Type IV pili trigger episymbiotic association of Saccharibacteria with its bacterial host.

Recent characterization of the obligate episymbiont Saccharibacteria (TM7) belonging to the candidate phyla radiation (CPR) has expanded the extent of microbial diversity. However, the episymbiotic lifestyle of TM7 is still underexploited due to the deficiency of cultivated representatives. Here, we describe gene-targeted TM7 cultivation guided by repurposing epicPCR (emulsion, paired isolation, and concatenation PCR) to capture in situ TM7‒host associations. Using this method, we obtained a novel Saccharibacteria isolate TM7i and its host Leucobacter aridicollis J1 from Cicadae Periostracum, the castoff shell of cicada. Genomic analyses and microscopic characterizations revealed that TM7i could bind to J1 through twitching-like motility mediated by type IV pili (T4P). We further showed that the inhibition of T4P extrusion suppressed the motility and host adherence of TM7i, resulting in its reduced growth. However, the inactivation of T4P had little effect on the growth of TM7i that had already adhered to J1, suggesting the essential role of T4P in host recognition by TM7i. By capturing CPR‒host association and elaborating the T4P-dependent episymbiotic association mechanism, our studies shed light on the distinct yet widespread lifestyle of CPR bacteria.

[Element relationship and extension path of clinical evidence knowledge map with Chinese patent medicine].

This study aims to construct the element relationship and extension path of clinical evidence knowledge map with Chinese patent medicine, providing basic technical support for the formation and transformation of the evidence chain of Chinese patent medicine and providing collection, induction, and summary schemes for massive and disorganized clinical data. Based on the elements of evidence-based PICOS, the conventional construction methods of knowledge graph were collected and summarized. Firstly, the data entities related to Chinese patent medicine were classified, and entity linking was performed(disambiguation). Secondly, the study associated and classified the attribute information of the data entity. Finally, the logical relationship between entities was constructed, and then the element relationship and extension path of the knowledge map conforming to the characteristics of clinical evidence of Chinese patent medicine were summarized. The construction of the clinical evidence knowledge map of Chinese patent medicine was mainly based on process design and logical structure, and the element relationship of the knowledge map was expressed according to the PICOS principle and evidence level. The extension path crossed three levels(model layer, data layer application, and new evidence application), and the study gradually explored the path from disease, core evaluation indicators, Chinese patent medicine, core prescriptions, syndrome and treatment rules, and medical case comparison(evolution law) to new drug research and development. In this study, the top-level design of the construction of the clinical evidence knowledge map of Chinese patent medicine has been clarified, but it still needs the joint efforts of interdisciplinary disciplines. With the continuous improvement of the map construction technology in line with the characteristics of TCM, the study can provide necessary basic technical support and reference for the development of the TCM discipline.

Response of Microbial Communities on Cathode with Different Potentials in a Single-Chamber Reactor.

Microbes use both organic and inorganic compounds as electron donors, with different electronic potentials, to produce energy required for growth in environments. Conventional studies on the effects of different electron donors on microbial community has been extensively studied with a set cathode potential. However, it remains under-researched how a microbial community response to the different redox potentials in different environments. Here, we incubated a lake sediment in a single-chamber reactor equipped with three working electrodes, i.e., with potentials of - 0.29 V, - 0.05 V versus standard hydrogen electrode and open-circuit, respectively. Results reveal that the structure of bacterial communities was highly similar for all closed-circuit electrodes (- 0.29 V, - 0.05 V), while differing significantly from those on open-circuit electrodes. We also show that specific bacteria were preferentially enriched by different electrode potentials, i.e., Pseudomonas and Rhodobacter preferentially grew on - 0.05 V and - 0.29 V cathode potentials, Azospirillum and Bosea preferentially grew on - 0.05 V; while Ferrovibrio, Hydrogenophaga, Delftia, and Sphingobium preferentially grew on - 0.29 V. In addition, microorganisms selectively enriched on open-circuit electrodes possess higher connectivity and closer relationship than microorganisms selectively enriched on closed-circuit electrode.

Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump.

Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from a Dietzia sp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes.

MiR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of airway smooth muscle cells by regulating the PTEN/Akt pathway.

BACKGROUND: Recent studies have shown that the up-regulation of microRNA miR-328-3p expression increases seasonal allergy and asthma symptoms in children, but the specific mechanism remains unclear. Therefore, the aim of this study was to explore the role and mechanism of -miR-328-3p in transforming growth factor (TGF)-ß1-induced airway smooth muscle cells (ASMCs). METHODS: The effect of TGF-ß1 on the expression of miR-328-3p in ASMCs was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cells proliferation, migration, and inflammatory factors in TGF-ß1-induced ASMCs were measured by cell counting kit-8 (CCK-8), transwell, and enzyme-linked immunosorbent assay (ELISA), respectively. Besides, TargetScan was used to predict phosphatase and tensin homolog (PTEN), the downstream target of miR-328-3p; double-luciferase reporter assay, western blot, and qRT-PCR were used to verify the targeting relationship between miR-328-3p and PTEN; western blot was also used to examine the effects of PTEN and miR-328-3p knockdown on the expression levels of PTEN, Akt, and p-Akt proteins. RESULTS: The expression of miR-328-3p was up-regulated in TGF-ß1-induced ASMCs. Knockdown of miR-328-3p significantly inhibited proliferation, migration, and inflammation of ASMCs induced by TGF-ß1 and decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. The dual--luciferase reporter assay results confirmed that PTEN was a target gene of miR-328-3p. Moreover, inhibition of PTEN expression reversed the inhibitory effect of low miR-328-3p expression on -TGF-ß1-induced ASMC's proliferation, migration, and inflammation. In comparison to the knockdown of miR-328-3p alone, the simultaneous knockdown of miR-328-3p with PTEN decreased PTEN protein expression levels and increased p-Akt/Akt ratio in TGF-ß1-induced ASMCs. CONCLUSION: Through regulating the expression of PTEN and the activity of Akt signaling pathway, miR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of ASMCs.

Comparative transcriptome analysis reveals different adaptation mechanisms for degradation of very long-chain and normal long-chain alkanes in Dietzia sp. DQ12-45-1b.

Hydrocarbon-degrading bacteria typically metabolize a broad range of alkane substrates, but global metabolic characteristics of strains growing on alkane substrates in different chain lengths remain unclear. In this study, we analysed the transcriptional profiles of a hydrocarbon degrading bacterium, Dietzia sp. DQ12-45-1b, during growth on octacosane (C28), hexadecane (C16) and glucose as the sole carbon sources. Our results highlight that C16 and C28 induced common genes of core alkane degradation pathways in DQ12-45-1b, whereas transcriptional patterns of genes related to lipid metabolism, energy metabolism, biomass synthesis, and metal ion transportation were distinct. In addition, the transcriptional differences of genes related to glyoxylate shunt (GS) as well as growth phenotypes of mutant strain with defects in GS demonstrated that GS is essential for C16 degradation, though it is dispensable for C28 degradation in DQ12-45-1b. These results demonstrate that DQ12-45-1b cells exhibited considerable metabolic flexibility by using various mechanisms during growth on alkane substrates in different chain lengths. This study advances our knowledge of microbial hydrocarbon degradation and provides valuable information for the application of alkane-degrading bacteria in bioremediation and microbial enhanced oil recovery.

The Roles of the Two-Component System, MtrAB, in Response to Diverse Cell Envelope Stresses in Dietzia sp. DQ12-45-1b.

Two-component systems (TCSs) act as common regulatory systems allowing bacteria to detect and respond to multiple environmental stimuli, including cell envelope stress. The MtrAB TCS of Actinobacteria is critical for cell wall homeostasis, cell proliferation, osmoprotection, and antibiotic resistance, and thus is found to be highly conserved across this phylum. However, how precisely the MtrAB TCS regulates cellular homeostasis in response to environmental stress remains unclear. Here, we show that the MtrAB TCS plays an important role in the tolerance to different types of cell envelope stresses, including environmental stresses (i.e., oxidative stress, lysozyme, SDS, osmotic pressure, and alkaline pH stresses) and envelope-targeting antibiotics (i.e., isoniazid, ethambutol, glycopeptide, and ß-lactam antibiotics) in Dietzia sp. DQ12-45-1b. An mtrAB mutant strain exhibited slower growth compared to the wild-type strain and was characterized by abnormal cell shapes when exposed to various environmental stresses. Moreover, deletion of mtrAB resulted in decreased resistance to isoniazid, ethambutol, and ß-lactam antibiotics. Further, Cleavage under targets and tagmentation sequencing (CUT&Tag-seq) and electrophoretic mobility shift assays (EMSAs) revealed that MtrA binds the promoters of genes involved in peptidoglycan biosynthesis (ldtB, ldtA, murJ), hydrolysis (GJR88_03483, GJR88_4713), and cell division (ftsE). Together, our findings demonstrated that the MtrAB TCS is essential for the survival of Dietzia sp. DQ12-45-1b under various cell envelope stresses, primarily by controlling multiple downstream cellular pathways. Our work suggests that TCSs act as global sensors and regulators in maintaining cellular homeostasis, such as during episodes of various environmental stresses. The present study should shed light on the understanding of mechanisms for bacterial adaptivity to extreme environments. IMPORTANCE The multilayered cell envelope is the first line of bacterial defense against various extreme environments. Bacteria utilize a large number of sensing and regulatory systems to maintain cell envelope homeostasis under multiple stress conditions. The two-component system (TCS) is the main sensing and responding apparatus for environmental adaptation. The MtrAB TCS highly conserved in Actinobacteria is critical for cell wall homeostasis, cell proliferation, osmoprotection, and antibiotic resistance. However, how MtrAB works with regard to signals impacting changes to the cell envelope is not fully understood. Here, we found that in the Actinobacterium Dietzia sp. DQ12-45-1b, a TCS named MtrAB is pivotal for ensuring normal cell growth as well as maintaining proper cell morphology in response to various cell envelope stresses, namely, by regulating the expression of cell envelope-related genes. Our findings should greatly advance our understanding of the adaptive mechanisms responsible for maintaining cell integrity in times of sustained environmental shocks.

[Clinical evidence analysis report of Chinese patent medicine in 2020].

The present study collected, collated, analyzed, and evaluated randomized controlled trial(RCT) of Chinese patent medicine published in Chinese and English journals in 2020, and summarized clinical evidence of Chinese patent medicine in stages, providing references for follow-up clinical research and evidence transformation and application. On the basis of the collection in the Traditional Chinese Medicine(TCM) Clinical Evidence Database System(EVDS), CNKI, Wanfang, SinoMed, Cochrane Library, PubMed, and EMbase were searched for RCTs of Chinese patent medicine published in 2020, and their research characteristics and methodological quality were analyzed and evaluated. A total of 1 285 research papers on Chinese patent medicine(1 257 in Chinese/28 in English) were included, involving 146 054 patients and 639 Chinese patent medicines, including 526 oral drugs, 68 injections, and 45 external drugs. A total of 412 diseases in 23 types were involved, which were dominated by circulatory system diseases and respiratory system diseases, specifically, cerebral infarction and angina pectoris. The sample size ranged from 20 cases to 2 673 cases, and 57.67% of RCTs had samples sizes less than 100. Single-center trials were the main ones, and multi-center trials only accounted for 4.75%(n=61). In terms of methodological quality, 52.91% of the RCTs had unclear descriptions or incorrect application of randomization methods, and the implementation of allocation concealment and blinding methods has not been paid much attention. In conclusion, compared with the conditions in 2019, the number of RCTs published in 2020 has decreased, and the research interest in respiratory diseases has increased, while the quality control in the process of research design and implementation has not been improved. Therefore, it is necessary to strengthen the methodological training of researchers and promote the output of high-quality research evidence.

[Clinical trials and evaluation of Chinese patent medicine for heart failure].

The present study systematically collected, analyzed, and evaluated randomized controlled trial(RCT) of Chinese patent medicine in the treatment of heart failure to provide references for follow-up clinical research design, guideline update, and policy formulation, and promote the improvement of clinical evidence quality. On the basis of the collection in the Traditional Chinese Medicine(TCM) Clinical Evidence Database System(EVDS), CNKI, VIP, Wanfang, SinoMed, PubMed, and Web of Science were searched for RCTs of Chinese patent medicine in the treatment of heart failure from database inception to December 31, 2020. The di-sease type, publication time, sample size, intervention/control setting, course of treatment, evaluation indexes, and methodological quality were analyzed and evaluated. A total of 1 631 RCTs were included, including 1 622 in Chinese and 9 in English. It was first published in 1995, with the largest number of publications in 2016. There were only 56 RCTs(3.43%) with a sample size≥200. Seventy-eight types of Chinese patent medicines were involved, including 49 types of oral drugs and 29 types of injections. There were 34 intervention/control protocols, which were dominated by Chinese patent medicine+conventional treatment vs conventional treatment, accounting for 28.51%(n=465). About 94.0% of RCTs reported the course of treatment, mainly 14-56 days. The evaluation indexes were mainly physical and chemical tests and symptoms/signs, and left ventricular ejection fraction(LVEF) was the most frequently used measurement index. In enumeration indexes, clinical efficacy(response rate) was used the most frequently. Methodologically, 92.0% of the research subjects were rated as high risk of blindness. There were only 13 RCTs(0.80%) reporting registered information. It is necessary to further standardize the design, implementation, and quality control of clinical studies in order to improve the quality of evidence and avoid research waste.

Membrane vesicles from a Dietzia bacterium containing multiple cargoes and their roles in iron delivery.

Membrane vesicles (MVs) released from bacteria act as extracellular vehicles carrying various functional cargoes between cells. MVs with different cargoes play multiple roles in stress adaptation, nutrient acquisition and microbial interactions. However, previous studies have primarily focused on MVs from Gram-negative bacteria, while the characteristics of cargoes in MVs from Gram-positive bacteria and their involvement in microbial interactions remain to be elucidated. Here, we used a Gram-positive strain, Dietzia sp. DQ12-45-1b from Corynebacteriales, to analyse the characteristics and functions of MVs. We identified the 'antioxidant' canthaxanthin is stored within MVs by LC-MS/MS. In addition, nearly the entire genomic content of strain DQ12-45-1b are evenly distributed in MVs, suggesting that MVs from DQ12-45-1b might involve in horizontal gene transfer. Finally, the mycobactin-type siderophores were detected in MVs. The iron-loaded MVs effectively mediate iron binding and delivery to homologous bacteria from the order Corynebacteriales, but not to more distantly related species from the orders Pseudomonadales, Bacillales and Enterobacterales. These results revealed that the iron-loaded MVs are shared between homologous species. Together, we report the Gram-positive bacterium Dietzia sp. DQ12-45-1b released MVs that contain canthaxanthin, DNA and siderophores and prove that MVs act as public goods between closely related species.

Pan-genomic analysis reveals that the evolution of Dietzia species depends on their living habitats.

The bacterial genus Dietzia is widely distributed in various environments. The genomes of 26 diverse strains of Dietzia, including almost all the type strains, were analysed in this study. This analysis revealed a lipid metabolism gene richness, which could explain the ability of Dietzia to live in oil related environments. The pan-genome consists of 83,976 genes assigned into 10,327 gene families, 792 of which are shared by all the genomes of Dietzia. Mathematical extrapolation of the data suggests that the Dietzia pan-genome is open. Both gene duplication and gene loss contributed to the open pan-genome, while horizontal gene transfer was limited. Dietzia strains primarily gained their diverse metabolic capacity through more ancient gene duplications. Phylogenetic analysis of Dietzia isolated from aquatic and terrestrial environments showed two distinct clades from the same ancestor. The genome sizes of Dietzia strains from aquatic environments were significantly larger than those from terrestrial environments, which was mainly due to the occurrence of more gene loss events during the evolutionary progress of the strains from terrestrial environments. The evolutionary history of Dietzia was tightly coupled to environmental conditions, and iron concentrations should be one of the key factors shaping the genomes of the Dietzia lineages.

Isolation and characterization of the novel Pseudomonas stutzeri bacteriophage 8P.

Bacteriophage 8P was isolated with a Pseudomonas stutzeri strain isolated from an oil reservoir as its host bacterium. The phage genome comprises 63,753 base pairs with a G+C content of 64.35. The phage encodes 63 predicted proteins, and 27 of them were functionally assigned. No tRNA genes were found. Comparative genomics analysis showed that 8P displayed some relatedness to F116-like phages (78% identity, 20% query coverage). The genome has very low sequence similarity to the other phage genomes in the GenBank database and Viral Sequence Database. Based on whole-genome analysis and transmission electron microscopy imaging, 8P is proposed to be a member of a new species in the genus Hollowayvirus, family Podoviridae.

A novel temperate phage, vB_PstS-pAN, induced from the naphthalene-degrading bacterium Pseudomonas stutzeri AN10.

A novel temperate phage named vB_PstS-pAN was induced by mitomycin C treatment from the naphthalene-degrading bacterium Pseudomonas stutzeri AN10. The phage particles have icosahedral heads and long non-contractile tails, and vB_PstS-pAN can therefore be morphologically classified as a member of the family Siphoviridae. The whole genome of vB_PstS-pAN is 39,466 bp in length, with an 11-nt 3' overhang cohesive end. There are 53 genes in the vB_PstS-pAN genome, including genes responsible for phage integration, replication, morphogenesis, and bacterial lysis. The vB_PstS-pAN genome has low similarity to other phage genomes in the GenBank database, suggesting that vB_PstS-pAN is a novel member of the family Siphoviridae.

The Impacts of the Occupants on the Bacterial Communities of Classrooms.

In modern societies, people spend most of their time in the built environment which harbors unique microbial assemblages with the potential to influence human health. However, how the occupants of buildings influence indoor microbial communities remains under-researched. Here, we investigated the diversities of the bacterial communities of a typical Chinese middle school to demonstrate the effects of occupant activities on bacterial communities inside classrooms. The results showed that samples taken from classrooms exhibited higher microbial diversity compared to samples collected from public areas such as gym and restaurant, suggesting the occupant activities could increase the diversities of the indoor microbial communities. Moreover, we also found that the duration of occupation strongly influence the presence/absence of phylogenetic lineages of the bacterial communities, the type of occupants, on the other hand, affect the relative abundances of bacterial taxa. In addition, samples taken from classrooms with longer occupation time exhibited a better fit to the Sloan Neutral Community Model for Prokaryotes, suggesting that room occupation influences the assembly process of microbial communities. In conclusion, our study demonstrates that the duration of occupation and the type of occupants influence the microbiome of the built environment.

Isolation Chip Increases Culturable Bacterial Diversity and Reduces Cultivation Bias.

Conventional cultivation methods, including petri dish plating, are selective and biased to enrich specific microorganisms, such as big population and fast-growing bacteria. In this study, we evaluated the ability of isolation chip (ichip) to reduce cultivation bias. We used the ichip and petri dish plating methods to cultivate bacteria from soil contaminated with (contaminated soil) or without (natural soil) crude oil. Ichip improved the richness and evenness of bacterial isolates in both the natural and contaminated soil samples. Using the petri dish plating method, Pseudomonas and Lysinibacillus isolates were found to be the most abundant, with over 50% of the relative abundance in the natural and oil-polluted soil-cultured communities, respectively. In comparison, using the ichip method, the isolates with the highest relative abundances were from Bacillus and Aeromonas in natural and contaminated soil-cultured communities, which only accounted for 20% and 28% of the total isolates, respectively. Interestingly, the evenness and richness of the bacteria varied slightly between the natural and oil-polluted soil samples, indicating that ichip had the ability to reduce the cultivation bias. In addition, oil selective pressure enriched the functional bacteria isolated using the petri dish plating method. In summary, ichip allows bacteria to grow evenly, as well as allowing for substance exchange between the environment and single cells. As such, it is a very good method for increasing culturable bacterial diversity and reducing cultivation bias.

Metabolic Exchange with Non-Alkane-Consuming Pseudomonas stutzeri SLG510A3-8 Improves n-Alkane Biodegradation by the Alkane Degrader Dietzia sp. Strain DQ12-45-1b.

Biodegradation of alkanes by microbial communities is ubiquitous in nature. Interestingly, the microbial communities with high hydrocarbon-degrading performances are sometimes composed of not only hydrocarbon degraders but also nonconsumers, but the synergistic mechanisms remain unknown. Here, we found that two bacterial strains isolated from Chinese oil fields, Dietzia sp. strain DQ12-45-1b and Pseudomonas stutzeri SLG510A3-8, had a synergistic effect on hexadecane (C16 compound) biodegradation, even though P. stutzeri could not utilize C16 individually. To gain a better understanding of the roles of the alkane nonconsumer P. stutzeri in the C16-degrading consortium, we reconstructed a two-species stoichiometric metabolic model, iBH1908, and integrated in silico prediction with the following in vitro validation, a comparative proteomics analysis, and extracellular metabolomic detection. Metabolic interactions between P. stutzeri and Dietzia sp. were successfully revealed to have importance in efficient C16 degradation. In the process, P. stutzeri survived on C16 metabolic intermediates from Dietzia sp., including hexadecanoate, 3-hydroxybutanoate, and α-ketoglutarate. In return, P. stutzeri reorganized its metabolic flux distribution to fed back acetate and glutamate to Dietzia sp. to enhance its C16 degradation efficiency by improving Dietzia cell accumulation and by regulating the expression of Dietzia succinate dehydrogenase. By using the synergistic microbial consortium of Dietzia sp. and P. stutzeri with the addition of the in silico-predicted key exchanged metabolites, diesel oil was effectively disposed of in 15 days with a removal fraction of 85.54% ± 6.42%, leaving small amounts of C15 to C20 isomers. Our finding provides a novel microbial assembling mode for efficient bioremediation or chemical production in the future.IMPORTANCE Many natural and synthetic microbial communities are composed of not only species whose biological properties are consistent with their corresponding communities but also ones whose chemophysical characteristics do not directly contribute to the performance of their communities. Even though the latter species are often essential to the microbial communities, their roles are unclear. Here, by investigation of an artificial two-member microbial consortium in n-alkane biodegradation, we showed that the microbial member without the n-alkane-degrading capability had a cross-feeding interaction with and metabolic regulation to the leading member for the synergistic n-alkane biodegradation. Our study improves the current understanding of microbial interactions. Because "assistant" microbes showed importance in communities in addition to the functional microbes, our findings also suggest a useful "assistant-microbe" principle in the design of microbial communities for either bioremediation or chemical production.

Pseudomonas jilinensis sp. nov., Isolated from Oil Production Water of Jilin Oilfield in China.

A Gram-stain-negative, rod-shaped bacterial strain JS15-10A1T was isolated from oil production water. Its optimum growth was observed at 37 °C, pH 7.0, and 3% (w/v) NaCl. The 16S rRNA gene sequence of strain JS15-10A1T showed the highest similarities with Pseudomonas parafulva CB-1T (97.6%) and P. fulva IAM 1529T (97.5%). In addition, phylogenetic analyses based on multilocus sequence analyses with concatenating 16S rRNA, gyrB, rpoD, and rpoB genes indicated that strain JS15-10A1T was a member of genus Pseudomonas but discriminated from other species. Furthermore, whole-genome analyses revealed that average nucleotide identities and in silico DNA-DNA hybridization values of strain JS15-10A1T against its closest relatives were all below 76.7% and 21.1%, respectively. The major cellular fatty acids of strain JS15-10A1T were summed feature 8 (C18:1ω7c/C18:1ω6c), C16:0, C12:0, C17:0 cyclo, summed feature 3 (C16:1ω7c/C16:1ω6c), C12:0 3-OH, C10:0 3-OH, and C19:0 cyclo ω8c. The predominant quinone was ubiquinone Q-9. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown amino-lipid, and two unidentified lipids. The genome DNA G + C content was 60.0 mol%. On the basis of phylogenetic, phenotypic, physiological, and chemotaxonomic analyses, it can be concluded that strain JS15-10A1T represents a novel species in genus Pseudomonas, for which the name Pseudomonas jilinensis sp. nov. is proposed. The type strain is JS15-10A1T (= CGMCC 1.16072T = LMG 30036T).

Pseudomonas Saliphila sp. nov., a Bacterium Isolated from Oil-Well Production Water in Qinghai Oilfield of China.

Strain 16W4-4-3 T was isolated from the oil-well production water in Qinghai Oilfield, China. Cells were Gram-stain-negative, rod-shaped, catalase- and oxidase-positive, facultatively anaerobic and motile by single polar flagellum. The 16S rRNA gene sequences of strain 16W4-4-3 T showed the highest similarities with Pseudomonas profundi M5T (98.8%), P. pelagia CL-AP6T (98.0%), P. salina XCD-X85T (97.7%), and P. sabulinigri J64T (97.5%). The phylogenetic trees based on multilocus sequence analyses with concatenating 16S rRNA, gyrB, rpoD and rpoB genes suggested that this strain should be affiliated to the genus Pseudomonas but remotely related from other species. In addition, whole genome analyses revealed that the digital DNA-DNA hybridization values and average nucleotide identities of strain 16W4-4-3 T against its close relatives were all below 28.8% and 86.5%, respectively. Furthermore, the isolate had totally different whole cell protein profile as compared to those of other species. Major fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C17:0cyclo. Major isoprenoid quinone was ubiquinone (Q-9), and major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The DNA G + C content was 58.5 mol%. Therefore, phenotypic, phylogenetic, genomic, chemotaxonomic, and proteomic traits showed that the isolate represented a novel species of the genus Pseudomonas, the name Pseudomonas saliphila sp. nov. is proposed. Type strain is 16W4-4-3 T (= CGMCC 1.13350 T = KCTC 72619 T).

Single-Homology-Arm Linear DNA Recombination by the Nonhomologous End Joining Pathway as a Novel and Simple Gene Inactivation Method: a Proof-of-Concept Study in Dietzia sp. Strain DQ12-45-1b.

Nonhomologous end joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNA in vivo Here, we established a proof-of-concept single-homology-arm linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNA in vivo by NHEJ could be used to generate nonreplicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach in Dietzia sp. strain DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA single homolog to the sequence in the chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% of prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, an Escherichia coli strain also showed NHEJ activity when the Ku and LigD of Dietzia sp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for nonmodel microorganisms.IMPORTANCE Many nonmodel Gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway in Dietzia sp. DQ12-45-1b was evaluated and was used to successfully knock out 11 genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the single-homology-arm linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse nonmodel prokaryotic organisms.

Clonal Characteristics of Circulating B Lymphocyte Repertoire in Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by elevated serum anti-mitochondrial Ab and lymphocyte-mediated bile duct damage. This study was designed to reveal the clonal characteristics of B lymphocyte repertoire in patients with PBC to facilitate better understanding of its pathogenesis and better management of these patients. Using high-throughput sequencing of Ig genes, we analyzed the repertoire of circulating B lymphocytes in 43 patients with PBC, and 34 age- and gender-matched healthy controls. Compared with healthy controls, PBC patients showed 1) a gain of 14 new clones and a loss of 8 clones; 2) a significant clonal expansion and increased relative IgM abundance, which corresponded with the elevated serum IgM level; 3) a significant reduction of clonal diversity and somatic hypermutations in class-switched sequences, which suggested a general immunocompromised status; 4) the reduction of clonal diversity and enhancement of clonal expansion were more obvious at the cirrhotic stage; and 5) treatment with ursodeoxycholic acid could increase the clonal diversity and reduce clonal expansion of the IgM repertoire, with no obvious effect on the somatic hypermutation level. Our data suggest that PBC is a complex autoimmune disease process with evidence of B lymphocyte clonal gains and losses, Ag-dependent ogligoclonal expansion, and a generally compromised immune reserve. This new insight into the pathogenesis of PBC opens up the prospect of studying disease-relevant B cells to better diagnose and treat this devastating disease.