Alanine-scanning mutagenesis of plasmatocyte spreading peptide identifies critical residues for biological activity

Plasmatocyte spreading peptide (PSP) is a 23-amino acid cytokine that induces a class of insect immune cells called plasmatocytes to spread on foreign surfaces. The structure of PSP consists of a disordered N terminus (residues 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide bridge between Cys(7) and Cys(19), hydrophobic interactions, and a short beta-hairpin. Structural comparisons also indicate that the core region of PSP adopts an epidermal growth factor (EGF)-like fold very similar to the C-terminal subdomain of EGF-like module 5 of thrombomodulin. To identify residues important for plasmatocyte spreading activity, we bioassayed PSP mutants in which amino acids were either replaced with alanine or deleted. Within the well-defined core of PSP, alanine replacement of Cys(7) and Cys(19) (C7.19A) eliminated all activity. Alanine replacement of Arg(13) reduced activity approximately 1000-fold in comparison to wild-type PSP, whereas replacement of the other charged residues (Asp(16), Arg(18), Lys(20)) surrounding Cys(19) diminished activity to a lesser degree. The point mutants Y11A, T14A, T22A, and F23A had activity identical or only slightly reduced to that of wild-type PSP. The mutant PSP-(7-23) lacked the entire unstructured domain of PSP and was found to have no plasmatocyte spreading activity. Surprisingly, E1A and N2A had higher activity than wild-type PSP, but F3A had almost no activity. We thus concluded that the lack of activity for PSP-(7-23) was largely due to the critical importance of Phe(3). To determine whether reductions in activity correlated with alterations in tertiary structure, we compared the C7.19A, R13A, R18A, and F3A mutants to wild-type PSP by NMR spectroscopy. As expected, the simultaneous replacement of Cys(7) and Cys(19) profoundly affected tertiary structure, but the R13A, R18A, and F3A mutants did not differ from wild-type PSP. Collectively, these results indicate that residues in both the unstructured and structured domains of PSP are required for plasmatocyte-spreading activity.     

Culture-independent study of bacterial communities in tropical river sediment

Ubiquitous microbial communities in river sediments actively govern organic matter decomposition, nutrient recycling, and remediation of toxic compounds. In this study, prokaryotic diversity in two major rivers in central Thailand, the Chao Phraya (CP) and the Tha Chin (TC) distributary was investigated. Significant differences in sediment physicochemical properties, particularly silt content, were noted between the two rivers. Tagged 16S rRNA sequencing on a 454 platform showed that the sediment microbiomes were dominated by Gammaproteobacteria and sulfur/sulfate reducing Deltaproteobacteria, represented by orders Desulfobacteriales and Desulfluromonadales together with organic degraders Betaproteobacteria (orders Burkholderiales and Rhodocyclales) together with the co-existence of Bacteroidetes predominated by Sphingobacteriales. Enrichment of specific bacterial orders was found in the clayey CP and silt-rich TC sediments, including various genera with known metabolic capability on decomposition of organic matter and xenobiotic compounds. The data represent one of the pioneered works revealing heterogeneity of bacteria in river sediments in the tropics.     

N-terminal residues of plasmatocyte-spreading peptide possess specific determinants required for biological activity.

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. The tertiary structure of PSP consists of an unstructured N terminus (residues 1-6) and a well structured core (residues 7-23). A prior study indicated that deletion of the N terminus from PSP eliminated all biological activity. Alanine substitution of the first three residues (Glu(1)-Asn(2)-Phe(3)) further indicated that only replacement of Phe(3) resulted in a loss of activity equal to the N-terminal deletion mutant. Here, we characterized structural determinants of the N terminus. Adding a hydroxyl group to the aromatic ring of Phe(3) (making a Tyr) greatly reduced activity, whereas the addition of a fluorine (p-fluoro) did not. Substitutions that changed the chirality or replaced the aromatic ring of Phe(3) with a branched aliphatic chain (making a Val) also greatly decreased activity. The addition of a methylene group to Val (making a Leu) partially restored activity, whereas the removal of a methylene group from Phe (phenyl-Gly) eliminated all activity. These results indicated that a branched carbon chain with a methylene spacer at the third residue is the minimal structural motif required for activity. The deletion of Glu(1) also eliminated activity. Additional experiments identified the charged N-terminal amine and backbone of Glu(1) as key determinants for activity.     

Comparative Analysis of Microbial Profiles in Cow Rumen Fed with Different Dietary Fiber by Tagged 16S rRNA Gene Pyrosequencing

The ruminal microbiome of cattle plays an important role not only in animal health and productivity but also in food safety and environment. Microbial profiles of rumen fluid obtained from dairy cows fed on three different fiber/starch diet compositions were characterized. Tagged 16S rRNA gene pyrosequencing and statistical analysis revealed that the dominant ruminal bacteria shared by all three sample groups belonged to phyla Bacteroidetes, Firmicutes, and Proteobacteria. However, the relative abundance of these bacterial groups was markedly affected by diet composition. In animals fed with a high fiber diet, the fibrolytic and cellulolytic bacteria Lachnospiraceae, Ruminococcaceae, and Fibrobacteraceae were found in highest abundance compared with animals fed other diets with lower fiber content. The polysaccharide-degrading Prevotellaceae and Flavobacteriaceae bacteria were most abundant in the rumen of cows fed on diet with the highest starch content. These data highlight the ruminal microbiome’s ability to adapt to feed composition and also provide a basis for the development of feed formulation systems designed to improve livestock productivity.