Expression profiling in reference bacteria: dreams and reality

Profiling of gene expression in bacteria is now being used to uncover unknown genes expressed in particular genetic backgrounds or environmental conditions. Obtaining the best possible information from the expected avalanche of such experiments will require standardization of both experimental approach and statistical analysis. The first such experiments reveal challenges, pitfalls and reasonable solutions.     

The inverse autotransporters of Yersinia ruckeri,YrInv and YrIlm,contribute to biofilm formation and virulence

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface-exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin-like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM.     

The FTO A/T Polymorphism and Elite Athletic Performance: A Study Involving Three Groups of European Athletes

Objective

The FTO A/T polymorphism (rs9939609) is a strong candidate to influence obesity-related traits. Elite athletes from many different sporting disciplines are characterized by low body fat. Therefore, the aim of this study was to assess whether athletic status is associated with the FTO A/T polymorphism.

Subjects and Methods

A large cohort of European Caucasians from Poland, Russia and Spain were tested to examine the association between FTO A/T polymorphism (rs9939609) and athletic status. A total of 551 athletes were divided by type of sport (endurance athletes, n = 266 vs. sprint/power athletes, n = 285) as well as by level of competition (elite-level vs. national-level). The control group consisted of 1,416 ethnically-matched, non-athletic participants, all Europeans. Multinomial logistic regression analyses were conducted to assess the association between FTO A/T genotypes and athletic status/competition level.

Results

There were no significantly greater/lesser odds of harbouring any type of genotype when comparing across athletic status (endurance athletes, sprint/power athletes or control participants). These effects were observed after controlling for sex and nationality. Furthermore, no significantly greater/lesser odds ratios were observed for any of the genotypes in respect to the level of competition (elite-level vs. national-level).

Conclusion

The FTO A/T polymorphism is not associated with elite athletic status in the largest group of elite athletes studied to date. Large collaborations and data sharing between researchers, as presented here, are strongly recommended to enhance the research in the field of exercise genomics.     

Sialic Acid-Containing Lipopolysaccharides of Salmonella O48 Strains—Potential Role in Camouflage and Susceptibility to the Bactericidal Effect of Normal Human Serum

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.     

Infiltration with <Emphasis Type="Italic">Agrobacterium</Emphasis> — the method for stable transformation avoiding tissue culture

A number of in planta transformation protocols that avoid long culture under sterile conditions were developed for Arabidopsis thaliana. The most widely used methods are based on vacuum infiltration and floral dip. These methods were adapted for transformation of other species as well. Successful in planta transformations of alfalfa, radish, pakchoi and petunia were reported recently. In this short review we present several modified procedures originally developed for Arabidopsis thaliana and in some cases adapted to other species. We emphasize the crucial parameters involved in in planta transformation. We also describe here the studies attempting to shed light on the mechanisms and estimating the cellular target of transformation, which may help in transforming new plant species.     

From macro to lab-scale: Changes in bacterial community led to deterioration of EBPR in lab reactor

A laboratory scale sequencing batch reactor (SBR), fed with synthetic wastewater containing a mixture of organic compounds, was operated for nearly six months. Despite maintaining the same operational conditions, a deterioration of enhanced biological phosphorus removal (EBPR) occurred after 40 days of SBR operation. The P_rel/C_upt ratio decreased from 0.28 to 0.06 P-mol C-mol^?1, and C requirements increased from 11 to 32 mg C h^?1 g^?1 of mixed liquor suspended solids. A FISH analysis showed that the percentage of Accumulibacter in an overall community of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) dropped from 93% to 13%. An increase in abundance of Gammaproteobacteria (from 2.6% to 22%) and Alphaproteobacteria (from 1.8% to 10%) was observed. The number of Competibacter increased from 0.5% to nearly 9%. Clusters 1 and 2 of Defluviicoccus-related GAOs, not detected before deterioration, constituted 35% and 27% of Alphaproteobacteria, respectively. We concluded that lab-scale experiments should not be extended implicitly to full-scale EBPR systems because some bacterial groups are detected mainlyin lab-scale reactors. Well-defined, lab-scale operational conditions reduce the number of ecological niches available to bacteria.     

Vernalization and photoperiod-related changes in the DNA methylation state in winter and spring rapeseed

Vernalization-induced flowering is an effect of the epigenetic regulation of gene expression through DNA methylation and histone modifications. Vernalization-mediated silencing of a floral repressor through histone modifications was shown in Arabidopsis thaliana. However, for Brassica napus L., the mechanism underlying vernalization is unclear, and the roles of DNA methylation and histone modifications have not been established. This study revealed the profiles of changes in the DNA methylation state during vernalization (after 14, 35, 56 days) and the subsequent growth in long- or short-day photoperiods (after 2, 7, 14 days) in the winter and spring rapeseed using TLC and MSAP techniques. TLC analysis showed a significant decrease in the amount of 5-methylcytosine (m⁵C) in genomic DNA in both cultivars at the beginning of vernalization, but upon its termination, the winter rape showed a reduced level of m⁵C contrary to a significantly increased level in the spring rape. MSAP analysis revealed that winter and spring rapeseed differed in the MSAP loci which were demethylated/methylated in the course of the experiment and presented diverse profiles of changes in the methylation state. The winter rape showed permanent demethylations at 69.2 % of MSAP loci in the course of vernalization that were mostly preserved upon its termination. The spring rape showed similar numbers of demethylations and methylations that were mainly transient. The study provides evidence of the role of DNA methylation in vernalization for rapeseed and for the significant prevalence of demethylations at the beginning of vernalization, which is necessary for the transition to reproductive growth.