bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-cycle-specific oscillation in the composition of chromatophore membrane in Rhodospirillum rubrum
Authors: Myers C R , Collins M L
Published: November 2016

Synchrony in phototrophic cultures of Rhodospirillum rubrum was induced by stationary-phase cycling or by alterations in light intensity. Intracytoplasmic chromatophore membranes were prepared by differential centrifugation. Analysis of the composition of chromatophores obtained from cells at different times indicated that the protein/bacteriochlorophyll a ratio was constant throughout the cell cycle but that the protein/phospholipid ratio oscillated. This cell-cycle-dependent fluctuation in chromatophore membrane composition was reflected in the buoyant densities of the isolated chromatophores.
Suppressors of the secY24 mutation: identification and characterization of additional ssy genes in Escherichia coli
Authors: Shiba K , Ito K , Yura T
Published: November 2016

We previously reported (Shiba et al., J. Bacteriol. 160:696-701, 1984) the isolation and characterization of the mutation (ssy) that suppresses the protein export defect due to the secY24(Ts) mutation and causes cold-sensitive growth of Escherichia coli. This report describes more systematic isolation of ssy mutations. Among temperature-resistant revertants of the secY24 mutant, 65 mutants were found to be cold sensitive. These cold-sensitive mutations have been classified by genetic mapping. Twenty-two mutations fell into the ssyA class previously described. The remaining mutations were located at five new loci: ssyB at 9.5 min between tsx and lon; ssyD around 3 min; ssyE at 72.5 min near secY; ssyF at 20.5 min within rpsA; and ssyG at 69.0 min near argG. Two predominant classes, ssyA and ssyB, are probably affected in protein synthesis at the elongation step, whereas the ssyF mutant contained an altered form of ribosomal protein S1 (the gene product of rpsA). These cold-sensitive ssy mutations which suppress secY24 may define genes whose function is somehow involved in the secY-dependent protein secretion mechanism. However, the existence of multiple suppressor loci makes it unlikely that all of these genes specify additional components of the export machinery. A delicate balance may exist between the systems for synthesizing and exporting proteins.
Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: localization and function of its attenuators
Authors: Matsumoto Y , Shigesada K , Hirano M , Imai M
Published: November 2016

We present evidence that the expression of rho is regulated by rho-dependent attenuation of transcription. Gene fusion analysis with nested series of deletions of rho indicated that the transcription of rho is attenuated in a rho-dependent manner in the leader region and that neither a read-through transcription from the upstream gene, trxA, nor a modulation of transcription initiation of the rho promoter is involved in the self-control of rho. S1 mapping and Northern hybridization analyses localized at least six transcription attenuation or termination sites in the region ranging from the 3' end of the trxA structural gene to the middle of the rho structural gene. Among them, the most upstream site overlapping the rho promoter sequence was assigned to the terminator for the trxA gene, and the second and third sites, mapping about 80 and 50 nucleotides upstream from the start codon of rho, were suggested to function as the major attenuation sites for regulation of the rho expression. Further, the start points of the trxA and rho RNAs were determined in an in vitro transcription system to be located 111 nucleotides (U) and 255 nucleotides (G) upstream from their respective start codons. These results necessitate revisions of previous predictions on the sites of transcriptional signals in the trxA and rho genes (S. Brown, B. Albrechtsen, S. Pedersen, and P. Klemm, J. Mol. Biol. 162:283-298, 1982; C.-J. Lim, D. Geraghty, and J. A. Fuchs, J. Bacteriol. 163:311-316, 1985; B.J. Wallace and S.R. Kushner, Gene 32:399-408, 1984).
Mutations specifically affecting ligand interaction of the Trg chemosensory transducer
Authors: Park C , Hazelbauer G L
Published: November 2016

The Trg transducer mediates chemotactic response to galactose and ribose by interacting, respectively, with sugar-occupied galactose- and ribose-binding proteins. Adaptation is linked to methylation of specific glutamyl residues of the Trg protein. This study characterized two trg mutations that affect interaction with binding protein ligands but do not affect methylation or adaptation. The mutant phenotypes indicated that the steady-state activity of methyl-accepting sites is independent of ligand-binding activity. The mutation trg-8 changed arginine 85 to histidine, and trg-19 changed glycine 151 to aspartate. The locations of the mutational changes provided direct evidence for functioning of the amino-terminal domain of Trg in ligand recognition. Cross-inhibition of tactic sensitivity by the two Trg-linked attractants implies competition for a common site on Trg. However, the single amino acid substitution caused by trg-19 greatly reduced the response to galactose but left unperturbed the response to ribose. Thus Trg must recognize the two sugar-binding proteins at nonidentical sites, and the complementary sites on the respective binding proteins should differ. trg-8 mutants were substantially defective in the response to both galactose and ribose. An increase in cellular content of Trg-8 protein improved the response to galactose but not to ribose. It appears that Trg-8 protein is defective in the generation of the putative conformational change induced by ligand interaction. The asymmetry of the mutational defect implies that functional separation of interaction sites could persist beyond the initial stage of ligand binding.
Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3'----5' exonuclease (proofreading) activity
Authors: Fowler R G , Schaaper R M , Glickman B W
Published: November 2016

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).
Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis
Authors: Monod M , Denoya C , Dubnau D
Published: November 2016

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.
Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light
Authors: Mason J M , Setlow P
Published: November 2016

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.
Diverse effects of the MalE-LacZ hybrid protein on Escherichia coli cell physiology
Authors: Ito K , Akiyama Y , Yura T , Shiba K
Published: November 2016

The hybrid protein between the periplasmic maltose-binding protein and the cytoplasmic beta-galactosidase (the MalE-LacZ hybrid protein) was previously shown to block the export of envelope proteins when synthesized in large amounts. Now we show that the hybrid protein exerts another major effect on the cell, that is, induction of the heat shock proteins. This latter effect was dependent on the htpR gene product but independent of the function of the signal sequence on the hybrid protein. On the other hand, the previously reported induction of the SecA protein by the hybrid protein was independent of htpR and may be caused by the reduced protein export ability of the cell. The functional htpR gene is essential for viability of the cell in which the basal level of the hybrid protein is synthesized, whereas in the absence of the hybrid protein htpR is dispensable at low temperature. These results indicate that the hybrid protein somehow generates a signal or stress that is similar to what the cell experiences at elevated temperatures.
Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli
Authors: Barron A , May G , Bremer E , Villarejo M
Published: November 2016

Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation.
Penicillin-binding site on the Escherichia coli cell envelope
Authors: Amaral L , Lee Y , Schwarz U , Lorian V
Published: November 2016

The binding of 35S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the "cell envelope" obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. At low pH, PBPs 1b, 1c, 2, and 3 demonstrated the greatest amount of binding. At high pH, these PBPs bound the least amount of penicillin. PBPs 1a and 5/6 exhibited the greatest amount of binding at pH 10 and the least amount at pH 4. With the exception of PBP 5/6, the effect of pH on the binding of penicillin was direct. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin. These observations suggest that a molecule of penicillin forms simultaneous bonds between its S at position 1 and sulfhydryl groups of PBP 5 and between its C-7 and free epsilon amino groups of PBP 5.
Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli
Authors: Biek D P , Cohen S N
Published: November 2016

We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element Tn10 into recD resulted in increased concatemerization and loss of pSC101 and ColE1-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coli chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.
Transposition of Tn917 in Bacillus megaterium
Authors: Bohall N A , Vary P S
Published: November 2016

Transposon Tn917, carried on plasmid pTV1, was introduced into Bacillus megaterium and transposed efficiently and apparently randomly. Insertional mutations included at least eight different auxotrophic loci, two carbon source loci, and sporulation loci. One trp::Tn917 mutation was further verified as an insertion by both reversion and transduction.
Iron respiration-driven proton translocation in aerobic bacteria
Authors: Short K A , Blakemore R P
Published: November 2016

Washed cell suspensions of Aquaspirillum magnetotacticum MS-1, A. itersonii E12639, Bacillus subtilis 6633, and Escherichia coli CSH27 translocated protons in response to the added oxidant O2 or NO3-, with triphenylmethylphosphonium bromide as the permeant ion. Iron respiration-driven proton translocation was observed in A. magnetotacticum MS-1, B. subtilis, and E. coli but not in a nonmagnetic strain of A. magnetotacticum (strain NM-1A) or with A. itersonii. Proton translocation to Fe3+ was totally inhibited by 500 microM NaN3 or 0.5 microM carbonyl cyanide m-chlorophenylhydrazone.
Mutational analysis of bacteriophage lambda lysis gene S
Authors: Raab R , Neal G , Garrett J , Grimaila R , Fusselman R , Young R
Published: November 2016

A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected. DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene. Thirty-three different missense alleles were generated. Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus. A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence. In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues. These regions resemble canonical membrane-spanning domains. In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm. The mutational pattern is largely consistent with the model. Charge changes within the putative imbedded regions render the protein nonfunctional. Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule. Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions. Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis. Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one. The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.
Heterospecific transformation among cyanobacteria
Authors: Stevens S E , Porter R D
Published: November 2016

Heterospecific transformation occurred between cyanobacteria currently classified in either the genus Synechococcus or Synechocystis. Cyanobacterial strains 73109 and 6906 were capable of physiological transformation.
Galactose metabolism in Rhizobium meliloti L5-30
Authors: Arias A , Cerveñansky C
Published: November 2016

Data from previous studies of Rhizobium meliloti mutants have been consistent with the catabolism of hexoses via the Entner-Doudoroff pathway. However, galactose metabolism was not impaired in those mutants. We show here by enzymatic assay and by identification of a galactose mutant lacking 2-keto-3-deoxy-6-phosphogalactonate aldolase that the De Ley-Doudoroff pathway is used for galactose metabolism. Mutants in this pathway have not been previously reported for any organism.
Transglycosylase and endopeptidase participate in the degradation of murein during autolysis of Escherichia coli
Authors: Kitano K , Tuomanen E , Tomasz A
Published: November 2016

The cell wall degradation products released from Escherichia coli during autolysis triggered by cephaloridine or trichloroacetic acid were isolated and characterized. Murein was selectively lost from the disaccharide tetrapeptides and the bisdisaccharide tetrapeptide components. Two major autolytic products accounted for more than 85% of the released material. Compound 1 (60 to 80% of released material) was a disaccharide tetrapeptide monomer containing a 1,6-anhydromuramic acid residue. Compound 2 (15 to 30% of released material) was a mixture of a tritripeptide and a tritetrapeptide without hexosamines. Taken together the findings suggest that autolytic cell wall degradation in E. coli is selective and involves the activity of both the hydrolytic transglycosylase and an endopeptidase. Upon release, at least some of the wall components were also exposed to the activity of the N-acetylmuramic acid-L-alanine amidase.
Isolation and characterization of new facultatively alkalophilic strains of Bacillus species
Authors: Guffanti A A , Finkelthal O , Hicks D B , Falk L , Sidhu A , Garro A , Krulwich T A
Published: November 2016

Four facultatively alkalophilic isolates were purified from enrichment cultures initiated with lime-treated garden soil. Four isolates, OF1, OF3, OF4, and OF6, were obligately aerobic, spore-forming, gram-positive, motile rods which were capable of growth at both pH 7.5 and pH 10.5. Strains OF1 and OF6 grew best at the lower pH value; and whereas growth of these strains at pH 10.5 was completely dependent on added Na+, growth at pH 7.5 was only partially dependent on added Na+. Strains OF3 and OF4 grew better at pH 10.5 than at pH 7.5, with strain OF3 growing modestly over its entire pH range, while OF4 grew well. Growth of OF3 and OF4 was completely dependent on added Na+ at both pH 7.5 and pH 10.5. DNA-DNA hybridization studies indicated that OF1 and OF6 are closely related strains but are not related to the other isolates, Bacillus subtilis, or two previously studied obligately alkalophilic bacilli. OF3 was unrelated to any of the other organisms examined in the study, whereas OF4 showed complete homology with obligately alkalophilic Bacillus firmus RAB. All four isolates maintained a cytoplasmic pH that was considerably lower than the external pH when the latter was 10.5. Although substantial transmembrane electrical potentials were observed, the total electrochemical proton gradient (delta mu H+) was low at pH 10.5 in all the strains. By contrast, delta mu H+ was substantial at pH 7.5 and at that pH was composed entirely of an electrical potential. These results are in contrast to previous findings that obligately alkalophilic bacilli generate only small electrical potentials at near neutral pH. All the isolates exhibited substantial rates of respiration as measured by oxygen consumption. Neither respiration nor NADH oxidation by everted membrane vesicles was significantly stimulated by Na+. Analyses of reduced versus oxidized difference spectra of membranes from OF4 showed that the total membrane cytochrome content was considerably higher in cells grown at pH 10.5 than at pH 7.5, with the levels of c- and a-type cytochromes exhibiting the largest pH-dependent differences. Initial examination of membrane protein profiles on gel electrophoresis also indicated a number of changes in pattern in each isolate, depending on the growth pH.
Nitrogenase promoter-lacZ fusion studies of essential nitrogen fixation genes in Bradyrhizobium japonicum I110
Authors: Yun A C , Noti J D , Szalay A A
Published: November 2016

DNA fragments containing either the nifD or nifH promoter and 5' structural gene sequences from Bradyrhizobium japonicum I110 were fused in frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of beta-galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild type and Fix mutants generated by transposon Tn5 mutagenesis and identified in the accompanying paper. No nif-positive regulatory mutants were identified from among an array of Fix- mutants in which Tn5 was inserted 9 kilobase pairs upstream of the nifDK operon and within the 18-kilobase-pair region separating the nifDK and nifH operons. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Interestingly, the level of beta-galactosidase activity expressed from the nifH promoter was twice that expressed from the nifD promoter, suggesting that the normal cellular level of the nifH gene product in bacteroids is in a 2:1 ratio with the nifD gene product instead of in the 1:1 stoichiometry of the nitrogenase enzyme complex.
Acquisition of glucose by Rickettsia prowazekii through the nucleotide intermediate uridine 5'-diphosphoglucose
Authors: Winkler H H , Daugherty R M
Published: November 2016

The ability of Rickettsia prowazekii to transport potential sources of the glucose moiety of bacterial polysaccharides was determined. Transport was determined both by filtration assays and by centrifugation through nonaqueous layers. Uridine 5'-diphosphoglucose (UDPG) was transported, whereas glucose was not transported; the uptake of glucose phosphates, although greater than that for glucose, was markedly lower than the transport of UDPG. Furthermore, the activities of hexokinase and phosphoglucomutase, enzymes required for the metabolism of glucose and glucose 6-phosphate, were undetectable in rickettsial extracts. The uptake of UDPG had an extended time course and did not reach a plateau until 60 min. The maximum rate of uptake was 340 pmol/min per mg of protein, and the rate was half-maximal at a UDPG concentration of 220 microM. Measurement of true influx of UDPG was complicated by the low activity of this transport system and the metabolism of the UDPG. The uptake of labeled UDPG was markedly inhibited by a 10-fold excess of uridine monophosphate, uridine diphospho-N-acetylglucosamine, and uridine diphospho-N-acetylgalactosamine but not by a variety of other structurally related compounds.
Nucleotide sequence of the btuCED genes involved in vitamin B12 transport in Escherichia coli and homology with components of periplasmic-binding-protein-dependent transport systems
Authors: Friedrich M J , de Veaux L C , Kadner R J
Published: November 2016

The products of the btuCED region of the Escherichia coli chromosome participate in the transport of vitamin B12 across the cytoplasmic membrane. The nucleotide sequence of the 3,410-base-pair HindIII-HincII DNA fragment carrying a portion of the himA gene and the entire btuCED region was determined. Comparison of the location of the open reading frames with the gene boundaries defined by transposon insertions allowed the assignment of polypeptide products to gene sequences. The btuC product is a highly nonpolar integral membrane protein of molecular weight 31,683. The distribution of hydrophobic regions suggests the presence of numerous membrane-spanning domains. The btuD product is a relatively polar but membrane-associated polypeptide of Mr 27,088 and contains segments bearing extensive homology to the ATP-binding peripheral membrane constituents of periplasmic binding protein-dependent transport systems. Other regions of this protein are similar to portions of the outer membrane vitamin B12 receptor. The btuE product (Mr 20,474) appears to have a periplasmic location. It has the mean hydropathy of a soluble protein but lacks an obvious signal sequence. The cellular locations and structural and sequence homologies of the Btu polypeptides point to the similarity of these three proteins to components of binding protein-dependent transport systems. However, the dependence on a periplasmic vitamin B12-binding protein has not yet been demonstrated.
Control of lysogeny and immunity of Bacillus subtilis temperate bacteriophage SP beta by its d gene
Authors: McLaughlin J R , Wong H C , Ting Y E , Van Arsdell J N , Chang S
Published: November 2016

The d gene from the Bacillus subtilis temperate bacteriophage SP beta was isolated. When introduced into an SP beta-sensitive strain of B. subtilis, the cloned d gene directed the synthesis of a 22-kilodalton protein and conferred on the host immunity to SP beta phage. A frameshift mutation, designated d2, was introduced into the cloned d gene, and it was subsequently crossed back into the SP beta phage genome. The resulting SP beta phage grew lytically and formed clear plaques on sensitive bacteria. Although the cloned d gene confers immunity to the host, we could not detect complementation of the d gene by mixed infection with SP beta d2 and various SP beta c mutants. The nucleotide sequence of the 1,033-base-pair PstI-to-EcoRI fragment containing the d gene was determined; it includes an open reading frame that could potentially encode a protein of 227 amino acids. The gene was mapped within the PstI H fragment on the phage genome, which positions the d gene about 25 kilobases from the right end of the phage genome. It is transcribed from right to left.
Characteristics of sterol uptake in Saccharomyces cerevisiae
Authors: Lorenz R T , Rodriguez R J , Lewis T A , Parks L W
Published: November 2016

A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were termed sterol depleted. When cultured on 11 micrograms of sterol ml-1 or more, the cells contained a maximal cellular free-cholesterol concentration of 6.8 nmol/mg (dry weight) and were termed free sterol saturated. Cells with free-sterol concentrations below the maximal level were capable of accumulating free sterol from the medium. The capacity of the cells for cholesterol uptake was inversely proportional to the initial intracellular concentration. The uptake of sterol was shown to be a nonactive process that is independent of cellular energy sources or viability. The intracellular transport of sterol for esterification is not sensitive to anti-microtubule agents.
Lipid interconversions in aging Mycoplasma capricolum cultures
Authors: Gross Z , Rottem S
Published: November 2016

During the progression of Mycoplasma capricolum cultures from the early exponential to the stationary phase of growth, a decrease in the phospholipid-to-protein ratio and increases in both the unsaturated-to-saturated fatty acid ratio and the diphosphatidylglycerol (DPG)-to-phosphatidylglycerol (PG) ratio were found. The freedom of motion of spin-labeled fatty acids incorporated into the membrane remained unchanged throughout the growth cycle. The increase in DPG was almost stoichiometric with the decrease in PG. Furthermore, exogenous PG added to the medium was incorporated by the cells and partially converted to DPG. The DPG that was accumulated upon aging was always more unsaturated than the PG. This accumulation was enhanced in palmitic acid-poor media, but was inhibited even in aged cells when the cells were grown in palmitic acid-rich media, suggesting that the accumulation of DPG upon aging was associated with changes in the fatty acid composition of membrane lipids rather than with the transition of the cells from the exponential- to stationary-growth phase.
Control of sensitivity to inactivation by H2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus
Authors: Sammartano L J , Tuveson R W , Davenport R
Published: November 2016

Mutations in the Escherichia coli katF gene (hydroperoxidase II) result in sensitivity to inactivation by H2O2 and broad-spectrum near-UV (NUV; 300 to 400 nm) radiation. Another mutation, nur, originally described as conferring sensitivity to inactivation by broad-spectrum and monochromatic NUV, also confers sensitivity to inactivation by H2O2. Genetic analysis via transduction suggests that the nur mutation allele of the katF locus. As previously reported for broad-spectrum and monochromatic NUV wavelengths, the sensitivity of a particular strain to H2O2 inactivation is also independent of the recA and uvrA alleles. Extracts of nur and katF strains lack catalase (hydroperoxidase II) as revealed by polyacrylamide gels stained for such activity, which is consistent with the genetic results.
cmp, a cis-acting plasmid locus that increases interaction between replication origin and initiator protein
Authors: Gennaro M L , Novick R P
Published: November 2016

pT181, a 4.4-kilobase multicopy plasmid of Staphylococcus aureus, encodes a trans-acting initiator protein, RepC, which was rate limiting for replication. Deletions in a 500-base-pair region of the plasmid external to the minimal replicon decreased the ability of the plasmid to compete with a coexisting incompatible plasmid. These deletions, which define a region called cmp (for competition), appeared to affect the interaction of RepC and the plasmid origin of replication. However, in the homoplasmid state the deletions affected neither copy number nor plasmid stability. The Cmp phenotype is orientation independent, and cmp defects could not be complemented in trans.
Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium
Authors: Owttrim G W , Colman B
Published: November 2016

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.
Cloning and expression of the Acinetobacter calcoaceticus mutarotase gene in Escherichia coli
Authors: Gatz C , Altschmied J , Hillen W
Published: November 2016

This article describes the cloning of the mutarotase gene from Acinetobacter calcoaceticus and its expression in Escherichia coli. Purification of mutarotase (EC 5.1.3.3) led to a single polypeptide of 40 kilodaltons. The sequences of 27 N-terminal and 76 C-terminal amino acids were determined. From six amino acids of the N-terminal and seven amino acids of the C-terminal portion of the protein, the sequences of two oligonucleotides were deduced. These were synthesized and used as gene probes. Completely restricted chromosomal DNA from A. calcoaceticus was size fractioned, and only fractions hybridizing with the gene probes were used to construct gene banks enriched for the mutarotase determinant. With the N-terminal gene probe, a bank of 6- to 7-kilobase-pair BclI fragments in pBR327 was obtained. A total of 1,200 candidates were screened by colony hybridization followed by dot-blot analysis of purified plasmids from positive candidates and subsequent Southern blot analysis of the respective restricted plasmids, and 500 base pairs (bp) from the 5' end of the mutarotase gene were isolated by this procedure. The 3' portion of the gene was isolated from a gene bank containing 1,500-bp-long HindIII fragments inserted in M13mp11. This bank was screened by dot-blot analysis of single-stranded phage DNA with the C-terminal gene probe. The isolated gene fragments were fused at a common restriction site in their overlapping region to yield the complete mutarotase gene. High-level expression of mutarotase in E. coli was achieved when the gene was placed under transcriptional control of the phage lambda promoter pL. More than 90% of mutarotase activity was found in the culture medium. The E. coli-derived mutarotase was purified and shown to be identical to the A. calcoaceticus-derived product with respect to the molecular weight and N-terminal amino acid sequence. The expression of mutarotase in E. coli was increased 200-fold in comparison to that the wild-type A. calcoaceticus.
Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes
Authors: Shimotsu H , Henner D J
Published: November 2016

In Bacillus subtilis, the extracellular enzyme levansurcrase is synthesized in the presence of sucrose. A termination structure between the transcription start site and the structural gene was the apparent site for regulation by sucrose of transcription into the structural gene. Sequence analysis of the sacB leader region from two strains constitutive for levansucrase synthesis showed a single base change in the stem of this termination structure. This single base change also led to the constitutive synthesis of a sacB'-'lacZ fusion, whereas the wild-type sacB'-'lacZ fusion was induced by the addition of sucrose. S1 nuclease mapping of sacB transcripts with probes labeled either within the termination structure or 3' to the termination structure showed that sucrose did not increase the number of transcripts extending into the termination structure; however, sucrose did increase the number of transcripts extending past the termination structure. Two pleiotropic mutations which affect the expression of levansucrase, sacQ36 hyperproducing [sacQ36(Hy)] and sacU32(Hy), were separately introduced into the strain carrying the sacB'-'lacZ fusion. These mutations each increased the expression levels of the sacB'-'lacZ fusion. S1 mapping showed increased levels of transcript initiating at the sacB promoter in strains with the sacQ36(Hy) and sacU32(Hy) mutations. This increased transcription appeared to be independent of the sucrose-regulated transcription termination, suggesting the existence of at least two different mechanisms for the regulation of sacB expression.
Organization and expression of genetic determinants for synthesis and assembly of type 51 R bodies
Authors: Kanabrocki J A , Quackenbush R L , Pond F R
Published: November 2016

Type 51 R bodies are produced by all bacterial endosymbionts (Caedibacter taeniospiralis) of Paramecium tetraurelia that confer the hump-killer trait upon their hosts. Type 51 R-body synthesis by C. taeniospiralis is required for expression of the hump-killer trait. The genetic determinants for type 51 R-body synthesis by C. taeniospiralis 47 have been cloned and expressed in Escherichia coli. In this communication we describe three species of polypeptides required for R-body synthesis and the organization of their genetic determinants. Each polypeptide species is controlled by a separate gene that is expressed as an independent transcriptional unit possessing regulatory signals that are recognized by E. coli. Two polypeptide species of 10 and 18 kilodaltons are required for R-body synthesis but apparently are not structural subunits. The third polypeptide species (13 kilodaltons) is the major structural subunit. R-body assembly involves polymerization reactions that result in high-molecular-mass polypeptide complexes, primarily composed of the 13-kilodalton polypeptide species, that appear to be the result of covalent cross-linking between structural subunits. The results presented here have been suggested to apply to the assembly and structure of all type 51 R bodies, but not necessarily to other R-body types.
Spontaneous deletion of a 20-kilobase DNA segment carrying genes specifying isopropylbenzene metabolism in Pseudomonas putida RE204
Authors: Eaton R W , Timmis K N
Published: November 2016

The genes encoding isopropylbenzene metabolism in Pseudomonas putida RE204 are readily lost in two ways: by loss (curing) of plasmid pRE4 which specifies the catabolic pathway and by deletion from pRE4 of an approximately 20-kilobase segment of DNA carrying the catabolic genes. The presence of DNA sequences at the ends of the catabolic gene region sharing homology with one another suggests that the deletions result from recombination events between these homologous sequences.
Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster
Authors: Suh J W , Boylan S A , Price C W
Published: November 2016

We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a lambda gt11 expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding S11) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation.
Assignment of symbiotic developmental phenotypes to common and specific nodulation (nod) genetic loci of Rhizobium meliloti
Authors: Debellé F , Rosenberg C , Vasse J , Maillet F , Martinez E , Dénarié J , Truchet G
Published: November 2016

Rhizobium meliloti nodulation (nod) genes required for specific infection and nodulation of alfalfa have been cloned. Transposon Tn5 mutagenesis defined three nod regions spanning 16 kilobases of the pSym megaplasmid. Genetic and cytological studies of 62 nodulation-defective mutants allowed the assignment of symbiotic developmental phenotypes to common and specific nod loci. Root hair curling was determined by both common (region I) and specific (region III) nod transcription units; locus IIIb (nodH gene) positively controlled curling on the homologous host alfalfa, whereas loci IIIa (nodFE) and IIIb (nodH) negatively controlled curling on heterologous hosts. Region I (nodABC) was required for bacterial penetration and infection thread initiation in shepherd's crooks, and the nodFE transcription unit controlled infection thread development within the alfalfa root hair. In contrast, induction of nodule organogenesis, which can be triggered from a distance, seemed to be controlled by common nodABC genes and not to require specific nod genes nodFE and nodH. Region II affected the efficiency of hair curling and infection thread formation.
Endonuclease IV (nfo) mutant of Escherichia coli
Authors: Cunningham R P , Saporito S M , Spitzer S G , Weiss B
Published: November 2016

A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.