No abstract

A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202-312. This single gene mutation was located between 490 and 491.6 kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cytoplasmic synthesis of haemolysin.

We have investigated the role of DnaJ in protein degradation by examining the degradation of intrinsically unstable proteins by Lon protease in vivo. In Escherichia coli, Lon protease is responsible for the rate-limiting step in degradation of highly unstable proteins such as SulA, RcsA, and N protein, as well as for about 50% of the rapid degradation of abnormal proteins such as canavanine-containing proteins. We found that Londependent degradation of both SulA and N protein was unaffected in cells lacking functional DnaJ, whereas Lon-dependent turnover of canavanine-containing proteins was slower in dnaJ mutant cells. DnaJ also affected the slow SulA degradation seen in the absence of Lon. The rate of degradation of RcsA was reduced in dnaJ mutants, but both Lon-dependent and Lon-independent degradation was affected; abnormal, canavanine-containing proteins were similarly affected. Both the RcsA that accumulated in dnaJ mutant cells and the SulA that accumulated in lon dnaJ mutant cells was aggregated. The abnormal proteins that partitioned to the insoluble pellet became solubilized over time in dnaJ ؉ cells but not in dnaJ ؊ cells. Therefore, the cochaperone DnaJ is not essential for Lon-dependent degradation and may act in protein turnover only as an accessory factor helping to maintain substrates in a soluble form. Such an accessory factor is apparently necessary for abnormal proteins and for RcsA. The relative rates of degradation and aggregation of specific protein targets may determine the importance of the chaperone systems in turnover of a given protein.

The hha gene of Escherichia coli was identified as modulating the expression of the haemolysin (hly) genes encoded by the recombinant plasmid pANN202-312. hha mutants harbouring plasmid pANN202-312 showed increased haemolysin production. The product of the hha gene, the Hha protein, shows strong homology to the YmoA protein of Yersinia enterocolitica, which plays a role in the thermoregulation of various Y. enterocolitica virulence genes. We show in this study that the Hha protein modulates the expression of haemolysin at the transcriptional level in cells harbouring plasmid pANN202-312. In addition, hha mutants show alterations in the level of plasmid DNA supercoiling. This suggests that the hha mutation increases haemolysin expression through changes in the DNA topology. This hypothesis is supported by our finding that gyr mutations, inhibitors of DNA gyrase such as novobiocin, or growth in conditions reported to reduce levels of negative supercoiling, such as low osmolarity medium, increase haemolysin production.

(15,32).A number of ISs have been described and analyzed in some detail. Galas and Chandler (15) list more than 50 different ISs for gram-negative bacteria. The complete nucleotide sequences of at least 30 ISs are available, and comparisons which suggest evolutionary relationships have been produced. Homologous ISs, detected mainly by conservation of specific motifs among their presumed transposases, have been grouped in families. The two most conspicuous families are the IS3 family (13, 30) and the IS4 family (32). The IS3 family includes a number of ISs sharing extended homology, particularly in a 50-amino-acid segment of the C-terminal end of their transposases, the signature of which is DG2Y5 and 26 amino acids later N32E36K43 (13, 30). The best-known members of this family are IS2, IS13, IS51, IS150, IS600, IS629, IS861, IS3411, and IS6110. IS21 is also related to this group since it possesses the N32E36K43 motif. They share not only the same signature but also a common genetic organization. All constituent members have two open reading frames (ORFs), with the start of the second one overlapping the terminus of the first in the -1 phase. It is thought that the active transposase is produced by slippage of the first ORF to the second during translation (25). The IS4 family is also very widespread. Its diagnostic signature, YE7K14, is found in IS4, IS10, IS50, IS186, IS231, and ISHJ and also in the more distantly related elements IS26, IS52, and IS903 (32). In this case, there is a common genetic organization, since the members of the IS4 family contain a long ORF covering almost all of the coding capacity of the element. However, there are still many well-known ISs, such as IS1, IS5, IS30, and IS200, etc., which do not display a significant relationship with either of these families or among themselves.IS91 was isolated from a hemolysin plasmid of Escherichia coli and found to occur in multiple copies in a number of Hly plasmids of different incompatibility groups (35). It shows * Corresponding author. the recombinational properties of an IS element (9) and is probably involved in the evolutionary spread of the hly genes (36). IS91 is particularly interesting because it shows an absolute insertion specificity for the sequences GAAC and CAAG and does not duplicate the target DNA upon insertion (10, 23). We report the DNA sequence of IS91 and identify the transposase gene by transposon insertion mutagenesis. We also show that IS91 is similar to IS801 and discuss the implications of this similarity for the previously proposed mechanism of IS801 transposition. MATERUILS AND METHODSStrains and plasmids. The strains used are listed in Table 1, and plasmids are listed in Table 2.Standard genetic techniques. Transformations were carried out by the method of Chung and Miller (5). For mating experiments, donor and recipient strains were grown overnight in Luria-Bertani (LB) broth (20) with appropriate antibiotics for plasmid selection. Donors were then diluted 1:10 into fresh LB broth and grown for 2 h. Then 0.5 ml o...