Viability of Escherichia coli topA mutants lacking DNA topoisomerase I

The viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri. This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E. coli. We find that E. coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42 degrees C without any compensatory mutations. These topA(-) cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA. In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9766-9771), E. coli cells lacking both type IA DNA topoisomerases I and III are found to be nonviable, indicating that the two type IA enzymes share a critical cellular function.     

The genetic control of DNA supercoiling in Salmonella typhimurium

We have elucidated the genetic control of DNA supercoiling in Salmonella typhimurium. The level of superhelix density is controlled by two classes of genes. The only member of the first class is topA, the structural gene for topoisomerase I. The second class, tos, (topoisomerase one suppressor) consists of at least two genes, one of which is linked to gyrA, the structural gene for the topoisomerase subunit of DNA gyrase. Deletions of topA result in oversupercoiling of plasmid DNA. These mutations do not require the acquisition of second-site compensatory mutations to allow cell growth, in contrast to the situation in Escherichia coli. However, tos mutations, unlinked to topA, have been isolated which reduce plasmid superhelix density. We conclude that the level of DNA supercoiling in S. typhimurium is a dynamic balance between the effects of the gene products of topA (relaxation) and tos (supercoiling) which act independently of each other. Using a variety of combinations of these mutations we have constructed a series of isogenic strains, each of which has a different but precisely defined level of plasmid supercoiling; the series as a whole provides a wide range of supercoiling both above and below the wild-type level.     

Structural and genetic relationships of two pairs of closely related O-antigens of Escherichia coli and Salmonella enterica: E. coli O11/S. enterica O16 and E. coli O21/S. enterica O38

The O-antigen is a part of the lipopolysaccharide molecule present in the outer membrane of Gram-negative bacteria, and is essential for the full function of the microorganisms. Salmonella enterica and Escherichia coli are taxonomically closely related species. In this study, the O-antigen structures of S. enterica O16 and O38 and E. coli O11 were determined. Salmonella enterica O38 and E. coli O21 were found to have identical O-antigen structures, whereas S. enterica O16 and E. coli O11 had closely related structures, differing only in the presence of a lateral glucose residue and O-acetylation of a mannose residue in the former. The O-antigen gene clusters of S. enterica O16 and O38 and E. coli O11 were sequenced and analyzed together with that of E. coli O21 retrieved from the GenBank. Each S. enterica/E. coli pair was found to contain the same set of genes organized in the same manner and to share 56-78% overall DNA identity. These data suggest that the O-antigen gene clusters of each pair studied originated from a common ancestor. Thus, it has become evident that in the past, the degree of relatedness between the O-antigens of S. enterica and E. coli was underestimated.     

DNA supercoiling in vivo

DNA topoisomerase mutants of Escherichia coli and Saccharomyces cerevisiae were used to study the topological state of intracellular DNA. In E. coli, it is shown that switching off the gene topA encoding DNA topoisomerase I leads to an increase in the degree of negative supercoiling of intracellular DNA and inhibition of the growth of the cells: a d(pCpG)16.d(pCpG)16 sequence on a plasmid is also shown to flip from a right-handed B-helical structure to a left-handed Z-helical structure in vivo when topA is switched off. In S. cerevisiae, the topological state of intracellular DNA is little affected by the cellular levels of the topoisomerases.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Relationships of the Escherichia coli O157, O111, and O55 O-antigen gene clusters with those of Salmonella enterica and Citrobacter freundii, which express identical O antigens

Escherichia coli O157, Salmonella enterica O30, and Citrobacter freundii F90 have identical O-antigen structures, as do E. coli O55 and S. enterica O50. The O-antigen gene cluster sequences for E. coli O157 and E. coli O55 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages. We determined sequences for S. enterica O30 and C. freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E. coli O157 cluster. We also determined the sequence of the S. enterica O50 O-antigen gene cluster and compared it to the sequence of the previously described E. coli O55 cluster. For both the S. enterica O30-C. freundii F90-E. coli O157 group and the S. enterica O50-E. coli O55 group of O antigens, the gene clusters have identical or nearly identical organizations. The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes. Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis. We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.     

The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.     

The Escherichia coli O111 and Salmonella enterica O35 gene clusters: gene clusters encoding the same colitose-containing O antigen are highly conserved

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. Escherichia coli and Salmonella enterica each have many forms of O antigen, but only three are common to the two species. It has been found that, in general, O-antigen genes are of low GC content. This deviation in GC content from that of typical S. enterica or E. coli genes (51%) is thought to indicate that the O-antigen DNA originated in species other than S. enterica or E. coli and was captured by lateral transfer. The O-antigen structure of Salmonella enterica O35 is identical to that of E. coli O111, commonly found in enteropathogenic E. coli strains. This O antigen, which has been shown to be a virulence factor in E. coli, contains colitose, a 3,6-dideoxyhexose found only rarely in the Enterobacteriaceae. Sequencing of the O35-antigen gene cluster of S. enterica serovar Adelaide revealed the same gene order and flanking genes as in E. coli O111. The divergence between corresponding genes of these two gene clusters at the nucleotide level ranges from 21.8 to 11.7%, within the normal range of divergence between S. enterica and E. coli. We conclude that the ancestor of E. coli and S. enterica had an O antigen identical to the O111 and O35 antigens, respectively, of these species and that the gene cluster encoding it has survived in both species.