15 16 17 18 Running Tittle: SEM images of E. coli strains harboring the pks genomic island 19 Abstract 49Colibactin is a natural product made by numerous strains of E. coli that harbor the pks 50 genomic island. The deletion of one of the genes within the pks island, the peptidase 51 clbP, has been found to disrupt the maturation of colibactin, thus promoting the 52 accumulation in the periplasmic space of numerous biosynthesis intermediates, some of 53 which have been characterized chemically. To date, no one has reported the effect of 54 such an accumulation of intermediates on the cellular morphology of the producing E. 55 coli bacterium. In this report, we describe the scanning electron microscopy (SEM) 56 images of numerous clinical isolates of E. coli harboring the pks island, collected from 57Puerto Rico hospitals. We have observed that the wild type isolates that harbor the pks 58 island display lesions on the bacterial envelope surface. These lesions are absent in 59 isolates lacking the pks island. To determine whether this phenotype is associated with 60 colibactin production, we deleted the clbP gene from the extraintestinal pathogenic E. 61 coli strain IHE3034, thus disrupting its ability to make colibactin. The wild-type IHE3034 62 displayed a spherical shape with no envelope lesions, and was practically 63 indistinguishable from the ΔclbP deletion mutant. To our knowledge, this work provides 64 the first SEM images of a pks deletion mutant. 65 66 67 68 69 70 71 87 88 89 90 91 92 93 94

Botryococcus braunii (B. braunii) is a green photosynthetic microalga that is highly investigated due to its capacity of hydrocarbon accumulation, secretion, and synthesis. However, its photosynthetic apparatus has not been elucidated. Photosystem I (PSI) is a membrane‐bound pigment‐protein complex that mediates the electron transfer from plastocyanin on the lumenal side to ferredoxin on the cytoplasmic side. In this study, our purpose was to isolate, purify and characterize PSI core subunits and light harvesting proteins. After the initial isolation via differential ultracentrifugation and solubilization of thylakoid membranes with 1% of n‐dodecyl β‐D‐maltoside, the resulting supernatant was loaded onto a Toyopearl DEAE‐650 ion exchange chromatography column. The purified PSI fraction obtained was pooled and concentrated using an Amicon centrifugal filter unit. The resulting protein concentration was 1 mg/ml, and the PSI characteristic 677 nm absorption maximum was attained. The biophysical characterization was performed via UV/Vis and fluorescence spectroscopy, native and SDS‐polyacrylamide gel electrophoresis, and MALDI‐MS, with the characteristic peaks and molecular weight results of the proteins and subunits found in PSI. Our characterization results show that PSI has been successfully purified, which may provide the fundamental knowledge of the function and possible applications of this integral membrane protein complex in B. braunii.Support or Funding InformationThis research was funded by NIH Research Initiative for Scientific Enhancement (RISE) Program Grant No. 5R25GM061151‐14 and DOD Contract No. W911NF‐11‐1‐0218.

The biosynthesis of polyunstaurated fatty acids (PUFA) in certain bacterial species, is regulated by processes that are not fully understood. One possible factor in the modulation of gene expression in PUFA biosynthesis is pfaR, a putative transcriptional regulator whose gene is encoded directly upstream of pfaA in some bacterial species but entirely absent in others. Here we report the expression of pfaR in Escherichia coli together with its purification by Ni2+ affinity chromatography. Our results show that the majority of pfaR is produced as an insoluble protein that can be reconstituted from inclusion bodies. After inclusion body reconstitution, pfaR remains amenable for affinity purification. Additional bioinformatics analyses indicate the presence of a conserved winged helix‐turn‐helix (wHTH) domain flanked by a number of conserved motifs which include a predicted protein‐protein interaction site. Sequence alignments and motif structure analyses reveal evolutionary divergence in this family of transcriptional regulators, consistent with the likely DNA‐binding function of pfaR. Currently, we are in the process of analyzing pfaR secondary structure using circular dichroism. Future work will include functional analysis using systematic evolution of ligands by exponential enrichment.Support or Funding InformationThis work was supported by the National Institutes of Health [R25 GM061838, R25GM061151‐ 15, 5T34GM007821‐37, T36‐GM‐095335]; and the NationalScience Foundation [CHE0953254].This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

All gram‐negative bacteria secrete outer membrane vesicles (OMV) that have several functions, among them the transportation of bacterial virulence and survival factors. It has been found by other groups that certain OMV promote aberrant growth in mammalian cells upon contact. OMV provide an insoluble secretion pathway for gram‐negative bacteria and are responsible for the release of outer membrane and periplasm content out of the cell. The main objective of this investigation is to compare the composition and biological activity of OMV isolated from diverse clinical strains of E. coli. We will assess morphological effects of OMV from selected clinical isolates of E. coli on HeLa cells. Furthermore, we will also measure the lipid composition of OMV for the different strains and mutants by GC/MS. As preliminary results, we observed a significant difference in the lipid composition of the IHE3034 (pks+ strain) OMV and the IHE3034‐ΔclbP OMV. The clbP gene is presumed to be involved in the biosynthesis of colibactin. We observed that the deletion of clbP peptidase causes a decrease in the percentage of 12:0 fatty acids and an increase of 16:0 fatty acids in OMV, but not in the cellular membrane. These results begin to establish a correlation between the presence of colibactin genes and events at the bacterial cell surface.Support or Funding InformationNSF grant CHE0953254 NIH grant R25GM061838 (MBRS‐RISE program) to Ramón Gómez‐Moreno