Methods for studying microbial acid stress responses: from molecules to populations.

The study of how micro-organisms detect and respond to different stresses has a long history of producing fundamental biological insights while being simultaneously of significance in many applied microbiological fields including infection, food and drink manufacture, and industrial and environmental biotechnology. This is well illustrated by the large body of work on acid stress. Numerous different methods have been used to understand the impacts of low pH on growth and survival of micro-organisms, ranging from studies of single cells to large and heterogeneous populations, from the molecular or biophysical to the computational, and from well-understood model organisms to poorly defined and complex microbial consortia. Much is to be gained from an increased general awareness of these methods, and so the present review looks at examples of the different methods that have been used to study acid resistance, acid tolerance, and acid stress responses, and the insights they can lead to, as well as some of the problems involved in using them. We hope this will be of interest both within and well beyond the acid stress research community.

Two of the three groEL homologues in Rhizobium leguminosarum are dispensable for normal growth.

Although many bacteria contain only a single groE operon encoding the essential chaperones GroES and GroEL, examples of bacteria containing more than one groE operon are common. The root-nodulating bacterium Rhizobium leguminosarum contains at least three operons encoding homologues to Escherichia coli GroEL, referred to as Cpn60.1, Cpn60.2 and Cpn60.3, respectively. We report here a detailed analysis of the requirement for and relative levels of these three proteins. Cpn60.1 is present at higher levels than Cpn60.2, and Cpn60.3 protein could not be detected under any conditions although the cpn60.3 gene is transcribed under anaerobic conditions. Insertion mutations could not be constructed in cpn60.1 unless a complementing copy was present, showing that this gene is essential for growth under the conditions used here. Both cpn60.2 and cpn60.3 could be inactivated with no loss of viability, and a double cpn60.2 cpn60.3 mutant was also constructed which was fully viable. Thus only Cpn60.1 is required for growth of this organism.

The chaperonins: perspectives from the Archaea.

Heat-shock protein (Hsp) 60 chaperones are almost ubiquitous and almost always essential. They can be divided on the basis of sequence homology into two broad types: group I (found in bacteria, mitochondria and chloroplasts) and group II (found in Archaea and the eukaryotic cytosol). Of the two, the group I chaperones are the better understood. Data on their structure, mechanism of action and cellular role will be briefly presented. The group II chaperones are less well studied. In eukaryotes they form large complexes with 8-fold symmetry containing eight different subunits, all of which are essential. They appear to have a major role in the folding of actin and tubulin, although they may also act on other substrates. No crystal structures are available for these complexes. The situation in the Archaea is simpler, with organisms containing between one and three genes for these chaperones. A 2.6 A structure exists for one archaeal group II chaperone complex. Some progress has been made in defining the reaction cycle of the archaeal group II chaperones and this has shown that they have some properties distinct from the group I chaperones. To date, the in vivo role and importance of the archaeal group II Hsp60 chaperones has not been determined. We have now shown that in the halophilic archaeon Haloferax volcanii not all the genes for these proteins are essential. Further analysis of these proteins in the Archaea should be very productive in yielding more information about these important chaperones and their cellular functions.

Microbial molecular chaperones.

Protein folding in the cell, long thought to be a spontaneous process, in fact often requires the assistance of molecular chaperones. This is thought to be largely because of the danger of incorrect folding and aggregation of proteins, which is a particular problem in the crowded environment of the cell. Molecular chaperones are involved in numerous processes in bacterial cells, including assisting the folding of newly synthesized proteins, both during and after translation; assisting in protein secretion, preventing aggregation of proteins on heat shock, and repairing proteins that have been damaged or misfolded by stresses such as a heat shock. Within the cell, a balance has to be found between refolding of proteins and their proteolytic degradation, and molecular chaperones play a key role in this. In this review, the evidence for the existence and role of the major cytoplasmic molecular chaperones will be discussed, mainly from the physiological point of view but also in relationship to their known structure, function and mechanism of action. The two major chaperone systems in bacterial cells (as typified by Escherichia coli) are the GroE and DnaK chaperones, and the contrasting roles and mechanisms of these chaperones will be presented. The GroE chaperone machine acts by providing a protected environment in which protein folding of individual protein molecules can proceed, whereas the DnaK chaperones act by binding and protecting exposed regions on unfolded or partially folded protein chains. DnaK chaperones interact with trigger factor in protein translation and with ClpB in reactivating proteins which have become aggregated after heat shock. The nature of the other cytoplasmic chaperones in the cell will also be reviewed, including those for which a clear function has not yet been determined, and those where an in vivo chaperone function has still to be proven, such as the small heat shock proteins IbpA and IbpB. The regulation of expression of the genes of the heat shock response will also be discussed, particularly in the light of the signals that are needed to induce the response. The major signals for induction of the heat shock response are elevated temperature and the presence of unfolded protein within the cell, but these are sensed and transduced differently by different bacteria. The best characterized example is the sigma 32 subunit of RNA polymerase from E. coli, which is both more efficiently translated and also transiently stabilized following heat shock. The DnaK chaperones modulate this effect. However, a more widely conserved system appears to be typified by the HrcA repressor in Bacillus subtilis, the activity of which is modulated by the GroE chaperone machine. Other examples of regulation of molecular chaperones will also be discussed. Finally, the likely future research directions for molecular chaperone biology in the post-genomic era will be briefly evaluated.

Trp203 mutation in GroEL promotes a self-association reaction: a hydrodynamic study.

A combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge is used to investigate the hydrodynamic integrity and increased self-association interactions of the mutant GroEL Y203W when compared to the wild-type GroEL molecule, which may be derived from increased hydrophobic exposure caused by the mutation. Sedimentation velocity has revealed that three distinct species were present throughout the concentration ranges used, corresponding to 14-mer (GroEL "super monomer") and 28-mer ("super dimer") subunit compositions with a small amount of 42-mer ("super trimer"), which, from the relative concentration of each species, would give an estimated weight average molecular weight of (1.0 +/- 0.1) x 10(6) Da. Sedimentation equilibrium gave an apparent weight average molecular weight (Mw,app) of (910,000 +/- 5000) Da, which is in agreement with these findings. These results are in contrast to wild-type GroEL which, in excellent agreement with the previous findings of Behlke and co-workers, revealed a single species with an Mw,app of (805,000 +/- 5200) Da and a sedimentation coefficient s(0)20,w of (21.6 +/- 0.3) S. We therefore conclude that the tryptophan mutation at the Y203 location causes a significant degree of self-association of the GroEL 14-mer assembly (with dimer and trimer present). These findings would appear to correlate well with the findings of Gibbons et al., who showed an increase in hydrophobic exposure due to this mutation.

Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants.

Protein disulfide isomerase (PDI) exhibits both an oxido-reductase and an isomerase activity on proteins containing cysteine residues. These activities arise from two active sites, both of which contain pairs of redox active cysteines. We have developed two simple in vivo assays for these activities of PDI, based on the demonstration that PDI can complement both a dsbA mutation and a dsbC mutation when expressed to the periplasm of Escherichia coli. We constructed a variety of mutants in and around the active sites of PDI and analysed them using these complementation assays. Our analysis showed that the active site amino acid residues have a major role in determining the activities exhibited by PDI, particularly the N-terminal cysteine of the N-terminal active site. The roles of the histidine residue at position 38 and the glutamic acid residue at position 30 were also studied using these assays. The results show that these two in vivo assays should be useful for rapid screening of mutants in PDI prior to purification and detailed biochemical analysis.

A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL.

Single-point mutants of GroEL were constructed with tryptophan replacing a tyrosine residue in order to examine nucleotide-induced structural transitions spectrofluorometrically. The tyrosine residues at positions 203, 360, 476 and 485 were mutated. Of these, the probe at residue 485 gave the clearest fluorescence signals upon nucleotide binding. The probe at 360 reported similar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated timescales, all of which precede hydrolysis of the nucleotide. All four of these rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is proposed to be a T-to-R transition. The next kinetic phase is a rearrangement to the open state identified by electron cryo-microscopy and this we designate an R to R* transition. Both of these rearrangements can occur when only a single ring of GroEL is loaded with ATP, and the results are consistent with the occupied ring behaving in a concerted, cooperative manner. At higher ATP concentrations both rings can be loaded with the nucleotide and the R to R* transition is accelerated. The resultant GroEL:ATP14 species can then undergo two final rearrangements, RR*-->[RR](+)-->[RR](#). These final slow steps are completely blocked when ADP occupies the second ring, i.e. it does not occur in the GroEL:ATP7:ADP7 or the GroEL:ATP7 species. All equilibrium and kinetic data conform to a minimal model in which the GroEL ring can exist in five distinct states which then give rise to seven types of oligomeric conformer: TT, TR, TR*, RR, RR*, [RR](+) and [RR](#), with concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by inter-ring contacts. This kinetic behaviour is consistent with the GroEL ring passing through distinct functional states in a binding-encapsulation-folding process, with the T-form having high substrate affinity (binding), the R-form being able to bind GroES but retaining substrate affinity (encapsulation), and the R*-form retaining high GroES affinity but allowing the substrate to dissociate into the enclosed cavity (folding). ADP induces only one detectable rearrangement (designated T to T*) which has no properties in common with those elicited by ATP. However, asymmetric ADP binding prevents ATP occupying both rings and, hence, restricts the system to the T*T, T*R and T*R* complexes.

Chaperone activity of a chimeric GroEL protein that can exist in a single or double ring form.

The molecular chaperone GroEL is a protein complex consisting of two rings each of seven identical subunits. It is thought to act by providing a cavity in which a protein substrate can fold into a form that has no propensity to aggregate. Substrate proteins are sequestered in the cavity while they fold, and prevented from diffusion out of the cavity by the action of the GroES complex, that caps the open end of the cavity. A key step in the mechanism of action of GroEL is the transmission of a conformational change between the two rings, induced by the binding of nucleotides to the GroEL ring opposite to the one containing the polypeptide substrate. This conformational change then leads to the discharge of GroES from GroEL, enabling polypeptide release. Single ring forms of GroEL are thus predicted to be unable to chaperone the folding of GroES-dependent substrates efficiently, since they are unable to discharge the bound GroES and unable to release folded protein. We describe here a detailed functional analysis of a chimeric GroEL protein, which we show to exist in solution in equilibrium between single and double ring forms. We demonstrate that whereas the double ring form of the GroEL chimera functions effectively in refolding of a GroES-dependent substrate, the single ring form does not. The single ring form of the chimera, however, is able to chaperone the folding of a substrate that does not require GroES for its efficient folding. We further demonstrate that the double ring structure of GroEL is likely to be required for its activity in vivo.

GroEL protects the sarcoplasmic reticulum Ca(++)-dependent ATPase from inactivation in vitro.

The molecular chaperone, GroEL, facilitates correct protein folding and inhibits protein aggregation. The function of GroEL is often, though not invariably, dependent on the co-chaperone, GroES, and ATP. In this study it is shown that GroEL alone substantially reduces the inactivation of purified Ca(++)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum. In the absence of GroEL, the enzyme became completely inactive in about 45-60 hours when kept at 25 degrees C, while in the presence of an equimolar amount of GroEL, the enzyme remained approximately 80% active even after 75 hours. Equimolar amounts of BSA or lysozyme were unable to protect the enzyme from inactivation under identical conditions. Analysis by SDS-PAGE showed GroEL was acting by blocking the aggregation of ATPase at 25 degrees C. GroEL was not as effective in protection at -20 degrees C or 4 degrees C. These results are discussed in the context of current models of the GroEL mechanism.

Mutations in dsbA and dsbB, but not dsbC, lead to an enhanced sensitivity of Escherichia coli to Hg2+ and Cd2+.

The Dsb proteins are involved in disulfide bond formation, reduction and isomerisation in a number of Gram-negative bacteria. Mutations in dsbA or dsbB, but not dsbC, increase the proportion of proteins with free thiols in the periplasm compared to wild-type. We investigated the effects of mutations in these genes on the bacterial resistance to mercuric and cadmium salts. Mutations in genes involved primarily in disulfide formation (dsbA and dsbB) generally enhanced the sensitivity to Hg2+ and Cd2+ while a mutation of the dsbC gene (primarily an isomerase of disulfide bonds) had no effect. Mutations of the dsb genes had no effect on the expression of the mercury-resistance determinants of the transposon Tn501.

An arginine residue (Arg101), which is conserved in many GroEL homologues, is required for interactions between the two heptameric rings.

Homologous recombination was used to construct a series of hybrid chaperonin genes, containing various lengths of Escherichia coli groEL replaced by the equivalent region from the homologous cpn60-1 gene of Rhizobium leguminosarum. Analysis of proteins produced by these hybrids showed that many of them formed structures with properties consistent with their being single heptameric rings under some conditions, as opposed to the double ring form in which both the GroEL and the Cpn60-1 proteins are found. By determining precise cross-over points, two regions in Cpn60-1 were defined which appeared to be critical for ring-ring interactions. Within one of these regions is a highly conserved arginine residue (Arg101), which we hypothesised to interact with a residue or residues toward the C terminus of the protein, this contact being required for double rings to form. To test this hypothesis, we mutagenised this residue from arginine to threonine in chaperonin genes from two different species of Rhizobium. In both cases, proteins which ran on non-denaturing gels as single rings were produced. Conversion of Arg101 to serine also had the same effect, whereas conversion of Arg101 to lysine did not. Two different single rings created by homologous recombination could be converted back to double rings by changing the threonine, which naturally occurs at this position in E. coli GroEL, back to arginine. The in vivo properties of the proteins were investigated by complementation following deletion of the chromosomal copy of the groEL gene, and by monitoring the ability of cells expressing the hybrid proteins to plate bacteriophage. Most of the hybrid and mutant proteins were functional in these assays, despite their altered properties compared to wild-type GroEL.

In vivo activities of GroEL minichaperones.

Fragments encompassing the apical domain of GroEL, called minichaperones, facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL. We have identified the smallest minichaperone that is active in vitro in chaperoning the refolding of rhodanese and cyclophilin A: GroEL(193-335). This finding raises the question of whether the minichaperones are active under more stringent conditions in vivo. The smallest minichaperones complement two temperature-sensitive Escherichia coli groEL alleles, EL44 and EL673, at 43 degreesC. Although they cannot replace GroEL in cells in which the chromosomal groEL gene has been deleted by P1 transduction, GroEL(193-335) enhances the colony-forming ability of such cells when limiting amounts of GroEL are expressed from a tightly regulated plasmid. Surprisingly, we found that overexpression of GroEL prevents plaque formation by bacteriophage lambda and inhibits replication of the lambda origin-dependent plasmid, Lorist6. The minichaperones also inhibit Lorist6 replication, but less markedly. The complex quaternary structure of GroEL, its central cavity, and the structural allosteric changes that take place on the binding of nucleotides and GroES are not essential for all of its functions in vivo.

Deletion of Escherichia coli groEL is complemented by a Rhizobium leguminosarum groEL homologue at 37 degrees C but not at 43 degrees C.

Bacterial Cpn60 proteins (homologues to the Escherichia coli GroEL protein) are often examined for function by testing their ability to complement a temperature sensitive mutation in the E. coli groEL gene. Such tests suffer from two drawbacks: the Cpn600 protein may come from a strain with a lower optimum growth temperature than E. coli, and the requirements for successful complementation in E. coli are likely to be more stringent at 43 degrees C than at lower temperatures. Here we describe the construction of a strain of E. coli where the chromosomal gene for the essential molecular chaperone GroEL has been deleted, with GroEL being expressed from a tightly regulated plasmid borne copy of the gene. The deletion can be transduced into strains expressing heterologous Cpn60 proteins, to test for complementation at any temperature. We show that a Cpn60 protein from the bacterium Rhizobium leguminosarum can function to allow E. coli growth at 37 degrees C but not at 43 degrees C. By switching off the plasmid borne groEL gene, the effects of progressive depletion of GroEL protein from E. coli cells can also be monitored at any temperature.

Induction of alpha/beta interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine.

The propensity of two Chlamydia trachomatis strains (L2/434/Bu [biovar LGV] and E/DK20/ON [biovar trachoma]) to induce putative host defense responses upon infection of McCoy (mouse) cell cultures was examined. Both strains induced production of alpha/beta interferon and nitric oxide (NO) by McCoy cells. NO synthesis was mediated by the inducible isoform of NO synthase as indicated by the ability of cycloheximide or the arginine analog NG-monomethyl-L-arginine to abolish NO production; the extent of the response was dependent upon the dose of chlamydiae applied. Incubation of McCoy cells with chloramphenicol prior to infection reduced NO production by strain 434 but not by DK20, suggesting that initial chlamydial metabolism was essential to induction by the LGV strain. Antibody inhibition studies indicated that NO synthesis was dependent upon production of alpha/beta interferon and induction via lipopolysaccharide. Overall, our findings show that chlamydiae are capable of the induction of interferon and NO in murine fibroblasts in the absence of exogenous cytokines. However, the role of NO as an antichlamydial effector could not be clearly demonstrated since treatment with an arginine analog, while suppressing NO production, gave no consistent enhancement of infected cell numbers.

Co-expression of human protein disulphide isomerase (PDI) can increase the yield of an antibody Fab' fragment expressed in Escherichia coli.

Secretion to the periplasm of Escherichia coli enables production of many eukaryotic extracellular proteins in a soluble form. The complex disulphide bond arrangement of such proteins is probably a major factor in determining the low yield of correctly folded product observed in many cases. Here we show that co-expression of human protein disulphide isomerase increased the yield of a monoclonal antibody Fab' fragment in the periplasm of E. coli.

Human protein disulfide isomerase functionally complements a dsbA mutation and enhances the yield of pectate lyase C in Escherichia coli.

Human PDI was expressed to the Escherichia coli periplasm, by using a plasmid encoded ompA-PDI fusion under the control of the trp promoter. Periplasmic extracts were shown to contain active PDI using the scrambled ribonuclease assay. PDI activity was also demonstrated by complementation of two phenotypes associated with a dsbA mutation. Alkaline phosphatase activity, which is reduced in dsbA cells, was restored to wild type levels by PDI. PelC, a pectate lyase from Erwinia carotovora, was shown to be DsbA dependent in E. coli. PDI was able to restore its activity to that seen in wild type cells. Increased expression of PDI was found to increase the yield of active PelC above that seen in wild type cells. PDI also enhanced the yield of PelC in DsbA- cells but only in the presence of exogenous oxidized glutathione. PDI is thus able to functionally substitute for DsbA in the folding of disulfide-bonded proteins in the bacterial periplasm and to enhance the yield of highly expressed protein when the ability of the E. coli periplasm to fold protein may be saturated. However, our results suggest that the activities of DsbA and PDI in vivo may be different.

The roles of molecular chaperones in vivo.

Table 1 summarizes the families of chaperones mentioned in this review, and lists their proposed functions. Many of these proteins are named in the accompanying review of Burston and Clarke. Molecular chaperones are proteins which interact with other proteins and help them to reach their final, active conformation. They appear to do this by binding them in an unfolded or partially folded state and subsequently releasing them in an altered form. This property may endow them with several essential or important roles in addition to helping newly synthesized proteins to fold correctly, such as repairing damaged proteins and assisting proteins in membrane translocation. To confirm that a given protein has molecular chaperone activity in vivo, it is necessary to show that interactions between the chaperone and other proteins do occur in the cell, and that loss of the molecular chaperone leads to the accumulation of inactive or precursor protein. The hsp70 protein family are highly conserved and ubiquitous. Genetic studies confirm that their depletion leads to the accumulation of inactive precursor or other proteins, and immunochemical studies show they associate with nascent polypeptides. They are implicated not only in protein folding, but also in protein transport across membranes and reactivation of heat-damaged proteins. The hsp60 proteins are also ubiquitous and very similar in sequence. Those found in bacteria and organelles, such as mitochondria (the GroEL family), are essential at all temperatures, and particularly after heat shock. Their loss or depletion leads to the formation of protein aggregates and eventual cell death. A co-chaperone protein (GroES) is required for their function. Cytosolic homologues (the TCP1 family) are also essential, though not heat-shock induced; they are believed to have a chaperone role in tubulin assembly and their actual role in the cell may be much broader. Many other proteins may have a chaperone function in vivo. Such a function may be specific to a particular substrate (such as the PapD protein in E. coli); others may be more general (such as hsp90 and SecB). Evidence is still needed to demonstrate whether all those proteins which show chaperone behaviour in vitro actually have such a role in vivo. It seems likely that different classes of chaperone may overlap in their specificity, and it is certain that the various proteins classed as molecular chaperones fulfil a wide variety of roles in the cell.

Rhizobium leguminosarum contains multiple chaperonin (cpn60) genes.

We have examined the heat shock response of Rhizobium leguminosarum. After normal growth at 28 degrees C, a 10 min heat shock at 37 degrees C induced the synthesis of proteins with approximate M(r) values of 90,000, 70,000, 60,000, 58,000, 19,000, 17,000 and 13,000. A monoclonal antibody raised against the E. coli Cpn60 cross-reacted with proteins of M(r) 60,000 and 58,000 in R. leguminosarum, suggesting that both were Cpn60 homologues. Hybridization of an E. coli cpn60 probe to total DNA from Rhizobium leguminosarum also showed evidence for at least two cpn60 homologues. One of these was cloned and completely sequenced, and showed close homology to cpn60 sequences from other prokaryotes. The expression of this gene in E. coli failed to complement a cpn60 mutation, either for growth at high temperature or for growth of bacteriophage lambda. Hybridization of total R. leguminosarum DNA with a probe from this gene revealed the presence of a third putative cpn60 gene. Two further hybridizing clones were analysed and found to consist of two additional cpn60 sequences plus upstream regions containing putative cpn10 genes.

Regulation of transcription in Escherichia coli from the mer and merR promoters in the transposon Tn501.

We report studies on deletion mutants of the regulatory region of the mercuric ion resistance (mer) genes of transposon Tn501, isolated from Pseudomonas aeruginosa. Transcription of the mer genes in Escherichia coli from the promoter Pmer is regulated both positively (in the presence of mercuric salts) and negatively (in their absence) by the product of the merR gene. The merR gene is transcribed divergently with respect to the other mer genes, and negatively regulates its own synthesis. The experiments described here suggest that both positive and negative regulation by MerR, as well as its autoregulation, are largely mediated by MerR binding to a single site on DNA. This site contains a hyphenated dyad symmetrical sequence centred 24 base-pairs before the start of the mer transcript. Additional sites may be involved in full repression of the mer and merR promoters. Studies on deletions of the Pmer promoter show that the -35 sequence is not required for constitutive activity. An alternative -10 sequence may be used in the absence of the -35 and normal -10 sequences, but the properties of a point mutation indicate that, in the presence of the -35 sequence, the normal -10 sequence is required for promoter activity. A model for the regulation of expression of the mercury resistance genes by mercuric ions and the MerR protein is discussed.