Jesus Garcia-Gil

Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes

Abstract. The phylogenetic diversity of green nonsulfur bacteria in nine stratified freshwater lakes was investigated. A set of oligonucleotide primers was developed that permitted the selective amplification of 16S rRNA gene sequences of this group. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced, which yielded a total of 19 novel sequence types. Ten of the sequences were related to those of different cultivated members of the Chloroflexus assemblage, whereas nine fell into the T78 group of environmental clones. For the latter subgroup of the green nonsulfur bacteria, no molecular isolate from freshwater plankton has been reported so far. Several of the sequence types occurred in more than one lake, indicating that not only relatives of the Chloroflexus assemblage, but also bacteria of the clone T78 group represent indigenous bacteria of nonthermal stratified freshwater ecosystems. Our results indicate that the natural diversity in the phylum of the green nonsulfur bacteria has been significantly underestimated in the past.

Similarity and Divergence among Adherent-Invasive Escherichia coli and Extraintestinal Pathogenic E. coli Strains

ABSTRACT Adherent-invasive Escherichia coli (AIEC) pathovar strains, which are associated with Crohns disease, share many genetic and phenotypic features with extraintestinal pathogenic E. coli (ExPEC) strains, but little is known about the level of genetic similarity between the two pathovars. We aimed to determine the frequency of strains with the “AIEC phenotype” among a collection of ExPEC strains and to further search for a common phylogenetic origin for the intestinal and extraintestinal AIEC strains. The adhesion, invasion, and intramacrophage replication capabilities (AIEC phenotype) of 63 ExPEC strains were determined. Correlations between virulence genotype and AIEC phenotype and between intestinal/extraintestinal origin, serotype, and phylogroup were evaluated for the 63 ExPEC and 23 intestinal AIEC strains. Phylogenetic relationships between extraintestinal and intestinal AIEC strains were determined using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. Only four (6.35%) ExPEC strains, belonging to the O6:H1, O83:H1, and O25:H4 serotypes, were classified as having an AIEC phenotype. These strains were found to be genetically related to some intestinal AIEC strains of the same serotypes as revealed by MLST. No particular virulence gene sets correlated with the intestinal/extraintestinal origin of the strains or with the AIEC phenotype, whereas the gene sets did correlate with the serogroup. We identified two intestinal AIEC strains and one extraintestinal AIEC strain belonging to the O25:H4 serotype that also belonged to the emerging and virulent clonal group ST131. In conclusion, the ExPEC and AIEC pathovars share similar virulence gene sets, and certain strains are phylogenetically related. However, the majority of ExPEC strains did not behave like AIEC strains, thus confirming that the AIEC pathovar possesses virulence-specific features that, to date, are detectable only phenotypically.

Identification of the bacteriochlorophyll homologues of Chlorobium phaeobacteroides strain UdG6053 grown at low light intensity

Detailed APCI LC-MS/MS analysis using an improved HPLC separation reveals the green sulphur bacterium Chlorobium phaeobacteroides strain UdG6053 to contain a wider range of distinct bacteriochlorophyll homologues than has been previously recognised in Chlorobiaceae. The diversity in the homologue distribution is confirmed as arising from differences in the extent of alkylation of the macrocycle and variation in the nature of the esterifying alcohol and a novel series of bacteriochlorophyll structures has been recognised. Homologues containing esterifying alcohols other than farnesol, a number of which have not previously been reported in Chlorobiaceae, are present in high relative abundance. Confirmation of the structures of the esterifying alcohols has been obtained by hydrolysis and analysis by GC-MS.

Fast energy transfer between BChl d and BChl c in chlorosomes of the green sulfur bacterium Chlorobium limicola.

We have studied energy transfer in chlorosomes of Chlorobium limicola UdG6040 containing a mixture of about 50% bacteriochlorophyll (BChl) c and BChl d each. BChl d-depleted chlorosomes were obtained by acid treatment. The energy transfer between the different pigment pools was studied using both steady-state and time-resolved fluorescence spectroscopy at room temperature and low temperature. The steady-state emission of the intact chlorosome originated mainly from BChl c, as judged by comparison of fluorescence emission spectra of intact and BChl d-depleted chlorosomes. This indicated that efficient energy transfer from BChl d to BChl c takes place. At room temperature BChl c/d to BChl a excitation energy transfer (EET) was characterized by two components of 27 and 74 ps. At low temperature we could also observe EET from BChl d to BChl c with a time constant of approximately 4 ps. Kinetic modeling of the low temperature data indicated heterogeneous fluorescence kinetics and suggested the presence of an additional BChl c pool, E790, which is more or less decoupled from the baseplate BChl a. This E790 pool is either a low-lying exciton state of BChl c which acts as a trap at low temperature or alternatively represents the red edge of a broad inhomogeneous absorption band of BChl c. We present a refined model for the organization of the spatially separated pigment pools in chlorosomes of Cb. limicola UdG6040 in which BChl d is situated distal and BChl c proximal with respect to the baseplate.

Evidence for spatially separate bacteriochlorophyll c and bacteriochlorophyll d pools within the chlorosomal aggregate of the green sulfur bacterium Chlorobium limicola

We have studied the organization of the bacteriochlorophylls (BChl) in isolated chlorosomes of the green sulfur bacterium Chlorobium limicola UdG6040 containing about 50% BChl d and BChl c each. When the chlorosomes are treated in acidic buffer (pH 3.0) two phases in the conversion from BChl to bacteriopheophytin (BPhe) are observed as evidenced by the changes in the absorption spectrum. In the early phase the pheophytinization of BChl d occurs much faster than that of BChl c. In the later phase BChl c and BChl d are converted at similar rates. The delayed BChl c conversion observed in intact chlorosomes is interpreted in terms of spatial separation within the same chlorosome that makes BChl d more accessible to reaction with acid than BChl c. This was supported by acid treatment of in vitro pigment-lipid aggregates which showed that the pheophytinization of aggregates consisting of only BChl c or BChl d takes place with the same rate. Moreover in mixed in vitro aggrega tes where BChl d and BChl c are supposed to be scrambled the two pigments are converted to BPhe simultaneously. Acid treatment of hexanol exposed chlorosomes indicates that the spatial separation of BChl d and BChl c within the chlorosomes is maintained even if the excitonic interaction between BChls has been disturbed by hexanol. Based on these findings it is suggested that BChl d and BChl c in the chlorosome are located distal and proximal, respectively, relative to the chlorosome baseplate.

Anti-tumour Necrosis Factor Treatment with Adalimumab Induces Changes in the Microbiota of Crohn's Disease

BACKGROUND The composition of the intestinal microbiota is altered in Crohns disease [CD] patients. The objective of this study was to evaluate the qualitative and quantitative changes in the microbiota of CD patients in 3 months of treatment with adalimumab [ADA], and determine whether or not these changes are produced towards the recovery of the normal, healthy-like microbiota. METHODS The microbiota composition, and the Faecalibacterium prausnitzii / Escherichia coli quantitative relationship as dysbiosis indicator, were studied at baseline [T0], one month [T1], and 3 months [T3] after starting treatment using a polymerase chain reaction-denaturing gradient gel electrophoresis [PCR-DGGE] of 16S rRNA gene fragments and quantitative PCR, respectively, in rectal mucosal biopsies from 15 CD patients and four healthy subjects. RESULTS T0 and T3 fingerprints were different in all patients; whereas T1 and T3 presented similar patterns. Recovered phylogroups were Firmicutes [79.1%], Bacteroides [12.5%], and Actinobacteria [6.25%]. The prevalence of E. coli decreased during treatment. Relative E. coli loads in CD samples were significantly reduced at every analysed step [T1 and T3] [p < 0.005] whereas no significant changes were observed in relative F. prausnitzii counts. CONCLUSION Treatment with ADA induces short-term changes in the microbiota composition which seem to parallel the partial recovery of the gut bacterial ecology, with recovery parameters tending to eubiosis recovery. The quantitative determination of dysbiosis-representative bacteria, such as E. coli, may provide a fast and reliable indicator of the healing state of the intestinal mucosa.

Adherent-Invasive Escherichia coli Phenotype Displayed by Intestinal Pathogenic E. coli Strains from Cats, Dogs, and Swine

ABSTRACT The adherent-invasive Escherichia coli (AIEC) pathotype, which has been associated with Crohns disease, shows similar traits to human and animal extraintestinal pathogenic E. coli (ExPEC) with respect to their phylogenetic origin and virulence gene profiles. Here, we demonstrate that animal ExPEC strains generally do not share the AIEC phenotype. In contrast, this phenotype is very frequent among animal intestinal pathogenic E. coli (InPEC) strains, particularly of feline and canine origin, that genetically resemble ExPEC. These results strengthen the particular identity and disease specificity of the AIEC pathotype and the putative role animals might play in the transmission of AIEC-like strains to humans.

Phosphorus deficiency and kinetics of alkaline phosphatase in isolates and natural populations of phototrophic sulphur bacteria.

Phosphorus deficiency was analysed in the oxic-anoxic gradient of the karstic sulphurous lakes Vilar and Sisó during the stratification period. The distribution of planktonic photosynthetic populations along a vertical gradient coincided with an increase in alkaline phosphatase activity (APA). A multiple stepwise correlation analysis of data yielded a positive correlation of APA with planktonic phototrophic populations. MUF-P hydrolysis saturation curves were used to estimate the enzyme kinetics. High-affinity phosphatases (i.e. low K(M) saturation constant) coincided with the oxic-anoxic gradient and progressively declined through both the epi- and the hypolimnion. Changes in the K(M) values are likely due to phosphate inhibition and the contribution of different planktonic populations in the induction of alkaline phosphatases. Extremely low organic phosphorus turnover times (as short as 0.37 h) were also estimated in the gradient zone, indicating a high dependence of the bacterial populations on organic phosphate esters. Phosphatase saturation kinetics revealed K(M) values from 0.53 to 8.45 microM MUF-P, perfectly matching those found in the isolates Thiocapsa sp. UdG3513, Chlorobium limicola UdG6050 and UdG6055 and Chlorobium phaeobacteroides CL1401. The results obtained indicate that a relevant adaptation of sulphur phototrophic bacteria may occasionally face periods of phosphate limitation despite thriving in nutrient-rich anoxic waters.

Structure and Function of Chlorosomes of Chlorobium Limicola UdG 6040 Containing Both Bchl c and Bchl d

The organization of light-harvesting pigments is essential for the funneling of excitation energy towards the photosynthetic reaction center. In green bacteria the light-harvesting chlorosomes are oblong flattened bodies attached the inner side of the cytoplasmic membrane. The chlorosome is surrounded by a lipid monolayer and filled with BChl c, d or e organized in large rod shaped aggregates. Moreover a small amount of BChl a is situated in the so-called base plate, the part of the lipid envelope facing the cytoplasmic membrane. The chlorosomal BChl a functions as an intermediate acceptor in the energy transfer from the bulk BChls towards the reaction centers [1].

P319 - Biofilm formation of adherent-invasive Escherichia coli (AIEC): a novel phenotypic trait that could be related to AIEC pathogenesis

P319 Biofilm formation of adherent-invasive Escherichia coli (AIEC): a novel phenotypic trait that could be related to AIEC pathogenesis M. Martinez-Medina1 *, P. Naves2, J. Blanco3, X. Aldeguer4, M. Blanco3, C. Ponte2, F. Soriano2, J. Garcia-Gil1. 1Laboratori de Microbiologia Molecular, Departament de Biologia, University of Girona, Girona, Spain, 2Department of Medical Microbiology and Antimicrobial Chemotherapy, Fundacion Jimenez Diaz-Capio, Madrid, Spain, 3Laboratorio de Referencia de E.coli, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain, 4Departament de Gastroenterologia, Hospital Dr. Josep Trueta, Girona, Spain