Appl Microbiol Biotechnol, 2000 Sep, 54(3), 302 - 10 Enslaved bacteria as new hope for plant biotechnologists; Hager M et al.; The most distinguishing feature of the plant cell is a DNA-containing organelle that sets plants apart from all other organisms: the chloroplast . Compelling evidence supports an endosymbiotic origin for chloroplasts . According to this theory, chloroplasts are descendants of formerly free-living cyanobacterial ancestors which entered an endosymbiotic relationship with a pre-eukaryotic cell and were ultimately integrated into the metabolism of the host cell . Chloroplasts retain many prokaryotic features and their gene expression system still closely resembles that of their eubacterial ancestors . During the past decade, our knowledge about chloroplast biology has benefited immensely from a most remarkable methodological breakthrough: the development of transformation technologies for chloroplast genomes . Moreover, recent advances in the manipulation of higher plant chloroplast genomes have created unprecedented opportunities for the genetic engineering of plants and promise to overcome many of the problems associated with conventional transgenic technologies . This review describes the state of the art in genetic engineering of higher plant chloroplast genomes and highlights the tremendous potential of these technologies for the biotechnology of the future. J Biol Chem, 2001 Jan 12, 276(2), 1361 - 8 Structural and catalytic similarities between nucleotide pyrophosphatases/phosphodiesterases and alkaline phosphatases; Gijsbers R et al.; Nucleotide pyrophosphatases/phosphodiesterases (NPPs) generate nucleoside 5'-monophosphates from a variety of nucleotides and their derivatives . Here we show by data base analysis that these enzymes are conserved from eubacteria to higher eukaryotes . We also provide evidence for the existence of two additional members of the mammalian family of ecto-NPPs . Homology searches and alignment-assisted mutagenesis revealed that the catalytic core of NPPs assumes a fold similar to that of a superfamily of phospho-/sulfo-coordinating metalloenzymes comprising alkaline phosphatases, phosphoglycerate mutases, and arysulfatases . Mutation of mouse NPP1 in some of its predicted metal-coordinating residues (D358N or H362Q) or in the catalytic site threonine (T238S) resulted in an enzyme that could still form the nucleotidylated catalytic intermediate but was hampered in the second step of catalysis . We also obtained data indicating that the ability of some mammalian NPPs to auto(de)phosphorylate is due to an intrinsic phosphatase activity, whereby the enzyme phosphorylated on Thr-238 represents the covalent intermediate of the phosphatase reaction . The results of site-directed mutagenesis suggested that the nucleotide pyrophosphatase/phosphodiesterase and the phosphatase activities of NPPs are mediated by a single catalytic site. Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11348 - 53 Heat shock protein 60 sequence comparisons: duplications, lateral transfer, and mitochondrial evolution; Karlin S et al.; Heat shock proteins 60 (GroEL) are highly expressed essential proteins in eubacterial genomes and in eukaryotic organelles . These chaperone proteins have been advanced as propitious marker sequences for tracing the evolution of mitochondrial (Mt) genomes . Similarities among HSP60 sequences based on significant segment pair alignment calculations are used to deduce associations of sequences taking into account GroEL functional/structural domain differences and to relate HSP60 duplications pervasive in alpha-proteobacterial lineages to the dynamics of lateral transfer and plasmid integration . Multiple alignments with consensuses are determined for 10 natural groups . The group consensuses sharpen the similarity contrasts among individual sequences . In particular, the Mt group matches best with the classical alpha-proteobacteria and closely with Rickettsia but significantly worse with the rickettsial groups Ehrlichia and Orientia . However, across broad protein sequence comparisons, there appears to be no consistent prokaryote whose protein sequences align best with animal Mt genomes . There are plausible scenarios indicating that the nuclear-encoded HSP60 (and HSP70) sequences functioning in Mt are results of lateral transfer and are probably derived from an alpha-proteobacterium . This hypothesis relates to the plethora of duplicated HSP60 sequences among the classical alpha-proteobacteria contrasted with no duplications of HSP60 among other clades of proteobacterial genomes . Evolutionary relations are confounded by differential selection pressures, convergence, variable mutational rates, site variability, and lateral gene transfer. J Periodontol, 2000 Sep, 71(9), 1387 - 94 Detection of major putative periodontopathogens in Korean advanced adult periodontitis patients using a nucleic acid-based approach; Choi BK et al.; BACKGROUND: Although extensive microbial analyses have been performed from subgingival plaque samples of periodontitis patients, systematic analysis of subgingival microbiota has not been carried out in a Korean population so far . The purpose of this study was to describe the prevalence of major putative periodontopathogens in Korean patients by culture-independent methods . METHODS: A total of 244 subgingival plaque samples (5 sites in each participant) were taken from 29 advanced adult periodontitis (AP) patients and 20 periodontally healthy subjects . AP samples were obtained from the 4 deepest periodontal pockets (> or =6 mm probing depth {PD}) and 1 healthy site (< or =3 mm PD) in each patient . Polymerase chain reaction (PCR) of 16S ribosomal DNA (rDNA) of subgingival plaque bacteria was performed with eubacterial primers . Aliquots of PCR products were then applied on nylon membranes and hybridized with specific oligonucleotide probes labeled with digoxigenin . RESULTS: All diseased sites harbored Fusobacterium sp., while Porphyromonas gingivalis, Treponema sp., and Bacteroides forsythus were detected in more than 96% of 116 diseased sites . Peptostreptococcus micros, Actinobacillus actinomycetemcomitans, and Prevotella intermedia were present in 82%, 74%, and 71% of diseased sites, respectively . In sites of periodontally healthy subjects, Fusobacterium sp . was present in the highest proportion (58%) . Treponema sp., P . gingivalis, and B . forsythus were detected in 22%, 18%, and 18% of healthy sites, respectively . P . micros, P . intermedia, and A . actinomycetemcomitans were found in 8%, 2%, and 1% of healthy sites, respectively . The prevalence of the periodontopathogens, with the exceptions of Fusobacterium sp . and B . forsythus, was significantly higher in the healthy sites of periodontitis subjects than in the healthy sites of periodontally healthy subjects (P <0.05) . CONCLUSIONS: Using highly sensitive methods relying on 16S ribosomal RNA-based oligonucleotide probes, we confirmed the strong association of 7 putative periodontopathogens with AP patients in a Korean population . With the exceptions of Fusobacterium sp . and B . forsythus, all the periodontopathogens were significantly more associated with the healthy sites of periodontitis subjects than in the healthy sites of periodontally healthy subjects. Microbiology, 2000 Oct, 146 ( Pt 10), 2573 - 81 The carboxyl terminus of the Bacillus subtilis SecA is dispensable for protein secretion and viability; van Wely KH et al.; The Escherichia coli secretion-dedicated chaperone SecB targets a subset of proteins to the translocase by interacting with the carboxyl (C-) terminus of SecA . This region of SecA is highly conserved in Eubacteria, but despite its presence in the Bacillus subtilis SecA, the B . subtilis genome does not appear to contain a gene for a clear homologue of SecB . Deletion of the C-terminus of the B . subtilis SecA yields cells that have normal viability, but that exhibit a response reminiscent of oxidative stress and the loss of a number of secretory proteins from the culture supernatant . Semi-quantitative RT-PCR demonstrates that these proteins are expressed at lower levels . The C-terminus of SecA fused to glutathione S:-transferase (GST) specifically binds a cytosolic protein, termed MrgA . This protein has been reported to function in relation to oxidative stress, but deletion of the mrgA gene does not result in a secretion defect nor does it cause an oxidative stress response . It is concluded that the C-terminus of the B . subtilis SecA is not essential for secretion and viability. Mikrobiologiia, 2000 Jul-Aug, 69(4), 499 - 505 {Inorganic polyphosphates and phosphohydrolases from Halobacterium salinarium}; Andreeva NA et al.; Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 mumol Pi/g wet biomass by the time it entered the growth retardation phase . The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase . The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days) . The polyphosphate content of H . salinarium cells was close to that of yeasts and eubacteria . The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities of H . salinarium cells were several times lower than those of the majority of eubacteria . The specific activity of pyrophosphatase, the most active hydrolase of H . salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period . Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol . The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa . The effective Km of this enzyme with respect to pyrophosphate was 115 microM. J Biol Chem, 2000 Dec 29, 275(52), 41350 - 7 TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway; Jongbloed JD et al.; The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC . Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy . The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium . Using a proteomic approach, we show that PhoD of B . subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway . Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process . Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway . Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B . subtilis. Mol Microbiol, 2000 Sep, 37(6), 1342 - 56 Tyr-326 plays a critical role in controlling SecA-preprotein interaction; Kourtz L et al.; SecA is an essential ATP-dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane . A region containing residues 267-340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA . To elucidate the function of this region further, we isolated mutants using a combination of region-specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure . Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr-326 was found to be critical for SecA function . secA mutants with non-conservative substitutions at Tyr-326 showed strong protein secretion defects in vivo and were completely defective for SecA-dependent translocation ATPase activity in vitro . The SecA-Y326 mutant proteins were normal in their membrane, SecYE and nucleotide-binding properties . However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays . Our results indicate that the region containing Tyr-326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA. Eur J Biochem, 2000 Oct, 267(19), 6012 - 8 The archaeal elongation factor 1alpha bound to GTP forms a ternary complex with eubacterial and eukaryal aminoacyl-tRNA; Raimo G et al.; The archaeal Sulfolobus solfataricus elongation factor 1alpha (SsEF-1alpha) bound to GTP or to its analogue guanyl-5'-yl imido diphosphate {Gpp(NH)p} formed a ternary complex with either Escherichia coli Val-tRNAVal or Saccharomyces cerevisiae Phe-tRNAPhe as demonstrated by gel-shift and gel-filtration experiments . Evidence of such an interaction also came from the observation that SsEF-1alphaz.rad;Gpp(NH)p was able to display a protective effect against either the spontaneous deacylation or the digestion of aminoacyl-tRNA by RNase A . Protection against the deacylation of aminoacyl-tRNA allowed evaluatation of the affinity of SsEF-1alphaz . rad;Gpp(NH)p for both aminoacyl-tRNAs used . The K'd values of the ternary complex containing S . cerevisiae Phe-tRNAPhe or E . coli Val-tRNAVal were 0.3 microM and 4.4 microM, respectively . In both cases, the affinity of SsEF-1alphaz.rad;Gpp(NH)p for aminoacyl-tRNA was three orders of magnitude lower than that of the homologous eubacterial ternary complexes, but comparable with the affinity shown by the ternary complex involving eukaryal EF-1alpha {Negrutskii, B.S . & El'skaya, A.V . (1998) Prog . Nucleic Acids Res . 60, 47-77} . As already observed with eukaryal EF-1alpha, SsEF-1alpha in its GDP-bound form was also able to protect the ester bond of aminoacyl-tRNA, even though with a 10-fold lower efficiency compared with SsEF-1alphaz.rad;Gpp(NH)p . The overall results indicated that the archaeal elongation factor 1alpha shares several properties with eukaryal EF-1alpha but not with eubacterial EF-Tu. Acta Microbiol Pol, 2000, 49(1), 19 - 29 Sequence, structural, and evolutionary analysis of prokaryotic ribosomal protein L11 methyltransferases; Bujnicki JM; The Escherichia coli PrmA enzyme catalyzes methylation of the large ribosomal subunit protein L11 . Database homology searches, multiple sequence alignment, and structure prediction allowed to dissect the primary structure of PrmA into two domains and assign putative functional or structural roles to invariant or highly conserved residues . Evolutionary relationships within the PrmA family were also analyzed . The topology of the branching order agrees to a large extent with the consensus phylogeny of Eubacteria, with the exception of beta and epsilon subdivisions of Proteobacteria, which most probably had their original prmA genes replaced by copies acquired via the lateral gene transfer from gamma-Proteobacteria and some close relative of the ancestor of gramnegative bacteria, respectively. Mol Membr Biol, 2000 Apr-Jun, 17(2), 75 - 94 Nucleobase transporters (review); de Koning H et al.; Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells . In addition, they can be used as nitrogen sources in plants and many microorganisms . Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins . Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals . A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level . Very recently, nucleobase transporters have been identified in plants . In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead . In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP . The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants . Interestingly, mammalian ascorbate transporters are homologous to NAT sequences . The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships . Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance. Aust Vet J, 2000 Aug, 78(8), 549 - 59 Bacterial infection of the lower respiratory tract in 34 horses; Racklyeft DJ et al.; OBJECTIVE: To investigate associations between the bacteriology and aspects of history, clinical presentation, outcome and pathology of lower respiratory tract disease of 34 horses . PROCEDURE: Detailed aerobic and anaerobic bacteriological investigations were performed on clinical specimens from horses with pneumonia, lung abscessation and necrotic pneumonia with or without pleurisy in an attempt to identify those bacteria that might contribute to the initiation and progression of infection . RESULTS: Bacteria were cultured from 33 of the 34 horses . In ten cases, only aerobic/facultatively anaerobic isolates were cultured while aerobic/facultatively anaerobic bacteria and obligately anaerobic bacteria were isolated in the other 23 cases . Moderate to large numbers of anaerobic bacteria were isolated only when the estimated duration of illness was at least five days . Bacteria were not cultured from 12 of the pleural fluid samples but were always cultured from pulmonary samples (either transtracheal aspirates from live horses or pulmonary lesions at necropsy) . Streptococcus equi subsp zooepidemicus was isolated in the three cases where only one bacterial species was cultured . In the other 30 cases, multiple species were isolated . These included most often and in greatest numbers, Streptococcus equi subsp zooepidemicus, Pasteurellaceae, Escherichia coli, anaerobic cocci, Eubacterium fossor, Bacteroides tectum, Prevotella heparinolytica, Fusobacterium spp, and pigmented members of the genera Prevotella and Porphyromonas . Aerobic/facultatively anaerobic organisms were isolated from 97% of horses, while obligately anaerobic organisms were cultured from 68% of horses . CONCLUSION: There was no association between the isolation of any specific bacterium and the outcome of disease . However, obligately anaerobic bacteria (such as anaerobic cocci, Bacteroides tectum, P heparinolytica and Fusobacterium spp) and the facultatively anaerobic species Escherichia coli, were recovered more commonly from horses that died or were euthanased than from those that survived . There was an association between failure of horses to recover from pleuropneumonia and delay in diagnosis and initiation of treatment. FEMS Microbiol Rev, 2000 Oct, 24(4), 367 - 402 The phylogeny of proteobacteria: relationships to other eubacterial phyla and eukaryotes; Gupta RS; The evolutionary relationships of proteobacteria, which comprise the largest and phenotypically most diverse division among prokaryotes, are examined based on the analyses of available molecular sequence data . Sequence alignments of different proteins have led to the identification of numerous conserved inserts and deletions (referred to as signature sequences), which either are unique characteristics of various proteobacterial species or are shared by only members from certain subdivisions of proteobacteria . These signature sequences provide molecular means to define the proteobacterial phyla and their various subdivisions and to understand their evolutionary relationships to the other groups of eubacteria as well as the eukaryotes . Based on signature sequences that are present in different proteins it is now possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low-G+C Gram-positive-->high-G+C Gram-positive-->Deinococcus-Thermus (green nonsulfur bacteria)-->cyanobacteria-->Spirochetes-->Chlamydia-Cytophaga-Aquifex -green sulfur bacteria-->Proteobacteria-1 (epsilon and delta)-->Proteobacteria-2 (alpha)-->Proteobacteria-3 (beta)-->Proteobacteria-4 (gamma) . An unexpected but important aspect of the relationship deduced here is that the main eubacterial phyla are related to each other linearly rather than in a tree-like manner, suggesting that the major evolutionary changes within Bacteria have taken place in a directional manner . The identified signatures permit placement of prokaryotes into different groups/divisions and could be used for determinative purposes . These signatures generally support the origin of mitochondria from an alpha-proteobacterium and provide evidence that the nuclear cytosolic homologs of many genes are also derived from proteobacteria. J Biol Chem, 2000 Dec 22, 275(51), 40211 - 7 The crystal structure of the Escherichia coli MobA protein provides insight into molybdopterin guanine dinucleotide biosynthesis; Lake MW et al.; The molybdenum cofactor (Moco) is found in a variety of enzymes present in all phyla and comprises a family of related molecules containing molybdopterin (MPT), a tricyclic pyranopterin with a cis-dithiolene group, as the invariant essential moiety . MPT biosynthesis involves a conserved pathway, but some organisms perform additional reactions that modify MPT . In eubacteria, the cofactor is often present in a dinucleotide form combining MPT and a purine or pyrimidine nucleotide via a pyrophosphate linkage . In Escherichia coli, the MobA protein links a guanosine 5'-phosphate to MPT forming molybdopterin guanine dinucleotide . This reaction requires GTP, MgCl(2), and the MPT form of the cofactor and can efficiently reconstitute Rhodobacter sphaeroides apo-DMSOR, an enzyme that requires molybdopterin guanine dinucleotide for activity . In this paper, we present the crystal structure of MobA, a protein containing 194 amino acids . The MobA monomer has an alpha/beta architecture in which the N-terminal half of the molecule adopts a Rossman fold . The structure of MobA has striking similarity to Bacillus subtilis SpsA, a nucleotide-diphospho-sugar transferase involved in sporulation . The cocrystal structure of MobA and GTP reveals that the GTP-binding site is located in the N-terminal half of the molecule . Conserved residues located primarily in three signature sequence motifs form crucial interactions with the bound nucleotide . The binding site for MPT is located adjacent to the GTP-binding site in the C-terminal half of the molecule, which contains another set of conserved residues presumably involved in MPT binding. EMBO J, 2000 Sep 1, 19(17), 4796 - 805 Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation; Fagegaltier D et al.; Decoding of UGA selenocysteine codons in eubacteria is mediated by the specialized elongation factor SelB, which conveys the charged tRNA(Sec) to the A site of the ribosome, through binding to the SECIS mRNA hairpin . In an attempt to isolate the eukaryotic homolog of SelB, a database search in this work identified a mouse expressed sequence tag containing the complete cDNA encoding a novel protein of 583 amino acids, which we called mSelB . Several lines of evidence enabled us to establish that mSelB is the bona fide mammalian elongation factor for selenoprotein translation: it binds GTP, recognizes the Sec-tRNA(Sec) in vitro and in vivo, and is required for efficient selenoprotein translation in vivo . In contrast to the eubacterial SelB, the recombinant mSelB alone is unable to bind specifically the eukaryotic SECIS RNA hairpin . However, complementation with HeLa cell extracts led to the formation of a SECIS-dependent complex containing mSelB and at least another factor . Therefore, the role carried out by a single elongation factor in eubacterial selenoprotein translation is devoted to two or more specialized proteins in eukaryotes. Biochem J, 2000 Sep 15, 350 Pt 3, 609 - 29 Tetrahydrofolate and tetrahydromethanopterin compared: functionally distinct carriers in C1 metabolism; Maden BE; In most organisms, tetrahydrofolate (H(4)folate) is the carrier of C(1) fragments between formyl and methyl oxidation levels . The C(1) fragments are utilized in several essential biosynthetic processes . In addition, C(1) flux through H(4)folate is utilized for energy metabolism in some groups of anaerobic bacteria . In methanogens and several other Archaea, tetrahydromethanopterin (H(4)MPT) carries C(1) fragments between formyl and methyl oxidation levels . At first sight H(4)MPT appears to resemble H(4)folate at the sites where C(1) fragments are carried . However, the two carriers are functionally distinct, as discussed in the present review . In energy metabolism, H(4)MPT permits redox-flux features that are distinct from the pathway on H(4)folate . In the reductive direction, ATP is consumed in the entry of carbon from CO(2) into the H(4)folate pathway, but not in entry into the H(4)MPT pathway . In the oxidative direction, methyl groups are much more readily oxidized on H(4)MPT than on H(4)folate . Moreover, the redox reactions on H(4)MPT are coupled to more negative reductants than the pyridine nucleotides which are generally used in the H(4)folate pathway . Thermodynamics of the reactions of C(1) reduction via the two carriers differ accordingly . A major underlying cause of the thermodynamic differences is in the chemical properties of the arylamine nitrogen N(10) on the two carriers . In H(4)folate, N(10) is subject to electron withdrawal by the carbonyl group of p-aminobenzoate, but in H(4)MPT an electron-donating methylene group occurs in the corresponding position . It is also proposed that the two structural methyl groups of H(4)MPT tune the carrier's thermodynamic properties through an entropic contribution . H(4)MPT appears to be unsuited to some of the biosynthetic functions of H(4)folate, in particular the transfer of activated formyl groups, as in purine biosynthesis . Evidence bearing upon whether H(4)MPT participates in thymidylate synthesis is discussed . Findings on the biosynthesis and phylogenetic distribution of the two carriers and their evolutionary implications are briefly reviewed . Evidence suggests that the biosynthetic pathways to the two carriers are largely distinct, suggesting the possibility of (ancient) separate origins rather than divergent evolution . It has recently been discovered that some eubacteria which gain energy by oxidation of C(1) compounds contain an H(4)MPT-related carrier, which they are thought to use in energy metabolism, as well as H(4)folate, which they are thought to use for biosynthetic reactions. Front Biosci, 2000 Sep 01, 5, D821 - 36 Archaeal peptidyl prolyl cis-trans isomerases (PPIases); Maruyama T et al.; PPIases are ubiquitous in living organisms . While 3 families of PPIases, cyclophilin (CyP), FK506 binding protein (FKBP) and parvulin (Pvn), have been studied in detail in Eukarya and Bacteria (eubacteria), little is known about archaeal PPIases . Among 2 cyclophilins found in Archaea, only Halobacterium cyclophilin (HcCyP19) has been characterized . It is a cyclosporin A (CsA) sensitive CyP with a MW of 19.4kDa . The PPIase activity and CsA sensitivity of this CyP is higher at higher salt concentration in the medium . No parvulin or its homolog has been found in Archaea . Two types of FKBPs, 26-30kDa long type and 17-18 kDa short type FKBP, have been found in Archaea . While the N-terminal regions of these 2 type FKBPs are similar to each other, the long type archaeal FKBP has an additional ca . 100 amino-acid sequence at its C-terminal region . In comparison with human HsFKBP12, the N-terminal region of the archaeal FKBP has 2 insertion sequences in the regions corresponding to Bulge and Flap of HsFKBP12 . A short type archaeal FKBP from Methanococcus thermolithotrophicus has been shown to have not only a PPIase activity but also a chperone like activity, which includes protein refolding and aggregation suppressing activities with regard to protein folding intermediates . Mutational analysis revealed that this chaperone-like activity was independent of the PPIase activity, and that the insertion sequence in the region corresponding to the Flap seemed to be important. J Mol Biol, 2000 Sep 1, 301(5), 1221 - 35 The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno-molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase; Hanzelmann P et al.; Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo(plus) CODH) and low intracellular molybdenum content (Mo(minus) CODH) at 2.25 A and 2.35 A resolution, respectively . The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model . The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II {2Fe-2S} clusters and the flavin-adenine dinucleotide . Unlike the CODH from O . carboxidovorans, the enzyme from H . pseudoflava reveals a unique post-translationally modified C(gamma)-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop . In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H . pseudoflava CODH is a Tyr193 in the O . carboxidovorans CODH . The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein . Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion . The structure of the catalytically inactive Mo(minus) CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized . The 5'-CDP residue was present in Mo(minus) CODH, whereas the Mo-pyranopterin moiety was absent . In Mo(plus) CODH the selenium faces the Mo ion and flips away from the Mo site in Mo(minus) CODH . The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo(plus) and Mo(minus) CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues . Appl Environ Microbiol, 2000 Sep, 66(9), 4058 - 67 Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose; Fernandez AS et al.; Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure . Replicated methanogenic bioreactor communities with two different community structures were established . The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones . One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA . The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA . In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure . The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis . A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S . A . Hashsham, A . S . Fernandez, S . L . Dollhopf, F . B . Dazzo, R . F . Hickey, J . M . Tiedje, and C . S . Criddle, Appl . Environ . Microbiol . 66:4050-4057, 2000). J Bacteriol, 2000 Sep, 182(18), 5147 - 52 Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus; Wilding EI et al.; Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus . In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S . aureus . Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model . The mvaA gene was cloned from S . aureus DNA and expressed with an N-terminal His tag in Escherichia coli . The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated . Unlike most other HMG-CoA reductases, the S . aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme . Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H) . In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H) . pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S . aureus enzyme . Fluvastatin inhibited competitively with HMG-CoA, with a K(i) of 320 microM, over 10(4) higher than that for a class I HMG-CoA reductase . Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci. J Cell Sci, 2000 Sep, 113 ( Pt 18), 3141 - 50 Eukaryotic signal transduction via histidine-aspartate phosphorelay; Thomason P et al.; Transmembrane signal transduction is a feature common to all eukaryotic and prokaryotic cells . We now understand that a subset of the signalling mechanisms used by eukaryotes and prokaryotes are not just similar in principle, but actually use homologous proteins . These are the histidine-aspartate phosphorelays, signalling systems of eubacterial origin, now known to be widespread in eukaryotes outside the animal kingdom . Genome projects are revealing that His-Asp phosphorelays are present as multigene families in lower eukaryotes and in plants . A major challenge is to understand how these 'novel' signal transduction systems form integrated networks with the more familiar signalling mechanisms also present in eukaryotic cells . Already, phosphorelays have been characterised that regulate MAP kinase cascades and the cAMP/PKA pathway . The probable absence of His-Asp phosphorelays from animals has generated interest in their potential as targets for anti-microbial therapy, including antifungals . Recent findings suggest that this approach holds promise. Tsitologiia, 2000, 42(6), 519 - 29 {Cell division genes and proteins in bacterial cells}; Kukekova AV et al.; In this review, genes and proteins involved in cytokinesis and cell proliferation of cell-wall bacteria and mycoplasms are considered . We hope that this comparative analysis of genes and proteins of phylogenetically distant bacteria, including the minimal cells of mycoplasmas, can be useful for understanding the basic principles of prokaryotic cell division . The ftsZ gene was found among representatives of all bacterial groups . The recent data indicate that FtsZ protein plays the central role in the process of bacterial cell division . FtsZ protein was revealed in all Eubacterial groups (including mycoplasmas), in Archaebacteria and chloroplasts, All FtsZ proteins are able to form protofilaments as a result of polymerization in vitro and demonstrate GTF-ase activity . On the base of these properties and some similarities in amino acid sequences with tubulins, it has been suggested that FtsZ protein is an evolutionary ancestor of Eukaryotic tubulins . On the earliest stage of bacterial cytokinesis FtsZ protein assembles into a submembranous Z-ring which encircles bacterial cell in the predivisional site . Some other bacterial proteins take part in stabilization and contraction of the Z-ring, which is considered as a cytoskeleton-like bacterial structure. Mutat Res, 2000 Aug 30, 460(3-4), 301 - 18 DNA ligases in the repair and replication of DNA; Timson DJ et al.; DNA ligases are critical enzymes of DNA metabolism . The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA.Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD(+) . Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar . Higher organisms express a variety of different ligases, which appear to be targetted to specific functions . DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining . Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase . The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known . Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority. Biochimie, 2000 Jun-Jul, 82(6-7), 537 - 48 The role of sigma factors in plastid transcription; Allison LA; Expression of plastid genes is controlled at both transcriptional and post-transcriptional levels in response to developmental and environmental signals . In many cases this regulation is mediated by nuclear-encoded proteins acting in concert with the endogenous plastid gene expression machinery . Transcription in plastids is accomplished by two distinct RNA polymerase enzymes, one of which resembles eubacterial RNA polymerases in both subunit structure and promoter recognition properties . The holoenzyme contains a catalytic core composed of plastid-encoded subunits, assembled with a nuclear-encoded promoter-specificity factor, sigma . Based on examples of transcriptional regulation in bacteria, it is proposed that differential activation of sigma factors may provide the nucleus with a mechanism to control expression of groups of plastid genes . Hence, much effort has focused on identifying and characterizing sigma-like factors in plants . While fractionation studies had identified several candidate sigma factors in purified RNA polymerase preparations, it was only 4 years ago that the first sigma factor genes were cloned from two photosynthetic eukaryotes, both of which were red algae . More recently this achievement has extended to the identification of families of sigma-like factor genes from several species of vascular plants . Now, efforts in the field are directed at understanding the roles in plastid transcription of each member of the rapidly expanding plant sigma factor gene family . Recent results suggest that accumulation of individual sigma-like factors is controlled by light, by plastid type and/or by a particular stage of chloroplast development . These data mesh nicely with accumulating evidence that the core sigma-binding regions of plastid promoters mediate regulated transcription in response to light-regime and plastid type or developmental state . In this review I will outline progress made to date in identifying and characterizing the sigma-like factors of plants, and in dissecting their potential roles in chloroplast gene expression. Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1655 - 63 Phylogeny of 33 ribosomal and six other proteins encoded in an ancient gene cluster that is conserved across prokaryotic genomes: influence of excluding poorly alignable sites from analysis; Hansmann S et al.; Thirty-nine proteins encoded in a large gene cluster that is well-conserved in gene content and gene order across 18 sequenced prokaryotic genomes were extracted, aligned and subjected to phylogenetic analysis . In individual analyses of the alignments, only two probable examples of lateral gene transfer between archaea and eubacteria were detected, involving the genes for ribosomal protein Rpl23 and adenylate kinase . Amino acid sequences for 35 of the 39 proteins were concatenated to yield a data set of 9087 amino acid positions per genome . Many of these proteins, 33 of which are ribosomal proteins, are not highly conserved across distantly related organisms and thus contain many regions that are difficult to align . Phylogenetic analyses were performed with subsets of the concatenated data from which the most highly variable sites had been iteratively removed, using the number of different amino acids that occur at a given site as a criterion of variability . Glycine, which has a strong influence on protein structure, tended to be more frequent at the most conserved (least polymorphic) sites . With most subsets of the data, the proteins from the cyanobacterium Synechocystis tended to branch with their homologues from gram-positive bacteria . The results indicate that excluding only a few percentage of poorly alignable sites from phylogenetic analysis can have a severe impact upon the phylogeny inferred and that bootstrap support for branches can fluctuate substantially, depending upon which sites are excluded. Mol Microbiol, 2000 Aug, 37(3), 528 - 41 The partition system of multidrug resistance plasmid TP228 includes a novel protein that epitomizes an evolutionarily distinct subgroup of the ParA superfamily; Hayes F; The segregational stability of bacterial, low-copy-number plasmids is promoted primarily by active partition . The plasmid-specified components of the prototypical P1 plasmid partition system consist of two proteins, ParA (44.3 kDa) and ParB (38.5 kDa), which, in conjunction with integration host factor, form a nucleoprotein complex at the plasmid partition site, parS . This complex is the probable substrate for the directed temporal and spatial intracellular movement of plasmids before cell division . The genetic organization of the partition cassette of the multidrug resistance plasmid TP228 differs markedly from that of the P1 paradigm . The TP228 system includes a novel member (ParF; 22.0 kDa) of the ParA superfamily of ATPases, of which the P1 ParA protein is the archetype . However, the ParF protein and its immediate relatives form a discrete subgroup of the ParA superfamily, which evolutionarily is more related to the MinD subgroup of cell division proteins than to ParA of P1 . The TP228 and P1 partition modules differ further in that the former does not include a parB homologue, but does specify a protein (ParG; 8.6 kDa) unrelated to ParB . Homologues of the parF gene are widely disseminated on eubacterial genomes, suggesting that ParF-mediated partition may be a common mechanism by which plasmid segregational stability is achieved. Mol Microbiol, 2000 Jul, 37(2), 254 - 62 The Escherichia coli aquaporin-Z water channel; Calamita G; The membrane pathway of the rapid fluxes of water by which microorganisms adapt promptly to abrupt changes in environmental osmolality have begun to be understood since the discovery of the Escherichia coli aquaporin-Z water channel, AqpZ . As in animals and plants, aquaporins are variously represented among microorganisms, in which 31 homologous genes have already been identified in eubacteria, Archaea, fungi and protozoa . The AqpZ channel is selectively permeable to water, although other functions are not excluded . Consistent with a conservation over the course of evolution, AqpZ and AQP1, a human counterpart, share similar structures . The aqpZ gene is growth phase and osmotically regulated . AqpZ has a role in both the short- and the long-term osmoregulatory response and is required by rapidly growing cells . AqpZ-like proteins seem to be necessary for the virulence expressed by some pathogenic bacteria . Microbial aquaporins are also likely to be involved in spore formation and/or germination . Additional roles may still be unknown . The use of AqpZ as a model system will continue to provide insight into the understanding of the importance of aquaporins. J Biol Chem, 2000 Oct 13, 275(41), 31838 - 46 Molecular characterization of lantibiotic-synthesizing enzyme EpiD reveals a function for bacterial Dfp proteins in coenzyme A biosynthesis; Kupke T et al.; The lantibiotic-synthesizing flavoprotein EpiD catalyzes the oxidative decarboxylation of peptidylcysteines to peptidyl-aminoenethiols . The sequence motif responsible for flavin coenzyme binding and enzyme activity is conserved in different proteins from all kingdoms of life . Dfp proteins of eubacteria and archaebacteria and salt tolerance proteins of yeasts and plants belong to this new family of flavoproteins . The enzymatic function of all these proteins was not known, but our experiments suggested that they catalyze a similar reaction like EpiD and/or may have similar substrates and are homododecameric flavoproteins . We demonstrate that the N-terminal domain of the Escherichia coli Dfp protein catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine . This reaction is essential for coenzyme A biosynthesis. FEMS Microbiol Ecol, 2000 Jul 1, 33(1), 11 - 19 Simultaneous detection of the establishment of seed-inoculated Pseudomonas fluorescens strain DR54 and native soil bacteria on sugar beet root surfaces using fluorescence antibody and in situ hybridization techniques; Lubeck PS et al.; Colonization at sugar beet root surfaces by seedling-inoculated biocontrol strain Pseudomonas fluorescens DR54 and native soil bacteria was followed over a period of 3 weeks using a combination of immunofluorescence (DR54-targeting specific antibody) and fluorescence in situ hybridization (rRNA-targeting Eubacteria EUB338 probe) techniques with confocal laser scanning microscopy . The dual staining protocol allowed cellular activity (ribosomal number) to be recorded in both single cells and microcolonies of strain DR54 during establishment on the root . After 2 days, the population density of strain DR54 reached a constant level at the root basis . From this time, however, high cellular activity was only found in few bacteria located as single cells, whereas all microcolony-forming cells occurring in aggregates were still active . In contrast, a low density of strain DR54 was observed at the root tip, but here many of the bacteria located as single cells were active . The native population of soil bacteria, comprising a diverse assembly of morphologically different forms and size classes, initiated colonization at the root basis only after 2 days of incubation . Hence the dual staining protocol allowed direct microscopic studies of early root colonization by both inoculant and native soil bacteria, including their differentiation into active and non-active cells and into single or microcolony-forming cells. Mikrobiologiia, 2000 May-Jun, 69(3), 396 - 406 {Heliobacterium sulfidophilum sp . Nov . and Heliobacterium undosum sp . Nov.: sulfide-oxidizing Heliobacteria from thermal sulfidic springs}; Briantseva IA et al.; Two new species of heliobacteria isolated from cyanobacterial mats of two alkaline sulfidic hot springs are formally described . Strains BR4 and BG29 are assigned to anoxygenic phototrophic bacteria of the family Heliobacteriaceae, since they possess the unique properties of this taxon: strict anaerobiosis, formation of bacteriochlorophyll g, the lack of extensive intracytoplasmic membranes and chlorosomes, an unusual cell wall structure, and phylogenetic relatedness to the low G + C gram-positive eubacteria . Based on the 16S rDNA sequence similarity, strains BR4 and BG29 are assigned to the genus Heliobacterium and described as two new species of this genus: Heliobacterium sulfidophilum sp . nov . and Heliobacterium undosum sp . nov . The G + C content of the DNA is 51.3 mol % in Hbt . sulfidophilum and 57.2-57.7 mol % in Hbt . undosum . The cells of Hbt . sulfidophilum are rods, and the cells of Hbt . undosum are slightly twisted spirilla or short rods . Both new bacteria are motile by peritrichous flagella . Hbt . sulfidophilum produces endospores . The new bacteria are strict anaerobes growing photoheterotrophically on a limited range of organic compounds . In the dark, they can switch from photosynthesis to the slow fermentation of pyruvate . Biotin is required as a growth factor . Both species are highly tolerant to sulfide (up to 2 mM at pH 7.5) and oxidize it photoheterotrophically to elemental sulfur; photoautotrophic growth was not observed . The temperature optimal for growth of Hbt . sulfidophilum and Hbt . undosum is 30-35 degrees C, and the optimal pH is 7-8. Structure Fold Des, 2000 Jul 15, 8(7), 709 - 18 Insights into molybdenum cofactor deficiency provided by the crystal structure of the molybdenum cofactor biosynthesis protein MoaC; Wuebbens MM et al.; BACKGROUND: The molybdenum cofactor (Moco) is an essential component of a large family of enzymes involved in important transformations in carbon, nitrogen and sulfur metabolism . The Moco biosynthetic pathway is evolutionarily conserved and found in archaea, eubacteria and eukaryotes . In humans, genetic deficiencies of enzymes involved in this pathway trigger an autosomal recessive and usually deadly disease with severe neurological symptoms . The MoaC protein, together with the MoaA protein, is involved in the first step of Moco biosynthesis . RESULTS: MoaC from Escherichia coli has been expressed and purified to homogeneity and its crystal structure determined at 2 A resolution . The enzyme is organized into a tightly packed hexamer with 32 symmetry . The monomer consists of an antiparallel, four-stranded beta sheet packed against two long alpha helices, and its fold belongs to the ferredoxin-like family . Analysis of structural and biochemical data strongly suggests that the active site is located at the interface of two monomers in a pocket that contains several strictly conserved residues . CONCLUSIONS: Asp128 in the putative active site appears to be important for catalysis as its replacement with alanine almost completely abolishes protein activity . The structure of the Asp128-->Ala variant reveals substantial conformational changes in an adjacent loop . In the human MoaC ortholog, substitution of Thr182 with proline causes Moco deficiency, and the corresponding substitution in MoaC severely compromises activity . This residue is located near the N-terminal end of helix alpha4 at an interface between two monomers . The MoaC structure provides a framework for the analysis of additional dysfunctional mutations in the corresponding human gene. Pac Symp Biocomput . 2000;:6-17. Enzyme evolution explained (sort of); Dean AM et al.; Sites in proteins evolve at markedly different rates; some are highly conserved, others change rapidly . We have developed a maximum likelihood method to identify regions of a protein that evolve rapidly or slowly relative to the remaining structure . We also show that solvent accessibility and distance from the catalytic site are major determinants of evolutionary rate in eubacterial isocitrate dehydrogenases . These two variables account for most of the rate heterogeneity not ascribable to stochastic effects. Mund Kiefer Gesichtschir, 2000 May, 4(3), 153 - 8 {Pathogen spectrum and resistance status of exclusively anaerobic odontogenic infections}; Eckert AW et al.; Out of 440 dentogenic pyogenic infections, 171 exclusively caused by anaerobes were investigated to understand the importance of anaerobic bacteria in dental pyogenic processes better . Grampositive anaerobic bacteria dominated . The predominant grampositive isolates in monoinfections were Peptostreptococci and in the case of mixed infections, strains of the genus Eubacterium . Strains of Prevotella and Porphyromonas dominated the gramnegative anaerobic spectra . The resistance to penicillin was very low . Altogether, only one strain of Prevotella oris and one strain of Prevotella oralis showed resistance to penicillin. J Agric Food Chem, 2000 Jul, 48(7), 2874 - 6 Identification of cysteinylated aroma precursors of certain volatile thiols in passion fruit juice; Tominaga T et al.; Together with 3-mercaptohexan-1-ol and 3-mercaptohexyl acetate, already known to contribute to the aroma of passion fruit (Passiflora edulis), 3-mercapto-3-methylbutan-1-ol and 3-mercapto-3-methylbutyl acetate have been identified for the first time in this fruit . 3-Mercaptohexan-1-ol and 3-mercapto-3-methylbutan-1-ol may be produced in vitro from nonvolatile extracts of this fruit by the enzymatic action of a cell-free extract of Eubacterium limosum, which has a beta-lyase activity on S-cysteine conjugates (EC 4.4.1.13) . This release strongly suggests that these volatile thiols are present in combined form, as S-cysteine conjugates . It was possible to identify the precursor of 3-mercaptohexan-1-ol as S-(3-hexan-1-ol)-L-cysteine, in the form of trimethylsilylated derivatives from the juice of this fruit, using GC/MS analysis . The presence of free and combined forms of these volatile thiols in this fruit has now been demonstrated. Eur J Clin Microbiol Infect Dis, 2000 May, 19(5), 388 - 92 Diagnostic value of cytokine assays in cerebrospinal fluid in culture-negative, polymerase chain reaction-positive bacterial meningitis; Lorino G et al.; Analysis of bacterial DNA using a polymerase chain reaction performed with broad-range eubacterial 16S rDNA primers may yield a diagnosis of bacterial meningitis in cases where Gram staining of cerebrospinal fluid (CFS), antigen detection techniques or culture fail . Since this PCR technique occasionally gives false-positive results due to contamination of samples or laboratory reagents, a study was performed to establish the diagnostic value of assaying concentrations of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in 90 CSF samples . A high correlation was found between a positive PCR result and the concentrations of TNF-alpha and IL-10, indicating that cytokine assays may be used as a confirmatory test . The findings suggested that a combination of the PCR technique, amplicon sequencing and assay of TNF-alpha and IL-10 concentrations in CSF is a reliable and cost-effective procedure for diagnosis of culture-negative bacterial meningitis. Arch Microbiol, 2000 May-Jun, 173(5-6), 339 - 45 Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum; Le Henaff M et al.; The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin . In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using {35S}cysteine, {35S}methionine, {14C}myristic acid (14C-14:0), {14C}palmitic acid (14C-16:0), and globomycin . SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with 14C-16:0 only (O-ester-linked acyl chains), and the mature form with both 14C-labelled fatty acids (O-ester-linked + amide-linked chains) . Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain . However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components . The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e . is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria . On the basis of these findings, a biogenesis pathway for spiralin is proposed. Biochemistry, 2000 Jul 11, 39(27), 8039 - 46 Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase that compensate for the formylation defect of a mutant tRNA aminoacylated with lysine; Li Y et al.; The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria such as Escherichia coli . In addition to the determinants for formylation present in the initiator tRNA, the nature of the amino acid attached to the tRNA is also important for formylation . We showed previously that a mutant tRNA aminoacylated with lysine was an extremely poor substrate for formylation . As a consequence, it was essentially inactive in initiation of protein synthesis in E . coli . In contrast, the same tRNA, when aminoacylated with methionine, was a good substrate for formylation and was, consequently, quite active in initiation . Here, we report on the isolation of suppressor mutations in MTF which compensate for the formylation defect of the mutant tRNA aminoacylated with lysine . The suppressor mutant has glycine 178 changed to glutamic acid . Mutants with glycine 178 of MTF changed to aspartic acid, lysine, and leucine were generated and were found to be progressively weaker suppressors . Studies on allele specificity of suppression using different mutant tRNAs as substrates suggest that the Gly178 to Glu mutation compensates for the nature of the amino acid attached to the tRNA . We discuss these results in the framework of the crystal structure of the MTF.fMet-tRNA complex published recently. Crit Rev Microbiol, 2000, 26(2), 111 - 31 The natural evolutionary relationships among prokaryotes; Gupta RS; Two contrasting and very different proposals have been put forward to account for the evolutionary relationships among prokaryotes . The currently widely accepted three domain proposal by Woese et al . (Proc . Natl . Acad . Sci . USA (1990) 87: 4576-4579) calls for the division of prokaryotes into two primary groups or domains, termed archaebacteria (Archaea) and eubacteria (Bacteria), both of which are suggested to have originated independently from a universal ancestor . However, this proposal, which is based primarily on genes involved in the information transfer processes, is inconsistent with the ultrastructural characteristics of prokaryotes as well as with many gene phylogenies and provides no explanation as to how the structural and molecular differences seen between these groups arose and how other prokaryotic taxa are related or evolved from the common ancestor . It also postulates that the last common ancestor of all organisms was a hypothetical entity lacking a cell membrane, which is contrary to the basic requirement of a cell membrane to define and separate all forms of life from the surrounding environment . A second alternate proposal for the evolutionary relationships among prokaryotes has emerged from extensive analyses of numerous conserved inserts and deletions found in various proteins (Gupta, R . S., Microbiol . Mol . Biol . Rev . (1998)62: 1435-1491; FEMS Microbiol . Rev . (2000) 24: in press . This proposal points to a specific relationship between archaebacteria and gram-positive bacteria, both of which are prokaryotes bounded by a single cell membrane (monoderm prokaryotes) . Gram-negative bacteria, which are bounded by two different membranes (diderm prokaryotes), are indicated to comprise a structurally and phylogenetically distinct taxa originating from gram-positive bacteria . This proposal postulates that the earliest prokaryote was a gram-positive bacteria from which both archaebacteria and diderm prokaryotes evolved by normal evolutionary mechanisms in response to the strong selection pressure exerted by antibiotics produced by certain groups of gram-positive bacteria . This proposal accounts for both the molecular as well structural differences seen among the main groups of prokaryotes by known evolutionary mechanisms without invoking any hypothetical process or entity and thus is a closer representation of the natural relationships among prokaryotes than the proposal for two distinct domains . Based on this new proposal, it is now possible to logically deduce the branching order of different prokaryotic taxa from the common ancestor, which is as follows: Gram-positive bacteria (Low G + C) (<=> Archaebacteria) => Gram-positive bacteria (High G + C) (<=> Archaebacteria)=> Deinococcus-Thermus => Green nonsulfur bacteria => Cyanobacteria => Spirochetes => Chlamydia- Cytophaga-Green sulfur bacteria => Proteobacteria-1 (epsilon, delta)=> Proteobacteria-2 (alpha) => Proteobacteria-3 (beta) => Proteobacteria-4 (gamma) . A surprising but very important aspect of the relationship deduced here is that the main eubacterial phyla are related to each other linearly rather than in a tree-like manner, suggesting that the major evolutionary changes within prokaryotes (bacteria) have occurred in a directional manner. Plant Physiol, 2000 Jul, 123(3), 883 - 94 Complementary expression of two plastid-localized sigma-like factors in maize; Lahiri SD et al.; The eubacterial-like RNA polymerase of plastids is composed of organelle-encoded core subunits and nuclear-encoded sigma-factors . Families of sigma-like factors (SLFs) have been identified in several plants, including maize (Zea mays) and Arabidopsis . In vitro import assays determined that at least two of the maize sigma-like proteins have functional chloroplast transit peptides and thus are likely candidates for chloroplast transcriptional regulators . However, the roles of individual SLFs in chloroplast transcription remain to be determined . We have raised antibodies against the unique amino-terminal domains of two maize SLFs, ZmSig1 and ZmSig3, and have used these specific probes to examine the accumulation of each protein in different maize tissues and during chloroplast development . The expression of ZmSig1 is tissue specific and parallels the light-activated chloroplast development program in maize seedling leaves . Its accumulation in mature chloroplasts however, is not affected by subsequent changes in the light regime . It is interesting that the expression profile of ZmSig3 is complementary to that of ZmSig1 . It accumulates in non-green tissues, including roots, etiolated seedling leaves, and the basal region of greening seedling leaves . The nonoverlapping expression patterns of these two plastid-localized SLFs suggest that they may direct differential expression of plastid genes during chloroplast development. J Virol, 2000 Aug, 74(15), 7079 - 84 A hypothesis for DNA viruses as the origin of eukaryotic replication proteins; Villarreal LP et al.; The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria . We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution . We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote . We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses . Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus . Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses . We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction. Mol Cell, 2000 Apr, 5(4), 761 - 6 Multiple functions of an evolutionarily conserved RNA binding domain; Vilardell J et al.; Ribosomal protein L30 of Saccharomyces cerevisiae binds to a distinct RNA structure to inhibit the splicing and the translation of its own transcript . Remarkably, the ortholog of L30 from the archaeon Sulfolobus acidocaldarius binds specifically to the same RNA fragment and inhibits splicing both in vitro and in vivo . Indeed, expression of Sulfolobus L30 in yeast severely reduces growth by limiting production of the endogenous L30 . This conservation of binding specificity implies that the target of regulation in the RPL30 transcript mimics a site in the rRNA that has been conserved for more than a billion years . We identify this site, whose location suggests that L30, which has no apparent eubacterial ortholog, is responsible for establishing the orientation of a key bridge between the large and small ribosomal subunits. Eur J Biochem, 2000 Jul, 267(14), 4583 - 92 Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family; Amiche M et al.; A 32-residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to homogeneity using a four-step protocol . Mass spectral analysis and sequencing of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ . This peptide proved to be bactericidal towards mollicutes (wall-less eubacteria) and Gram-positive eubacteria, and also, though to a lesser extent, towards Gram-negative eubacteria . Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin . Observation of bacterial cells using reflected light fluorescence microscopy after DNA-staining was consistent with a mechanism of cell killing based upon the alteration of membrane permeability rather than membrane solubilization, very likely by forming ion-conducting channels through the plasma membrane . CD spectroscopy and secondary structure predictions indicated that dermatoxin assumes an amphipathic alpha-helical conformation in low polarity media which mimic the lipophilicity of the membrane of target microorganisms . PCR analysis coupled with cDNA cloning and sequencing revealed that dermatoxin is expressed in the skin, the intestine and the brain . Preprodermatoxin from the brain and the intestine have the same sequence as the skin preproform except for two amino-acid substitutions in the preproregion of the brain precursor . The dermatoxin precursor displayed the characteristic features of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp . Precursors of this family have a common N-terminal preproregion followed by markedly different C-terminal domains that give rise to 19-34-residue peptide antibiotics named dermaseptins B and phylloxin, and to the D-amino-acid-containing opioid heptapeptides dermorphins and deltorphins . Because the structures and cidal mechanisms of dermatoxin, dermaseptins B and phylloxin are very different, dermatoxin extends the repertoire of structurally and functionally diverse peptides derived from the rapidly evolving C-terminal domains of precursors of the dermaseptins family. Appl Environ Microbiol, 2000 Jul, 66(7), 2748 - 58 The secondary endosymbiotic bacterium of the pea aphid Acyrthosiphon pisum (Insecta: homoptera); Fukatsu T et al.; The secondary intracellular symbiotic bacterium (S-symbiont) of the pea aphid Acyrthosiphon pisum was investigated to determine its prevalence among strains, its phylogenetic position, its localization in the host insect, its ultrastructure, and the cytology of the endosymbiotic system . A total of 14 aphid strains were examined, and the S-symbiont was detected in 4 Japanese strains by diagnostic PCR . Two types of eubacterial 16S ribosomal DNA sequences were identified in disymbiotic strains; one of these types was obtained from the primary symbiont Buchnera sp., and the other was obtained from the S-symbiont . In situ hybridization and electron microscopy revealed that the S-symbiont was localized not only in the sheath cells but also in a novel type of cells, the secondary mycetocytes (S-mycetocytes), which have not been found previously in A . pisum . The size and shape of the S-symbiont cells were different when we compared the symbionts in the sheath cells and the symbionts in the S-mycetocytes, indicating that the S-symbiont is pleomorphic under different endosymbiotic conditions . Light microscopy, electron microscopy, and diagnostic PCR revealed unequivocally that the hemocoel is also a normal location for the S-symbiont . Occasional disordered localization of S-symbionts was also observed in adult aphids, suggesting that there has been imperfect host-symbiont coadaptation over the short history of coevolution of these organisms. EMBO J, 1983, 2(8), 1291 - 4 Archaebacteria and eukaryotes possess DNA-dependent RNA polymerases of a common type; Huet J et al.; DNA-dependent RNA polymerases of archaebacteria not only resemble the nuclear RNA polymerases of eukaryotes rather than the eubacterial enzymes in their complex component patterns but also show striking immunochemical, i.e., structural, homology with the eukaryotic polymerases at the level of single components . Thus, eukaryotic and archaebacterial RNA polymerases are indeed of the same type, distinct from the eubacterial enzymes, which, however, are also derived from a common ancestral structure. Nucleic Acids Res, 2000 Jun 15, 28(12), 2431 - 8 The global intrinsic curvature of archaeal and eubacterial genomes is mostly contained in their dinucleotide composition and is probably not an adaptation; Merino E et al.; Until now, the genomic DNA of all eubacteria analyzed has been hyper-curved, its global intrinsic curvature being higher than that of a random sequence . In contrast, that rule failed for archaea or eukaryotes, which could be either hypo- or hyper-curved . The existence of the rule suggested that, at least for eubacteria, global intrinsic curvature is adaptive . However, the present results from analyzing 21 eubacterial and six archaeal genomes argue against adaptation . First, there are two eubacterial exceptions to the former rule . More significantly, we found that the dinucleotide composition of the genome alone (which lacks all sequence information) is enough to determine the genome curvature . Additional evidence against adaptation came from showing that the global curvature of bacterial genomes could not have evolved under either of two complementary models of curvature selection: (i) that curvature is selected locally from unbiased variability; (ii) that curvature is established globally through the selection of a curvature-altering mutational bias . We found that the observed relationship between curvature and dinucleotide composition is incompatible with model (i) . We also found that, contrary to the predictions of model (ii), the dinucleo-tide compositions of bacterial genomes were not statistically special in their curvature-related properties (when compared to stochastically generated dinucleotide compositions). Nucleic Acids Res, 2000 Jun 15, 28(12), 2342 - 52 Evolutionary appearance of genes encoding proteins associated with box H/ACA snoRNAs: cbf5p in Euglena gracilis, an early diverging eukaryote, and candidate Gar1p and Nop10p homologs in archaebacteria; Watanabe Y et al.; A reverse transcription-polymerase chain reaction (RT-PCR) approach was used to clone a cDNA encoding the EUGLENA: gracilis homolog of yeast Cbf5p, a protein component of the box H/ACA class of snoRNPs that mediate pseudouridine formation in eukaryotic rRNA . Cbf5p is a putative pseudouridine synthase, and the EUGLENA: homolog is the first full-length Cbf5p sequence to be reported for an early diverging unicellular eukaryote (protist) . Phylogenetic analysis of putative pseudouridine synthase sequences confirms that archaebacterial and eukaryotic (including EUGLENA:) Cbf5p proteins are specifically related and are distinct from the TruB/Pus4p clade that is responsible for formation of pseudouridine at position 55 in eubacterial (TruB) and eukaryotic (Pus4p) tRNAs . Using a bioinformatics approach, we also identified archaebacterial genes encoding candidate homologs of yeast Gar1p and Nop10p, two additional proteins known to be associated with eukaryotic box H/ACA snoRNPs . These observations raise the possibility that pseudouridine formation in archaebacterial rRNA may be dependent on analogs of the eukaryotic box H/ACA snoRNPs, whose evolutionary origin may therefore predate the split between Archaea (archaebacteria) and Eucarya (eukaryotes) . Database searches further revealed, in archaebacterial and some eukaryotic genomes, two previously unrecognized groups of genes (here designated 'PsuX' and 'PsuY') distantly related to the Cbf5p/TruB gene family. Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 6954 - 9 The chimeric eukaryote: origin of the nucleus from the karyomastigont in amitochondriate protists; Margulis L et al.; We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes . A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium . The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur . Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium "Thiodendron latens." By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved . This "earliest branching protist" that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts . The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector . As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei . The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera) . This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa. Genome Res, 2000 Jun, 10(6), 744 - 57 Conservation of DNA regulatory motifs and discovery of new motifs in microbial genomes; McGuire AM et al.; Regulatory motifs can be found by local multiple alignment of upstream regions from coregulated sets of genes, or regulons . We searched for regulatory motifs using the program AlignACE together with a set of filters that helped us choose the motifs most likely to be biologically relevant in 17 complete microbial genomes . We searched the upstream regions of potentially coregulated genes grouped by three methods: (1) genes that make up functional pathways; (2) genes homologous to regulons from a well-studied species (Escherichia coli); and (3) groups of genes derived from conserved operons . This last group is based on the observation that genes making up homologous regulons in different species are often assorted into coregulated operons in different combinations . This allows partial reconstruction of regulons by looking at operon structure across several species . Unlike other methods for predicting regulons, this method does not depend on the availability of experimental data other than the genome sequence and the locations of genes . New, statistically significant motifs were found in the genome sequence of each organism using each grouping method . The most significant new motif was found upstream of genes in the methane-metabolism functional group in Methanobacterium thermoautotrophicum . We found that at least 27% of the known E . coli DNA-regulatory motifs are conserved in one or more distantly related eubacteria . We also observed significant motifs that differed from the E . coli motif in other organisms upstream of sets of genes homologous to known E . coli regulons, including Crp, LexA, and ArcA in Bacillus subtilis; four anaerobic regulons in Archaeoglobus fulgidus (NarL, NarP, Fnr, and ModE); and the PhoB, PurR, RpoH, and FhlA regulons in other archaebacterial species . We also used motif conservation to aid in finding new motifs by grouping upstream regions from closely related bacteria, thus increasing the number of instances of the motif in the sequence to be aligned . For example, by grouping upstream sequences from three archaebacterial species, we found a conserved motif that may regulate ferrous ion transport that was not found in individual genomes . Discovery of conserved motifs becomes easier as the number of closely related genome sequences increases. J Bacteriol, 2000 Jun, 182(12), 3383 - 93 Salmonella enterica serovar typhimurium peptidase B is a leucyl aminopeptidase with specificity for acidic amino acids; Mathew Z et al.; Peptidase B (PepB) of Salmonella enterica serovar Typhimurium is one of three broad-specificity aminopeptidases found in this organism . We have sequenced the pepB gene and found that it encodes a 427-amino-acid (46.36-kDa) protein, which can be unambiguously assigned to the leucyl aminopeptidase (LAP) structural family . PepB has been overexpressed and purified . The active enzyme shows many similarities to other members of the LAP family: it is a heat-stable (70 degrees C; 20 min) hexameric ( approximately 270-kDa) metallopeptidase with a pH optimum of 8.5 to 9.5 . A detailed study of the substrate specificity of the purified protein shows that it differs from other members of the family in its ability to hydrolyze peptides with N-terminal acidic residues . The preferred substrates for PepB are peptides with N-terminal Asp or Glu residues . Comparison of the amino acid sequence of PepB with those of other LAPs leads to the conclusion that PepB is the prototype of a new LAP subfamily with representatives in several other eubacterial species and to the prediction that the members of this family share the ability to hydrolyze peptides with N-terminal acidic residues . Site-directed mutagenesis has been used to show that this specificity appears to be determined by a single Lys residue present in a sequence motif conserved in all members of the subfamily. J Mol Biol, 2000 Jun 16, 299(4), 907 - 30 A DNA structural atlas for Escherichia coli; Pedersen AG et al.; We have performed a computational analysis of DNA structural features in 18 fully sequenced prokaryotic genomes using models for DNA curvature, DNA flexibility, and DNA stability . The structural values that are computed for the Escherichia coli chromosome are significantly different from (and generally more extreme than) that expected from the nucleotide composition . To aid this analysis, we have constructed tools that plot structural measures for all positions in a long DNA sequence (e.g . an entire chromosome) in the form of color-coded wheels . We find that these "structural atlases" are useful for the discovery of interesting features that may then be investigated in more depth using statistical methods . From investigation of the E . coli structural atlas, we discovered a genome-wide trend, where an extended region encompassing the terminus displays a high of level curvature, a low level of flexibility, and a low degree of helix stability . The same situation is found in the distantly related Gram-positive bacterium Bacillus subtilis, suggesting that the phenomenon is biologically relevant . Based on a search for long DNA segments where all the independent structural measures agree, we have found a set of 20 regions with identical and very extreme structural properties . Due to their strong inherent curvature, we suggest that these may function as topological domain boundaries by efficiently organizing plectonemically supercoiled DNA . Interestingly, we find that in practically all the investigated eubacterial and archaeal genomes, there is a trend for promoter DNA being more curved, less flexible, and less stable than DNA in coding regions and in intergenic DNA without promoters . This trend is present regardless of the absolute levels of the structural parameters, and we suggest that this may be related to the requirement for helix unwinding during initiation of transcription, or perhaps to the previously observed location of promoters at the apex of plectonemically supercoiled DNA . We have also analyzed the structural similarities between groups of genes by clustering all RNA and protein-encoding genes in E . coli, based on the average structural parameters . We find that most ribosomal genes (protein-encoding as well as rRNA genes) cluster together, and we suggest that DNA structure may play a role in the transcription of these highly expressed genes . Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6658 - 63 A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family; Smith JS et al.; The yeast Sir2 protein, required for transcriptional silencing, has an NAD(+)-dependent histone deacetylase (HDA) activity . Yeast extracts contain a NAD(+)-dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted . This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD(+) in all species tested . The yeast NPT1 gene, encoding an important NAD(+) synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci . Null mutants in this gene have significantly reduced intracellular NAD(+) concentrations and have phenotypes similar to sir2 null mutants . Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels . The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states. Arch Pharm Res, 2000 Apr, 23(2), 172 - 7 Biotransformation of glycyrrhizin by human intestinal bacteria and its relation to biological activities; Kim DH et al.; The relationship between the metabolites of glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronide, GL) and their biological activities was investigated . By human intestinal microflora, GL was metabolized to 18beta-glycyrrhetinic acid (GA) as a main product and to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) as a minor product . The former reaction was catalyzed by Eubacterium L-8 and the latter was by Streptococcus LJ-22 . Among GL and its metabolites, GA and GAMG had more potent in vitro anti-platelet aggregation activity than GL . GA also showed the most potent cytotoxicity against tumor cell lines and the potent inhibitory activity on rotavirus infection as well as growth of Helicobacter pylori . GAMG, the minor metabolite of GL, was the sweetest. J Biol Chem, 2000 Jun 2, 275(22), 16820 - 6 Identification and characterization of human mitochondrial tryptophanyl-tRNA synthetase; Jorgensen R et al.; A full-length cDNA clone encoding the human mitochondrial tryptophanyl-tRNA synthetase (h(mt)TrpRS) has been identified . The deduced amino acid sequence shows high homology to both the mitochondrial tryptophanyl-tRNA synthetase ((mt)TrpRS) from Saccharomyces cerevisiae and to different eubacterial forms of tryptophanyl-tRNA synthetase (TrpRS) . Using the baculovirus expression system, we have expressed and purified the protein with a carboxyl-terminal histidine tag . The purified His-tagged h(mt)TrpRS catalyzes Trp-dependent exchange of PP(i) in the PP(i)-ATP exchange assay . Expression of h(mt)TrpRS in both human and insect cells leads to high levels of h(mt)TrpRS localizing to the mitochondria, and in insect cells the first 18 amino acids constitute the mitochondrial localization signal sequence . Until now the human cytoplasmic tryptophanyl-tRNA synthetase (hTrpRS) was thought to function as the h(mt)TrpRS, possibly in the form of a splice variant . However, no mitochondrial localization signal sequence was ever detected and the present identification of a different (mt)TrpRS almost certainly rules out that possibility . The h(mt)TrpRS shows kinetic properties similar to human mitochondrial phenylalanyl-tRNA synthetase (h(mt)PheRS), and h(mt)TrpRS is not induced by interferon-gamma as is hTrpRS. J Biol Chem, 2000 Aug 18, 275(33), 25102 - 8 Conserved serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis; Tjalsma H et al.; Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane . Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalent, but structurally quite different, forming two distinct SPase families that share only few conserved residues . P-type SPases were, so far, exclusively identified in eubacteria and organelles, whereas ER-type SPases were found in the three kingdoms of life . Strikingly, the presence of ER-type SPases appears to be limited to sporulating Gram-positive eubacteria . The present studies were aimed at the identification of potential active site residues of the ER-type SPase SipW of Bacillus subtilis, which is required for processing of the spore-associated protein TasA . Conserved serine, histidine, and aspartic acid residues are critical for SipW activity, suggesting that the ER-type SPases employ a Ser-His-Asp catalytic triad or, alternatively, a Ser-His catalytic dyad . In contrast, the P-type SPases employ a Ser-Lys catalytic dyad (Paetzel, M., Dalbey, R . E., and Strynadka, N . C . J . (1998) Nature 396, 186-190) . Notably, catalytic activity of SipW was not only essential for pre-TasA processing, but also for the incorporation of mature TasA into spores. FEBS Lett, 2000 May 19, 473(3), 328 - 32 Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli; Rodriguez-Concepcion M et al.; An alternative mevalonate-independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet . In this work, E . coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) . In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium. Proteins, 2000 Jun 1, 39(4), 417 - 20 Do more complex organisms have a greater proportion of membrane proteins in their genomes? Stevens TJ, Arkin IT. One may speculate that higher organisms require a proportionately greater abundance of membrane proteins within their genomes in order to furnish the requirements of differentiated cell types, compartmentalization, and intercellular signalling . With the recent availability of several complete prokaryotic genome sequences and sufficient progress in many eukaryotic genome sequencing projects, we seek to test this hypothesis . Using optimized hydropathy analysis of proteins in several, diverse proteomes, we show that organisms of the three domains of life-Eukarya, Eubacteria, and Archaea-have similar proportions of alpha-helical membrane proteins within their genomes and that these are matched by the complexity of the aqueous components. Adv Enzymol Relat Areas Mol Biol, 2000, 74, 129 - 84 The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes; Mehta PK et al.; The pyridoxal-5-phosphate-dependent enzymes (B6 enzymes) that act on amino acid substrates are of multiple evolutionary origin . The numerous common mechanistic features of B6 enzymes thus are not historical traits passed on from a common ancestor enzyme but rather reflect evolutionary or chemical necessities . Family profile analysis of amino acid sequences supported by comparison of the available three-dimensional (3-D) crystal structures indicates that the B6 enzymes known to date belong to four independent evolutionary lineages of homologous (or more precisely paralogous) proteins, of which the alpha family is by far the largest . The alpha family (with aspartate aminotransferase as the prototype enzyme) includes enzymes that catalyze, with several exceptions, transformations of amino acids in which the covalency changes are limited to the same carbon atom that carries the amino group forming the imine linkage with the coenzyme (i.e., Calpha in most cases) . Enzymes of the beta family (tryptophan synthase beta as the prototype enzyme) mainly catalyze replacement and elimination reactions at Cbeta . The D-alanine aminotransferase family and the alanine racemase family are the two other independent lineages, both with relatively few member enzymes . The primordial pyridoxal-5-phosphate-dependent enzymes apparently were regio-specific catalysts that first diverged into reaction-specific enzymes and then specialized for substrate specificity . Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages . Comparison of sequences from eukaryotic, archebacterial, and eubacterial species indicates that the functional specialization of most B6 enzymes has occurred already in the universal ancestor cell . The cofactor pyridoxal-5-phosphate must have emerged very early in biological evolution; conceivably, organic cofactors and metal ions were the first biological catalysts . In attempts to stimulate particular steps of molecular evolution, oligonucleotide-directed mutagenesis of active-site residues and directed molecular evolution have been applied to change both the substrate and reaction specificity of existent B6 enzymes . Pyridoxal-5-phosphate-dependent catalytic antibodies were elicited with a screening protocol that applied functional selection criteria as they might have been operative in the evolution of protein-assisted pyridoxal catalysis. J Biol Chem, 2000 May 12, 275(19), 14031 - 7 Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium; Fillinger S et al.; Bacillus subtilis possesses two similar putative phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding genes, gap (renamed gapA) and gapB . A gapA mutant was unable to grow on glycolytic carbon sources, although it developed as well as the wild-type strain on gluconeogenic carbon sources . A gapB mutant showed the opposite phenotype . Purified GapB showed a 50-fold higher GAPDHase activity with NADP(+) than with NAD(+), with K(m) values of 0.86 and 5.7 mm, respectively . lacZ reporter gene fusions revealed that the gapB gene is transcribed during gluconeogenesis and repressed during glycolysis . Conversely, gapA transcription is 5-fold higher under glycolytic conditions than during gluconeogenesis . GAPDH activity assays in crude extracts of wild-type and mutant strains confirmed this differential expression pattern at the enzymatic level . Genetic analyses demonstrated that gapA transcription is repressed by the yvbQ (renamed cggR) gene product and indirectly stimulated by CcpA . Thus, the same enzymatic step is catalyzed in B . subtilis by two enzymes specialized, through the regulation of their synthesis and their enzymatic characteristics, either in catabolism (GapA) or in anabolism (GapB) . Such a dual enzymatic system for this step of the central carbon metabolism is described for the first time in a nonphotosynthetic eubacterium, but genomic analyses suggest that it could be a widespread feature. Mol Microbiol, 2000 Apr, 36(2), 278 - 89 Intrinsic instability of the essential cell division protein FtsL of Bacillus subtilis and a role for DivIB protein in FtsL turnover; Daniel RA et al.; Cell division in most eubacteria is driven by an assembly of about eight conserved division proteins . These proteins form a ring structure that constricts in parallel with the formation of the division septum . Here, we show that one of the division proteins, FtsL, is highly unstable . We also show that the protein is targeted to the ring structure and that targeting occurs in concert with the recruitment of several other membrane-associated division proteins . FtsL stability is further reduced in the absence of DivIB protein (probably homologous to E . coli FtsQ) at high temperature, suggesting that DivIB is involved in the control of FtsL turnover . The reduced stability of FtsL may explain the temperature dependence of divIB mutants, because their phenotype can be suppressed by overexpression of FtsL . The results provide new insights into the roles of the FtsL and DivIB proteins in bacterial cell division. J Clin Microbiol, 2000 May, 38(5), 1747 - 52 Contamination and sensitivity issues with a real-time universal 16S rRNA PCR; Corless CE et al.; A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system . During the development of this PCR, problems were noted with the use of this gene as an amplification target . Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry . This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry . In an attempt to overcome this problem, several methodologies were applied . Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity . With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction . Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA . Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical. Rheumatology (Oxford), 2000 Mar, 39(3), 274 - 82 What determines arthritogenicity of bacterial cell wall? A study on Eubacterium cell wall-induced arthritis; Zhang X et al.; OBJECTIVE: To study what determines the arthritogenicity of the bacterial cell wall (CW) using Eubacterium CW-induced arthritis in the rat . METHODS: Eubacterium aerofaciens, previously reported as arthritogenic, and E . limosum and E . alactolyticum, known as non-arthritogenic, were used . Gas chromatography-mass spectrometry (GC-MS) was applied to analyse the chemical composition of the bacterial cell wall . Cellular immune response was measured by concanavalin A (Con A) stimulation and FACScan analysis . Also, serum antibodies against the injected cell wall were determined . RESULTS: Unexpectedly, from the two strains of E . aerofaciens used only one proved to be arthritogenic (with a CW inducing chronic arthritis after a single intraperitoneal injection), even though these two strains were 100% identical by 16S rDNA analysis . CW of the other E . aerofaciens strain induced only transient acute arthritis; CW of E . limosum and E . alactolyticum induced weak signs of acute arthritis . Based on the GC-MS analysis and on the results published previously, putative structures of peptidoglycan (PG) in the four CW preparations are presented . It is apparent that the presence of lysine in position 3 of the PG stem peptide contributes to arthritogenicity but is alone not decisive . Both strains of E . aerofaciens were immunosuppressive, when tested by Con A response at 2 weeks after CW injection . Such an immunosuppression was not observed after injection of CW from E . limosum or E . alactolyticum . FACScan analysis for six T cell markers and studies on serum antibody responses did not reveal any differences in the effect of the four bacterial strains used . CONCLUSIONS: The results obtained suggest that the chemical structure of PG present in the bacterial CW is decisive in determining arthritogenicity/non-arthritogenicity . Therefore, from two bacterial strains belonging to normal human intestinal flora and 100% identical by 16S rDNA analysis, one proved to be arthritogenic and the other non-arthritogenic. RNA, 2000 Apr, 6(4), 584 - 97 Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation; Verrier SB et al.; In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation . An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes . Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR) . Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells . For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF) . When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect . A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt . Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect . Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes . Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA. Mikrobiologiia, 2000 Mar-Apr, 69(2), 163 - 74 {About nannobacteria}; Vainshtein MB et al.; Bacterial nanocells 0.2-0.3 micron in size and hundreds of a cubic micron in volume have been revealed in natural habitats and obtained in pure cultures . The taxonomic analysis of naturally occurring nanobacteria showed that they belong to the known taxa of the kingdom Eubacteria . The results of the cytological investigation of nanocells suggest that they are universally formed in response to stress impacts. Cytogenet Cell Genet, 2000, 88(1-2), 43 - 9 Cloning and characterization of human FTCD on 21q22.3, a candidate gene for glutamate formiminotransferase deficiency; Solans A et al.; We have identified a new human gene, FTCD, which maps to chromosome 21q22.3 and encodes the enzyme formiminotransferase cyclodeaminase, an intermediate metabolism enzyme that links histidine catabolism to folate metabolism . The major cDNA encodes a protein containing 541 amino acid residues and shows 84% identity with porcine FTCD . Several other cDNAs have been isolated, which may result from alternative splicing events and have the potential to code for three different protein isoforms . The gene is highly expressed in human fetal and adult liver . The two FTCD protein domains show high sequence similarity to two distinct open reading frames from eubacterial genomes, suggesting that eukaryotic FTCD appeared through a gene fusion event . Defects in the glutamate formiminotransferase pathway have been documented, and the deficiency is presumed to be inherited as an autosomal recessive trait . The sequence reported here may be helpful in identifying the primary defect in glutamate formiminotransferase deficiency and establishing a molecular diagnosis . J Biol Chem, 2000 Apr 21, 275(16), 11829 - 35 Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding; Dickson R et al.; Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, alpha and beta . The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated . Toward this goal, we have cloned the alpha and beta subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments . In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure . However, in the presence of MgATP, the beta subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10 . In contrast, alpha subunits only assemble in the presence of beta subunits . Although beta and alpha/beta 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different . Similar to the authentic chloroplast protein, the reconstituted alpha/beta 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10 . In contrast, the folding reaction mediated by the reconstituted beta 14-mers is only efficient with mitochondrial cpn10 . The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system. Genes Dev, 2000 Apr 1, 14(7), 777 - 82 Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation; Chen X et al.; The genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an ortholog of an RNA-binding protein known as the Ro 60-kD autoantigen . This protein, which was previously identified only in higher eukaryotes, is normally bound to small RNAs known as Y RNAs . We show that the Ro protein ortholog Rsr contributes to the resistance of D . radiodurans to UV irradiation . Rsr binds several small RNAs, encoded upstream of rsr, that accumulate following UV irradiation . One of these RNAs resembles a Y RNA . These results suggest that Ro RNPs could similarly contribute to the recovery of higher cells following UV irradiation. J Biol Chem, 2000 Jul 28, 275(30), 23113 - 9 Mutational analysis of beta '260-309, a sigma 70 binding site located on Escherichia coli core RNA polymerase; Arthur TM et al.; In eubacteria, the final sigma subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters . Previously, our laboratory defined a region of the beta' subunit that interacts with final sigma(70) in vitro . This region of beta' contained heptad repeat motifs indicative of coiled coils . In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260-309 of beta', to look at disruption of the final sigma(70)-core interaction . Several of the mutants were defective for binding final sigma(70) in vitro . Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in which the mutant beta' is the sole source of beta' subunit for the cell . All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for Efinal sigma(70) holoenzyme formation . Several of the mutants were also defective for holoenzyme assembly with various minor final sigma factors . In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E . A., Minakhin, L., Richter, C., Severinov, K . , and Darst, S . A . (1999) Cell 98, 811-824), the region homologous to beta'(260-309) of Escherichia coli forms a coiled coil . Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil. J Biol Chem, 2000 Jul 28, 275(30), 22888 - 94 The additional 165 amino acids in the B protein of Escherichia coli DNA gyrase have an important role in DNA binding; Chatterji M et al.; DNA gyrase is the only enzyme known to negatively supercoil DNA . The enzyme is a heterotetramer of A(2)B(2) subunit composition . Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes . The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases . In Escherichia coli, no function has so far been attributed to this stretch . In this study, we have tried to assess the function of this region both in vivo and in vitro . A deletant (GyrBDelta160) lacking this region is non-functional in vivo . The holoenzyme reconstituted from gyrase A (GyrA) and GyrBDelta160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro . GyrBDelta160 retains its ability to bind to GyrA and novobiocin . However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding . The intrinsic ATPase activity of the GyrBDelta160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA . These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability of E . coli gyrase. J Bacteriol, 2000 May, 182(9), 2629 - 34 Heterologous expression of bacterial Epoxyalkane:Coenzyme M transferase and inducible coenzyme M biosynthesis in Xanthobacter strain Py2 and Rhodococcus rhodochrous B276; Krum JG et al.; Coenzyme M (CoM) (2-mercaptoethanesulfonic acid) biosynthesis is shown to be coordinately regulated with the expression of the enzymes of alkene and epoxide metabolism in the propylene-oxidizing bacteria Xanthobacter strain Py2 and Rhodococcus rhodochrous strain B276 . These results provide the first evidence for the involvement of CoM in propylene metabolism by R . rhodochrous and demonstrate for the first time the inducible nature of eubacterial CoM biosynthesis. J Bacteriol, 2000 May, 182(9), 2536 - 43 Peptidase E, a peptidase specific for N-terminal aspartic dipeptides, is a serine hydrolase; Lassy RA et al.; Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase . PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes . A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family . To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE . The sequence comparison suggests that PepE is a serine hydrolase . We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity . Conversion of Ser120 to Cys leads to severely reduced (10(4)-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile . These results suggest that PepE is the prototype of a new family of serine peptidases . The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available. Biochim Biophys Acta, 2000 Apr 25, 1491(1-3), 196 - 204 DRG represents a family of two closely related GTP-binding proteins; Li B et al.; In a previous publication we identified a novel human GTP-binding protein that was related to DRG, a developmentally regulated GTP-binding protein from the central nervous system of mouse . Here we demonstrate that both the human and the mouse genome possess two closely related drg genes, termed drg1 and drg2 . The two genes share 62% sequence identity at the nucleotide and 58% identity at the protein level . The corresponding proteins appear to constitute a separate family within the superfamily of the GTP-binding proteins . The DRG1 and the DRG2 mRNA are widely expressed in human and mouse tissues and show a very similar distribution pattern . The human drg1 gene is located on chromosome 22q12, the human drg2 gene on chromosome 17p12 . Distantly related species including Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae also possess two drg genes . In contrast, the genomes of archaebacteria (Halobium, Methanococcus, Thermoplasma) harbor only one drg gene, while eubacteria do not seem to contain any . The high conservation of the polypeptide sequences between distantly related organisms indicates an important role for DRG1 and DRG2 in a fundamental pathway. Plant Physiol, 2000 Apr, 122(4), 1057 - 71 Coordinate regulation of the nuclear and plastidic genes coding for the subunits of the heteromeric acetyl-coenzyme A carboxylase; Ke J et al.; Plastidic acetyl-coenzyme A (CoA) carboxylase (ACCase) catalyzes the first committed reaction of de novo fatty acid biosynthesis . This heteromeric enzyme is composed of one plastid-coded subunit (beta-carboxyltransferase) and three nuclear-coded subunits (biotin carboxy-carrier, biotin carboxylase, and alpha-carboxyltransferase) . We report the primary structure of the Arabidopsis alpha-carboxyltransferase and beta-carboxyltransferase subunits deduced from nucleotide sequences of the respective genes and/or cDNA . Co-immunoprecipitation experiments confirm that the alpha-carboxyltransferase and beta-carboxyltransferase subunits are physically associated . The plant alpha-carboxyltransferases have gained a C-terminal domain relative to eubacteria, possibly via the evolutionary acquisition of a single exon . This C-terminal domain is divergent among plants and may have a structural function rather than being essential for catalysis . The four ACCase subunit mRNAs accumulate to the highest levels in tissues and cells that are actively synthesizing fatty acids, which are used either for membrane biogenesis in rapidly growing tissues or for oil accumulation in developing embryos .