bluuebunny/crossref_metadata_2025 huggingface dataset:
Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis
Authors: Lee J J , Smith H O
Published: November 2016

The four restriction enzymes ApaI (5'-GGGCCC), EagI (5'-CGGCCG), NaeI (5'-GCCGGC), and SmaI (5'-CCCGGG) were found to produce distributions of DNA fragment sizes useful for mapping of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. ApaI produced 21 fragments (range, 1.6 to 305 kilobases [kb]), EagI yielded 30 fragments (0.6 to 339 kb), NaeI produced 32 fragments (2.3 to 290 kb), and SmaI yielded 16 fragments (6.0 to 377 kb). Summation of the fragment lengths in each digest yielded estimates for the size of the H. influenzae chromosome ranging from 1,834 kb.
Nucleotide sequence of traQ and adjacent loci in the Escherichia coli K-12 F-plasmid transfer operon
Authors: Wu J H , Ippen-Ihler K
Published: November 2016

The F tra operon region that includes genes trbA, traQ, and trbB was analyzed. Determination of the DNA sequence showed that on the tra operon strand, the trbA gene begins 19 nucleotides (nt) distal to traF and encodes a 115-amino-acid, Mr-12,946 protein. The traQ gene begins 399 nt distal to trbA and encodes a 94-amino-acid, Mr-10,867 protein. The trbB gene, which encodes a 179-amino-acid, Mr-19,507 protein, was found to overlap slightly with traQ; its start codon begins 11 nt before the traQ stop codon. Protein analysis and subcellular fractionation of the products expressed by these genes indicated that the trbB product was processed and that the mature form of this protein accumulated in the periplasm. In contrast, the protein products of trbA and traQ appeared to be unprocessed, membrane-associated proteins. The DNA sequence also revealed the presence of a previously unsuspected locus, artA, in the region between trbA and traQ. The artA open reading frame was found to lie on the DNA strand complementary to that of the F tra operon and could encode a 104-amino-acid, 12,132-dalton polypeptide. Since this sequence would not be expressed as part of the tra operon, the activity of a potential artA promoter region was assessed in a galK fusion vector system. In vivo utilization of the artA promoter and translational start sites was also examined by testing expression of an artA-beta-galactosidase fusion protein. These results indicated that the artA gene is expressed from its own promoter.
Homology between virF, the transcriptional activator of the Yersinia virulence regulon, and AraC, the Escherichia coli arabinose operon regulator
Authors: Cornelis G , Sluiters C , de Rouvroit C L , Michiels T
Published: November 2016

Virulent yersiniae (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) restrict their growth at 37 degrees C in rich medium deprived of calcium. This property, called calcium dependency, correlates with the secretion of Yersinia outer membrane proteins (Yops) and with pathogenicity. It is mediated by a 70-kilobase plasmid called pYV. The structural genes of the Yops (yop genes), as well as genes involved in the control of their expression (vir genes), have been localized on pYV. In this communication we show that virF encodes a transcriptional activator controlling the yop regulon. This activator is a 30,879-dalton protein related to AraC, the regulator of the Escherichia coli and Salmonella typhimurium arabinose operons. We also show in this paper that transcription of virF is thermodependent and presumably autoregulated. virF is thus responsible for the effect of temperature on the production of the Yops. Finally, we show that virF activates transcription of the yop genes independently of the presence of calcium ions. The role of calcium therefore remains unaccounted for.
Nucleotidylation, not phosphorylation, is the major source of the phosphotyrosine detected in enteric bacteria
Authors: Foster R , Thorner J , Martin G S
Published: November 2016

The majority of the phosphotyrosine recovered from partial acid hydrolysates of 32P-labeled Escherichia coli is derived from a single prominent protein. We show here by biochemical, genetic, and immunological criteria that this protein is actually glutamine synthetase adenylylated (not phosphorylated) at tyrosine. Furthermore, all of the phosphotyrosine detectable in partial acid hydrolysates of 32P-labeled Salmonella typhimurium was eliminated in a strain deficient in both glutamine synthetase and uridylyltransferase, an enzyme which uridylylates the regulatory protein PII at a tyrosine residue. These results suggest that protein-tyrosine phosphorylation represents a rare modification in eubacterial cells.
Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18
Authors: Misono H , Yonezawa J , Nagata S , Nagasaki S
Published: November 2016

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.
Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790
Authors: Higgins M L , Glaser D , Dicker D T , Zito E T
Published: November 2016

Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly.
Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae
Authors: Egilmez N K , Jazwinski S M
Published: November 2016

The life spans of individual Saccharomyces cerevisiae cells were determined microscopically by counting the number of buds produced by each cell to provide a measure of the number of cell generations (age) before death. As the cells aged, their generation times increased five- to sixfold. The generation times of daughter cells were virtually identical to those of their mothers throughout the life spans of the mothers. However, within two to three cell divisions after the daughters were detached from their mothers by micromanipulation, their generation times reverted to that characteristic of their own age. Recovery from the mother cell effect was also observed when the daughters were left attached to their mothers. The results suggest that senescence, as manifested by the increase in generation time, is a phenotypically dominant feature in yeast cells and that it is determined by a diffusible cytoplasmic factor(s) that undergoes turnover. This factor(s) appeared to be transmitted by a cell not only to its daughter, but also indirectly to its granddaughter. In separate studies, it was determined that the induced deposition of chitin, the major component of the bud scar, in the yeast cell wall had no appreciable effect on life span. We raise the possibility that the cytoplasmic factor(s) that appears to mediate the "senescent phenotype" is a major determinant of yeast life span. This factor(s) may be the product of age-specific gene expression.
Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus
Authors: Sommer J M , Newton A
Published: November 2016

We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus. pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act. pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was required during an overlapping period from 0.6 to 0.95 of the cell cycle to activate flagellum rotation as well as to enable loss of the flagellum and stalk formation by swarmer cells after division. The third pleiotropic gene, pleD, is described here for the first time. A pleD mutation was identified as a bypass suppressor of a temperature-sensitive pleC allele. Strains containing this mutation were highly motile, did not shed the flagellum or form stalks, and retained motility throughout the cell cycle. Since pleD was required to turn off motility and was a bypass suppressor of pleC, we conclude that it acts after the pleA and pleC gene functions in the cell cycle. No mutants defective in both flagellum biosynthesis and stalk formation were identified. Consequently, we propose that the steps required for formation of swarmer cells and subsequent development into stalked cells are organized into at least two developmental pathways: a pleA-dependent sequence of events, responsible for flagellum biosynthesis in predivisional cells, and a pleC-pleD-dependent sequence, responsible for flagellum activation in predivisional cells and loss of motility and stalk formation in progeny swarmer cells.
Amino acid sequence and length requirements for assembly and function of the colicin A lysis protein
Authors: Howard S P , Cavard D , Lazdunski C
Published: November 2016

The roles of the various parts of the mature colicin A lysis protein (Cal) in its assembly into the envelope and its function in causing "quasi-lysis," the release of colicin A, and the activation of phospholipase A were investigated. By using cassette mutagenesis, many missense mutations were introduced into the highly conserved portion of the lysis protein. In vitro mutagenesis was also used to introduce stop codons after amino acids 16 and 18 and a frameshift mutation at amino acid 17 of the mature Cal sequence. The processing and modification of the mutants were identical to those of the wild type, except for the truncated Cal proteins, which were neither acylated nor processed. Thus, the carboxy-terminal half of Cal must be present (or replaced by another peptide) for the proper processing and assembly of the protein. However, the specific sequence of this region is not required for the membrane-damaging function of the protein. Furthermore, the sequence specificity for even the conserved amino acids of the amino-terminal half of the protein is apparently exceedingly relaxed, since only those mutant Cal proteins in which a highly conserved amino acid has been replaced by a glutamate were impaired in their function.
Structural and functional analysis of transcriptional control of the Rhodobacter capsulatus puf operon
Authors: Adams C W , Forrest M E , Cohen S N , Beatty J T
Published: November 2016

We report data indicating that the Rhodobacter capsulatus puf operon promoter and the site for its oxygen regulation are located more than 700 base pairs upstream from the previously identified puf genes and have identified the nucleotide sequences that constitute these control signals. A model is proposed in which a polycistronic transcript at least 3.4 kilobases in length is initiated near the O2-regulated promoter and is processed posttranscriptionally by endonucleolytic cleavage at multiple sites, yielding discrete mRNA segments that are degraded at different rates. A newly identified gene (pufQ), which includes a hydrophobic domain having some similarity to domains of the products of the pufL and pufM genes, begins 313 nucleotides into the puf transcript and is located entirely within the most rapidly degraded segment of the transcript. A previously identified puf transcript segment encoding structural proteins for photosynthetic membrane complexes persists after degradation of the most 5' region of the transcript and is itself subject to segmentally specific degradation. Our results suggest a model in which differential expression of the multiple genes encoded by the puf operon is at least in part attributable to major differences in the rates of decay of the various segments of puf mRNA.
Characterization of three choline transport activities in Rhizobium meliloti: modulation by choline and osmotic stress
Authors: Pocard J A , Bernard T , Smith L T , Le Rudulier D
Published: November 2016

Choline has both a nutritional and osmoregulatory role in Rhizobium meliloti (T. Bernard, J. A. Pocard, B. Perroud, and D. Le Rudulier, Arch. Microbiol. 143:359-364, 1986). In view of this fact, choline transport was studied in R. meliloti 102F34 to determine how the rate of choline uptake is modulated. The effects of the cultural conditions on the kinetics of transport are presented. A high-affinity activity and a low-affinity activity were found in cells grown in minimal medium. The addition of 0.3 M NaCl or other osmolytes to the medium resulted in a marked decrease in the high-affinity activity, whereas the low-affinity activity remained fairly constant. Furthermore, results from osmotic upshock and downshock experiments indicate that the response of the cell to high osmolarity is rapid; hence, the mechanism of regulation by salt likely does not involve gene induction. A second high-affinity transport activity was induced by choline itself. Like the constitutive low-affinity transport activity, this activity was not greatly altered when the cells were grown in media of elevated osmotic strength. We conclude that although all three kinetically distinct transport systems are efficient at low osmolarity, only the induced high- and low-affinity activities are important for osmoregulation. The characteristics of the three transport activities from R. meliloti are compared with those from other bacterial species that use choline for growth and/or osmoregulation.
Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194
Authors: Bergemann A D , Whitley J C , Finch L R
Published: November 2016

The complete nucleotide sequence of pADB201, a 1.7-kilobase cryptic plasmid from Mycoplasma mycoides subsp. mycoides, is reported. The sequence contains a single large open reading frame capable of coding for a polypeptide of up to 198 codons long. The sequence of the putative polypeptide shows significant similarity to that of the repF gene product of staphylococcal plasmid pE194.
Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp
Authors: Ishizaki T , Hirayama N , Shinkawa H , Nimi O , Murooka Y
Published: November 2016

The nucleotide sequence of a 2.1-kilobase-pair fragment containing the Streptomyces choA gene, which codes a secreted cholesterol oxidase, was determined. A single open reading frame encodes a mature cholesterol oxidase of 504 amino acids, with a calculated Mr of 54,913. The leader peptides extend over 42 amino acids and have the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core near the signal cleavage site. Analyses of the total amino acid composition and amino acid sequencing of the first 21 amino acids from the N terminus of the purified extracellular enzyme agree with the values deduced from nucleotide sequencing data.
Altered induction of the adaptive response to alkylation damage in Escherichia coli recF mutants
Authors: Volkert M R
Published: November 2016

Escherichia coli recF mutants are hypermutable when treated with methyl methanesulfonate (G. C. Walker, Mol. Gen. Genet. 152:93-103, 1977). In this study, methylation hypermutability of recF mutant strains was examined, and it was found that recF+ is required for normal induction of the adaptive response to alkylation damage. Although this regulatory effect of recF mutations results in reduced levels of enzymes that specifically repair methyl lesions in DNA, it only partially explains the hypermutability. Further examination showed that methylation hypermutability of recF mutant strains required a functional umuDC operon, a component of the SOS response. These results lead to the hypothesis that methylation hypermutability results from the effects of recF mutations on the induction of both the SOS response and the adaptive response.
Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells
Authors: Marvin H J , ter Beest M B , Hoekstra D , Witholt B
Published: November 2016

Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicles. No fusion activity was observed when the EDTA treatment was omitted. Fusion could be elicited at low pH and by a combination of a higher pH and Ca2+. The low-pH-induced fusion was composed of a fast and a slow reaction. The latter and the Ca2+-induced fusion could be completely inhibited by trypsin treatments of the EDTA-treated cells, which also resulted in the simultaneous disappearance of two outer membrane protein bands (50 and 58 kilodaltons) and the appearance of proteins banding at 22, 52, and 54 kilodaltons. The most efficient fusion was obtained with negatively charged liposomes composed of cardiolipin. In contrast to the Ca2+-induced fusion, fusion was observed at low pH with small unilamellar vesicles containing lipids with decreased negative charge (phosphatidylserine). Fluorescent and phase-contrast microscopy revealed that essentially all bacteria were engaged in fusion. We propose that a Ca2+-EDTA treatment of E. coli cells results in the appearance of phospholipids and the exposure of a protein(s) in the outer leaflet of the outer membrane, both of which could mediate fusion with liposomes.
Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58
Authors: von Bodman S B , McCutchan J E , Farrand S K
Published: November 2016

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.
Bacillus subtilis mutant allele sup-3 causes lysine insertion at ochre codons: use of sup-3 in studies of translational attenuation
Authors: Mulbry W W , Ambulos N P , Lovett P S
Published: November 2016

The mutation sup-3 in Bacillus subtilis suppresses ochre (TAA) mutations at each of three codons in the 5' end of the cat-86 coding sequence. The suppressor is shown to insert lysine at ochre codons. The efficiency of suppression by sup-3 is about 15%, as determined by changing a cat-86 Lys codon (codon 12) to an ochre codon and measuring the level of CAT in the suppressor-containing strain. The results obtained are discussed in light of previous observations that ochre mutations at cat leader codons 2 and 3 can be phenotypically suppressed by sup-3, whereas ochre mutations at leader codons 4 and 5 cannot. Translation of the cat leader is essential to inducible expression of cat. Our data support the interpretation that the nature of amino acids 2 through 5 of the leader peptide contributes to determining whether chloramphenicol can stall a ribosome in the leader, which in turn leads to induction of cat expression.
Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa
Authors: Donald L J , Molgat G F , Duckworth H W
Published: November 2016

The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted.
DNA replication in Escherichia coli mutants that lack protein HU
Authors: Ogawa T , Wada M , Kano Y , Imamoto F , Okazaki T
Published: November 2016

DNA replication in Escherichia coli cells lacking protein HU was studied. HU has been suggested to be involved in the initiation of replication from in vitro studies. The isolated HU mutants, however, are viable under normal growth conditions (M. Wada, Y. Kano, T. Ogawa, T. Okazaki, and F. Imamoto, J. Mol. Biol. 204:581-591, 1988). Chromosomal replication in the mutants appeared to be normal with respect to bidirectional replication from oriC and to its dependence on dnaA and some other dna gene products. No significant defect was observed in DNA synthesis in vitro with a crude enzyme fraction prepared from the mutant cells. These results, along with the earlier in vitro studies, suggest that other histonelike protein(s) may substitute for HU in the initiation of replication in the mutant cells. Minichromosomes were more unstable in the mutants. In the absence of either the mioC promoter, from which transcription enters oriC, or the DnaA box (DnaA protein-binding site) just upstream of the mioC promoter, the minichromosomes were especially unstable in the HU mutant and were integrated into the chromosomal oriC region under conditions selective for the plasmid-harboring cells.
Identification and analysis of transcriptional regulatory signals for the kil and kor loci of Streptomyces plasmid pIJ101
Authors: Stein D S , Kendall K J , Cohen S N
Published: November 2016

Four regulated promoters that direct the transcription of genes (i.e., korA, tra, kilB, and korB) involved in the transfer of the Streptomyces plasmid pIJ101 were isolated following the in vitro fusion of plasmid DNA fragments to a promoterless gene encoding the S. lividans extracellular enzyme beta-galactosidase. Introduction of pIJ101 into cells carrying each of these promoter-lac fusions resulted in decreased lac expression. The sites of initiation of transcription by the promoter sequences were identified by primer extension experiments, and the DNA sequences specifically required for promoter activity and regulation by pIJ101-encoded functions were determined by deletion analysis. The data obtained indicate that the korB locus encodes a repressor that regulates its own transcription, as well as transcription of the kilB promoter; korA and tra are transcribed from overlapping divergent promoters that are coregulated by the korA gene product. Common DNA sequence domains within coregulated promoters allowed the identification of putative binding sites for each of the kor gene products.
Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair
Authors: Thomas S M , Sedgwick S G
Published: November 2016

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli.
Structure and function of a menaquinone involved in electron transport in membranes of Clostridium thermoautotrophicum and Clostridium thermoaceticum
Authors: Das A , Hugenholtz J , Van Halbeek H , Ljungdahl L G
Published: November 2016

Clostridium thermoaceticum and Clostridium thermoautotrophicum contain the same menaquinone. Its structure, determined by thin-layer chromatography, UV absorption spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy, was found to be MK-7 (2-methyl-3-heptaprenyl-1,4-naphthoquinone). The menaquinone is located in the cytoplasmic membranes and is involved in redox reactions of two b-type cytochromes present in the clostridia. These reactions were studied with right-side-out membranes prepared from C. thermoautotrophicum by using CO as an electron donor. In intact membranes, both cytochromes were reduced, whereas after inactivation of the menaquinone by exposure of the membranes to UV irradiation, reduction of the low-potential cytochrome (Eo', -200 mV) but not of the high-potential cytochrome (Eo', -48 mV) occurred. The reduction of the high-potential cytochrome in UV-irradiated membranes was restored following the addition of oxidized menaquinone and with an excess of CO. The addition of oxidized menaquinone to reduced membranes resulted initially in a preferential oxidation of the low-potential cytochrome. The results obtained indicate that the menaquinone acts between the two b-type cytochromes in an electron transport chain.
Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: genome size, fragment identification, and gene localization
Authors: Suwanto A , Kaplan S
Published: November 2016

Four restriction endonucleases, AseI (5'-ATTAAT), SpeI (5'-ACTAGT), DraI (5'-TTTAAA), and SnaBI (5'-TACGTA), generated DNA fragments of suitable size distributions for mapping the genome of Rhodobacter sphaeroides by transverse alternating field electrophoresis. AseI produced 17 fragments, ranging in size from 3 to 1,105 kilobases (kb), SpeI yielded 16 fragments (12 to 1,645 kb), DraI yielded at least 25 fragments (6 to 800 kb), and SnaBI generated 10 fragments (12 to 1,225 kb). A total genome size of approximately 4,400 +/- 112 kb was determined by summing the fragment lengths in each of the digests generated by using the different restriction endonucleases. The total genomic DNA consisted of chromosomal DNA (3,960 +/- 112 kb) and the five endogenous plasmids (approximately 450 kb total) whose cognate DNA fragments have been unambiguously identified. A number of genes have been physically mapped to the AseI-generated restriction endonuclease fragments of total genomic DNA by Southern hybridization analysis with either homologous or heterologous specific gene probes or, in the case of several auxotrophic and pigment-biosynthetic mutants apparently generated by Tn5, a Tn5-specific probe. Other genes have been mapped by a comparison with wild-type patterns of the electrophoretic banding patterns of the AseI-digested genomic DNA derived from mutants generated by the insertion of either kanamycin or spectinomycin-streptomycin resistance cartridges. The relative orientations, distance, and location of the pufBALMX, puhA, cycA, and pucBA operons have also been determined, as have been the relative orientations between prkB and hemT and between prkA and the fbc operon.
The tyrT locus of Escherichia coli exhibits a regulatory function for glycine metabolism
Authors: Michelsen U , Bösl M , Dingermann T , Kersten H
Published: November 2016

The tyrT locus in Escherichia coli codes for two gene copies of tRNA(1Tyr). Both genes are organized in one operon, which has a unique structure. The two tRNA genes are separated by a spacer segment highly homologous to a part of a unit which is repeated three times in the distal portion of the locus. This operon also contains coding capacity for a small basic protein. A genomic deletion of this locus was constructed and marked by a kanamycin resistance cassette. Deletion mutants exhibited a characteristic phenotype when cells were shifted from rich medium to minimal medium. The cells entered a transient lag phase, apparently resulting from specific glycine starvation. This phenotype involved stringent response and was therefore not observed in relA derivatives. The genomic deletion was complemented in trans by a plasmid-borne tyrT locus. From deletion mapping, it can be concluded that a product of the tyrT operon is responsible for complementation. However, neither the tRNA(1Tyr) nor the proposed basic protein is the complementation-competent entity.
The cytochrome bc1 complex of Rhodobacter sphaeroides can restore cytochrome c2-independent photosynthetic growth to a Rhodobacter capsulatus mutant lacking cytochrome bc1
Authors: Davidson E , Prince R C , Haith C E , Daldal F
Published: November 2016

Plasmids encoding the structural genes for the Rhodobacter capsulatus and Rhodobacter sphaeroides cytochrome (cyt) bc1 complexes were introduced into strains of R. capsulatus lacking the cyt bc1 complex, with and without cyt c2. The R. capsulatus merodiploids contained higher than wild-type levels of cyt bc1 complex, as evidenced by immunological and spectroscopic analyses. On the other hand, the R. sphaeroides-R. capsulatus hybrid merodiploids produced only barely detectable amounts of R. sphaeroides cyt bc1 complex in R. capsulatus. Nonetheless, when they contained cyt c2, they were capable of photosynthetic growth, as judged by the sensitivity of this growth to specific inhibitors of the photochemical reaction center and the cyt bc1 complex, such as atrazine, myxothiazol, and stigmatellin. Interestingly, in the absence of cyt c2, although the R. sphaeroides cyt bc1 complex was able to support the photosynthetic growth of a cyt bc1-less mutant of R. capsulatus in rich medium, it was unable to do so when C4 dicarboxylic acids, such as malate and succinate, were used as the sole carbon source. Even this conditional ability of R. sphaeroides cyt bc1 complex to replace that of R. capsulatus for photosynthetic growth suggests that in the latter species the cyt c2-independent rereduction of the reaction center is not due to a structural property unique to the R. capsulatus cyt bc1 complex. Similarly, the inability of R. sphaeroides to exhibit a similar pathway is not due to some inherent property of its cyt bc1 complex.
Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol
Authors: Chen Y M , Lu Z , Lin E C
Published: November 2016

L-1,2-Propanediol is an irretrievable end product of L-fucose fermentation by Escherichia coli. Selection for increased aerobic growth rate on propanediol results in the escalation of basal synthesis of the NAD+-linked oxidoreductase encoded by fucO, a member of the fuc regulon for the utilization of L-fucose. In general, when fucO becomes constitutively expressed, two other simultaneous changes occur: the fucA gene encoding fuculose-1-phosphate aldolase becomes constitutively expressed and the fucPIK operon encoding fucose permease, fucose isomerase, and fuculose kinase becomes noninducible. In the present study, we show that fucO and fucA form an operon which is divergently transcribed from the adjacent fucPIK operon. In propanediol-positive and fucose-negative mutants the cis-controlling region shared by the operons fucAO and fucPIK is lengthened by 1.2 kilobases. DNA hybridization identified the insertion element to be IS5. This element, always oriented in the same direction with the left end (the BglII end) proximal to fucA, apparently causes constitutive expression of fucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.
Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida
Authors: Harayama S , Rekik M , Ngai K L , Ornston L N
Published: November 2016

The meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds. In this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route. In the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the actions of 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxovalerate aldolase. In this study, the isomerase, decarboxylase, and hydratase encoded in the TOL plasmid pWW0 of Pseudomonas putida mt-2 were purified and characterized. The 28-kilodalton isomerase was formed by association of extremely small identical protein subunits with an apparent molecular weight of 3,500. The decarboxylase and the hydratase were 27- and 28-kilodalton polypeptides, respectively, and were copurified by high-performance-liquid chromatography with anion-exchange, hydrophobic interaction, and gel filtration columns. The structural genes for the decarboxylase (xylI) and the hydratase (xylJ) were cloned into Escherichia coli. The elution profile in anion-exchange chromatography of the decarboxylase and the hydratase isolated from E. coli XylI+XylJ- and XylI-XylJ+ clones, respectively, were different from those isolated from XylI+ XylJ+ bacteria. This suggests that the carboxylase and the hydratase form a complex in vivo. The keto but not the enol form of 4-oxalocrotonate was a substrate for the decarboxylase. The product of decarboxylation was 2-hydroxypent-2,4-dienoate rather than its keto form, 2-oxopent-4-enoate. The hydratase acts on the former but not the latter isomer. Because 2-hydroxypent-2,4-dienoate is chemically unstable, formation of a complex between the decarboxylase and the hydratase may assure efficient transformation of this unstable intermediate in vivo.
The acylated precursor form of the colicin A lysis protein is a natural substrate of the DegP protease
Authors: Cavard D , Lazdunski C , Howard S P
Published: November 2016

The acylated precursor form of the colicin A lysis protein (pCalm) is specifically cleaved by the DegP protease into two acylated fragments of 6 and 4.5 kilodaltons (kDa). This cleavage was observed after globomycin treatment, which inhibits the processing of pCalm into mature colicin A lysis protein (Cal) and the signal peptide. The cleavage took place in lpp, pldA, and wild-type strans carrying plasmids which express the lysis protein following SOS induction and also in cells containing a plasmid which expresses it under the control of the tac promoter. Furthermore, the DegP protease was responsible for the production of two acylated Cal fragments of 3 and 2.5 kDa in cells carrying plasmids which overproduce the Cal protein, without treatment with globomycin. DegP could also cleave the acylated precursor form of a mutant Cal protein containing a substitution in he amino-terminal portion of the protein, but not that of a mutant Cal containing a frameshift mutation in its carboxyl-terminal end. The functions of Cal in causing protein release, quasi-lysis, and lethality were increased in degP41 cells, suggesting that mature Cal was produced in higher amounts in the mutant than in the wild type. These effects were limited in cells deficient in phospholipase A. Interactions between the DegP protease and phospholipase A were suggested by the characteristics of degP pldA double mutants.
Tandem arrangement of photolyase and superoxide dismutase genes in Halobacterium halobium
Authors: Takao M , Kobayashi T , Oikawa A , Yasui A
Published: November 2016

A DNA fragment containing the photolyase gene was cloned from Halobacterium halobium. The deduced amino acid sequence is highly similar to those of four known photolyases from eubacteria and a eucaryote. The cloned gene expressed in Escherichia coli cells the survival of UV-irradiated host cells by photoreactivation. These results indicate that photolyases of eucaryotes, eubacteria, and archaebacteria are derived from a common origin. In this cloned DNA fragment, two additional open reading frames (ORFs), ORF 151 and ORF 200, were found in the 5' and 3' adjacent flanking regions of the photolyase gene. ORF 200 shows unequivocal amino acid sequence homology to all known manganese and iron superoxide dismutases. Northern (RNA) hybridization analysis of H. halobium RNA revealed the existence of three transcripts, one of which covered all three ORFs, indicating that photolyase and superoxide dismutase are partly cotranscribed in this bacterium.
Mutational analysis of the histidine operon promoter of Salmonella typhimurium
Authors: Shand R F , Blum P H , Holzschu D L , Urdea M S , Artz S W
Published: November 2016

We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.
Ti plasmid-controlled chromosome transfer in Agrobacterium tumefaciens
Authors: Dessaux Y , Petit A , Ellis J G , Legrain C , Demarez M , Wiame J M , Popoff M , Tempe J
Published: November 2016

In octopine-type A. tumefaciens R10, transfer of chromosomal arginine degradation genes (arc genes) was observed under conditions in which Ti plasmid transfer took place. However, transconjugants that had acquired the arc genes but not the Ti plasmid were recovered. During this process, several other chromosomal genes, such as genes encoding phage resistances or genes complementing a galactose utilization mutation or a glycine-serine auxotrophy, were transferred from strain R10 to the recipient.
Structural similarity among Escherichia coli FtsW and RodA proteins and Bacillus subtilis SpoVE protein, which function in cell division, cell elongation, and spore formation, respectively
Authors: Ikeda M , Sato T , Wachi M , Jung H K , Ishino F , Kobayashi Y , Matsuhashi M
Published: November 2016

The Escherichia coli cell division gene ftsW (2 min) was cloned and sequenced. It encodes a hydrophobic protein(s) with 414 and/or 384 amino acid residues. The deduced amino acid sequence and the hydropathy profile of the protein showed high homology with those of the E. coli RodA protein functioning in determination of the cell shape and the Bacillus subtilis SpoVE protein functioning in spore formation. Probably similar functional membrane proteins are involved in these three cell cycle process.
Inhibition of growth of ftsQ, ftsA, and ftsZ mutant cells of Escherichia coli by amplification of a chromosomal region encompassing closely aligned cell division and cell growth genes
Authors: Jung H K , Ishino F , Matsuhashi M
Published: November 2016

Amplification of a 2.6-kilobase chromosomal fragment of the mra region of Escherichia coli encompassing the ftsI(pbpB) gene and an open reading frame upstream with lethal to E. coli strains with mutations of the flanking cell division genes ftsQ, ftsA, and ftsZ. A shortened fragment in which the major portion of ftsI was deleted also had lethal effects on ftsQ and ftsZ mutants.
Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control
Authors: Liu J D , Parkinson J S
Published: November 2016

Mutations at the Escherichia coli pcnB locus reduce the copy number of ColE1-like plasmids. We isolated additional mutations in this gene and conducted a preliminary characterization of its product. F-prime elements carrying the pcnB region were constructed and used to show that the mutations were recessive. The wild-type pcnB gene was cloned into a low-copy-number plasmid, and its nucleotide sequence was determined. The sequence analysis indicated that pcnB is probably the first gene in an operon that contains one or more additional genes of unknown function. The pcnB locus should encode a polypeptide of 47,349 daltons (Da). A protein of this size was observed in minicells carrying a pcnB+ plasmid, and transposon insertions and deletions that truncated this protein generally abolished pcnB function. One exceptional transposon insertion at the promoter-distal end of the pcnB gene truncated the 47-kDa protein by about 20% but did not abolish complementation activity, indicating that the C-terminus of the PcnB product is dispensable. The deduced amino acid sequence of PcnB revealed numerous charged residues and, with 10% arginines, an overall basic character, suggesting that PcnB might interact with DNA or RNA in a structural capacity. Disruption of the pcnB gene by insertional mutagenesis caused a reduction in growth rate, indicating that PcnB has an important cellular function.
Comparison of glucose uptake kinetics in different yeasts
Authors: Does A L , Bisson L F
Published: November 2016

The kinetics of glucose uptake were investigated in laboratory wild-type strains of Saccharomyces cerevisiae of differing genetic backgrounds, in other species of Saccharomyces, and in other yeasts, both fermentative and respiratory. All yeasts examined displayed more than one uptake system for glucose. Variations in apparent Km values, velocity of uptake, and effects of glucose concentration on carrier activity were observed. The three type strains for the species S. cerevisiae, Saccharomyces bayanus, and Saccharomyces carlsbergensis gave distinctive patterns, and each of the laboratory strains was similar to one or another of the type strains. Other fermentative yeasts (Pichia guillermondi and Pichia strasburgensis) regulated glucose uptake in a manner similar to that of Saccharomyces spp. Such was not true for the respiratory yeasts investigated, Pichia heedi and Yarrowia lipolytica, which did not demonstrate glucose repression of carrier activity; this finding suggests that this mechanism of control of transporter activity may be associated with fermentative ability.
Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum
Authors: Freudenberg W , Dietrichs D , Lebertz H , Andreesen J R
Published: November 2016

The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-14C]riboflavin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The molecular mass of the native enzyme could be determined by several methods to be 68 kilodaltons, and an enzyme with a molecular mass of 34.5 kilodaltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of cell extracts separated by sodium dodecyl sulfate-polyacrylamide or linear polyacrylamide gel electrophoresis resulted in a single fluorescent band. NADPH instead of NADH was the preferred electron donor of this lipoamide dehydrogenase. This was also indicated by Michaelis constants of 0.085 mM for NADPH and 1.1 mM for NADH at constant lipoamide and enzyme concentrations. The enzyme exhibited no thioredoxin reductase, glutathione reductase, or mercuric reductase activity. Immunological cross-reactions were obtained with cell extracts of Clostridium cylindrosporum, Clostridium sporogenes, Clostridium sticklandii, and bacterium W6, but not with extracts of other glycine- or purine-utilizing anaerobic or aerobic bacteria, for which the lipoamide dehydrogenase has already been characterized.