The products of the groES and groEL genes of Escherichia coli, constituting the groE operon, are known to be required for growth at high temperature (42°C) and are members of the heat shock regulon. Using a genetic approach, we examined the requirement for these gene products for bacterial growth at low temperature (17 to 30°C). To do this, we constructed various groES groEL heterodiploid derivative strains. By inactivating one of the groE operons by a polar insertion, it was shown by bacteriophage P1 transduction that at least one of the groE genes was essential for growth at low temperature. Further P1 transduction experiments with strains that were heterodiploid for only one of the groE genes demonstrated that both groE gene products were required for growth at low temperature, which suggested a fundamental role for the groE proteins in E. coli growth and physiology.The groES (mopA) and groEL (mopB) genes of Escherichia coli form an operon located at 94.2 min on the standard genetic map (2). They were first defined by mutations affecting the morphogenesis of several bacteriophages, including X, T4, and T5 (see reference 6 for a review). Both of the groE gene products have been shown to be essential for bacteriophage X head assembly (6)(7)(8)33) and for bacteriophage T5 tail assembly (8,40). In addition, the groEL gene product has been shown to be required for proper T4 head assembly (6,7,29,32). Some alleles of both genes were subsequently shown to also result in thermosensitive bacterial growth at 42°C, affecting both DNA and RNA synthesis (ts mutants; 36). Both the bacterial temperature-sensitive phenotype and inability to propagate bacteriophage A always contransduced, which demonstrated that the groE gene products are required for bacterial growth at least at high temperature (7). Although the exact role of these gene products in cell physiology remains to be determined, several of their properties are known at the physiological and molecular levels. The groES and groEL genes code for 10,368-and 57,259-Mr acidic polypeptides, respectively, found at high intracellular levels (about 2% of total cell proteins at 37°C; 12, 26, 33). Furthermore, as members of the heat shock regulon, the intracellular levels of their products increase with temperature through a positive transcriptional control exerted by the rpoH (U32) gene product (10).The products of some of the heat shock genes are either totally indispensable (e.g., rpoD, which codes for the Cr70 subunit of E. coli RNA polymerase; 26), dispensable (e.g., lon, which codes for an ATP-dependent protease [22,26]

As a typical mesophile, Escherichia coli can maintain balanced growth between approximately 10 and 49°C (19). In the range of approximately 21 to 37°C, the rate of E. coli growth varies as a simple function of temperature (19). Raising the temperature above 40°C leads to progressively slower growth rates and changes in the cellular content of many proteins (30, 31). The adaptation processes that occur on a shift to high temperature include an increased expression of a set of genes, called heat shock genes, many of which are highly conserved among procaryotic and eucaryotic organisms. These genes are dispersed throughout the chromosome, and their products perform various functions in the cell, most of which are not clearly understood thus far (12,16,30,31,34). The transcription of the majority of these genes is positively regulated by the product of the rpoH gene (previously known also as htpR or hin; for a review, see references 30 and 31), the ar32 subunit of the RNA polymerase holoenzyme (6,17). Some of these genes are also essential for bacterial growth under normal temperature conditions, for example, the rpoD gene, which codes for the cr70 subunit of RNA polymerase (30, 31), or grpE, which is essential for the replication of bacteriophage X but which plays an otherwise unknown role in E. coli physiology (D. Ang and C. Georgopoulos, submitted for publication). Several heat shock genes, like lysU, which codes for an alternate form of lysyl-tRNA synthetase (30,31,38), or lon, which codes for an ATP-dependent protease (26), are not absolutely essential for bacterial growth. There is also a class of heat shock genes that is conditionally dispensable at low temperatures; i.e., deletion mutants can be constructed at low temperatures but they grow poorly and rapidly accumulate extragenic suppressors (e.g., dnaK [7a] and dnaJ [S. Sell and C. Georgopoulos, unpublished data]).Apart from the canonical heat shock genes, the rpoH regulatory gene itself is indispensable for cell adaptation to high temperatures (9). It is also known from two-dimensional electrophoresis of total E. coli proteins that there are other * Corresponding author.

Although genetic and biochemical evidence has established that GroES is required for the full function of the molecular chaperone, GroEL, little is known about the molecular details of their interaction. GroES enhances the cooperativity of ATP binding and hydrolysis by GroEL (refs 4, 5) and is necessary for release and folding of several GroEL substrates. Here we report that native GroES has a highly mobile and accessible polypeptide loop whose mobility and accessibility are lost upon formation of the GroES/GroEL complex. In addition, lesions present in eight independently isolated mutant groES alleles map in the mobile loop. Studies with synthetic peptides suggest that the loop binds in a hairpin conformation at a site on GroEL that is distinct from the substrate-binding site. Flexibility may be required in the mobile loops on the GroES seven-mer to allow them to bind simultaneously to sites on seven GroEL subunits, which may themselves be able to adopt different arrangements, and thus to modulate allosterically GroEL/substrate affinity.

In 1994, an outbreak of Enterobacter sakazakii infections occurred in a neonatal intensive care unit in France from 5 May to 11 July. During the outbreak, 13 neonates were infected with E. sakazakii, resulting in 3 deaths. In addition, four symptomless neonates were colonized by E. sakazakii. The strains were subjected to 16S rRNA gene sequence analysis, genotyped using pulsed-field gel electrophoresis, and phenotyped for a range of enzyme activities. E. sakazakii was isolated from various anatomical sites, reconstituted formula, and an unopened can of powdered infant formula. A fourth neonate died from septic shock, attributed to E. sakazakii infection, during this period. However, 16S rRNA gene sequence analysis revealed that the organism was Enterobacter cloacae. There were three pulsotypes of E. sakazakii associated with infected neonates, and three neonates were infected by more than one genotype. One genotype matched isolates from unused prepared formula and unfinished formula. However, no pulsotypes matched the E. sakazakii strain recovered from an unopened can of powdered infant formula. One pulsotype was associated with the three fatal cases, and two of these isolates had extended-spectrum ␤-lactamase activity. It is possible that E. sakazakii strains differ in their pathogenicities, as shown by the range of symptoms associated with each pulsotype.Enterobacter sakazakii is an opportunistic pathogen associated with the ingestion of reconstituted infant formula and is a rare cause of neonatal meningitis, necrotizing enterocolitis (NEC), and sepsis (9, 10, 11, 23). Such cases often occur among low-birth-weight preterm neonates, who are generally more susceptible to gram-negative bacterial sepsis and endotoxemia associated with NEC (1, 26). The International Commission on Microbiological Specifications for Foods (14) has ranked E. sakazakii as a "severe hazard for restricted populations, life-threatening or substantial chronic sequelae or long duration." A number of reported E. sakazakii outbreaks have been attributed to contaminated reconstituted infant formula (4,7,13,18,31). Bowen and Braden (4) reviewed 46 cases of invasive E. sakazakii infections and showed a link between symptoms and birth weight but did not consider cases of NEC.The virulence of E. sakazakii has been studied by Pagotto et al. (23) and Mange et al. (21), who showed the presence of enterotoxins and adhesion to brain cells, respectively. Townsend et al. demonstrated the translocation of E. sakazakii and other intestinal bacteria across the rat intestinal wall in response to the presence of lipopolysaccharide (28). They also demonstrated that E. sakazakii causes chronic-patterned inflammation in the neonatal rat brain, invades capillary endothelial brain cells, is taken up by macrophages, and induces anti-inflammatory cytokine (interleukin-10) expression in vitro and in vivo at various levels according to strain (29). However, these publications did not report the individual case details associated with the isolates under study. Therefor...

We have investigated the role of three IS911‐specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by −1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB‐mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site‐specific intramolecular transposition event. We present a model which explains both intra‐ and intermolecular transposition events in terms of a single reaction mechanism of the ‘cut and paste’ type.

The expression of an increasing number of genes of both prokaryotic and eukaryotic origin has been shown to be regulated at the translational level by programmed (sequence-specific) ribosomal frameshifting. Among these are the bacterial insertion sequences IS1 and two members of the widely distributed IS3-family, IS150 and IS911. Frameshifting provides a means of specifying several proteins with different functions using a minimum of genetic information. In this review, we survey present understanding of the way in which frameshifting is integrated into the overall control of transposition activity in these elements.

No abstract

Members of the IS3 family of insertion sequences are found in a wide range of bacteria. At least 10 members of this family carry two major open reading frames: a small upstream frame (0 phase), and a longer downstream frame in the -1 phase. The downstream frame shows significant similarity at the amino acid level. A highly conserved region of this frame also exhibits notable similarity with a region of the integrase (endonuclease) domain of retroviruses. Although the overall transposition mechanism of the insertion sequence and retroviral elements is certainly different, the two groups may share additional common features, including a -1 frameshift resulting in the production of a fusion protein.