Biofactors, 1993 May, 4(2), 95 - 104 Hypusine: its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation; Park MH et al.; The amino acid, hypusine {N epsilon-(4-amino-2-hydroxybutyl) lysine}, a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology eIF-4D), is formed post-translationally in two enzymatic steps: (i) transfer of the 4-aminobutyl moiety of the polyamine spermidine to the epsilon-amino group of a single specific lysine residue in the eIF-5A precursor protein to form an intermediate, deoxyhypusine, and (ii) subsequent hydroxylation in this 4-aminobutyl portion . Hypusine is produced soon after the translation of eIF-5A mRNA; the modification is essentially irreversible . Hypusine is found in all eukaryotes examined as well as in archaebacteria; it does not occur in eubacteria . The protein containing hypusine from each species displays a high degree of amino acid identity; the sequence of amino acids surrounding the hypusine residue is strictly conserved, suggesting the importance of the hypusine modification throughout evolution . Expression of one of the two yeast eIF-5A genes is required for survival and the lysine codon at the site of hypusine synthesis is vital for yeast growth . The precise cellular function of eIF-5A remains to be elucidated; however, eIF-5A stimulates methionyl-puromycin synthesis in a model assay for translation initiation and eIF-5A precursors containing lysine in place of hypusine are inactive in this assay . This provides evidence that the hypusine modification is needed for eIF-5A activity . In view of the important role of hypusine in eIF-5A and because of the narrow specificities of the enzymes involved in formation of this unusual amino acid, the hypusine biosynthetic steps offer promising targets for intervention in cellular proliferation . Spermidine analogs that are inhibitors of deoxyhypusine synthase in vitro also cause inhibition of hypusine formation in cells, together with a reduction in protein synthesis and in cell growth . In addition, certain metal chelating inhibitors of deoxyhypusine hydroxylase exhibit anti-proliferative effects by arresting mammalian cells at the G1/S boundary of the cell cycle . These results lay the foundation for the potential regulation of cellular events through the application of specific and potent inhibitors of hypusine biosynthesis. Plant Physiol, 1993 May, 102(1), 155 - 63 Treatment of pea (Pisum sativum L.) protoplasts with DNA-damaging agents induces a 39-kilodalton chloroplast protein immunologically related to Escherichia coli RecA; Cerutti H et al.; Organisms must have efficient mechanisms of DNA repair and recombination to prevent alterations in their genetic information due to DNA damage . There is evidence for DNA repair and recombination in plastids of higher plants, although very little is known at the biochemical level . Many chloroplast proteins are of eubacterial ancestry, suggesting that the same could be true for the components of a DNA repair and recombination system . A 39-kD protein, immunologically related to Escherichia coli RecA, is present in chloroplasts of pea (Pisum sativum L.) . Bandshift gel assays suggest that it binds single-stranded DNA . Its steady-state level is increased by several DNA-damaging agents . These results are consistent with it being a plastid homolog of E . coli RecA protein, presumably involved in DNA repair and recombination, and with the existence of an SOS-like response in pea leaf cells . Experiments with protein synthesis inhibitors suggest that the 39-kD chloroplast protein is encoded in the nucleus. FEMS Microbiol Lett, 1993 Apr 15, 108(3), 255 - 8 Aspartame as a source of essential phenylalanine for the growth of oral anaerobes; Wyss C; Phenylalanine and aspartic acid requirements were determined for 13 species of oral bacteria using the chemically defined medium OMIZ-W1 . None of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Eikenella corrodens, Selenomonas sputigena, Treponema pectinovorum, T . socranskii, or Wolinella recta required either of these amino acid constituents of aspartame (L-aspartyl-L-phenylalanine methylester) . Phenylalanine was essential for the growth of Capnocytophaga gingivalis, Eubacterium timidum, Fusobacterium nucleatum, Porphyromonas gingivalis, T . denticola, and T . vincentii, while aspartic acid was not required . With the exception of E . timidum, all phenylalanine-dependent strains could grow when the free amino acid was replaced by aspartame at concentrations at least 10-fold lower than those used for aspartame as an artificial sweetener. Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3511 - 5 Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli; Tanaka K et al.; The rpoS gene of Escherichia coli encodes a putative RNA polymerase sigma factor that is considered to be the central regulator of gene expression in stationary phase . The gene product (sigma 38) was overproduced using the cloned rpoS gene and purified to homogeneity . Reconstituted RNA polymerase holoenzyme (E sigma 38) was found to recognize in vitro a number of typical sigma 70-type promoters, including the lacUV5 and trp promoters . Some, however, were recognized exclusively or preferentially by E sigma 70, whereas at least one, fic, was favored by E sigma 38 . Thus E . coli promoters can be classified into three groups: the first group is recognized by E sigma 70 and E sigma 38, but the second and third groups are recognized substantially by either E sigma 70 or E sigma 38 alone . In contrast to other minor sigma factors, sigma 38 shares a set of amino acid sequences common among the principal sigma factors of eubacteria and is therefore a member of the RpoD-related protein family . The intracellular level of sigma 38 was demonstrated to increase in vivo upon entry into stationary phase . These results together indicate that sigma 38 is a second principal sigma factor in stationary-phase E . coli. Mol Microbiol, 1993 Apr, 8(1), 1 - 4 Mitochondrial transcription: is a pattern emerging? Jaehning JA. Despite the striking similarities of RNA polymerases and transcription signals shared by eubacteria, archaebacteria and eukaryotes, there has been little indication that transcription in mitochondria is related to any previously characterized model . Only in yeast has the subunit structure of the mitochondrial RNA polymerase been determined . The yeast enzyme is composed of a core related to polymerases from bacteriophage T7 and T3, and a promoter recognition factor similar to bacterial sigma factors . Soluble systems for studying mitochondrial transcript initiation in vitro have been described from several organisms, and used to determine consensus sequences at or near transcription start sites . Comparison of these sequences from fungi, plants, and amphibians with the T7/T3 promoter suggests some intriguing similarities . Mammalian mitochondrial promoters do not fit this pattern but instead appear to utilize upstream sites, the target of a transcriptional stimulatory factor, to position the RNA polymerase . The recent identification of a possible homologue of the mammalian upstream factor in yeast mitochondria may indicate that a pattern will eventually be revealed relating the transcriptional machineries of all eukaryotic mitochondria. Int J Syst Bacteriol, 1993 Apr, 43(2), 302 - 4 Eubacterium saphenum sp . nov., isolated from human periodontal pockets {corrected}; Uematsu H et al.; A new species, Eubacterium saphenum {corrected} sp . nov., established on the basis of the results of DNA-DNA hybridization, was proposed for strains isolated from human periodontal pockets . Differential characteristics are given. J Clin Microbiol, 1993 Apr, 31(4), 1001 - 2 UV red fluorescence of Eubacterium lentum; Mosca A et al.; Twenty-nine clinical isolates of Eubacterium lentum and two type species were evaluated for the ability to fluoresce under UV light . Twenty-one of the 29 isolates and both of the reference strains showed orange-to-red fluorescence . This fluorescence did not require blood or hemin in the culture media and did not fade upon air exposure . The fluorescent pigment, after extraction by 1 N NaOH, showed peak excitation at a wavelength of around 400 nm . The capacity of E . lentum to produce fluorescence may be a useful and time-sparing laboratory aid for its identification. J Bacteriol, 1993 Apr, 175(7), 1871 - 8 Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium; Kovacs SA et al.; Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA . snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems . This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis . These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap . Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera . S . leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates . Thin-layer chromatography of S . leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E . coli immunoprecipitates . Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B . subtilis, as well . This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells. Oral Microbiol Immunol, 1993 Apr, 8(2), 105 - 10 Identification of Actinobacillus actinomycetemcomitans: polymerase chain reaction amplification of lktA-specific sequences; Goncharoff P et al.; Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis . Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis . In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A . actinomycetemcomitans . Specific oligonucleotide primers LKT2 and LKT3 were designed to hybridize to the A . actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A . actinomycetemcomitans virulence factor . The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A . actinomycetemcomitans strains tested . In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers . These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubacterial 16S ribosomal DNA (rDNA) . PCR amplifications of all bacterial species tested, including A . actinomycetemcomitans, yielded 16S rDNA-specific DNA fragments . Furthermore, each bacterial species tested, with the exception of A . actinomycetemcomitans, failed to amplify lktA sequences . The LKT and RRN primers were used in further PCR experiments to detect A . actinomycetemcomitans directly from gingival fluid samples . The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A . actinomycetemcomitans. Mol Microbiol, 1993 Apr, 8(2), 243 - 52 Complex transcription of an operon encoding the SalI restriction-modification system of Streptomyces albus G; Alvarez MA et al.; High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G . The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream . Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1 . The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity . Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent. Infect Agents Dis, 1993 Apr, 2(2), 87 - 99 Why is Chlamydia sensitive to penicillin in the absence of peptidoglycan? Moulder JW. Most eubacteria are sensitive to penicillin because the antibiotic inhibits synthesis of peptidoglycan, an essential constituent of their cell walls . A few eubacteria have no measurable peptidoglycan, and, with one exception, they are not susceptible to penicillin . The exception is the genus Chlamydia whose members are just as sensitive to penicillin as peptidoglycan-containing bacteria . A numbers of ways to resolve this anomaly, penicillin sensitivity without peptidoglycan, are proposed . It is concluded that there are serious objections to each one and that the chlamydial anomaly remains unresolved . However, examination of the relation between penicillin and chlamydiae is useful because it reveals how little is known of the evolutionary history of penicillin, penicillin-binding proteins, and peptidoglycan. Curr Microbiol, 1993 Apr, 26(4), 233 - 7 Aspects of energy-yielding metabolism in the aphid, Schizaphis graminum, and its endosymbiont: detection of gene fragments potentially coding for the ATP synthase beta-subunit and glyceraldehyde-3-phosphate dehydrogenase; Clark MA et al.; Specialized cells within the aphid, Schizaphis graminum, contain intracellular, vesicle-enclosed eubacterial endosymbionts (Buchnera aphidicola) . Using oligonucleotide probes derived from conserved sequences of the ATP synthase beta-subunit and glyceraldehyde-3-phosphate dehydrogenase, and the polymerase chain reaction (PCR), we have amplified, cloned, and sequenced three DNA fragments . Amino acid sequence similarity indicated that two of these fragments corresponded to endosymbiont and host genes potentially coding for the beta-subunit of ATP synthase . The host gene fragment contained two putative introns . The third DNA fragment corresponded to a portion of a gene coding for a glyceraldehyde-3-phosphate dehydrogenase that was highly related to one of the enzymes from Escherichia coli (GapA) . These results indicate that B . aphidicola may have an ATP synthase and consequently could synthesize ATP from a proton motive force generated within the intracellular vesicles of host cells containing the endosymbionts . The detection of a gene fragment coding for a protein similar to glyceraldehyde-3-phosphate dehydrogenase suggests the presence of this glycolytic enzyme in the endosymbiont and its involvement in energy-yielding metabolism. Science, 1993 Mar 26, 259(5103), 1892 - 6 Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast; Story RM et al.; RecA protein is essential in eubacteria for homologous recombination and promotes the homologous pairing and strand exchange of DNA molecules in vitro . Recombination proteins with weak sequence similarity to bacterial RecA proteins have been identified in bacteriophage T4, yeast, and other higher organisms . Analysis of the primary sequence relationships of DMC1 from Saccharomyces cerevisiae and UvsX of T4 relative to the three-dimensional structure of RecA from Escherichia coli suggests that both proteins are structural homologs of bacterial RecA proteins . This analysis argues that proteins in this group are members of a single family that diverged from a common ancestor that existed prior to the divergence of prokaryotes and eukaryotes. Nucleic Acids Res, 1993 Mar 25, 21(6), 1449 - 55 The sequence of 28S ribosomal RNA varies within and between human cell lines; Leffers H et al.; The primary structure of 28S ribosomal RNA constitutes a conserved core which is similar among most 23S-like rRNAs and expansion segments which occur at specific positions in the sequence . The expansion segments account for most of the size difference between prokaryotic (archaeal and eubacterial) and eukaryotic rRNAs and they exhibit a sequence variation which is unique among rRNAs . We have investigated the sequence variation of one of the expansion segments, V8, by sequencing a total of 111 V8 segments from 9 different human cell lines and tissues and have found 35 different variants . The variation occur mainly at two 'hot spots' which are separated by 170 nucleotides in the primary sequence but are neighbours in the secondary structure . The sequence of V8 segments varies both within and between human cell lines and tissues . The implications for the evolution of the eukaryotic 28S rRNA are discussed together with possible functions of the expansion segments . We also present a secondary structure model for the V8 segment based on comparative sequence analysis and chemical and enzymatic foot printing. Nucleic Acids Res, 1993 Mar 25, 21(6), 1335 - 8 Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum; Inagaki Y et al.; In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal . Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted . To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA {mRNA(UAA)}, UAG {mRNA(UAG)} and UGA {mRNA(UGA)} in-frame . In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released . Upon addition of the E.coli S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction . These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum. J Biol Chem, 1993 Mar 25, 268(9), 6097 - 106 Old Yellow Enzyme . The discovery of multiple isozymes and a family of related proteins; Stott K et al.; Using fast protein liquid chromatography, we have separated native Old Yellow Enzyme from Brewer's Bottom Yeast into three distinct fractions . Two of these fractions are homodimeric forms of the enzyme while the third is the corresponding heterodimeric form . One of these homodimeric fractions is identical in every respect to OYE1, originally cloned from Brewer's Bottom Yeast (Saito, K., Thiele, D . J., Davio, M., Lockridge, O., and Massey, V . (1991) J . Biol . Chem . 266, 20720-20724) . We have cloned, sequenced, and expressed a second Old Yellow Enzyme gene from Saccharomyces cerevisiae, showing close similarity, but not identity, with OYE1 . Native Old Yellow Enzyme samples were also affinity-purified from a strain of S . cerevisiae and an OYE deletion mutant constructed from it . A total of at least seven isozymes of Old Yellow Enzyme have been discovered, each having slightly different characteristics ranging from surface charge to NADPH dehydrogenase activities with different electron acceptors, as well as N-terminal amino acid sequence . In addition, both recombinant enzymes showed considerable similarity to two proteins in the GenBank/EMBL data bank, a 60,000-dalton bile acid-inducible polypeptide in Eubacterium sp . (Mallonee, D . H., White, W . B., and Hylemon, P . B . (1990) J . Lipid Res . 172, 7011-7019) and a 72,000-dalton NADH oxidase in Thermoanaerobium brockii. Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2305 - 9 From elongator tRNA to initiator tRNA; Varshney U et al.; We show that the two most important properties needed for a tRNA to function in initiation in Escherichia coli are its ability to be formylated and its ability to bind to the ribosomal P site . This conclusion is based on conversion of two different elongator tRNAs to ones that can act as initiators in E . coli . We transplanted the features unique to E . coli and eubacterial initiator tRNAs to E . coli elongator methionine tRNA (tRNA(Met)) along with an anticodon sequence change and analyzed their activities in initiation in E . coli . Introduction of a C1.A72 mismatch at the end of the acceptor stem of tRNA(Met), which generates the minimal features necessary for formylation, produces a tRNA with very low activity in initiation . Subsequent introduction of three consecutive G.C base pairs at the bottom of the anticodon stem, which is necessary for ribosomal P site binding, produces a tRNA with significant activity in initiation . Furthermore, introduction of the features necessary for formylation and for ribosomal P site binding into E . coli elongator glutamine tRNA produces a tRNA that initiates protein synthesis in E . coli. J Mol Biol, 1993 Mar 5, 230(1), 57 - 76 Gene organization, primary structure and RNA processing analysis of a ribosomal RNA operon in Lactococcus lactis; Chiaruttini C et al.; Southern blot analysis of genomic DNA of the mesophilic lactic bacterium Lactococcus lactis subsp . lactis strain IL1403, illuminated six rRNA gene clusters . Each cluster contains one copy each of three rRNA genes, displaying the typical eubacterial organization of physically linked 16 S, 23 S and 5 S rRNA genes . Five of the six rRNA clusters were cloned into plasmid pBR322 . One recombinant plasmid, pSLCM6, containing a 6500 base-pair genomic DNA fragment, was characterized by physical mapping and the sequences encoding rRNAs and tRNAs were localized by Southern hybridization . This fragment contains a single operon composed of one promoter, a leader sequence, a 16 S rRNA gene, a tRNA(Ala) gene, a 23 S rRNA gene, a 5 S rRNA gene and a tRNA(Asn) gene . S1 nuclease mapping and primer extension analysis of in vivo transcripts localized one transcriptional initiation site 150 base-pairs upstream from the start of the 16 S rRNA gene . These procedures also suggest that this transcript is processed by an RNAse III-like activity similar to Bacillus subtilis; i.e . the L . lactis nuclease might be sequence-specific . The chronology of specific cleavages occurring during the maturation process of the precursor transcript is described . One interesting observation is that the regions flanking the 16 S and 23 S rRNAs containing the primary processing sites are identical and contain sequences that could be involved in transcriptional antitermination . S1 mapping of the 3' ends of in vivo transcripts indicate that a terminator-like sequence a few base-pairs downstream from the distal tRNA(Asn) gene is inefficient in arresting transcription. J Mol Biol, 1993 Mar 5, 230(1), 51 - 6 Unassigned or nonsense codons in Micrococcus luteus; Kano A et al.; We previously reported that in Micrococcus luteus, a Gram-positive eubacterium with high genomic G + C content, certain codons ending with A did not appear in coding frames, including termination sites, and tRNAs that translate these codons were not detected . These facts suggest that at least some of them are unassigned (nonsense) codons, i.e . not assigned to any amino acid or to any stop signal . We have investigated whether AGA and AUA, universal Arg and Ile codons, respectively, are really unassigned codons by using a cell-free extract prepared from M . luteus and synthetic messenger RNAs . Translation of synthetic mRNA containing in-frame AGA codons does not result in "read-through" to codons beyond the AGA codons, i.e . translation ceases at codon AGA . Essentially the same result was obtained with mRNA containing AUA in-frame . A sucrose-gradient centrifugation profile of the reaction mixture has shown that practically all of the peptides that have been synthesized are attached to 70 S ribosomes . When in-frame AGA or AUA codons are replaced by UGA codons in mRNA, no read-through occurs beyond UGA, just as in the case of AGA or AUA . However, the synthesized peptide is released from the 70 S ribosomes . These data suggest that AGA and AUA are unassigned codons and differ from UGA in that they are not used for termination. J Biol Chem, 1993 Mar 5, 268(7), 4643 - 50 Cross-linked amino acids in the protein pairs L3-L19 and L23-L29 of Bacillus stearothermophilus ribosomes after treatment with diepoxybutane; Herwig S et al.; Treatment of native 50 S ribosomal subunits of Bacillus stearothermophilus with the homobifunctional cross-linking reagent diepoxybutane generated two cross-linked protein pairs, L3-L19 and L23-L29, which were isolated and identified . The analysis of the cross-linking sites at the amino acid level in both protein pairs is presented . Using a combination of sequence analysis and mass spectrometry it could be demonstrated that His-28 in protein L3 and the N-terminal amino acids Met-1, His-2, and His-3 in protein L19 are involved in forming the cross-link L3-L19 . Within the pair L23-L29 Met-1 in protein L23 and Lys-4 in protein L29 were identified as cross-linking sites employing a similar approach . Comparison of our data with results derived from other cross-linking experiments showed that in general the structural organization of the ribosomes in eubacteria (the Gram-positive B . stearothermophilus and the Gram-negative Escherichia coli) has been conserved to quite an extent during evolution but that the fine structures differ slightly . By mass spectrometry the specificity of diepoxybutane and its cleaving mechanism using sodium periodate could be examined . In addition the complete amino acid sequence of protein L19 of B . stearothermophilus has been determined and revealed 58% identical amino acid residues to the homologous E . coli protein L19. FEMS Immunol Med Microbiol, 1993 Mar, 6(2-3), 213 - 7 Black-pigmented gram-negative anaerobes in endodontic infections; Haapasalo M; Necrotic dental root canal infections are polymicrobial infections dominated by anaerobic bacteria . The number of different species in one canal is usually low, approx . 4-7 species . The species isolated most frequently belong to the genera Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Eubacterium and Streptococcus . The frequency of isolation of black-pigmented Gram-negative anaerobes in endodontic infections varies from 25% to > 50% . Pr . intermedia is the most commonly found pigmented species, followed by Pr . denticola and two Porphyromonas species, P . gingivalis and P . endodontalis . Several studies have shown that P . gingivalis and P . endodontalis are closely related to the presence of acute symptoms in endodontic infections, whereas other black-pigmented Gram-negative anaerobes are not . However, several other species may also be involved in acute infections . Moreover, Porphyromonas species have occasionally been isolated from cases with no symptoms . Although Porphyromonas spp . are clearly related to symptoms at the beginning of therapy, they are not important for the prognosis of the treatment. Plant Mol Biol, 1993 Mar, 21(5), 779 - 87 Secondary structure and phylogeny of the chloroplast 23S rRNA gene from the brown alga Pylaiella littoralis; Somerville CC et al.; The entire nucleotide sequence of a 23S rRNA gene from the brown alga Pylaiella littoralis (L.) Kjellm has been determined . The predicted length of the 23S rRNA is 2948 nucleotides, including the 4.5S rRNA-like region at the 3' end of the molecule . The putative transcript has been folded into a secondary structure by comparison to existing structure models, and the predicted helical regions were inspected by identifying compensatory downstream base changes . The 23S rRNA secondary structure presented here has features that are unique to P . littoralis (no other chromophyte or red algal 23S rRNA sequences are yet available), but has none of the features specific to the chloroplast rRNAs of green plants and green algae . The Pylaiella sequence was aligned with analogous plastidial and eubacterial gene sequences, and the alignment was used to construct a phylogenetic tree . The plastidial sequences formed a coherent cluster closely associated with the 23S rRNA of the cyanobacterium Anacystis nidulans . Within the plastid group, the P . littoralis sequence was most closely related to that of Euglena gracilis confirming earlier analyses based upon 16S rRNA sequences. J Clin Microbiol, 1993 Mar, 31(3), 646 - 52 Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens; Meier A et al.; Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens . By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination . Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems . However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA . Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents . The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured. J Bacteriol, 1993 Mar, 175(5), 1514 - 23 Cloning, characterization, and functional expression in Escherichia coli of chaperonin (groESL) genes from the phototrophic sulfur bacterium Chromatium vinosum; Ferreyra RG et al.; A recombinant lambda phage which was able to propagate in groE mutants of Escherichia coli was isolated from a Chromatium vinosum genomic DNA library . A 4-kbp SalI DNA fragment, isolated from this phage and subcloned in plasmid vectors, carried the C . vinosum genes that allowed lambda growth in these mutants . Sequencing of this fragment indicated the presence of two open reading frames encoding polypeptides of 97 and 544 amino acids, respectively, which showed high similarity to the molecular chaperones GroES and GroEL, respectively, from several eubacteria and eukaryotic organelles . Expression of the cloned C . vinosum groESL genes in E . coli was greatly enhanced when the cells were transferred to growth temperatures that induce the heat shock response in this host . Coexpression in E . coli of C . vinosum groESL genes and the cloned ribulose bisphosphate carboxylase/oxygenase genes from different phototrophic bacteria resulted in an enhanced assembly of the latter enzymes . These results indicate that the cloned DNA fragment encodes C . vinosum chaperonins, which serve in the assembly process of oligomeric proteins . Phylogenic analysis indicates a close relationship between C . vinosum chaperonins and their homologs present in pathogenic species of the gamma subdivision of the eubacterial division Proteobacteria. Res Microbiol, 1993 Mar-Apr, 144(3), 187 - 200 The histidine operon of Azospirillum brasilense: organization, nucleotide sequence and functional analysis; Fani R et al.; A 3457-base pair fragment of Azospirillum brasilense DNA which complemented mutations in the hisA and hisF genes of Escherichia coli was sequenced . The sequence analysis revealed the presence of six major contiguous open reading frames (ORF) . The comparison of the predicted amino acid sequence of these ORF with those encoded by the eubacterial, archaebacterial and eukaryotic his genes sequenced thus far revealed that four of them have a significant degree of homology with the E . coli hisH, hisA, hisF and the C-terminal domain of the hisI gene products . S1 mapping experiments indicated that the putative transcription start site coincided with the AUG translational initiation codon of the hisBd gene, the first gene of the A . brasilense his operon . Downstream from the last ORF, a sequence was identified which functions as a Rho-independent transcription terminator . Comparison of amino acid sequences, gene order and organization and evolutionary aspects of the A . brasilense his cluster are discussed. Curr Microbiol, 1993 Mar, 26(3), 161 - 6 The genome of the non-cultured, bacterial-like organism associated with citrus greening disease contains the nusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase; Villechanoux S et al.; We have recently cloned three DNA fragments (In-2.6, In-1.0, and In-0.6) of the non-cultured, bacterial-like organism (BLO) associated with citrus greening disease . Nucleotide sequence determination has shown that fragment In-2.6 is part of the rplKAJL-rpoBC gene cluster, a well-known operon in eubacteria . The DNA fragment upstream of and partially overlapping with In-2.6 could be isolated and was shown to be the nusG gene . In Escherichia coli, nusG is also immediately upstream of rplKAJL-rpoBC . Fragment In-1.0 carries the gene for a bacteriophage type DNA polymerase . Fragment In-0.6 could not be identified . When In-2.6 was used, at high stringency, as a probe to detect greening BLO strains in infected plants, hybridization was obtained with all Asian strains tested, but not with the African strain examined . At lower stringencies, In-2.6 was able to detect also the African strain . The implications of these results in the taxonomical position of the greening BLO are discussed. Curr Microbiol, 1993 Mar, 26(3), 129 - 32 Congruent evolution between whiteflies (Homoptera: Aleyrodidae) and their bacterial endosymbionts based on respective 18S and 16S rDNAs; Campbell BC; Whiteflies (family Aleyrodidae) possess heritable eubacterial endosymbionts sustained in specialized organ-like structure called mycetomes . Comparisons of distances between the ash whitefly, Siphoninus phillyreae, and two biotypes ("A" and "B") of the sweetpotato whitefly, Bemisia tabaci, based on sequence analysis of genes for 18S rRNAs (rDNAs), were equivalent to the distances represented by the 16S rDNAs of their respective endosymbionts . This finding indicates that evolutionary divergence in whitefly hosts and their endosymbionts is congruent . The nucleotide sequences of the 18S rDNAs and endosymbiont 16S rDNAs indicate the two biotypes of B . tabaci are the same species. Appl Environ Microbiol, 1993 Mar, 59(3), 687 - 94 Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria; Herrick JB et al.; We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR) . PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture . However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P . putida G7 NAH7 plasmid was detected by PCR amplification . Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P . putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments . Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers . The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations . Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P . putida G7 . The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments. FEBS Lett, 1993 Feb 22, 318(1), 61 - 4 Evidence for a Rieske-type FeS center in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius; Anemuller S et al.; A high-potential iron-sulfur cluster with characteristics similar to a Rieske-type center was detected in the plasma membrane of Sulfolobus acidocaldarius by EPR spectroscopy . In the reduced form this center has g-values of gz = 2.031, gy = 1.890 and gx = 1.725 (gav = 1.88, rhombicity = 0.37) and its reduction potential at pH 7.4 was determined to be +325 +/- 10 mV . The The archaebacterial cluster exhibits some unique properties, in comparison to eubacterial and eukaryotic Rieske-type centers . First, the reduction potential is pH-dependent in the range from pH 6.7 to 8.2 . Second, the typical inhibitor of Rieske FeS centers, DBMIB, had no effect on the g-values of this cluster . The center is reducible by both NADH and succinate in the presence of cyanide, an inhibitor of the terminal oxidases . The possible role of a Rieske-type center in an organism lacking any c-type cytochromes is discussed. Eur J Biochem, 1993 Feb 15, 212(1), 41 - 9 Glucose-6-phosphate dehydrogenase . Structure-function relationships and the Pichia jadinii enzyme structure; Jeffery J et al.; The primary structure of glucose-6-phosphate dehydrogenase from the yeast Pichia jadinii (formerly Candida utilis) has been determined . It consists of a 495-residue, N-terminally acetylated protein chain . The structure shows extensive differences from those of the corresponding mammalian, fruit fly, and bacterial enzymes (52-68% residue non-identities), but also from that of another yeast, Saccharomyces cerevisiae (38%) . A eubacterial type and a yeast type of glucose-6-phosphate dehydrogenase are discerned, in addition to the known mammalian type . They are distinguished from each other, from the mammalian type, and the insect enzyme, on the basis of both specific residues and pattern differences . The distribution of residues conserved in all forms locates short segments in which identities are closely grouped . Approximately 50% of these segments correspond to predicted turns and appear to mark the principal folds characteristic of the enzyme's tertiary structure . A region in the N-terminal part of the protein chain has characteristics suggestive of a coenzyme-binding site, while, in the middle third, another functionally important segment may be related to glucose-6-phosphate binding and catalysis. Gene, 1993 Feb 14, 124(1), 67 - 74 Domain V of Giardia lamblia large-subunit rRNA: structure of the peptidyl transferase loop from an early-branching eukaryote and correlation with antibiotic sensitivity; Edlind TD et al.; Large subunit rRNA (LSR) sequences that have been implicated in peptide bond formation form a specific secondary structure called the peptidyl transferase loop (PTL) . Although well conserved, the PTLs of eubacteria, archaebacteria, and eukaryotes have several distinct differences . These differences correlate with different sensitivities to peptidyl transferase and translocase inhibitors . To shed light on the basis for these kingdom-specific differences in PTL structure and function, we have analyzed the sequence and secondary structure of LSR domain V, which contains the PTL, from Giardia lamblia . This parasitic protozoan derives from a very early branch in eukaryotic evolution, and its rRNA was previously shown to have bacteria-like features . In vitro and cell-free systems were also used to test the sensitivity of G . lamblia protein synthesis to specific PTL-targeted inhibitors . Our results indicate that the PTL structure and inhibitor sensitivity typical of higher eukaryotes is conserved in G . lamblia . However, several adjacent domain V sequences more closely resemble archaebacterial rRNA, confirming the 'primitive' nature of G . lamblia rRNA . Thus, the eukaryotic PTL has been conserved over a vast evolutionary period . We speculate that the eukaryotic PTL is primordial and employs specific RNA-RNA interactions to catalyze protein synthesis . Three potential interactions were identified. Nucleic Acids Res, 1993 Feb 11, 21(3), 649 - 55 Zinc finger-like motifs in rat ribosomal proteins S27 and S29; Chan YL et al.; The primary structures of the rat 40S ribosomal subunit proteins S27 and S29 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed by determination of amino acid sequences in the proteins . Ribosomal protein S27 has 83 amino acids and the molecular weight is 9,339 . Hybridization of cDNA to digests of nuclear DNA suggests that there are 4-6 copies of the S27 gene; the mRNA for the protein is about 620 nucleotides in length . Ribosomal protein S29 has 55 amino acids and the molecular weight is 6,541 . There are 14-17 copies of the S29 gene and its mRNA is about 500 nucleotides in length . Rat ribosomal protein S29 is related to several members of the archaebacterial and eubacterial S14 family of ribosomal proteins . S27 and S29 have zinc finger-like motifs as do other proteins from eukaryotic, archaebacterial, eubacterial, and mitochondrial ribosomes . Moreover, ribosomes and ribosomal subunits appear to contain zinc and iron as well. J Biol Chem, 1993 Feb 5, 268(4), 2828 - 35 Sarcina ventriculi synthesizes very long chain dicarboxylic acids in response to different forms of environmental stress; Jung S et al.; Changes in the composition of membrane lipids in a strictly anaerobic, facultative acidophilic eubacterium, Sarcina ventriculi, were studied in response to various forms of environmental stress . Changes in lipid composition and structure occurred in response to changes in environmental pH . At neutral pH, the predominant membrane fatty acids ranged in chain length from C14 to C18 . However, when cells were grown at pH 3.0, a family of unique very long chain fatty acids containing 32-36 carbon atoms was synthesized and accounted for 50% of the total membrane fatty acids . These acids were identified as very long chain alpha,omega-dicarboxylic acids ranging in length from 28 to 36 carbons by electron impact mass spectrometry of methyl and (perdeuterio) methyl ester derivatives . These methyl esters all bore a vicinal dimethyl group toward the center of the chain . The assignment of the structures was confirmed by isolating one of the very long chain unusual fatty acids as the ester form after methanolysis and performing further analyses including 1H and 13C NMR spectroscopy and Fourier transform infrared spectroscopy . Coupling this information with the data from gas chromatography/mass spectrometry analysis, the exact structure was confirmed as alpha,omega-15,16-dimethyltricotanedioate dimethyl ester . Addition of alcohols, either metabolic (0.25 M ethanol) or nonmetabolic (0.05 M butanol) to cells grown at pH 7.0, or thermal stress (growth temperature at pH 7.0 was raised from 37 to 45 or 55 degrees C) also resulted in the synthesis of these very long chain fatty acids . Synthesis of these very long chain alpha,omega-dicarboxylic acids was reversed by reducing the temperature back to 37 degrees C . S . ventriculi is also unusual in that the membrane components are not the usual phospholipid components but appear to be predominantly glycolipids. J Biol Chem, 1993 Feb 5, 268(4), 2755 - 61 The primary structure of rat ribosomal protein L23a . The application of homology search to the identification of genes for mammalian and yeast ribosomal proteins and a correlation of rat and yeast ribosomal proteins; Suzuki K et al.; The amino acid sequence of the rat 60 S ribosomal subunit protein L23a was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein L23a has 156 amino acids and a molecular weight of 17,684 . Hybridization of the L23a cDNA to digests of nuclear DNA suggests that there are 18-20 copies of the L23a gene . The mRNA for the protein is about 600 nucleotides in length . Rat L23a is related to the yeast Saccharomyces cerevisiae L25, to the archaebacterial Methanococcus vannielii L23, to eubacterial Escherichia coli L23, and to other members of the L23 family of ribosomal proteins . A novel application of a routine homology search procedure was employed to identify a nucleotide sequence that could be used to design an oligodeoxynucleotide probe to screen a library for a cDNA that encodes rat L23a; this same procedure uncovered a number of previously unidentified genes for yeast ribosomal proteins in the GenBank DNA data base . In a correlation of rat and yeast ribosomal proteins 48 pairs are shown to be related. Mol Cell Probes, 1993 Feb, 7(1), 7 - 17 Amplification of rDNA loci to detect and type Neisseria meningitidis and other eubacteria; McLaughlin GL et al.; In 1991-92, Neisseria meningitidis group C was isolated from the blood of eight students in Urbana, Illinois, USA, and from the cerebrospinal fluid of one student from a nearby community, Decatur, Illinois . These and other bacterial species were analysed by PCR fingerprinting using primers selected from the ribosomal (r)DNA loci . A rDNA primer pair spanning a region within the 16S rDNA amplified a predicted 280 base pair (bp) DNA fragment from Neisseria spp . and fragments of different sizes for other genera . This primer pair specifically detected a carrier of N . meningitidis in a small clinical battery . Identity of the fragment was confirmed by restriction endonuclease analysis . A 600 bp fragment was also amplified from the 16S-23S internal transcribed spacer (ITS) of N . meningitidis; amplification from six other genera yielded different-sized fragments . Digestion of the ITS fragment from N . meningitidis with Alu I revealed three patterns; pattern I was found only for serogroup C isolates, and it was the dominant pattern among recent isolates with the exception of the one from Decatur . The isolate from Decatur yielded pattern III which suggested a non-clonal relationship to the seven isolates from Urbana . Patterns II and III were more prevalent in isolates from the 1960's and 1980's . PCR-based analysis of these loci can complement the techniques which are currently used for the detection and typing of these and other eubacteria. J Bacteriol, 1993 Feb, 175(3), 669 - 73 Initial steps in the anaerobic degradation of 3,4,5-trihydroxybenzoate by Eubacterium oxidoreducens: characterization of mutants and role of 1,2,3,5-tetrahydroxybenzene; Haddock JD et al.; Chemical mutagenesis and antibiotic enrichment techniques were used to isolate five mutant strains of the obligate anaerobe Eubacterium oxidoreducens that were unable to grow on 3,4,5-trihydroxybenzoate (gallate) . Two strains could not transform gallate and showed no detectable gallate decarboxylase activity . Two other strains transformed gallate to pyrogallol and dihydrophloroglucinol but lacked the hydrolase activity responsible for ring cleavage . A fifth strain accumulated pyrogallol, although it contained adequate levels of the enzymes proposed for the complete transformation of gallate to the ring cleavage product . The conversion of pyrogallol to phloroglucinol by cell extract of the wild-type strain was dependent on the addition of 1,2,3,5-tetrahydroxybenzene or dimethyl sulfoxide . This activity was induced by growth on gallate, while the other enzymes involved in the initial reactions of gallate catabolism were constitutively expressed during growth on crotonate . The results confirm the initial steps in the pathway previously proposed for the metabolism of gallate by E . oxidoreducens, except for the conversion of pyrogallol to phloroglucinol. J Bacteriol, 1993 Feb, 175(3), 583 - 8 Retrotransfer in Escherichia coli conjugation: bidirectional exchange or de novo mating? Heinemann JA, Ankenbauer RG. DNA can be transferred among eubacteria and to plants and fungi by related, plasmid-mediated processes collectively referred to as bacterial conjugation . Conjugation occurs between cells in contact with one another and results in the unidirectional delivery of DNA from a bacterial donor to a recipient . Recent experiments that have reexamined the directionality of DNA flow during conjugation have come to different conclusions, some suggesting that genetic material also flows from recipient cells into the donor and that this process, termed retrotransfer, is likewise directed by donor-encoded functions . Given that bacteria are perhaps united with all living creatures by conjugation, the possibility of gene flow into donor bacteria during conjugation raises interesting evolutionary and biocontainment issues . Here we report that plasmid transmission from bacterial recipients to donors is not a donor-mediated event . Movement of genetic material from recipients to donors was inhibited by streptomycin, which does not inhibit the conjugative donor, indicating that retrotransfer requires gene expression in recipients . Furthermore, retrotransfer was reduced in matings mediated by plasmids that encode strong entry exclusion, to a similar degree as matings between two donors . Therefore we suggest that retrotransfer is in fact newly initiated conjugation between transconjugants and donors. Appl Environ Microbiol, 1993 Feb, 59(2), 417 - 21 Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA; Leff LG et al.; Resistance to kanamycin and neomycin in the bacterial assemblage of a coastal plain stream was detected by growth of colonies on media containing antibiotics . Three of 184 kanamycin-resistant colonies hybridized with a probe containing the nptII gene from transposon Tn5; the nptII gene encodes the enzyme neomycin phosphotransferase and conveys resistance to kanamycin and neomycin . In one of these isolates, the homologous gene was cloned and shown to confer resistance to a kanamycin-sensitive Escherichia coli strain . Since enumeration of bacteria by acridine orange direct counts revealed that less than 0.2% of the bacteria present were cultivated, direct examination of environmental DNA was used to assess abundance of sequences that hybridize to the nptII gene . To examine the resistance potential of bacteria that were not cultured, total DNA was extracted from environmental samples and hybridized with specific probes . The relative amount of eubacterial DNA in each sample was determined by using a eubacterial specific rDNA probe . Then, the abundance of sequences that hybridize to the eubacterial neomycin phosphotransferase gene was determined by hybridization and expressed relative to the total eubacterial DNA in the assemblage . Relative gene abundance was significantly different among assemblages from different habitats (leaves, midchannel sediments, and bank sediments) but did not differ among stream sites. Mol Microbiol, 1993 Feb, 7(4), 577 - 84 Identification and characterization of IS1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family; Bhugra B et al.; Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes . We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements . By subcloning a single strain of M . pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event . The nucleotide sequences of the element, designated IS1138, and the target site into which it inserted were determined . IS1138 consists of 1288 bp with 18 bp perfect terminal inverted repeats . Sequence analysis of the target site before and after insertion of IS1138 identified a 3 bp duplication of target DNA flanking the element . The predicted amino acids encoded by the major open reading frame of IS1138 share significant similarity with the transposases of the IS3 family . Southern hybridization analysis indicates that repetitive sequences similar to IS1138 are present in most, if not all, strains of M . pulmonis, but IS1138-like sequences were not detected in other mycoplasmal species. Gene, 1993 Jan 15, 123(1), 25 - 32 Nucleotide sequences of the trpI, trpB, and trpA genes of Pseudomonas syringae: positive control unique to fluorescent pseudomonads; Auerbach S et al.; A 904-bp probe from Pseudomonas aeruginosa was used to identify the trpB, trpA and trpI genes of Pseudomonas syringae . Transcription initiation at the P . syringae trpBA promoter in vitro was activated by the P . aeruginosa TrpI protein in the presence of indoleglycerol phosphate . Thus, trpB and trpA are regulated positively in three species of fluorescent pseudomonads, P . aeruginosa, P . putida, and P . syringae, but in no other eubacteria so far investigated {Crawford, Annu . Rev . Microbiol . 43 (1989) 567-600} . In addition to conservation of protein-coding sequences, there is a high degree of nucleotide sequence identity in the intergenic control region that includes the divergent trpI and trpBA promoters, especially in the binding sites for TrpI protein . Differences in patterns of codon usage distinguish the trpI genes of P . syringae and P . putida from P . aeruginosa trpI and from the trpB and trpA genes of all three species. Gene, 1993 Jan 15, 123(1), 75 - 80 Allele-specific structure probing of plasmid-derived 16S ribosomal RNA from Escherichia coli; Powers T et al.; Biochemical analysis of Escherichia coli ribosomes containing mutant 16S or 23S (r)ribosomal RNAs, produced via cloned rDNA genes on multicopy plasmids, has been hindered by the background of wild-type (wt) ribosomes containing host-derived rRNA . Here, we describe a method for the construction of unique priming sites in 16S rRNA that allow allele-specific structure probing of ribosomes containing plasmid-encoded RNA . Phenotypically silent mutations, designed to mimic related eubacterial sequences, have been introduced into four phylogenetically variable regions in the 16S rDNA gene that allow inspection of several 16S rRNA functional sites . When oligodeoxyribonucleotides complementary to these altered sequences are used to prime cDNA synthesis in primer extension reactions using reverse transcriptase, only plasmid-derived 16S rRNA is used as a template, thus rendering the wt background invisible . Unexpectedly, we were unable to introduce silent mutations into one nonconserved helix in 16S rRNA, suggesting that constraints in addition to Watson-Crick pairing are important in this region. Biochem Mol Biol Int, 1993 Jan, 29(1), 25 - 31 A small novel chloroplast ribosomal protein (S31) that has no apparent counterpart in the E . coli ribosome; Schmidt J et al.; Higher plant chloroplast ribosomes contain several novel protein components whose homologues are not present in the eubacterial E . coli ribosome, indicating a complex evolution of the chloroplast translational apparatus following the endosymbiotic event . Here we describe the isolation and characterization of a new small protein from spinach chloroplast ribosome which has, based on the amino acid sequence and immunological data, no counterpart in the E . coli ribosome . We suggest a nomenclature suitable for this protein and other novel proteins in the chloroplast ribosome. Ital J Biochem, 1993 Jan-Feb, 42(1), 1 - 11 Properties of the purified elongation factor 2 in the thermoacidophilic archaebacterium Sulfolobus solfataricus; Raimo G et al.; The elongation factor 2 (aEF-2) has been purified to homogeneity from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus . It is the only target protein which is ADP-ribosylated by diphtheria toxin in presence of NAD and this modification abolishes its property to support poly(Phe) synthesis in vitro . The factor is constituted by a single polypeptide chain with a relative molecular mass of 78,000 and an isoelectric point of 5.9 . aEF-2 is resistant to heat denaturation as shown by the fact that its capability to be ADP-ribosylated was only 10% reduced after 4 h treatment at 80 degrees C . Its amino acid composition does not reveal significant differences with that of analogous factors in other sources; nevertheless, the deviation function indicates that aEF-2 is related to Sulfolobus acidocaldarius and eukaryotes EF-2 more than to eubacterial EF-G or other archaebacterial EF-2. Vet Microbiol, 1993 Jan, 34(1), 89 - 95 Chromosomal DNA probes for the identification of Bacteroides tectum and Bacteroides fragilis from the oral cavity of cats; Love DN et al.; A dot-blot hybridisation assay using high molecular weight DNA as whole chromosomal probes was used to differentiate Bacteroides tectum from Bacteroides fragilis . 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes . The whole chromosomal probes were specific--differentiating B . tectum from B . fragilis and both from a variety of other species (including other members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses . However, even at very high stringencies, B . tectum homology groups I, II and III were not distinguishable from one another using either 32P-labelled or DIG-labelled probes . Thus, DIG-labelled whole chromosome probes directed against cellular DNA released directly onto nitrocellulose membranes is considered a useful method for diagnostic veterinary laboratories wishing to identify B . tectum and distinguish it from B . fragilis and other oral anaerobic flora of cats. Mol Microbiol, 1993 Jan, 7(2), 159 - 65 SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane; Oliver DB; Recent insight into the biochemical mechanisms of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure . SecA appears to promote these events by direct recognition of the preprotein or preprotein-SecB complex, binding to inner-membrane anionic phospholipids, insertion into the membrane bilayer and association with the preprotein translocator, SecY/SecE . ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states . As a component likely to catalyse a rate-determining step in protein secretion, SecA synthesis is co-ordinated with the activity of the protein export pathway . This form of negative regulation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell . A precise biochemical scheme for SecA-dependent catalysis of protein export and the details of secA regulation appear to be close at hand . The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways. J Bacteriol, 1993 Jan, 175(1), 133 - 40 Proteins antigenically related to methyl-accepting chemotaxis proteins of Escherichia coli detected in a wide range of bacterial species; Morgan DG et al.; The four methyl-accepting chemotaxis proteins of Escherichia coli, often called transducers, are transmembrane receptor proteins that exhibit substantial identity among the sequences of their cytoplasmic domains . Thus, antiserum raised to one of these proteins recognizes the others and might be expected to recognize related proteins in other bacteria . We used antiserum raised to the transducer Trg in immunoblot experiments to probe a wide range of bacterial species for the presence of antigenically related proteins . Such proteins were detected in over 20 different species, representing 6 of the 11 eubacterial phyla defined by analysis of rRNA sequences as well as one archaebacterial group . Species containing proteins antigenically related to the transducers of E . coli included members of all four subdivisions of the phylum in which E . coli is placed, members of four of the six subdivisions of spirochetes, and two gliding bacteria . These observations provide substantial support for the notion that methyl-accepting taxis proteins are widely distributed among the diversity of bacterial species. Protein Sci, 1993 Jan, 2(1), 20 - 30 Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi, and eubacteria; Reizer J et al.; We demonstrate that three integral membrane receptors of mammals--the ecotropic retroviral leukemia receptor (ERR), the human retroviral receptor (HRR), and the T-cell early activator (Tea)--are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC), which catalyze solute uniport, solute:cation symport, or solute:solute antiport in yeast, fungi, and eubacteria . Interestingly, the ERR membrane protein was recently shown to function as a cation:amino acid cotransporter . A binary sequence similarity matrix and an evolutionary tree of the 14 members of this family, illustrating their sequence similarities and divergences, were constructed . Other proteins, including the developmentally controlled GerAII spore germination protein of Bacillus subtilis and the acetylcholine receptor of Drosophila melanogaster gave sequence comparison scores of a sufficiently large magnitude to suggest (but not to establish) a common evolutionary origin with members of the APC family . We report an extended and corrected Tea cDNA sequence and show that the mammalian Tea and ERR encoding genes are differentially expressed in tissues and cell lines . Furthermore, the two mammalian cDNA sequences hybridize with other vertebrate and yeast genomic DNAs under stringent conditions . These observations support the notion that cell surface receptor proteins in mammals are transport proteins that share a common origin with transport proteins of single-celled organisms . Thus, permeases of essential metabolites may function pathologically as viral receptors. Antimicrob Agents Chemother, 1993 Jan, 37(1), 46 - 50 Coumermycin A1 inhibits growth and induces relaxation of supercoiled plasmids in Borrelia burgdorferi, the Lyme disease agent; Samuels DS et al.; Coumermycin A1 is an inhibitor of DNA gyrase, an enzyme that catalyzes supercoiling of DNA and is required for bacterial DNA replication . We have investigated the activity of this coumarin antibiotic on Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease . B . burgdorferi was more susceptible than many other eubacteria to coumermycin as well as novobiocin, another coumarin antibiotic; this contrasted with its relative resistance to the DNA gyrase inhibitors nalidixic acid, oxolinic acid, and ciprofloxacin . Coumermycin at 0.2 micrograms/ml inhibited the growth of B . burgdorferi B31 in BSK II medium . A 100-fold-lower concentration induced the relaxation of two negatively supercoiled circular plasmids within 2 h . Plasmid supercoiling was restored within 2 h of removal of coumermycin . These results suggest that B . burgdorferi has a DNA gyrase and that this enzyme's activity is required for growth . Furthermore, structural analogs of coumermycin may be considered as treatments for Lyme disease. Biosystems, 1993, 30(1-3), 113 - 35 Statistical analysis of L-tuple frequencies in eubacteria and organelles; Sitnikova TL et al.; This work is an attempt to study the structural features and evolutionary patterns of nucleotide sequences by analyzing their 1- through 4-plet frequencies and statistical relations between them . We present mathematical apparatus for this analysis . In particular, we introduce criteria to estimate the degree of homogeneity of L-plet composition in a given set of sequences and the dependence of the L-plet frequencies on the composition of lower orders . We apply these criteria to the study of eubacteria, mitochondria and chloroplasts . We demonstrate that L-plet frequencies are quite useful for revealing evolutionary relationship between DNA sequences and that the non-random distribution is more typical for doublets than to triplets . Non-randomness of triplet composition is more characteristic to coding than to non-coding regions, while no significant differences in dinucleotide composition can be observed . The obtained results can be used for revealing possible mechanisms of the codon usage phenomena. Antonie Van Leeuwenhoek, 1993, 63(3-4), 275 - 88 Carbohydrate transport in bacteria under environmental conditions, a black box? Lengeler JW. A typical eubacterium carries a battery of substrate transport systems which are the ultimate pacemakers for growth . These systems reflect a billion year old selection for coping with rapidly changing conditions in the environment and each of them is optimised for specific growth conditions . Metabolic pathways in combination with transport systems can be interpreted as transient sensory systems, where a transport system corresponds to a sensor for external stimuli . Characteristics is a tightly linked common control between a carbohydrate metabolic pathway and the corresponding transport system . Many of the observed growth phenomena are a direct result of adaptation and regulation of transport capacity to rapid changes in environmental conditions . Some of the better understood examples are discusses . Nevertheless, knowledge on bacterial carbohydrate transport under environmental conditions as documented in the literature is still scarce. Biosystems, 1993, 31(2-3), 127 - 30; discussion 130-3 Holobionts, hybrids, and cladistic classification; Zrzavy J et al.; David P . Mindell assumes that incorporation of the holobionts (evolutionarily stable symbiotic complexes) and hybrids into phylogenetic trees necessarily distorts the hierarchical structure of cladistic classification, and must lead to reformulation of some basic cladistic concepts (BioSystems, 27, 53-62, 1992) . He does not regard the Eukarya and Eubacteria as monophyletic taxa, the former being 'polyphyletic', the latter 'paraphyletic' . We attempt to show that the existence of holobionts/hybrids is not a cladistic problem . Cladistics is a systematic methodology, not a system of evolutionary hypotheses; cladograms are statements about distribution of shared characters (synapomorphies) and, consequently, the cladograms are always branching and hierarchically structured . A taxon is monophyletic if it has a single root in the cladogram, not a single ancestor 'in reality' . Cladistic methodology does not provide a clue for distinguishing whether a conflicting distribution of the potential synapomorphies is due to reticulation (e.g., symbiogenesis) or convergent evolution . Consequently, both Eubacteria and Eukarya either are or are not monophyletic taxa if they are or are not determined to be so during cladistic analysis. Biosystems, 1993, 31(2-3), 111 - 9 Horizontal transfer of ATPase genes--the tree of life becomes a net of life; Hilario E et al.; An ancient gene duplication gave rise to the catalytic and non-catalytic subunits of each of the three types of proton pumping ATPases: vacuolar, archaebacterial and eubacterial . Previously, this gene duplication has been used to root the universal tree of life . However, recent findings of archaebacterial type ATPases in eubacteria and of eubacterial type in an archaebacterium suggested that both types of ATPases may have been already present in the last common ancestor . Here we show that a phylogenetic analysis of these ATPase subunits indicates that this conclusion is premature . We suggest that horizontal gene transfer can explain the data . In addition, we show that the analysis of glutamate dehydrogenases data neither affirm nor contradict any particular placement of the last common ancestor in the universal tree of life . The prevalence and the mode of horizontal gene transfer is discussed. Reprod Nutr Dev, 1993, 33(6), 577 - 84 Effect of Eubacterium limosum, a ruminal hydrogenotrophic bacterium, on the degradation and fermentation of cellulose by 3 species of rumen anaerobic fungi; Bernalier A et al.; The degradation and fermentation of cellulose filter paper were studied in axenic cultures of 3 species of rumen anaerobic fungi, Neocallimastix frontalis, Piromyces communis and Caecomyces communis, and in cocultures containing 1 of these fungal strains and Eubacterium limosum, a hydrogenotrophic rumen bacterial species . When E limosum was introduced into fungal cultures a slight decrease in fungal cellulolytic activity was observed . The end products of the fermentation of cellulose found in the cocultures were different from those found in the fungal monocultures . E limosum used formate and part of the hydrogen produced by the fungi and probably created a shift in the metabolism of the fungi resulting in a reduction of lactate and ethanol production. FASEB J, 1993 Jan, 7(1), 7 - 14 Recent studies of ribonuclease P; Altman S et al.; RNase P is an essential enzyme that is required for the biosynthesis of tRNA . It is composed of RNA and protein subunits . The RNA subunit of the enzyme derived from eubacterial sources can carry out the catalytic function by itself in vitro . Current studies of RNase P focus on structure-function relationships with respect to interactions of the RNA subunit with its substrates and with respect to the determination of the kinetic parameters of the reaction, the role of the protein component, and the rules governing recognition of substrates. FEBS Lett, 1992 Dec 21, 314(3), 211 - 4 Nucleotide sequence of the genes for ribosomal proteins HS15 and HSH from Haloarcula marismortui: an archaeon-specific gene cluster; Arndt E et al.; The nucleotide sequences of the genes for two ribosomal proteins, HS15 and HSH, from the archaeon Haloarcula marismortui, have been determined . The genes were found in a cluster together with another open reading frame with a probable regulatory function . HS15 and HSH have counterparts in eucarya . HS15 is significantly homologous to S19 from frog (Xenopus laevis) . HSH is related to S37 from yeast (Saccharomyces cerevisiae) and S27 from fly (Drosophila melanogaster), as well as to other members of the S27 family . Eubacterial counterparts were not found, suggesting that these proteins are 'extra proteins' that are absent in eubacterial ribosomes. J Biol Chem, 1992 Dec 15, 267(35), 25304 - 8 The primary structure of rat ribosomal protein S5 . A ribosomal protein present in the rat genome in a single copy; Kuwano Y et al.; The amino acid sequence of the rat 40 S ribosomal subunit protein S5 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by the determination, directly from the protein, of 17 residues near the NH2 terminus . S5 has 204 amino acids; the molecular weight is 22,863 . The protein designated S5a has the same amino acid sequence as S5 except that it lacks the NH2-terminal 5 residues . It is not known whether the conversion of a portion of S5 to S5a is physiological or fortuitous . The mRNA for S5 has about 820 nucleotides . Hybridization of the S5 cDNA to digests of nuclear DNA indicates that the rat genome has only a single copy of the gene; this is in distinction to the mouse and human genomes which have three to six copies of the S5 gene . Rat ribosomal protein S5 is related to the eubacteria, the arachaebacteria, and the chloroplast family of S7 ribosomal proteins . There is a peptide of 16 residues at the carboxyl terminus of S5 that is highly conserved in 18 species spanning the three kingdoms and chloroplasts. Nucleic Acids Res, 1992 Dec 11, 20(23), 6331 - 7 Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria; Vioque A; The genes encoding the RNA subunit of ribonuclease P from the unicellular cyanobacterium Synechocystis sp . PCC 6803, and from the heterocyst-forming strains Anabaena sp . PCC 7120 and Calothrix sp . PCC 7601 were cloned using the homologous gene from Anacystis nidulans (Synechococcus sp . PCC 6301) as a probe . The genes and the flanking regions were sequenced . The genes from Anabaena and Calothrix are flanked at their 3'-ends by short tandemly repeated repetitive (STRR) sequences . In addition, two other sets of STRR sequences were detected within the transcribed regions of the Anabaena and Calothrix genes, increasing the length of a variable secondary structure element present in many RNA subunits of ribonuclease P from eubacteria . The ends of the mature RNAs were determined by primer extension and RNase protection . The predicted secondary structure of the three RNAs studied is similar to that of Anacystis and although some idiosyncrasies are observed, fits well with the eubacterial consensus. Biochem Int, 1992 Dec, 28(4), 725 - 34 Relationship between substrate activity and pKa value of phenols on sulfotransferase from Eubacterium A-44; Konishi-Imamura L et al.; The relationship between the kinetics of the enzyme activity and the structural features of phenolic donor and of acceptor substrates was investigated with a sulfotransferase from Eubacterium A-44, a human intestinal bacterium . The enzyme catalyzed the transfer of the sulfate group from the sulfate esters of phenol having a lower pKa to phenols having a higher pKa . When the Km values for acceptor substrates were measured at their optimal pH, a linear plot for log10Km versus the pKa with a slope of 0.615 was obtained . In addition, it is considered that the effect of pH on the Km values for the various acceptors is due to ionization of free enzyme . The kinetic behavior of bacterial sulfotransferase differed from that of mammalian phenol sulfotransferase. J Bacteriol, 1992 Dec, 174(24), 8008 - 15 Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus; Breitwieser A et al.; Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca . 10-nm-thick peptidoglycan-containing layer and a ca . 10-nm-thick S layer . Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan . This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria . Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells . In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain . In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca . 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer . The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant . It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane . Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside. EMBO J, 1992 Dec, 11(12), 4369 - 78 Isolation and cloning of Omp alpha, a coiled-coil protein spanning the periplasmic space of the ancestral eubacterium Thermotoga maritima; Engel AM et al.; We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima . In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body . Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54 . The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment . The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes . Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues . Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils. Nucleic Acids Res, 1992 Nov 25, 20(22), 5919 - 25 Evolutionary conserved nucleotides within the E.coli 4.5S RNA are required for association with P48 in vitro and for optimal function in vivo; Wood H et al.; E.coli 4.5S RNA is homologous to domain IV of eukaryotic SPR7S RNA, the RNA component of the signal recognition particle . The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54 . In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, arachaebacteria and eukaryotes . This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop . This conserved region is a candidate for the SRP54/P48 binding site . To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence . Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro . Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects . This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48 . On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo. J Theor Biol, 1992 Nov 7, 159(1), 53 - 65 Macroevolution of plasmids: a model for plasmid speciation; Sykora P; A new evolutionary model for diversification in plasmid incompatibility groups (plasmid speciation) is suggested . The model is based on the formation of plasmid cointegrates from two compatible plasmids . The existence of plasmid cointegrates is well known, however, their potential key role in plasmid macroevolution has not yet been recognized . In a hypothesis presented here, one of the rep genes is supposed to be relaxed from selection in plasmid cointegrates and thus becomes free to accumulate mutations . These mutations can lead to a change in incompatibility specificity . Evidence supporting this hypothesis comes from the common occurrence of multi-replicon plasmids in nature as well as from experimental studies on plasmid cointegrate formation . A more speculative extension of this model hypothesizes an evolutionary scenario for origin of the eubacterial single-replicon genome and the eukaryotic multi-replicon genome, as well as the place of plasmids and viruses in this picture. Gene, 1992 Nov 2, 121(1), 155 - 60 Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase; Marks GL et al.; The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors . Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73) . R . prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria . Comparison of the major sigma factors from Escherichia coli and R . prowazekii revealed 44.9% aa identity . R . prowazekii sigma 73 produced in E . coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis . This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein . Despite a similarity to the E . coli sigma 70 encoded by rpoD, R . prowazekii sigma 73 did not complement E . coli rpoD temperature-sensitive mutants. J Exp Biol, 1992 Nov, 172, 137 - 47 Evolution and isoforms of V-ATPase subunits; Gogarten JP et al.; The structure of V- and F-ATPases/ATP synthases is remarkably conserved throughout evolution . Sequence analyses show that the V- and F-ATPases evolved from the same enzyme that was already present in the last common ancestor of all known extant life forms . The catalytic and non-catalytic subunits found in the dissociable head groups of both V-ATPases and F-ATPases are paralogous subunits, i.e . these two types of subunits evolved from a common ancestral gene . The gene duplication giving rise to these two genes (i.e . those encoding the catalytic and non-catalytic subunits) pre-dates the time of the last common ancestor . Similarities between the V- and F-ATPase subunits and an ATPase-like protein that is implicated in flagellar assembly are evaluated with regard to the early evolution of ATPases . Mapping of gene duplication events that occurred in the evolution of the proteolipid, the non-catalytic and the catalytic subunits onto the tree of life leads to a prediction of the likely quaternary structure of the encoded ATPases . The phylogenetic implications of V-ATPases found in eubacteria are discussed . Different V-ATPase isoforms have been detected in some higher eukaryotes, whereas others were shown to have only a single gene encoding the catalytic V-ATPase subunit . These data are analyzed with respect to the possible function of the different isoforms (tissue-specific, organelle-specific) . The point in evolution at which the different isoforms arose is mapped by phylogenetic analysis. Appl Environ Microbiol, 1992 Nov, 58(11), 3622 - 9 Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture; DiStefano TD et al.; Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture . This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system . Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures . The effect of vancomycin on dechlorination was more complex . Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could . These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen . Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool . This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised. FEMS Microbiol Lett, 1992 Nov 1, 77(1-3), 161 - 8 Analysis of genomic DNA from Chlamydia trachomatis for Dam and Dcm methylation; Wagar EA et al.; Chlamydia trachomatis is a Gram-negative eubacterium with a dimorphic developmental cycle and obligate intracellular growth in the eucaryotic host . The Dam transmethylase of Escherichia coli methylates at the N6 position of adenine in the sequence 5'-GATC-3' and the Dcm transmethylase adds methyl groups to the C5 position of the internal cytosines in the sequences 5'-CCWGG-3' . In contrast to E . coli, C . trachomatis DNA appears to have unmethylated Dam sites and only low level Dcm methylation. Biosci Biotechnol Biochem, 1992 Nov, 56(11), 1797 - 800 Purification and characterization of a DNA-dependent RNA polymerase from Pseudomonas putida; Fujita M et al.; DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from Pseudomonas putida . The enzyme had the typical composition of beta',beta,alpha, and sigma subunits of eubacterial RNA polymerases . The molecular masses of the subunits were 156,000 Da, 151,000 Da, 87,000 Da, and 42,000 Da, respectively, as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The NH2-terminal amino acid residues of the alpha subunit had a marked homology with those of the alpha subunit of Escherichia coli RNA polymerase . The enzyme activity was dependent on ribonucleoside triphosphates, Mg2+, and a DNA template, and was inhibited in vitro by rifampicin . The enzyme activity was maximal in the presence of 10 mM MgCl2 . In an in vitro transcription assay using the tac promoter-controlled DNA as a template, the RNA polymerase of P . putida initiated transcription at the same site as that of E . coli. Can J Microbiol, 1992 Nov, 38(11), 1091 - 6 Bacterial conjugation: everybody's doin' it; Frost LS; Recent evidence suggests that bacterial conjugation is ubiquitous in the bacterial world and that DNA transfer between different genera-kingdoms is possible . It has also been demonstrated that many bacterial gene transfer systems resemble each other at the molecular level and that others are a blend of two or more of these systems . Thus, in the absence of a sexual cycle, bacterial conjugation, along with bacteriophages, transposons, and natural transformation systems, forms a potent force for gene dissemination and repair in the eubacteria and simple eukaryotes. J Endod, 1992 Nov, 18(11), 558 - 61 The relationship between clinical symptoms and anaerobic bacteria from infected root canals; Hashioka K et al.; The purpose of this study was to investigate the correlation between the composition of bacterial flora from infected root canals and clinical symptoms . The materials evaluated consisted of 28 teeth from 25 patients with apical periodontitis . Eubacterium were found to be significantly related to acute or chronic clinical symptoms and Peptococcus, Peptostreptococcus, and Porphyromonas gingivalis to subacute clinical symptoms . We suggested that Peptococcus, Peptostreptococcus, Eubacterium, Porphyromonas, and Bacteroides were significantly related to percussion pain; Porphyromonas and Bacteroides were significantly related to odor in the infected root canals . Many Bacteroides were isolated from most of the infected root canals. Gene, 1992 Oct 12, 120(1), 33 - 41 The sequence of the gene encoding elongation factor Tu from Chlamydia trachomatis compared with those of other organisms; Cousineau B et al.; Nucleotide (nt) sequences encoding the elongation factor Tu (EF-Tu), tRNA(Thr) and tRNA(Trp) from Chlamydia trachomatis have been determined . The environment of the EF-Tu-encoding gene (tuf), between two tRNA gene sequences, suggests that it is part of a tufB locus . The nt sequence and the deduced amino acid (aa) sequence were aligned with comparable sequences from other organisms and the resulting data bases were used to infer phylogenies . Phylogenetic trees based on aa sequences and nt sequences are similar, but not completely congruent with rRNA gene-based phylogenies . Both the nt and aa sequence trees concur on the early divergence of Thermotoga and Chlamydia from the bacterial root . The aa alignment highlights the presence of four unique Cys residues in the chlamydial sequence which are found at strictly conserved positions in other sequences . Further peculiarities of the chlamydial and eubacterial sequences have been mapped to the X-ray crystallographic structure of the protein. Oral Microbiol Immunol, 1992 Oct, 7(5), 285 - 90 Coaggregation studies of the Eubacterium species; George KS et al.; Eubacterium species are gram-positive anaerobic rods that are frequently isolated from subgingival plaque of periodontal pockets . Five Eubacterium species were tested for their ability to coaggregate with 33 oral bacterial strains . Using visual and turbidimetric assays, coaggregation was observed among Eubacterium brachy, Eubacterium nodatum, Eubacterium alactolyticum and Eubacterium limosum strains only when tested with Fusobacterium nucleatum strains; Eubacterium saburreum displayed only weak coaggregation ability . Coaggregation between F . nucleatum and the Eubacterium species was observed over a wide range of concentrations of each organism . The F . nucleatum strains contained a heat labile and the Eubacterium species a heat stabile coaggregation receptor . Arginine, histidine, lysine and glycine inhibited the coaggregation between F . nucleatum and the Eubacterium species . Sugars and other amino acids tested did not inhibit the observed coaggregation . Rabbit anti-F . nucleatum serum completely inhibited coaggregation, but anti-E . brachy serum and normal rabbit serum did not . As these anaerobic microorganisms are frequently isolated from the same oral lesions, the surface interactions observed may be important in the pathogenesis of these polymicrobic infections. Oral Microbiol Immunol, 1992 Oct, 7(5), 257 - 62 Associations between microbial species in dental root canal infections; Sundqvist G; The existence of commensal or antagonistic relationships between microorganisms in the root canals of teeth with apical periodontitis was investigated . Samples were taken from 65 infected human root canals and were analysed according to species, frequency of occurrence and proportion of the total isolated flora . The most frequent species were Fusobacterium nucleatum, Prevotella intermedia, Peptostreptococcus micros, Peptostreptococcus anaerobius, Eubacterium alactolyticum, Eubacterium lentum and Wolinella recta . An odds ratio system was used to calculate positive or negative associations between the isolated bacteria . Strong positive associations were found between F . nucleatum and P . micros, Porphyromonas endodontalis, Selenomonas sputigena and W . recta . There was also a positive association between P . intermedia and P . micros, P . anaerobius and the eubacteria . In general, species of streptococci, Propionibacterium propionica, Capnocytophaga ochracea and Veillonella parvula showed no or negative associations with the other bacteria . The results are consistent with the concept of a special and selective environment occurring in the root canal that is due, in part, to the cooperative as well as antagonistic nature of the relationships between bacteria in the root canal. Appl Environ Microbiol, 1992 Oct, 58(10), 3413 - 6 Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction; Bruce KD et al.; Specific DNA sequences from native bacterial populations present in soil, sediment, and sand samples were amplified by using the polymerase chain reaction with primers for either "universal" eubacterial 16S rRNA genes or mercury resistance (mer) genes . With standard amplification conditions, 1.5-kb rDNA fragments from all 12 samples examined and from as little as 5 micrograms of soil were reproducibly amplified . A 1-kb mer fragment from one soil sample was also amplified . The identity of these amplified fragments was confirmed by DNA-DNA hybridization. Mol Microbiol, 1992 Oct, 6(19), 2797 - 804 Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2); Takano E et al.; Transcription of redD, the activator gene required for production of the red-pigmented antibiotic undecylprodigiosin by Streptomyces coelicolor A3(2), showed a dramatic increase during the transition from exponential to stationary phase . The increase in redD expression was followed by transcription of redX, a biosynthetic structural gene, and the appearance of the antibiotic in the mycelium, and coincided with the intracellular appearance of ppGpp . However, ppGpp production elicited either by nutritional shift-down of, or addition of serine hydroxamate to, exponentially growing cultures had no stimulatory effect on redD transcription . The presence of redD on a multicopy plasmid resulted in elevated levels of the redD transcript and production of redX and undecylprodigiosin during exponential growth; the normal growth-phase-dependent production of undecylprodigiosin appeared to be mediated entirely through the redD promoter, which shows limited similarity to the consensus sequence for the major class of eubacterial promoters. J Gen Virol, 1992 Oct, 73 ( Pt 10), 2763 - 6 Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins; Koonin EV et al.; It is demonstrated, by means of computer-assisted analysis, that C1 protein involved in the replication of geminivirus DNA is related to the rolling circle replication initiator proteins of eubacterial plasmids, particularly the plasmids of the pMV158 family . Three sequence motifs conserved in the geminivirus and plasmid replication proteins were delineated, one of them encompassing the Tyr residue that presumably forms a covalent linkage to DNA . These findings are compatible with the results of recent analyses of geminivirus replicative intermediates suggesting a rolling circle mechanism for geminivirus DNA replication . It is hypothesized that C1 protein initiates the rolling circle replication of geminivirus DNA by nicking a specific site in the virus-sense DNA and covalently linking to the 5' side of the nick . The putative rolling circle replication initiator domain comprises the N-terminal portion of C1, whereas its C-terminal part is a putative helicase domain . By analogy with prokaryotic systems, it is speculated that the replication initiator domain and the helicase domain function coordinately . The possibility of the origin of geminiviruses from prokaryotic circular ssDNA replicons is discussed. J Bacteriol, 1992 Oct, 174(19), 6317 - 20 Characterization of the gill symbiont of Thyasira flexuosa (Thyasiridae: Bivalvia) by use of polymerase chain reaction and 16S rRNA sequence analysis; Distel DL et al.; Comparative molecular sequence (16S rRNA) analysis methods were used to identify and characterize the symbionts of Thyasira flexuosa independently of pure culture techniques and to compare these symbionts with the previously reported putative symbiont isolate, Thiobacillus thyasiris TG-2 (A . P . Wood and D . P . Kelly, Arch . Microbiol . 152:160-166, 1989) . Polymerase chain reaction amplification using 16S rRNA primers specific for eubacteria was used to amplify a single unique sequence from the gill tissue of T . flexuosa . This sequence is phylogenetically most closely related to the 16S rRNA genes of known symbionts of lucinid clams and is distinct from those determined for strain TG-2 and other known bacteria . Strain TG-2 most closely resembles a free-living, chemolithoautotrophic bacterium known to be associated with the surfaces of thiotrophic bivalve shells, suggesting that this strain is a contaminant and not the authentic intracellular symbiont of T . flexuosa. J Bacteriol, 1992 Oct, 174(19), 6103 - 8 Evidence that a plasmid from a hyperthermophilic archaebacterium is relaxed at physiological temperatures; Charbonnier F et al.; A plasmid of 3.45 kb (pGT5) was recently discovered in a strain of hyperthermophilic archaebacterium which was isolated from samples collected in a deep-sea hydrothermal vent . This strain (GE5) grows within a temperature range of 68 to 101.5 degrees C, and we show here that it contains a strong ATP-dependent reverse gyrase activity (positive DNA supercoiling) . By comparison with eubacterial plasmids of known superhelical densities, we estimated the superhelical density of the archaebacterial plasmid pGT5 to be -0.026 at 25 degrees C . The equation which relates the change of the rotation angle of the DNA double helix with temperature was validated at 95 degrees C, the optimal growth temperature of the GE5 strain . Considering these new data, the superhelical density of plasmid pGT5 was calculated to be -0.006 at the physiological temperature of 95 degrees C, which is close to the relaxed state . This finding shows that the DNA topology of a plasmid isolated from a hyperthermophilic archaebacterium containing reverse gyrase activity is strikingly different from that of typical eubacterial plasmids. Eur J Biochem, 1992 Oct 1, 209(1), 351 - 5 Cloning and sequencing of the gene for the cytoplasmic inorganic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum; Richter OM et al.; The gene (ppa) from the thermoacidophilic archaebacterium Thermoplasma acidophilum, encoding the cytoplasmic pyrophosphatase, has been cloned . Two degenerate oligonucleotide probes, synthesized according to the N-terminal amino acid sequence of the isolated protein, were used to screen subgenomic libraries . The DNA-derived amino acid sequence of the archaebacterial enzyme allows, for the first time, comparative studies of cytoplasmic pyrophosphatases to be extended to all three urkingdoms . The archaebacterial pyrophosphatase more closely resembles the eubacterial enzymes on the basis of sequence similarity and subunit size . The majority of amino acid residues considered to be essential for hydrolysis of pyrophosphate seem to have been conserved throughout evolution, as inferred from the results of an alignment of sequences from all three urkingdoms. Eur J Biochem, 1992 Oct 1, 209(1), 343 - 9 Purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum; Richter OM et al.; Cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium Thermoplasma acidophilum . The enzyme was purified to electrophoretic homogeneity by combining ion-exchange and affinity-chromatographic separations . This soluble pyrophosphatase probably consists of six identical subunits, since SDS/PAGE gave an estimate of about 22 kDa for a single subunit and size-exclusion chromatography under non-denaturing conditions indicates a molecular mass of 110 +/- 5 kDa . The two most prominent catalytic features of this enzyme are the absolute requirement for divalent cations for catalytic action, Mg2+ conferring the highest activity, and the pronounced specificity for PPi . The catalytic behavior apparently follows simple Michaelis-Menten kinetics with a Km of about 7 microM for PPi and a specific activity of about 1200 U/mg at 56 degrees C . Surprisingly, maximum activity could be observed at 85 degrees C which is more than 20 degrees C above the temperature for optimal growth . Several cytoplasmic extracts of eubacteria and archaebacteria have been probed with a polyclonal antiserum raised against the purified archaebacterial protein . The only noticeable cross-reactivity could be detected with an extract from the methanogen Methanosarcina barkeri although this probably does not reflect the inferred phylogenetic relationship between methanogens and Thermoplasma acidophilum. Gene, 1992 Sep 21, 119(1), 113 - 8 Sequence analysis of a DNA fragment from Buchnera aphidicola (an endosymbiont of aphids) containing genes homologous to dnaG, rpoD, cysE, and secB; Lai CY et al.; The aphid, Schizaphis graminum, contains a prokaryotic, obligately intracellular endosymbiont, Buchnera aphidicola, which is necessary for the survival of the host . A recent study of Bu . aphidicola 16S rRNA has indicated that it is a member of the gamma-3 subdivision of the eubacterial class, Proteobacteria, which includes Escherichia coli . In order to further characterize the endosymbiont and establish its similarity to free-living eubacteria and/or organelles, we have cloned and sequenced a 4534-bp DNA fragment containing dnaG-rpoD-cysE-secB . The deduced amino acid (aa) sequence identity to the homologous E . coli proteins ranged from 47 to 80% . The close proximity of the pair, dnaG-rpoD, to the pair, cysE-secB, on the Bu . aphidicola DNA, differed from E . coli, in which these two pairs of genes are 14 min apart on the bacterial chromosome . The results of past physiological studies of the endosymbiont were consistent with the presence and function of DNA primase (DnaG), sigma factor (RpoD) and components of the secretory system (SecB) . Comparison of the deduced aa sequence of Bu . aphidicola CysE (serine acetyltransferase, a key allosterically regulated enzyme in cysteine biosynthesis) with the E . coli wild-type enzyme and a mutant defective in feedback inhibition suggested that the endosymbiont CysE may not be regulated . By analogy with E . coli, the lack of feedback inhibition may lead to overproduction of cysteine by the endosymbiont . The results of this and previous investigations indicate that Bu . aphidicola has many of the properties of free-living bacteria and not of organelles. Scand J Immunol, 1992 Sep, 36(3), 497 - 506 Immunohistology of joint inflammation induced in rats by cell wall fragments of Eubacterium aerofaciens; Kool J et al.; After a single intraperitoneal injection of cell wall fragments of Eubacterium aerofaciens, a main resident from the human intestinal flora, an acute arthritis develops within 2 days which is followed by a chronic arthritis that lasts at least 90 days . In an earlier report the histological appearance of the joint inflammation during this period has been described . In this study we investigated in more detail the cell types that are involved in the development of arthritis by using cell-type-specific monoclonal antibodies in an immunohistological assay . In the acute phase of arthritis, T-helper cells appeared in the synovial tissue together with ED1-positive (ED1+) and ED3-positive (ED3+) macrophages . After a temporary decline at day 12 all macrophage subsets, as well as T-helper cells, reappeared or increased again at day 33 . Later, in the chronic phase (days 47-90), an increased number of ED1-positive (ED1+) cells in the synovial tissue and a decreased number of ED2-positive (ED2+) cells in the synovial lining was the most prominent finding when compared with control rats . These results indicate that, apart from T lymphocytes, macrophages also play an important role in the development and continuation of chronic arthritis in this model. Acta Stomatol Belg, 1992 Sep, 89(3), 155 - 62 Susceptibility of anaerobic microorganisms to hypothiocyanite produced by lactoperoxidase; Courtois P et al.; The susceptibility of Capnocytophaga ochracea, Eikenella corrodens, Eubacterium yurii, Fusobacterium nucleatum, Peptostreptococcus micros, Prevotella intermedia, Selenomonas sputigena, Wolinella recta to hypothiocyanite (OSCN-) produced by the lactoperoxidase system was tested . Results showed a decrease of bacterial survival rate after OSCN- exposure, with an intra- and inter-species variability from 0 to 95% for C . ochracea, 34-100% for E . corrodens, 0-83% for E . yurii, 1-15% for F . nucleatum, 8-61% for P . micros, 0-100% for P . intermedia, 0-44% for S . sputigena and 0-8% for W . recta . The survival rate did not correlate with the NADH/OSCN- oxidoreductase activity present in the lysed bacteria (r = -0.3248; N = 15; NS). Plasmid, 1992 Sep, 28(2), 170 - 6 DNA sequence and analysis of a cryptic 4.2-kb plasmid from the filamentous cyanobacterium, Plectonema sp . strain PCC 6402; Perkins DR et al.; The 4194-bp plasmid, pRF1, from Plectonema sp . Strain PCC 6402 was completely sequenced and analyzed . Seven potential open reading frames were identified . The predicted amino acid sequence of open reading frame C (ORF C) had identities of 34, 29, and 25% with Rep B from the Staphylococcus aureus plasmid, pUB110; Rep from the Bacillus amyloliquefaciens plasmid, pFTB14; and protein A from the S . aureus plasmid, pC194, respectively . A 75-amino-acid region conserved in these proteins (Rep B, Rep, and protein A) also was highly conserved in ORF C with identities of 45, 37, and 40%, respectively . Significantly, 16 of the 21 amino acids conserved in Rep B, Rep, and protein A were found at the same positions in ORF C . This ORF may encode a replication protein that includes a region conserved in some eubacteria . Additional structural features include a 425-bp region that contains palindromes, tandem repeats, and short direct repeats which may correspond to the origin of replication . An 18-bp inverted repeat was located between two open reading frames, A and G. J Gen Microbiol, 1992 Sep, 138 ( Pt 9), 1875 - 80 Cloning and physical mapping of the Staphylococcus aureus rplL, rpoB and rpoC genes, encoding ribosomal protein L7/L12 and RNA polymerase subunits beta and beta'; Aboshkiwa M et al.; Using segments of the Escherichia coli rpoB and rpoC genes as heterologous probes, we have identified and cloned an 8.3 kb PstI fragment from the Staphylococcus aureus genome containing the rpoB and rpoC genes, which respectively encode the beta and beta' subunits of DNA-directed RNA polymerase . This region is almost certainly equivalent to the rif locus, located near to fus at interval 12/13 on the S . aureus linkage map . Limited DNA sequencing revealed the gene order rpoB-rpoC (transcribed from left to right) and identified the rplL gene, encoding ribosomal protein L7/L12, upstream of rpoB . This and other evidence suggests that the rpoBC genes of S . aureus form part of a large gene cluster encoding components of the transcription and translation apparatus which is well-conserved in other eubacteria. Curr Opin Dent, 1992 Sep, 2, 66 - 71 Bacteriology of periodontal disease; Russell RR; The microbial flora associated with the periodontal tissues in health and disease is extremely complex, and much research is being directed toward identifying those species that may be etiologic agents or that can be used as prognostic indicators . Recent work has resulted in changes in the taxonomic position of several periodontal species and the recognition that several others, particularly species of Eubacterium and Peptostreptococcus, as well as a novel oral spirochete, may be important in disease . New rapid techniques for identifying and enumerating the periodontal flora are being applied to cross-sectional and longitudinal studies to assess the significance of the various species; the DNA probe and immunologic detection methods demonstrate both advantages and limitations when compared with methods based on determining the predominant cultivable flora. Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 58 - 64 The primary structure of rat ribosomal protein L3; Kuwano Y et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L3 was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein L3 has 403 amino acids and has a molecular weight of 46,106 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 9 copies of the L3 gene . The mRNA for the protein is about 1,400 nucleotides in length . Rat L3 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes. Biochim Biophys Acta, 1992 Aug 17, 1132(1), 94 - 6 The complete nucleotide sequence of the gene (rpoD1) encoding the principal sigma factor of the RNA polymerase from the cyanobacterium Synechococcus sp . strain PCC7942; Tanaka K et al.; The complete nucleotide sequence of rpoD1 gene from Synechococcus PCC7942 has been determined . The nucleotide data have indicated the presence of an open reading frame of 1155 base pairs encoding a polypeptide which shares the framework structure for principal sigma factors of eubacterial strains. Eur J Biochem, 1992 Aug 1, 207(3), 877 - 85 Primary structures of ribosomal proteins L3 and L4 from Bacillus stearothermophilus; Herwig S et al.; Ribosomal proteins L3 and L4 were purified to homogeneity from total protein isolated from the 50S subunit of Bacillus stearothermophilus by reversed-phase high-performance liquid chromatography (RP-HPLC) . Amino acid sequences of both proteins were determined by automated N-terminal sequence analysis and sequencing of internal peptides . Using oligonucleotides deduced from the N-terminal region of protein L3 as hybridization probes, a DNA fragment coding for proteins L3, L4 and the N-terminal part of protein L23 has been identified, cloned and sequenced . The organization of the genes is identical to that found in the S10 operon of Escherichia coli . Comparison of the sequences of proteins L3 and L4 with those of other organisms revealed that all proteins of the L3 family are highly conserved . On the other hand, the archaebacterial L4 proteins show no significant sequence similarity to the E . coli L4 protein whereas the L4 protein of B . stearothermophilus is significantly similar to all of the L4 proteins and thus justifies the membership of all the L4 proteins in one protein family . The results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes and possible functional domains of proteins L3 and L4. J Dent Res, 1992 Aug, 71(8), 1509 - 15 Immunoglobulins in milk from cows immunize