A transposon insertion mutant of Mycobacterium fortuitum attenuated in virulence and persistence in a murine infection model that is complemented by Rv3291c of Mycobacterium tuberculosis.

Mycobacterium fortuitum is a non-tubercular fast growing pathogenic mycobacteria whose virulence factors have not been studied. Infection of M. fortuitum ATCC 6841 in a murine infection model leads to spinning of the head in 8-12 days after infection, 20-25% mortality and a constant bacillary load in the kidney of mice, suggesting persistence. From a TnphoA insertion library, a mutant MT13 was isolated which was attenuated in virulence with lesser bacterial burden, milder and delayed spinning of the head and no mortality of mice. The significant feature of the mutant was its failure to persist in kidney and thus the persistent bacillary load characteristic exhibited by the wild type strain was not observed. The insertion of transposon in MT13 was mapped in a region of the genome, which showed homology to Rv3291c of M. tuberculosis, annotated as a transcriptional regulatory factor and reported to be up regulated in nutrient starvation and anaerobic persistent states. Complementation of MT13 with rv3291c resulted in restoration of wild type characteristics including persistence in kidney suggesting the role of a Rv3291c homolog in the virulence and persistence of M. fortuitum.

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast).

We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using two-dimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in other types of ribosomes. Orthologues of E. coli L25 and L30 are absent in spinach plastid ribosome. 25 of the plastid 50 S RPs are encoded in the nuclear genome and synthesized on cytosolic ribosomes, whereas eight of the plastid RPs are encoded in the plastid organelle genome and synthesized on plastid ribosomes. Sites for transit peptide cleavages in the cytosolic RP precursors and formyl Met processing in the plastid-synthesized RPs were established. Post-translational modifications were observed in several mature plastid RPs, including multiple forms of L10, L18, L31, and PSRP-5 and N-terminal/internal modifications in L2, L11 and L16. Comparison of the RPs in gradient-purified 70 S ribosome with those in the 30 and 50 S subunits revealed an additional protein, in approximately stoichiometric amount, specific to the 70 S ribosome. It was identified to be plastid ribosome recycling factor. Combining with our recent study of the proteins in plastid 30 S subunit (Yamaguchi, K., von Knoblauch, K., and Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465), we show that spinach plastid ribosome comprises 59 proteins (33 in 50 S subunit and 25 in 30 S subunit and ribosome recycling factor in 70 S), of which 53 are E. coli orthologues and 6 are plastid-specific proteins (PSRP-1 to PSRP-6). We propose the hypothesis that PSRPs were evolved to perform functions unique to plastid translation and its regulation, including protein targeting/translocation to thylakoid membrane via plastid 50 S subunit.

Synthesis and preliminary pharmacological investigations of 1-(1,2-dihydro-2-acenaphthylenyl)piperazine derivatives as potential atypical antipsychotic agents in mice.

In research towards the development of new atypical antipsychotic agents, one strategy is that the dopaminergic system can be modulated through manipulation of the serotonergic system. The synthesis and preliminary pharmacological evaluation of a series of potential atypical antipsychotic agents based on the structure of 1-(1,2-dihydro-2-acenaphthylenyl)piperazine (7) is described. Compound 7e, 5-{2-[4-(1,2-dihydro-2-acenaphthylenyl)piperazinyl]ethyl}-2,3-dihy dro-1H- indol-2-one, from this series showed significant affinities at the 5-HT1A and 5-HT2A receptors and moderate affinity at the D2 receptor. 7e exhibits a high reversal of catalepsy induced by haloperidol indicating its atypical antipsychotic nature.

Different consequences of incorporating chloroplast ribosomal proteins L12 and S18 into the bacterial ribosomes of Escherichia coli.

We have incorporated chloroplast ribosomal proteins (R-proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)-dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coil S18, 74 residues), containing a repeating, chloroplast-specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 30S subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coil ribosomes by a chloroplast R-protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12-lacking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)-dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a 'chloroplast loop' present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R-proteins concerned with specific functions are more divergent than their assembly properties. We have previously described methods suitable for overproduction and purification of chloroplast R-proteins that are encoded in organellar DNA (approximately 20), but that gave poor yield for those encoded in the nuclear DNA (approximately 45). Here we describe a method that overcomes this problem and allows the purification of nucleus-encoded chloroplast R-proteins in milligram quantities.

Evolution of the NH2- and COOH-terminal extensions of chloroplast ribosomal protein S18. Nucleotide sequence of pea and rye chloroplast rps 18 genes.

An unusual, variably repeated heptapeptide motif is present in most chloroplast ribosomal protein S18 sequences (Weglöhner and Subramanian, FEBS Lett. 269, 193-197, 1991), whereas it is absent in bacterial, cyanelle, and in the chloroplast S18 of the lower plant liverwort. In order to understand the evolution of this higher plant-specific motif, we have cloned and sequenced chloroplast rps18 genes from pea, a dicot plant of the large legume family and rye, a monocot plant with temperature-sensitive chloroplast ribosome formation. The derived amino acid sequence of pea S18 protein shows two and that of rye seven repeats of this motif. We also show that a different heptapeptide motif is discernible in the recently published chloroplast S18 sequence of Pinus thunbergii (a gymnosperm), which can however be derived convergently from a putative progenitor of angiosperm-gymnosperm chloroplast S18. The presence of a 3-fold repeat of an asparagine-rich heptapeptide in the C-terminal extensions of all cereal S18 is also shown here. The results are further discussed in terms of possible origin of these repeats and the ribosomal protein evolution in general.

Recognition of novel and divergent higher plant chloroplast ribosomal proteins by Escherichia coli ribosome during in vivo assembly.

Architecture of higher plant chloroplast ribosomes involves additional protein domains over that found in the Escherichia coli ribosome, although the rRNAs in these two kinds of ribosomes are very similar in length and sequence (Subramanian, A. R. (1993) Trends Biochem. Sci. 18, 177-180). Here, we show that two chloroplast-specific protein domains (a novel chloroplast ribosomal protein of the 30 S subunit, called Psrp-1 or S22, and a divergent protein of the 50 S subunit with long terminal extensions and low homology to its E. coli counterpart, L21) are both incorporated in E. coli ribosomes and polysomes when their gene constructs are expressed in E. coli. Also, the 67-residue NH2-terminal extension in chloroplast L21 by itself is incorporated. Thus, our results indicate preexisting binding sites for novel chloroplast-specific ribosomal proteins/domains on eubacterial ribosomes. Additionally, we observed cleavage of the chloroplast-targeting transit peptide (present in the expressed Psrp-1 precursor), indicating protease(s) of the required specificity in E. coli cells. The expression of chloroplast L21 with its NH2-terminal extension was inhibitory to E. coli growth, suggesting a drastic effect of the latter on some property of L21. Expression of Psrp-1 was neutral, consistent with a function only in chloroplast translation. Based on analysis of the assembly of Psrp-1 and various L21 fragments in E. coli ribosomes, a general model for studying ribosomal protein-ribosome interactions is suggested.

Protein substitution in chloroplast ribosome evolution. A eukaryotic cytosolic protein has replaced its organelle homologue (L23) in spinach.

The chloroplast translational system differs from the eubacterial ones in containing several ribosomal proteins (RPs) that have no apparent homologues in eubacteria, and in having their RP genes distributed in two cellular genome compartments. The genes maintained in the organelle genome encode mainly ribosome assembly proteins. The discovery in spinach and related plants (Caryophyllidae) of a disrupted chloroplast gene encoding the ribosome assembly protein L23 raised speculations about the transfer of the functional rpl23 gene to the nucleus or the evolutionary loss of L23 protein requirement. To solve this problem, we overexpressed in E. coli the intact rpl23 gene from corn (Zea mays), purified the protein and raised antibodies. Based on immunoanalysis, we show that a prokaryotic-type L23 protein is absent in spinach. Concomittantly we have isolated a new protein from spinach chloroplast 50 S ribosomal subunits and determined its amino acid sequence. The data revealed an unexpectedly high sequence identity to the eukaryotic family of cytosolic L23 proteins (reported from yeast, trypanosome and rat), with conservation of a peptide motif responsible for the specific interaction of these proteins with domain III of 26 S and 23 S rRNA. We propose that the prokaryotic-type L23 protein in the chloroplast ribosomes of Caryophyllidae has been replaced by a homologue of the eukaryotic cytosolic L23 family. These results represent the first case of a protein (gene) substitution in chloroplast ribosome evolution, and open a new view on how the nuclear genome could progressively exert stronger control over the chloroplast translational system. We also describe experiments on the incorporation of chloroplast L23 into E. coli ribosomes, its effect on cell growth, and an unexpected immuno cross-reaction between two chloroplast RP families.

Multicopy GTPase center protein L12 of Arabidopsis chloroplast ribosome is encoded by a clustered nuclear gene family with the expressed members closely linked to tRNA(Pro) genes.

A conserved architectural feature of ribosomes is a protuberance (the stalk) in the large subunit, essential for ribosomal interactions with translational factors and GTP hydrolysis and generated by two dimers of L12, the only multicopy protein in ribosomes. In higher plants, the rpl12 gene for chloroplast L12 is located in the nucleus. We report here the cloning and sequencing of this nuclear gene from Arabidopsis thaliana, revealing the first gene family for a chloroplast ribosomal protein (RP). A single cluster/haploid genome of three rpl12 genes is located in the sequenced 9.1-kilobase region of the Arabidopsis genome. Two of the rpl12 genes encode identical mature proteins, and the third encodes a 25% divergent RP, although the chloroplast-targeting transit peptide in each is distinct. The rpl12 genes encoding identical RPs are closely linked at their 5' ends to identical cytosolic tRNA(Pro) genes with a < 250-base pair spacer. Reverse transcriptase polymerase chain reaction experiments with total RNA isolated from Arabidopsis (and characterization of several L12 cDNA clones) show that only the tRNA-linked rpl12 genes are expressed. We also show (by polymerase chain reaction experiments with isolated total DNA) that this tRNA(Pro)-rpl12 gene linkage is conserved in spinach (inferred to contain a single gene copy) indicating its importance. The previously described enhanced translation of spinach L12 mRNA from its two tandem AUG codons and the two functional rpl12 genes in Arabidopsis probably provide two mechanisms for generating the four copies of L12/chloroplast ribosome, qualitatively different from those attempted in eubacteria.

A novel operon organization involving the genes for chorismate synthase (aromatic biosynthesis pathway) and ribosomal GTPase center proteins (L11, L1, L10, L12: rplKAJL) in cyanobacterium Synechocystis PCC 6803.

Many of the ribosomal protein (RP) genes in both bacterial and chloroplast genomes occur, for reasons not yet understood, in operons that include nonribosomal genes. Here we report such an operon organization in a cyanobacterium (Synechocystis PCC6803) involving the genes for four RPs that are important in the GTPase function of the ribosome and the aroC gene encoding chorismate synthase, a key enzyme in the shikimate pathway for biosynthesis of aromatic amino acids and cell wall components. The Synechocystis aroC encodes a 362-amino-acid residue protein which is 52, 60, and 68% identical to two eubacterial (both 52%), yeast, and a higher plant (Corydalis) chorismate synthase, respectively. The gene was overexpressed in Escherichia coli, and the gene product was shown to cross-react with antibodies to Corydalis chorismate synthase; it also complemented an aroC-lacking E. coli strain. The Synechocystis rpl1 and rpl11 genes encode polypeptides of 237 and 141 amino acid residues, respectively, also with high sequence identities to the corresponding RP sequences from other eubacteria and higher plant chloroplasts. The gene order is shown to be: rpl11-86bp spacer-rpl1-460bp spacer-rpl10-87-bp spacer-rpl12-206bp spacer-aroC. Southern and Northern blot analyses of Synechocystis DNA and RNA, respectively, revealed a single cluster of these genes per genome which is transcribed from a common promoter to an unusually long, approximately 9500-nucleotide transcript. Several constructs of the cyanobacterial aroC and rpl12 genes were made and expressed in E. coli to examine the mechanisms for their very differential expression from a polycistronic mRNA (e.g. four copies L12/ribosome; chorismate synthase, a non-abundant protein). These results present the first biochemical/molecular genetic evidence of shikimate pathway in the cyanobacterial group.

Molecular genetics of chloroplast ribosomal proteins.

Chloroplasts contain a complete translational apparatus which, in land plants, synthesizes the 80 or so polypeptides encoded by the organelle's own small genome. Recent molecular genetic studies have revealed much about the chloroplast ribosomal proteins (RPs). Some of these proteins are encoded by the chloroplast genome and others by the nuclear genome. Many of these genes have now been cloned and characterized, including some that have no prokaryotic homologues.

Co-transcription pattern of an introgressed operon in the maize chloroplast genome comprising four ATP synthase subunit genes and the ribosomal rps2.

Several examples of the introduction of a gene from one gene complex into another (introgression) are found when chloroplast RP gene clusters are compared to those in Escherichia coli or cyanobacteria. Here we describe the transcript pattern of one such cluster from maize (Zea mays) that includes the genes for 4 subunits of the thylakoid ATP synthase (atpI, H, F, A) and the rps2 gene. Twelve transcript species covering the size range from 7,000 to 800 nt were identified in RNA isolated from dark-grown and greening maize seedlings, and several of them were characterized by reverse transcription analysis. A major species of 6,200 nt, with its 5' end at 181 nt upstream of the initiating ATG of rps2, contained the transcripts of all the 5 genes. Two further sets of transcripts having their 5' ends ca. 120 and 50 nt upstream of the initiation codons of the atpI and atpH genes were also identified. Thus, this plastid gene cluster in maize is functionally organized as an operon with additional regulatory features to allow for increased accumulation of mRNAs for the thylakoid components.

Nucleotide sequence of maize chloroplast rpl32: completing the apparent set of plastid ribosomal protein genes and their tentative operon organization.

By sequencing the rpl32 gene, we have characterized the apparent complete set of the RP genes in Zea mays plastid genome. Key data for these 21 genes (total of 26 gene copies) and the proteins encoded by them are presented, and the operon organization is discussed on the basis of available transcription data. A nomenclature for the inferred 13 operons is suggested.

Chloroplast rps15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning non-chloroplast-encoded RNA polymerase.

Transcription of plastid genes and transcript accumulation were investigated in white leaves of the albostrians mutant of barley (Hordeum vulgare) and in heat-bleached leaves of rye (Secale cereale) as well as in normal green leaves of both species. Cells of white leaves of the mutant and cells of heat-bleached leaves bear undifferentiated plastids lacking ribosomes and, consequently, plastid translation products, among them the subunits of a putative chloroplast RNA polymerase encoded by the plastid genes rpoA, B, C1 and C2. The following results were obtained. (i) Plastid genes are transcribed despite the lack of chloroplast gene-encoded RNA polymerase subunits. The plastid origin of these transcripts was proven. This finding provides evidence for the existence of a plastid RNA polymerase encoded entirely by nuclear genes. (ii) Transcripts of the rpo genes and of rps15, but not of genes involved in photosynthesis and related processes (psbA, rbcL, atpI-H), were abundantly accumulated in ribosome-deficient plastids. In contrast, chloroplasts accumulated transcripts of photosynthetic, but not of the rpo genes. (iii) Differences in transcript accumulation between chloroplasts and ribosome-deficient plastids are due to different relative transcription rates and different transcript stability. (iv) The observed differences in transcription are not caused by an altered pattern of methylation of plastid DNA. Thus, the prokaryotic plastid genome of higher plants is transcribed by two RNA polymerases. The observed differences in transcription between chloroplasts and undifferentiated plastids might reflect different functions of the two enzymes.

A small novel chloroplast ribosomal protein (S31) that has no apparent counterpart in the E. coli ribosome.

Higher plant chloroplast ribosomes contain several novel protein components whose homologues are not present in the eubacterial E. coli ribosome, indicating a complex evolution of the chloroplast translational apparatus following the endosymbiotic event. Here we describe the isolation and characterization of a new small protein from spinach chloroplast ribosome which has, based on the amino acid sequence and immunological data, no counterpart in the E. coli ribosome. We suggest a nomenclature suitable for this protein and other novel proteins in the chloroplast ribosome.

Purification and characterization of seven chloroplast ribosomal proteins: evidence that organelle ribosomal protein genes are functional and that NH2-terminal processing occurs via multiple pathways in chloroplasts.

Putative genes for 21 ribosomal proteins (RPs) have been identified in the chloroplast DNA of four plants by nucleotide sequencing and homology comparison but few of the gene products have been characterized. Here we report the purification and N-terminal sequencing of seven proteins from the spinach chloroplast ribosome. The data show them to be the homologues of Escherichia coli RPs L20, L32, L33, L36, S12, S16 and S19, and thus support the view that their genes identified in the chloroplast DNA represent functional genes. The initiating methionine residue was not detected in the mature protein in most cases but it was present in S16, indicating that only the formyl group is removed in this case. This result and the previously reported finding of N-methyl alanine at the N-terminus of chloroplast L2 indicate the existence of multiple N-terminal processing pathways in the chloroplast.

Effect of E. coli ribosomal protein S1 on the fidelity of the translational elongation step: reading and misreading of poly(U) and poly(dT).

Ribosomal protein S1 was selectively removed from E. coli ribosomes by affinity chromatography and the effect of added S1 on the translation of poly(dT) [which is read as poly(U) in the presence of neomycin] and on the misreading of poly(U) and poly(dT) were examined. S1 enhances the translation of poly(dT) at low template concentration, which is similar to the effect of S1 on poly(U) translation. The misreading of poly(dT) by E. coli ribosomes is at a lower level than is the case with poly(U). This low misreading is the same for "S1-dependent" and "S1-independent" modes of translation. On the other hand, the misreading of poly(U) is significantly reduced when S1 is present. These results thus indicate that S1 not only facilitates the binding of mRNA to the ribosome as already known, but also plays a role in the correct codon-dependent selection of aminoacyl-tRNA.