bluuebunny/crossref_metadata_2025 huggingface dataset:
The Bacillus subtilis spo0B stage 0 sporulation operon encodes an essential GTP-binding protein
Authors: Trach K , Hoch J A
Published: November 2016

Transcription of the spo0B gene and genes downstream of it was investigated by S1 nuclease protection experiments. The spo0B gene was transcribed from a single promoter, and this transcript extended through a gene, obg, coding for a 47,668 Mr protein. Transcription of this operon ended in a stem-loop structure. The sequence of the deduced obg protein contained a region with homology to known GTP-binding proteins in the nucleotide-binding regions. The amino-terminal portion of this protein showed homology to mammalian collagen, suggesting a structural role. The purified obg protein was shown to bind [alpha-32P]GTP in vitro. Several attempts to inactivate the obg gene were unsuccessful, indicating that the obg gene product was essential for growth. The possible function of this protein and its relationship to RAS-like proteins and sporulation was discussed. Immediately downstream of the obg gene were two genes involved in phenylalanine biosynthesis, pheB and pheA. The pheA gene coded for monofunctional prephenate dehydratase, on the basis of the high homology of the deduced amino acid sequence to prephenate dehydratases of bacterial origin. The sequence of the pheB gene product was not homologous to chorismate mutase, and its function remains unknown. Transcription of the phe genes was shown to begin at the stem-loop structure between obg and pheB. The possibility was entertained that phe gene transcription arises from processing or antitermination of the spo0B transcript.
Cloning, purification, and properties of Candida albicans thymidylate synthase
Authors: Singer S C , Richards C A , Ferone R , Benedict D , Ray P
Published: November 2016

The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIII DNA fragment and was shown to be expressed in a Thy- strain of Escherichia coli. The nucleotide sequence of the TS gene predicted a protein of 315 amino acids with a molecular weight of 36,027. The gene was cloned into a T7 expression vector in E. coli, allowing purification of large amounts of C. albicans TS. It was also purified from a wild-type C. albicans strain. Comparison of several enzyme properties including analysis of amino-terminal amino acid sequences showed the native and cloned C. albicans TS to be the same.
Preparation and characterization of monoclonal antibodies against native membrane-bound penicillin-binding protein 1B of Escherichia coli
Authors: Den Blaauwen T , Wientjes F B , Kolk A H , Spratt B G , Nanninga N
Published: November 2016

We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.
DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23
Authors: Lewis P J , Wake R G
Published: November 2016

Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).
Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin
Authors: Miyakawa T , Oka Y , Tsuchiya E , Fukui S
Published: November 2016

A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated. The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2+ and CaM. The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist. When the kinase was incubated in the presence of ATP, Ca2+, and CaM before the assay, the enzyme showed activity even in the presence of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and TFP. The conversion to this Ca2+- and CaM-independent form occurred very rapidly under the incubation conditions required for protein phosphorylation by the kinase. At the highest level of conversion, Ca2+- and CaM-independent kinase activity, which was measured in the presence of EGTA and TFP, was nearly equal to the total kinase activity, which was measured in the presence of Ca2+ and CaM. A protein with a molecular weight of 58,000 was the major species that was phosphorylated in a Ca2+- and CaM-dependent manner by incubation of the CaM affinity-purified proteins with [gamma-32P]ATP. The protein kinase activity of the protein with the same molecular weight was demonstrated by in situ protein phosphorylation in sodium dodecyl sulfate-polyacrylamide gels by using casein as the substrate, after removal of the detergent from electrophoresed CaM-binding proteins. These data indicate that phosphorylation of the kinase is responsible for the conversion of enzyme activity. Enzyme regulation by this mode may play an important role in integrating cellular functions during the cell cycle. A possible role for the Ca2+-and CaM-dependent protein kinase in the signal transduction of the mating pheromone alpha factor is also discussed.
Genetic suppression of a dnaG mutation in Escherichia coli
Authors: Katayama T , Murakami Y , Wada C , Ohmori H , Yura T , Nagata T
Published: November 2016

Escherichia coli strains with a temperature-sensitive mutation, dnaG2903, in the primase-encoding gene spontaneously reverted to the temperature-insensitive phenotype at a high frequency. Many of the reversions were caused by extragenic sdg suppressors. About 100 independently isolated sdg suppressors were analyzed. They fall into two classes. The sdgA mutations were genetically mapped very close to and upstream of the dnaG gene and were found to be cis dominant. DNA sequencing of two of them revealed that G----A and C----A base substitutions had occurred 43 and 62 bases, respectively, upstream of the dnaG start codon. This region represents a transcriptional terminator thought to contribute to control of dnaG gene expression. The other class of suppressor, sdgB, seemed to comprise mutant alleles in the rpoB gene coding for the beta subunit of RNA polymerase core enzyme. Some of them were initially isolated as rifampin-resistant mutants. Both the sdgA and sdgB suppressors were found to increase the transcriptional activity of dnaG. This finding and other observations led to the proposition that sdgA and sdgB suppress the phenotype caused by dnaG2903 by overproducing the mutated primase; the quantitative oversupply may compensate for the qualitative defect of the dnaG2903 primase. An alternative mechanism of suppression by sdgB is discussed.
Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells
Authors: Hiraga S , Niki H , Ogura T , Ichinose C , Mori H , Ezaki B , Jaffé A
Published: November 2016

To study the chromosomal partitioning mechanism in cell division, we have isolated a novel type of Escherichia coli mutants which formed anucleate cells, by using newly developed techniques. One of them, named mukA1, is not lethal and produces normal-sized anucleate cells at a frequency of 0.5 to 3% of total cells in exponentially growing populations but does not produce filamentous cells. Results suggest that the mutant is defective in the chromosome positioning at regular intracellular positions and fails frequently to partition the replicated daughter chromosomes into both daughter cells, resulting in production of one anucleate daughter cell and one with two chromosomes. The mukA1 mutation causes pleiotropic effects: slow growth, hypersensitivity to sodium dodecyl sulfate, and tolerance to colicin E1 protein, in addition to anucleate cell formation. Cloning of the mukA gene indicates that the mukA1 mutation is recessive and that the mukA gene is identical to the tolC gene coding for an outer membrane protein.
Induction of saccharolytic enzymes by sucrose in Bacillus subtilis: evidence for two partially interchangeable regulatory pathways
Authors: Steinmetz M , Le Coq D , Aymerich S
Published: November 2016

Sucrose induces two saccharolytic enzymes in Bacillus subtilis, an intracellular sucrase and an extracellular levansucrase, encoded by sacA and sacB, respectively. It was previously shown that the sacY gene encodes a positive regulator involved in a sucrose-dependent antitermination upstream from the sacB coding sequence. We show here that the sacY product is not absolutely required for sacB induction: a weak but significant induction can be observed in strains harboring a sacY deletion. The sacY-independent induction was altered by mutations located in the sacP and sacT loci but was observed in both sacU+ and sacU32 genetic backgrounds. These results suggest that B. subtilis has two alternative systems allowing sacB induction by sucrose. Both systems also seem to be involved in sacA induction.
Site-directed mutation of the Escherichia coli ada gene: effects of substitution of methyl acceptor cysteine-321 by histidine in Ada protein
Authors: Tano K , Bhattacharyya D , Foote R S , Mural R J , Mitra S
Published: November 2016

Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins. In mutant I the methyl acceptor Cys-321 for O6-methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted. Neither mutant protein had O6-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69. Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein. On the other hand, mutant II protein was inducible by N-methyl-N'-nitro-N-nitrosoguanidine, although to a smaller extent than the wild-type protein was, and the phosphotriester-DNA methyltransferase activity appeared to reside in 24- to 30-kilodalton cleavage products. Mutant I protein could be produced under lac promoter control, and its cleavage products, unlike those of mutant II protein, tended to aggregate. These results indicate that (i) Cys-321 cannot be replaced or transposed with the nucleophilic amino acid histidine for O6-methylguanine-DNA methyltransferase function, (ii) single amino acid replacement or transposition at the O6-methylguanine methyl acceptor site can have a profound effect on the in vivo stability and regulatory function of the Ada protein, and (iii) the integrity of the protein may not be absolutely needed for its transcription-activation function.
Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli
Authors: Atlung T , Nielsen A , Hansen F G
Published: November 2016

A plasmid carrying a regulator gene, designated appY, was found in the screening of an Escherichia coli gene library for clones overproducing AppA, an acid phosphatase which is induced as a culture approaches the stationary phase. In cells containing multicopy plasmids carrying the appY gene, the expression of the chromosomal appY gene was stimulated 10- to 40-fold in the stationary phase and more than 100-fold during exponential growth. The appA plasmid also changed the rate of synthesis of more than 30 other proteins in a growth-phase-dependent way. The appY gene was mapped to 13 min on the E. coli genetic map. The position of the appY gene on the 4.9-kilobase HindIII fragment of the original clone was located by Tn5 mutagenesis and deletion analysis, and the nucleotide sequence of a 1.9-kilobase region containing the gene was determined. The appY gene product was identified as a weakly expressed 243-amino-acid polypeptide which contains a stretch of 20 amino acids showing very good similarity to the conserved DNA-binding domain of repressors and transcriptional activators.
Suppression of the Escherichia coli rpoH opal mutation by ribosomes lacking S15 protein
Authors: Yano R , Yura T
Published: November 2016

Several suppressors (suhD) that can specifically suppress the temperature-sensitive opal rpoH11 mutation of Escherichia coli K-12 have been isolated and characterized. Unlike the parental rpoH11 mutant deficient in the heat shock response, the temperature-resistant pseudorevertants carrying suhD were capable of synthesizing sigma 32 and exhibiting partial induction of heat shock proteins. These strains were also cold sensitive and unable to grow at 25 degrees C. Genetic mapping and complementation studies permitted us to localize suhD near rpsO (69 min), the structural gene for ribosomal protein S15. Ribosomes and polyribosomes prepared from suhD cells contained a reduced level (ca. 10%) of S15 relative to that of the wild type. Cloning and sequencing of suhD revealed that an IS10-like element had been inserted at the attenuator-terminator region immediately downstream of the rpsO coding region. The rpsO mRNA level in the suhD strain was also reduced to about 10% that of wild type. Apparently, ribosomes lacking S15 can actively participate in protein synthesis and suppress the rpoH11 opal (UGA) mutation at high temperature but cannot sustain cell growth at low temperature.
Termination sites T1 and T2 from the Escherichia coli chromosome inhibit DNA replication in ColE1-derived plasmids
Authors: Pelletier A J , Hill T M , Kuempel P L
Published: November 2016

Inhibition sites T1 and T2 from the Escherichia coli terminus functioned with the same characteristics in ColE1-derived plasmids and in the chromosome. These characteristics included polarity and dependence on tus, a trans-acting factor required for inhibition. Inhibition in the terminus region of the R6K plasmid was also tus dependent.
Temperature-sensitive sec mutants of Escherichia coli: inhibition of protein export at the permissive temperature
Authors: Ito K , Hirota Y , Akiyama Y
Published: November 2016

Phenotypes of secY and secA temperature-sensitive mutants at permissive (low) temperature have been examined. The secY24 mutant was found to be extremely susceptible to export inhibition by a basal-level synthesis of the MalE-LacZ 72-47 hybrid protein or to overproduction of a normal secretory protein such as maltose-binding protein or beta-lactamase. Comparison of this phenotype of secY24 with those of the secY100 and secA51 mutants under similar conditions suggested that MalE-LacZ protein and overproduced secretory protein do not nonspecifically enhance the partial secretion defect but act synergistically with secY24 to inhibit protein export.
Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6
Authors: Ishii M , Igarashi Y , Kodama T
Published: November 2016

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).
Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain
Authors: Ichige A , Matsutani S , Oishi K , Mizushima S
Published: November 2016

Two gene transfer systems were established for a marine bacterium, Vibrio sp. strain 60. One was generalized transduction with a newly isolated bacteriophage, As3, and the other was conjugal gene transfer by the use of newly constructed transposon-facilitated recombination (Tfr) donors. As3 transduced various chromosomal markers at frequencies of 10(-4) to 10(-6). Tfr donors, which were constructed by introducing transposon Tn10 into both plasmid RP4 and the chromosome, mediated the polarized transfer of chromosomal genes from the sites of Tn10 insertion on the chromosome. By means of these gene transfer systems, a genetic map of the vibrio chromosome was constructed.
Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles
Authors: Couto L B , Friedberg E C
Published: November 2016

Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R. Fleer, W. Siede, and E. C. Friedberg, J. Bacteriol. 169:4884-4892, 1987). In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E. coli. The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele. We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E. coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4. These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide. This region of the gene may be important both for the toxic effect of the Rad4 protein in E. coli and for its role in DNA repair in S. cerevisiae.
The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp
Authors: Kitada M , Onda K , Horikoshi K
Published: November 2016

The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the same conditions, the pH homeostasis was not observed in the cells of nonalkalophilic mutant, and the cytoplasmic pH immediately rose to pH 10. On the other hand, the results of the rapid acidification from pH 9 to 7 showed that the internal pH was maintained as more basic than the external pH in a neutral medium in both strains. The Na+/H+ antiport system has been characterized by either the effect of Na+ on delta pH formation or 22Na+ efflux in Na+-loaded right-side-out membrane vesicles of strain N-6. Na+- or Li+-loaded vesicles exhibited a reversed delta pH (inside acid) after the addition of electron donors (ascorbate plus tetramethyl-p-phenylenediamine) at both pH 7 and 9, whereas choline-loaded vesicles generated delta pHs of the conventional orientation (inside alkaline). 22Na+ was actively extruded from 22Na+-loaded vesicles whose potential was negative at pH 7 and 9. The inclusion of carbonyl cyanide m-chlorophenylhydrazone inhibited 22Na+ efflux in the presence of electron donors. These results indicate that the Na+/H+ antiport system in this strain operates electrogenically over a range of external pHs from 7 to 10 and plays a role in pH homeostasis at the alkaline pH range. The pH homeostasis at neutral ph was studied in more detail. K+ -depleted cells showed no delta pH (acid out) in the neutral conditions in the absence of K+, whereas these cells generated a delta pH if K+ was present in the medium. This increase of internal pH was accompanied by K+ uptake from the medium. These results suggest that electrogenic K+ entry allows extrusion of H+ from cells by the primary proton pump at neutral pH.
Induction and metabolite regulation of levanase synthesis in Bacillus subtilis
Authors: Martin I , Debarbouille M , Klier A , Rapoport G
Published: November 2016

Levanase expression in Bacillus subtilis was studied by using transcriptional and translational fusions. It was shown that the degradative products of levan or inulin and low concentrations of fructose were able to induce levanase expression. In the wild-type strain and in a constitutive overproducing sacL mutant, levanase synthesis was repressed by glucose or fructose. This catabolite repression was partially abolished in the derepressed alpha-amylase gra-26 mutant. The levanase gene (sacC) appears to be the distal gene of an operon transcribed from a fructose-inducible promoter. Deletion mapping experiments and primer extension analysis revealed a transcriptional start point located 2.7 kilobases upstream from the sacC gene. Two constitutive sacL mutations were shown to be closely linked by transformation to the sacC gene. The sacL6 and sacL8 mutations were mapped in the promoter-proximal region of the operon.
Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF
Authors: Biek D P , Cohen S N
Published: November 2016

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.
Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: mutations in the topA gene allow pSC101 replication in the absence of IHF
Authors: Biek D P , Cohen S N
Published: November 2016

Integration host factor (IHF), encoded by the himA and himD genes, is a histonelike DNA-binding protein that participates in many cellular functions in Escherichia coli, including the maintenance of plasmid pSC101. We have isolated and characterized a chromosomal mutation that compensates for the absence of IHF and allows the maintenance of wild-type pSC101 in him mutants, but does not restore IHF production. The mutation is recessive and was found to affect the gene topA, which encodes topoisomerase I, a protein that relaxes negatively supercoiled DNA and acts in concert with DNA gyrase to regulate levels of DNA supercoiling. A previously characterized topA mutation, topA10, could also compensate for the absence of IHF to allow pSC101 replication. IHF-compensating mutations affecting topA resulted in a large reduction in topoisomerase I activity, and plasmid DNA isolated from such strains was more negatively supercoiled than DNA from wild-type strains. In addition, our experiments show that both pSC101 and pBR322 plasmid DNAs isolated from him mutants were of lower superhelical density than DNA isolated from Him+ strains. A concurrent gyrB gene mutation, which reduces supercoiling, reversed the ability of topA mutations to compensate for a lack of him gene function. Together, these findings indicate that the topological state of the pSC101 plasmid profoundly influences its ability to be maintained in populations of dividing cells and suggest a model to account for the functional interactions of the him, rep, topA, and gyr gene products in pSC101 maintenance.
Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes
Authors: Yelton D B , Peng S L
Published: November 2016

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.
Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase
Authors: Lee H S , Hancock R E , Ingraham J L
Published: November 2016

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.
Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli
Authors: Kilstrup M , Meng L M , Neuhard J , Nygaard P
Published: November 2016

Addition of purines to the growth medium of Escherichia coli represses synthesis of cytosine deaminase (codA) and enzymes of purine de novo synthesis. After Tn10 mutagenesis, mutants displaying derepressed levels of cytosine deaminase in the presence of hypoxanthine were isolated. One of these had simultaneously acquired resistance to the hypoxanthine analog 6-mercaptopurine. The mutation purR6::Tn10 was shown to affect de novo synthesis of the purine enzymes glutamine phosphoribosylpyrophosphate amidotransferase (purF) and phosphoribosyl glycinamide synthetase (purD). The mutation was mapped by P1 transduction at 36 min on the E. coli linkage map. A plasmid containing the purR region was obtained by complementation of the purR6::Tn10 mutation. By comparing the restriction maps of the cloned fragment and the E. coli chromosome, the purR gene was found to be located very close to the lpp gene (36.3 min).
Comparison of tellurite resistance determinants from the IncP alpha plasmid RP4Ter and the IncHII plasmid pHH1508a
Authors: Walter E G , Taylor D E
Published: November 2016

The tellurite resistance (Ter) determinants of the IncHII plasmid pHH1508a and the broad host range IncP alpha plasmid RP4Ter were cloned into pUC8, creating plasmids pDT1364 and pDT1558, respectively. The Ter region of pDT1364 was localized to a 1.25-kilobase region by using Tn1000 insertion mutagenesis. Insertions of Tn1000 into pDT1558 which resulted in tellurite sensitivity spanned 1.75 kilobases of DNA. No similarity between the restriction maps of these two plasmids was observed, and no homology could be detected by DNA-DNA hybridization. Expression in an in vitro transcription-translation system showed that pDT1364 encoded two polypeptides with molecular masses of 23 and 12 kilodaltons (kDa) which were not expressed by pUC8. Some of the Tn1000 insertion mutants did not express the 23-kDa protein. pDT1558 encoded a 40-kDa polypeptide which was not expressed by pUC8. Both Ter determinants were expressed constitutively. Our findings suggest that the mechanisms of Ter encoded by these two plasmids are different.
Streptokinase mutations relieving Escherichia coli K-12 (prlA4) of detriments caused by the wild-type skc gene
Authors: Müller J , Reinert H , Malke H
Published: November 2016

A novel phenotype is described for Escherichia coli K-12 carrying the prlA4 allele determining a membrane component of the protein export mechanism. It is manifest as transformation deficiency for plasmids containing the cloned group C streptococcal streptokinase gene, skc. Streptokinase plasmid mutations relieving the prlA4 strain of this deficiency fell into three classes. Class 1 included skc::IS5 insertions, with IS5 integrated in a region encoding the Skc signal sequence and inactivating skc. Class 2 included IS1 insertions leaving skc intact but reducing skc expression, presumably by altering the function of the skc promoter as judged by an insertion site close to the -35 region. The most interesting class, 3, included skc deletions removing the entire signal sequence or a tetrapeptide from its hydrophobic core. The tetrapeptide deletion reduced the size, hydrophobicity, and predicted alpha-helicity of the central region of the Skc signal sequence but facilitated the export of mature Skc in both the wild type and the prlA4 mutant. These findings indicate that the incompatibility between prlA4 and skc is related to deleterious effects of the Skc signal sequence. The tetrapeptide deletion may function by altering the conformation of the signal sequence so as to render interaction with both the PrlA wild-type protein and the PrlA4 mutant protein less detrimental to the export mechanism. These findings also provide an explanation for the difficulties encountered in cloning streptokinase genes in E. coli plasmids and maintaining their structural stability.
Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa
Authors: Rivera M , McGroarty E J
Published: November 2016

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.
Onset of penicillin-induced bacteriolysis in staphylococci is cell cycle dependent
Authors: Maidhof H , Johannsen L , Labischinski H , Giesbrecht P
Published: November 2016

Synchronously growing staphylococci were treated with "lytic" concentrations of penicillin at different stages of their division cycle. Coulter Counter measurements and light microscopy were used to determine the onset of bacteriolysis. Independent of the stage of the division cycle at which penicillin was added, (i) the cells were always able to perform the next cell division; (ii) the following division, however, did not take place; and (iii) instead, at this time, when the onset of the subsequent cell separation was observed in control cultures, lysis of the penicillin-treated cells occurred. These results support a recent model (P. Giesbrecht, H. Labischinski, and J. Wecke, Arch. Microbiol. 141:315-324, 1985) explaining penicillin-induced bacteriolysis of staphylococci as the result of a special morphogenetic mistake during cross wall formation.
Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775
Authors: Singer J T , Earley S
Published: November 2016

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.
Movement of multiple DNA units between Myxococcus xanthus cells
Authors: Starich T , Zissler J
Published: November 2016

Myxococcus xanthus YS produces particles (Mx alpha particles) that transmit genetic information between cells. Mx alpha particles might be viruses, although no host able to sustain lytic growth of Mx alpha has been discovered. The particles could be detected by their ability to transduce a Tn5 transposon tag to recipient bacteria. DNA from purified particles hybridized to a limited number of DNA restriction fragments of strain YS, suggesting that Mx alpha particles contain only specific DNA sequences. A set of Tn5 insertions residing in the transducible region provided genetic markers for cloning cellular DNA packaged by Mx alpha. A map of this region showed that transducible DNA comprised multiple units of approximately 80 kilobases each. Individual units share DNA homology but are divergent in the location of restriction sites. Other wild-type isolates of Myxococcus species contained DNA sequences with homology to Mx alpha DNA, indicating that Mx alpha DNA is widespread in nature. Experiments on the transfer of Mx alpha DNA in strain YS suggested that DNA transfer is enhanced during the developmental cycle.
Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity
Authors: Franklin N C , Doelling J H
Published: November 2016

The N protein of bacteriophage lambda (N lambda) modifies Escherichia coli RNA polymerase in such a way that it transcribes through termination signals, a process called antitermination. N antitermination normally occurs only if the template contains a specific utilization or nut site upstream of the terminators and only in the presence of host-encoded Nus proteins. The lambda-related phages 21 and P22 produce N analogs, N21 and N22, but these require different nut sites and show a different pattern of functional interaction with one of the Nus factors, NusA, according to whether this protein is of E. coli or Salmonella origin (NusAEc or NusASal). We report the overproduction of N lambda, N21, or N22, each of which was induced by isopropyl-beta-D-thiogalactopyranoside at 37 degrees C from its cloned position downstream from ptac on a high-expression plasmid, each in a host that provided NusAEc or NusASal. Overproduction of each of these N proteins resulted in relaxed specificity for nut, which was shown by the ability to complement N mutants of heterologous phages; NusA specificity was determined by the N type that was present in these complementation tests. We also observed that excess N was able to suppress transcriptional polarity in the particular case of cloned 'trpA, the last gene of the tryptophan operon, although there was no effect on polarity within chromosomal trpE. Such polarity is attributed to the presence of cryptic intragenic terminators that become exposed in the absence of translation. Because there is no known nut site cis to 'trpA, we suggest that the 'trpA segment itself fortuitously contains a nut sequence that is able to function with excess N of any of the types tested and with either NusAEc or NusASal. We also found that excess N of any specificity, or even inactive N with missense mutation, could cause an increase in the level of NusAEc or NusASal, possibly because interaction between N and NusA, but independent of nut, whether functional or not, interferes with the autoregulation of NusA synthesis. These observations highlight the importance of protein concentration for the specificity of interactions both with other proteins and with nucleic acids. They also indicate that the interaction between N and NusA requires nut participation both for specificity and functionality.
Hypervariable DNA fingerprinting in Escherichia coli: minisatellite probe from bacteriophage M13
Authors: Huey B , Hall J
Published: November 2016

Extensive restriction-fragment-length polymorphism was revealed in Escherichia coli strains by using a region of the bacteriophage M13 genome as a DNA hybridization probe. This variation was observed across natural strains, in clinical samples, and to a lesser extent in laboratory strains. The sequence in M13 which revealed this fingerprint pattern was a region of the gene III coat protein, which contains two clusters of a 15-base-pair repeat. Oligonucleotides made to a consensus of these repeats also revealed the fingerprint profile. While this consensus sequence has significant homology to the lambda chi site sequence, an oligonucleotide made of the chi sequence did not reveal polymorphic fingerprint patterns in E. coli. The strain variation revealed by the M13 and M13-derived oligonucleotide probes will be useful for bacterial characterization and should find use in studies of bacterial evolution and population dynamics. The findings raise questions about what these repeated sequences are and why they are so variable.
Gene encoding the alpha core subunit of Bacillus subtilis RNA polymerase is cotranscribed with the genes for initiation factor 1 and ribosomal proteins B, S13, S11, and L17
Authors: Boylan S A , Suh J W , Thomas S M , Price C W
Published: November 2016

We describe the genetic and transcriptional organization of the promoter-distal portion of the Bacillus subtilis alpha operon. By DNA sequence analysis of the region surrounding rpoA, the gene for the alpha core subunit of RNA polymerase, we identified six open reading frames by the similarity of their products to their counterparts in the Escherichia coli transcriptional and translational apparatus. Gene order in this region, given by gene product, was IF1-B-S13-S11-alpha-L17. Gene order in E. coli is similar but not identical: SecY-B-S13-S11-S4-alpha-L17. The B. subtilis alpha region differed most strikingly from E. coli in the presence of IF1 and the absence of ribosomal protein S4, which is the translational regulator of the E. coli alpha operon. In place of the gene for S4, B. subtilis had a 177-base-pair intercistronic region containing two possible promoter sequences. However, experiments with S1 mapping of in vivo transcripts, gene disruptions in the alpha region, and a single-copy transcriptional fusion vector all suggested that these possible promoters were largely inactive during logarithmic growth, that the major promoter for the alpha operon lay upstream from the region cloned, and that the genes in the IF1 to L17 interval were cotranscribed. Thus, the transcriptional organization of the region resembles that of E. coli, wherein the alpha operon is transcribed primarily from the upstream spc promoter, but the absence of the S4 gene suggests that the translational regulation of the region may differ more fundamentally.
Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation
Authors: Kornitzer D , Teff D , Altuvia S , Oppenheim A B
Published: November 2016

The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII gene is located in the leftward operon, which is transcribed from the pL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression.
Regulation of isocitrate dehydrogenase by phosphorylation in Escherichia coli K-12 and a simple method for determining the amount of inactive phosphoenzyme
Authors: Edlin J D , Sundaram T K
Published: November 2016

In several Escherichia coli K-12 strains grown on a limiting concentration of glucose, isocitrate dehydrogenase (IDH) was inactivated about 90% after cessation of growth upon exhaustion of the glucose. Such inactivation has been previously observed in several E. coli strains but not in E. coli K-12 (unless acetate was added to the bacterial culture when growth ceased). IDH was inactivated 75 to 80% in all E. coli K-12 strains we examined during growth on acetate. The inactivation involved phosphorylation of the enzyme and is considered to be a regulatory mechanism facilitating metabolite flow along the glyoxylate shunt. Phospho-IDH interacted with antibodies to enzymatically active IDH. We have devised a method, based on this immunological cross-reaction, for determining the proportions of active and inactive (phospho-) IDH in cell extracts.
Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al
Authors: Cali B M , Micca J L , Stewart V
Published: November 2016

We isolated Mu dI1734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas- phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. beta-Galactosidase synthesis in nas+/phi(nas-lacZ) merodiploid strains was induced by nitrate or nitrite and was inhibited by exogenous ammonia or by anaerobiosis. beta-Galactosidase synthesis in phi(nas-lacZ) haploid (Nas-) strains was nearly constitutive during nitrogen-limited aerobic growth and uninducible during anaerobic growth. A general nitrogen control regulatory mutation (ntrB4) allowed nitrate induction of phi(nas-lacZ) expression during anaerobic growth. This and other results suggest that the apparent anaerobic inhibition of phi(nas-lacZ) expression was due to general nitrogen control, exerted in response to ammonia generated by anaerobic (respiratory) nitrate reduction.