Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists

Protozoan grazing is a major mortality factor faced by bacteria in the environment. Vibrio cholerae, the causative agent of the disease cholera, is a natural inhabitant of aquatic ecosystems, and its survival depends on its ability to respond to stresses, such as predation by heterotrophic protists. Previous results show that grazing pressure induces biofilm formation and enhances a smooth to rugose morphotypic shift, due to increased expression of Vibrio polysaccharide (VPS). In addition to negatively controlling vps genes, the global quorum sensing (QS) regulator, HapR, plays a role in grazing resistance as the ΔhapR strain is efficiently consumed while the wild type (WT) is not. Here, the relative and combined contributions of VPS and QS to grazing resistance were investigated by exposing VPS and HapR mutants and double mutants in VPS and HapR encoding genes at different phases of biofilm development to amoeboid and flagellate grazers. Data show that the WT biofilms were grazing resistant, the VPS mutants were less resistant than the WT strain, but more resistant than the QS mutant strain, and that QS contributes to grazing resistance mainly in mature biofilms. In addition, grazing effects on biofilms of mixed WT and QS mutant strains were investigated. The competitive fitness of each strain in mixed biofilms was determined by CFU and microscopy. Data show that protozoa selectively grazed the QS mutant in mixed biofilms, resulting in changes in the composition of the mixed community. A small proportion of QS mutant cells which comprised 4% of the mixed biofilm biovolume were embedded in grazing resistant WT microcolonies and shielded from predation, indicating the existence of associational protection in mixed biofilms. © 2013 Sun et al

Development of Novel Drugs from Marine Surface Associated Microorganisms

While the oceans cover more than 70% of the Earth’s surface, marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds (“bioactives”) to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactives because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surfaces of marine eukaryotes. Chemically driven interactions are also important for the establishment of cross-relationships between microbes and their eukaryotic hosts, in which organisms producing antimicrobial compounds (“antimicrobials”), may protect the host surface against over colonisation in return for a nutrient rich environment. As is the case for bioactive discovery in general, progress in the detection and characterization of marine microbial bioactives has been limited by a number of obstacles, such as unsuitable culture conditions, laborious purification processes, and a lack of de-replication. However many of these limitations are now being overcome due to improved microbial cultivation techniques, microbial (meta-) genomic analysis and novel sensitive analytical tools for structural elucidation. Here we discuss how these technical advances, together with a better understanding of microbial and chemical ecology, will inevitably translate into an increase in the discovery and development of novel drugs from marine microbial sources in the future

Next-generation studies of microbial biofilm communities

© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. As we look into the future of microbial biofilm research, there is clearly an emerging focus on communities rather than populations. This represents an essential change in direction to more accurately understand how and why microorganisms assemble into communities, as well as the functional implications for such a life style. For example, current research studies shows that communities display emergent properties or functions that are not predicted from the individual single species populations, including elevated stress tolerance and resistance to antibiotics. Models for mixed species biofilms can be very simple, comprised only a handful of species or can be extremely species rich, with hundreds or thousands of species present. The future holds much promise for this area of research, where investigators will increasingly be able to resolve, at the molecular and biochemical levels, interspecies relationships and mechanisms of interaction. The outcome of these studies will greatly enhance our understanding of the ecological and evolutionary factors that drive community function in natural and engineered systems

In situ grazing resistance of Vibrio cholerae in the marine environment

Previous laboratory experiments revealed that Vibrio cholerae A1552 biofilms secrete an antiprotozoal factor that prevents Rhynchomonas nasuta from growing and thus prevents grazing losses. The antiprotozoal factor is regulated by the quorum-sensing response regulator, HapR. Here, we investigate whether the antiprotozoal activity is ecologically relevant. Experiments were conducted in the field as well as under field-like conditions in the laboratory to assess the grazing resistance of V. cholerae A1552 and N16961 (natural frameshift mutation in hapR) biofilms to R. nasuta and Cafeteria roenbergensis. In laboratory experiments exposing the predators to V. cholerae grown in seawater containing high and low glucose concentrations, we determined that V. cholerae biofilms showed increased resistance towards grazing by both predators as glucose levels decreased. The relative resistance of the V. cholerae strains to the grazers under semi-field conditions was similar to that observed in situ. Therefore, the antipredator defense is environmentally relevant and not lost when biofilms are grown in an open system in the marine environment. The hapR mutant still exhibited some resistance to both predators and this suggests that V. cholerae may coordinate antipredator defenses by a combination of density-dependent regulation and environmental sensing to protect itself from predators in its natural habitat. © 2011 Federation of European Microbiological Societies

Analyse av pengestrømmer fra offentlig sektor til helseanlegg i Mosambik, Ghana og Uganda

Denne oppgaven tar for seg undersøkelser av pengestrømmer fra offentlig sektor til helseanlegg (PETS) i Mosambik, Ghana og Uganda. Jeg har tatt med FNs helserelaterte tusenårsmål, og ønsker å se om PETS kan fungere som et nyttig verktøy for å oppnå målene. Undersøkelsene viser at registreringer og datamateriale i helsesektoren i de tre landene er svært mangelfull. Offentlige utgifter i form av medisiner og medisinsk utstyr distribueres til helseanleggene i naturalia, og verdiene er som regel ukjent. Dette gir rom for lekkasje, slik at registreringer av helseutgifter på sentralt hold ikke samsvarer med resultater på tjenestenivå. Undersøkelsene fra de tre landene kan være effektive dersom myndighetene tar funnene til etterretning og iverksetter reformer og endringer for å løse problemene. Dersom dette blir gjort kan PETS være et nyttig verktøy for å oppnå FNs tusenårsmål

Community Structure and Functional Gene Profile of Bacteria on Healthy and Diseased Thalli of the Red Seaweed Delisea pulchra

Disease is increasingly viewed as a major factor in the ecology of marine communities and its impact appears to be increasing with environmental change, such as global warming. The temperate macroalga Delisea pulchra bleaches in Southeast Australia during warm summer periods, a phenomenon which previous studies have indicated is caused by a temperature induced bacterial disease. In order to better understand the ecology of this disease, the bacterial communities associated with threes type of samples was investigated using 16S rRNA gene and environmental shotgun sequencing: 1) unbleached (healthy) D. pulchra 2) bleached parts of D. pulchra and 3) apparently healthy tissue adjacent to bleached regions. Phylogenetic differences between healthy and bleached communities mainly reflected relative changes in the taxa Colwelliaceae, Rhodobacteraceae, Thalassomonas and Parvularcula. Comparative metagenomics showed clear difference in the communities of healthy and diseased D. pulchra as reflected by changes in functions associated with transcriptional regulation, cation/multidrug efflux and non-ribosomal peptide synthesis. Importantly, the phylogenetic and functional composition of apparently healthy tissue adjacent to bleached sections of the thalli indicated that changes in the microbial communities already occur in the absence of visible tissue damage. This shift in unbleached sections might be due to the decrease in furanones, algal metabolites which are antagonists of bacterial quorum sensing. This study reveals the complex shift in the community composition associated with bleaching of Delisea pulchra and together with previous studies is consistent with a model in which elevated temperatures reduce levels of chemical defenses in stressed thalli, leading to colonization or proliferation by opportunistic pathogens or scavengers

Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di- GMP) is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. Current methodologies for the quantification of c-di-GMP are typically based on chemical extraction, representing end point measurements. Chemical methodologies also fail to take into consideration the physiological heterogeneity of the biofilm and thus represent an average c-di-GMP concentration across the entire biofilm. To address these problems, a ratiometric, image-based quantification method has been developed based on expression of the green fluorescence protein (GFP) under the control of the c-di-GMPresponsive cdrA promoter (Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., and Tolker-Nielsen, T. (2012) Appl. Environ. Microbiol. 78, 5060-5069). The methodology uses the cyan fluorescent protein (CFP) as a biomass indicator and the GFP as a c-di-GMP reporter. Thus, the CFP/GFP ratio gives the effective c-di-GMP per biomass. A binary mask was applied to alleviate background fluorescence, and fluorescence was calibrated against known c-di-GMP concentrations. Using flow cells for biofilm formation, c-di-GMP showed a non-uniform distribution across the biofilm, with concentrated hot spots of c-di- GMP. Additionally, c-di-GMP was found to be localized at the outer boundary of mature colonies in contrast to a uniform distribution in early stage, small colonies. These data demonstrate the application of a method for the in situ, real time quantification of c-di-GMP and show that the amount of this biofilmregulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time

Environmental cues and genes involved in establishment of the superinfective Pf4 phage of Pseudomonas aeruginosa

© 2014 Hui, Mai-Prochnow, Kjelleberg, McDougald and Rice. Biofilm development in Pseudomonas aeruginosa is in part dependent on a filamentous phage, Pf4, which contributes to biofilm maturation, cell death, dispersal and variant formation, e.g., small colony variants (SCVs). These biofilm phenotypes correlate with the conversion of the Pf4 phage into a superinfection (SI) variant that reinfects and kills the prophage carrying host, in contrast to other filamentous phage that normally replicate without killing their host. Here we have investigated the physiological cues and genes that may be responsible for this conversion. Flow through biofilms typically developed SI phage approximately days 4 or 5 of development and corresponded with dispersal. Starvation for carbon or nitrogen did not lead to the development of SI phage. In contrast, exposure of the biofilm to nitric oxide, H2O2 or the DNA damaging agent, mitomycin C, showed a trend of increased numbers of SI phage, suggesting that reactive oxygen or nitrogen species (RONS) played a role in the formation of SI phage. In support of this, mutation of oxyR, the major oxidative stress regulator in P. aeruginosa, resulted in higher level of and earlier superinfection compared to the wild-type (WT). Similarly, inactivation of mutS, a DNA mismatch repair gene, resulted in the early appearance of the SI phage and this was four log higher than the WT. In contrast, loss of recA, which is important for DNA repair and the SOS response, also resulted in a delayed and decreased production of SI phage. Treatments or mutations that increased superinfection also correlated with an increase in the production of morphotypic variants. The results suggest that the accumulation of RONS by the biofilm may result in DNA lesions in the Pf4 phage, leading to the formation of SI phage, which subsequently selects for morphotypic variants, such as SCVs

'Big things in small packages: The genetics of filamentous phage and effects on fitness of their host'

© FEMS 2015. This review synthesizes recent and past observations on filamentous phages and describes how these phages contribute to host phentoypes. For example, the CTXφ phage of Vibrio cholerae encodes the cholera toxin genes, responsible for causing the epidemic disease, cholera. The CTXφ phage can transduce non-toxigenic strains, converting them into toxigenic strains, contributing to the emergence of new pathogenic strains. Other effects of filamentous phage include horizontal gene transfer, biofilm development, motility, metal resistance and the formation of host morphotypic variants, important for the biofilm stress resistance. These phages infect a wide range of Gram-negative bacteria, including deep-sea, pressure-adapted bacteria. Many filamentous phages integrate into the host genome as prophage. In some cases, filamentous phages encode their own integrase genes to facilitate this process, while others rely on host-encoded genes. These differences are mediated by different sets of 'core' and 'accessory' genes, with the latter group accounting for some of the mechanisms that alter the host behaviours in unique ways. It is increasingly clear that despite their relatively small genomes, these phages exert signficant influence on their hosts and ultimately alter the fitness and other behaviours of their hosts