bluuebunny/crossref_metadata_2025 huggingface dataset:
Unique modification of adenine in genomic DNA of the marine cyanobacterium Trichodesmium sp. strain NIBB 1067
Authors: Zehr J P , Ohki K , Fujita Y , Landry D
Published: November 2016

The genomic DNA of the marine nonheterocystous nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067 was found to be highly resistant to DNA restriction endonucleases. The DNA was digested extensively by the restriction enzyme DpnI, which requires adenine methylation for activity. The DNA composition, determined by high-performance liquid chromatography (HPLC), was found to be 69% AT. Surprisingly, it was found that a modified adenine which was not methylated at the usual N6 position was present and made up 4.7 mol% of the nucleosides in Trichodesmium DNA (15 mol% of deoxyadenosine). In order for adenine residues to be modified at this many positions, there must be many modifying enzymes or at least one of the modifying enzymes must have a degenerate recognition site. The reason(s) for this extensive methylation has not yet been determined but may have implications for the ecological success of this microorganism in nature.
The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa
Authors: Aguilar O M , Grasso D H
Published: November 2016

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment. Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv- Nod- mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli IlvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv- and Nod-) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.
Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate transcription factor sigma B of Bacillus subtilis
Authors: Boylan S A , Thomas M D , Price C W
Published: November 2016

We describe a general, in vivo method for identifying Bacillus subtilis genes controlled by specific, nonessential regulatory factors. We establish the use of this approach by identifying, isolating, and characterizing a gene dependent on sigma B, an alternate transcription factor which is found early in stationary phase but which is not essential for sporulation. The method relies on two features: (i) a plate transformation technique to introduce a null mutation into the regulatory gene of interest and (ii) random transcriptional fusions to a reporter gene to monitor gene expression in the presence and absence of a functional regulatory product. We applied this genetic approach to isolate genes comprising the sigma B regulon. We screened a random Tn917lacZ library for fusions that required an intact sigma B structural gene (sigB) for greatest expression, converting the library strains from wild-type sigB+ to sigB delta::cat directly on plates selective for chloramphenicol resistance. We isolated one such fusion, csbA::Tn917lacZ (csb for controlled by sigma B), which mapped between hisA and degSU on the B. subtilis chromosome. We cloned the region surrounding the insertion, identified the csbA reading frame containing the transposon, and found that this frame encoded a predicted 76-residue product which was extremely hydrophobic and highly basic. Primer extension and promoter activity experiments identified a sigma B-dependent promoter 83 bp upstream of the csbA coding sequence. A weaker, tandem, sigma A-like promoter was likewise identified 28 bp upstream of csbA. The csbA fusion was maximally expressed during early stationary phase in cells grown in Luria broth containing 5% glucose and 0.2% glutamine. This timing of expression and medium dependence were very similar to those for ctc, the only other recognized gene dependent on sigma B.
Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E sigma F: identification of features of good E sigma F-dependent promoters
Authors: Sun D , Fajardo-Cavazos P , Sussman M D , Tovar-Rojo F , Cabrera-Martinez R M , Setlow P
Published: November 2016

Translational lacZ fusions to forespore genes of Bacillus subtilis were not expressed in spoIIAC (sigma F) or spoIIIE mutants when the lacZ fusions were integrated at the loci of the same genes or at the SP beta locus. However, some of these genes, including gerA, gpr, spoIIIG (sigma G), and sspE, were expressed in spoIIIE mutants and spoIIIE spoIIIG double mutants (but not in spoIIAC mutants) when the lacZ fusions were integrated at the amyE locus. When tested, the beta-galactosidase made in these mutants was found only in the forespore, and the 5' ends of the mRNAs produced in these mutants were identical to those in a Spo+ background. Analysis of the in vitro transcription of forespore genes by RNA polymerase containing sigma F (E sigma F) revealed a direct correlation between good in vitro transcription by E sigma F and expression at the amyE locus in spoIIIE mutants. This result suggests that forespore genes are transcribed by E sigma F in spoIIIE and spoIIIE spoIIIG mutants. Comparison of the promoter regions of genes transcribed well and poorly by E sigma F in vivo and in vitro showed that good transcription by E sigma F was correlated with G residues at positions -15 and -16, a purine residue at position -13, and a T residue at position -7 relative to the start site of transcription. The importance of these residues in sigma F recognition was confirmed by analysis of the E sigma F-dependent transcription in vivo and in vitro of mutant ssp genes.
Characterization of cytochromes c550 and c555 from Bradyrhizobium japonicum: cloning, mutagenesis, and sequencing of the c555 gene (cycC)
Authors: Tully R E , Sadowsky M J , Keister D L
Published: November 2016

The major soluble c-type cytochromes in cultured cells of Bradyrhizobium japonicum USDA 110 comprised a CO-reactive c555 (Mr, approximately 15,500) and a non-CO-reactive c550 (Mr, approximately 12,500). Levels of cytochrome per gram of soluble protein in aerobic, anaerobic, and symbiotic cells were 32, 21, and 30 nmol, respectively, for c555 and 31, 44, and 65 nmol, respectively, for c550. The midpoint redox potentials (Em,7) of the purified cytochromes were +236 mV for c555 and +277 mV for c550. The CO reactivity of c555 was pH dependent, with maximal reactivity at pH 10 or greater. Rabbit antiserum was produced against purified c555 and used to screen a B. japonicum USDA 110 genomic DNA expression library in lambda gt11 for a downstream portion of the c555 gene (cycC). This sequence was then used to probe a cosmid library for the entire c555 locus. The nucleotide sequence shows an open reading frame of 149 amino acids, with an apparent signal sequence at the N terminus and a heme-binding site near the C terminus. The deduced amino acid sequence is similar to those of the cytochromes c556 of Rhodopseudomonas palustris and Agrobacterium tumefaciens. The cycC gene was mutagenized by insertion of a kanamycin resistance cassette and homologously recombined into the B. japonicum genome. The resulting mutant made no c555 but made normal amounts of c550. The levels of membrane cytochromes were unaffected. The mutant and wild type exhibited identical phenotypes when used to nodulate plants of soybean (Glycine max L. Merr.), with no significant differences in nodule number, nodule mass, or total amount of N2 fixed.
The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain: purification, structure, and interaction with synthetic membranes
Authors: Jagannadham M V , Rao V J , Shivaji S
Published: November 2016

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain was purified to homogeneity from methanol extracts of dried cells by reverse-phase liquid chromatography and was designated P-3. On the basis of the UV-visible, infrared, mass, and 1H nuclear magnetic resonance spectra of P-3, it was identified as bisdehydro-beta-carotene-2-carboxylic acid. The pigment interacted with synthetic membranes of phosphatidylcholine and dimyristoyl phosphatidylcholine and stabilized the membranes. These results also indicate that P-3 is different from canthaxanthin, the major carotenoid pigment from a mesophilic M. roseus strain.
Characterization of endoglucanase A from Clostridium cellulolyticum
Authors: Fierobe H P , Gaudin C , Belaich A , Loutfi M , Faure E , Bagnara C , Baty D , Belaich J P
Published: November 2016

A construction was carried out to obtain a high level of expression in Escherichia coli of the gene celCCA, coding for the endoglucanase A from Clostridium cellulolyticum (EGCCA). The enzyme was purified in two forms with different molecular weights, 51,000 and 44,000. The smaller protein was probably the result of proteolysis, although great care was taken to prevent this process from occurring. Evidence was found for the loss of the conserved reiterated domains which are characteristic of C. thermocellum and C. cellulolyticum cellulases. The two forms were extensively studied, and it was demonstrated that although they had the same pH and temperature optima, they differed in their catalytic properties. The truncated protein gave the more efficient catalytic parameters on carboxymethyl cellulose and showed improved endoglucanase characteristics, whereas the intact enzyme showed truer cellulase characteristics. The possible role of clostridial reiterated domains in the hydrolytic activity toward crystalline cellulose is discussed.
Specificity of SOS mutagenesis in native M13lacI phage
Authors: Yatagai F , Horsfall M J , Glickman B W
Published: November 2016

Base substitutions account for 90% of all forward mutations sequenced in unmodified M13lacI DNA grown in both UV-irradiated and nonirradiated hosts. The principal effect of SOS induction was an increase in the contribution of transversions, in particular A.T----T.A events.
Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi
Authors: Brusca J S , McDowall A W , Norgard M V , Radolf J D
Published: November 2016

Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracellularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins.
Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates
Authors: van der Meer J R , Eggen R I , Zehnder A J , de Vos W M
Published: November 2016

Pseudomonas sp. strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes. The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined. The sequence contained five large open reading frames, which were all colinear. The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities. The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity. The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa). The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clcABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134. The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three. Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place. TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol. ClcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol.
Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Brahamsha B , Haselkorn R
Published: November 2016

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 responds to combined nitrogen deprivation by forming specialized nitrogen-fixing cells at regular intervals along the filament. Genetic and biochemical studies have indicated that regulation of gene expression during differentiation occurs at the transcriptional level. As part of a characterization of RNA polymerase during differentiation, the gene encoding the 52-kDa principal sigma factor of the Anabaena sp. strain PCC 7120 vegetative-cell RNA polymerase was isolated by using an oligonucleotide probe based on the sequence of the N-terminal seven amino acids of the purified protein. sigA codes for a 390-amino-acid polypeptide that has a predicted molecular weight of 45,641. The amino acid sequence of the polypeptide encoded by sigA contains four regions corresponding to conserved domains of the principal RNA polymerase sigma factors of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). Thus, although the subunit composition of cyanobacterial RNA polymerase core differs from that of other eubacteria (G. J. Schneider and R. Haselkorn, J. Bacteriol. 170:4136-4140, 1988), the principal sigma factor of at least one cyanobacterium is typically eubacterial. In contrast to sigma 70 and sigma 43 operon organization, sigA is monocistronic and encodes two transcripts of 1.7 and 2.2 kb. The abundance of the 1.7-kb transcript remains constant under both nitrogen-replete and nitrogen-limiting conditions, whereas the 2.2-kb transcript is induced following the removal of combined nitrogen. Continued or enhanced transcription of sigA under nitrogen starvation conditions is consistent with the observation that the principal RNA polymerase in differentiating cells contains SigA.
Isolation and characterization of Escherichia coli mutants able to utilize the novel pentose L-ribose
Authors: Trimbur D E , Mortlock R P
Published: November 2016

Wild-type strains of Escherichia coli were unable to utilize L-ribose for growth. However, L-ribose-positive mutants could be isolated from strains of E. coli K-12 which contained a ribitol operon. L-ribose-positive strains of E. coli, isolated after 15 to 20 days, had a growth rate of 0.22 generation per h on L-ribose. Growth on L-ribose was found to induce the enzymes of the L-arabinose and ribitol pathways, but only ribitol-negative mutants derived from strains originally L-ribose positive lost the ability to grow on L-ribose, showing that a functional ribitol pathway was required. One of the mutations permitting growth on L-ribose enabled the mutants to produce constitutively an NADPH-linked reductase which converted L-ribose to ribitol. L-ribose is not metabolized by an isomerization to L-ribulose, as would be predicted on the basis of other pentose pathways in enteric bacteria. Instead, L-ribose was metabolized by the reduction of L-ribose to ribitol, followed by the conversion to D-ribulose by enzymes of the ribitol pathway.
Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells
Authors: Fry I J , Becker-Hapak M , Hageman J H
Published: November 2016

Although calcium ions are crucial in a variety of bacterial processes, including spore development, reports of calmodulin in procaryotes have been few. We have purified to homogeneity a calmodulinlike protein (CaLP) from sporulating cells of Bacillus subtilis grown in a chemically defined sporulation medium; purification involved heat treatment, fractionation with ammonium sulfate, affinity chromatography, and gel filtration on high-performance columns. The protein was eluted from a phenothiazine affinity column in a calcium ion-dependent manner, stained poorly with Coomassie blue and silver stain dyes, bound poorly to nitrocellulose filters, and was not an inhibitor of the major intracellular serine proteinase. It stimulated bovine brain phosphodiesterase in a dose- and Ca2(+)-dependent manner and stimulated NAD kinase from peas in a dose-dependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzyme-linked immunoabsorbance assays. The amino acid composition data showed it to be distinctly different from eucaryotic calmodulins, having particularly high levels of serine and glycine. The pI of the protein was estimated to be 4.9 to 5.0. The molecular weight was estimated to be 23,000 or 25,000, based on amino acid composition and detergent gel electrophoresis, respectively. The protein reacted with rhodamine isothiocyanate, which blocked its enzyme-activating capacity and greatly increased its electrophoretic mobility and Coomassie dye-binding ability.
Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium
Authors: Collins L V , Hackett J
Published: November 2016

The rfc gene of Salmonella typhimurium was located in a 1.75-kb HindIII fragment and restored wild-type lipopolysaccharide synthesis ability to both an older rfc point mutant and new rfc::IS10 mutants. DNA sequencing of the HindIII fragment revealed an open reading frame which could encode a protein of 407 amino acids with an Mr of 47,472 and also revealed potential translation signals. Modulator codons accounted for 12.5% of the total codon content, providing a possible explanation for the nondetectability of the protein in subcellular systems. Secondary structure analysis suggested the presence of transmembrane beta-sheet structures, implying a possible role for the protein in translocation of hydrophilic O-antigen-containing materials. Salmonella strains of groups A, B, and D1 contained rfc-homologous DNA, but strains of groups C1, C2, C3, D2, and E2 did not.
Construction and analysis of deletions in the structural gene (uvrD) for DNA helicase II of Escherichia coli
Authors: Washburn B K , Kushner S R
Published: November 2016

DNA helicase II, the product of the uvrD gene, has been implicated in DNA repair, replication, and recombination. Because the phenotypes of individual uvrD alleles vary significantly, we constructed deletion-insertion mutations in the uvrD gene to determine the phenotype of cells lacking DNA helicase II. Deletion mutants completely lacking the protein, as well as one which contains a truncated protein retaining the ATP-binding site, remained viable. However, they were sensitive to UV light and exhibited elevated levels of homologous recombination and spontaneous mutagenesis. In addition, mutations mapping in or near rep which allow construction of rep uvrD double mutants at a high frequency were isolated.
The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III
Authors: Hansson M , Rutberg L , Schröder I , Hederstedt L
Published: November 2016

We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.
The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene
Authors: Cooley M B , D'Souza M R , Kado C I
Published: November 2016

The ros chromosomal gene is present in octopine and nopaline strains of Agrobacterium tumefaciens as well as in Rhizobium meliloti. This gene encodes a 15.5-kDa protein that specifically represses the virC and virD operons in the virulence region of the Ti plasmid. The ros gene was cloned from a genomic bank by electroporation and complementation in Agrobacterium cells. Reporter fusion to the ros gene indicates that the level of transcription is controlled in part by autoregulation. A consensus inverted repeat sequence present in the ros promoter and in the virC and virD promoters of pTiC58, pTiA6, and pRiA4b suggests that a specific Ros binding site exists in these promoters. In the virC and virD promoter region, this binding site is within a cluster of vir box consensus sequences in which the VirG protein binds. This suggests possible binding competition between Ros and VirG at the virC and virD promoters. That the Ros protein binds DNA is suggested by the presence of a 'zinc finger' consensus sequence in the protein.
Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome
Authors: Kulakauskas S , Wikström P M , Berg D E
Published: November 2016

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.
Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation
Authors: Keasling J D , Palsson B O , Cooper S
Published: November 2016

F plasmid replication during the Escherichia coli division cycle was investigated by using the membrane-elution technique to produce cells labeled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The F plasmid replicated, like the minichromosome, during a restricted portion of the bacterial division cycle; i.e., F plasmid replication is cell-cycle specific. The F plasmid replicated at a different time during the division cycle than a minichromosome present in the same cell. F plasmid replication coincided with doubling in the rate of enzyme synthesis from a plasmid-encoded gene. When the cell cycle age of replication of the F plasmid was determined over a range of growth rates, the cell size at which the F plasmid replicated followed the same rules as did replication of the bacterial chromosome--initiation occurred when a constant mass per origin was achieved--except that the initiation mass per origin for the F plasmid was different from that for the chromosome origin. In contrast, the high-copy mini-R6K plasmid replicated throughout the division cycle.
Increased ATP-dependent proteolytic activity in lon-deficient Escherichia coli strains lacking the DnaK protein
Authors: Kroh H E , Simon L D
Published: November 2016

Extracts made from Escherichia coli null dnaK strains contained elevated levels of ATP-dependent proteolytic activity compared with levels in extracts made from dnaK+ strains. This ATP-dependent proteolytic activity was not due to Lon, Clp, or Alp-associated protease. Comparison of the levels of ATP-dependent proteolytic activity present in lon rpoH dnaK mutants and in lon rpoH dnaK+ mutants showed that the level of ATP-dependent proteolytic activity was elevated in the lon rpoH dnaK mutant strain. These findings suggest that DnaK negatively regulates a new ATP-dependent proteolytic activity, independently of sigma 32. Other results indicate that an ATP-dependent proteolytic activity was increased in a lon alp strain after heat shock. It is not yet known whether the same protease is associated with the increased ATP-dependent proteolytic activity in the dnaK mutants and in the heat-shocked lon alph strain.
Formation of factor 390 by cell extracts of Methanosarcina barkeri
Authors: van de Wijngaard W M , vermey P , van der Drift C
Published: November 2016

Cell extracts of Methanosarcina barkeri converted coenzyme F420 in an ATP-dependent reaction to the adenylylated derivative factor 390. Although it was reported previously (L. M. Gloss and R. P. Hausinger, BioFactors 1:237-240, 1988) that whole cells were unable to perform this conversion, we observed the conversion in 7 of 11 extracts, all of which were prepared from different batches of cells.
Molecular cloning of the 5-aminolevulinic acid dehydratase gene from Rhodobacter sphaeroides
Authors: Delaunay A M , Huault C , Balangé A P
Published: November 2016

A hemB mutant of Escherichia coli was used to clone the gene encoding 5-aminolevulinic acid dehydratase from Rhodobacter sphaeroides after physiological complementation of the mutation. A 2.9-kb DNA fragment was obtained and cloned in both orientations into the unique PstI restriction site of pUC19. This recombinant plasmid encodes a protein (Mr 39,000) that is immunoreactive with antibodies raised against the enzyme from higher plants.
The single-stranded-DNA-binding protein encoded by the Escherichia coli F factor can complement a deletion of the chromosomal ssb gene
Authors: Porter R D , Black S
Published: November 2016

Genes encoding single-stranded-DNA-binding proteins (SSBs) are carried by a variety of large self-transmissible plasmids, and it previously has been shown that these plasmid-borne genes can complement conditional lethal alleles of the ssb gene on the Escherichia coli chromosome for cellular viability. We have tested one of the plasmid-borne ssb genes, the ssf gene from the E. coli F factor, for its ability to complement total deletion of the chromosomal ssb gene for viability. We have found that ssf can complement the ssb deletion, but only when it is present on a high-copy-number plasmid. Cells that are totally dependent on the F-factor-encoded SSB for viability manifest growth properties indicative of problems in DNA replication.
Hydrogen-oxidizing electron transport components in the hyperthermophilic archaebacterium Pyrodictium brockii
Authors: Pihl T D , Black L K , Schulman B A , Maier R J
Published: November 2016

The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105 degrees C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by S0 to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Q10 or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.
Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius
Authors: Stutzman-Engwall K J , Otten S L , Hutchinson C R
Published: November 2016

Two DNA segments, dnrR1 and dnrR2, from the Streptomyces peucetius ATCC 29050 genome were identified by their ability to stimulate secondary metabolite production and resistance. When introduced into the wild-type ATCC 29050 strain, the 2.0-kb dnrR1 segment caused a 10-fold overproduction of epsilon-rhodomycinone, a key intermediate of daunorubicin biosynthesis, whereas the 1.9-kb dnrR2 segment increased production of both epsilon-rhodomycinone and daunorubicin 10- and 2-fold, respectively. In addition, the dnrR2 segment restored high-level daunorubicin resistance to strain H6101, a daunorubicin-sensitive mutant of S. peucetius subsp. caesius ATCC 27952. Analysis of the sequence of the dnrR1 fragment revealed the presence of two closely situated open reading frames, dnrI and dnrJ, whose deduced products exhibit high similarity to the products of several other Streptomyces genes that have been implicated in the regulation of secondary metabolism. Insertional inactivation of dnrI in the ATCC 29050 strain with the Tn5 kanamycin resistance gene abolished epsilon-rhodomycinone and daunorubicin production and markedly decreased resistance to daunorubicin. Sequence comparison between the products of dnrIJ and the products of the Streptomyces coelicolor actII-orf4, afsR, and redD-orf1 genes and of the Streptomyces griseus strS, the Saccharopolyspora erythraea eryC1, and the Bacillus stearothermophilus degT genes reveals two families of putative regulatory genes. The members of the DegT, DnrJ, EryC1, and StrS family exhibit some of the features characteristic of the protein kinase (sensor) component of two-component regulatory systems from other bacteria (even though none of the sequences of these four proteins show a significant overall or regional similarity to such protein kinases) and have a consensus helix-turn-helix motif typical of DNA binding proteins. A helix-turn-helix motif is also present in two of the proteins of the other family, AfsR and RedD-Orf1. Both sets of Streptomyces proteins are likely to be trans-acting factors involved in regulating secondary metabolism.
In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA
Authors: Masker W
Published: November 2016

A double-strand break in a bacteriophage T7 genome significantly reduced the ability of that DNA to produce viable phage when the DNA was incubated in an in vitro DNA replication and packaging system. When a homologous piece of T7 DNA (either a restriction fragment or T7 DNA cloned into a plasmid) that was by itself unable to form a complete phage was included in the reaction, the break was repaired to the extent that many more viable phage were produced. Moreover, repair could be completed even when a gap of about 900 nucleotides was put in the genome by two nearby restriction cuts. The repair was accompanied by acquisition of a genetic marker that was present only on the restriction fragment or on the T7 DNA cloned into a plasmid. These data are interpreted in light of the double-strand gap repair mode of recombination.
Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan
Authors: Watanabe T , Kasahara N , Aida K , Tanaka H
Published: November 2016

Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.
A zinc finger protein from Candida albicans is involved in sucrose utilization
Authors: Kelly R , Kwon-Chung K J
Published: November 2016

A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans.
Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS
Authors: Fetzner S , Müller R , Lingens F
Published: November 2016

The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS were purified to homogeneity. Yellow component B is a monomer (Mr, 37,500) with NADH-acceptor reductase activity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Component B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 mol of iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2. Component B was reduced by the addition of NADH. Red-brown component A (Mr, 200,000 to 220,000) is an iron-sulfur protein containing 5.8 mol of iron and 6.0 mol of acid-labile sulfide. The isoelectric point was within the range of pH 4.5 to 5.3. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. Component A consisted of nonidentical subunits alpha (Mr, 52,000) and beta (Mr, 20,000). It contained approximately equimolar amounts of alpha and beta, and cross-linking studies suggested an alpha 3 beta 3 subunit structure of component A. The NADH- and Fe(2+)-dependent enzyme system was named 2-halobenzoate 1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate to catechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogs with electron-withdrawing substituents at the ortho position were transformed preferentially.
Lamina, a novel multicellular form of Methanosarcina mazei S-6
Authors: Mayerhofer L E , Macario A J , de Macario E C
Published: November 2016

A novel multicellular form of Methanosarcina mazei S-6 is described. It was termed lamina, and it formed during the exponential growth phase when packets or single cells were grown in 40 mM trimethylamine and a total concentration of 8.3 to 15.6 mM Ca2+ and/or Mg2+, in cultures that were not shaken. A distinct molecular event represented by the increment in expression and a spatial redistribution of an antigen during lamina formation is documented.
Characterization of lipopolysaccharide fractions and their interactions with cells and model membranes
Authors: Yeh H Y , Jacobs D M
Published: November 2016

The role of the length of the O-antigen polysaccharide side chain of bacterial lipopolysaccharide (LPS) in biological and model membrane systems was investigated. LPS from Salmonella typhimurium ATCC 14028 was chromatographed on a Sephadex G-200 column in the presence of sodium deoxycholate and separated into three fractions on the basis of molecular size. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot (immunoblot), and chemical analyses indicated that these fractions differed from each other primarily in the number of repeating units in the O-antigen polysaccharide side chain. In a biological system fractions 2 and 3 had the same effects to induce mitogenesis in murine lymphocytes, but fraction 1 was less effective than the other two fractions. In a model membrane system, LPS induced changes in small unilamellar vesicles (SUVs) which were measured by changes in the behavior of a fluorescent probe, 1,6-diphenylhexa-1,3,5-triene (DPH), and interaction of increasing amounts of all LPS fractions with SUVs gradually increased DPH anisotropy. Fractions 2 and 3 had similar effects on the SUVs as detected by changes in DPH anisotropy, while fraction 1 had almost twice as much activity as the other two fractions. These results suggest that the polysaccharide side chain of LPS may modulate the ability of biologically active lipid A to interact with cells and model membranes. In addition, factors other than changes in membrane fluidity may play a role in mediating LPS-induced cell activation.
New minC mutations suggest different interactions of the same region of division inhibitor MinC with proteins specific for minD and dicB coinhibition pathways
Authors: Mulder E , Woldringh C L , Tétart F , Bouché J P
Published: November 2016

Proper positioning of division sites in Escherichia coli requires balanced expression of minC, minD, and minE gene products. Previous genetic analysis has shown that either MinD or an apparently unrelated protein, DicB, cooperates with MinC to inhibit division. We have isolated and sequenced minC mutations that suppress division inhibition caused by overproduction of either DicB or MinD proteins. Most missense mutations were located in the amino acid 160 to 200 region of MinC (231 amino acids). Some mutations exhibited preferential resistance to one or the other coinhibitor, suggesting that two distinct proteins, possibly MinD and DicB themselves, interact in slightly different manners with the same region of MinC to promote division inhibition.
Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion
Authors: Cooper T G , Kovari L , Sumrada R A , Park H D , Luche R M , Kovari I
Published: November 2016

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.
Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli
Authors: Srivastava S K , Cannistraro V J , Kennell D
Published: November 2016

Crude extracts from Escherichia coli were screened for any broad-specificity endoribonuclease after the cell proteins were fractionated by size. In a mutant lacking the gene for RNase I (molecular mass, 27,156 Da), the only such activities were also in the size range of 23 to 28 kDa. Fractionation by chromatography on a strong cation-exchange resin revealed only two activities. One of them eluted at a salt concentration expected for RNase M and had the specificity of RNase M. It preferred pyrimidine-adenosine bonds, could not degrade purine homopolymers, and had a molecular mass of approximately 27 kDa (V. J. Cannistraro and D. Kennell, Eur. J. Biochem. 181:363-370, 1989). A second fraction, eluting at a higher salt concentration, was active against any phosphodiester bond but was about 100 times less active than are RNase I and RNase I* (a form of RNase I) in the wild-type cell. On the basis of sizing-gel chromatography, this enzyme had a molecular mass of approximately 24 kDa. We call it RNase R (for residual). RNase R is not an abnormal product of the mutant rna gene; a cell carrying many copies of that gene on a plasmid did not synthesize more RNase R. Our search for broad-specificity endoribonucleases was prompted by the expectation that the primary activities for mRNA degradation are expressed by a relatively small number of broad-specificity RNases. If correct, the results suggest that the endoribonucleases for this major metabolic activity reside in the 24- to 28-kDa size range. Endoribonucleases with much greater specificity must have as primary functions the processing of specific RNA molecules at a very limited number of sites as steps in their biosynthesis. In exceptional cases, these endoribonucleases inactivate a specific message that has such a site, and they can also effect total mRNA metabolism indirectly by a global disturbance of the cell physiology. It is suggested that a distinction be made between these processing and degradative activities.
Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1
Authors: Chistoserdova L V , Lidstrom M E
Published: November 2016

The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.