaling molecules used in the quorum sensing (QS) of Gram-negative bacteria. Some bacteria interfere with the QS system using AHL-inactivating enzymes, commonly known as quorum-quenching (QQ) enzymes. We have recently isolated a new QQ bacterium showing high resistance to multiple ß-lactam antibiotics, and its QQ enzyme (MacQ) confers ß-lactam antibiotic resistance and exhibits QQ activities. This observation suggests the possibility of isolating novel QQ bacteria from ß-lactam antibiotic-resistant bacteria. In this direction, we attempted to isolate penicillin G (PENG)-resistant bacteria from penicillin-contaminated river sediments and activated sludge treating penicillin-containing wastewater and characterize their QQ activities. Of 19 PENG-resistant isolates, six isolates showed high QQ activity toward a broad range of AHLs, including AHLs with 3-oxo substituents. Five of the six AHL-degraders showed AHL-acylase activity and hydrolyzed the amide bond of AHLs, whereas the remaining one strain did not show AHL-acylase activity, suggesting that this isolate may likely possess alternative degradation mechanism such as AHL-lactonase activity hydrolyzing the lactone ring of AHLs. The 16S rRNA gene sequence analysis results categorized these six AHL-degrading isolates into at least five genera, namely, Sphingomonas (Alphaproteobacteria), Diaphorobacter (Betaproteobacteria), Acidovorax (Betaproteobacteria), Stenotrophomonas (Gammaproteobacteria), and Mycobacterium (Actinobacteria); of these, Mycobacterium sp. M1 has never been known as QQ bacteria. Moreover, multiple ß-lactam antibiotics showed high minimum inhibitory concentrations (MICs) when tested against all of isolates. These results strongly demonstrate that a wide variety of ß-lactam antibiotic-resistant bacteria possess QQ activities. Although the genetic and enzymatic elements are yet unclear, this study may infer the functional and evolutionary correlation between ß-lactam antibiotic resistance and QQ activities.

Ecological impact assessment of a bioaugmentation site on remediation of chlorinated ethylenes by multi-omics analysis.

Bioremediation may affect the ecological system around bioremediation sites. However, little is known about how microbial community structures change over time after the initial injection of degraders. In this study, we have assessed the ecological impact of bioaugmentation using metagenomic and metatranscriptomic approaches to remove trichlorinated ethylene/cis-dichloroethylene (TCE/cDCE) by Rhodococcus jostii strain RHA1 as an aerobic chemical compound degrader. Metagenomic analysis showed that the number of organisms belonging to the genus Rhodococcus, including strain RHA1, increased from 0.1% to 76.6% of the total microbial community on day 0 at the injection site. Subsequently, the populations of strain RHA1 and other TCE/cDCE-degrading bacteria gradually decreased over time, whereas the populations of the anaerobic dechlorinators Geobacter and Dehalococcoides increased at later stages. Metatranscriptomic analysis revealed a high expression of aromatic compound-degrading genes (bphA1-A4) in strain RHA1 after RHA1 injection. From these results, we concluded that the key dechlorinators of TCE/cDCE were mainly aerobic bacteria, such as RHA1, until day 1, after which the key dechlorinators changed to anaerobic bacteria, such as Geobacter and Dehalococcocides, after day 6 at the injection well. Based on the α-diversity, the richness levels of the microbial community were increased after injection of strain RHA1, and the microbial community composition had not been restored to that of the original composition during the 19 days after treatment. These results provide insights into the assessment of the ecological impact and bioaugmentation process of RHA1 at bioremediation sites.

Draft Genome Sequence of Pseudomonas citronellolis LA18T, a Bacterium That Uses Levulinic Acid.

Pseudomonas citronellolis LA18T catabolizes levulinic acid (LA) from cellulosic biomass hydrolysate via acetyl-coenzyme A (acetyl-CoA) and propionyl-CoA. This study reports the 7.22-Mbp draft genome sequence of P. citronellolis LA18T. The draft genome sequence will aid the study of the LA catabolic pathway, which will allow for more applications of LA-utilizing bacteria.

Pseudomonas furukawaii sp. nov., a polychlorinated biphenyl-degrading bacterium isolated from biphenyl-contaminated soil in Japan.

Strain KF707T was isolated from a biphenyl-contaminated site in Kitakyushu, Japan. Analysis of 16S rRNA gene sequences, retrieved from the whole-genome sequence, revealed that the isolate was closely related to members of the genus Pseudomonas, sharing the highest sequence similarities with Pseudomonas balearica strain SP1402T (DSM 6083) (97.8 %). The DNA G+C chromosome and plasmid content of strain KF707T were 65.5 and 60.5 mol%. The major cellular fatty acids were iso-C15 :  0 and C16 : 1ω7c/C16 : 1ω6c. Polyphasic analysis indicated that strain KF707T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas furukawaii sp. nov. is proposed. The type strain is KF707T (=DSM 10086T=NBRC 110670T).

Bifunctional quorum-quenching and antibiotic-acylase MacQ forms a 170-kDa capsule-shaped molecule containing spacer polypeptides.

Understanding the molecular mechanisms of bacterial antibiotic resistance will help prepare against further emergence of multi-drug resistant strains. MacQ is an enzyme responsible for the multi-drug resistance of Acidovorax sp. strain MR-S7. MacQ has acylase activity against both N-acylhomoserine lactones (AHLs), a class of signalling compounds involved in quorum sensing, and ß-lactam antibiotics. Thus, MacQ is crucial as a quencher of quorum sensing as well as in conferring antibiotic resistance in Acidovorax. Here, we report the X-ray structures of MacQ in ligand-free and reaction product complexes. MacQ forms a 170-kDa capsule-shaped molecule via face-to-face interaction with two heterodimers consisting of an α-chain and a ß-chain, generated by the self-cleaving activity of a precursor polypeptide. The electron density of the spacer polypeptide in the hollow of the molecule revealed the close orientation of the peptide-bond atoms of Val20SP-Gly21SP to the active-site, implying a role of the residues in substrate binding. In mutational analyses, uncleaved MacQ retained degradation activity against both AHLs and penicillin G. These results provide novel insights into the mechanism of self-cleaving maturation and enzymatic function of N-terminal nucleophile hydrolases.

Insights into the genomic plasticity of Pseudomonas putida KF715, a strain with unique biphenyl-utilizing activity and genome instability properties.

Pseudomonas putida KF715 exhibits unique properties in both catabolic activity and genome plasticity. Our previous studies revealed that the DNA region containing biphenyl and salycilate metabolism gene clusters (termed the bph-sal element) was frequently deleted and transferred by conjugation to closely related P. putida strains. In this study, we first determined the complete nucleotide sequence of the KF715 genome. Next, to determine the underlying cause of genome plasticity in KF715, we compared the KF715 genome with the genomes of one KF715 defective mutant, two transconjugants, and several P. putida strains available from public databases. The gapless KF715 genome sequence revealed five replicons: one circular chromosome, and four plasmids. Southern blot analysis indicated that most of the KF715 cell population carries the bph-sal element on the chromosome whereas a small number carry it on a huge plasmid, pKF715A. Moreover, the bph-sal element is present stably on the plasmid and did not integrate into the chromosome of its transconjugants. Comparative genome analysis and experiments showed that a number of diverse putative genetic elements are present in KF715 and are likely involved in genome rearrangement. These data provide insights into the genetic plasticity and adaptability of microorganisms for survival in various ecological niches.

Draft Genome Sequence of a Caffeine-Utilizing Bacterium, Cupriavidus sp. Strain D384.

Cupriavidus sp. D384 was isolated from forest soil in Japan and is known to utilize caffeine as a sole source of carbon and energy. We report here the 6,835,230-bp genome sequence for this strain, which contains 6,116 predicted coding sequences, including gene operon for alkaloid degradation.

A Novel Quorum-Quenching N-Acylhomoserine Lactone Acylase from Acidovorax sp. Strain MR-S7 Mediates Antibiotic Resistance.

N-Acylhomoserine lactone acylase (AHL acylase) is a well-known enzyme responsible for disrupting cell-cell communication (quorum sensing) in bacteria. Here, we isolated and characterized a novel and unique AHL acylase (designated MacQ) from a multidrug-resistant bacterium, Acidovorax sp. strain MR-S7. The purified MacQ protein heterologously expressed in Escherichia coli degraded a wide variety of AHLs, ranging from C6 to C14 side chains with or without 3-oxo substitutions. We also observed that AHL-mediated virulence factor production in a plant pathogen, Pectobacterium carotovorum, was dramatically attenuated by coculture with MacQ-overexpressing Escherichia coli, whereas E. coli with an empty vector was unable to quench the pathogenicity, which strongly indicates that MacQ can act in vivo as a quorum-quenching enzyme and interfere with the quorum-sensing system in the pathogen. In addition, this enzyme was found to be capable of degrading a wide spectrum of ß-lactams (penicillin G, ampicillin, amoxicillin, carbenicillin, cephalexin, and cefadroxil) by deacylation, clearly indicating that MacQ is a bifunctional enzyme that confers both quorum quenching and antibiotic resistance on strain MR-S7. MacQ has relatively low amino acid sequence identity to any of the known acylases (<39%) and has among the broadest substrate range. Our findings provide the possibility that AHL acylase genes can be an alternative source of antibiotic resistance genes posing a threat to human health if they migrate and transfer to pathogenic bacteria.IMPORTANCEN-Acylhomoserine lactones (AHLs) are well-known signal molecules for bacterial cell-cell communication (quorum sensing), and AHL acylase, which is able to degrade AHLs, has been recognized as a major target for quorum-sensing interference (quorum quenching) in pathogens. In this work, we succeeded in isolating a novel AHL acylase (MacQ) from a multidrug-resistant bacterium and demonstrated that the MacQ enzyme could confer multidrug resistance as well as quorum quenching on the host organism. Indeed, the purified MacQ protein was found to be bifunctional and capable of degrading not only various AHL derivatives but also multiple ß-lactam antibiotics by deacylation activities. Although quorum quenching and antibiotic resistance have been recognized to be distinct biological functions, our findings clearly link the two functions by discovering the novel bifunctional enzyme and further providing the possibility that a hitherto-overlooked antibiotic resistance mechanism mediated by the quorum-quenching enzyme may exist in natural environments and perhaps in clinical settings.

A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach.

Volume 31, no. 4, Page 435-441, 2016Page 435 Year of "Published online" is incorrect. It should be corrected as follows:incorrect: 2015correct: 2016 This correction has been completed on the electronic file of the present paper available at J-STAGE and PMC.The editorial board expresses sincere apology for the misprinting. Microbes and Environments editorial board.

Draft Genome Sequence of Caenibacillus caldisaponilyticus B157T, a Thermophilic and Phospholipase-Producing Bacterium Isolated from Acidulocompost.

Caenibacillus caldisaponilyticus B157T (= NBRC 111400T = DSM 101100T), in the family Sporolactobacillaceae, was isolated from acidulocompost as a thermophilic and phospholipid-degrading bacterium. Here, we report the 3.36-Mb draft genome sequence, with a G+C content of 51.8%, to provide the genetic information coding for phospholipases.

Complete Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Pseudomonas putida KF715 (NBRC 110667) Isolated from Biphenyl-Contaminated Soil.

Pseudomonas putida KF715 (NBRC 110667) utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report a complete genome sequence of the KF715 strain, which comprises a circular chromosome and four plasmids. Biphenyl catabolic genes were located on the largest plasmid, pKF715A.

Electrodialytic separation of levulinic acid catalytically synthesized from woody biomass for use in microbial conversion.

Levulinic acid (LA) is produced by the catalytic conversion of a variety of woody biomass. To investigate the potential use of desalting electrodialysis (ED) for LA purification, electrodialytic separation of levulinate from both reagent and cedar-derived LA solution (40-160 g L-1 ) was demonstrated. When using reagent LA solution with pH5.0-6.0, the recovery rates of levulinate ranged from 68 to 99%, and the energy consumption for recovery of 1 kg of levulinate ranged from 0.18 to 0.27 kWh kg-1 . With cedar-derived LA solution (pH6.0), good agreement in levulinate recovery (88-99%), and energy consumption (0.18-0.22 kWh kg-1 ) were observed in comparison to the reagent LA solutions, although a longer operation time was required due to some impurities. The application of desalting ED was favorable for promoting microbial utilization of cedar-derived LA. From 0.5 mol L-1 of the ED-concentrated sodium levulinate solution, 95.6% of levulinate was recovered as LA calcium salt dihydrate by crystallization. This is the first report on ED application for LA recovery using more than 20 g L-1 LA solutions (40-160 g L-1 ). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:448-453, 2017.

A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach.

A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly derived from a bacterium within the phylum Deinococcus-Thermus. Purified BopA exhibited maximum activity at pH 7.5 with bilirubin as its substrate and was found to be active over a markedly broad pH range from 6 to 11. It also showed notable thermostability; its activity remained intact even after a heat treatment at 90°C for 60 min. This enzyme is a thermostable-bilirubin oxidase that exhibits markedly higher thermostability than that previously reported for laccases.

Draft Genome Sequence of Burkholderia stabilis LA20W, a Trehalose Producer That Uses Levulinic Acid as a Substrate.

Burkholderia stabilis LA20W produces trehalose using levulinic acid (LA) as a substrate. Here, we report the 7.97-Mb draft genome sequence of B. stabilis LA20W, which will be useful in investigations of the enzymes involved in LA metabolism and the mechanism of LA-induced trehalose production.

Screening, identification, and characterization of α-xylosidase from a soil metagenome.

A novel α-xylosidase, MeXyl31, was isolated and characterized from a soil metagenomic library. The amino acid sequence of MeXyl31 showed a slight homology with other characterized α-xylosidases. The optimal pH and temperature of recombinant MeXyl31 were pH 5.5 and 45°C, respectively. Recombinant MeXyl31 had a higher α-xylosidase activity toward pNP α-d-xylopyranoside than pNP α-d-glucopyranoside, isoprimeverose, and other xyloglucan oligosaccharides. The kcat/Km value toward pNP α-d-xylopyranoside was about 750-fold higher than that of isoprimeverose. MeXyl31 activity was strongly inactivated in the presence of zinc and copper ions. MeXyl31 is the first α-xylosidase isolated from the metagenome and, relative to other xyloglucan oligosaccharides, shows higher activity toward pNP α-d-xylopyranoside.

Caenibacillus caldisaponilyticus gen. nov., sp. nov., a thermophilic, spore-forming and phospholipid-degrading bacterium isolated from acidulocompost.

A thermophilic and phospholipid-degrading bacterium, designated strain B157T, was isolated from acidulocompost, a garbage compost processed under acidic conditions at moderately high temperature. The organism was Gram-stain-positive, aerobic, spore-forming and rod-shaped. Growth was observed to occur at 40-65 °C and pH 4.8-8.1 (optimum growth: 50-60 °C, pH 6.2). The strain was catalase- and oxidase-positive. The cell wall contained meso-diaminopimelic acid, alanine, glutamic acid and galactose. The predominant respiratory quinone was menaquinone-7 (MK-7) and the major fatty acids were anteiso-C17 : 0 and iso-C17 : 0. Comparative 16S rRNA gene sequence analysis showed that strain B157T was related most closely to Tuberibacillus calidus 607T (94.8 % identity), and the phylogenetic analysis revealed that it belonged to the family Sporolactobacillaceae. The DNA G+C content was determined as 51.8 mol%. In spite of many similarities with the type strains of members of the family Sporolactobacillaceae, genotypic analyses suggest that strain B157T represents a novel species of a new genus, Caenibacilluscaldisaponilyticus gen. nov., sp. nov. The type strain of Caenibacilluscaldisaponilyticus is B157T (=NBRC 111400T=DSM 101100T).