Barend Kraal

ssgA is essential for sporulation of Streptomyces coelicolor A3(2) and affects hyphal development by stimulating septum formation.

The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant of Streptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novel whi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for ssgA in Streptomyces cell division.

Limited proteolysis of alfalfa mosaic virus: Influence on the structural and biological function of the coat protein

Abstract Limited tryptic digestion of intact alfalfa mosaic virus resulted in the quantitative removal of 27 amino acids from the N-terminal portions of the coat protein subunits. The release of this peptide material, which contains a relatively high number of basic residues, causes a breakdown of the bacilliform viral components into spherical nucleoprotein particles. From this it was concluded that the proteolytic cleavage interferes with protein-RNA interactions in the virus, but not with protein-protein interactions. The release of the N-terminal peptide also destroyed the capacity of the coat protein to activate the alfalfa mosaic virus genome. This supports the hypothesis that this activation is accomplished by a specific interaction of the coat protein with alfalfa mosaic virus RNAs.

Unlocking Streptomyces spp. for Use as Sustainable Industrial Production Platforms by Morphological Engineering

ABSTRACT Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.

The Elongation Factor EF-Tu and Its Two Encoding Genes

Publisher Summary The chapter discusses all of the current knowledge concerning EF-Tu. It discusses some of the recent insights that emerge from an integration of genetic, biochemical, and biophysical studies of this interesting protein. The considerable amount of structural information regarding EF-Tu and its encoding genes and the construction of plasmids harboring these genes has opened new avenues toward an understanding of EF-Tu function at the molecular level. Structural alterations in the polypeptide chain have been brought about by mutagenesis; the functional consequences will be reviewed in this chapter. The availability of E.coli mutants, specifically altered in tufA or tufB or in both, also enabled the study of the regulation of the expression of these genes. These studies have led to the conclusion that the expression of tufA and tuft3 is coordinately regulated, but that two distinct mechanisms control the expression of the two genes. They have also indicated that EF-Tu itself is involved in the regulation of the expression of tufB, but not in that of tufA. The evidence supporting the hypothesis that EF-Tu acts as an autogenous repressor inhibiting tufB expression is discussed in this chapter.

Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors.

Read‐through of nonsense codons has been studied in wild‐type Escherichia coli cells and in cells harbouring mutant species of the elongation factor EF‐Tu. The two phenomena differ essentially. Readthrough of UGA in wild‐type cells is reduced by inactivation of tufB but is restored to the original level by introducing into the cell plasmid‐borne EF‐Tu. This shows that the natural UGA leakiness is dependent on the intracellular concentration of EF‐Tu. Strains of E. coli harbouring mutant species of the elongation factor EF‐Tu suppress the nonsense codons UAG, UAA and UGA. Suppression shows a codon context dependence. It requires the combined action of two different EF‐Tu species: EF‐TuAR(Ala 375‐‐‐‐Thr) and EF‐TuBo(Gly 222‐‐‐‐Asp). Cells harbouring EF‐TuAR(Ala 375‐‐‐‐Thr) and wild‐type EF‐TuB, or wild‐type EF‐TuA and EF‐TuBo(Gly 222‐‐‐‐Asp) do not display suppressor activity. These data demonstrate that mutated tuf genes form an additional class of nonsense suppressors. The requirement for two different mutant EF‐Tu species raises the question whether translation of sense codons also occurs by the combined action of two EF‐Tu molecules on the ribosome.

tRNA-Like Structure Regulates Translation of Brome Mosaic Virus RNA

ABSTRACT For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3′ poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLSTYMV of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3′ TLSBMV (about 200 nucleotides) with determinants for tyrosylation. We discovered TLSBMV-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLSBMV tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLSBMV in trans. Intriguingly, a subdomain of the TLSBMV could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLSBMV during the BMV infection cycle.

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF‐Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala‐375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF‐Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross‐linked to Lys‐357, also located on domain II but structurally very far from Ala‐375, these data suggest that the replacements alter the relative position of domains I and II. The Ala‐375 replacements also lower the dissociation rates of the binary complexes EF‐Tu.GTP and the binding constants for EF‐Tu.GTP and Phe‐tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly‐222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF‐Tu. Gly‐222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF‐Tu to sustain protein synthesis. It increases the dissociation rate of EF‐Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF‐Tu.GDP does not bind to the ribosome, in contrast to its wild‐type counterpart. A possible explanation is now open for experimental verification.

Effects of increased and deregulated expression of cell division genes on the morphology and on antibiotic production of streptomycetes

This paper describes the effects of increased expression of the cell division genes ftsZ, ftsQ, and ssgA on the development of both solid- and liquid-grown mycelium of Streptomyces coelicolor and Streptomyces lividans. Over-expression of ftsZ in S. coelicolor M145 inhibited aerial mycelium formation and blocked sporulation. Such deficient sporulation was also observed for the ftsZ mutant. Over-expression of ftsZ also inhibited morphological differentiation in S. lividans 1326, although aerial mycelium formation was less reduced. Furthermore, antibiotic production was increased in both strains, and in particular the otherwise dormant actinorhodin biosynthesis cluster of S. lividans was activated in liquid- and solid-grown cultures. No significant alterations were observed when the gene dosage of ftsQ was increased. Analysis by transmission electron microscopy of an S. coelicolor strain over-expressing ssgA showed that septum formation had strongly increased in comparison to wild-type S. coelicolor, showing that SsgA clearly influences Streptomyces cell division. The morphology of the hyphae was affected such that irregular septa were produced with a significantly wider diameter, thereby forming spore-like compartments. This suggests that ssgA can induce a process similar to submerged sporulation in Streptomyces strains that otherwise fail to do so. A working model is proposed for the regulation of septum formation and of submerged sporulation.

Functional evidence for D- and T-loop interactions in tmRNA

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNAAla and also has a clover‐leaf structure folded similarly as in canonical tRNAs. Here we propose the L‐shape of tmRNA to be stabilized by two tertiary interactions between its D‐ and T‐loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G13 and U342. Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.

Translational regulation by modifications of the elongation factor Tu

EF-Tu fromE. coli, one of the superfamily of GTPase switch proteins, plays a central role in the fast and accurate delivery of aminoacyl-tRNAs to the translating ribosome. An overview is given about the regulatory effects of methylation, phosphorlation and phage-induced cleavage of EF-Tu on its function. During exponential growth, EF-Tu becomes monomethylated at Lys56 which is converted to Me2Lys upon entering the stationary phase. Lys56 is in the GTPase switch-1 regions (residues 49–62), a strongly conserved site involved in interactions with the nucleotide and the 5′ end of tRNA. Methylation was found to attenuate GTP hydrolysis and may thus enhance translational accuracy.In vivo 5–10% of EF-Tu is phosphorylated at Thr382 by a ribosome-associated kinase. In EF-Tu-GTP, Thr382 in domain 3 has a strategic position in the interface with domain 1; it is hydrogen-bonded to Glu117 that takes part in the switch-2 mechanism, and is close to the T-stem binding site of the tRNA, in a region known for many kirromycin-resistance mutations. Phosphorylation is enhanced by EF-Ts, but inhibited by kirromycin. In reverse, phosphorylated EF-Tu has an increased affinity for EF-Ts, does not bind kirromycin and can no longer bind aminoacyl tRNA. Thein vivo role of this reversibles modification is still a matter of speculation. T4 infection ofE. coli may trigger a phage-exclusion mechanism by activation of Lit, a host-encoded proteinase. As a result, EF-Tu is cleaved site-specifically between Gly59-Ile60 in the switch-1 region. Translation was found to drop beyond a minimum level. Interestingly, the identical sequence in the related EF-G appeared to remain fully intact. Although the Lit cleavage-mechanism may eventually lead to programmed cell death, the very efficient prevention of phage multiplication may be caused by a novel mechanisms ofin cis inhibition of late T4 mRNA translation.