Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

Whole-genome sequencing and analysis of Chryseobacterium arthrosphaerae from Rana nigromaculata.

Chryseobacterium arthrosphaerae strain FS91703 was isolated from Rana nigromaculata in our previous study. To investigate the genomic characteristics, pathogenicity-related genes, antimicrobial resistance, and phylogenetic relationship of this strain, PacBio RS II and Illumina HiSeq 2000 platforms were used for the whole genome sequencing. The genome size of strain FS91703 was 5,435,691 bp and GC content was 37.78%. A total of 4,951 coding genes were predicted; 99 potential virulence factors homologs were identified. Analysis of antibiotic resistance genes revealed that strain FS91703 harbored 10 antibiotic resistance genes in 6 categories and 2 multidrug-resistant efflux pump genes, including adeG and farA. Strain FS91703 was sensitive to ß-lactam combination drugs, cephem, monobactam and carbapenems, intermediately resistant to phenicol, and resistant to penicillin, aminoglycosides, tetracycline, fluoroquinolones, and folate pathway inhibitors. Phylogenetic analysis revealed that strain FS91703 and C. arthrosphaerae CC-VM-7T were on the same branch of the phylogenetic tree based on 16 S rRNA; the ANI value between them was 96.99%; and the DDH values were 80.2, 72.2 and 81.6% by three default calculation formulae. These results suggested that strain FS91703 was a species of C. arthrosphaerae. Pan-genome analysis showed FS91703 had 566 unique genes compared with 13 other C. arthrosphaerae strains, and had a distant phylogenetic relationship with the other C. arthrosphaerae strains of the same branch in phylogenetic tree based on orthologous genes. The results of this study suggest that strain FS91703 is a multidrug-resistant and highly virulent bacterium, that differs from other C. arthrosphaerae strains at the genomic level. The knowledge about the genomic characteristics and antimicrobial resistance of strain FS91703 provides valuable insights into this rare species, as well as guidance for the treatment of the disease caused by FS91703 in Rana nigromaculata.

Chryseobacterium gotjawalense sp. nov. Isolated from Soil in the Volcanic Forest Gotjawal, Jeju Island.

Strain wdc7T, a rod-shaped bacterium, was isolated from soil in the Gotjawal Forest on Jeju Island, South Korea. Strain wdc7T was Gram stain-negative, facultatively anaerobic, catalase- and oxidase positive, yellow pigmented, and non-flagellated. It grew at 4-37 °C and pH 5.0-8.0 in 0-3% (w/v) NaCl. 16S rRNA gene sequencing analysis revealed that strain wdc7T belonged to the genus Chryseobacterium and was most closely related to Chryseobacterium salivictor NBC 122T, with a sequence similarity of 98.51%. Menaquinone 6 was the sole respiratory quinone, and C15:0 anteiso, C15:0 iso, and summed feature 9 were the major fatty acids. The genome length was 3.30 Mbp, with a 37% G + C content. Average amino acid identity, average nucleotide identity, and digital DNA-DNA relatedness between strain wdc7T and C. salivictor NBC 122T were 93.52%, 92.80%, and 49.7%, respectively. Digital genomic and polyphasic analyses showed that strain wdc7T likely represented a new species of the genus Chryseobacterium. We proposed the name Chryseobacterium gotjawalense sp. nov., with wdc7T (= KCTC 92440T = JCM 35602T) as the type strain.

A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish.

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 µM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

First case of Chryseobacterium gallinarum bloodstream infection: a diagnostic and therapeutic challenge for an emerging pathogen.

Chryseobacterium spp. belongs to the Flavobacteriaceae family and is a rod-shaped gram-negative, glucose non-fermenting, non-motile bacterium ubiquitous in the environment. In humans, Chryseobacterium may be responsible for infections such as urinary tract infections (UTI) and ventriculitis with a pathogenic burden increasing in recent years. Chryseobacterium gallinarum was isolated for the first time in 2014 in a pharyngeal scrape sample of chicken and, until now, only one case of human UTI has been described in a pregnant 20-year-old Indian patient. Herein, we report the first case of bloodstream infection caused by C. gallinarum in a 67-year-old female burn patient, correctly identified by 16S-rRNA sequencing and successfully treated with cefepime and fosfomycin.

Chryseobacterium luquanense sp. nov., a casein-hydrolysing bacterium from the Jiaozi Mountain in Yunnan, PR China.

Strain KC 927T was isolated during an investigation of the soil bacteria diversity on Jiaozi Mountain, central Yunnan, Southwest China. The strain was Gram-stain-negative, rod-shaped, non-motile, oxidase-negative, catalase-positive and aerobic. Results of 16S rRNA gene alignment and phylogenetic analysis indicated that strain KC 927T was a member of the genus Chryseobacterium and closely related to Chryseobacterium caseinilyticum GCR10T (98.4%), Chryseobacterium piscicola DSM 21068T (98.3 %) and 'Chryseobacterium formosus' CCTCC AB 2015118T (97.9 %). With a genome size of 4 348 708 bp, strain KC 927T had 33.5 mol% DNA G+C content and contained 4012 protein-coding genes and 77 RNA genes. The average nucleotide identity and digital DNA-DNA hybridization values between strain KC 927T and C. caseinilyticum GCR10T, C. piscicola DSM 21068T and 'C. formosus' CCTCC AB 2015118T were 80.1, 79.6 and 90.7 %, and 25.5, 23.6 and 42.0 %, respectively. The main polar lipid of strain KC 927T was phosphatidylethanolamine and the respiratory quinone was MK-6. The major fatty acids (≥10 %) were iso-C15 : 0, iso-C17 : 1 ω9c and iso-C17 : 0 3-OH. Evidence from phenotypic, phylogenetic and chemotaxonomic analyses support that strain KC 927T represents a new species of the genus Chryseobacterium, for which the name Chryseobacterium luquanense sp. nov. is proposed. The type strain is KC 927T (=CGMCC 1.18760T=JCM 35707T).

Genotypic and phenotypic analysis of Elizabethkingia meningoseptica in bullfrog Rana catesbeiana isolated in Taiwan.

Elizabethkingia meningoseptica is a hazardous bacterium for agriculture production and human health. The present study identified E. meningoseptica from the bullfrog, human and reference strain BCRC 10677 by API 20NE, 50S ribosome protein L27 sequencing and pulse field gel electrophoresis to differentiate isolates of E. meningoseptica from aquatic animals and humans. All isolates from bullfrogs and humans were identified as E. meningoseptica by DNA sequencing with 98.8%-100% sequence identity. E. meningoseptica displayed significant genetic diversity when analysed using pulsed-field gel electrophoresis (PFGE). There were six distinct pulsotypes, including one pulsotype found in bullfrog isolates and five pulsotypes found in human isolates. However, E. meningoseptica from bullfrog exhibited one genotype only by PFGE. Overall, molecular epidemiological analysis of PFGE results indicated that the frog E. meningoseptica outbreaks in Taiwan were produced by genetically identical clones. The bullfrog isolates were not genetically related to other E. meningoseptica from human and reference isolates. This research provided the first comparisons of biochemical characteristics and genetic differences of E. meningoseptica from human and bullfrog isolates.

Novel Antibiotic Resistance Genes Identified by Functional Gene Library Screening in Stenotrophomonas maltophilia and Chryseobacterium spp. Bacteria of Soil Origin.

As one of the most diverse habitats of microorganisms, soil has been recognised as a reservoir of both antibiotics and the antibiotic resistance genes (ARGs). Bacteria naturally inhabiting soil or water often possess innate ARGs to counteract the chemical compounds produced by competitors living in the same environment. When such bacteria are able to cause infections in immunocompromised patients, their strong innate antibiotic resistance mechanisms make treatment difficult. We generated functional gene libraries using antibiotic-resistant Stenotrophomonas maltophilia and Chryseobacterium spp. bacteria isolated from agricultural soils in Lithuania to select for the genetic determinants responsible for their resistance. We were able to find novel variants of aminoglycoside and ß-lactam resistance genes, with ß-lactamases isolated from the Chryseobacterium spp. functional gene library, one of which is a variant of IND-like metallo-ß-lactamase (MBL) IND-17 and the other of which is a previously uncharacterised MBL we named CHM (Chryseobacterium metallo ß-lactamase). Our results indicate that soil microorganisms possess a diversity of ARG variants, which could potentially be transferred to the clinical setting.

Chryseobacterium paridis sp. nov., an endophytic bacterial species isolated from the root of Paris polyphylla Smith var. yunnanensis.

A Gram-negative, yellow-pigmented, rod-shaped bacterial strain YIM B02567T was isolated from the root of Paris polyphylla Smith var. yunnanensis in China. Strain YIM B02567T grew optimally at 25-30 °C and at pH 7.0 in the absence of NaCl on nutrient agar. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain YIM B02567T belong to the genus Chryseobacterium, and was closely related to Chryseobacterium piperi CTMT and Chryseobacterium soli DSM 19298T. Whole genome sequencing indicated that the genome size was 4,774,612 bp and with a G + C content of 34.5 mol%. Values of the ANI and the dDDH between strain YIM B02567T and its closely related Chryseobacterium species were below 81.72% and 24.7%. Strain YIM B02567T contained menaquinone-6 as the sole isoprenoid quinone, anteiso-C15:0, iso-C17:1 ω9c and iso-C17:0 3-OH as major fatty acids and phosphatidylethanolamine as major polar lipid. Based on the polyphasic analyses, strain YIM B02567T could be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium. The isolate, therefore, represents a novel species, for which the name Chryseobacterium paridis sp. nov. is proposed. The type strain is YIM B02567T (= CGMCC 1.18657T).

Chryseobacterium cheonjiense sp. nov., isolated from forest soil.

A yellow-pigmented, non-motile and rod-shaped bacterium, designated RJ-7-14T was obtained from forest soil sampled at Cheonji-dong, Seogwipo-si, Jeju-do, South Korea. Cells were Gram-stain-negative and produced flexirubin type pigments. A phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain RJ-7-14T formed a lineage within the family Weeksellaceae and clustered as members of the genus Chryseobacterium. The closest members were Chryseobacterium geocarposphaerae DSM 27617T (98.2% sequence similarity), Chryseobacterium hispalense DSM 25574T (98.0%) and Chryseobacterium nepalense KACC 18907T (98.0%). The sequence similarity for other members was < 98.0%. The genome was 4,276,416 bp long with 9 scaffolds and 3779 protein-coding genes. The sole respiratory quinone was MK-6. The major cellular fatty acids were iso-C15:0, summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl), summed feature 3 (iso-C15:0 2-OH and/or C16: 1ω7c) and iso-C17:0 3-OH. The major polar lipid was phosphatidylethanolamine (PE). The DNA G + C content of the type strain was 37.2 mol%. In addition, the average nucleotide identity (ANIu) and in silico DNA-DNA hybridization (dDDH) relatedness values between strain RJ-7-14T and phylogenetically closest members were ≤ 88.2% and ≤ 35.0%, respectively, which were below the threshold values of 95-96% (for ANI) and 70% (for dDDH), suggesting the allocation of novel strain to a new species. Based on genomic, chemotaxonomic, phenotypic and phylogenetic analyses, strain RJ-7-14T represents novel species in the genus Chryseobacterium, for which the name Chryseobacterium cheonjiense sp. nov. is proposed. The type strain is RJ-7-14T (= KACC 21625T = NBRC 114362T).

Heterologous expression and characterisation of a keratinase produced by Chryseobacterium carnipullorum.

Chryseobacterium carnipullorum 9_R23581T, isolated from raw chicken meat, was evaluated for its potential to degrade keratin found in feathers. The focus of this study was to heterologously express and characterise a keratinolytic enzyme produced by C. carnipullorum. Chryseobacterium carnipullorum secretes proteolytic enzymes that have feather degrading capabilities during its exponential growth phase. This study concluded that the most likely main component of the keratinolytic enzymes of C. carnipullorum was peptidase M64, a serine-endopeptidase with a molecular weight in crude form of 49.46 kDa. Primers were designed on the selected gene of interest, which was amplified from the genome of C. carnipullorum (accession number NZ-FRCD01000002.1). The gene coding for peptidase M64 was further cloned, propagated and expressed in E. coli BL21 [DE3] cells. Purification was by Immobilised Metal Affinity Chromatography (IMAC). The molecular weight of the keratinase was about 50 kDa after purification while its optimum temperature and pH were 50 °C and 8.5, respectively. The activity of this keratinase was inhibited by phenylmethylsulfonyl fluoride (PMSF) and it was enhanced by the presence of divalent metal ions such as Mg2+ and Ca2+. Enzyme activity was further assayed by application to chicken feathers and observed degradation was an indication of keratinolytic potential.

Chryseobacterium potabilaquae sp. nov., Chryseobacterium aquaeductus sp. nov. and Chryseobacterium fistulae sp. nov., from drinking water systems.

A polyphasic taxonomic study was conducted on three strains isolated from drinking water systems that had previously been deposited as Chryseobacterium species at the Spanish Type Culture Collection in order to complete their classification. Strains CECT 9293T, CECT 9390T and CECT 9393T were isolated from sites in Barcelona, Spain, in the framework of a project aimed at generating the first MALDI-TOF database specific for bacteria present in water for human consumption. Their partial 16S rRNA sequences showed that their closest relatives among the type strains of Chryseobacterium exhibited 98 % similarity or less, supporting their taxonomic novelty. At the same time, comparison between them revealed that strains CECT 9293T and CECT 9393T could perhaps be related at the species level as they shared 99.5 % similarity. However, whole genome sequencing was performed and the subsequent calculation of relatedness indices, average nucleotide identity and estimated DNA-DNA hybridization, ruled out that possibility and confirmed instead that each of the strains should be considered a separate species in the genus Chryseobacterium. Having clarified their status, we also performed phylogenomic analyses and searched for possible environmental or non-type material sequences that could be related to any of them at the species level. In parallel, the strains were characterized phenotypically and compared to their closest relatives to determine diagnostic traits to support their formal proposal. The proposed species are Chryseobacterium potabilaquae sp. nov. with the type strain CECT 9293T (=LMG 32084T), Chryseobacterium aquaeductus sp. nov. with the type strain CECT 9390T (=LMG 32085T) and Chryseobacterium fistulae sp. nov. with the type strain CECT 9393T (=LMG 32086T).

Chryseobacterium salivictor sp. nov., a plant-growth-promoting bacterium isolated from freshwater.

A Gram-stain-negative, non-motile, rod-shaped bacterial strain, designated NBC 122T, was isolated from freshwater of the Nakdong River Republic of Korea. Growth occurred at pH 5.0-8.0 (optimum, pH 7.0), at 4-37 °C (optimum, 25 °C), and with 0-3.5% (w/v) NaCl (optimum, 1%). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain NBC 122T belongs to the genus Chryseobacterium and is most closely related to Chryseobacterium antarcticum AT1013T (97.85%). The average nucleotide identity and in silico DNA-DNA hybridization (DDH) values between strain NBC 122T and related Chryseobacterium species were 77.77-80.28 and 20.9-23.2%, respectively. The strain NBC 122T contained MK-6 as the major respiratory quinone. The major fatty acids included anteiso C15:0, iso C15:0, summed feature 9 (C17:1 iso ω9c, C16:0 10-methyl) and iso C17:0 3-OH, and the polar lipids were phosphatidylethanolamine, five unidentified aminolipids and six unidentified lipids. The DNA G + C content was 37.5 mol%. On the basis of a 16S rRNA gene phylogeny and comparative phenotypic analyses, strain NBC 122T represents a novel species in the genus Chryseobacterium, for which the name Chryseobacterium salivictor sp. nov. is proposed. The type strain is NBC122T (= KCTC 72248T = JCM 33980T).

Genomic and functional insights into the adaptation and survival of Chryseobacterium sp. strain PMSZPI in uranium enriched environment.

Metal enriched areas represent important and dynamic microbiological ecosystems. In this study, the draft genome of a uranium (U) tolerant bacterium, Chryseobacterium sp. strain PMSZPI, isolated from the subsurface soil of Domiasiat uranium ore deposit in Northeast India, was analyzed. The strain revealed a genome size of 3.8 Mb comprising of 3346 predicted protein-coding genes. The analysis indicated high abundance of genes associated with metal resistance and efflux, transporters, phosphatases, antibiotic resistance, polysaccharide synthesis, motility, protein secretion systems, oxidoreductases and DNA repair. Comparative genomics with other closely related Chryseobacterium strains led to the identification of unique inventory of genes which were of adaptive significance in PMSZPI. Consistent with the genome analysis, PMSZPI showed superior tolerance to uranium and other heavy metals. The metal exposed cells exhibited transcriptional induction of metal translocating PIB ATPases suggestive of their involvement in metal resistance. Efficient U binding (~90% of 100 µM U) and U bioprecipitation (~93-94% of 1 mM U at pH 5, 7 and 9) could be attributed as uranium tolerance strategies in PMSZPI. The strain demonstrated resistance to a large number of antibiotics which was in agreement with in silico prediction. Reduced gliding motility in the presence of cadmium and uranium, enhanced biofilm formation on uranium exposure and tolerance to 1.5 kGy of 60Co gamma radiation were perceived as adaptive responses in PMSZPI. Overall, the positive correlation observed between uranium/metal tolerance abilities predicted using genome analysis and the functional characterization reinforced the multifaceted adaptation strategies employed by PMSZPI for its survival in the soil of uranium ore deposit comprising of high concentrations of uranium and other heavy metals.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Differences in Clinical Manifestations, Antimicrobial Susceptibility Patterns, and Mutations of Fluoroquinolone Target Genes between Chryseobacterium gleum and Chryseobacterium indologenes.

Chryseobacterium infections are uncommon, and previous studies have revealed that Chryseobacterium gleum is frequently misidentified as Chryseobacterium indologenes We aimed to explore the differences in clinical manifestations and antimicrobial susceptibility patterns between C. gleum and C. indologenes The database of a clinical microbiology laboratory was searched to identify patients with Chryseobacterium infections between 2005 and 2017. Species were reidentified using 16S rRNA gene sequencing, and patients with C. gleum and C. indologenes infections were included in the study. A total of 42 C. gleum and 84 C. indologenes isolates were collected from consecutive patients. A significant increase in C. indologenes incidence was observed. C. gleum was significantly more associated with bacteremia than C. indologenes Patients with C. gleum infections had more comorbidities of malignancy and liver cirrhosis than those with C. indologenes infections. The overall case fatality rate was 19.8%. Independent risk factors for mortality were female sex and C. indologenes infection. These isolates were most susceptible to minocycline (73%), followed by trimethoprim-sulfamethoxazole (47.6%), tigecycline (34.1%), and levofloxacin (32.5%). C. gleum exhibited a significantly higher rate of susceptibility than C. indologenes to piperacillin, piperacillin-tazobactam, ceftazidime, tigecycline, and levofloxacin. Alterations in DNA gyrase subunit A were identified to be associated with fluoroquinolone resistance in C. indologenes No nonsynonymous substitutions were observed in the quinolone resistance-determining regions (QRDRs) of C. gleum Differences in epidemiology, clinical manifestations, and antimicrobial susceptibility patterns exist between C. gleum and C. indologenes Additional investigations are needed to explore the significance of these differences.

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

Draft genome and description of Chryseobacterium phocaeense sp. nov.: a new bacterial species isolated from the sputum of a cystic fibrosis patient.

Strain 6021061333T was isolated from the sputum of 16-year-old girl with cystic fibrosis following a pulmonary exacerbation. This bacterial strain could not be identified by our systematic MALDI-TOF mass spectrometry screening on a MicroFlex. This led to the sequencing of the 16S rRNA gene, which shows 97.83% sequence identity with Chryseobacterium kwangjuense strain KJ1R5T, the phylogenetic closely related type strain of a species with standing in nomenclature, which putatively classifies it as a new species. Colonies are yellow, circular and 0.5-1 mm in diameter after cultivation at 28 °C for 24 h on 5% sheep blood-enriched Colombia agar. Growth occurs at temperatures in the range of 28-37 °C (optimally at 28 °C). Strain 6021061333T is Gram-negative, non-motile and strictly aerobic bacillus. It is catalase and oxidase positive. The 4,864,678 bp-long genome, composed of five contigs, has a G+C content of 38.86%. Out of the 4427 predicted genes, 4342 were protein-coding genes and 85 were RNAs. The major fatty acids are branched (13-methyl-tetradecanoic acid and 15-methyl-hexadecenoic acid). Digital DNA-DNA hybridization (dDDH) estimation and average nucleotide identity (ANI) of the strain 6021061333T against genomes of the type strains of related species ranged between 23.60 and 50.40% and between 79.31 and 93.06%, respectively. According to our taxonogenomics results, we propose the creation of Chryseobacterium phocaeense sp. nov. that contains the type strain 6021061333T (= CSUR P2660, = CECT 9670).

Isolation and Characterization of Flavobacteriaceae from Farmed and Wild Nile Tilapia in Tanzania.

The present study was conducted to explore the occurrence of Flavobacteriaceae in wild Nile Tilapia Oreochromis niloticus (n = 108) collected from Lake Victoria and farmed Nile Tilapia (n = 187) collected from 12 ponds in the Morogoro region of Tanzania. The size of the ponds surveyed ranged from 130 to 150 m2 . Pond parameters and fish morphometric data were recorded during sampling. In total, 67 Flavobacterium-like isolates (n = 44 from farmed fish; n = 23 from wild fish) were identified on the basis of colony morphology and biochemical tests. Sequences from the 16S ribosomal RNA (rRNA) gene revealed that all 67 isolates belonged to the genera Flavobacterium and Chryseobacterium. Based on 16S rRNA nucleotide identity, 26 isolates showed high similarity with C. indologenes (99-100% identity), 16 showed similarity to C. joostei (98-99.9%), and 17 were similar to diverse species of Chryseobacterium (97-99%). Three isolates were similar to F. aquatile and three were similar to F. indicum, with 99-100% nucleotide identity in both cases, and two isolates were similar to F. oryzae (99-100% identity). The findings obtained in this study provide a baseline for future studies and contribute to an understanding of the threats presented by the aquatic Flavobacteriaceae reservoir toward the development of healthy fish farming in Tanzania. Such knowledge is vital for the development of a sustainable aquaculture industry in Tanzania that will contribute to increased food security.

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.

Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. We mutated distinguishing active-site residues to generate enzymes that had a common Zn2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of these pruned enzymes with a series of substrates. A substantial rate enhancement of ∼1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 107-108-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.