Growth and flagellation of Vibrio fischeri during initiation of the sepiolid squid light organ symbiosis

A pure culture of the luminous bacterium Vibrio fischeri is maintained in the light-emitting organ of the sepiolid squid Euprymna scolopes. When the juvenile squid emerges from its egg it is symbiont-free and, because bioluminescence is part of an anti-predatory behavior, therefore must obtain a bacterial inoculum from the surrounding environment. We document here the kinetics of the process by which newly hatched juvenile squids become infected by symbiosis-competent V. fischeri. When placed in seawater containing as few as 240 colony-forming-units (CFU) per ml, the juvenile became detectably bioluminescent within a few hours. Colonization of the nascent light organ was initiated with as few as 1 to 10 bacteria, which rapidly began to grow at an exponential rate until they reached a population size of approximately 10⁵ cells by 12 h after the initial infection. Subsequently, the number of bacteria in the established symbiosis was maintained essentially constant by a combination of both a >20-fold reduction in bacterial growth rate, and an expulsion of excess bacteria into the surrounding seawater. While V. fischeri cells are normally flagellated and motile, these bacteria did not elaborate these appendages once the symbiosis was established; however, they quickly began to synthesize flagella when they were removed from the light organ environment. Thus, two important biological characteristics, growth rate and flagellation, were modulated during establishment of the association, perhaps as part of a coordinated series of symbiotic responses.     

Population Structure of Vibrio fischeri within the Light Organs of Euprymna scolopes Squid from Two Oahu (Hawaii) Populations

We resolved the intraspecific diversity of Vibrio fischeri, the bioluminescent symbiont of the Hawaiian sepiolid squid Euprymna scolopes, at two previously unexplored morphological and geographical scales. These scales ranged from submillimeter regions within the host light organ to the several kilometers encompassing two host populations around Oahu. To facilitate this effort, we employed both novel and standard genetic and phenotypic assays of light-organ symbiont populations. A V. fischeri-specific fingerprinting method and five phenotypic assays were used to gauge the genetic richness of V. fischeri populations; these methods confirmed that the symbiont population present in each adult host's light organ is polyclonal. Upon statistical analysis of these genetic and phenotypic population data, we concluded that the characteristics of symbiotic populations were more similar within individual host populations than between the two distinct Oahu populations of E. scolopes, providing evidence that local geographic symbiont population structure exists. Finally, to better understand the genesis of symbiont diversity within host light organs, the process of symbiosis initiation in newly hatched juvenile squid was examined both experimentally and by mathematical modeling. We concluded that, after the juvenile hatches, only one or two cells of V. fischeri enter each of six internal epithelium-lined crypts present in the developing light organ. We hypothesize that the expansion of different, crypt-segregated, clonal populations creates the polyclonal adult light-organ population structure observed in this study. The stability of the luminous-bacterium-sepiolid squid mutualism in the presence of a polyclonal symbiont population structure is discussed in the context of contemporary evolutionary theory.     

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.

Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.     

Phylogeny and fitness of Vibrio fischeri from the light organs of Euprymna scolopes in two Oahu,Hawaii populations

The evolutionary relationship among Vibrio fischeri isolates obtained from the light organs of Euprymna scolopes collected around Oahu, Hawaii, were examined in this study. Phylogenetic reconstructions based on a concatenation of fragments of four housekeeping loci (recA, mdh, katA, pyrC) identified one monophyletic group (‘Group-A'') of V. fischeri from Oahu. Group-A V. fischeri strains could also be identified by a single DNA fingerprint type. V. fischeri strains with this fingerprint type had been observed to be at a significantly higher abundance than other strains in the light organs of adult squid collected from Maunalua Bay, Oahu, in 2005. We hypothesized that these previous observations might be related to a growth/survival advantage of the Group-A strains in the Maunalua Bay environments. Competition experiments between Group-A strains and non-Group-A strains demonstrated an advantage of the former in colonizing juvenile Maunalua Bay hosts. Growth and survival assays in Maunalua Bay seawater microcosms revealed a reduced fitness of Group-A strains relative to non-Group-A strains. From these results, we hypothesize that there may exist trade-offs between growth in the light organ and in seawater environments for local V. fischeri strains from Oahu. Alternatively, Group-A V. fischeri may represent an example of rapid, evolutionarily significant, specialization of a horizontally transmitted symbiont to a local host population.     

Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection

Using a novel cDNA microarray prepared from sources of actively responding immune system cells, we have investigated the changes in gene expression in the target tissue during the early stages of infection of neonatal chickens with infectious bursal disease virus. Infections of two lines of chickens previously documented as genetically resistant and sensitive to infection were compared in order to ascertain early differences in the response to infection that might provide clues to the mechanism of differential genetic resistance. In addition to major changes that could be explained by previously described changes in infected tissue, some differences in gene expression on infection, and differences between the two chicken lines, were observed that led to a model for resistance in which a more rapid inflammatory response and more-extensive p53-related induction of apoptosis in the target B cells might limit viral replication and consequent pathology. Ironically, the effect in the asymptomatic neonatal infection is that more-severe B-cell depletion is seen in the more genetically resistant chicken. Changes of expression of many chicken genes of unknown function, indicating possible roles in the response to infection, may aid in the functional annotation of these genes.     

Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 Å and 2.0 Å resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical “canonical” fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.     

Mutants of a lovastatin-hyperproducingAspergillus terreus deficient in the production of sulochrin

Summary A lovastatin-hyperproducing culture ofAspergillus terreus was shown to produce several co-metabolites extracted from whole broth. The predominant co-metabolite was the benzophenone, sulochrin, reported to arise from a polyketide biosynthetic pathway. This compound was targeted for elimination by classical mutagenesis and screening. A surface culture method employing microtiter, plates was used to ferment mutants for the primary screen. Qualitative determinations of lovastatin and sulochrin production were achieved by high-performance thin-layer chromatography. A mutant, strain AH6, which produced lovastatin titers equivalent to the parent culture and no detectable sulochrin was isolated. In addition, a lovastatin-hyperproducing mutant designated CB4 was capable of producing 16% more lovastatin and 30% less sulochrin than the parent culture in shake flask fermentations. In a pilot-scale 250-gallon fermentation, strain CB4 gave a 20% increase in lovastatin titer while producing 83% less sulochrin than the parent culture.     

Characterisation of Peptide Microarrays for Studying Antibody-Antigen Binding Using Surface Plasmon Resonance Imagery

Background

Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules.

Methodology/Principal Findings

We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65) and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van''t Hoff type analyses to provide comparative ΔG, ΔS and ΔH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding.

Conclusions

The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen interaction where good structural, mechanistic and immunological data are available. Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods. Furthermore, our data indicate that SPRi is well suited to a multiplexed immunoassay using GAD65 proteins, and may be applicable to other biomarkers.     

Reduction of ferric green rust by Shewanella putrefaciens

AIMS: To reduce carbonated ferric green rust (GR*) using an iron respiring bacterium and obtain its reduced homologue, the mixed Fe(II)-Fe(III) carbonated green rust (GR). METHODS AND RESULTS: The GR* was chemically synthesized by oxidation of the GR and was incubated with Shewanella putrefaciens cells at a defined [Fe(III)]/[cell] ratio. Sodium methanoate served as the sole electron donor. The GR* was quickly transformed in GR (iron reducing rate = 8.7 mmol l(-1) h(-1)). CONCLUSIONS: Ferric green rust is available for S. putrefaciens respiration as an electron acceptor. The reversibility of the GR redox state can be driven by bacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work suggests that GRs would act as an electronic balance in presence of bacteria. It provides also new perspectives for using iron reducing bacterial activity to regenerate the reactive form of GR during soil or water decontamination processes.