bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of menaquinone in inactivation and activation of the Bacillus cereus forespore respiratory system
Authors: Escamilla J E , Barquera B , Ramírez R , García-Horsman A , del Arenal P
Published: November 2016

The respiratory systems of the Bacillus cereus mother cell, forespore, and dormant and germinated spore were studied. The results indicated that the electron transfer capacity during sporulation, dormancy, and germination is related to the menaquinone levels in the membrane. During the maturation stages of sporulation (stages III to VI), forespore NADH oxidase activity underwent inactivation concomitant with a sevenfold decrease in the content of menaquinone and without major changes in the content of cytochromes and segment transfer activities. During the same period, NADH oxidase and menaquinone levels in the mother cell compartment steadily decreased to about 50% at the end of stage VI. Dormant spore membranes contained high levels of NADH dehydrogenase and cytochromes, but in the presence of NADH, they exhibited very low levels of O2 uptake and cytochrome reduction. Addition of menadione to dormant spore membranes restored NADH-dependent respiration and cytochrome reduction. During early germination, NADH-dependent respiration and cytochrome reduction were restored simultaneously with a fourfold increase in the menaquinone content; during germination, no significant changes in cytochrome levels or segment electron transfer activities of the respiratory system took place.
Allele replacement in Escherichia coli by use of a selectable marker for resistance to spectinomycin: replacement of the lexA gene
Authors: Hill S A , Little J W
Published: November 2016

We replaced the Escherichia coli lexA gene by a segment of DNA coding for resistance to spectinomycin and streptomycin. The use of this segment expands the range of selectable markers usable for allele replacement. The availability of this null lexA mutation will facilitate genetic analysis of lexA and the SOS regulon.
Isolation and characterization of Mycoplasma mycoides subsp. mycoides mutants deficient in nucleoside monophosphate transport
Authors: Youil R , Finch L R
Published: November 2016

To select mutants lacking dAMP uptake, log-phase cells of Mycoplasma mycoides subsp. mycoides Y were incubated with high-specific-activity [32P]dAMP and then stored several weeks at -20 degrees C to allow 32P decay before plating out. Mutants were screened for lack of labeling by [32P]dAMP. Two mutants were studied further by uptake and growth experiments with other nucleotides.
Analysis of the diphtheria tox promoter by site-directed mutagenesis
Authors: Boyd J , Murphy J R
Published: November 2016

By oligonucleotide-directed mutagenesis, we introduced alterations in the two putative -10 regions of the diphtheria tox promoter which are positioned at -50 and -56 from the GUG tox initiation signal. The -10 region positioned at -50 is favored in the expression of ADP-ribosyltransferase activity from the wild-type tox promoter in recombinant Escherichia coli; however, the promoter down mutation at position -50 is compensated for by increased activity of the -10 region positioned at -56.
Isolation of subunits from Methanosarcina barkeri ATPase: nucleotide-binding site in the alpha subunit
Authors: Inatomi K , Maeda M
Published: November 2016

The alpha (62,000-dalton) and beta (49,000-dalton) subunits of Methanosarcina barkeri ATPase were purified to homogeneity. The subunits and ATPase complex were trypsinized in the presence of various nucleotides. ATP and ADP changed the trypsin sensitivity of the alpha subunit in the complex and isolated forms, suggesting the presence of a nucleotide-binding site in the alpha subunit.
Localization and enumeration of fimbria-associated adhesins of Bacteroides loescheii
Authors: Weiss E I , London J , Kolenbrander P E , Hand A R , Siraganian R
Published: November 2016

Monoclonal antibodies that specifically inhibit coaggregation between Bacteroides loescheii PK1295 and its two gram-positive partners Streptococcus sanguis 34 and Actinomyces israelii PK14 were used to enumerate and localize two distinct types of fimbria-associated adhesins on the surface of B. loescheii. Binding studies with radiolabeled monoclonal antibodies indicated that a maximum (Bmax calculated from Scatchard plots) of approximately 400 adhesin molecules specific for S. sanguis and 310 adhesin molecules specific for A. israelii reside on the surface of the cell. Immunoelectron microscopy revealed that the adhesins were not an integral part of the fimbrial subunit; rather, they were usually found on the distal portion of the structures arranged in a random fashion.
Regulation of the fixA gene and fixBC operon in Bradyrhizobium japonicum
Authors: Gubler M , Hennecke H
Published: November 2016

The transcriptional start site of the Bradyrhizobium japonicum fixBC operon was identified by nuclease S1 mapping. It was located approximately 700 base pairs upstream of fixB and was preceded by a promoter sequence that showed strong homology to the B. japonicum fixA promoter and thus to the general nif consensus promoter sequence. Further transcript mapping experiments revealed that fixA and fixBC transcription in B. japonicum strictly depended on the presence of the regulatory gene nifA and on low oxygen partial pressure. Consistent with these data, chromosomally integrated fixA- and fixB-lacZ fusions expressed beta-galactosidase activity only in the wild type but not in a nifA mutant and only under microaerobic but not aerobic growth conditions. The presence of nifA accounted for a 19-fold and 44-fold activation of the fixA and fixB promoters, respectively. These results show that the fixA and fixBC genes are regulated in a way similar to that of the nitrogenase genes nifH and nifDK. A very peculiar finding was that the fixA and fixB promoters, when they were located on plasmids, could hardly be activated by the NifA protein, irrespective of whether this was tested in Escherichia coli or B. japonicum backgrounds. This is in clear contrast to the situation with nifH and nifD promoters.
Loss of the spacer loop sequence from the rrnB operon in the Escherichia coli K-12 subline that bears the relA1 mutation
Authors: Harvey S , Hill C W , Squires C , Squires C L
Published: November 2016

A polymorphism affecting the spacer region of the rrnB rRNA operon is described. Strains from a major Escherichia coli K-12 subbranch are missing a 106-nucleotide portion of the rrnB 16S-to-23S spacer, and a 20-nucleotide sequence is found in its place. We have called this mutant operon rrnB2. The rrnB2 spacer was most probably derived from either rrnC or rrnE. This alteration of rrnB may have occurred by a recombinational exchange or by gene conversion. In the genealogy of E. coli K-12 strains, the appearance of rrnB2 is associated with the spontaneous occurrence of the first relaxed mutation, but attempts to show a selective relationship between the two mutational events have had negative results. The sequences of the rrnG and rrnC 16S-to-23S spacers have also been determined and their comparisons to the other rrn operons encoding tRNAGlu2 are presented.
Analysis of the incompatibility determinants of I-complex plasmids
Authors: Nikoletti S , Bird P , Praszkier J , Pittard J
Published: November 2016

The isolation and characterization of minireplicons corresponding to group B and I-complex plasmids are reported. The molecular structures of the small RNAs that may play a major role in the replication control and incompatibility reactions of the plasmids are compared. A mutant plasmid with changed copy number and incompatibility specificity is described. This mutant had a single-base-pair substitution in the DNA region that codes for the small RNA.
Mobilization of Bacteroides plasmids by Bacteroides conjugal elements
Authors: Valentine P J , Shoemaker N B , Salyers A A
Published: November 2016

A 4.2-kilobase cryptic Bacteroides plasmid, pB8-51, is found in several colonic Bacteroides species. To determine whether pB8-51 is mobilized by any of the known Bacteroides conjugal elements, we constructed an Escherichia coli-Bacteroides shuttle vector, pVAL-1, which contains pB8-51. We constructed Bacteroides uniformis 0061 derivatives which carry pVAL-1 and various Bacteroides conjugal elements. The Bacteroides conjugal elements tested were six conjugal tetracycline resistance (Tcr) elements (which appear to be chromosomal), i.e., Tcr ERL, Tcr V479, Tcr Emr ERL, Tcr Emr 12256, Tcr Emr DOT, and Tcr Emr CEST, and the conjugal erythromycin resistance (Emr) plasmid pBF4. These Tcr conjugal elements have not been extensively characterized, except for Tcr ERL. All six Tcr elements tested mobilized pVAL-1 at high frequency (10(-3) to 10(-5)) from one Bacteroides strain to another or from a Bacteroides strain to E. coli. Pregrowth of the donors (containing one of the Tcr elements and pVAL-1) in 1 microgram of tetracycline per ml enhanced the transfer of pVAL-1 by 20- to 10,000-fold, depending on which Tcr element was present in the donor. An Ems derivative of pBF4 (pBF4 delta E2) mobilized pVAL-1 from one Bacteroides strain to another at a frequency of 10(-4) but did not mobilize pVAL-1 from a Bacteroides strain to E. coli as efficiently. Thus the Tcr conjugal elements and pBF4 recognize a mobilization region on pB8-51.
Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein
Authors: Andrup L , Atlung T , Ogasawara N , Yoshikawa H , Hansen F G
Published: November 2016

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.
Inhibition of the SOS response of Escherichia coli by the Ada protein
Authors: Vericat J A , Guerrero R , Barbé J
Published: November 2016

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.
Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri
Authors: Peinemann S , Müller V , Blaut M , Gottschalk G
Published: November 2016

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.
Replication patterns of multiple plasmids coexisting in Escherichia coli
Authors: Leonard A C , Helmstetter C E
Published: November 2016

The replication patterns of several plasmids were measured simultaneously during the cell division cycle of Escherichia coli B/r. F plasmids harboring oriS, both oriS and oriV, pSC101, and pBR322 were found to replicate at all stages of the cell division cycle with kinetics which were indistinguishable from one another and clearly different from the periodic synthesis of the minichromosomes pAL49 and pAL70.
New locus for exopolysaccharide overproduction in Escherichia coli K-12
Authors: Zinkewich-Péotti K , Fraser J M
Published: November 2016

A new locus for exopolysaccharide overproduction in Escherichia coli K-12 was mapped by insertion mutagenesis. A 66% linkage to serA, which is located at 62 min on the E. coli K-12 linkage map, was shown by P1 transduction. The polysaccharide produced by the mutant was isolated and was shown to be similar to colanic acid.
The methanoreductosome: a high-molecular-weight enzyme complex in the methanogenic bacterium strain Gö1 that contains components of the methylreductase system
Authors: Mayer F , Rohde M , Salzmann M , Jussofie A , Gottschalk G
Published: November 2016

The methanogenic bacterium strain Gö1 harbors a high-molecular-weight enzyme complex containing methyl coenzyme M methylreductase as revealed by immunoelectron microscopy. This complex consists of a spherelike, hollow head piece, in the wall of which a number of copies of the methyl coenzyme M methylreductase are located. It is named Rc (c indicates collector). Intimately bound to it is a group of additional subunits of unknown composition referred to as Rm (m indicates mediator). Electron microscopy of negatively stained samples indicated that Rm contains a functional pore or channel which connects the internal volume of Rc with the outside. The RcRm complex is named Rs (s indicates spherelike). This complex was often found detached from the inside of the cytoplasmic membrane when membrane vesicles were investigated. However, Rs was also seen attached to a third component of the complex located in the membrane, the attachment being mediated by Rm. This membrane part of the complex is designated Rt (t indicates translocator). It consists of subunits with unknown composition. When Rs is attached to the membrane, the pore in Rm appears to be plugged by Rt. This indicates that the internal volume in Rc is in contact, via the pore in Rm, with Rt. The RcRmRt complex is referred to as methanoreductosome. Functional implications of the structural organization of the methylreductase system are discussed in view of methane formation and the creation of a transmembrane proton gradient used by the cell for ATP synthesis.
Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii
Authors: Joerger R D , Bishop P E
Published: November 2016

A 3.8-kilobase-pair EcoRI fragment which corrects the mutations carried by the NifB- Azotobacter vinelandii strains CA30 and UW45 was cloned, and its nucleotide sequence was determined. Four complete open reading frames (ORFs) and two partial ORFs were found. The translation product of the first partial ORF is the carboxy-terminal end of a protein homologous to the nifA gene product from Klebsiella pneumoniae. A 285-base-pair sequence containing a potential nif promoter and nif regulatory sites separates this nifA gene from the first complete ORF which encodes a protein homologous to nifB gene products from K. pneumoniae and Rhizobium species. The Tn5 insertion in strain CA30 and the nif-45 mutation of strain UW45 are located within this nifB gene. The ORF downstream from nifB predicts an amino acid sequence with a cysteine residue pattern that is characteristic of ferredoxins. No similarities were found between the translation product of the third complete ORF and those of nif genes from other organisms. At the carboxy-terminal end of the predicted translation product of the fourth complete ORF, 30 of 60 amino acid residues were identical with the sequence of the nifQ gene product from K. pneumoniae. The partial ORF located at the end of the fragment encodes the N-terminal part of a potential protein with an unknown function. Northern (RNA) blot analysis indicated that transcripts from the region containing the four complete ORFs were NH4+ repressible and that the transcription products were identical in cells derepressed under conditions of Mo sufficiency or Mo deficiency or in the presence of vanadium. In contrast to the NifB- strain CA30, which is Nif- under all conditions, mutants that carry mutations affecting the C-terminal end of nifB or genes located immediately downstream from nifB, grew under all N2-fixing conditions. However, in the presence of Mo, most of the strains required 1,000 times the amount of molybdate that is sufficient for maximal growth of the wild-type strain CA under N2-fixing conditions. Growth data from strain CA37, which carries a Kanr insertion in nifQ, indicate that nifQ in A. vinelandii is not required for N2 fixation in the presence of V2O5 or under Mo-deficient conditions. Growth studies and acetylene reduction assays performed on two nifEN deletion strains showed that nifE and nifN are required for N2 fixation under Mo sufficiency, as previously observed (K. E. Brigle, M. C. Weiss, W. E. Newton, and D. R. Dean, J. Bacteriol. 169:1547-1553, 1987), but not under conditions of Mo deficiency or in the presence of 50 nM V2O5.
Construction and characterization of mutations in hupB, the gene encoding HU-beta (HU-1) in Escherichia coli K-12
Authors: Storts D R , Markovitz A
Published: November 2016

Plasmid pJMC21 contains Escherichia coli chromosomal DNA encoding Lon protease, HU-beta (HU-1), and an unidentified 67,000-dalton protein. A kanamycin resistance cassette was used in the construction of insertion and deletion mutations in hupB, the gene encoding HU-beta on plasmid pJMC21. The reconstructed plasmids were linearized and used to introduce hupB chromosomal mutations into JC7623 (recBC sbcBC). These mutations, as expected, mapped in the 9.8-min region of the E. coli chromosome by P1 transduction (16% linkage to proC+). Southern blot hybridization of chromosomal fragments verified that hupB+ was replaced by the mutant allele, with no indication of gene duplication. All the mutant strains had growth rates identical to that of wild-type E. coli, were resistant to UV irradiation and nitrofurantoin, and supported the in vivo transposition-replication of bacteriophage Mu, Mu lysogenization, Tn10 transposition from lambda 1098, and lambda replication-lysogenization. The only observable phenotypic variation was a reduced Mu plaque size on the hupB mutant strains; however, the yield of bacteriophage Mu in liquid lysates prepared from the mutant strains was indistinguishable from the yield for the wild type.
Purification and reconstitution of the electron transport components for 6-deoxyerythronolide B hydroxylase, a cytochrome P-450 enzyme of macrolide antibiotic (erythromycin) biosynthesis
Authors: Shafiee A , Hutchinson C R
Published: November 2016

The hydroxylation of 6-deoxyerythronolide B (6D) to erythronolide B, a step in the biosynthesis of the 14-membered macrolide antibiotic erythromycin A by Saccharopolyspora erythraea, is catalyzed by a cytochrome P-450 monooxygenase that requires two electron transport proteins for the function of this terminal hydroxylase (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210, 1987). Two flavoproteins and an iron-sulfur protein (erythrodoxin) were purified from S. erythraea CA340 and shown to act with 6D hydroxylase to catalyze the hydroxylation of (9R)-[9-3H]9-deoxo-9-hydroxy-6D in vitro in a suitably reconstituted system. The flavoproteins contained flavin adenine dinucleotide and exhibited characteristic absorption maxima at 356 and 456 nm. The one with an Mr of 47,000 showed NADPH-dependent diaphorase and cytochrome c reductase activity, and the other, with an Mr of 53,000 showed NADH-dependent activities of the same two types. Erythrodoxin contained acid-labile sulfur and iron, had an Mr of 27,500, and showed a broad absorption maximum between 394 and 404 nm. The sequence of its first 15 amino acids, except for position 12, was the same as that of the ferredoxin from Mycobacterium smegmatis.
Characterization of the promoter region of the Bacillus subtilis spoIIE operon
Authors: Guzmán P , Westpheling J , Youngman P
Published: November 2016

Mutations that define the spoIIE locus of Bacillus subtilis block sporulation at an early stage and recently were shown to prevent the proteolytic processing of sigma E (sigma 29) into its active form, an event that is believed to control critical changes in gene expression during the second hour of development. By taking advantage of two Tn917-mediated insertional mutations in spoIIE, we have cloned DNA spanning the locus. Gene disruption experiments with subcloned fragments transferred to integrational vectors revealed that the locus consisted of a single transcription unit about 2.5 kilobase pairs in size. Transcriptional lacZ fusions were used to show that expression of this transcription unit initiated at 1.5 h after the end of log-phase growth and depended upon the products of all spo0 loci. Expression was directed by a single promoter whose position was determined by high-resolution S1 protection mapping. A deletion analysis of the promoter region was also carried out, with novel integrational vectors based on derivatives of coliphage M13. The results indicated that a region of DNA extending from 183 to 118 base pairs upstream from the start point of transcription was required for full activity of the spoIIE promoter. The presumptive RNA polymerase-binding region of the promoter exhibited striking similarity to the spoIIG promoter and featured perfect but unusually spaced -10 and -35 consensus sequences for sigma A (sigma 43)-associated RNA polymerase.
Genetic organization and transcriptional analysis of a major gene cluster involved in siderophore biosynthesis in Pseudomonas putida WCS358
Authors: Marugg J D , Nielander H B , Horrevoets A J , van Megen I , van Genderen I , Weisbeek P J
Published: November 2016

In iron-limited environments, the plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358. The transcriptional organization and the iron-regulated expression of a major gene cluster involved in the biosynthesis and transport of pseudobactin 358 were analyzed in detail. The cluster comprises a region with a minimum length of 33.5 kilobases and contains at least five transcriptional units, of which some are relatively large. The directions of transcription of four transcriptional units were determined by RNA-RNA hybridization and by analysis in Escherichia coli minicells. The latter also demonstrated that large polypeptides were encoded by these transcriptional units. The results allowed us to localize several promoter regions on the DNA. The iron-dependent expression of at least two genes within this cluster appears to be regulated at the transcriptional level.
Molecular cloning and sequencing of a pectate lyase gene from Yersinia pseudotuberculosis
Authors: Manulis S , Kobayashi D Y , Keen N T
Published: November 2016

A pectate lyase gene (pelY) from Yersinia pseudotuberculosis was cloned in Escherichia coli DH-5 alpha. The gene was expressed in either orientation in pUC plasmids, indicating that the insert DNA carried a Y. pseudotuberculosis promoter which functioned in E. coli. However, when cloned in the orientation which placed the coding region downstream of the vector lac promoter, expression of pelY was nine times higher than it was in the opposite orientation and the growth of E. coli cells was inhibited. Nucleotide sequence analysis of the pelY gene disclosed an open reading frame of 1,623 base pairs (PLY). The peptide sequence at the amino-terminal end of the protein contains a typical signal peptide sequence, consistent with the observation that the mature PLY protein accumulated largely in the periplasmic space of E. coli. The pI of PLY produced in E. coli cells was 4.5, and its activity was inhibited 90% or more by EDTA. The enzyme macerated cucumber tissue about 1,000 times less efficiently than did PLe from Erwinia chrysanthemi EC16. The pelY gene has no sequence similarity to the pel genes thus far sequenced from Erwinia spp.
Oxacillin-induced inhibition of protein and RNA synthesis in a tolerant Staphylococcus aureus isolate
Authors: Jablonski P E , Mychajlonka M
Published: November 2016

A clinical isolate of Staphylococcus aureus was found to be tolerant (MBC much greater than MIC) to a number of beta-lactam antibiotics, including oxacillin. Biophotometric analysis showed that a number of concentrations of oxacillin were capable of stimulating rapid cellular lysis in this organism, but the extent of lysis was antibiotic concentration dependent and limited. Cell cultures treated with an antibiotic concentration yielding the maximum rate and extent of lysis were analyzed for protein and RNA synthesis by pulse-labeling techniques. RNA synthesis was initially stimulated and then severely inhibited. Protein synthesis was not inhibited initially; however, the increase in the rate of synthesis expected as the result of logarithmic growth was not observed. Instead, the antibiotic-treated culture maintained for approximately 50 min the rate of protein synthesis ongoing at the time of antibiotic addition. The rate of protein synthesis declined thereafter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein samples taken 1 and 3 h after antibiotic addition showed that the shutdown of protein synthesis was not coordinate but rather was suggestive of the operation of a stress regulon perhaps similar to those responsible for heat shock, SOS, and oxidation stress.
Induction of pathogenic-like responses in the legume Macroptilium atropurpureum by a transposon-induced mutant of the fast-growing, broad-host-range Rhizobium strain NGR234
Authors: Djordjevic S P , Ridge R W , Chen H C , Redmond J W , Batley M , Rolfe B G
Published: November 2016

Mutant strain ANU2861, a transposon Tn5 mutant of the fast-growing, broad-host-range Rhizobium strain ANU280 (NGR234 Smr Rfr) overproduces polysaccharide, is an ade auxotroph, and induces poorly developed nodules on Leucaena leucocephala and Lablab purpureus (H.C. Chen, M. Batley, J.W. Redmond, and B.G. Rolfe, J. Plant Physiol. 120:331-349, 1985). Strain ANU2861 cannot form nodules on Macroptilium atropurpureum Urb. (siratro) or on Desmodium intortum and D. uncinatum and the nonlegume Parasponia. The parent strain, ANU280, effectively nodulates all these legume species except Parasponia, on which it forms ineffective nodules. Ultrastructural examination of infection sites on the legume siratro showed that mutant strain ANU2861 caused root hair curling (Hac+ phenotype), some cortical cell division (Noi+), but no infection threads (Inf-). Localized cellular responses, known to occur in phytopathological interactions, were observed in electron micrographs of the epidermal tissue at or near the infection zone after inoculation with strain ANU2861 but not the wild-type parental strain. These include (i) the rapid (within 20 h) accumulation of osmiophilic droplets attached to membranes at potential sites of strain ANU2861 penetration and (after 48 h) in the epidermal cells in the immediate region of the curled root hairs, and (ii) localized cell death of the epidermal cells. In addition, strain ANU2861 can initiate a systemic response in split-root siratro plants which prevents the successful nodulation of strain ANU280. A 6.3-kilobase fragment of wild-type genomic DNA, which includes the site of Tn5 insertion in strain ANU2861, was cloned and introduced to strain ANU2861. All the phenotypic defects of the mutant strain were corrected by the introduction of this DNA fragment. This indicates that the original Tn5 insertion is responsible for the phenotype.
Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae
Authors: Simpson D A , Hausinger R P , Mulks M H
Published: November 2016

Each isolate of Neisseria gonorrhoeae produces one of two distinct immunoglobulin A1 (IgA1) proteases, type 1 or type 2, which are known to possess different cleavage specificities for peptide bonds in the hinge region of human IgA1. Both proteases were secreted into the culture medium throughout exponential growth; however, the activity level of the type 2 protease was 10-fold that observed for the type 1 enzyme. The type 2 protease was quite stable and resistant to a variety of inhibitors. In contrast, the type 1 enzyme was highly unstable and inhibited by low concentrations of metal chelators, salts, and thiol- or serine-specific chemical reagents. Both types of gonococcal IgA1 protease were purified from broth culture supernatants by a combination of anion-exchange, chromatofocusing, and molecular sieve chromatography techniques. The stable type 2 enzyme comprised a 114-kilodalton (kDa) peptide which converted to a still active 109-kDa peptide during isolation. In contrast, the type 1 protease possessed a 112-kDa peptide which did not convert to a smaller form and which could not be dissociated from peptides of 34 and 31 kDa without complete loss of enzyme activity.
Inducer expulsion in Streptococcus pyogenes: properties and mechanism of the efflux reaction
Authors: Sutrina S L , Reizer J , Saier M H
Published: November 2016

Expulsion of preaccumulated methyl-beta-D-thiogalactoside-phosphate (TMG-P) from Streptococcus pyogenes is a two-step process comprising intracellular dephosphorylation of TMG-P followed by rapid efflux of the intracellularly formed free galactoside (J. Reizer, M.J. Novotny, C. Panos, and M.H. Saier, Jr., J. Bacteriol. 156:354-361, 1983). The present study identifies the mechanism and the order and characterizes the temperature dependency of the efflux step. Unidirectional efflux of the intracellularly formed [14C]TMG was only slightly affected when measured in the presence of unlabeled TMG (25 to 400 mM) in the extracellular medium. In contrast, pronounced inhibition of net efflux was observed in the presence of relatively low concentrations (1 to 16 mM) of extracellular [14C]TMG. Since net efflux was nearly arrested when the external concentration of [14C]TMG approached the intracellular concentration of this sugar, we propose that a facilitated diffusion mechanism is responsible for efflux and equilibration of TMG between the intracellular and extracellular milieus. The exit reaction was markedly dependent upon temperature, exhibited a high energy of activation (23 kcal [ca. 96 kJ] per mol), and followed first-order kinetics, indicating that the permease mediating this efflux was not saturated under the conditions of expulsion employed.
Promoter determining the timing and spatial localization of transcription of a cloned Streptomyces coelicolor gene encoding a spore-associated polypeptide
Authors: Guijarro J , Santamaria R , Schauer A , Losick R
Published: November 2016

Streptomyces coelicolor is a filamentous, gram-positive bacterium that exhibits a complex cycle of morphological differentiation involving the formation of an aerial mycelium of multinucleoid hyphae which undergo septation to form long chains of spores. We report the identification of two proteins of 13 and 3 kilodaltons, designated SapA and SapB, respectively, that are produced during formation of the aerial mycelium and are found in assocation with purified, mature spores. We cloned the structural gene (sapA) for one of these spore-associated proteins. Nucleotide sequence analysis suggests that the 13-kilodalton polypeptide is derived from a larger pre- or preproprotein containing a leader sequence of 37 amino acids. Nuclease protection-hybridization analysis and experiments using the Vibrio harveyi, luciferase-encoding luxAB operon as a gene tag demonstrated that expression of sapA is controlled from a promoter contained within a region of less than 110 base pairs in length, whose transcription start site is located approximately 50 base pairs upstream from the initiation codon for the sapA open reading frame. Transcription of sapA was induced at the time of appearance of the aerial mycelium, and the level of sapA transcripts was significantly reduced in certain mutants blocked in aerial mycelium (bld) and or spore (whi) formation. As further evidence of the association of sapA transcription with morphological differentiation, experiments in which we monitored sapA transcription topographically by use of a sapA-luxAB operon fusion demonstrated a close spatial correlation between colony regions undergoing aerial mycelium formation and zones of sapA-promoted light emission.
Variation in the ability of Pseudomonas sp. strain B13 cultures to utilize meta-chlorobenzoate is associated with tandem amplification and deamplification of DNA
Authors: Rangnekar V M
Published: November 2016

Single-colony isolates of Pseudomonas sp. strain B13 were examined for their ability to utilize benzoate (Ben) and meta-chlorobenzoate (3CB) as the sole carbon source. Scoring of B13 cultures by the replica-plating technique revealed that under nonselective conditions, B13 spontaneously formed four different types of colonies: 3CB+ Ben+, 3CB+ Ben-, 3CB- Ben-, 3CB- Ben+. Successive testing of each of the four colony types showed that each produced the same four different types of single-colony isolates. Colonies of each class had characteristic phenotypic properties with respect to the accumulation of Ben or 3CB pathway intermediate products. The physical abundance of a 4.3-kilobase DNA encoding the first three enzymes of the chlorocatechol pathway correlated with the 3CB+ phenotype. Increased abundance of the 4.3-kilobase DNA fragment was the result of tandem amplification.
Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white
Authors: Roncero C , Valdivieso M H , Ribas J C , Durán A
Published: November 2016

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.
Transposition of IS50L activates downstream genes
Authors: Kendrick K E , Reznikoff W S
Published: November 2016

A transposition system constructed to detect the transposition of Tn5 to a site upstream of the lacZ gene has revealed that transposition of IS50L can activate downstream genes. Expression is apparently mediated by the NPTII promoter. Transposase produced either by IS50R or by the suppressed IS50L catalyzed transposition of IS50L.
Homocitrate cures the NifV- phenotype in Klebsiella pneumoniae
Authors: Hoover T R , Imperial J , Ludden P W , Shah V K
Published: November 2016

Dinitrogenase was isolated from a culture of a Klebsiella pneumoniae NifV- strain derepressed for nitrogenase in the presence of homocitrate. The enzyme isolated from this culture was identical to the wild-type dinitrogenase. These data provide in vivo evidence that the absence of homocitrate is responsible for the NifV- phenotype.
Hydrogen-mediated mannose uptake in Azotobacter vinelandii
Authors: Maier R J , Prosser J
Published: November 2016

Azotobacter vinelandii can grow mixotrophically with H2 plus mannose under N2-fixing conditions (T. Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). Mixotrophically grown cultures incubated in H2 transported mannose with a Vmax fourfold greater than that observed for cultures incubated in argon, but H2 did not change the apparent Km for mannose. Respiratory inhibitors, such as potassium cyanide, hydroxylamine, and p-chloromercuribenzoic acid, as well as the proton conductor carbonyl cyanide m-chlorophenyl-hydrazone inhibited mannose uptake. We suggest that one of the roles of H2 in mixotrophic metabolism is to supply energy that facilitates mannose transport.
A lacZ-pbpB gene fusion coding for an inducible hybrid protein that recognizes localized sites in the inner membrane of Escherichia coli
Authors: Ayala J A , Plá J , Desviat L R , de Pedro M A
Published: November 2016

An in-phase gene fusion consisting of the 5'-terminal 1,314 base pairs (bp) of the structural gene for beta-galactosidase (lacZ) and the 3'-terminal 1,644 bp of the structural gene coding for penicillin-binding protein 3 (pbpB) of Escherichia coli was constructed and cloned in the plasmid pDIAM64. The product of the fusion gene was a remarkably stable protein with an apparent molecular weight of 110,000 (p110) that retained the ability to covalently interact with beta-lactam antibiotics. The fusion protein was found associated with the membrane at low levels of induction, but it accumulated in the cytoplasm of cells induced for a long time as inclusion bodies of high density. Inclusion bodies were localized at defined positions corresponding to septal sites in all of the pDIAM64-containing strains tested except PAT84 and GD113 (which carry the ftsZ84 mutant allele). These findings indicate a possible role of the FtsZ protein in the integration of Pbp3 into the membrane and in septum localization during the cell division cycle.
Restriction and modification activities from Streptococcus lactis ME2 are encoded by a self-transmissible plasmid, pTN20, that forms cointegrates during mobilization of lactose-fermenting ability
Authors: Higgins D L , Sanozky-Dawes R B , Klaenhammer T R
Published: November 2016

A self-transmissible (Tra+) plasmid encoding determinants for restriction and modification activities (R+/M+) from Streptococcus lactis ME2 was isolated and characterized. The 28-kilobase (kb) plasmid (pTN20) was detected in lactose-fermenting (Lac+) transconjugants generated from matings between S. lactis N1, and ME2 variant, and a plasmid-free recipient, S. lactis LM2301. The plaquing efficiencies of prolate- and small isometric-headed phages were reduced on transconjugants containing either pTN20 (R+/M+ Tra+) or 100-kb plasmids encoding Lac+, R+/M+, and Tra+. Lac+ transconjugants which harbored pTR1040 (Lac+) and pTN20 (R+/M+) were phenotypically R-/M- and transferred Lac+ at low frequency in subsequent matings to give rise to 100-kb R+/M+ plasmids. R+/M+ activities and high-frequency conjugal transfer ability were detected in Lac+ transconjugants that contained pTR1041 (Lac+) and pTN20 (R+/M+). No 100-kb R+/M+ plasmids were recovered after these matings, suggesting that pTR1041 was mobilized by pTN20 through a process that resembled plasmid donation. pTR1041 was identical to pTR1040 but contained an additional 3.3-kb DNA fragment. These data suggested that phenotypic expression of R+/M+ and Tra+ is affected by coresident Lac+ plasmids. Restriction enzyme analysis and hybridization reactions demonstrated that the 100-kb R+/M+ plasmid was formed by a cointegration event between pTR1040 (Lac+) and pTN20 (R+/M+ Tra+) during conjugal transfer via a conductive-type process. This is the first report that defines self-transmissible restriction and modification plasmids in the lactic streptococci.
The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes
Authors: Sundquist A R , Fahey R C
Published: November 2016

An NADPH-specific disulfide reductase that is active with bis-gamma-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-gamma-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2+ resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2+ resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native GCR was found to be a dimeric flavoprotein of Mr 122,000 and to be more stable to heat when in buffer of very high ionic strength. DLD was chromatographed on columns of immobilized Cu2+ resin in buffer containing NaCl and in buffer containing (NH4)2SO4, the elution of DLD differing markedly in the two buffers. Purified DLD was found to be a heat-stable, dimeric flavoprotein of Mr 120,000 and to be very specific for NAD. The utility of immobilized-metal-ion affinity chromatography for the purification of halobacterial enzymes and the likely cellular function of GCR are discussed.
In vitro transcription from the late promoter of bacteriophage P4
Authors: Keener J , Dale E C , Kustu S , Calendar R
Published: November 2016

The late genes of satellite bacteriophage P4 are cotranscribed from a single promoter which shares little homology with known classes of Escherichia coli promoters (E. Dale, G. Christie, and R. Calendar, J. Mol. Biol. 192:793-803, 1986). In a coupled transcription-translation system, the P4 late gene promoter was activated by either the delta protein of P4 or the ogr protein of helper phage P2 in the absence of any other phage-encoded factor. delta-dependent transcription was inhibited by antibodies to the sigma 70 subunit of E. coli RNA polymerase but was restored by purified sigma 70, indicating that activation of transcription by the delta protein of P4 is dependent on the sigma 70 holoenzyme.
Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+
Authors: Thoms B , Wackernagel W
Published: November 2016

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.
Dominant lethal mutations in the dnaB helicase gene of Salmonella typhimurium
Authors: Maurer R , Wong A
Published: November 2016

A class of dominant lethal mutations in the dnaB (replicative helicase) gene of Salmonella typhimurium is described. The mutated genes, when present on multicopy plasmids, interfered with colony formation by Escherichia coli host strains with a functional chromosomal dnaB gene. The lethal phenotype was expressed specifically in supE (glutamine-inserting) host strains and not in Sup+ strains, because the mutant genes, by design, also possessed an amber mutation derived from a glutamine codon. Mutations located at 11 sites by deletion mapping and DNA sequence analysis varied in the temperature dependence and severity of their lethal effects. None of the mutations complemented a dnaB(Ts) host strain at high temperature (42 degrees C). Therefore, these nonfunctional DnaB proteins must engage some component(s) of the DNA replication machinery and inhibit replication. These mutations are predicted to confer limited, specific defects in either the catalytic activity of DnaB or the ability of DnaB to interact with one of its ligands such as DNA, nucleotide, or another replication protein. The variety of mutant sites and detailed phenotypes represented in this group of mutations may indicate the operation of more than one specific mechanism of lethality.
Is efficiency of suppressor tRNAs controlled at the level of ribosomal proofreading in vivo?
Authors: Faxén M , Kirsebom L A , Isaksson L A
Published: November 2016

Ribosomal rpsD mutations did not stimulate nonsense suppressor tRNAs in a general manner according to their increased ribosomal ambiguity and decreased proofreading efficiency. Streptomycin, which stimulates error production by blocking proofreading in vitro, did not increase efficiency of suppressor tRNAs in strains with normal or streptomycin-resistant (rpsL) ribosomes. It did so only in combination with one rpsL mutation which is associated with streptomycin pseudodependence.
Determination of gene products and coding regions from the murE-murF region of Escherichia coli
Authors: Maruyama I N , Yamamoto A H , Hirota Y
Published: November 2016

We report the cloning of murE and murF genes and the identification of their gene products. The murE and murF genes encode diaminopimelic acid- and D-alanyl-D-alanine-adding enzymes, respectively, and both genes are involved in cell wall peptidoglycan biosynthesis in Escherichia coli.