bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence and molecular characterization of pnlA, the structural gene for damage-inducible pectin lyase of Erwinia carotovora subsp. carotovora 71
Authors: Chatterjee A , McEvoy J L , Chambost J P , Blasco F , Chatterjee A K
Published: November 2016

In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N-terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins.
Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli
Authors: Braaten B A , Blyn L B , Skinner B S , Low D A
Published: November 2016

Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states. Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter. DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site. papI and papB are two regulatory genes in the pap operon. Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site. We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites. The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E. coli chromosome. The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter. E. coli containing mbf::mTn10 and phase-off mbf+ E. coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E. coli cells. These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses. Moreover, transposon insertion within mbf greatly reduced Pap pilus expression. The mbf locus was isolated on a low-copy-number cosmid, pMBF1. Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon. The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states.
Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity
Authors: Ikeda T , LaPorte D C
Published: November 2016

For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase. The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme's activity and thus forcing isocitrate through the bypass. This phosphorylation cycle is catalyzed by a bifunctional enzyme, IDH kinase/phosphatase, which is encoded by aceK. We have employed random mutagenesis to isolate novel alleles of aceK. These alleles were detected by the loss of ability to complement an aceK null mutation. The products of one class of these alleles retain IDH kinase activity but have suffered reductions in IDH phosphatase activity by factors of 200 to 400. Selective loss of the phosphatase activity also appears to have occurred in vivo, since cells expressing these alleles exhibit phenotypes which are reminiscent of strains lacking IDH; these strains are auxotrophic for glutamate. Assays of cell-free extracts confirmed that this phenotype resulted from nearly quantitative phosphorylation of IDH. The availability of these novel alleles of aceK allowed us to assess the significance of the precise control which is a characteristic of the IDH phosphorylation cycle in vivo. The fractional phosphorylation of IDH was varied by controlled expression of one of the mutant alleles, aceK3, in a wild-type strain. Reduction of IDH activity to 50% of the wild-type level did not adversely affect growth on acetate. However, further reductions inhibited growth, and growth arrest occurred when the IDH activity fell to 15% of the wild-type level. Thus, although wild-type cells maintain a precise effective IDH activity during growth on acetate, this precision is not critical.
The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans
Authors: Szekeres M , Droux M , Buchanan B B
Published: November 2016

The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form.
A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens
Authors: Zhang L H , Kerr A
Published: November 2016

Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h. A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A. tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate. The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer. Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations. The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium. The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF. Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound. CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation. Data suggest that CF might be an activator or derepressor in the conjugation system of A. tumefaciens. CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity.
The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation
Authors: Mysliwiec T H , Errington J , Vaidya A B , Bramucci M G
Published: November 2016

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.
Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences
Authors: Ng W L , Kothakota S , DasSarma S
Published: November 2016

Halobacterium-halobium NRC-1 harbors a 200-kb plasmid, pNRC100, which contains a cluster of genes for synthesis of buoyant gas-filled vesicles. Physical mapping of pNRC100 by using pulsed-field gel electrophoresis showed the presence of a large (35 to 38-kb) inverted repeat (IR) sequence. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single-copy region and the large single-copy region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. Additionally, the identities and approximate positions of 17 insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. The large IRs terminated at an ISH2 element at one end and an ISH3 element at the other end. pNRC100 is similar in structure to chloroplast and mitochondrial genomes, which contain large IRs and other large halobacterial and prokaryotic plasmids that are reservoirs of IS elements but lack the large IRs.
Partial characterization of a lysU mutant of Escherichia coli K-12
Authors: Hassani M , Saluta M V , Bennett G N , Hirshfield I N
Published: November 2016

The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity. Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent. When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed. The lysU mutation was transferred to other E. coli K-12 backgrounds by using P1 transduction. The lysU transductants behaved comparably to their lysU+ parents at different growth temperatures. Therefore, the LysU proteins does not appear to be essential for growth at high temperatures, at least under the conditions examined here. In addition, lysU transductants were found to be defective for inducible lysine decarboxylase, (LDC), inducible arginine decarboxylase (ADI), and melibiose utilization (Mel), which are all missing in GNB10181. Complementation of the above missing functions was achieved by using the Clarke-Carbon plasmids pLC4-5 (LysU LDC) and pLC17-38 (LysU Mel ADI). From these experiments, it appears that GNB10181 has suffered a chromosomal deletion between 93.4 and 93.7 min, which includes the lysU gene. By using plasmid pLC17-38, the position of ADI on two-dimensional gels was identified. Finally, lysS delta lysU double mutants were constructed which can potentially be used as positive selection agents for the isolation of LysRS genes from other sources.
Role of RpoH, a heat shock regulator protein, in Escherichia coli carbon starvation protein synthesis and survival
Authors: Jenkins D E , Auger E A , Matin A
Published: November 2016

Escherichia coli starvation proteins include several heat shock proteins whose induction by heat is controlled by the minor sigma factor, sigma 32. The level of sigma 32 increased in wild-type E. coli upon starvation, and three sigma 32-controlled heat shock proteins (DnaK, GroEL, and HtpG) were not induced during starvation in an isogenic delta rpoH strain, which is unable to synthesize sigma 32. Thus, sigma 32 plays a role in the induction of these proteins during both heat shock and starvation. The delta rpoH strain was more sensitive to starvation but could develop starvation-mediated cross protection against heat and oxidation.
Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae
Authors: Zinser E , Sperka-Gottlieb C D , Fasch E V , Kohlwein S D , Paltauf F , Daum G
Published: November 2016

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.
Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides
Authors: Falcone D L , Tabita F R
Published: November 2016

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes.
A unique repetitive DNA sequence in the Myxococcus xanthus genome
Authors: Fujitani S , Komano T , Inouye S
Published: November 2016

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.
A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK
Authors: Lennon E , Gutman P D , Yao H L , Minton K W
Published: November 2016

A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control.
Identification of the promoter region of the Escherichia coli major cold shock gene, cspA
Authors: Tanabe H , Goldstein J , Yang M , Inouye M
Published: November 2016

The major cold shock protein of Escherichia coli, CS7.4, is produced at a level of 13% of total protein synthesis upon a temperature shift from 37 to 10 degrees C. The transcription of its gene (cspA) was found to be tightly regulated and induced only at low temperature. In addition, the cspA mRNA was extremely unstable at 37 degrees C, so that CS7.4 production was hardly detected when the culture temperature was shifted from 15 degrees C to 37 degrees C. The transcription initiation site (+1) was identified. In vivo footprinting demonstrated that the region from bases -35 to -73 was protected from chemical modification, and gel mobility shift analysis showed that a cold-shocked cell extract contained a factor(s) specifically bound to the fragment containing the sequence between bases -63 and -92. This factor was synthesized de novo only at low temperature, and its synthesis was inhibited by chloramphenicol. Possible functions of this factor are discussed.
Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol
Authors: Kamlage B , Blaut M
Published: November 2016

Methanosarcina strain Gö1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain Gö1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.
Cloning and nucleotide sequence of the leucyl-tRNA synthetase gene of Bacillus subtilis
Authors: Vander Horn P B , Zahler S A
Published: November 2016

The leucyl-tRNA synthetase gene (leuS) of Bacillus subtilis was cloned and sequenced. A mutation in the gene, leuS1, increases the transcription and expression of the ilv-leu operion, permitting monitoring of leuS alleles. The leuS1 mutation was mapped to 270 degrees on the chromosome. Sequence analysis showed that the mutation is a single-base substitution, possibly in a monocistronic operon. The leader mRNA predicted by the sequence would contain a number of possible secondary structures and a T box, a sequence observed upstream of leader mRNA terminators of Bacillus tRNA synthetases and the B. subtilis ilv-leu operon. The DNA of the B. subtilis leuS open reading frame is 48% identical to the leuS gene of Escherichia coli and is predicted to encode a polypeptide with 46% identity to the leucyl-tRNA synthetase of E. coli.
Mutational analysis of signal transduction by ArcB, a membrane sensor protein responsible for anaerobic repression of operons involved in the central aerobic pathways in Escherichia coli
Authors: Iuchi S , Lin E C
Published: November 2016

In Escherichia coli, the expression of a group of operons involved in aerobic metabolism is regulated by a two-component signal transduction system in which the arcB gene specifies the membrane sensor protein and the arcA gene specifies the cytoplasmic regulator protein. ArcB is a large protein belonging to a subclass of sensors that have both a transmitter domain (on the N-terminal side) and a receiver domain (on the C-terminal side). In this study, we explored the essential structural features of ArcB by using mutant analysis. The conserved His-292 in the transmitter domain is indispensable, indicating that this residue is the autophosphorylation site, as shown for other homologous sensor proteins. Compression of the range of respiratory control resulting from deletion of the receiver domain and the importance of the conserved Asp-533 and Asp-576 therein suggest that the domain has a kinetic regulatory role in ArcB. There is no evidence that the receiver domain enhances the specificity of signal transduction by ArcB. The defective phenotype of all arcB mutants was corrected by the presence of the wild-type gene. We also showed that the expression of the gene itself is not under respiratory regulation.
Physical map of the genome of Rhodobacter capsulatus SB 1003
Authors: Fonstein M , Zheng S , Haselkorn R
Published: November 2016

A map of the chromosome of Rhodobacter capsulatus was constructed by overlapping the large restriction fragments generated by endonucleases AseI and XbaI. The analyses were done by hybridization of single fragments with the restriction fragments blotted from pulsed-field gels and by grouping cosmids of a genomic library of R. capsulatus into contigs, corresponding to the restriction fragments, and further overlapping of the contigs. A technical difficulty due to a repeated sequence made it necessary to use hybridization with cloned genes and prior knowledge of the genetic map in order to close the physical circle in a unique way. In all, 41 restriction sites were mapped on the 3.6-Mb circular genome and 22 genes were positioned at 26 loci of the map. Cosmid clones were grouped in about 80 subcontigs, forming two groups, one corresponding to the chromosome of R. capsulatus and the other corresponding to a 134-kb plasmid. cos site end labeling and partial digestion of cosmids were used to construct a high-resolution EcoRV map of the 134-kb plasmid. The same method can be extended to the entire chromosome. The cosmid clones derived in this work can be used as a hybridization panel for the physical mapping of new genes as soon as they are cloned.
Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG
Authors: Pazour G J , Ta C N , Das A
Published: November 2016

The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules. A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers. Mutants were isolated at a frequency of 1 in 10(7) to 10(8). Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype. One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106. In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52. Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype. In contrast, several substitutions at residue 106 led to a constitutive phenotype. The possible roles of the residues at positions 54 and 106 in VirG function are discussed.
Involvement of Fis protein in replication of the Escherichia coli chromosome
Authors: Filutowicz M , Ross W , Wild J , Gourse R L
Published: November 2016

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.
Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein
Authors: Arini A , Tuscan M , Churchward G
Published: November 2016

To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication.
The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids
Authors: Paterson E S , Iyer V N
Published: November 2016

The oriT region of the conjugative IncN plasmid pCU1 has been localized to a 669-bp sequence extending from pCU1 coordinates 8.48 to 9.15 kb. The nucleotide sequence of this region was determined. The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues. Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence. Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated. This specificity is encoded by the region lying clockwise of the BglII site at coordinate 3.3 on the pCU1 map. Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCU1 or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.
Identification of the gene (aroK) encoding shikimic acid kinase I of Escherichia coli
Authors: Løbner-Olesen A , Marinus M G
Published: November 2016

DNA sequence analysis has revealed that an unidentified open reading frame (ufr1) is present immediately preceding the aroB gene of Escherichia coli. The predicted protein product of urf1 contains a consensus ATP-binding-site sequence and shows 34% amino acid homology to shikimate kinase II in a 97-amino-acid region. Inactivation of urf1 by insertion of an antibiotic resistance gene had a polar effect on aroB, indicating that these two genes constitute a transcriptional unit. The auxotrophic requirements of a strain mutant for both urf1 and aroL (encoding shikimate kinase II) are consistent with shikimate kinase deficiency. We propose that urf1 encodes shikimate kinase I and that it be designated aroK.
Characterization of amino acid aminotransferases of Methanococcus aeolicus
Authors: Xing R Y , Whitman W B
Published: November 2016

Four aminotransferases were identified and characterized from Methanococcus aeolicus. Branched-chain aminotransferase (BcAT, EC 2.6.1.42), aspartate aminotransferase (AspAT, EC 2.6.1.1), and two aromatic aminotransferases (EC 2.6.1.57) were partially purified 175-, 84-, 600-, and 30-fold, respectively. The apparent molecular weight, substrate specificity, and kinetic properties of the BcAT were similar to those of other microbial BcATs. The AspAT had an apparent molecular weight of 162,000, which was unusually high. It had also a broad substrate specificity, which included activity towards alanine, a property which resembled the enzyme from Sulfolobus solfataricus. An additional alanine aminotransferase was not found in M. aeolicus, and this activity of AspAT could be physiologically significant. The apparent molecular weights of the aromatic aminotransferases (ArAT-I and ArAT-II) were 150,000 and 90,000, respectively. The methanococcal ArATs also had different pIs and kinetic constants. ArAT-I may be the major ArAT in methanococci. High concentrations of 2-ketoglutarate strongly inhibited valine, isoleucine, and alanine transaminations but were less inhibitory for leucine and aspartate transaminations. Aromatic amino acid transaminations were not inhibited by 2-ketoglutarate. 2-Ketoglutarate may play an important role in the regulation of amino acid biosynthesis in methanococci.
Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni
Authors: Brown K L , Whiteley H R
Published: November 2016

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.
Characterization of spoIVA, a sporulation gene involved in coat morphogenesis in Bacillus subtilis
Authors: Roels S , Driks A , Losick R
Published: November 2016

We report the cloning and characterization of the Bacillus subtilis sporulation locus spoIVA, mutations at which cause an unusual defect in spore formation in which the coat misassembles as swirls within the mother cell. We show that spoIVA is a single gene of 492 codons that is capable of encoding a polypeptide of 55 kDa. Transcription of spoIVA is induced at about the second hour of sporulation by the regulatory protein sigma E from two closely spaced promoters designated P1 and P2. Experiments in which the upstream promoter P1 was removed show that transcription of spoIVA from P2 is sufficient for efficient spore formation. Based on these and other findings, we infer that the spoIVA gene product is a morphogenetic protein; we discuss its role in the deposition of coat polypeptides around the developing forespore.
Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo
Authors: Iost I , Guillerez J , Dreyfus M
Published: November 2016

We show that in Escherichia coli at 32 degrees C, the T7 RNA polymerase travels over the lacZ gene about eightfold faster than ribosomes travel over the corresponding mRNA. We discuss how the T7 phage might exploit this high rate in its growth optimization strategy and how it obviates the possible drawbacks of uncoupling transcription from translation.
CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase, which is involved in the synthesis of the major wall teichoic acid in Bacillus subtilis 168, is encoded by tagF (rodC)
Authors: Pooley H M , Abellan F X , Karamata D
Published: November 2016

Assays of CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (CGPTase) (EC 2.7.8.12) in membranes isolated from Bacillus subtilis 168 wild type and 11 strains bearing conditional lethal thermosensitive mutations in tagB, tagD, or tagF revealed that CGPTase deficiency was associated only with mutant tagF alleles. In vitro, thermosensitivity of CGPTase strongly suggests that the structural gene for this enzyme is tagF. We discuss apparent discrepancies between biochemical evidence favoring a membrane location for TagF and a previous report that suggested a cytoplasmic location based on sequence analysis.
Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene
Authors: Mérida A , Flores E , Florencio F J
Published: November 2016

The glnA gene from Synechocystis sp. strain PCC 6803 was cloned by hybridization with the glnA gene from Anabaena sp. strain PCC 7120, and a deletion-insertion mutation of the Synechocystis gene was generated in vitro. A strain derived from Synechocystis sp. strain PCC 6803 which contained integrated into the chromosome, in addition to its own glnA gene, the Anabaena glnA gene was constructed. From that strain, a Synechocystis sp. glnA mutant could be obtained by transformation with the inactivated Synechocystis glnA gene; this mutant grew by using Anabaena glutamine synthetase and was not a glutamine auxotroph. A Synechocystis sp. glnA mutant could not be obtained, however, from the wild-type Synechocystis sp. The Anabaena glutamine synthetase enzyme was subject to ammonium-promoted inactivation when expressed in the Synechocystis strain but not in the Anabaena strain itself.
Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica)
Authors: Upadhyaya N M , Glare T R , Mahanty H K
Published: November 2016

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.
Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene
Authors: Blanche F , Thibaut D , Famechon A , Debussche L , Cameron B , Crouzet J
Published: November 2016

Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-3H]S-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr congruent to 27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr congruent to 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 +/- 0.2 microM for precorrin-6x and 23.5 +/- 3.5 microM for NADPH and a Vmax value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand.
Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the cobL gene product
Authors: Blanche F , Famechon A , Thibaut D , Debussche L , Cameron B , Crouzet J
Published: November 2016

A protein catalyzing methylation at C-5 and C-15 and decarboxylation of the acetic acid side chain at C-12 on precorrin-6y to yield precorrin-8x was purified to homogeneity from a recombinant strain of Pseudomonas denitrificans. It was sequenced at the N terminus and shown to be encoded by the cobL gene.
Replication of the R6K plasmid during the Escherichia coli cell cycle
Authors: Keasling J D , Palsson B O , Cooper S
Published: November 2016

The cell-cycle replication pattern of the R6K plasmid has been investigated by using the membrane-elution technique to produce cells labelled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The high-copy plasmid R6K replicates exponentially in a cell-cycle-independent manner. A mini-R6K plasmid deleted for the ori alpha origin of replication also replicates, exponentially in a cell-cycle-independent manner.
Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo
Authors: Gutowski J C , Schreier H J
Published: November 2016

In vivo dimethyl sulfate footprinting of the Bacillus subtilis glnRA regulatory region under repressing and derepressing conditions demonstrated that the GlnR protein, encoded by glnR, interacts with two sites situated within and adjacent to the glnRA promoter. One site, glnRAo1, between positions -40 and -60 relative to the start point of transcription, is a 21-bp symmetrical element that has been identified as essential for glnRA regulation (H. J. Schreier, C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi, J. Mol. Biol. 220:241-253, 1991). The second site, glnRAo2, is a quasisymmetrical element having partial homology to glnRAo1 and is located within the promoter between positions -17 and -37. The symmetry and extent of modifications observed for each site during repression and derepression indicated that GlnR interacts with the glnRA regulatory region by binding to both sites in approximately the same manner. Experiments using potassium permanganate to probe open complex formation by RNA polymerase demonstrated that transcriptional initiation is inhibited by GlnR. Furthermore, distortion of the DNA helix within glnRAo2 occurred upon GlnR binding. While glutamine synthetase, encoded by glnA, has been implicated in controlling glnRA expression, analyses with dimethyl sulfate and potassium permanganate ruled out a role for glutamine synthetase in directly influencing transcription by binding to operator and promoter regions. Our results suggested that inhibition of transcription from the glnRA promoter involves GlnR occupancy at both glnRAo1 and glnRAo2. In addition, modification of bases within the glnRAo2 operator indicated that control of glnRA expression under nitrogen-limiting (derepressing) conditions included the involvement of a factor(s) other than GlnR.
Analysis of a Caulobacter crescentus gene cluster involved in attachment of the holdfast to the cell
Authors: Kurtz H D , Smith J
Published: November 2016

Caulobacter crescentus firmly adheres to surfaces with a structure known as the holdfast, which is located at the flagellar pole of swarmer cells and at the stalk tip in stalked cells. A three-gene cluster (hfaAB and hfaC) is involved in attachment of the holdfast to the cell. Deletion and complementation analysis of the hfaAB locus revealed two genes in a single operon; both were required for holdfast attachment to the cell. Sequence analysis of the hfaAB locus showed two open reading frames with the potential to encode proteins of 15,000 and 26,000 Da, respectively. A protein migrating with an apparent size of 21 kDa in gel electrophoresis was encoded by the hfaA region when expressed in Escherichia coli under the control of the lac promoter, but no protein synthesis could be detected from the hfaB region. S1 nuclease analysis indicated that transcription of the hfaAB locus was initiated from a region containing a sequence nearly identical to the consensus for C. crescentus sigma 54-dependent promoters. In addition, a sequence with some similarity to ftr sequences (a consensus sequence associated with other Caulobacter sigma 54-dependent genes) was identified upstream of the hypothesized sigma 54 promoter. At least one of the hfaAB gene products was required for maximal transcription of hfaC. The sequence of hfaB showed some similarity to that of transcriptional activators of other bacteria. The C-terminal region of the putative gene product HfaA was found to be homologous to PapG and SmfG, which are adhesin molecules of enteropathogenic E. coli and Serratia marcescens, respectively. This information suggests that the protein encoded by the hfaA locus may have a direct role in the attachment of the holdfast to the cell, whereas hfaB may be involved in the positive regulation of hfaC.
Propagation of pSC101 plasmids defective in binding of integration host factor
Authors: Biek D P , Cohen S N
Published: November 2016

Integration host factor (IHF), a multifunctional protein of E. coli, normally is required for the replication of plasmid pSC101. T. T. Stenzel, P. Patel, and D. Bastia (Cell 49:709-717, 1987) have reported that IHF binds to a DNA locus near the pSC101 replication origin and enhances a static bend present in this region; mutation of the IHF binding site affects the plasmid's ability to replicate. We report here studies indicating that the requirement for IHF binding near the pSC101 replication origin is circumvented partially or completely by (i) mutation of the plasmid-encoded repA (replicase) gene or the chromosomally encoded topA gene, (ii) the presence on the plasmid of the pSC101 partition (par) locus, or (iii) replacement of the par locus by a strong transcriptional promoter. With the exception of the repA mutation, the factors that substitute for a functional origin region IHF binding site are known to alter plasmid topology by increasing negative DNA supercoiling, as does IHF itself. These results are consistent with the proposal that IHF binding near the pSC101 replication origin promotes plasmid replication by inducing a conformational change leading to formation of a repA-dependent DNA-protein complex. A variety of IHF-independent mechanisms can facilitate formation of the putative replication-initiation complex.