bluuebunny/crossref_metadata_2025 huggingface dataset:
Translational attenuation control of ermSF, an inducible resistance determinant encoding rRNA N-methyltransferase from Streptomyces fradiae
Authors: Kamimiya S , Weisblum B
Published: November 2016

An inducible resistance determinant, ermSF, from the tylosin producer Streptomyces fradiae NRRL 2338 has been cloned, sequenced, and shown to confer inducible macrolide-lincosamide-streptogramin B resistance when transferred to Streptomyces griseofuscus NRRL 23916. From mapping studies with S1 nuclease to locate the site of transcription initiation, the ermSF message contains a 385-nucleotide 5' leader sequence upstream from the 960-nucleotide major open reading frame that encodes the resistance determinant. On the basis of the potential secondary structure that the ermSF leader can assume, a translational attenuation model similar to that for ermC is proposed. The model is supported by mutational analysis involving deletions in the proposed attenuator. By analysis with restriction endonucleases, ermSF is indistinguishable from the tlrA gene described by Birmingham et al. (V. A. Birmingham, K. L. Cox, J. L. Larson, S. E. Fishman, C. L. Hershberger, and E. T. Seno, Mol. Gen. Genet. 204:532-539, 1986) which comprises one of at least three genes from S. fradiae that can confer tylosin resistance when subcloned into S. griseofuscus. When tested for inducibility, ermSF appears to be strongly induced by erythromycin, but not by tylosin.
Roles of bacteriochlorophyll and carotenoid synthesis in formation of intracytoplasmic membrane systems and pigment-protein complexes in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114
Authors: Iba K , Takamiya K , Toh Y , Nishimura M
Published: November 2016

Synthesis of bacteriochlorophyll and carotenoids was inhibited in an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114, by alpha, alpha'-dipyridyl and diphenylamine. Formation of two pigment-protein complexes, reaction center-B870 (RC-B870) and B806, and development of the intracytoplasmic membranes of the cells were studied by spectral analysis and electron microscopy. Inhibition of bacteriochlorophyll synthesis by alpha, alpha'-dipyridyl, which was accompanied by a decrease in carotenoid synthesis, suppressed formation of intracytoplasmic membranes in the cells. Growth under illumination had a similar effect on formation of pigments and membranes. On the other hand, inhibition of carotenoid synthesis by diphenylamine did not suppress either development of the membrane system or bacteriochlorophyll synthesis. Formation of RC-B870 and B806 complexes, however, was differentially affected by blockage of carotenoid synthesis. In the presence of diphenylamine, the B806 complex was formed in a much smaller amount than the RC-B870 complex. These results suggest that, in Erythrobacter sp. strain OCh114, bacteriochlorophyll plays an essential role in intracytoplasmic membrane development, and carotenoids are important for assembly of pigment-protein complexes.
Genetic analysis of the iron uptake region of the Vibrio anguillarum plasmid pJM1: molecular cloning of genetic determinants encoding a novel trans activator of siderophore biosynthesis
Authors: Tolmasky M E , Actis L A , Crosa J H
Published: November 2016

Clones carrying the iron uptake region of the Vibrio anguillarum plasmid pJM1 were subjected to insertion mutagenesis, using transposon Tn3::HoHo1 which carries a promoterless lacZ gene and can thus generate lacZ transcriptional fusions if inserted downstream from an indigenous promoter. Four classes of insertion mutants were obtained based on the level of expression of components of the iron uptake system, and six genetic units were defined according to the phenotype of the mutants. Five of the six genetic units were crucial for biosynthesis of the siderophore anguibactin. Insertions in the remaining genetic unit led to an iron uptake-deficient phenotype and showed either reduced levels of the outer membrane protein OM2 as well as anguibactin activity or a complet shutoff of both OM2 and anguibactin biosyntheses. Analysis of beta-galactosidase production by cells carrying the lacZ fusion derivatives identified iron-regulated and constitutive transcriptional units as well as their orientation in the genetic units. Molecular cloning of pJM1 plasmid DNA noncontiguous to the iron uptake region also identified genetic determinants for a trans-acting factor required for full expression of anguibactin activity. Evidence obtained from bioassays, spectrophotometric measurements, and the lacZ fusion mutants suggested that the trans-acting factor is a novel activator of siderophore biosynthesis at the transcriptional level.
Phosphoglycerate kinase gene from Zymomonas mobilis: cloning, sequencing, and localization within the gap operon
Authors: Conway T , Ingram L O
Published: November 2016

The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids, including the N-terminal methionine, with a molecular weight of 41,384. This promoterless gene is located 225 base pairs downstream from the gap gene and is part of the gap operon. Previous studies have shown that the specific activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase do not change coordinately in Z. mobilis, although the two enzymes appear to be under the control of a common promoter. The translated amino acid sequence for the Z. mobilis phosphoglycerate kinase is less conserved than those of eucaryotic genes. A comparison of known sequences for phosphoglycerate kinase revealed a high degree of conservation of structure with 102 amino acid positions being retained by all. In general, the amino acid positions at the boundaries of beta-sheet and alpha-helical regions and those connecting these regions were more highly conserved than the amino acid positions within regions of secondary structure.
Expression and DNA sequence of the cloned bacteriophage T4 dCMP hydroxymethylase gene
Authors: Thylén C
Published: November 2016

A plasmid expressing the cloned bacteriophage T4 gene 42 gave the same levels of complementation of gene 42 mutants in a polarity-suppressing rho mutant as in a rho+ host. A reading frame likely corresponding to gene 42 and putative promoter and terminator sequences were identified in the partial sequence of the cloned fragment.
Association of the virD2 protein with the 5' end of T strands in Agrobacterium tumefaciens
Authors: Young C , Nester E W
Published: November 2016

The soil bacterium Agrobacterium tumefaciens can incite tumors in many dicotyledonous plants by transferring a portion (T-DNA) of its Ti plasmid into susceptible plant cells. The T-DNA is flanked by border sequences that serve as recognition sites for specific cleavage by an endonuclease that comprises two virD-encoded proteins (VirD1 and VirD2). After cleavage, both double-stranded, nicked T-DNA molecules and single-stranded T-DNA molecules (T strands) were present. We have determined that a protein is tightly associated with, and probably covalently attached to, the 5' end of the T strands. Analysis of deletion derivatives in Escherichia coli, immunoprecipitation, and a procedure combining immunoblot and nucleic acid hybridization data identified this protein as the gene product of virD2.
Effect of molybdenum and tungsten on synthesis and composition of formate dehydrogenase in Methanobacterium formicicum
Authors: May H D , Patel P S , Ferry J G
Published: November 2016

The influence of sodium molybdate and sodium tungstate on formate dehydrogenase activity was studied in H2-CO2-grown cultures of Methanobacterium formicicum. Depletion of molybdate from the growth medium resulted in a 75-fold decrease of intracellular molybdenum and a 35-fold decrease in enzyme activity; however, growth rate and cell yields were not influenced. By using an indirect enzyme-linked immunoassay, the amount of formate dehydrogenase approximated 3% of the total protein in cells grown in the presence of molybdate. Molybdenum-starved cells contained approximately 15-fold less formate dehydrogenase protein; Western blot (immunoblot) analysis revealed that both subunits of the enzyme were synthesized. Molybdenum starvation resulted in an increase in the amount of mRNA that hybridized to fdh-specific DNA. The results indicated an inverse relationship between the amount of transcript and the amount of formate dehydrogenase protein detected in response to molybdenum starvation. The addition of 1 mM tungstate to molybdate-containing media resulted in nearly complete loss of enzyme activity and decreased the intracellular concentration of molybdenum 10-fold. Cells grown in the presence of tungstate synthesized high amounts of inactive formate dehydrogenase and contained mRNA that hybridized to fdh-specific DNA in amounts similar to that in cells grown with sufficient molybdate. Inactive formate dehydrogenase, purified from cells grown in the presence of tungstate, had the same subunit composition and contained amounts of molybdopterin cofactor, albeit metal-free, comparable to those in the active enzyme.
Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120
Authors: McCarn D F , Whitaker R A , Alam J , Vrba J M , Curtis S E
Published: November 2016

A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli. The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATP synthase. This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E. coli. A unique feature of the Anabaena atp1 cluster is overlap between the coding regions for atpF and atpD. The atp1 cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1. The deduced translation products for the Anabaena sp. strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E. coli.
Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional sigma 32
Authors: Calendar R , Erickson J W , Halling C , Nolte A
Published: November 2016

Escherichia coli K-12 strain 285c contains a short deletion mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase. The sigma 70 protein encoded by this allele (rpoD285) unstable, and this instability leads to temperature-sensitive growth. Pseudorevertants of 285c that can grow at high temperature contain mutations in the rpoH gene (encoding the heat shock sigma factor sigma 32), and their mutant sigma 70 proteins have increased stability. We characterized the alterations in three of these rpoH alleles. rpoH111 was a point mutation resulting in a single amino acid substitution. rpoH107 and rpoH113, which are known to be incompatible with rpoD+, altered the restriction map of rpoH. rpoH113 was deleted for 72 base pairs of the rpoH gene yet retained some sigma 32 activity. rpoH107 had two IS1 elements that flanked an unknown DNA segment of more than 6.4 kilobases inserted in the rpoH promoter region. The insertion decreased the amount of rpoH mRNA to less than 0.5% of the wild-type level at 30 degrees C. However, the mRNA from several heat shock promoters was decreased only twofold, suggesting that the strain has a significant amount of sigma 32.
Differential regulation of synthesis of multiple forms of specific endoglucanases by Trichoderma reesei QM9414
Authors: Messner R , Gruber F , Kubicek C P
Published: November 2016

A method consisting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent detection of endoglucanases by blotting with a polyclonal antibody against endoglucanase I was used to investigate the effect of induction and carbon catabolite derepression on the synthesis of multiple forms of endoglucanase I by Trichoderma reesei. Five forms appeared upon growth on cellulose, whereas four and only two appeared upon growth on lactose (carbon catabolite derepression) and induction by sophorose in a resting cell system, respectively. All endoglucanases detected resembled endoglucanase I in their specificity, since they exhibited no activity toward xylan or paranitrophenyl-beta-D-lactobioside. A small (25-kilodalton) endoglucanase only appeared during growth on cellulose. None of the multiple forms arose by postsecretional modification. The results indicate that sophorose may not be the only compound mediating cellulose induction of the specific endoglucanases in T. reesei.
Transformation in Bacillus subtilis: involvement of the 17-kilodalton DNA-entry nuclease and the competence-specific 18-kilodalton protein
Authors: Vosman B , Kuiken G , Kooistra J , Venema G
Published: November 2016

A protein complex, consisting of a 17-kilodalton (kDa) nuclease and an 18-kDa protein, is believed to be involved in the binding and entry of donor DNA during transformation of Bacillus subtilis (H. Smith, K. Wiersman, S. Bron, and G. Venema, J. Bacteriol. 156:101-108, 1983). In this paper, the nucleotide sequences of the genes encoding both the nuclease and the 18-kDa protein are presented. The genes are encoded by a 904-base-pair PstI-HindIII fragment. The open reading frames encoding both proteins are partly overlapping. A B. subtilis mutant was constructed by insertion of a Cmr marker into the gene encoding the nuclease. This mutant lacked the competence-specific nuclease activity and the 18-kDa protein but retained 5% residual transformation. The total DNA association of the mutant was higher than that of the wild-type cells, and DNA entry was reduced to 30% of the wild-type level. These results suggest that an alternative pathway exists for the internalization of transforming DNA. A mutant, exclusively deficient for the 18-kDa protein, previously suggested to be involved in the binding of transforming DNA, was constructed by insertion of a kanamycin resistance gene into the coding sequence of the gene. Since the mutant showed wild-type DNA-binding activity, the 18-kDa protein is probably not involved in the binding of donor DNA to competent cells. The transforming activity of the mutant was reduced to 25% of the wild-type level, indicating that the 18-kDa protein has a function in the transformation process. In vitro experiments showed that the 18-kDa protein is capable of inhibiting the activity of the competence-specific nuclease. Its possible role in transformation is discussed.
Penicillin-binding proteins of bdellovibrios
Authors: Park J T , Mahadevan S
Published: November 2016

We examined the predacious gram-negative bacterium Bdellovibrio bacteriovorous 109J and free-living strains 109J-A1 and 109J-KA1 derived therefrom for penicillin-binding proteins (PBPs). We compared their PBPs with those of the host bacterium, Escherichia coli, and with those of a facultatively predacious bdellovibrio, B. stolpii UKi2, grown axenically. The multiple PBPs of the 109J strains and of UKi2 differed from each other and from those of E. coli, which suggests that screening for PBPs may be a convenient way to determine to what extent the bdellovibrios may represent a diverse group of organisms. A method for labeling furazlocillin and cefaperizone with iodine-125 is also described.
Klebsiella pneumoniae origin of replication (oriC) is not active in Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides
Authors: O'Neill E A , Bender R A
Published: November 2016

A DNA fragment carrying genes encoding the conjugal transfer system of the broad host range plasmid RK2 was inserted into a plasmid carrying the chromosomal origin of replication (oriC) from Klebsiella pneumoniae. The resulting plasmid, pEON1, was readily transferred between gram-negative bacteria and carried two potential origins of replication: oriC and the replication origin from pBR322 (oriPBR). Although pEON1 could be transferred to Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides, pEON1 was not maintained in these strains. However, an oriC-containing plasmid was maintained in these nonenteric bacteria when an RK2 origin of replication was present on the plasmid. Thus, the inability of pEON1 to be established in a nonenteric bacterium represents a failure of oriC to function as an origin of replication rather than a toxic effect of oriC. The initiation potential of the chromosomal origin of replication from K. pneumoniae appears to be realized only in enteric bacteria.
Germination of Saccharomyces cerevisiae ascospores without trehalose mobilization as revealed by in vivo 13C nuclear magnetic resonance spectroscopy
Authors: Donnini C , Puglisi P P , Vecli A , Marmiroli N
Published: November 2016

Saccharomyces cerevisiae ascospores germinate in the presence of acetate without any detectable trehalose degradation, as revealed by high-resolution nuclear magnetic resonance spectroscopy and by a standard colorimetric assay. The results presented here substantiate the hypothesis that in S. cerevisiae trehalose supplies energy during dormancy of the spores and not during the germination process.
Identification of a celE-binding protein and its potential role in induction of the celE gene in Thermomonospora fusca
Authors: Lin E S , Wilson D B
Published: November 2016

Thermomonospora fusca cellulase E5 is encoded by the celE gene. This gene appears to be regulated at the transcriptional level by both induction and repression, and three putative closely linked promoters have been located by S1 mapping. To study its regulatory mechanism, a gel retardation assay was used to identify a protein in T. fusca cell extracts that interacted specifically with the DNA fragment containing the celE promoters. It was found that the binding activity appeared only when cellulase synthesis was induced, and it therefore resembled an activator protein involved in cellulase induction. DNase I footprinting identified the target sequence for this protein as a 21-base-pair sequence downstream from the putative celE promoters. The level of this protein was measured in two cellulase constitutive mutants, and the results suggest a complex control for celE induction.
Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate
Authors: Chaudhry G R , Huang G H
Published: November 2016

A Flavobacterium sp. (strain 50001), capable of degrading 2,4-dichlorophenoxyacetate (2,4-D), 2-methyl-4-chlorophenoxyacetate, and 2-chlorobenzoate and imparting resistance to mercury, harbored a degradative plasmid, pRC10. Cured strains of the Flavobacterium sp. lost the plasmid as well as the ability to degrade these chlorinated compounds. Comparison of this plasmid with the well-characterized 2,4-D-degradative plasmid pJP4 from Alcaligenes eutrophus showed regions of homology between the two plasmids. Restriction fragments of plasmid pRC10 which shared homology with the regions conferring 2,4-D-degradative genes (tfd) of plasmid pJP4 were cloned into a broad-host-range plasmid and studied in Pseudomonas putida. From the results obtained, the cloned DNA fragment expressed the genes for 2,4-D monooxygenase (tfdA) and 2,4-dichlorophenol hydroxylase (tfdB). In spite of the similarity in function, the size (45 kilobases) and restriction pattern of plasmid pRC10 were considerably different from those of pJP4 (80 kilobases). This may be due to the difference in the microbial background during evolution of the two plasmids.
Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli
Authors: Jenkins D E , Schultz J E , Matin A
Published: November 2016

Glucose- or nitrogen-starved cultures of Escherichia coli exhibited enhanced resistance to heat (57 degrees C) or H2O2 (15 mM) challenge, compared with their exponentially growing counterparts. The degree of resistance increased with the time for which the cells were starved prior to the challenge, with 4 h of starvation providing the maximal protection. Protein synthesis during starvation was essential for these cross protections, since chloramphenicol addition at the onset of starvation prevented the development of thermal or oxidative resistance. Starved cultures also demonstrated stronger thermal and oxidative resistance than did growing cultures adapted to heat, H2O2, or ethanol prior to the heat or H2O2 challenge. Two-dimensional gel electrophoresis of 35S-pulse-labeled proteins showed that subsets of the 30 glucose starvation proteins were also synthesized during heat or H2O2 adaptation; three proteins were common to all three stresses. Most of the common proteins were among the previously identified Pex proteins (J.E. Schultz, G. I. Latter, and A. Matin, J. Bacteriol. 170:3903-3909, 1988), which are independent of cyclic AMP positive control for their induction during starvation. Induction of starvation proteins dependent on cyclic AMP was not important in these cross protections, since a delta cya strain of E. coli K-12 exhibited the same degree of resistance to heat or H2O2 as the wild-type parent did during both growth and starvation.
Construction of delta aroA his delta pur strains of Salmonella typhi
Authors: Edwards M F , Stocker B A
Published: November 2016

Salmonella typhi strains with two deletion mutations, each causing an attenuating auxotrophy, have been constructed from strains Ty2 and CDC 10-80 for possible use as oral-route live vaccines. An aroA(serC)::Tn10 transposon insertion was first transduced from a Salmonella typhimurium donor into each wild-type S. typhi strain. Transductants of the Aro- SerC- phenotype were treated with transducing phage grown on an S. typhimurium strain with an extensive deletion at aroA; selection for SerC+ yielded transductants, some of which were delta aroA. A his mutation was next inserted into a delta aroA strain in each line by two steps of transduction. Two deletions affecting de novo purine biosynthesis were used as second attenuating mutations: delta purHD343, causing a requirement for hypoxanthine (or any other purine) and thiamine, and delta purA155, causing an adenine requirement. The purHD343 deletion was introduced into the delta aroA his derivatives of each strain by cotransduction with purH::Tn10, and the purA155 deletion was introduced into the CDC 10-80 delta aroA his derivative by cotransduction with an adjacent silent Tn10 insertion by selection for tetracycline resistance. Tetracycline-sensitive mutants of each of the three delta aroA his delta pur strains were isolated by selection for resistance to fusaric acid. The tetracycline-sensitive derivative of the CDC 10-80 delta aroA his delta purA155 strain, designated 541Ty, and its Vi-negative mutant, 543Ty, constitute the candidate oral-route live-vaccine strains used in a recent volunteer trail (M. M. Levine, D. Herrington, J. R. Murphy, J. G. Morris, G. Losonsky, B. Tall, A. A. Lindberg, S. Stevenson, S. Baqar, M. F. Edwards, and B. A. D. Stocker, J. Clin. Invest. 79:885-902, 1987). Tetracycline-sensitive mutants of the delta aroA his delta purHD derivative of strains Ty2 and CDC 10-80 may also be appropriate as live vaccines but have not been tested as such.
Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export
Authors: Watanabe T , Hayashi S , Wu H C
Published: November 2016

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.
Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit
Authors: Dunlap P V , Greenberg E P
Published: November 2016

Expression of the Vibrio fischeri luminescence genes (lux genes) requires two transcriptional activators: the V. fischeri luxR gene product with autoinducer and the cyclic AMP (cAMP) receptor protein (CRP) with cAMP. It has been established that autoinducer and the luxR gene product are required for transcriptional activation of the luxICDABE operon, which contains a gene required for autoinducer synthesis and genes required for light emission. However, the role of cAMP-CRP in the induction of luminescence is not clear. We examined transcriptional control of the lux genes in Escherichia coli, using catabolite repression mutants carrying lux DNA-containing plasmids. Transcriptional fusions between the lacZ gene on Mu dI and luxR were used to assess luxR promoter activity, and the luxAB genes (which encode the two luciferase subunits) were used as a natural reporter of luxICDABE promoter activity. A plasmid containing luxR under control of the cAMP-CRP-independent tac promoter was constructed to direct the synthesis of the luxR gene product in cells containing compatible luxR::Mu dI insertion mutant plasmids. In E. coli, cAMP-CRP activated transcription of luxR and concurrently decreased luxICDABE transcription. In the presence of relatively high levels of the luxR gene product, cAMP and CRP were not required for induction of the luxICDABE operon. The luxR gene product in the presence of autoinducer activated transcription of the luxICDABE operon, as has been shown previously, and we demonstrate that it also decreased luxR transcription. Apparently, control of the V. fischeri luminescence genes involves a regulatory circuit in which cAMP and CRP activate luxR transcription and in turn the luxR gene product activates transcription of the operon responsible for light emission (uxICDABE). Furthermore, in lux gene regulation cAMP-CRP and autoinducer-LuxR protein appear to function as transcriptional antagonists.
Organization and temporal expression of a flagellar basal body gene in Caulobacter crescentus
Authors: Hahnenberger K M , Shapiro L
Published: November 2016

Caulobacter crescentus assembles a single polar flagellum at a defined time in the cell cycle. The protein components of the flagellar hook and filament are synthesized just prior to their assembly. We demonstrated that the expression of a gene, flaD, that is involved in the formation of the flagellar basal body is under temporal control and is transcribed relatively early in the cell cycle, before the hook and flagellin genes are transcribed. Thus, the order of flagellar gene transcription reflects the order of assembly of the protein components. A mutation in the flaD gene results in the assembly of a partial basal body which is missing the outermost P and L rings as well as the external hook and filament (K.M. Hahnenberger and L. Shapiro, J. Mol. Biol. 194:91-103, 1987). The flaD gene was cloned and characterized by nucleotide sequencing and S1 nuclease protection assays. In contrast to the protein components of the hook and filament, the protein encoded by the flaD gene contains a hydrophobic leader peptide. The predicted amino acid sequence of the leader peptide of flaD is very similar to the leader peptide of the flagellar basal body P ring of Salmonella typhimurium (M. Homma, Y. Komeda, T. Iino, and R.M. Macnab, J. Bacteriol. 169:1493-1498, 1987).
A hybrid toxin from bacteriophage f1 attachment protein and colicin E3 has altered cell receptor specificity
Authors: Jakes K S , Davis N G , Zinder N D
Published: November 2016

A hybrid protein was constructed in vitro which consists of the first 372 amino acids of the attachment (gene III) protein of filamentous bacteriophage f1 fused, in frame, to the carboxy-terminal catalytic domain of colicin E3. The hybrid toxin killed cells that had the F-pilus receptor for phage f1 but not F- cells. The activity of the hybrid protein was not dependent upon the presence of the colicin E3 receptor, BtuB protein. The killing activity was colicin E3 specific, since F+ cells expressing the colicin E3 immunity gene were not killed. Entry of the hybrid toxin was also shown to depend on the products of tolA, tolQ, and tolR which are required both for phage f1 infection and for entry of E colicins. TolB protein, which is required for killing by colicin E3, but not for infection by phage f1, was also found to be necessary for the killing activity of the hybrid toxin. The gene III protein-colicin E3 hybrid was released from producing cells into the culture medium, although the colicin E3 lysis protein was not present in those cells. The secretion was shown to depend on the 18-amino-acid-long gene III protein signal sequence. Deletion of amino acids 3 to 18 of the gene III moiety of the hybrid protein resulted in active toxin, which remained inside producing cells unless it was mechanically released.
Location of sites that inhibit progression of replication forks in the terminus region of Escherichia coli
Authors: Pelletier A J , Hill T M , Kuempel P L
Published: November 2016

We used a Southern hybridization assay to locate precisely the sites at which DNA replication is arrested in the terminus region of the Escherichia coli chromosome. The assay was based on the properties of restriction fragments that contain stalled replication forks. Replication forks that entered the terminus from the clockwise direction with respect to the genetic map were inhibited near manA at a site called T2, which we located at kilobase 442 on the physical map of Bouché (J. P. Bouché, J. Mol. Biol. 154:1-20, 1982). Those that entered the terminus region traveling in the counterclockwise direction were inhibited near pyrF at a site called T1, which we located at kilobase 90. In each case we found only a single, precise site of arrest. Inhibition at T1 was not detectable in our assay in strains lacking the trans-acting locus tus, which is located near T2 and is required for T1 to function. We demonstrated that the sites of inhibition are also used during termination of replication in exponentially growing, wild-type cells. In all previous studies on the terminus of E. coli, inhibition has only been detected in strains that were modified so that the origin used was placed near the terminus to force the use of the sites of inhibition.
Sensitivity of some marine bacteria, a moderate halophile, and Escherichia coli to uncouplers at alkaline pH
Authors: MacLeod R A , Wisse G A , Stejskal F L
Published: November 2016

The inhibitory effects of uncouplers on amino acid transport into three marine bacteria, Vibrio alginolyticus 118, Vibrio parahaemolyticus 113, and Alteromonas haloplanktis 214, into a moderate halophile, Vibrio costicola NRC 37001, and into Escherichia coli K-12 were found to vary depending upon the uncoupler tested, its concentration, and the pH. Higher concentrations of all of the uncouplers were required to inhibit transport at pH 8.5 than at pH 7.0. The protonophore carbonyl cyanide m-chlorophenylhydrazone showed the greatest reduction in inhibitory capacity as the pH was increased, carbonyl cyanide p-trifluoromethoxyphenylhydrazone showed less reduction, and 3,3',4',5-tetrachlorosalicylanilide was almost as effective as an inhibitor of amino acid transport at pH 8.5 as at pH 7.0 for all of the organisms except A. haloplanktis 214. Differences between the protonophores in their relative activities at pHs 7.0 and 8.5 were attributed to differences in their pK values. 3,3',4',5-Tetrachlorosalicylanilide, carbonyl cyanide m-chlorophenylhydrazone, 2-heptyl-4-hydroxyquinoline-N-oxide, and NaCN all inhibited Na+ extrusion from Na+-loaded cells of V. alginolyticus 118 at pH 8.5. The results support the conclusion that Na+ extrusion from this organism at pH 8.5 occurs as a result of Na+/H+ antiport activity. Data are presented indicating the presence in V. alginolyticus 118 of an NADH oxidase which is stimulated by Na+ at pH 8.5.
Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64
Authors: Komano T , Toyoshima A , Morita K , Nisioka T
Published: November 2016

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.
Conjugative plasmid transfer from Enterococcus faecalis to Escherichia coli
Authors: Trieu-Cuot P , Carlier C , Courvalin P
Published: November 2016

The possibility of transfer of genetic information by conjugation from gram-positive to gram-negative bacteria was investigated with a pBR322-pAM beta 1 chimeric plasmid, designated pAT191. This shuttle vector, which possesses the tra functions of the streptococcal plasmid pAM beta 1, was conjugatively transferred from Enterococcus faecalis to Escherichia coli with an average frequency of 5 x 10(-9) per donor colony formed after mating.
Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacterium Anabaena sp. strain PCC 7120 and Rhizobium meliloti
Authors: Mulligan M E , Buikema W J , Haselkorn R
Published: November 2016

The nucleotide sequence of a region located downstream of the nifB gene, both in the cyanobacterium Anabaena sp. strain PCC 7120 and in Rhizobium meliloti, has been determined. This region contains a gene (fdxN) whose predicted polypeptide product strongly resembles typical bacterial ferredoxins. Cyanobacteria have not previously been shown to contain bacterial-type ferredoxins. The presence of this gene suggests that nitrogen-fixing cyanobacteria have at least four distinct ferredoxins.
Purification of Rts1 RepA protein and binding of the protein to mini-Rts1 DNA
Authors: Kamio Y , Itoh Y , Terawaki Y
Published: November 2016

RepA protein, essential for the replication of plasmid Rts1, was purified, and its binding to mini-Rts1 subregions was examined by a DNase I protection assay. RepA protected the incI and incII iterons, a region immediately upstream of the repA promoter, and a 10-base-pair region located between the most external incII iteron and a GATC box. The protection was less efficient when preheated RepA was used.
Detection and subcellular localization of two Sym plasmid-dependent proteins of Rhizobium leguminosarum biovar viciae
Authors: de Maagd R A , Wijffelman C A , Pees E , Lugtenberg B J
Published: November 2016

The previously described Sym plasmid-dependent 24-kilodalton rhi protein of Rhizobium leguminosarum biovar viciae was localized in the cytosol fraction. Another Sym plasmid-dependent protein of 50 kilodaltons is secreted into the growth medium, and its expression is dependent on both the nodD gene and a nod gene inducer.
Three-dimensional structure of an open form of the surface layer from the fish pathogen Aeromonas salmonicida
Authors: Dooley J S , Engelhardt H , Baumeister W , Kay W W , Trust T J
Published: November 2016

Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.
Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis
Authors: Watanabe W , Sampei G , Aiba A , Mizobuchi K
Published: November 2016

It has been shown that the Escherichia coli purE locus specifying 5'-phosphoribosyl-5-amino-4-imidazole carboxylase in de novo purine nucleotide synthesis is divided into two cistrons. We cloned and determined a 2,449-nucleotide sequence including the purE locus. This sequence contains two overlapped open reading frames, ORF-18 and ORF-39, encoding proteins with molecular weights of 18,000 and 39,000, respectively. The purE mutations of CSH57A and DCSP22 were complemented by plasmids carrying ORF-18, while that of NK6051 was complemented by plasmids carrying ORF-39. Thus, the purE locus consists of two distinct genes, designated purE and purK for ORF-18 and ORF-39, respectively. These genes constitute a single operon. A highly conserved 16-nucleotide sequence, termed the PUR box, was found in the upstream region of purE by comparing the sequences of the purF and purMN operons. We also found three entire and one partial repetitive extragenic palindromic (REP) sequences in the downstream region of purK. Roles of the PUR box and REP sequences are discussed in relation to the genesis of the purEK operon.
The REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis
Authors: Larimer F W , Perry J R , Hardigree A A
Published: November 2016

The REV1 gene of Saccharomyces cerevisiae is required for normal induction of mutations by physical and chemical agents. We have determined the sequence of a 3,485-base-pair segment of DNA that complements the rev1-1 mutant. Gene disruption was used to confirm that this DNA contained the REV1 gene. The sequenced segment contains a single long open reading frame, which can encode a polypeptide of 985 amino acid residues. The REV1 transcript is 3.1 kilobase pairs in length. Frameshift mutations introduced into the open reading frame yielded a Rev-phenotype. A base substitution, encoding Gly-193 to Arg-193, was found in this open reading frame in rev1-1. Deletion mutants, lacking segments of the 5' region of REV1, had intermediate mutability relative to REV1 and rev1-1; a complete deletion exhibited lower mutability than rev1-1. REV1 is not an essential gene. An in-frame fusion of the 5' end of the REV1 open reading frame to the lacZ gene produced beta-galactosidase activity constitutively. The predicted REV1 protein is hydrophilic, with a predicted pI of 9.82. No homologies to RAD1, RAD2, RAD3, RAD7, or RAD10 proteins were noted. A 152-residue internal segment displayed 25% identity with UMUC protein.
Characterization and sequence analysis of the scrA gene encoding enzyme IIScr of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system
Authors: Sato Y , Poy F , Jacobson G R , Kuramitsu H K
Published: November 2016

The Streptococcus mutans GS-5 scrA gene coding for enzyme IIScr of the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS) was localized upstream from the scrB gene coding for sucrose-6-phosphate hydrolase activity after Mu dE transposon mutagenesis of plasmid pMH613. The cloned scrA gene product was identified as a 68-kilodalton protein by minicell analysis after isolation of the gene in plasmid pD4. In addition, the membrane fraction from Escherichia coli cells containing pD4 exhibited sucrose PTS activity upon complementation with enzyme I and HPr from strain GS-5. The nucleotide sequence of the scrA region revealed that this gene was located immediately upstream from the scrB gene and divergently transcribed from the opposite DNA strand. The scrA gene was preceded by potential Shine-Dalgarno and promoterlike sequences and was followed by a transcription terminator-like sequence. The scrA gene coded for an enzyme IIScr protein of 664 amino acid residues with a calculated molecular weight of 69,983. This enzyme IIScr protein was larger than the comparable proteins from Bacillus subtilis and E. coli containing sucrose-metabolizing plasmid pUR400. The 491-amino-acid N-terminal sequence of the S. mutans enzyme IIScr was homologous with the B. subtilis and E. coli sequences, and the 173-amino-acid C-terminal sequence of the S. mutans protein was also homologous with the Salmonella typhimurium enzyme IIIGlc and the 162-amino-acid C terminus of E. coli enzyme IIBgl. These results suggest that the sucrose PTS system of S. mutans is enzyme III independent.
Isolation and nucleotide sequencing of an aminocyclitol acetyltransferase gene from Streptomyces rimosus forma paromomycinus
Authors: López-Cabrera M , Pérez-González J A , Heinzel P , Piepersberg W , Jiménez A
Published: November 2016

A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Streptomyces rimosus forma paramomycinus NRRL 2455 was cloned in the Streptomyces plasmid pIJ702 and expressed in Streptomyces lividans 1326. Subcloning experiments located the aacC7 structural gene on a 1.05-kilobase DNA sequence. The direction of transcription of aacC7 was determined by using riboprobes synthesized in vitro from a DNA fragment internal to the gene. A DNA segment encoding the AAC(3)-VII activity and comprising 1,495 base pairs was sequenced. The aacC7 gene was located in an open reading frame of 864 base pairs that encoded a polypeptide of Mr 31,070, consistent with the Mr (32,000) of the AAC(3)-VII enzyme as determined by physicochemical methods. High-resolution S1 nuclease mapping suggested that transcription starts at or near the A residue of the ATG initiator codon. A DNA fragment from the 5' region of aacC7 had promoter activity in the promoter-probe plasmid pIJ486. The -10 and -35 regions of this fragment showed limited sequence resemblance to other Streptomyces promoters. The primary structure of the AAC(3)-VII enzyme showed strong homology with those of the AAC(3)-III and AAC(3)-IV enzymes encoded by plasmids in gram-negative bacterial genera. Upstream of the aacC7 gene was an open reading frame of 357 nucleotides which did not appear to be involved in controlling the expression of the aacC7 gene.
Biochemical characterization of two cloned resistance determinants encoding a paromomycin acetyltransferase and a paromomycin phosphotransferase from Streptomyces rimosus forma paromomycinus
Authors: Pérez-González J A , López-Cabrera M , Pardo J M , Jiménez A
Published: November 2016

The mechanism conferring resistance to paromomycin in Streptomyces rimosus forma paromomycinus, the producing organism, was studied at the level of both protein synthesis and drug-inactivating enzymes. Ribosomes prepared from this organism grown in either production or nonproduction medium were fully sensitive to paromomycin. A paromomycin acetyltransferase and a paromomycin phosphotransferase, both characteristic of the producer, were highly purified from extracts prepared from two Streptomyces lividans transformants harboring the relevant genes inserted in pIJ702-derived plasmids. In vitro, paromomycin was inactivated by either activity. In vivo, however, S. lividans clones containing the gene for either enzyme inserted in the low-copy-number plasmid pIJ41 were resistant to only low levels of paromomycin. In contrast, an S. lividans transformant containing both genes inserted in the same pIJ41-derived plasmid displayed high levels of resistance to paromomycin. These results indicate that both genes are required to determine the high levels of resistance to this drug in the producing organism. Paromomycin is doubly modified by the enzymes. However, whereas acetylparomomycin was a poorer substrate than paromomycin for the phosphotransferase, phosphorylparomomycin was modified more actively than was the intact drug by the acetyltransferase. These findings are discussed in terms of both a permeability barrier to paromomycin and the possible role(s) of the two enzymes in the biosynthetic pathway of this antibiotic.
Expression of the Rhodobacter sphaeroides cytochrome c2 structural gene
Authors: Brandner J P , McEwan A G , Kaplan S , Donohue T J
Published: November 2016

A Rhodobacter sphaeroides mutant (CYCA1) lacking cytochrome c2 (cyt c2) was previously constructed (T. J. Donohue, A. G. McEwan, S. Van Doren, A. R. Crofts, and S. Kaplan, Biochemistry, 27: 1918-1924, 1988) by a combination of in vivo and in vitro molecular genetic techniques. CYCA1 was incapable of photosynthetic growth (PS-); in this presentation, we show that chemoheterotrophically grown CYCA1 contained significant quantities of a high potential soluble c-type cytochrome(s) with an alpha band of approximately 554 nm which had previously gone undetected under these physiological conditions in wild-type cells. In addition, the PS- phenotype of CYCA1 can be complemented in trans with stable low-copy-number (approximately 5 to 9 per R. sphaeroides genome) broad-host-range plasmids containing the wild-type cyt c2 structural gene (cycA) and upstream regulatory sequences. cyt c2 and cycA-specific mRNA levels were elevated in both the wild type and CYCA1 derivatives harboring intact cycA genes in trans, presumably as a result of increased gene dosage. Although photosynthetically grown wild-type cells contained approximately twofold more cycA-specific transcripts than chemoheterotrophically grown cells, there was an approximately four- to sevenfold increase in cyt c2 levels under photosynthetic conditions. Similarly, complemented CYCA1 strains contained between 1.3- and 2.3-fold more cycA mRNA under photosynthetic conditions than under chemoheterotrophic conditions and had 6- to 12-fold higher steady-state levels of cyt c2 under the same physiological conditions. These data are discussed in terms of possible posttranscriptional control over cyt c2.