Proteomic Analysis of Neisseria gonorrhoeae Biofilms Shows Shift to Anaerobic Respiration and Changes in Nutrient Transport and Outermembrane Proteins

Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with 13C6-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions

The Pseudomonas aeruginosa Chemotaxis Methyltransferase CheR1 Impacts on Bacterial Surface Sampling

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa

The Impact of Religious Commitment on Women’s Sexual Self-Esteem

Religious commitment is associated with decreased sexual activity, poor sexual satisfaction, and sexual guilt, particularly among women. The purpose of this paper was to investigate how religious commitment is related to sexual self-esteem among women. Participants included 196 female undergraduate students, 87 % of whom identified as Christian. Participants completed the Sexual Self-Esteem Inventory for Women (SSEI-W), Religious Commitment Inventory-10, Revised Religious Fundamentalism Scale, Brief Sexual Attitudes Scale, and a measure of their perception of God’s view of sex. Results suggested that women with high religious commitment held more conservative sexual attitudes. Significant relationships between religious commitment and two subscales (moral judgment and attractiveness) of the SSEI-W revealed that women with high religious commitment were less likely to perceive sex as congruent with their moral values and simultaneously reported significantly greater confidence in their sexual attractiveness. A significant relationship between religious commitment and overall sexual self-esteem was found for women whose religion of origin was Catholicism, such that those with higher religious commitment reported lower sexual self-esteem. A hierarchical regression analysis revealed that high religious commitment and perception that God viewed sex negatively independently predicted lower sexual selfesteem, as related to moral judgment. Implications of the findings are provided

DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides

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

Low spatial structure and selection against secreted virulence factors attenuates pathogenicity in Pseudomonas aeruginosa

Bacterial opportunistic pathogens are feared for their difficult-to-treat nosocomial infections and for causing morbidity in immunocompromised patients. Here, we study how such a versatile opportunist, Pseudomonas aeruginosa, adapts to conditions inside and outside its model host Caenorhabditis elegans, and use phenotypic and genotypic screens to identify the mechanistic basis of virulence evolution. We found that virulence significantly dropped in unstructured environments both in the presence and absence of the host, but remained unchanged in spatially structured environments. Reduction of virulence was either driven by a substantial decline in the production of siderophores (in treatments without hosts) or toxins and proteases (in treatments with hosts). Whole-genome sequencing of evolved clones revealed positive selection and parallel evolution across replicates, and showed an accumulation of mutations in regulator genes controlling virulence factor expression. Our study identifies the spatial structure of the non-host environment as a key driver of virulence evolution in an opportunistic pathogen