bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary perspectives on multiresistance beta-lactamase transposons
Authors: Lafond M , Couture F , Vézina G , Levesque R C
Published: November 2016

A series of intragenic DNA probes, encoding the major part of the transposase resolvase and inverted repeats of transposons Tn3, Tn21, and Tn2501, were used in hybridization assays for homologous DNA sequences in 18 transposons studied. The tnpA and tnpR probes detected extensive homology with Tn3-like and Tn21-like elements for 11 transposons. This high degree of homology was confirmed with the 38- and 48-base-pair inverted-repeat oligonucleotide probes of Tn3, Tn21, and Tn2501. The Southern-type gel hybridization experiments localized the tnpA-homologous sequences on the physical DNA maps constructed. The genetic and physical maps of the transposons were compared, as were their nucleic acid sequence homologies. These comparisons suggested a subfamily of mobile elements distinct from but related to the Tn21 group. Based on these results, an evolutionary model is proposed and a pedigree is presented for the genesis of multiresistance beta-lactamase transposons.
Evolution of a tRNA operon in gamma purple bacteria
Authors: Giroux S , Cedergren R
Published: November 2016

Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum.
Nucleotide sequence and expression in Escherichia coli of the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene of Pseudomonas mevalonii
Authors: Anderson D H , Rodwell V W
Published: November 2016

The mva operon of Pseudomonas mevalonii encodes two enzymes that can convert internalized mevalonate into acetoacetate and acetyl-coenzyme A (CoA). The promoter-proximal gene of this operon is mvaA, the structural gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (EC 1.1.1.88). The cloning, characterization, and expression of mvaA has been reported (M. J. Beach and V. W. Rodwell, J. Bacteriol. 171:2994-3001, 1989). We report here the nucleotide sequence of another gene of this operon, mvaB, its expression in Escherichia coli, and its identification as the structural gene for HMG-CoA lyase (EC 4.1.3.4). P. mevalonii HMG-CoA lyase is a cytosolic protein with 301 amino acid residues and a molecular weight of 31,600. This represents the first reported sequence of an HMG-CoA lyase from any source.
Transcription events in the origin of replication of plasmid pSC101
Authors: Manen D , Izaurralde E , Churchward G , Caro L
Published: November 2016

Insertion mutations were isolated in the origin fragment of the plasmid pSC101 after random cleavage with DNase I. The replication properties of the resulting plasmids confirmed previous findings and extended the characterization of the essential regions. Using these plasmids, we analyzed by various methods the transcription events in the pSC101 origin. In addition to the mRNA of repA, a gene coding for the self-regulated RepA protein which is essential for replication of the plasmid, we characterized a transcript, which we called RNA Y, that runs in the opposite direction and that starts in the middle of the second repeated sequence in the origin region. Like the self-regulated repA mRNA, RNA Y is weakly expressed. It does not code for a complete protein within the origin fragment but may do so in the wild-type plasmid. We also found indications for one or, possibly, two small RNA species, called RNA X, which run in the same direction as RepA and which are partially complementary to RNA Y. We postulate that RNA Y and, possibly, RNA X are implicated in the initiation of replication of pSC101.
Biochemical and genetic analyses of acetoin catabolism in Alcaligenes eutrophus
Authors: Fründ C , Priefert H , Steinbüchel A , Schlegel H G
Published: November 2016

In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used Tn5::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth. The transposon-harboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A. eutorphus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks. The insertions of Tn5 were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants. The native DNA fragments were cloned from a lambda L47 or from a cosmid gene bank. Evidence is provided that fragments A (21 kilobase pairs [kb]) and C (7.7 kb) are closely linked in the genome; the insertions of Tn5 covered a region of approximately 5 kb. Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown. In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of Tn5::mob. No structural gene could be assigned to the D or E fragments. In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb). This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein. The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.
Biosynthesis of caldariellaquinone in Sulfolobus spp
Authors: Zhou D , White R H
Published: November 2016

The biosynthesis of caldariellaquionone (CQ) was studied in species of Sulfolobus by measuring the incorporation of stable isotopically labeled tyrosines into CQ. By feeding a series of tyrosines labeled with deuterium or 13C and then measuring the extent and position at which label was incorporated into CQ by mass spectrometry, it was shown that more than 95% of the label was incorporated into the benzo[b]thiophen-4,7-quinone moiety of CQ. From the labeling experiments, it is concluded that the benzo[b]thiophen-4,7-quinone is derived as an intact unit from all of the carbons of tyrosine except C-1.
xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter
Authors: Ingram C , Brawner M , Youngman P , Westpheling J
Published: November 2016

We describe the development of a convenient and sensitive reporter gene system for Streptomyces spp. based on the use of a promoterless copy of the xylE gene of Pseudomonas putida. The xylE gene product is a catechol dioxygenase, which converts the colorless substrate catechol to an intensely yellow hydroxymuconic semialdehyde. A promoterless copy of xylE was placed under the transcriptional control of galP1, a glucose-repressed and galactose-induced promoter from Streptomyces lividans, and its expression was examined in bacterial colonies on agar plates or in liquid cultures grown in the presence of glucose or galactose as the sole carbon source. On plates, colonies of bacteria grown on galactose turned bright yellow within a few minutes of being sprayed with a solution of catechol, whereas colonies on glucose-containing plates remained white or only slightly colored, even after extensive incubation. Activity of galP1-xylE fusions was conveniently measured in crude cell extracts with a simple colorimetric assay and was shown to faithfully reflect intracellular RNA levels, as determined by quantitative dot blots. Moreover, differences in expression levels of xylE fusions driven by mutant galP1 promoters were readily apparent in color reactions on plates. The properties of xylE as a reporter gene thus make it suitable not only for quantitatively monitoring expression of regulated promoters in Streptomyces spp. but also for recovering mutations that alter the expression levels of promoters of interest.
A cryptic fimbrial gene in Serratia marcescens
Authors: Moriya T , Kawabata S , Mizunoe Y , Amako K
Published: November 2016

The gene coding for the mannose-sensitive hemagglutinating fimbriae in Serratia marcescens US5 was cloned into Escherichia coli K4 with a cosmid vector system. One of the transformants, US5-1, expressed two morphologically distinct fimbriae, one that was 5-nm wide and one that was 3-nm wide. The latter fimbria was morphologically and serologically indistinguishable from that of strain US5. Genetic analysis of transformant US5-1 showed that the gene responsible for the 5-nm-wide fimbriae was located more than 10 kilobases away from the gene responsible for the 3-nm-wide fimbriae. The molecular sizes of the subunits of these two fimbriae, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 19 kilodaltons for the 3-nm-wide fimbriae and 20 kilodaltons for the 5-nm-wide fimbriae. Serologically, the 5-nm-wide fimbriae did not cross-react with monoclonal or polyclonal antibodies raised against the mannose-sensitive hemagglutinating fimbriae of strain US5. Strain EL101, which expressed only the 5-nm-wide fimbriae, did not agglutinate chicken or human erythrocytes. These experimental results suggest that the gene for the 5-nm-wide fimbriae is cryptic in strain US5 and is expressed in E. coli K4 only after it is moved by transformation.
Phenotypic characterization of the archaebacterial genus Sulfolobus: comparison of five wild-type strains
Authors: Grogan D W
Published: November 2016

Though amenable to routine manipulation and a popular subject of molecular genetic and biochemical studies on archaebacteria, the genus Sulfolobus has remained poorly described in phenotypic terms. To delineate their physiological capabilities and diversity, five laboratory strains, including type strains of the described species Sulfolobus acidocaldarius and S. solfataricus, were compared with respect to a variety of growth and biochemical parameters, including component profile of the surface-layer cell wall, inhibitors of growth, growth rate as a function of temperature and pH, and compounds used as sole sources of carbon or nitrogen. Motility and photoregulated production of an orange pigment were detected in all five strains tested. The results provide new criteria for distinguishing Sulfolobus strains as well as potential tools for the physiological and genetic manipulation of these extreme thermophiles.
Mutations affecting regulation of cobinamide biosynthesis in Salmonella typhimurium
Authors: Andersson D I , Roth J R
Published: November 2016

Transcription of the genes for cobalamin biosynthesis is reduced during aerobic growth. We isolated and characterized mutants that showed a 2- to 90-fold increase in aerobic expression of the cobinamide biosynthesis (CobI) genes, depending on the particular mutation and growth conditions. Four different classes of mutations were characterized. All mutations (CobRI through CobRIV) were cis-acting, dominant mutations that mapped near the promoter end of the CobI operon. Two of these classes of mutations (III and IV) caused an increase in anaerobic as well as aerobic transcription of the CobII and CobIII operons; this led to increased biosynthesis of cobalamin under anaerobic growth conditions. A recessive mutation (cobF) mapping far from the CobI operon increased anaerobic CobI operon expression by about fourfold.
Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein
Authors: Mulbry W W , Karns J S
Published: November 2016

The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.
Homology between hydroxybutyryl and hydroxyacyl coenzyme A dehydrogenase enzymes from Clostridium acetobutylicum fermentation and vertebrate fatty acid beta-oxidation pathways
Authors: Youngleson J S , Jones D T , Woods D R
Published: November 2016

The enzymes NAD-dependent beta-hydroxybutyryl coenzyme A dehydrogenase (BHBD) and 3-hydroxyacetyl coenzyme A (3-hydroxyacyl-CoA) dehydrogenase are part of the central fermentation pathways for butyrate and butanol production in the gram-positive anaerobic bacterium Clostridium acetobutylicum and for the beta oxidation of fatty acids in eucaryotes, respectively. The C. acetobutylicum hbd gene encoding a bacterial BHBD was cloned, expressed, and sequenced in Escherichia coli. The deduced primary amino acid sequence of the C. acetobutylicum BHBD showed 45.9% similarity with the equivalent mitochondrial fatty acid beta-oxidation enzyme and 38.4% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of the bifunctional enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase from rat peroxisomes. The pig mitochondrial 3-hydroxyacyl-CoA dehydrogenase showed 31.7% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of the bifunctional enzyme from rat peroxisomes. The phylogenetic relationship between these enzymes supports a common evolutionary origin for the fatty acid beta-oxidation pathways of vertebrate mitochondria and peroxisomes and the bacterial fermentation pathway.
Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity
Authors: Hanna P C , Wnek A P , McClane B A
Published: November 2016

Clostridium perfringens type A enterotoxin (CPE) causes the symptoms associated with C. perfringens food poisoning. To determine whether the C-terminal half of CPE contains receptor-binding activity, the 3' half of the cpe structural gene was cloned with an Escherichia coli expression vector system. E. coli lysates containing the expressed C-terminal CPE fragment (CPEfrag) were then assayed for CPE-like serologic, receptor-binding, and cytotoxic activities. CPEfrag was shown to contain an epitope located at or near the receptor-binding domain of the CPE molecule. Competitive-binding studies showed specific competition for CPE receptors between CPE and CPEfrag lysates. CPEfrag lysates did not cause cytotoxicity in Vero (African green monkey kidney) cells. However, preincubation with CPEfrag lysates specifically protected Vero cells from subsequent CPE challenge. This indicates that CPEfrag recognizes the physiologic receptor which mediates CPE cytotoxicity. Collectively, these studies indicate that the C-terminal half of CPE contains a receptor-binding domain but additional amino acid sequences appear to be required for CPE cytotoxicity.
Cloning and expression of a Bacillus subtilis division initiation gene for which a homolog has not been identified in another organism
Authors: Harry E J , Wake R G
Published: November 2016

The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector. The two genes were separated by approximately 3 kilobases. The gene in which the ts-1 mutation resides was shown to be the same as the B. subtilis homolog of the Escherichia coli ftsZ gene. The other gene was named divIB. It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound.
The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer
Authors: Toro N , Datta A , Carmi O A , Young C , Prusti R K , Nester E W
Published: November 2016

In Agrobacterium tumefaciens, a cis-active 24-base-pair sequence adjacent to the right border of the T-DNA, called overdrive, stimulates tumor formation by increasing the level of T-DNA processing. Recent results from our laboratory have suggested that the virC operon which enhances T-DNA processing probably does so because the VirC1 protein interacts with overdrive (N. Toro, A. Datta, M. Yanofsky, and E. W. Nester, Proc. Natl. Acad. Sci. USA 85:8558-8562, 1988). We report here the purification of the VirC1 protein from cells of Escherichia coli harboring a plasmid containing the coding sequences of the virC locus of the octopine Ti plasmid. By gel mobility shift and DNase I footprinting assays, we showed that this purified virC1 gene product binds to overdrive but not to the right border of T-DNA.
Localization of mannoprotein in Cryptococcus neoformans
Authors: Vartivarian S E , Reyes G H , Jacobson E S , James P G , Cherniak R , Mumaw V R , Tingler M J
Published: November 2016

Cell wall mannoprotein of nonpathogenic yeasts is surface exposed, since the cells are agglutinated by concanavalin A and antimannoprotein antibodies. However, nonencapsulated cells of Cryptococcus neoformans were agglutinated neither by concanavalin A nor by antimannoprotein antibodies. Immunogold electron microscopy located most mannoprotein in the inner cell wall. Chemical analysis of purified cell walls showed the lack of mannose, xylose, and galactose residues. These data indicate that cryptococcal mannoprotein recovered from the cultural supernatant is a nonstructural element of the cell wall.
Overproduction of truncated subunit a of H+-ATPase causes growth inhibition of Escherichia coli
Authors: Eya S , Maeda M , Tomochika K , Kanemasa Y , Futai M
Published: November 2016

Genes (uncB) for wild-type and mutant a subunits of Escherichia coli H+-ATPase (F0F1) were cloned into recombinant plasmids. The subunits were expressed under the control of a weak promoter of the unc operon at 30 degrees C and strong promoters of lambda phage at 42 degrees C. At 30 degrees C, the wild type and a truncated (Glu-269----end) a subunit complemented the defect of the a subunit mutant KF24A (Trp-111----end), whereas the other mutant subunits (Trp-111----end, Trp-231----end, Gln-252----end, and a subunit with a deletion of residues 21 to 227) did not. Three mutant subunits (Trp-231----end, Gln-252----end, and Glu-269----end) and the wild-type a subunit caused growth inhibition associated with cell elongation, an uneven distribution of membrane proteins, and an altered septum structure when they were expressed at 42 degrees C. These phenomena were not observed with the other mutant subunits, suggesting that overproduction of the middle region (between residues 111 and 230) of the a subunit causes growth inhibition.
Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus
Authors: Yoh M , Honda T , Miwatani T , Tsunasawa S , Sakiyama F
Published: November 2016

Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.
Characterization in vitro of the defect in a temperature-sensitive mutant of the protein subunit of RNase P from Escherichia coli
Authors: Baer M F , Wesolowski D , Altman S
Published: November 2016

We have studied the assembly of Escherichia coli RNase P from its catalytic RNA subunit (M1 RNA) and its protein subunit (C5 protein). A mutant form of the protein subunit, C5A49, has been purified to apparent homogeneity from a strain of E. coli carrying a thermosensitive mutation in the rnpA gene. The heat inactivation kinetics of both wild-type and mutant holoenzymes are similar, an indication of equivalent thermal stability. However, when the catalytic efficiencies of the holoenzymes were compared, we found that the holoenzyme containing the mutant protein had a lower efficiency of cleavage than the wild-type holoenzyme at 33, 37, and 44 degrees C. We then explored the interaction of M1 RNA and C5 protein during the assembly of the holoenzyme. The yield of active holoenzyme obtained by reconstitution with wild-type M1 RNA and C5A49 protein in vitro can be considerably enhanced by the addition of excess M1 RNA, just as it can be in vivo. We concluded that the Arg-46----His-46 mutation in the C5A49 protein affects the ability of the protein to participate with M1 RNA in the normal assembly process of RNase P.
Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli
Authors: Asai Y , Katayose Y , Hikita C , Ohta A , Shibuya I
Published: November 2016

The Escherichia coli pgsA3 allele encoding a defective phosphatidylglycerophosphate synthase is lethal for all but certain strains. Genetic analysis of such strains has revealed that the lethal effect is fully suppressed by the lack of the major outer membrane lipoprotein that consumes phosphatidylglycerol for its maturation.
Developmental potential of Frankia vesicles
Authors: Schultz N A , Benson D R
Published: November 2016

The ability of nitrogenase-containing Frankia sp. strain CpI1 vesicles to regrow vegetative hyphae is demonstrated. Vesicles attached to hyphae in N2-fixing CpI1 cultures and sucrose gradient-isolated vesicles exhibited hyphal outgrowths when incubated in certain defined liquid media. Single or multiple hyphal extensions grew out from the vesicles.
Pyrogallol-to-phloroglucinol conversion and other hydroxyl-transfer reactions catalyzed by cell extracts of Pelobacter acidigallici
Authors: Brune A , Schink B
Published: November 2016

Permeabilized cells and cell extracts of Pelobacter acidigallici catalyzed the conversion of pyrogallol (1,2,3-trihydroxybenzene) to phloroglucinol (1,3,5-trihydroxybenzene) in the presence of 1,2,3,5-tetrahydroxybenzene. Pyrogallol consumption by resting cells stopped after lysis by French press or mild detergent (cetyltrimethylammonium bromide [CTAB]) treatment. Addition of 1,2,3,5-tetrahydroxybenzene to the assay mixture restored pyrogallol consumption and led to stoichiometric phloroglucinol accumulation. The stoichiometry of pyrogallol conversion to phloroglucinol was independent of the amount of tetrahydroxybenzene added. The tetrahydroxybenzene concentration limited the velocity of the transhydroxylation reaction, which reached a maximum at 1.5 mM tetrahydroxybenzene (1 U/mg of protein). Transhydroxylation was shown to be reversible. The equilibrium constant of the reaction was determined, and the free-energy change (delta G degree') of phloroglucinol formation from pyrogallol was calculated to be -15.5 kJ/mol. Permeabilized cells and cell extracts also catalyzed the transfer of hydroxyl moieties between other hydroxylated benzenes. Tetrahydroxybenzene and hydroxyhydroquinone participated as hydroxyl donors and as hydroxyl acceptors in the reaction, whereas pyrogallol, resorcinol, and phloroglucinol were hydroxylated by both donors. A novel mechanism deduced from these data involves intermolecular transfer of the hydroxyl moiety from the cosubstrate (1,2,3,5-tetrahydroxybenzene) to the substrate (pyrogallol), thus forming the product (phloroglucinol) and regenerating the cosubstrate.
Determination of the packaging capacity of bacteriophage VWB
Authors: Anné J , Van Mellaert L , Eyssen H
Published: November 2016

VWB is a temperate bacteriophage whose chromosome has cohesive ends. VWB can stably package modified chromosomes that contain insertions of up to about 4 kilobases of foreign DNA. Phage particles containing extra DNA differ from the wild type in their increased sensitivity to chelating agents. Because of these properties, VWB is a promising cloning vector for Streptomyces venezuelae.
Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Authors: Kido N , Ohta M , Kato N
Published: November 2016

A rapid and easy method for staining lipopolysaccharides with ethidium bromide is described. Lipopolysaccharides could be visualized by ethidium bromide with almost the same sensitivity as found with the silver-staining method in less than 30 min. The ethidium bromide-staining method was particularly suitable for staining lipopolysaccharides possessing acidic O-specific polysaccharides, which were poorly visualized by silver staining.
Methanogenesis involving a novel carrier of C1 compounds in Methanogenium tationis
Authors: Raemakers-Franken P C , Kortstee A J , van der Drift C , Vogels G D
Published: November 2016

The pathway of CO2 reduction to methane in Methanogenium tationis and Methanogenium thermophilicum is similar to that observed in other methanogens. In M. tationis a novel pterin, tatiopterin, is present. This pterin appears to be a structural and functional analog of methanopterin and sarcinapterin. Folate could not substitute for tatiopterin.
Gene replacement and expression of foreign DNA in mycobacteria
Authors: Husson R N , James B E , Young R A
Published: November 2016

A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms.
Expression of Rhizobium leguminosarum CFN42 genes for lipopolysaccharide in strains derived from different R. leguminosarum soil isolates
Authors: Brink B A , Miller J , Carlson R W , Noel K D
Published: November 2016

Two mutant derivatives of Rhizobium leguminosarum ANU843 defective in lipopolysaccharide (LPS) were isolated. The LPS of both mutants lacked O antigen and some sugar residues of the LPS core oligosaccharides. Genetic regions previously cloned from another Rhizobium leguminosarum wild-type isolate, strain CFN42, were used to complement these mutants. One mutant was complemented to give LPS that was apparently identical to the LPS of strain ANU843 in antigenicity, electrophoretic mobility, and sugar composition. The other mutant was complemented by a second CFN42 lps genetic region. In this case the resulting LPS contained O-antigen sugars characteristic of donor strain CFN42 and reacted weakly with antiserum against CFN42 cells, but did not react detectably with antiserum against ANU843 cells. Therefore, one of the CFN42 lps genetic regions specifies a function that is conserved between the two R. leguminosarum wild-type isolates, whereas the other region, at least in part, specifies a strain-specific LPS structure. Transfer of these two genetic regions into wild-type strains derived from R. leguminosarum ANU843 and 128C53 gave results consistent with this conclusion. The mutants derived from strain ANU843 elicited incompletely developed clover nodules that exhibited low bacterial populations and very low nitrogenase activity. Both mutants elicited normally developed, nitrogen-fixing clover nodules when they carried CFN42 lps DNA that permitted synthesis of O-antigen-containing LPS, regardless of whether the O antigen was the one originally made by strain ANU843.
A family of genes encode the multiple forms of the Saccharomyces cerevisiae ribosomal proteins equivalent to the Escherichia coli L12 protein and a single form of the L10-equivalent ribosomal protein
Authors: Newton C H , Shimmin L C , Yee J , Dennis P P
Published: November 2016

The budding yeast Saccharomyces cerevisiae contains a family of genes that encodes four different but related small acidic ribosomal proteins designated L12eIA, L12eIB, L12eIIA, and L12eIIB and a single larger protein designated L10e. These proteins are equivalent (e) to the L12 and L10 proteins of Escherichia coli that assemble as a 4:1 complex onto the large ribosomal subunit. The five yeast genes (or their cDNAs) have been cloned and sequenced (M. Remacha, M. T. Saenz-Robles, M. D. Vilella, and J. P. G. Ballesta, J. Biol. Chem. 263:9044-9101, 1988; K. Mitsui and K. Tsurugi, Nucleic Acids Res. 16:3573, 3574, and 3575, 1988; this work). Here, the transcripts of these genes were characterized and quantitated and the proteins they encode were compared and aligned. Four of the genes, L12eIA, -IB, -IIA, and L10e, are uninterrupted, whereas the L12eIIB gene contains a 301-nucleotide-long intron between codons 38 and 39. The transcripts derived from each of these genes were analyzed by Northern (RNA) hybridization, primer extension, and S1 nuclease protection. All five genes are expressed, albeit at different levels. The transcript levels are coordinate and exhibit growth rate-dependent regulation in rich (glucose) and poor (ethanol) media. The five yeast proteins each contain a highly conserved acidic carboxy terminus of about 20 residues in length. This domain of unknown function is also present in archaebacterial but absent from eubacterial L10e and L12e proteins. Comparisons of the factor-binding domains in the yeast and other eucaryotic and archaebacterial L12e proteins indicate that the original duplication to produce the type I and II genes was a very ancient event. The evolutionary relationships between the eucaryotic, archaebacterial, and eubacterial L10e and L12e genes (and proteins) are discussed.
Expression of the Pseudomonas aeruginosa toxA positive regulatory gene (regA) in Escherichia coli
Authors: Hamood A N , Iglewski B H
Published: November 2016

The regA gene is a positive regulatory gene that regulates toxin A production in Pseudomonas aeruginosa at the transcriptional level. The product of the regA gene was examined in Escherichia coli with the expression vector pT7-7. A 1.3-kilobase AvaI-HindIII fragment containing the regA gene was cloned into the pT7-7 vector. A recombinant plasmid (pAH1) encoded a 29-kilodalton protein. The molecular weight of this protein correlated closely with the predicted molecular weight of the RegA protein. Production of the RegA protein in E. coli required both an E. coli promoter and an E. coli ribosome-binding site. Two in-frame deletion derivatives in which certain regions of the regA gene were expressed from the T7 promoter encoded 26- and 18-kilodalton fusion proteins, respectively. The RegA protein and the two fusion proteins were localized to the inner membrane of E. coli. Neither RegA protein nor the two fusion proteins showed DNA-binding activity to the 410-base-pair fragment containing the upstream region of toxA when synthesized in E. coli.
A Rhizobium leguminosarum mutant defective in symbiotic iron acquisition
Authors: Nadler K D , Johnston A W , Chen J W , John T R
Published: November 2016

Iron acquisition by symbiotic Rhizobium spp. is essential for nitrogen fixation in the legume root nodule symbiosis. Rhizobium leguminosarum 116, an ineffective mutant strain with a defect in iron acquisition, was isolated after nitrosoguanidine mutagenesis of the effective strain 1062. The pop-1 mutation in strain 116 imparted to it a complex phenotype, characteristic of iron deficiency: the accumulation of porphyrins (precursors of hemes) so that colonies emitted a characteristic pinkish-red fluorescence when excited by UV light, reduced levels of cytochromes b and c, and wild-type growth on high-iron media but low or no growth in low-iron broth and on solid media supplemented with the iron scavenger dipyridyl. Several iron(III)-solubilizing agents, such as citrate, hydroxyquinoline, and dihydroxybenzoate, stimulated growth of 116 on low-iron solid medium; anthranilic acid, the R. leguminosarum siderophore, inhibited low-iron growth of 116. The initial rate of 55Fe uptake by suspensions of iron-starved 116 cells was 10-fold less than that of iron-starved wild-type cells. Electron microscopic observations revealed no morphological abnormalities in the small, white nodules induced by 116. Nodule cortical cells were filled with vesicles containing apparently normal bacteroids. No premature degeneration of bacteroids or of plant cell organelles was evident. We mapped pop-1 by R plasmid-mediated conjugation and recombination to the ade-27-rib-2 region of the R. leguminosarum chromosome. No segregation of pop-1 and the symbiotic defect was observed among the recombinants from these crosses. Cosmid pKN1, a pLAFR1 derivative containing a 24-kilobase-pair fragment of R. leguminosarum DNA, conferred on 116 the ability to grow on dipyridyl medium and to fix nitrogen symbiotically. These results indicate that the insert cloned in pKN1 encodes an element of the iron acquisition system of R. leguminosarum that is essential for symbiotic nitrogen fixation.
Transcriptional analysis of the flagellar regulon of Salmonella typhimurium
Authors: Kutsukake K , Ohya Y , Iino T
Published: November 2016

In Salmonella typhimurium, nearly 50 genes are involved in flagellar formation and function and constitute at least 13 different operons. In this study, we examined the transcriptional interaction among the flagellar operons by combined use of Mu d1(Apr Lac) cts62 and Tn10 insertion mutants in the flagellar genes. The results showed that the flagellar operons can be divided into three classes: class I contains only the flhD operon, which is controlled by the cAMP-CAP complex and is required for expression of all of the other flagellar operons; class II contains seven operons, flgA, flgB, flhB, fliA, fliE, fliF, and fliL, which are under control of class I and are required for the expression of class III; class III contains five operons, flgK, fliD fliC, motA, and tar. This ordered cascade of transcription closely parallels the assembly of the flagellar structure. In addition, we found that the fliD defect enhanced expression of the class III operons. This suggests that the fliD gene product may be responsible for repression of the class III operons in the mutants in the class II genes. These results are compared with the cascade model of the flagellar regulon of Escherichia coli proposed previously (Y. Komeda, J. Bacteriol. 170:1575-1581, 1982).
Modification of dinitrogenase reductase in the cyanobacterium Anabaena variabilis due to C starvation and ammonia
Authors: Ernst A , Reich S , Böger P
Published: November 2016

In the heterocystous cyanobacterium Anabaena variabilis, a change in nitrogenase activity and concomitant modification of dinitrogenase reductase (the Fe protein of nitrogenase) was induced either by NH4Cl at pH 10 (S. Reich and P. Böger, FEMS Microbiol. Lett. 58:81-86, 1989) or by cessation of C supply resulting from darkness, CO2 limitation, or inhibition of photosystem II activity. Modification induced by both C limitation and NH4Cl was efficiently prevented by anaerobic conditions. Under air, endogenously stored glycogen and added fructose protected against modification triggered by C limitation but not by NH4Cl. With stored glycogen present, dark modification took place after inhibition of respiration by KCN. Reactivation of inactivated nitrogenase and concomitant demodification of dinitrogenase reductase occurred after restoration of diazotrophic growth conditions. In previously C-limited cultures, reactivation was also observed in the dark after addition of fructose (heterotrophic growth) and under anaerobiosis upon reillumination in the presence of a photosynthesis inhibitor. The results indicate that modification of dinitrogenase reductase develops as a result of decreased carbohydrate-supported reductant supply of the heterocysts caused by C limitation or by increased diversion of carbohydrates towards ammonia assimilation. Apparently, a product of N assimilation such as glutamine is not necessary for modification. The increase of oxygen concentration in the heterocysts is a plausible consequence of all treatments causing Fe protein modification.
Nucleotide sequence and transcriptional analysis of the Escherichia coli agp gene encoding periplasmic acid glucose-1-phosphatase
Authors: Pradel E , Marck C , Boquet P L
Published: November 2016

The nucleotide sequence of the agp gene, which encodes a periplasmic glucose-1-phosphatase, was determined. The deduced amino acid sequence corresponds to a 413-amino-acid-residue polypeptide with a typical hydrophobic signal sequence of 22 amino acids. The mature protein lacks the N-terminal signal peptide and has a calculated Mr of 43,514. Its promoter was defined by primer extension of the mRNA made in vivo. Like many genes under positive control, its -35 promoter region does not match the consensus. The agp gene is both preceded and followed by transcription termination signals, so it appears to be transcribed as a single unit.
Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC
Authors: Essar D W , Eberly L , Crawford I P
Published: November 2016

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.
Oligomerization of the bacteriophage lambda S protein in the inner membrane of Escherichia coli
Authors: Zagotta M T , Wilson D B
Published: November 2016

Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S--beta-galactosidase fusion protein) demonstrated that the bacteriophage lambda S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle. Between 100 and 1,000 molecules of S protein per cell were present at the time of phage-induced lysis. Western blots of chemically cross-linked membranes from induced lysogens showed a ladder of bands at 18, 24, 32, and 42 kilodaltons (the S-protein monomer ran at 8 kilodaltons) that reacted with anti-S-protein antibody. Thus, the S protein appears to reside in the inner membrane as a multimer, and the molecular weights of the cross-linked species are consistent with those of S-protein homopolymers. Sodium dodecyl sulfate-resistant dimers were also detected when S protein was purified by immunoprecipitation.
Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli
Authors: Guzzo J , Murgier M , Filloux A , Lazdunski A
Published: November 2016

Pseudomonas virulence is thought to depend on multiple characteristics, including the production of an extracellular alkaline protease. We report the isolation, from a PAO1 DNA genomic bank, of a cosmid carrying the structural gene coding for alkaline protease. By in vivo mutagenesis using transposon Tn1735, which functions as a transposable promoter, the expression of an 8.8-kilobase DNA fragment under control the tac promoter was obtained. When expressed in Escherichia coli, active alkaline protease was synthesized and secreted to the extracellular medium in the absence of cell lysis.
Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia
Authors: Wood M S , Lory C , Lessie T G
Published: November 2016

We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRP1::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-beta-D-thiogalactopyranoside. Lac+ variants of the pGC91.14-containing strains which formed beta-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.
Effect of altering GATC sequences in the plasmid ColE1 primer promoter
Authors: Patnaik P K , Merlin S , Polisky B
Published: November 2016

Plasmid ColE1 has three recognition sites for the Escherichia coli DNA adenine methylase in the immediate upstream region of the primer promoter. Two of these sites are conserved among all plasmid relatives of ColE1 and constitute parts of an inverted repeat that can conceivably form a cruciform structure. Recent experiments have indicated that hemimethylated ColE1-type plasmids are inefficiently replicated after transformation (D. W. Russell and N. Zinder, Cell 50:1071-1079, 1987). By mutating the three methylation sites, we found that disruption of all three GATC sites was necessary for complete relief of the hemimethylation-mediated inhibition of replication in vivo. We also found that these three methylation sites acted in a position-specific manner. The putative cruciform, if present, did not play a regulatory role in the hemimethylation-mediated inhibition of replication.
A novel Bacillus subtilis gene involved in negative control of sporulation and degradative-enzyme production
Authors: Honjo M , Nakayama A , Fukazawa K , Kawamura K , Ando K , Hori M , Furutani Y
Published: November 2016

We have cloned a 2.5-kilobase fragment of the Bacillus subtilis genomic DNA which caused the reduction of extracellular and cell-associated protease levels when present in high copy number. This fragment, in multicopy, was also responsible for reduced levels of alpha-amylase, levansucrase, alkaline phosphatase, and sporulation inhibition. The gene relevant to this pleiotropic phenotype is referred to as pai. By DNA sequencing, two open reading frames--ORF1 and ORF2, encoding polypeptides of 172 and 207 amino acid residues, respectively--were found. These open reading frames seemed to form an operon. Deletion analysis revealed that an entire region for ORF1 and ORF2 was necessary for the pai phenotype. In addition, it was observed that the presence of the pai gene, in multicopy, caused overproduction of two proteins (molecular masses, 21 and 24 kilodaltons [kDa]). Analyses of the N-terminal amino acid sequences of these two proteins suggested that they were products of ORF1 and ORF2. Disruption of the pai gene at ORF1 in the genomic DNA resulted in the release of repression on protease synthesis and sporulation in glucose-enriched (2%) medium. The mutant carrying insertional disruption at ORF2 could not be constructed, suggesting that the ORF2 product, the 24-kDa protein, is essential for growth. The 21-kDa protein contains a helix-turn-helix domain observed in other DNA-binding proteins. Chromosomal mapping of pai indicated that this gene is located close to thr-5. These results suggest that the pai gene is a novel transcriptional-regulation gene involved in glucose repression.
Identification of the mcrD gene product and its association with component C of methyl coenzyme M reductase in Methanococcus vannielii
Authors: Sherf B A , Reeve J N
Published: November 2016

A mcrD-lacZ gene fusion has been constructed and expressed under lacP control in Escherichia coli. Antibodies raised against the product of this gene fusion have been used in Western blotting (immunoblotting) to demonstrate the gene product of mcrD (gpmcrD) in Methanococcus vannielii. The alpha, beta, and gamma subunit polypeptides of component C of methyl coenzyme M reductase (MR) were coprecipitated with gpmcrD when bound by antibodies raised either against MR or against gpmcrD-lacZ. This association of MR and gpmcrD did not withstand polyacrylamide gel electrophoresis under nondenaturing conditions.