Ana Babic

Direct Visualization of Horizontal Gene Transfer

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.

Autonomous plasmid-like replication of a conjugative transposon.

Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra‐chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid‐like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1‐encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid‐like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations.

Efficient Gene Transfer in Bacterial Cell Chains

ABSTRACT Horizontal gene transfer contributes to evolution and the acquisition of new traits. In bacteria, horizontal gene transfer is often mediated by conjugative genetic elements that transfer directly from cell to cell. Integrative and conjugative elements (ICEs; also known as conjugative transposons) are mobile genetic elements that reside within a host genome but can excise to form a circle and transfer by conjugation to recipient cells. ICEs contribute to the spread of genes involved in pathogenesis, symbiosis, metabolism, and antibiotic resistance. Despite its importance, little is known about the mechanisms of conjugation in Gram-positive bacteria or how quickly or frequently transconjugants become donors. We visualized the transfer of the integrative and conjugative element ICEBs1 from a Bacillus subtilis donor to recipient cells in real time using fluorescence microscopy. We found that transfer of DNA from a donor to a recipient appeared to occur at a cell pole or along the lateral cell surface of either cell. Most importantly, we found that when acquired by 1 cell in a chain, ICEBs1 spread rapidly from cell to cell within the chain by additional sequential conjugation events. This intrachain conjugation is inherently more efficient than conjugation that is due to chance encounters between individual cells. Many bacterial species, including pathogenic, commensal, symbiotic, and nitrogen-fixing organisms, harbor ICEs and grow in chains, often as parts of microbial communities. It is likely that efficient intrachain spreading is a general feature of conjugative DNA transfer and serves to amplify the number of cells that acquire conjugative mobile genetic elements. IMPORTANCE Conjugative elements contribute to horizontal gene transfer and the acquisition of new traits. They are largely responsible for spreading antibiotic resistance in bacterial communities. To study the cell biology of conjugation, we visualized conjugative DNA transfer between Bacillus subtilis cells in real time using fluorescence microscopy. In contrast to previous predictions that transfer would occur preferentially from the donor cell pole, we found that transfer of DNA from a donor to a recipient appeared to occur at a cell pole or along the lateral cell surface of either cell. Most importantly, we found that when acquired by 1 cell in a chain, the conjugative DNA spread rapidly from cell to cell within the chain through sequential conjugation events. Since many bacterial species grow naturally in chains, this intrachain transfer is likely a common mechanism for accelerating the spread of conjugative elements within microbial communities. Conjugative elements contribute to horizontal gene transfer and the acquisition of new traits. They are largely responsible for spreading antibiotic resistance in bacterial communities. To study the cell biology of conjugation, we visualized conjugative DNA transfer between Bacillus subtilis cells in real time using fluorescence microscopy. In contrast to previous predictions that transfer would occur preferentially from the donor cell pole, we found that transfer of DNA from a donor to a recipient appeared to occur at a cell pole or along the lateral cell surface of either cell. Most importantly, we found that when acquired by 1 cell in a chain, the conjugative DNA spread rapidly from cell to cell within the chain through sequential conjugation events. Since many bacterial species grow naturally in chains, this intrachain transfer is likely a common mechanism for accelerating the spread of conjugative elements within microbial communities.

Construction of an improved RP4 (RK2)-based conjugative system

In many bacteria, gene replacement can only be achieved using RP4-based conjugative systems where exogenous DNA is delivered to the recipient on conditionally replicating plasmids carrying an oriT(RP4). In the commonly used strains SM10 and S17-1 conjugative functions are provided by an RP4 plasmid inserted in the chromosome. Those strains, besides mobilizing oriT(RP4)-carrying plasmids, also transfer their own chromosomal genes, which can be inconvenient for several reasons. We describe here a new version of an RP4-based conjugative system that allows for efficient transfer of mobilizable plasmids without the transfer of chromosomal genes, providing fully controlled and efficient exogeneous DNA delivery to non-transformable bacteria.

Prediagnostic Plasma 25-Hydroxyvitamin D and Pancreatic Cancer Survival

PURPOSE Although vitamin D inhibits pancreatic cancer proliferation in laboratory models, the association of plasma 25-hydroxyvitamin D [25(OH)D] with patient survival is largely unexplored. PATIENTS AND METHODS We analyzed survival among 493 patients from five prospective US cohorts who were diagnosed with pancreatic cancer from 1984 to 2008. We estimated hazard ratios (HRs) for death by plasma level of 25(OH)D (insufficient, < 20 ng/mL; relative insufficiency, 20 to < 30 ng/mL; sufficient ≥ 30 ng/mL) by using Cox proportional hazards regression models adjusted for age, cohort, race and ethnicity, smoking, diagnosis year, stage, and blood collection month. We also evaluated 30 tagging single-nucleotide polymorphisms in the vitamin D receptor gene, requiring P < .002 (0.05 divided by 30 genotyped variants) for statistical significance. RESULTS Mean prediagnostic plasma level of 25(OH)D was 24.6 ng/mL, and 165 patients (33%) were vitamin D insufficient. Compared with patients with insufficient levels, multivariable-adjusted HRs for death were 0.79 (95% CI, 0.48 to 1.29) for patients with relative insufficiency and 0.66 (95% CI, 0.49 to 0.90) for patients with sufficient levels (P trend = .01). These results were unchanged after further adjustment for body mass index and history of diabetes (P trend = .02). The association was strongest among patients with blood collected within 5 years of diagnosis, with an HR of 0.58 (95% CI, 0.35 to 0.98) comparing patients with sufficient to patients with insufficient 25(OH)D levels. No single-nucleotide polymorphism at the vitamin D receptor gene met our corrected significance threshold of P < .002; rs7299460 was most strongly associated with survival (HR per minor allele, 0.80; 95% CI, 0.68 to 0.95; P = .01). CONCLUSION We observed longer overall survival in patients with pancreatic cancer who had sufficient prediagnostic plasma levels of 25(OH)D.

2-Aminopurine Allows Interspecies Recombination by a Reversible Inactivation of the Escherichia coli Mismatch Repair System

2-Aminopurine treatment of Escherichia coli induces a reversible phenotype of DNA mismatch repair deficiency. This transient phenotype results in a 300-fold increase in the frequency of interspecies conjugational recombination with a Salmonella enterica serovar Typhimurium Hfr donor. This method can be used for the generation of biodiversity by allowing recombination between diverged genes and genomes.

Cigarette Smoking and Pancreatic Cancer Survival

Purpose Cigarette smoking is associated with increased incidence of pancreatic cancer. However, few studies have prospectively evaluated the association of smoking with patient survival. Patients and Methods We analyzed survival by smoking status among 1,037 patients from two large US prospective cohort studies diagnosed from 1986 to 2013. Among 485 patients from four prospective US cohorts, we also evaluated survival by prediagnostic circulating levels of cotinine, a metabolite of nicotine that is proportional to tobacco smoke exposure. On the basis of prediagnosis cotinine levels, we classified patients as nonsmokers (< 3.1 ng/mL), light smokers (3.1-20.9 ng/mL), or heavy smokers (≥ 21.0 ng/mL). We estimated hazard ratios (HRs) for death by using Cox proportional hazards models, with adjustment for age, sex, race/ethnicity, body mass index, diabetes status, diagnosis year, and cancer stage. Results The multivariable-adjusted HR for death was 1.37 (95% CI, 1.11 to 1.69) comparing current smokers with never smokers ( P = .003). A statistically significant negative trend in survival was observed for increasing pack-years of smoking ( Ptrend = .008), with HR for death of 1.49 (95% CI, 1.05 to 2.10) for > 60 pack-years of smoking versus never smoking. Survival among former smokers was similar to that for never smokers, regardless of time since quitting. Heavy smokers defined by prediagnostic circulating cotinine levels had a multivariable-adjusted HR for death of 1.76 (95% CI, 1.23 to 2.51) compared with nonsmokers. Among patients with circulating cotinine levels measured within 5 years before diagnosis, heavy smokers had a multivariable-adjusted HR for death of 2.47 (95% CI, 1.24 to 4.92) compared with nonsmokers. Conclusion Cigarette smoking was associated with a reduction in survival among patients with pancreatic cancer.

TNF-α expression, risk factors, and inflammatory exposures in ovarian cancer: evidence for an inflammatory pathway of ovarian carcinogenesis?☆☆☆

Inflammatory cytokines, like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), are elevated in ovarian cancer. Differences in cytokine expression by histologic subytpe or ovarian cancer risk factors can provide useful insight into ovarian cancer risk and etiology. We used ribonucleic acid in situ hybridization to assess TNF-α and IL-6 expression on tissue microarray slides from 78 epithelial ovarian carcinomas (51 serous, 12 endometrioid, 7 clear cell, 2 mucinous, 6 other) from a population-based case-control study. Cytokine expression was scored semiquantitatively, and odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using polytomous logistic regression. TNF-α was expressed in 46% of the tumors, whereas sparse IL-6 expression was seen in only 18% of the tumors. For both markers, expression was most common in high-grade serous carcinomas followed by endometrioid carcinomas. Parity was associated with a reduced risk of TNF-α-positive (OR, 0.3; 95% CI, 0.1-0.7 for 3 or more children versus none) but not TNF-α-negative tumors (P heterogeneity=.02). In contrast, current smoking was associated with a nearly 3-fold increase in risk of TNF-α-negative (OR, 2.8; 95% CI, 1.2-6.6) but not TNF-α-positive tumors (P heterogeneity = .06). Our data suggest that TNF-α expression in ovarian carcinoma varies by histologic subtype and provides some support for the role of inflammation in ovarian carcinogenesis. The novel associations detected in our study need to be validated in a larger cohort of patients in future studies.

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096-111. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Circulating Exosomes in Pancreatic Cancer: Will They Succeed on the Long, Littered Road to Early Detection Marker?

Pancreatic cancer is the fourth leading cause of cancer death in the US and is projected to be the second leading cause of cancer death by 2020. The high mortality of pancreatic ductal adenocarcinoma (PDAC),3 the most common form of pancreatic cancer, is largely a consequence of diagnosis at an advanced stage, when the tumor can no longer be surgically resected for cure. Nevertheless, symptoms rarely develop with early disease, and established risk factors for PDAC—tobacco smoking, obesity, personal history of chronic pancreatitis or diabetes, and family history of pancreatic cancer—are insufficient to risk-stratify the population to facilitate disease screening. Despite extensive research efforts and many potential candidates, no noninvasive biomarker has reached the clinic with utility for early detection of PDAC. Experimental studies indicate that more than a decade elapses from formation of the founder malignant clone to a patients diagnosis, suggesting a window of opportunity for early detection. With this issue in mind, 2 recent studies have exposed new and interesting biology related to the release of exosomes from pancreatic tumors into peripheral blood (1, 2), raising the possibility of a noninvasive tool for risk stratification and earlier PDAC diagnosis. Exosomes are small, extracellular membrane–enclosed vesicles released from most cell types. Because they initially form from intracellular compartments, they contain nucleic acids and proteins, which can be transferred to other cells upon fusion with their extracellular membrane. Although the full spectrum of exosome biology remains to be defined, it is increasingly clear that exosomes are secreted from cancer cells at higher rates than from healthy cells and are important in facilitating cancer progression and spread (3). By use of proteomics, Melo et al. (1) compared exosomes secreted from cancer cell lines and nontumorigenic cells and identified glypican 1 (GPC1) as a membrane-bound protein preferentially present …