Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus.

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins' folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S-RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach.

For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here we describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections.

Production of p-amino-L-phenylalanine (L-PAPA) from glycerol by metabolic grafting of Escherichia coli.

BACKGROUND: The non-proteinogenic aromatic amino acid, p-amino-L-phenylalanine (L-PAPA) is a high-value product with a broad field of applications. In nature, L-PAPA occurs as an intermediate of the chloramphenicol biosynthesis pathway in Streptomyces venezuelae. Here we demonstrate that the model organism Escherichia coli can be transformed with metabolic grafting approaches to result in an improved L-PAPA producing strain. RESULTS: Escherichia coli K-12 cells were genetically engineered for the production of L-PAPA from glycerol as main carbon source. To do so, genes for a 4-amino-4-deoxychorismate synthase (pabAB from Corynebacterium glutamicum), and genes encoding a 4-amino-4-deoxychorismate mutase and a 4-amino-4-deoxyprephenate dehydrogenase (papB and papC, both from Streptomyces venezuelae) were cloned and expressed in E. coli W3110 (lab strain LJ110). In shake flask cultures with minimal medium this led to the formation of ca. 43 ± 2 mg l-1 of L-PAPA from 5 g l-1 glycerol. By expression of additional chromosomal copies of the tktA and glpX genes, and of plasmid-borne aroFBL genes in a tyrR deletion strain, an improved L-PAPA producer was obtained which gave a titer of 5.47 ± 0.4 g l-1 L-PAPA from 33.3 g l-1 glycerol (0.16 g L-PAPA/g of glycerol) in fed-batch cultivation (shake flasks). Finally, in a fed-batch fermenter cultivation, a titer of 16.7 g l-1 L-PAPA was obtained which is the highest so far reported value for this non-proteinogenic amino acid. CONCLUSION: Here we show that E. coli is a suitable chassis strain for L-PAPA production. Modifying the flux to the product and improved supply of precursor, by additional gene copies of glpX, tkt and aroFBL together with the deletion of the tyrR gene, increased the yield and titer.

A simple and reliable method to conduct and monitor expression cassette integration into the Escherichia coli chromosome.

We report a method for the integration of expression cassettes into the Escherichia coli chromosome using rare and dispensable sugar degradation gene loci as sites for integration. Clones carrying successfully recombined DNA fragments in the chromosome are easily screened using a solid differential medium containing the respective sugar compound. As an example for the heterologous expression of a complex natural product biosynthesis pathway, we show the stepwise chromosomal integration of the zeaxanthin biosynthesis pathway from Pantoea ananatis into E. coli.

Homologous npdGI genes in 2,4-dinitrophenol- and 4-nitrophenol-degrading Rhodococcus spp.

Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F(420) reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F(420). Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F(420)-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F(420)-dependent glucose-6-phosphate dehydrogenases and F(420)-dependent N(5),N(10)-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F(420)-dependent enzymes involved in catabolism.

npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation.

Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol (picric acid) or 2,4-dinitrophenol (2,4-DNP) as sole nitrogen source. A gene cluster involved in picric acid degradation was recently identified. The functional assignment of three of its genes, npdC, npdG and npdI, and the tentative functional assignment of a fourth one, npdH, is reported. The genes were expressed in Escherichia coli as His-tag fusion proteins that were purified by Ni-affinity chromatography. The enzyme activity of each protein was determined by spectrophotometry and HPLC analyses. NpdI, a hydride transferase, catalyses a hydride transfer from reduced F420 to the aromatic ring of picric acid, generating the hydride sigma-complex (hydride Meisenheimer complex) of picric acid (H(-)-PA). Similarly, NpdI also transformed 2,4-DNP to the hydride sigma-complex of 2,4-DNP. A second hydride transferase, NpdC catalysed a subsequent hydride transfer to H(-)-PA, to produce a dihydride sigma-complex of picric acid (2H(-)-PA). All three reactions required the activity of NpdG, an NADPH-dependent F420 reductase, for shuttling the hydride ions from NADPH to F420. NpdH converted 2H(-)-PA to a hitherto unknown product, X. The results show that npdC, npdG and npdI play a key role in the initial steps of picric acid degradation, and that npdH may prove to be important in the later stages.