Binding of Plasminogen to Streptococcus suis Protein Endopeptidase O Facilitates Evasion of Innate Immunity in Streptococcus suis

The Gram-positive bacterial species Streptococcus suis is an important porcine and human pathogen that causes severe life-threatening diseases associated with high mortality rates. However, the mechanisms by which S. suis evades host innate immunity remain elusive, so identifying novel virulence factors involved in immune evasion is crucial to gain control over this threatening pathogen. Our previous work has shown that S. suis protein endopeptidase O (SsPepO) is a novel fibronectin-binding protein. Here, we identified that recombinant SsPepO binds human plasminogen in a dose-dependent manner. Moreover, the binding of SsPepO and plasminogen, upon the activation of urokinase-type plasminogen activator, generated plasmin, which could cleave complement C3b, thus playing an important role in complement control. Additionally, a SspepO-deficient mutant showed impaired adherence to plasminogen as well as impaired adherence to and invasion of rat brain microvascular endothelial cells compared with the wildtype strain. We further found that the SspepO-deficient mutant was efficiently killed by human serum and blood. We also confirmed that the SspepO-deficient mutant had a lower mortality rate than the wildtype strain in a mouse model. In conclusion, these results indicate that SsPepO is a novel plasminogen-binding protein that contributes to S. suis immune evasion.

Engineered fluoride sensitivity enables biocontainment and selection of genetically-modified yeasts

Biocontainment systems are needed to neutralize genetically modified organisms (GMOs) that pose ecological threats outside of controlled environments. In contrast, benign selection markers complement GMOs with reduced fitness. Benign selection agents serve as alternatives to antibiotics, which are costly and risk spread of antibiotic resistance. Here, we present a yeast biocontainment strategy leveraging engineered fluoride sensitivity and DNA vectors enabling use of fluoride as a selection agent. The biocontainment system addresses the scarcity of platforms available for yeast despite their prevalent use in industry and academia. In the absence of fluoride, the biocontainment strain exhibits phenotypes nearly identical to those of the wildtype strain. Low fluoride concentrations severely inhibit biocontainment strain growth, which is restored upon introduction of fluoride-based vectors. The biocontainment strategy is stringent, easily implemented, and applicable to several eukaryotes. Further, the DNA vectors enable genetic engineering at reduced costs and eliminate risks of propagating antibiotic resistance.

An Optimal Lysis Time Maximizes Bacteriophage Fitness in Quasi-continuous Culture

ABSTRACTOptimality models have a checkered history in evolutionary biology. While optimality models have been successful in providing valuable insight into the evolution of a wide variety of biological traits, a common objection is that optimality models are overly simplistic and ignore organismal genetics. We revisit evolutionary optimization in the context of a major bacteriophage life history trait, lysis time. Lysis time refers to the period spanning phage infection of a host cell and its lysis, whereupon phage progeny are released. Lysis time, therefore, directly determines phage fecundity assuming progeny assembly rate is maximized. Noting that previous tests of lysis time optimality rely on batch culture, we implemented a quasi-steady state system to observe productivity of a panel of isogenic phage λ mutants differing in lysis time. We report that λ phage productivity in our experiments is maximized around an optimal lysis time of 65 min, which is the lysis time of the λ “wildtype” strain. We discuss this finding in light of recent results that lysis time variation is also minimized in the λ “wildtype” strain.

The Consequences of Egg Adaptation in the H3N2 Component to the Immunogenicity of Live Attenuated Influenza Vaccine

Adaptation in egg-passaged vaccine strains may cause reduced vaccine effectiveness due to altered antigenicity of the influenza haemagglutinin. We tested whether egg adaptation modified serum and mucosal antibody responses to the A(H3N2) component in the Live Attenuated Influenza Vaccine (LAIV). Twice as many children seroconverted to an egg-adapted H3N2 than the equivalent wildtype strain. Seroconversion to the wildtype strain was greater in children seronegative pre-LAIV, whereas higher mucosal IgA responses to wildtype antigen were observed if seropositive prior to vaccination. Sequencing of virus from nasopharyngeal swabs from 7 days post-LAIV showed low sequence diversity and no reversion of egg-adaptive mutations.

Dihydrofolate reductase (DHFR) reporter enzyme assay for Haloferax volcanii

The dihydrofolate reductase (DHFR) is routinely used a reporter enzyme for H. volcanii. The DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate and the concomitant oxidation of NADPH to NADP+. This leads to a reduction of extinction at 340 nm, which is measured to quantify the DHFR activity. To avoid background, it is best to use an H. volcanii strain with a deletion of the chromosomal dhfr gene, which is available upon request ([email protected]). However, the expression level of the chromosomal dhfr gene is very low, so that it is also possible to use the wildtype strain and subtract the DHFR background level. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the dhfr gene in a translational fusion with the gene of interest.

Polyhydroxyalkanoate granule accumulation makes optical density measurement an unreliable method for bacterial growth assessment inBurkholderia thailandensis

Optical density (OD) measurement is the standard method used in microbiology for estimating bacterial concentrations in cultures. However, most studies do not compare these measurements with viable cell counts and assume that they reflect the real cell concentration.Burkholderia thailandensiswas recently identified as a polyhydroxyalkanoate (PHA) producer. PHA biosynthesis seems to be coded by an ortholog of theCupriavidus necator phaCgene. When growing cultures of wildtype strain E264 and an isogenicphaC- mutant, we noted a difference in their OD600values, although viable cell counts indicated similar growth. Investigating the cellular morphologies of both strains, we found that under our conditions the wildtype strain was full of PHA granules, deforming the cells, while the mutant contained no granules. These factors apparently affected the light scattering, making the OD600values no longer representative of cell density. We show a direct correlation between OD600values and the accumulation of PHA. We conclude that OD measurement is unreliable for growth evaluation ofB. thailandensisbecause of PHA production. This study also suggests thatB. thailandensiscould represent an excellent candidate for PHA bioproduction. Correlation between OD measurements and viable cell counts should be verified on any study realized inB. thailandensis.

Influence of Modified Atmosphere Packaging on the Survival of Salmonella Enteritidis PT 8 on the Surface of Chilled Chicken Legs

The aim of this study was to find whether low numbers of Salmonella in the presence of natural microflora will survive on the surface of chicken legs stored in 30% CO2/70% N2 and 20% CO2/80% O2. In four experiments, a total of 240 pieces of chicken leg were inoculated with a Salmonella Enteritidis, PT 8, wildtype strain resulting in initial concentrations of approximately 4 log, 2.5 log, 1.5 log and 0.5 log cells per piece and kept under selected modified atmospheres for 14 days. Counts of Salmonella were determined by the Most Probable Number method (MPN) at days 0, 3, 7, 10 and 14 of storage. No significant increase or decrease in Salmonella numbers was observed in the atmosphere of 20% CO2/80% O2. In the atmosphere of 30% CO2/70% N2 there was a significant decrease in cell numbers at days 10 and 14; however, this decrease was proved only in experiments with an initial Salmonella concentration of 4 log and 1.5 log cells per piece. We proved that even low numbers of S. Enteritidis in the presence of natural microflora survive well on the surface of poultry stored at 3 °C in both modified atmospheres we tested. In the case of temperature abuse even products with low initial numbers of Salmonella may constitute a health risk for consumers.

Comparison of the behaviour of a transformed hygromycin resistant strain of Trichoderma atroviride (M1057hygR) with the wildtype strain (M1057)

The biocontrol isolate Trichoderma atroviride M1057 and a transformed hygromycin resistant biotype (M1057hygR) were compared using biological control rhizosphere competence and antibiosis studies to determine whether the transformed biotype performed in a similar manner to the wildtype strain In an onion growth chamber trial using soil naturally infested with the onion white rot pathogen Sclerotium cepivorum there was no significant difference (P>005) in the level of disease control given by the two T atroviride strains Similarly populations of T atroviride M1057 and M1057hygR were equivalent (P>005) in the rhizosphere of onion seedlings There was no significant difference (P>005) between the mycelial growth rates of S cepivorum when grown on agar amended with culture filtrate of T atroviride M1057 and M1057hygR Thus T atroviride M1057hygR has similar biological attributes to the wildtype isolate and can be used in future field studies looking at the population ecology of the biological control agent

Herbicide Resistance and Growth of D1 Ala251 Mutants in Chlamydomonas

We elucidated the effects of substituting seven amino acids for Ala at residue 251 of the Chlamydomonas reinhardtii D1 protein on herbicide resistance and photoautotrophic growth. Ala251 has been suggested to play a key role in the structural integrity and function of the stromal loop between transmembrane helices IV and V of D1 and has previously been shown to affect resistance to “classical” PSII specific herbicides. Sensitive and rapid microtiter assays were employed to compare herbicide resistance and photoautotrophic growth in the various mutants. Substitution of Ala251 by Ile, Leu or Val conferred resistance to the PSII herbicides atrazine, bromacil and metribuzin but not to DCMU, and impaired photoautotrophic growth in high and low light. Compared to an otherwise isogenic wildtype strain, the lie and Val mutants exhibited nearly identical levels of herbicide resistance and reduced growth while the Leu mutant had even slower growth and higher levels of herbicide resistance. In contrast Cys, Pro, Ser and Gly mutants were phenotypically indistinguishable from wildtype in terms of herbicide sensitivity and photoautotrophic doubling times. Collectively the seven Ala251 mutations differed markedly from an Ala mutant (dr-1) at the well characterized Ser264 D1 residue in terms of herbicide resistance and photoautotrophic growth

A COMPARATIVE STUDY OF THE PRODUCTION OF RNA BYROUND SPOREMUTATIONS INASCOBOLUS IMMERSUS

Ninety-nine spontaneous round spore mutants from Ascobolus immersus were grouped into two independent series designated 828 and 1034. Series 828 is a complex locus with various alleles. Total RNA characterization of three mutants from series 828 and one from series 1034 was done. Significant quantitative differences in RNA content were found between the mutant strains and the wildtype strain S2. In the case of the particular mutant strains, it seems that the expression on RNA synthesis by heteroallelic strains is identical; while it varies for an independent locus it is still responsible for the same morphological aberration in ascospore size.