The C terminus of sigma(32) is not essential for degradation by FtsH.

A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.

Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol.

We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In DeltarpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5-10% and 20-30% of total protein aggregated at 30 degrees C and 42 degrees C respectively. The aggregates contained 350-400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30 degrees C, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low-level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to DeltarpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30 degrees C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK-depleted cells. At 42 degrees C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42 degrees C.

Disruption of the genes for ClpXP protease in Salmonella enterica serovar Typhimurium results in persistent infection in mice, and development of persistence requires endogenous gamma interferon and tumor necrosis factor alpha.

The enteric pathogen Salmonella enterica serovar Typhimurium, similar to other facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophages of the host organism by producing a set of stress proteins that are also induced by various environmental stresses. The stress-induced ClpXP protease is a member of the ATP-dependent proteases, which are known to be responsible for more than 90% of all proteolysis in Escherichia coli. To investigate the contribution of the ClpXP protease to the virulence of serovar Typhimurium we initially cloned the clpP and clpX operon from the pathogenic strain serovar Typhimurium chi3306 and then created insertional mutations in the clpP and/or clpX gene. The Delta clpP and Delta clpX mutants were used to inoculate BALB/c mice by either the intraperitoneal or the oral route and found to be limited in their ability to colonize organs of the lymphatic system and to cause systemic disease in the host. A variety of experiments were performed to determine the possible reasons for the loss of virulence. An oxygen-dependent killing assay using hydrogen peroxide and paraquat (a superoxide anion generator) and a serum killing assay using murine serum demonstrated that all of the serovar Typhimurium Delta clpP and Delta clpX mutants were as resistant to these killing mechanisms as the wild-type strain. On the other hand, the macrophage survival assay revealed that all these mutants were more sensitive to the intracellular environment than the wild-type strain and were unable to grow or survive within peritoneal macrophages of BALB/c mice. In addition, it was revealed that the serovar Typhimurium ClpXP-depleted mutant was not completely cleared but found to persist at low levels within spleens and livers of mice. Interferon gamma-deficient mice and tumor necrosis factor alpha-deficient mice failed to survive the attenuated serovar Typhimurium infections, suggesting that both endogenous cytokines are essential for regulation of persistent infection with serovar Typhimurium.

The heat shock response of Escherichia coli.

A large variety of stress conditions including physicochemical factors induce the synthesis of more than 20 heat shock proteins (HSPs). In E. coli, the heat shock response to temperature upshift from 30 to 42 degrees C consists of the rapid induction of these HSPs, followed by an adaptation period where the rate of HSP synthesis decreases to reach a new steady-state level. Major HSPs are molecular chaperones, including DnaK, DnaJ and GrpE, and GroEL and GroES, and proteases. They constitute the two major chaperone systems of E. coli (15-20% of total protein at 46 degrees C). They are important for cell survival, since they play a role in preventing aggregation and refolding proteins. The E. coli heat shock response is positively controlled at the transcriptional level by the product of the rpoH gene, the heat shock promoter-specific sigma32 subunit of RNA polymerase. Because of its rapid turn-over, the cellular concentration of sigma32 is very low under steady-state conditions (10-30 copies/cell at 30 degrees C) and is limiting for heat shock gene transcription. The heat shock response is induced as a consequence of a rapid increase in sigma32 levels and stimulation of sigma32 activity. The shut off of the response occurs as a consequence of declining sigma32 levels and inhibition of sigma32 activity. Stress-dependent changes in heat shock gene expression are mediated by the antagonistic action of sigma32 and negative modulators which act upon sigma32. These modulators are the DnaK chaperone system which inactivate sigma32 by direct association and mediate its degradation by proteases. Degradation of sigma32 is mediated mainly by FtsH (HflB), an ATP-dependent metallo-protease associated with the inner membrane. There is increasing evidence that the sequestration of the DnaK chaperone system through binding to misfolded proteins is a direct determinant of the modulation of the heat shock genes expression. A central open question is the identity of the binding sites within sigma32 for DnaK, DnaJ, FtsH and the RNA polymerase, and the functional interplay between these sites. We have studied the role of two distinct regions of sigma32 in its activity and stability control: region C and the C-terminal part. Both regions are involved in RNA polymerase binding.

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB.

UNLABELLED: We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (

Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network.

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid binding, and of the disaggregation/refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.

Trigger factor and DnaK cooperate in folding of newly synthesized proteins.

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.

Role of region C in regulation of the heat shock gene-specific sigma factor of Escherichia coli, sigma32.

Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma32 by stress-dependent association and mediates sigma32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma32 homologs, termed region C, was proposed to play a role in sigma32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma32 protein did not affect sigma32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma32 control by DnaK and FtsH. Instead, the sigma32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma32 to RNA polymerase. Region C thus confers on sigma32 a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli.

The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42 degrees C. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the IpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.

Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli.

The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the sigma32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes sigma32, and the FtsH protease, which is largely responsible for sigma32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose-controlled promoters. Small increases in levels of DnaK and the DnaJ co-chaperone (< 1.5-fold of wild type) resulted in decreased level and activity of sigma32 at intermediate temperature and faster shut-off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat-denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shut-off phase of the heat shock response. In contrast, a decrease in FtsH levels increased the sigma32 levels, but the accumulated sigma32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress-sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of sigma32 in vivo.

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of sigma32, an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades sigma32 and has a central role in the control of the sigma32 level. The ftsH null mutant was isolated, and establishment of the DeltaftsH mutant allowed us to investigate control mechanisms of the stability and the activity of sigma32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of sigma32 ( approximately 20-fold of the wild type), leading to the impaired downregulation of the level of sigma32. Surprisingly, however, DeltaftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of sigma32 when it is accumulated. Overproduction of DnaK/J reduces the activity of sigma32 in DeltaftsH cells without any detectable changes in the level of sigma32, indicating that the DnaK chaperone system is responsible for the activity control of sigma32 in vivo. In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of sigma32.

Improvement of the immunomagnetic separation method selective for Escherichia coli O157 strains.

Immunomagnetic separation is a useful enrichment method selective for Escherichia coli O157 cells against non-O157 E. coli cells from a preenrichment culture. However, E. coli cells are adsorbed onto a solid surface nonspecifically. With the conventional immunomagnetic separation method, this nonspecific adsorption interfered with immunomagnetic separation. It was found that this interference could be reduced with a low-ionic-strength solution. When immunomagnetic separation was carried out with this solution, the proportion of E. coli O157 cells to non-O157 E. coli cells increased from 9.6 to 31.4 times compared to the proportion obtained by the conventional immunomagnetic separation method. The effectiveness of this solution was successfully evaluated by the use of E. coli O157-spiked samples.

SpoVM, a small protein essential to development in Bacillus subtilis, interacts with the ATP-dependent protease FtsH.

The spoVM gene encodes a 26-amino-acid polypeptide that is essential for spore formation in Bacillus subtilis. A transposon insertion within the spoVM open reading frame has been shown to encode a chimeric protein which is biologically inactive and produces a phenotype identical to that of a deletion and insertion mutation. A genetic approach was used to identify possible interacting proteins, and the membrane-bound FtsH protease was identified. Mutations in ftsH suppressed the sporulation defect of certain spoVM mutants but not others. However, production of the mother cell sigma factors, sigmaE and sigmaK, was abnormal in the suppressed strains, and mutations in either spoVM or ftsH alone impaired sigma factor production and sporulation gene expression. Using FtsH purified from Escherichia coli, we demonstrated that in vitro (i) SpoVM inhibits FtsH protease activity and (ii) SpoVM is a substrate for the FtsH protease. We propose that during sporulation, SpoVM serves as a competitive inhibitor of FtsH activity. This interaction appears to be important for completion of the prespore engulfment step of sporulation, based on the phenotype of certain spoVM ftsH double mutants.

Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli.

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage lambda CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage lambda. Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor sigma32. Proteolysis of sigma32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of sigma32. Electron microscopy revealed that purified FtsH forms ring-shaped structures with a diameter of 6-7 nm.

The tolZ gene of Escherichia coli is identified as the ftsH gene.

Escherichia coli tolZ mutants are tolerant to colicins E2, E3, D, Ia, and Ib (Tol-), can grow on glucose but not on succinate or other nonfermentable carbon sources (Nfc-), and show temperature-sensitive growth (Ts). A 1.8-kb DNA fragment that complemented the tolZ mutation was cloned. The DNA fragment was sequenced, and one open reading frame was found. This frame was identical to a part of the E. coli FtsH protein, an ATP-dependent metalloprotease that binds to the cytoplasmic membrane. The tolZ gene was located at 69 min on the E. coli genetic map, and the mutation was complemented by a plasmid carrying the ftsH gene, indicating that the tolZ gene is identical to the ftsH gene. The mutated tolZ21 gene was also cloned and sequenced and was found to have a single base change that caused an amino acid alteration of His-418 to Tyr in the FtsH protein. The tolZ21 mutant showed Hfl- (high frequency of lysogenization) and Std- (stop transfer-defective) pheno-types, both of which are due to a mutation in the ftsH (hflB) gene. However, the ftsH1, ftsH101, and hflB29 mutants did not show Tol- and Nfc phenotypes. The tolZ21 mutant was found to have a suppressor mutation, named sfhC, which allowed cells to survive. The sfhC mutation alone caused no Tol-, Nfc-, Ts, or Hfl- phenotypes in the tolZ21 mutant.

A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32.

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress-dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter-specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase-bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ-sigma32 complexes are 5900- and 20-fold higher respectively than those of DnaK-sigma32 complexes in the absence of ATP. ATP destabilizes DnaK-sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK-bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK-DnaJ-sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.

Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

A Lactococcus lactis gene encodes a membrane protein with putative ATPase activity that is homologous to the essential Escherichia coli ftsH gene product.

A gene, encoding a protein homologous to an essential Escherichia coli protein, FtsH, was identified adjacent to the hpt gene and the trnA operon in the Gram-positive bacterium Lactococcus lactis. The deduced amino acid sequence of the gene product showed full-length similarity to FtsH of E. coli, Yme1p of Saccharomyces cerevisiae and a conserved region found in a new family of putative ATPases. In-frame fusions of L. lactis ftsH and phoA1 in E. coli, and immunodetection of the L. lactis FtsH protein in cell fractions using anti-E. coli FtsH serum showed that L. lactis ftsH was expressed and encodes a membrane protein. When contained on a high copy number plasmid, the L. lactis ftsH gene complemented the lethality of a delta ftsH3::kan mutation in E. coli at 37 degrees C and below, indicating that the L. lactis ftsH gene can functionally replace the E. coli ftsH gene to some extent. The resulting E. coli strain showed temperature sensitivity and salt sensitivity. A L. lactis mutant with an insertion into ftsH was salt-, heat- and cold-sensitive. These results suggest that FtsH is somehow involved in stress responses. Southern hybridization analysis indicated that genes homologous to ftsH of L. lactis were also present in Bacillus subtilis, and several Lactobacillus and Leuconostoc species, suggesting high conservation of ftsH in bacterial species.

Cell growth and lambda phage development controlled by the same essential Escherichia coli gene, ftsH/hflB.

The lambda phage choice between lysis and lysogeny is influenced by certain host functions in Escherichia coli. We found that the frequency of lambda lysogenization is markedly increased in the ftsH1 temperature-sensitive mutant. The ftsH gene, previously shown to code for an essential inner membrane protein with putative ATPase activity, is identical to hflB, a gene involved in the stability of the phage cII activator protein. The lysogenic decision controlled by FtsH/HflB is independent of that controlled by the protease HflA. Overproduction of FtsH/HflB suppresses the high frequency of lysogenization in an hflA null mutant. The FtsH/HflB protein, which stimulates cII degradation, may be a component of an HflA-independent proteolytic pathway, or it may act as a chaperone, maintaining cII in a conformation subject to proteolysis via such a pathway. Suppressor mutations of ftsH1 temperature-sensitive lethality, located in the fur gene (coding for the ferric uptake regulator), did not restore FtsH/HflB activity with respect to lambda lysogenization.

The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression.

The ftsH gene is essential for cell viability in Escherichia coli. We cloned and sequenced the wild-type ftsH gene and the temperature-sensitive ftsH1(Ts) gene. It was suggested that FtsH protein was an integral membrane protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding domain. The ftsH1(Ts) gene was found to have two base substitutions within the coding sequence corresponding to the amino acid substitutions Glu-463 by Lys and Pro-587 by Ala. Homology search revealed that an approximately 200-amino-acid domain, including the putative ATP-binding sequence, is highly homologous (35 to 48% identical) to the domain found in members of a novel, eukaryotic family of putative ATPases, e.g., Sec18p, Pas1p, CDC48p, and TBP-1, which function in protein transport pathways, peroxisome assembly, cell division cycle, and gene expression, respectively. Possible implications of these observations are discussed.