UV-light-induced mutability in Salmonella strains containing the umuDC or the mucAB operon: evidence for a umuC function

Multicopy plasmids carrying either the umuDC operon of Escherichia coli or its analog mucAB operon, were introduced into Ames Salmonella strains in order to analyze the influence of UmuDC and MucAB proteins on repair and mutability after UV irradiation. It was found that in uvr+ bacteria, plasmid pICV80:mucAB increased the frequency of UV-induced His+ revertants whereas pSE117:umuDC caused a smaller increase in UV mutagenesis. In delta uvrB bacteria, the protective role of pSE117 against UV killing was weak, and there was a great reduction in the mutant yield. In contrast, in these cells, pICV80 led to a large increase in both cell survival and mutation frequency. These results suggest that in Salmonella, as in E. coli, MucAB proteins mediate UV mutagenesis more efficiently than UmuDC proteins do. Plasmid pICV84:umuD+ C- significantly increased UV mutagenesis of TA2659: delta uvrB cells whereas in them, pICV77:mucA+ B- had no effect on mutability indicating the presence in Salmonella TA2659 of a gene functionally homologous to umuC.     

Reduced lactose operon expression in an E. coli mutant lacking leucyl-tRNA:protein transferase

A mutant of Escherichia coli deficient in L-leucyl-tRNA:protein leucyl transferase was found to have reduced specific activities for all three lactose operon proteins when compared to its parent or revertant strains. The effect was most pronounced in cells grown with glucose as the carbon source. Under these conditions, there was an additional polar effect, in which the distal gene product showed the greatest reduction in activity.     

Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168.

The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells. Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX. In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis. It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B. subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E. coli proteins. The B. subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection. The B. subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B. subtilis LH45, which is a subtilin-producing mutant of B. subtilis 168. B. subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B. subtilis ATCC 6633 (W. Liu and J. N. Hansen, J. Bacteriol. 173:7387-7390, 1991). Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633. Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium. Conversely, a mutant (LH45 deltaproU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine. We conclude that the proU-like locus in B. subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.     

Heterospecific expression of misrepair-enhancing activity of mucAB in Escherichia coli and Bacillus subtilis.

Enterobacterial plasmid genes mucAB, which possess error-prone repair activity, were cloned and sequenced independently of a sequence previously determined (K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, and G.C. Walker, Proc. Natl. Acad. Sci. USA 82:4331-4335, 1985). The survival- and mutation-enhancing activities of mucAB ligated to the MLSr promoter of a Bacillus subtilis plasmid in the shuttle vector pTE22R were expressed in B. subtilis as well as in Escherichia coli after mutagenic treatment. mucAB fragments with 5' deletions of various lengths up to the base sequence encoding Ala-26-Gly-27, the putative RecA-mediated cleavage site of the MucA protein, showed mutation-enhancing activity for noninducible lexA3 E. coli when ligated to the MLSr promoter in frame. This activity was lost by extending the deletion downstream. The formations of MucA and MucB proteins in B. subtilis and E. coli were demonstrated by Western blot (immunoblot) analysis. MucA cleavage in Rec+ B. subtilis was observed only after treatment with an alkylating agent and was not observed in RecA- and RecE- strains, whereas in E. coli cleavage was observed in Rec+ cells after treatment with either mitomycin C or an alkylating agent but was not detected in RecA- cells. Common activity of B. subtilis Rec and E. coli RecA in the induction of mutants is suggested.     

Aminotransferase from a deletion mutant in the histidine operon

The imidazolylacetolphosphate:l-glutamate aminotransferase from the deletion mutant hisHB22 has been partially characterized. Although genetic studies have not yet shown the deletion to involve the structural information for this enzyme, physical studies indicate that an abnormal enzyme is produced. Evidence is presented which, together with previous data on the characterization of the enzyme, indicates that the catalytic integrity of the enzyme is intact, and that the low specific activity seen in cell extracts is due to formation of an enzyme which has a reduced coenzyme content. It is suggested that this reduced coenzyme content is due primarily to a reduced affinity of the enzyme (nascent or apo-) for its coenzyme, and that the coenzyme must be incorporated into the enzyme at the moment of synthesis for formation of a functional protein.     

Insufficient expression of the ilv-leu operon encoding enzymes of branched-chain amino acid biosynthesis limits growth of a Bacillus subtilis ccpA mutant

Bacillus subtilis ccpA mutant strains exhibit two distinct phenotypes: they are defective in catabolite repression, and their growth on minimal media is strongly impaired. This growth defect is largely due to a lack of expression of the gltAB operon. However, growth is impaired even in the presence of glutamate. Here, we demonstrate that the ccpA mutant strain needs methionine and the branched-chain amino acids for optimal growth. The control of expression of the ilv-leu operon by CcpA provides a novel regulatory link between carbon and amino acid metabolism.     

Re-initiation of tryptophan operon expression in a promoter deletion strain of Salmonella typhimurium.

Summary A genetic and enzymological study was made of five spontaneous prototrophic revertants of a tryptophan auxotroph of Salmonella typhimurium which carries a deletion extending from the closely linked supX locus into the trp operator-promoter region. The revertants were found to have regained initiation of expression of all five trp genes. Recombinational tests showed that in each case the genetic change responsible for re-initiation is cotransducible with the trp-cysB region of the chromosome. Two different mechanisms leading to re-initiation of trp gene expression were established: (a) an extension of the limits of the original deletion resulting in the fusion of the trp structural genes with a nearby gene or gene set located outside the operator end of trp, and (b) translocation of a duplicate set of the trp structural genes to other chromosomal sites, located operator-distal to the normal trp operon, in such a manner that they are functionally fused to foreign genetic units. One revertant which arose by mechanism (a) was shown to have an extended deletion with one new terminus in trp and the other in the nearby cysB locus. All the revertants exhibit constitutive expression of the trp enzymes, with activities varying among strains from five to forty five times greater than the fully repressed wild type level. The protein product of trpA, the first structural gene of the operon, appears to have been partially damaged by the re-initiation event in at least two strains, while in the other strains, the enzyme appears in preliminary tests to be indistinguishable from that of wild type.     

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.