bluuebunny/crossref_metadata_2025 huggingface dataset:
Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus
Authors: Mollet B , Knol J , Poolman B , Marciset O , Delley M
Published: November 2016

Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food.
Molecular characterization of a negative regulator of Streptococcus sobrinus surface protein antigen gene
Authors: Takahashi I , Okahashi N , Hamada S
Published: November 2016

Mutans streptococci have been shown to give rise to variants in terms of expression of surface protein antigens by repeated subculturing of the organisms, which in turn induces changes in colonial morphologies. A 2,850-bp upstream region of the gene (pag) for a surface protein antigen, PAg, of Streptococcus sobrinus MT3791 was determined. Analysis of the nucleotide sequence revealed the existence of three open reading frames (ORFs) located upstream of the pag gene. ORF1 extended from an undetermined further upstream sequence to the termination codon TAG lying 1,943 bp upstream of the pag gene. ORF2, consisting of 609 bp lying 1,689 bp upstream of the pag gene, encoded a protein of 23,347 Da and a protein of 22,792 Da. The synthesis of these proteins (protein antigen regulators) was demonstrated by using the in vitro T7 RNA polymerase/promoter system. ORF3, extending from 314 bp upstream of the pag gene to 712 bp upstream of the pag gene, encoded a protein of 14,802 Da. Disruption of chromosomal ORF2 of parent strain MT3791 by allelic exchange resulted in isogenic mutants, termed PAREm-1 and PAREm-2, that synthesized larger amounts of cell-free and cell-associated PAg than did the parent strain. RNA dot blot analysis demonstrated that expression of PAg-specific mRNA transcripts by mutants PAREm-1 and PAREm-2 was about 32-fold higher than that by strain 3791. Mutants PAREm-1 and PAREm-2 were found to be more hydrophobic than strain MT3791. Resting cells of these mutants attached in larger numbers to saliva-coated hydroxyapatite than did those of the parent strain. These results suggest that protein antigen regulator regulates the expression of PAg gene in a negative fashion, affecting the colonization of tooth surfaces by the organism. Thus, ORF2 is concluded to be a negative regulator gene of PAg synthesis and was designated par.
Sequence of a class E tetracycline resistance gene from Escherichia coli and comparison of related tetracycline efflux proteins
Authors: Allard J D , Bertrand K P
Published: November 2016

We determined the nucleotide sequence of the class E tetA gene on plasmid pSL1456 from Escherichia coli SLH1456A. The deduced amino acid sequence of the class E TetA protein shows 50 to 56% identity with the sequences of five related TetA proteins (classes A through D and G). Hydrophobicity profiles identify 12 putative transmembrane segments with similar boundaries in all six TetA sequences. The N-terminal alpha domain of the six sequences is more highly conserved than the C-terminal beta domain; the central hydrophilic loop connecting the alpha and beta domains is the least conserved region. Amino acid residues that have been shown to be important for class B (Tn10) TetA function are conserved in all six TetA sequences. Unlike the class B tetA gene, the class D and E tetA genes do not exhibit a negative gene dosage effect when present on multicopy plasmids derived from pACYC177.
Purine metabolism by intracellular Chlamydia psittaci
Authors: McClarty G , Fan H
Published: November 2016

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.
A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells
Authors: Donnenberg M S , Yu J , Kaper J B
Published: November 2016

Enteropathogenic Escherichia coli (EPEC) is capable of attaching intimately to epithelial cells and effacing their microvilli. A chromosomal locus, eaeA (originally eae), is required for the intimate attachment aspect of this effect. We report the mapping of a region of the EPEC chromosome that is located immediately downstream of the eaeA gene and that is also necessary for intimate attachment. An isogenic in-frame deletion mutation in one of the open reading frames identified in this region was engineered. Because the resulting mutant, like an eaeA deletion mutant, is deficient in the ability to attach intimately to epithelial cells, the mutated gene is designated eaeB. Full activity is restored to the eaeB mutant when the cloned gene is reintroduced on a plasmid. The eaeB mutant remains capable of producing intimin, the product of the eaeA gene. No differences in the fractionation properties or electrophoretic mobility of intimin are apparent in the eaeB mutant. The product of the eaeB locus was identified by in vitro transcription-translation. The nucleotide sequence of the eaeB gene predicts a protein that contains a sequence motif common to several aminotransferase enzymes. These results indicate that the attaching and effacing effect is a complex phenotype dependent on a gene cluster present on the EPEC chromosome.
Characterization of an activity from the strict anaerobe Roseburia cecicola that degrades DNA when exposed to air
Authors: O'Connor L T , Savage D C
Published: November 2016

Roseburia cecicola is an obligately anaerobic bacterium that is extremely sensitive to oxygen. Genomic DNA isolated from cells exposed to air for even a brief period (< 5 min) is partially degraded, while DNA extracted from cells maintained in an anaerobic environment remains intact. Cells exposed to air for longer and longer periods yield DNA which is progressively degraded into fragments with decreasing sizes. Oxygen toxicity for this anaerobe appears to result, at least in part, from degradation of its genomic DNA. Cell lysates of the organism exhibited a similar ability to degrade exogenous sources of DNA when assayed in vitro under aerobic conditions. A substance that degrades both DNA and RNA when incubated aerobically was partially purified from such lysates. It has an approximate molecular weight of 2,800 and is unlikely to be a protein. It requires a reducing agent for activity and can be inhibited by catalase and peroxidase but not superoxide dismutase. The rate at which it degrades DNA in vitro can be enhanced by temperatures above 37 degrees C or by oxygen at partial pressures above atmospheric pressure. These results suggest that this substance degrades nucleic acids by a mechanism involving oxygen radicals.
Oar, a 115-kilodalton membrane protein required for development of Myxococcus xanthus
Authors: Martinez-Canamero M , Munoz-Dorado J , Farez-Vidal E , Inouye M , Inouye S
Published: November 2016

Myxococcus xanthus is a developmental gram-negative bacterium which forms multicellular fruiting bodies upon nutrient starvation. This bacterium was found to contain a 115-kDa membrane protein which separated with the inner membrane fraction by sucrose density gradient centrifugation. The gene for this protein was cloned, and its DNA sequence was determined. The deduced amino acid sequence consists of 1,061 residues. This protein contains a putative signal sequence and many short segments, found scattered throughout the entire protein, that have sequence similarities with OmpA, a major outer membrane protein of Escherichia coli. Thus, the gene was designated oar (OmpA-related protein). A second open reading frame was found 36 bases downstream of the oar termination codon. This open reading frame encodes a protein of 236 residues and contains a putative lipoprotein signal sequence. An aor disruption mutation (delta oar) showed no effect on vegetative growth but caused abnormal morphogenesis during development and reduced myxospore formation. When examined with a light microscope, delta oar cells were unable to aggregate on developmental agar, indicating that Oar is required for cellular adhesiveness during development.
Variation of the rfb gene clusters in Salmonella enterica
Authors: Xiang S H , Haase A M , Reeves P R
Published: November 2016

In order to explore the genetic variation of O antigens of Salmonella enterica, we surveyed 164 strains (132 serovars) belonging to 45 serogroups, using 25 mostly single-gene rfb DNA probes for colony hybridization. The results revealed that strains within a serogroup have very similar or identical rfb genes. At least three of the four rhamnose genes were detected in all 17 serogroups reported to contain rhamnose, and one or more were detected in three others. The likelihood of being detected decreased in the order rfbB, rfbC, rfbA, and rfbD, which is the map order, suggesting a gradient of divergence. Mannose pathway genes were much less conserved, and of 27 groups reported to contain mannose or mannose derivatives colitose or fucose, only 9 hybridized to the rfbM and rfbK probes. Dideoxyhexose genes were found only in groups reported to contain dideoxyhexoses. Group D2, which had not been studied previously, appears to resemble group D1, with the substitution of one gene from group E1 to give a change in one linkage. In contrast to sugar pathway genes, sugar transferase genes did not in general hybridize to strains of other groups outside the closely related groups A, B, and D, with the exception of the galactose transferase gene also shared by groups C2, C3, and all E groups.
Site-directed mutations in the relaxase operon of RP4
Authors: Cole S P , Lanka E , Guiney D G
Published: November 2016

Mutations were constructed by site-directed mutagenesis in the relaxase operon of the broad-host-range plasmid RP4. The mutations were constructed in smaller plasmids, recombined into the 60-kb RP4 plasmid, and tested for their ability to transfer. The relaxase operon contains the transfer genes traJ, traH, and traI, which are involved in nicking at the transfer origin to generate the single strand destined to be transferred to the recipient cell. In the first mutant, the C terminus of TraI was truncated, leaving TraH intact. This mutant decreased transfer by approximately 500-fold in Escherichia coli, and the traI mutation could be complemented by a wild-type copy of traI in trans in the donor. The traI mutation similarly decreased transfer between a variety of gram-negative bacteria. A site-specific mutation was made by the polymerase chain reaction-based unique-site mutagenesis procedure to alter the start site of traH. This mutation had no effect on intraspecific E. coli transfer but reduced transfer by up to sevenfold for some gram-negative bacteria. The traH mutation had no effect on plasmid stability. Thus, neither TraH nor the C terminus of TraI is required for conjugative transfer, but both increase mating efficiency in some hosts.
Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus
Authors: Wang E , Garcia M M , Blake M S , Pei Z , Blaser M J
Published: November 2016

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.
Patterns of gene expression in Bacillus subtilis colonies
Authors: Salhi B , Mendelson N H
Published: November 2016

Bacillus subtilis 5:7, a derivative of macrofiber-producing strain FJ7, carries the lacZ reporter gene within Tn917 at an unknown location in the host genome. Expression of the host gene carrying lacZ within colonies of 5:7 was observed by examining growth under different conditions in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). At a high plating density small colonies arose that expressed the host gene early and throughout the colony, whereas at a low density large colonies were produced that expressed the host gene late in development and only in cells forming a ring pattern close to the colony periphery. A highly regulated spatial and temporal gene expression pattern was observed in growth from cross-streaks, suggesting that gene expression is responsive to concentration gradient fields established by neighboring growth. Colonies cultured on agar blocks revealed that expression was governed by depletion of a medium component and also by the geometry of the substrate upon which the colonies grew. At least three factors influenced the control of expression: (i) the concentration of a diffusible component of the medium exhausted by cell growth, (ii) a spatial-temporal factor related to growth within the colony, and (iii) the geometry of the growth substrate.
Identification and transcriptional analysis of the Escherichia coli htrE operon which is homologous to pap and related pilin operons
Authors: Raina S , Missiakas D , Baird L , Kumar S , Georgopoulos C
Published: November 2016

We have characterized a new Escherichia coli operon consisting of two genes, ecpD and htrE. The ecpD gene encodes a 27-kDa protein which is 40% identical at the amino acid level to the pilin chaperone PapD family of proteins. Immediately downstream of the ecpD gene is the htrE gene. The htrE gene encodes a polypeptide of 95 kDa which is processed to a 92-kDa mature species. The HtrE protein is 38% identical to the type II pilin porin protein PapC. The ecpD htrE operon is located at 3.3 min on the genetic map, corresponding to the region from kbp 153 to 157 of the E. coli physical map. The htrE gene was identified on the basis of a Tn5 insertion mutation which resulted in a temperature-sensitive growth phenotype above 43.5 degrees C. The transcription of this operon is induced with a temperature shift from 22 to 37 or 42 degrees C but not to higher temperatures, e.g., 50 degrees C. Consistent with this result, the temperature-induced transcription was shown to be independent of the rpoH gene product (sigma 32). The transcription of this operon was further shown to require functional integration host factor protein, since himA or himD mutant bacteria possessed lower levels of ecpD htrE transcripts. Among the three transcriptional start sites discovered, one, defined by the P2 promoter, was found to be under the positive regulation of the katF (rpoS) gene, which encodes a putative sigma factor required for the transcription of many growth phase-regulated genes.
Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii
Authors: Etchebehere L C , Simon M N , Campanhã R B , Zapella P D , Véron M , Maia J C
Published: November 2016

Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.
Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64
Authors: Kim S R , Funayama N , Komano T
Published: November 2016

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.
Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii
Authors: Ismaiel A A , Zhu C X , Colby G D , Chen J S
Published: November 2016

Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants.
Expression analysis of cloned chromosomal segments of Escherichia coli
Authors: Sankar P , Hutton M E , VanBogelen R A , Clark R L , Neidhardt F C
Published: November 2016

The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentially with a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects. Selected E. coli chromosomal fragments from an ordered genomic library (Y. Kohara, K. Ikiyama, and K. Isono, Cell 50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizing its T7 promoter. The protein products were analyzed by two-dimensional gel electrophoresis. By using DNA sequence information, the gel migration was predicted for the protein products of open reading frames from these segments, and this information was used to identify gene products visualized as spots on two-dimensional gels. Even in the absence of DNA sequence information, this approach offers the opportunity to identify all gene products of E. coli and map their genes to within 10 kb on the E. coli genome; with sequence information, this approach can produce a definitive expression map of the E. coli genome.
Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains
Authors: Simons G , Nijhuis M , de Vos W M
Published: November 2016

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.
The outer membranes of Brucella spp. are not barriers to hydrophobic permeants
Authors: Martínez de Tejada G , Moriyón I
Published: November 2016

The patterns of susceptibility to hydrophobic and hydrophilic drugs and the uptake of the fluorescent probe N-phenyl-naphthylamine in Brucella spp., Haemophilus influenzae, Escherichia coli, and deep rough Salmonella minnesota mutants were compared. The results show that the outer membranes of smooth and naturally rough Brucella spp. do not represent barriers to hydrophobic permeants and that this absence of a barrier relates at least in part to the properties of Brucella lipopolysaccharide.
Genes for the establishment and maintenance of lysogeny by the temperate coliphage 186
Authors: Lamont I , Richardson H , Carter D R , Egan J B
Published: November 2016

To identify the genes in coliphage 186 that are required for lysogeny, we isolated clear-plaque mutants. Complementation studies and DNA sequencing identified two genes, the cI gene for the immunity maintenance repressor and the cII gene, which is required only for the establishment of lysogeny. One mutant carried a change in the LexA-binding site controlling expression of the antirepression protein Tum.
Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli
Authors: Thornton C G , Kumar G K , Haase F C , Phillips N F , Woo S B , Park V M , Magner W J , Shenoy B C , Wood H G , Samols D
Published: November 2016

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types: a hexameric central 12S subunit to which 6 outer 5S subunits are attached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl coenzyme A and pyruvate serve as substrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12S gene encodes a protein of 604 amino acids and of M(r) 65,545. The deduced sequence shows regions of extensive homology with the beta subunit of mammalian propionyl-CoA carboxylase as well as regions of homology with acetyl-CoA carboxylase from several species. Two genomic fragments were subcloned into pUC19 in an orientation such that the 12S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 12S. The Escherichia coli-expressed 12S was purified to apparent homogeneity by a three-step procedure and compared with authentic 12S from P. shermanii. Their quaternary structures were identical by electron microscopy, and the E. coli 12S preparation was fully active in the reactions catalyzed by this subunit. We conclude that we have cloned, sequenced, and expressed the 12S subunit which exists in a hexameric active form in E.coli.
Peptidyl transferase inhibition by the nascent leader peptide of an inducible cat gene
Authors: Gu Z , Rogers E J , Lovett P S
Published: November 2016

The site of ribosome stalling in the leader of cat transcripts is critical to induction of downstream translation. Site-specific stalling requires translation of the first five leader codons and the presence of chloramphenicol, a sequence-independent inhibitor of ribosome elongation. We demonstrate in this report that a synthetic peptide (the 5-mer) corresponding to the N-terminal five codons of the cat-86 leader inhibits peptidyl transferase in vitro. The N-terminal 2-, 3-, and 4-mers and the reverse 5-mer (reverse amino acid sequence of the 5-mer) are virtually without effect on peptidyl transferase. A missense mutation in the cat-86 leader that abolishes induction in vivo corresponds to an amino acid replacement in the 5-mer that completely relieves peptidyl transferase inhibition. In contrast, a missense mutation that does not interfere with in vivo induction corresponds to an amino acid replacement in the 5-mer that does not significantly alter peptidyl transferase inhibition. Our results suggest that peptidyl transferase inhibition by the nascent cat-86 5-mer peptide may be the primary determinant of the site of ribosome stalling in the leader. A model based on this concept can explain the site specificity of ribosome stalling as well as the response of induction to very low levels of the antibiotic inducer.
Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment
Authors: Cherest H , Thomas D , Surdin-Kerjan Y
Published: November 2016

The transsulfuration pathways allow the interconversion of homocysteine and cysteine with the intermediary formation of cystathionine. The various organisms studied up to now incorporate reduced sulfur into a three- or a four-carbon chain and use differently the transsulfuration pathways to synthesize sulfur amino acids. In enteric bacteria, the synthesis of cysteine is the first step of organic sulfur metabolism and homocysteine is derived from cysteine. Fungi are capable of incorporating reduced sulfur into a four-carbon chain, and they possess two operating transsulfuration pathways. By contrast, synthesis of cysteine from homocysteine is the only existing transsulfuration pathway in mammals. In Saccharomyces cerevisiae, genetic, phenotypic, and enzymatic study of mutants has allowed us to demonstrate that homocysteine is the first sulfur amino acid to be synthesized and cysteine is derived only from homocysteine (H. Cherest and Y. Surdin-Kerjan, Genetics 130:51-58, 1992). We report here the cloning of genes STR4 and STR1, encoding cystathionine beta-synthase and cystathionine gamma-lyase, respectively. The only phenotypic consequence of the inactivation of STR1 or STR4 is cysteine auxotrophy. The sequencing of gene STR4 has allowed us to compare all of the known sequences of transsulfuration enzymes and enzymes catalyzing the incorporation of reduced sulfur in carbon chains. These comparisons reveal a partition into two families based on sequence motifs. This partition mainly correlates with similarities in the catalytic mechanisms of these enzymes.
Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b: cloning, sequencing, and expression of the tyrosinase gene mepA
Authors: Mercado-Blanco J , García F , Fernández-López M , Olivares J
Published: November 2016

Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b (140 MDa). Transfer of this plasmid to GR4-cured derivatives or to Agrobacterium tumefaciens enables these bacteria to produce melanin. Sequence analysis of a 3.5-kb PstI fragment of plasmid pRmeGR4b has revealed the presence of a open reading frame 1,481-bp that codes for a protein whose sequence shows strong homology to two conserved regions involved in copper binding in tyrosinases and hemocyanins. In vitro-coupled transcription-translation experiments showed that this open reading frame codes for a 55-kDa polypeptide. Melanin production in GR4 is not under the control of the RpoN-NifA regulatory system, unlike that in R. leguminosarum bv. phaseoli 8002. The GR4 tyrosinase gene could be expressed in Escherichia coli under the control of the lacZ promoter. For avoiding confusion with mel genes (for melibiose), a change of the name of the previously reported mel genes of R. leguminosarum bv. phaseoli and other organisms to mep genes (for melanin production) is proposed.
Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions
Authors: Chen L , James L P , Helmann J D
Published: November 2016

We have cloned two metal-regulated genes (mrgA and mrgC) from Bacillus subtilis by using transposon Tn917-lacZ. Both were isolated as iron-repressible gene fusions, but the metal specificity and sensitivity of gene repression are distinct. Transcription of mrgA-lacZ is induced at the end of logarithmic-phase growth in minimal medium, and this induction is prevented by excess manganese, iron, cobalt, or copper. Limitation for metal ions is sufficient for mrgA-lacZ induction, since resuspension in medium lacking both manganese and iron rapidly induces transcription. Transcription of mrgC-lacZ is also induced by iron deprivation but is not repressed by added manganese or other metal ions. Expression of mrgC-lacZ and a 2,3-dihydroxybenzoic acid-based siderophore is repressed in parallel by iron, and in both cases, only iron effects repression. We have cloned and sequenced the promoter and regulatory regions of both mrgA and mrgC. Both genes are preceded by a predicted sigma A-dependent promoter element with overlapping sequences similar to the iron box consensus element for recognition by the Escherichia coli ferric uptake regulator protein (Fur). Mutation of the putative iron box for gene mrgC leads to partial derepression in iron-replete medium.
Mutations in the alpha subunit of RNA polymerase that affect the regulation of porin gene transcription in Escherichia coli K-12
Authors: Sharif T R , Igo M M
Published: November 2016

The two-component regulatory system consisting of OmpR and EnvZ controls the differential expression of major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12. We have isolated and characterized two mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that decrease the expression of OmpF. These mutations have a number of properties that distinguish them from previously isolated rpoA mutations that affect porin expression. The rpoA203 mutation decreases the expression of porin genes ompF and ompC and also decreases the expression of the malE and phoA genes. In contrast, rpoA207 decreases the expression of ompF but does not affect ompC, malE, or phoA transcription. Our results suggest that mutations at various positions in the alpha subunit may affect the OmpR-dependent transcription of ompF and ompC differently and may be useful for analyzing the mechanism underlying their differential expression in response to medium osmolarity.
High-molecular-weight protein 2 of Yersinia enterocolitica is homologous to AngR of Vibrio anguillarum and belongs to a family of proteins involved in nonribosomal peptide synthesis
Authors: Guilvout I , Mercereau-Puijalon O , Bonnefoy S , Pugsley A P , Carniel E
Published: November 2016

The iron-regulated irp2 gene is specific for the highly pathogenic Yersinia species and encodes high-molecular-weight protein 2 (HMWP2). Despite the established correlation between the presence of HMWP2 and virulence, the role of this protein is still unknown. To gain insight into the function of HMWP2, the entire coding sequence and the promoter of irp2 were sequenced. Two putative -35 and -10 promoter sequences were identified upstream of a large open reading frame, and two potential Fur-binding sites were found overlapping the second -35 box. The large open reading frame is composed of 6,126 nucleotides and may encode a protein of 2,035 amino acids (ca. 228 kDa) with a pI of 5.81. A signal sequence was not present at the N terminus of the protein. Despite the existence of 30 cysteine residues, carboxymethylation prevented the formation of most if not all disulfide bonds that otherwise occurred when the cells were sonicated. The protein was composed of three main domains: a central region of ca. 850 residues, bordered on each side by a repeat of 550 residues. A high degree of identity (44.5%) was found between HMWP2 and the protein AngR of Vibrio anguillarum. The central part of HMWP2 (after removal of a loop of 337 residues) also displayed significant homology with proteins belonging to the superfamily of adenylate-forming enzymes and, like them, possessed a putative ATP-binding motif that is also present in AngR. In addition, HMWP2 shared with the group of antibiotic and enterochelin synthetases a potential amino acid-binding site. Six consensus sequences defining the superfamily and four defining the family of synthetases were derived from the multiple alignment of the 30 sequences of proteins or repeated domains. A phylogenetic tree that was constructed showed that HMWP2 and AngR are in a family composed of Lys2, EntF, and the tyrocidine, gramicidin, and Beta-lactam synthetases. This finding suggests that HMWP2 may participate in the nonribosomal synthesis of small biologically active peptides.
Degradation of individual chromosomes in recA mutants of Escherichia coli
Authors: Skarstad K , Boye E
Published: November 2016

Rapidly growing wild-type Escherichia coli cells contain two, four, or eight fully replicated chromosomes after treatment with rifampin, reflecting that all replication origins are initiated simultaneously. Cells with defects in the timing of the initiation of replication may contain three, five, six, or seven fully replicated chromosomes after such treatment. This phenotype, termed the asynchrony phenotype, is also seen in recombination-deficient recA mutants. It is shown here that for recA strains, the phenotype can be explained by a selective and complete degradation of individual chromosomes. The selective degradation is largely recD dependent and is thus carried out by the RecBCD exonuclease.
Glucose uptake and catabolite repression in dominant HTR1 mutants of Saccharomyces cerevisiae
Authors: Ozcan S , Freidel K , Leuker A , Ciriacy M
Published: November 2016

Growth and carbon metabolism in triosephosphate isomerase (delta tpi1) mutants of Saccharomyces cerevisiae are severely inhibited by glucose. By using this feature, we selected for secondary site revertants on glucose. We defined five complementation groups, some of which have previously been identified as glucose repression mutants. The predominant mutant type, HTR1 (hexose transport regulation), is dominant and causes various glucose-specific metabolic and regulatory defects in TPI1 wild-type cells. HTR1 mutants are deficient in high-affinity glucose uptake and have reduced low-affinity transport. Transcription of various known glucose transporter genes (HXT1, HXT3, and HXT4) was defective in HTR1 mutants, leading us to suggest that HTR mutations affect a negative factor of HXT gene expression. By contrast, transcript levels for SNF3, which encodes a component of high-affinity glucose uptake, were unaffected. We presume that HTR1 mutations affect a negative factor of HXT gene expression. Multicopy expression of HXT genes or parts of their regulatory sequences suppresses the metabolic defects of HTR1 mutants but not their derepressed phenotype at high glucose concentrations. This suggests that the glucose repression defect is not a direct result of the metabolically relevant defect in glucose transport. Alternatively, some unidentified regulatory components of the glucose transport system may be involved in the generation or transmission of signals for glucose repression.
Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer
Authors: Hagège J , Pernodet J L , Sezonov G , Gerbaud C , Friedmann A , Guérineau M
Published: November 2016

pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined. Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids [aa]), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa). traSA is essential for pSAM2 intermycelial transfer and pock formation. It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101. TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment. spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer. A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype). The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family. Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer. However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.
Sequencing and characterization of a gene cluster encoding the enzymes for L-rhamnose metabolism in Escherichia coli
Authors: Moralejo P , Egan S M , Hidalgo E , Aguilar J
Published: November 2016

The sequencing of the EcoRI-HindIII fragment complementing mutations in the structural genes of the L-rhamnose regulon of Escherichia coli has permitted identification of the open reading frames corresponding to rhaB, rhaA, and rhaD. The deduced amino acid sequences gave a 425-amino-acid polypeptide corresponding to rhamnulose kinase for rhaB, a 400-amino-acid polypeptide corresponding to rhamnose isomerase for rhaA, and a 274-amino-acid polypeptide corresponding to rhamnulose-1-phosphate aldolase for rhaD. Transcriptional fusions of the three putative promoter regions to lacZ showed that only the rhaB leader region acted as a promoter, as indicated by the high beta-galactosidase activity induced by rhamnose, while no significant activity from the rhaA and rhaD constructions was detected. The rhaB transcription start site was mapped to -24 relative to the start of translation. Mutations in the catabolic genes were used to show that L-rhamnose may directly induce rhaBAD transcription.
Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication
Authors: Bidnenko V E , Gruss A , Ehrlich S D
Published: November 2016

We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+. All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu. These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication. We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination.
Cloning, sequencing, expression, and regulation of the structural gene for the copper/topa quinone-containing methylamine oxidase from Arthrobacter strain P1, a gram-positive facultative methylotroph
Authors: Zhang X , Fuller J H , McIntire W S
Published: November 2016

Deoxyoligonucleotides corresponding to amino acid sequences of methylamine oxidase and polyclonal anti-methylamine oxidase antibodies were used to probe Arthrobacter strain P1 plasmid and chromosomal DNA libraries. Two open reading frames, maoxI and maoxII, which are greater than 99% homologous, were cloned from the chromosomal library. The deduced amino acid sequences of the coding regions are identical except for two residues near the C termini. On the other hand, the 5'- and 3'-flanking regions of maoxI and maoxII are quite different. While either gene could code for methylamine oxidase, the dissimilarity in the 5'-flanking regions indicates that the genes are differently regulated. It was determined that maoxII alone encodes methylamine oxidase. The tyrosyl residue which is converted to topa quinone in the mature enzyme was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidase. Transcriptional start sites and possible regulatory elements were identified in the 5' region of maoxI and maoxII, and stem-loop structures were found in the 3'-flanking regions. High levels of methylamine oxidase are produced when Arthrobacter strain P1 is grown on methylamine alone or on glucose plus methylamine, but growth on LB medium plus methylamine resulted in very low production of the enzyme. Expression of maoxII from its own promoter in Escherichia coli grown on glucose or LB medium with or without methylamine gave the same level of production of methylamine oxidase.
A cryptic promoter in the O(R) region of bacteriophage lambda
Authors: Woody S T , Fong R S , Gussin G N
Published: November 2016

A cryptic promoter, designated P alpha, initiates transcription within the O(R) region of bacteriophage lambda. Transcription from P alpha proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site. P alpha is likely to compete with both PR and PRM for formation of open complexes, since it is only active when PR is mutated and can be suppressed by mutations that increase PRM activity. In addition, transcription initiation at P alpha is blocked by lambda repressor. Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P alpha was approximately 1/3 as strong as PRM. However, a P alpha mutation had little effect on KBkf (the association rate constant) for PRM. These observations can be explained by the finding that open complexes formed at P alpha are relatively unstable (half-life = 20 to 25 min). Dissociation of RNA polymerase from P alpha allows additional open complexes to form at PR or PRM, and thus the apparent strength of P alpha decreases with increasing preincubation times.
Pyrophosphate-dependent phosphofructo-1-kinase complements fructose 1,6-bisphosphatase but not phosphofructokinase deficiency in Escherichia coli
Authors: Kemp R G , Tripathi R L
Published: November 2016

The gene from Propionibacterium freudenreichii for PPi-dependent phosphofructo-1-kinase, an enzyme that is found in some bacteria, in a number of anaerobic protists, and in plants, complements the absence of fructose 1,6-bisphosphatase in Escherichia coli but does not complement the deficiency of the ATP-dependent phosphofructokinase.