Bacillus subtilis rRNA promoters are growth rate regulated in Escherichia coli.

rRNA promoters from the rrnB locus of Bacillus subtilis and from the rrnB locus of Escherichia coli were fused to the gene for chloramphenicol acetyltransferase (CAT). The level of expression of CAT in E. coli showed growth rate dependence when the CAT gene was linked to either E. coli or B. subtilis tandem promoters. The downstream promoter of the tandem Bacillus pair showed growth rate regulation, while the upstream promoter did not, whereas for the E. coli tandem promoters, only the upstream promoter was growth rate regulated.     

Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis

Summary Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNA originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.Abbreviations kop kilobase pairs - OcTase ornithine carbamoyl transferase - CPSase carbamoyl phosphate synthetase     

Expression of the genes for lysine biosynthesis of Bacillus subtilis in Escherichia coli cells

Hybrid plasmids pLRS33 and pLRB4 containing Bac. subtilis genes coding lysin biosynthesis were subjected to genetical analysis. It is shown that after pLRS33- and pLRB4- transformation of E. coli strains, auxotrophic relative to lysin and diaminopimelic acid, there occurs complementation of dapA, dapB, dapC, dapD, dapE, lysA mutations by plasmid pLRS33 and of dapC, dapB, lysA mutations by plasmid pLRB4. The plasmids are studied for their influence on the level of lysin and its precurror synthesis in E. coli strains.     

The Bacillus subtilis addAB genes are fully functional in Escherichia coli

An Escherichia coli recBCD deletion mutant was transformed with plasmids containing the Bacillus subtilis add genes. The transformants had relatively high ATP-dependent exonuclease- and ATP-dependent helicase activities, and their viability, the ability to repair u.v.-damaged DNA and the recombination in conjugation were nearly completely restored. The B. subtilis Add enzyme did not show Chi-activity in phage lambda recombination. The individual B. subtilis Add proteins were not able to form an enzymatically active complex with the E. coli RecB,C,D proteins, and they could not complement the recB,C,D deficiency. Evidence is presented that only two subunits are involved in the B. subtilis ATP-dependent exonuclease. This is in contrast to E. coli in which the RecBCD enzyme consists of three subunits.     

Serine biosynthesis and its regulation in Bacillus subtilis

Cell-free extracts of Bacillus subtilis strains GSY and 168 convert (14)C-phosphoglycerate to (14)C-serine phosphate and (14)C-serine. These reactions indicate a functional phosphorylated pathway for serine biosynthesis in these cells. The addition of serine to the incubation mixture inhibited the formation of both radioactive products. Extracts of mutant strains that require serine for growth lacked the capacity to synthesize serine phosphate, confirming that the phosphorylated pathway was the only functional pathway available for serine synthesis. Serine phosphate phosphatase and phosphoglycerate dehydrogenase activity were demonstrated in cell extracts, and the phosphoglycerate dehydrogenase was shown to be inhibited specifically by l-serine. The extent of serine inhibition increased when the temperature was raised from 25 to 37 C, and the thermal stability of the enzyme was enhanced by the presence of the inhibitor serine or the coenzyme reduced nicotinamide adenine dinucleotide. At 37 C the curve representing the relationship between phosphoglycerate concentration and enzyme velocity was biphasic, and the serine inhibition which was competitive at low substrate concentrations became noncompetitive at higher concentrations.     

Independent regulation of transport and biosynthesis of arginine in Escherichia coli K-12.

From an arginine auxotrophic strain, a mutant was isolated which is able to utilize d-arginine as a source of l-arginine and shows a high sensitivity to inhibition of growth by canavanine. Transport studies revealed a four- to five-fold increased uptake of arginine and ornithine in cells from the mutant strain. The kinetics of entry of arginine and ornithine evidenced elevated maximal influx values for the arginine- and ornithine-specific transport systems. A close parallel between arginine transport activity and arginine binding activity with one arginine-specific binding periplasmic protein in the mutant strongly suggests that such binding protein is a component of the arginine-specific permease. The affinity between arginine and the binder, isolated from the mutant cells, as well as the electrophoretic mobility of the protein, remain unchanged. The enhanced transport activity of arginine and ornithine with mutant cells is insensitive to repression by arginine or ornithine, whereas the biosynthesis of arginine-forming enzymes is normally repressible. When transport activity was examined in strains with mutations leading to derepression of arginine biosynthesis, the regulation of arginine transport was found to be normal. These studies support the conclusion that arginine transport and arginine biosynthesis, in Escherichia coli K-12, are not regulated in a concerted manner, although both systems may have components in common.     

MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli

Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.     

Identification and chromosomal distribution of DNA sequence segments conserved since divergence of Escherichia coli and Bacillus subtilis

DNA sequence segments conserved since divergence of Escherichia coli and Bacillus subtilis were identified, using the GenBank sequence database. Chromosomal locations of the conserved segments were compared between the two bacteria, and the following three features were observed. (1) Although the two genomes are nearly identical in size, chromosomal arrangements of the conserved segments are considerably different from each other. (2) In many cases, chromosomal locations of a conserved segment in the two species have deviated from each other by a multiple of 60°. (3) There are many instances in which a contiguous segment in one genome is split into two or more segments located at distinct positions in the other genome, and these split segments were found to tend to lie on the E. coli or B. subtilis genome separated by distances of multiples of 60°. On the basis of these observations, genome organizations of the two bacteria were discussed in terms of genome doublings as well as random chromosomal rearrangements.     

Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfpo and Escherichia coli entD genes.

In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin. We isolated two sets of B. subtilis DNA sequences that complemented the mutations of several E. coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes. One set contained DNA sequences that complemented only an entD mutation. The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations. The two sets of DNA sequences did not appear to overlap. AB. subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics. These data indicate that (i) at least five of the siderophore biosynthesis genes of B. subtilis can function in E. coli, (ii) the genetic organization of these siderophore genes in B. subtilis is similar to that in E. coli, and (iii) the B. subtilis entD homolog is required for efficient growth in low-iron medium. The nucleotide sequence of the B. subtilis DNA contained in plasmid pENTA22, a clone expressing the B. subtilis entD homolog, revealed the presence of at least two genes. One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B. subtilis and which is highly homologous to the E. coli entD gene. We present evidence that the E. coli entD and B. subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules.     

δ-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA

Abstract Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of δ-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis . The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.     

Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 microg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p(R) promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

Effect of Microwaves on Escherichia coli and Bacillus subtilis

Suspensions of Escherichia coli and Bacillus subtilis spores were exposed to conventional thermal and microwave energy at 2,450 MHz. The degrees of inactivation by the different energy sources were compared quantitatively. During the transient heating period by microwave energy, approximately a 6 log cycle reduction in viability was encountered for E. coli. This reduction was nearly identical to what is expected for the same time-temperature exposure to conventional heating. Heating of B. subtilis spores by conventional and microwave energy was also carried out at 100 C, in ice and for transient heating. The degree of inactivation by microwave energy was again identical to that by conventional heating. In conclusion, inactivation of E. coli and B. subtilis by exposure to microwaves is solely due to the thermal energy, and there is no per se effect of microwaves.     

Regulatory gene mutations affecting arginine biosynthesis in Escherichia coli

Summary Mutants of Escherichia coli with altered regulation of arginine biosynthesis were isolated. The alterations in all of the mutants with increased levels of the biosynthetic enzymes were found to map in the argR locus. The mutants were grouped into three classes based on their effect on the regulatory behavior. Complementation studies with stable merodiploid strains demonstrated that the derepressed synthesis in the mutants was recessive to wild-type regulation.     

Regulation of arginine biosynthesis,catabolism and transport in Escherichia coli

Amino Acids - Already very early, the study of microbial arginine biosynthesis and its regulation contributed significantly to the development of new ideas and concepts. Hence, the term...