The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli

The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation has yet to be established. To study the in vivo function of IscS in Escherichia coli, a strain lacking IscS was constructed and characterized. Using this iscS deletion strain, we have observed decreased specific activities for proteins containing [4Fe-4S] clusters from soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase). A specific role for IscS in in vivo Fe-S cluster assembly was demonstrated by showing that an Fe-S cluster independent mutant of FNR is unaffected by the lack of IscS. These data support the conclusion that, via its cysteine desulfurase activity, IscS provides the sulfur that subsequently becomes incorporated during in vivo Fe-S cluster synthesis. We also have characterized a growth phenotype associated with the loss of IscS. Under aerobic conditions the deletion of IscS caused an auxotrophy for thiamine and nicotinic acid, whereas under anaerobic conditions, only nicotinic acid was required. The lack of IscS also had a general effect on the growth of E. coli because the iscS deletion strain grew at half the rate of wild type in many types of media even when the auxotrophies were satisfied.     

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.     

Initiator proteins for the assembly of the 50S subunit from Escherichia coli ribosomes.

An rRNA-binding protein that binds to the rRNA independently of other proteins during the course of ribosomal assembly is termed "assembly initiator protein." In spite of the large number of rRNA-binding proteins (more than 17 out of 32 proteins have been identified in the case of the large ribosomal subunit), only a very small number of proteins should actually initiate ribosomal assembly for theoretical reasons. Here we demonstrate that only two of the L proteins derived from the large subunit (50S) function as assembly initiator proteins. Two different techniques are used to identify these initiator proteins: reconstitution experiments with purified proteins and pulse-chase experiments during in vitro assembly. Both methods independently identify L24 and L3 as initiator proteins for the 50S assembly. The existence of two initiator proteins (not just one) resolves an apparent contradiction--namely, that on the one hand, rRNA is synthesized in excess under unfavorable growth conditions, whereas on the other hand, rRNA-binding proteins should be available for translational control.     

Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor.

The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.     

Identification of spacer tRNA genes in individual ribosomal RNA transcription units of Escherichia coli.

Transfer RNA genes ("spacer tRNA genes") are present in the spacer region between 16S and 23S rRNA genes in Escherichia coli. We have analyzed spacer tRNA genes carried by seven rRNA operons with different chromosomal locations. Six of these were isolated on plasmids and one on a transducing phage. We found that, in addition to the two previously identified genes for tRNA2Glu and tRNAIIle, there is a spacer tRNA gene which codes for tRNAIBAla. Of the seven rRNA operons studied, three had both tRNAIBAla and tRNAIIle genes, and the remaining four had the tRNA2Glu gene in their spacers. In addition, genes for tRNAIAsp were found near the distal ends of two different rRNA operons.     

Localization and characterization of the gene encoding release factor RF3 in Escherichia coli.

Two protein release factors (RFs) showing codon specificity, RF1 and RF2, are known to be required for polypeptide chain termination in Escherichia coli. A third protein component has also been described that stimulates termination in vitro, but it has remained uncertain whether this protein, RF3, participates in termination in vivo or is essential to cell growth. We report (i) the purification and N-terminal sequencing of RF3; (ii) the isolation of transposon insertion mutants similar to miaD, a suppressor of a leaky UAA mutation affecting the gene miaA, leading to enhanced nonsense suppression; (iii) the localization of the affected gene on the physical map of the chromosome; and (iv) the cloning and sequencing of the wild-type gene, providing proof that it encodes the factor RF3. We designate the gene prfC. Two transposon insertions were shown to interrupt the coding sequence of prfC, at codons 287 and 426. The enhanced nonsense suppression in the insertion mutants shows that the product participates in termination in vivo. The isolation of such mutants strongly suggests that the gene product is not essential to cell viability, though cell growth is affected. RF3 is a protein with a molecular weight of 59,460 containing 528 amino acids and displays much similarity to elongation factor EF-G, a GTP binding protein necessary for ribosomal translocation, and other GTP binding proteins known or thought to interact with the ribosome.     

Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.

S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 45 degrees C express HS genes in vitro approximately 8 times better than extracts from cells at 33 degrees C. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% of that seen in extracts from cells at 33 degrees C. These results correlate well with the levels of HS sigma factor and normal sigma factor bound to RNA polymerase. Thus, there was an 8-fold increase in the HS sigma factor and a 60% decrease in the normal sigma factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS sigma factor could be accounted for by a 3-fold increase in the level of HS sigma factor mRNA during heat induction.     

Thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase.

Reaction microcalorimetry and potentiometry have been used to define the thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from its catalytic and regulatory subunits and the linkage between assembly and proton binding. Over the pH range 7-9.5 and the temperature range 15-30 degrees C, assembly is characterized by negative enthalpy and heat capacity changes and positive entropy changes. The dependence of the enthalpy and entropy changes on pH is complex; however, the negative heat capacity change results in both quantities becoming more negative with increasing temperature. Assembly is linked to the binding of protons; the effects observed can be fit to models involving six or more ionizable groups with pK values of 7.3-7.4, 8.5-8.8, and 9.2-9.5, which ionize cooperatively. Contributions from additional groups cannot be ruled out and are in fact expected. The overall pattern of thermodynamic effects implies a complex set of intersubunit interactions. Protonation reactions and increased hydrogen bonding are likely to be the major sources of the negative enthalpy change; however, the negative heat capacity change results primarily from changes in solvent structure associated with hydrophobic and electrostatic bond formation with changes in low-frequency vibrational modes making a secondary contribution. Similarly, the relatively small entropy change observed within the temperature range examined probably reflects the balance between positive contributions from increased hydrophobic and electrostatic bonding and negative contributions from increased hydrogen bonding and damping of low-frequency vibrational modes.     

Cluster of ribosomal protein genes in Escherichia coli containing genes for proteins S6, S18, and L9.

A mutant of Escherichia coli K-12 isolated for temperature-sensitive growth was found to harbor an alteration in robosomal protein L9. Because the chromosomal location of the structural gene for this protein (rplI) was not known, we mapped the mutation by using various Hfr strains. The fine mapping of this gene by Plkc phage-mediated transductions has revealed that it forms a gene cluster at 94 min on the E. coli genetic map together with the genes coding for two other ribosomal proteins, S6 (rpsF) and S18 (rpsR). Furthermore, the region of the E. coli genetic map containing this cluster was found to be shorter than previously estimated by approximately 2 min.     

Recombination genes on the Escherichia coli sex factor specific for transposable elements.

The Escherichia coli sex factor stimulates precise excision of transposons Tn5 and Tn10 from sites either within the bacterial chromosome or within the factor itself. We have isolated two kinds of mutations that affect this activity. The ferA mutations eliminate the stimulation; the ferB mutations enhance it in the presence of FerA+. We conclude that ferA defines a sex factor gene that stimulates precise excision. The ferB mutations also specifically increase the rate of recombination between two IS3 elements on F' lac-pro (F'128) in a reaction that requires the product of recA. The stimulation of this recombination by ferB also requires an active ferA gene, which implies that the ferA gene stimulates this reaction as well as precise excision. A ferA mutation was mapped at 84.2 kilobases on the F factor, and a ferB mutation was mapped at 82.5 kilobases. The fer mutants were obtained by an approach that permits the isolation of mutants affecting precise excision.