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A pale yellow bacterial strain, designated SDU1-6T, was isolated from a soil sample collected in the Jinan campus of Shandong University, Shandong Province, PR China. The strain was aerobic, motile, Gram-stain-negative and rod-shaped. Cellular growth occurred at 13–37 °C, pH 5.0–10.0 and with 0–0.1 % (w/v) NaCl, and the optimal growth was at 25 °C and pH 7.5. SDU1-6T had the highest 16S rRNA gene similarity of 95.42 % with Chryseolinea serpens DSM 24574T. The genome was 6 542 746 bp in length with 43.5 mol% DNA G+C content. The major fatty acids of SDU1-6T were C16 : 1ω5 and iso-C15 : 0, the major menaquinone was menaquinone-7 (MK-7) and the major polar lipid was phosphatidylethanolamine. On the basis of the polyphasic evidence and the 16S rRNA gene sequence, SDU1-6T represents a novel species of the genus Chryseolinea , for which the name Chryseolinea flava sp. nov. is proposed. The type strain is SDU1-6T (=CGMCC 1.13492T=JCM 32520T).

The ParABS partitioning system, a main driver of DNA segregation in bacteria, employs two proteins, ParA and ParB, for plasmid partition. The pMF1 plasmid from Myxococcus fulvus 124B02 has a par operon encoding a small acidic protein, ParC, in addition to type I ParA and ParB homologs. Here, we show that expression of parC upstream of parA (as in the natural case), but not ectopic expression, is essential for the plasmid inheritance in Myxococcus cells. Co-expression of parC upstream of parA was determined to form a soluble ParC–ParA heterodimer at a 1:1 ratio, while individual expression of parA or co-expression of parA with ectopic parC formed insoluble ParA proteins. Purified ParA proteins alone had no ATPase activity and was easily dimerized, while mixing ParA with ParC formed the ParC–ParA heterodimer with the ATPase and polymerization activities. Fusing ParC and ParA also produced soluble proteins and some chimeras restored the ATPase activity and plasmid inheritance. The results highlight that proximal location of parC before parA is critical to realize the functions of ParA in the partition of Myxococcus plasmid pMF1 and shed light on a new mechanism to realize a protein function by two separate proteins.

Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase and error-free template switching catalyzed by RecA, and their competition on the arrested fork affects bacterial SOS mutagenesis. DnaE2 is an error-prone TLS polymerase, and its functions require ImuA and ImuB. Here we investigated the function of imuA, imuB and dnaE2 in Myxococcus xanthus and found that imuA showed differences from imuB and dnaE2 in bacterial growth, resistance and mutation frequency. Transcriptomics analysis found that ImuA were associated with bacterial SOS response. Yeast-two-hybrid scanning revealed that ImuA interacted with RecA1 besides ImuB. Protein activity analysis proved that ImuA had no DNA binding activity, but inhibited the DNA binding and recombinase activity of RecA1. These findings highlight that ImuA not only participates in TLS by binding ImuB, but also inhibits the recombinase activity of RecA1 in M. xanthus, suggesting a role of ImuA in the two replication restart pathways.ImportanceDnaE2 is responsible for bacterial SOS mutagenesis in nearly one third of sequenced bacterial strains. However, its mechanism, especially the function of its accessory protein ImuA, is still unclear. Here we reported that M. xanthus ImuA might facilitate DnaE2 TLS by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the scientific problem of choosing one of the two restart pathways to repair the stalled replication fork.

Myxococcus xanthus DK1622 has two RecA genes, recA1 (MXAN_1441) and recA2 (MXAN_1388) with unknown differentiated functions. Herein, we showed that both recA genes were induced by ultraviolet (UV) irradiation, but the induction of recA1 was more delayed than that of recA2. Deletion of recA1 did not affect the growth, but significantly decreased the UV-irradiation survival, homologous recombination ability, and induction of the LexA-dependent SOS genes. In contrast, the deletion of recA2 markedly prolonged the lag phase of bacterial growth and DNA damage caused by hydrogen peroxide, but did not change the UV-radiation resistance and the SOS-gene inducibility. Protein activity analysis demonstrated that RecA1, but not RecA2, catalyzed DNA strand exchange and LexA-autocleavage in vitro. Transcriptomic analysis indicated that RecA2 has evolved to mainly regulate the gene expressions for cellular transportation and antioxidation. This is the first report of functional divergence of duplicated bacterial recA genes. The results highlight the evolutionary strategy of M. xanthus cells for the DNA homologous recombination and the evolution of genomes from simple to complex.

RecA is a ubiquitous multifunctional protein for bacterial homologous recombination and SOS response activation.Myxococcus xanthusDK1622 possesses tworecAgenes, and their functions and mechanisms are almost unclear. Here, we showed that the transcription ofrecA1(MXAN_1441) was less than one-tenth ofrecA2(MXAN_1388). Expressions of the tworecAgenes were both induced by ultraviolet (UV) irradiation, but in different periods. Deletion ofrecA1did not affect the growth, but significantly decreased the UV-irradiation survival, the homologous recombination ability, and the induction of the LexA-dependent SOS genes. Comparably, the deletion ofrecA2markedly prolonged the lag phase for cellular growth and antioxidation of hydrogen peroxide, but did not change the UV-irradiation resistance and the SOS-gene inducibility. The two RecA proteins are both DNA-dependent ATPase enzymes. We demonstrated that RecA1, but not RecA2, hadin vitroDNA recombination capacity and LexA-autolysis promotion activity. Transcriptomic analysis indicated that the duplicate RecA2 has evolved to mainly regulate the gene expressions for cellular transportation and antioxidation. We discuss the potential mechanisms for the functional divergence. This is the first time to clearly determine the divergent functions of duplicatedrecAgenes in bacterial cells. The present results highlight that the functional divergence of RecA duplicates facilitates the exertion of multiple RecA functions.Author summaryMyxobacteria has a large-size genome, contains many DNA repeats that are rare in the prokaryotic genome. It encodes bacterial RecA that could promote recombination between homologous DNA sequences. How myxobacteria avoid the undesired recombination between DNA repeats in its genome is an interesting question.M. xanthusencodes two RecA proteins, RecA1 (MXAN_1441) and RecA2 (MXAN_1388). Both RecA1 and RecA2 shows more than 60% sequence consistency withE. coliRecA (EcRecA) and can partly restore the UV resistance ofE. coli recAmutant. Here, our results proved their divergent functions of the two RecAs. RecA1 retains the ability to catalyze DNA recombination, but its basal expression level is very low. RecA2 basal expression level is high, but no recombination activity is detected in vitro. This may be a strategy forM. xanthusto adapt to more repetitive sequences in its genome and avoid incorrect recombination.HighlightsM. xanthushas tworecAs, which are expressed with significantly different levels. BothrecAs are inducible by UV irradiation, but in different stages.The absence ofrecA1reduces bacterial UV-irradiation resistance, while the absence ofrecA2delays bacterial growth and antioxidant capacity.RecA1 retains the DNA recombination and SOS induction abilities, while RecA2 has evolved to regulate the expression of genes for cellular transport and antioxidation.

Although plasmids provide additional functions for cellular adaptation to the environment, they also create a metabolic burden, which causes the host cells to be less competitive with their siblings. Low-copy-number plasmids have thus evolved several mechanisms for their long-term maintenance in host cells. pMF1, discovered in Myxococcus fulvus 124B02, is the only endogenous autonomously replicated plasmid yet found in myxobacteria. Here we report that a post-segregational killing system, encoded by a co-transcriptional gene pair of pMF1.19 and pMF1.20, is involved in maintaining the pMF1 plasmid in its host cells. We demonstrate that the protein encoded by pMF1.20 is a new kind of nuclease, which is able to cleave DNA in vitro. The nuclease activity can be neutralized by the protein encoded by pMF1.19 through protein–protein interaction, suggesting that the protein is an immune protein for nuclease cleavage. We propose that the post-segregational killing mechanism of the nuclease toxin and immune protein pair encoded by pMF1.20 and pMF1.19 is helpful for the stable maintenance of pMF1 in M. fulvus cells.

Background Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor survival. Recurrent genetic aberrations, such as chromosomal rearrangements and gene mutations, are currently used for patient classification and prognosis, and form the basis of our current understanding of pathogenic mechanisms. However, these markers incompletely predict disease behavior and outcomes. Alterations in DNA methylation patterns are a major hallmark of cancer and recent studies have demonstrated differential global DNA methylation patterns among AML patients. Here we sought to define a novel approach to classify AML patients using genome-wide DNA methylation patterns and to uncover the biological basis and features associated with these epigenetic patterns by integrating mutation, gene expression, and functional data. Methods We analyzed genome-wide DNA methylation data from Illumina arrays on 649 AML cases combined from the Beat AML consortium housed at the Oregon Health & Science University (OHSU) (n=226), The Ohio State University (n=27), TCGA (n=190), and other published studies (n=206). Small molecule inhibitor response data was obtained from the Beat AML consortium. RNA sequencing and survival analyses were performed by Kaplan-Meier analysis in the TCGA and OHSU cohorts. Results K-mediods based clustering on the 500 most-variable CpGs identified 13 distinct subtypes, we termed AML 'epitypes' (Fig. 1A). Comparative analyses incorporating normal hematopoietic cells revealed that AML epitypes retain unique combinations of biological features including the degree of myeloid development, chromatin landscapes, transcription factor binding and gene expression patterns/pathways. Eleven of 13 epitypes were associated with somatic genetic aberrations, i.e. mutations (or combinations thereof) in NPM1, IDH1/2, TET2, DNMT3A, CEBPA, and chromosomal rearrangements t(8;21), t(15;17) and inv(16) accounted for the majority of patients within each respective epitype. The remaining two epitypes were not associated with dominant genetic events, rather they clustered closely with normal CD34+ hematopoietic stem and progenitor cells. These 'stem-like' epigenetic patterns accounted for 27% of patients and showed limited correlation to other leukemic stem cell gene expression signatures. Stem-like epitypes demonstrated broad resistance to a range of small molecule inhibitors. Differential gene expression analysis between normal cells and epitypes revealed enrichment of the JAK-STAT pathway along with other inflammatory pathways, such as interleukin and interferon signaling, selectively in the stem-like epitypes. Patients with stem-like epitypes exhibited decreased overall survival relative to others independent of age (P We next investigated the impact of STAT pathway activation on AML epigenetic patterns. FLT3 mutations are known to selectively activate the STAT pathway in AML and are associated with inferior outcomes. Controlling for the distribution of FLT3 mutations across epitypes, we uncovered a hypomethylation signature enriched in STAT transcription factor binding and FLT3 mutations which we have termed the 'STAT hypomethylation signature' (SHS). SHS-positivity was not restricted to FLT3 mutations, as 34% of SHS+ AMLs lacked FLT3 mutations and, conversely, SHS was absent in 24% of FLT3 mutated patients. SHS-positivity did not impact outcome in the stem-like epitypes; however, within non-stem-like epitypes, SHS+ patients displayed significantly poorer outcomes (P=0.021; Fig 1C). Conclusion Applying our integrative approach of using global DNA methylation profiles to subclassify AML patients, we have uncovered new insights into potential pathogenic mechanisms by which genetic and epigenetic aberrations may contribute to discrete avenues of AML development. Distinct global DNA methylation patterns are commonly driven by genetic aberrations, while others were associated with stem cell-like features and inflammatory pathways. The identification of JAK/STAT pathway upregulation in the stem-like epitype and a subset of non-stem-like samples highlights that nearly half of AML patients exhibit evidence of JAK/STAT activation and experience poor outcomes. Disclosures Druker: Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Celgene: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Beat AML LLC: Other: Service on joint steering committee; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Gilead Sciences: Other: former member of Scientific Advisory Board; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees . Tyner:Petra: Research Funding; Janssen: Research Funding; Aptose: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Takeda: Research Funding; Syros: Research Funding; Array: Research Funding; AstraZeneca: Research Funding; Array: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Syros: Research Funding; Seattle Genetics: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Agios: Research Funding; Petra: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Agios: Research Funding. Byrd:Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau.

Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.

With a novel and universal strategy for the cloning of multiple DNA fragments, a complex synthetic vector (pVEC100), harboring the target DNA fragments in conventional 100-bp DNA ladder, was constructed for efficient and large-scale production of 100-bp DNA marker through bacteria fermentation, plasmid extraction and restrictive digestion. Since the restrictive digestion of complex vectors yields insufficient small DNA fragments, an innovative PCR model was developed as an alternative. The PCR model comprised a specially designed template vector and a unit amplification model for producing groups of small DNA fragments. The unit amplification model improved the efficiency of the PCR protocol and made it more economical and easier for small DNA fragment amplification. The approach presented in this paper – a unit cloning model for constructing complex synthetic vectors combined with the modular design of unit amplification by PCR – is a powerful method for preparing small DNA fragments of DNA molecular weight standards.

Myxococcus fulvus HW-1, a salt-tolerant bacterial strain, which was isolated from a coastal environment, changes its behavior with different salinities. To study the relationship between behavioral shifts and the adaption to oceanic conditions, the HW-1 strain was randomly mutagenized using transposon insertion, producing a dispersed-growing mutant, designated YLH0401. The mutant did not develop fruiting bodies and myxospores, was deficient in S-motility, produced less extracellular matrix and was less salt tolerant. The YLH0401 strain was determined to be mutated by a single insertion in a large gene of unknown function (7011 bp in size), which is located in a horizontally transferred DNA fragment. The gene is expressed during the vegetative growth stage, as well as highly and stably expressed during the development stage. This horizontally transferred gene may allow Myxococcus to adapt to oceanic conditions.

The mts locus in salt-tolerant Myxococcus fulvus HW-1 was found to be critical for gliding motility, fruiting-body formation, and sporulation. The homologous genes in Myxococcus xanthus are also important for social motility and fruiting-body development. The mts genes were determined to be involved in cell-cell cohesion in both myxobacterial species.

One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.

Thus far, pMF1 is the only endogenous myxobacterial plasmid whose replication mechanism is unclear. In this study, we determined that the plasmid replicates via the theta-mode. The pMF1.14 gene, located in the pMF1.13–pMF1.15 operon (repABC), encodes an essential replication initiation protein that was predicted to have no typical DNA/protein binding motifs but contains rich disordered regions. The pMF1 replication-related essential cis-acting DNA region, approximate 370 bp, was located within pMF1.14, and was found to contain several directly and inverted atypical repeats. The unique characteristics of the pMF1 replicon are suggested to be the reason for its strict narrow host range in Myxococcus cells.

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics.

One of the key limitations to genetic manipulation in myxobacteria is that the cells grow in clumps in liquid. A salt-tolerant strain HW-1 of Myxococcus fulvus was treated with UV irradiation and produced a completely dispersedly growing mutant UV684. There were no significant differences between the parent HW-1 and the mutant UV684 in terms of salt-tolerant growth. The mutant UV684 and the parent strain had the similar abilities of the fruiting body formation and S motility. Interestingly, the mutant exhibited high transformation/transposition efficiency with 10(5)-10(6) colony-forming units per microg DNA, which was about 10(3)-10(5) fold higher than HW-1. The results indicate that the mutation that led to dispersed growth in the UV684 mutant strain had a few impacts on social behavior, but greatly facilitated molecular genetic manipulation.

Monozygous twins share a common genotype. However, most monozygotic twin pairs are not identical; several types of phenotypic discordance may be observed, such as differences in susceptibilities to disease and a wide range of anthropomorphic features. There are several possible explanations for these observations, but one is the existence of epigenetic differences. To address this issue, we examined the global and locus-specific differences in DNA methylation and histone acetylation of a large cohort of monozygotic twins. We found that, although twins are epigenetically indistinguishable during the early years of life, older monozygous twins exhibited remarkable differences in their overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation, affecting their gene-expression portrait. These findings indicate how an appreciation of epigenetics is missing from our understanding of how different phenotypes can be originated from the same genotype.

Amplification of oncogenes is an important mechanism that can cause gene overexpression and contributes to tumor development. The identification of amplified regions might have both prognostic and therapeutic significance. We used primary lung carcinomas and lung cancer cell lines for restriction landmark genomic scanning (RLGS) to identify novel amplified sequences. Enhanced RLGS fragments that indicate gene amplification were observed in primary tumors and lung cancer cell lines of both non-small cell lung cancer and small cell lung cancer. We identified one novel amplicon on chromosome 11q22, in addition to previously reported amplicons that include oncogenes MYCC, MYCL1 and previously identified amplification of chromosomal regions 6q21 and 3q26-27. Amplification of 11q22 has been reported in other types of cancer and was refined to an approximately 1.19 Mbp region for which the complete sequence is available. Based on a patient sample with a small region of low-level amplification we were able to further narrow this region to 0.92 Mbp. Genes localized in this region include two inhibitors of apoptosis (cIAP1 and cIAP2). Immunohistochemistry and western blot analysis identified cIAP1 and cIAP2 as potential oncogenes in this region as both are overexpressed in multiple lung cancers with or without higher copy numbers.