Homologous cpn60 genes in Rhizobium leguminosarum are not functionally equivalent

Many bacteria possess 2 or more genes for the chaperonin GroEL and the cochaperonin GroES. In particular, rhizobial species often have multiple groEL and groES genes, with a high degree of amino-acid similarity, in their genomes. The Rhizobium leguminosarum strain A34 has 3 complete groE operons, which we have named cpn.1, cpn.2 and cpn.3. Previously we have shown the cpn. 1 operon to be essential for growth, but the two other cpn operons to be dispensable. Here, we have investigated the extent to which loss of the essential GroEL homologue Cpn60.1 can be compensated for by expression of the other two GroEL homologues (Cnp60.2 and Cpn60.3). Cpn60.2 could not be overexpressed to high levels in R. leguminosarum, and was unable to replace Cpn60.1. A strain that overexpressed Cpn60.3 grew in the absence of Cpn60.1, but the complemented strain displayed a temperature-sensitive phenotype. Cpn60.1 and Cpn60.3, when coexpressed in Escherichia coli, preferentially selfassembled rather than forming mixed heteroligomers. We conclude that, despite their high amino acid similarity, the GroEL homologues of R. leguminosarum are not functionally equivalent in vivo.     

Two of the three <Emphasis Type="Italic">groEL</Emphasis> homologues in <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> 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.Dedicated to the memory of Professor V. Javier Benedí, 1957–2002     

The unusual mycobacterial chaperonins: evidence for in vivo oligomerization and specialization of function

The pathogen Mycobacterium tuberculosis expresses two chaperonins, one (Cpn60.1) dispensable and one (Cpn60.2) essential. These proteins have been reported not to form oligomers despite the fact that oligomerization of chaperonins is regarded as essential for their function. We show here that the Cpn60.2 homologue from Mycobacterium smegmatis also fails to oligomerize under standard conditions. However, we also show that the Cpn60.2 proteins from both organisms can replace the essential groEL gene of Escherichia coli, and that they can function with E. coli GroES cochaperonin, as well as with their cognate cochaperonin proteins, strongly implying that they form oligomers in vivo. We show that the Cpn60.1 proteins, but not the Cpn60.2 proteins, can complement for loss of the M. smegmatis cpn60.1 gene. We investigated the oligomerization of the Cpn60.2 proteins using analytical ultracentrifugation and mass spectroscopy. Both form monomers under standard conditions, but they form higher order oligomers in the presence of kosmotropes and ADP or ATP. Under these conditions, their ATPase activity is significantly enhanced. We conclude that the essential mycobacterial chaperonins, while unstable compared to many other bacterial chaperonins, do act as oligomers in vivo, and that there has been specialization of function of the mycobacterial chaperonins following gene duplication.     

Affinity purification of fusion chaperonin Cpn60-(His)(6) from thermophilic bacterium Bacillus strain MS and its use in facilitating protein refolding and preventing heat denaturation

The cpn60 gene from Bacillus strain MS, which is highly homologous to Bacillus stearothermophilus, was cloned. Cpn60 with a hexahistidine affinity tag (His)(6) fused to its C-terminus (cpn60-(His)(6)) was overproduced in Escherichia coli. Cpn60-(His)(6) was expressed in a soluble form in E. coli. and purified to homogeneity in a single step by nickel chelate affinity chromatography. Cpn60-(His)(6) formed a tetradecamer and had ATPase activity. Cpn60-(His)(6) mediated refolding of guanidine hydrochloride unfolded pig heart malic dehydrogenase (MDH) and Thermus flavus MDH at 25 and 70 degrees C, respectively, in an ATP-dependent manner. In addition, cpn60-(His)(6) prevented heat denaturation of pig heart MDH and T. flavus MDH at 30 and 80 degrees C, respectively, in an ATP-dependent manner. Therefore, cpn60-(His)(6) facilitates protein refolding and prevents heat denaturation of proteins across a wide temperature range.     

Diverse bacteria isolated from root nodules of <Emphasis Type="Italic">Trifolium</Emphasis>, <Emphasis Type="Italic">Crotalaria</Emphasis> and <Emphasis Type="Italic">Mimosa</Emphasis> grown in the subtropical regions of China

We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system. However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of the proteins regulated by these two systems. Accession numbers: DQ173160 (hrcA operon); DQ173161 (rpoH gene).     

Characterization of the A2-A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection

The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2-A2rel gene clusters, the 5' and 3' DNA sequences flanking the A2-A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2-A2rel gene clusters knock-out mutants, it was not possible to delete all the A2-A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2-expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.     

Mycobacterium tuberculosis GroEL homologues unusually exist as lower oligomers and retain the ability to suppress aggregation of substrate proteins

Chaperonin-60s are large double ring oligomeric proteins with a central cavity where unfolded polypeptides undergo productive folding. In conjunction with their co-chaperonin, Chaperonin-60s bind non-native polypeptides and facilitate their refolding in an ATP-dependent manner. The ATPase activity of Chaperonin-60 is tightly regulated by the 10 kDa co-chaperonin. In contrast to most other bacterial species, Mycobacterium tuberculosis genome carries a duplicate set of cpn60 genes, one of which occurs on the groESL operon (cpn60.1), while the other is separately arranged on the chromosome (cpn60.2). Biophysical characterization of the mycobacterial proteins showed that these proteins exist as lower oligomers and not tetradecamers, an unexpected property much different from the other known Chaperonin-60s. Failure of the M.tuberculosis chaperonins to oligomerize can be attributed to amino acid mutations at the oligomeric interface. Rates of ATP hydrolysis of the M.tuberculosis chaperonins showed that these proteins possess a very weak ATPase activity. Both the M.tuberculosis chaperonins were partially active in refolding substrate proteins. Interestingly, their refolding activity was seen to be independent of the co-chaperonin and ATP. We hypothesize that the ATP independent chaperones might offer benefit to the pathogen by promoting its existence in the latent phase of its life cycle.     

Novel motifs in amino acid permease genes from Leishmania

Eight amino acid permease genes from the protozoan parasite Leishmania donovani (AAPLDs) were cloned, sequenced, and shown to be expressed in promastigotes. Seven of these belong to the amino acid transporter-1 and one to the amino acid polyamino-choline superfamilies. Using these sequences as well as known and characterized amino acid permease genes from all kingdoms, a training set was established and used to search for motifs, using the MEME motif discovery tool. This study revealed two motifs that are specific to the genus Leishmania, four to the family trypanosomatidae, and a single motif that is common between trypanosomatidae and mammalian systems A1 and N. Interestingly, most of these motifs are clustered in two regions of 50-60 amino acids. Blast search analyses indicated a close relationship between the L. donovani and Trypanosoma brucei amino acid permeases. The results of this work describe the cloning of the first amino acid permease genes in parasitic protozoa and contribute to the understanding of amino acid permease evolution in these organisms. Furthermore, the identification of genus-specific motifs in these proteins might be useful to better understand parasite physiology within its hosts.     

Expression of the chaperonin 10 gene during zebrafish development

We have isolated a cDNA encoding chaperonin 10 (cpn10) from the zebrafish. Using northern, western, and in situ hybridization analysis, we observed that the cpn10 gene is expressed uniformly and ubiquitously throughout embryonic development of the zebrafish. Upregulation of cpn10 expression was observed following exposure of zebrafish embryos to a heat shock of 1 hour at 37 degrees C compared to control embryos raised at 27 degrees C. The extracellular form of Cpn10 called early pregnancy factor (EPF), found in the serum of pregnant mammals, was not detected in the serum of either male or female zebrafish. These expression studies suggest that Cpn10 plays a general role in zebrafish development as well as being consistent with the hypothesis that EPF is involved in the embryo implantation process in mammals.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

The dimeric structure of the Cpn60.2 chaperonin of Mycobacterium tuberculosis at 2.8 Å reveals possible modes of function

Mycobacterium tuberculosis expresses two proteins (Cpn60.1 and Cpn60.2) that belong to the chaperonin (Cpn) family of heat shock proteins. Studies have shown that the two proteins have different functional roles in the bacterial life cycle and that Cpn60.2 is essential for cell viability and may be involved in M. tuberculosis pathogenicity. Cpn60.2 does not form a tetradecameric double ring, which is typical of other Cpns. We have determined the crystal structure of recombinant Cpn60.2 to 2.8 Å resolution by molecular replacement; the asymmetric unit (AU) contains a dimer, which is consistent with size-exclusion high-performance liquid chromatography and dynamic light-scattering measurements of the soluble recombinant protein. However, we suggest that the actual Cpn60.2 dimer may be different from that identified within the AU on the basis of surface contact stability, solvation free-energy gain, and functional aspects. Unlike the dimer found in the AU, which is formed through apical domain interactions, the dimeric form we propose here provides a free apical domain that is required for normal chaperone activity and may be involved in M. tuberculosis association with macrophages and arthrosclerosis plaque formation. Here we describe in detail the structural aspects that lead to Cpn60.2 dimer formation and prevent the formation of heptameric rings and tetradecameric double rings.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes

The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ～10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ～10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ～10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.     

Genomic organization and functional expression of differentially regulated cysteine protease genes of Leishmania donovani complex

For the first time, we report the genomic organization and characterization of Cathepsin L-like cysteine protease gene cluster from the members of Leishmania donovani complex. The cysteine protease gene cluster of Leishmania chagasi has five copies of tandemly arranged genes. The first gene (Ldccys1A) is identical to Ldccys1 cDNA and is predominantly expressed in promastigotes. The last gene (Ldccys1E) is identical to Ldccys1A with a 13 amino acids deletion in the mature domain, including one of the active site histidine residues and a truncated carboxyl terminal extension. It has a diverged 3' untranslated region and is also constitutively expressed in the parasite. Results from rapid amplification of cDNA ends (RACE) suggest that there are three different types of 3' untranslated regions, one of them is identical to that of Ldccys1A whereas another to Ldccys1E. The third one is also identical to Ldccys1A, but has a 154 nucleotides deletion near the polyA region and this gene is constitutively expressed. Gene organization and expression in L. donovani cluster is similar to that of L. chagasi. However, the last gene (Lddcys1F) is different from Ldccys1E as it lacks 13 amino acid deletions. Also, L. donovani possesses an additional copy of the gene (Lddcys1E), which is located away from the cluster. Furthermore, for the first time we have expressed full-length cysteine protease genes in an insect expression system. Ldccys1A and Ldccys1F cleaved gelatin whereas Ldccys1E was found to be inactive in gelatin assays.     

Characterisation of a new Leishmania META gene and genomic analysis of the META cluster

The META1 gene of Leishmania is upregulated in metacyclic promastigotes and encodes a 12 kDa virulence-related protein, conserved in all Leishmania species analysed. In this study, the genomic region adjacent to the Leishmania amazonensis META1 gene was characterised and compared to the Leishmania major META1 locus as well as to syntenic loci identified in Trypanosoma brucei and Trypanosoma cruzi. Three new genes expressed with increased abundance of steady state mRNA in L. amazonensis promastigotes were identified, two of which are upregulated in stationary phase promastigotes, sharing the pattern of expression previously described for the META1 mRNA. One of these new genes, named META2, encodes a polypeptide of 444 amino acid residues with a repetitive structure showing three repeats of the META domain (defined as a small domain family found in the Leishmania META1 protein and in bacterial proteins hypothetically secreted and/or implicated in motility) and a carboxyl-terminal region similar to several putative calpain-like proteins of Trypanosoma and Leishmania.     

Evolution of codon usage and base contents in kinetoplastid protozoans

In this study we analyze and compare the trends in codon usage in five representative species of kinetoplastid protozoans (Crithidia fasciculata, Leishmania donovani, L. major, Trypanosoma cruzi and T. brucei), with the purpose of investigating the processes underlying these trends. A principal component analysis shows that the G+C content at the third codon position represents the main source of codon-usage variation, both within species (among genes) and among species. The non- Trypanosoma species exhibit narrow distributions in codon usage, while both Trypanosoma species present large within-species heterogeneity. The three non-Trypanosoma species have very similar codon-usage preferences. These codon preferences are also shared by the highly expressed genes of T. cruzi and to a lesser degree by those of T. brucei. This leads to the conclusion that the codon preferences shared by these species are the ancestral ones in the kinetoplastids. On the other hand, the study of noncoding sequences shows that Trypanosoma species exhibit mutational biases toward A + T richness, while the non- Trypanosoma species present mutational pressure in the opposite direction. These data taken together allow us to infer the origin of the different codon-usage distributions observed in the five species studied. In C. fasciculata and Leishmania, both mutational biases and (translational) selection pull toward G + C richness, resulting in a narrow distribution. In Trypanosoma species the mutational pressure toward A + T richness produced a shift in their genomes that differentially affected coding and noncoding sequences. The effect of these pressures on the third codon position of genes seems to have been inversely proportional to the level of gene expression.      

c-fos and tumor necrosis factor gene expression in Leishmania donovani-infected macrophages.

Leishmania donovani is an obligate intracellular protozoan which resides in macrophages and impairs a number of macrophage functions. We have undertaken to study this host cell-parasite interaction by examining the ability of L. donovani to impair the transmission of information from the cell surface to the nucleus and thus influence normal gene expression. We demonstrate that, in response to lipopolysaccharide, expression of both the c-fos and tumor necrosis factor genes was impaired in L. donovani-infected macrophages. Indomethacin reversed the parasite-mediated downregulation of the tumor necrosis factor gene but not the c-fos gene, suggesting that the impaired expression of these two genes occurred through different mechanisms. Direct stimulation of protein kinase C with oleoyl-2-acetoyl-3-glycerol did not abrogate inhibition of c-fos gene expression by L. donovani; however, L929 cell-conditioned medium induced a similar level of c-fos gene expression in both infected and noninfected macrophages. Impairment of c-fos gene expression by L. donovani thus appeared to be selective, depending on the external stimuli used to induce its expression. These data argue that L. donovani was capable of impairing macrophage gene expression in a selective rather than a general manner.