bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstration of ferric L-parabactin-binding activity in the outer membrane of Paracoccus denitrificans
Authors: Bergeron R J , Weimar W R , Dionis J B
Published: November 2016

Under low-iron conditions, Paracoccus denitrificans excretes a catecholamine siderophore, L-parabactin, to sequester and utilize iron. In this report, we demonstrate the presence of stereospecific high-affinity ferric L-parabactin-binding activity associated with P. denitrificans membranes grown in low-iron medium. Isolated outer membrane components were shown to be three to four times higher in specific activity for ferric L-parabactin. The same amount of binding activity existed whether or not the radiolabel was present in the metal (55Fe) or the ligand (3H) portion of ferric parabactin chelate, suggesting that binding was to the intact complex. Ion-exchange chromatography of a Triton X-100-solubilized outer membrane mixture on DEAE-cellulose resulted in a 10-fold increase in binding activity relative to that present in whole membranes. Polypeptide profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of each stage of the purification showed that binding activity copurified with one or more of the low-iron-induced outer membrane proteins in the 80-kilodalton (kDa) region. Membrane proteins and [55Fe]ferric L-parabactin electrophoresed in nondenaturing gels demonstrated the presence of membrane component(s) which stereo-specifically bound ferric L-parabactin, thus providing independent confirmation of the binding assay results. Moreover, when the band labeled by [55Fe]ferric L-parabactin was excised and profiled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 80-kDa polypeptides were the major components present. These results demonstrate the presence of a high-affinity ferric L-parabactin receptor in P. denitrificans membranes and suggest that one or more of the 80-kDa low-iron-induced polypeptides are components of the ferric L-parabactin receptor.
Lipoprotein 28, an inner membrane protein of Escherichia coli encoded by nlpA, is not essential for growth
Authors: Yamaguchi K , Inouye M
Published: November 2016

Lipoprotein 28, an inner membrane protein of Escherichia coli encoded by nlpA, was found to be nonessential for cell growth. A deletion strain in which most of the coding region of the nlpA gene was replaced by a kanamycin resistance gene was able to grow under various conditions. No discernible differences between the wild type and the deletion strain were observed in terms of cell morphology and sensitivity to various chemicals, except for kanamycin.
Transcription of the celE gene in Thermomonospora fusca
Authors: Lin E S , Wilson D B
Published: November 2016

The steady-state level of celE mRNA (coding for cellulase E5) in Thermomonospora fusca YX was measured by Northern (RNA blot) hybridization under conditions causing induction or repression of cellulase synthesis. A good correlation was found between the mRNA level and the level of cellulase E5, suggesting that the T. fusca celE gene is regulated at the level of mRNA and, most likely, at the level of transcription. The 5' and 3' ends of the celE gene transcription unit were determined by S1 mapping with single-stranded DNA probes. These results showed that there were three species of celE mRNA in T. fusca YX with closely spaced 5' ends and identical 3' ends. The size of each mRNA was about 1.5 kilobases, from both the Northern and S1 data. This size is only slightly longer than that required to code for the 45-kilodalton E5 protein. In Escherichia coli D318 (celE), the 5' ends of the celE mRNAs are identical to those in T. fusca, but the 3' ends are located ca. 300 base pairs upstream of the T. fusca 3' end. The region where the putative celE promoters were located had some interesting features, including a 60-base-pair A + T-rich sequence and sequences resembling sigma 60 promoters.
Role of the 25-, 27-, and 29-kilodalton flagellins in Caulobacter crescentus cell motility: method for construction of deletion and Tn5 insertion mutants by gene replacement
Authors: Minnich S A , Ohta N , Taylor N , Newton A
Published: November 2016

Caulobacter crescentus incorporates two distinct, but related proteins into the polar flagellar filament: a 27-kilodalton (kDa) flagellin is assembled proximal to the hook and a 25-kDa flagellin forms the distal end of the filament. These two proteins and a third, related flagellin protein of 29 kDa are encoded by three tandem genes (alpha-flagellin cluster) in the flaEY gene cluster (S.A. Minnich and A. Newton, Proc. Natl. Acad. Sci. USA 84: 1142-1146, 1987). Since point mutations in flagellin genes had not been isolated their requirement for flagellum function and fla gene expression was not known. To address these questions, we developed a gene replacement protocol that uses cloned flagellin genes mutagenized by either Tn5 transposons in vivo or the replacement of specific DNA fragments in vitro by the antibiotic resistance omega cassette. Analysis of gene replacement mutants constructed by this procedure led to several conclusions. (i) Mutations in any of the three flagellin genes do not cause complete loss of motility. (ii) Tn5 insertions in the 27-kDa flagellin gene and a deletion mutant of this gene do not synthesize the 27-kDa flagellin, but they do synthesize wild-type levels of the 25-kDa flagellin, which implies that the 27-kDa flagellin is not required for expression and assembly of the 25-kDa flagellin; these mutants show slightly impaired motility on swarm plates. (iii) Mutant PC7810, which is deleted for the three flagellin genes in the flaEY cluster, does not synthesize the 27- or 29-kDa flagellin, and it is significantly more impaired for motility on swarm plates than mutants with defects in only the 27-kDa flagellin gene. The synthesis of essentially normal levels of 25-kDa flagellin by strain PC7810 confirms that additional copies of the 25-kDa flagellin map outside the flaEY cluster (beta-flagellin cluster) and that these flagellin genes are active. Thus, while the 29- and 27-kDa flagellins are not absolutely essential for motility in C. crescentus, their assembly into the flagellar structure is necessary for normal flagellar function.
Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene
Authors: Bever R A , Iglewski B H
Published: November 2016

The elastase structural gene (lasB) from Pseudomonas aeruginosa PAO1 has been previously cloned on an 8-kilobase (kb) DNA fragment. The lasB gene, cloned in both orientations in pUC18, produced elastase in Escherichia coli, indicating that its promoter and translation initiation sites are functional in E. coli. Deletion analysis further defined the location of the lasB gene to a 3.0-kb EcoRI-KpnI fragment (pRB1803). Elastase prepared from E. coli TB1 (pRB1803) corresponded in molecular weight to mature P. aeruginosa extracellular elastase (33,000). The lasB gene directed the synthesis of 54- and 50-kilodalton (kDa) proteins in a bacterial cell-free transcription-translation system. The 33-, 50-, and 54-kDa proteins reacted with elastase-specific antiserum. To further characterize the lasB gene, the nucleotide sequence of the 3.0-kb EcoRI-KpnI fragment was determined. This DNA fragment contained a 1,491-base-pair open reading frame encoding 498 amino acids, corresponding to a predicted molecular weight of 53,600. The deduced amino acid sequence contained a putative signal sequence followed by a large polypeptide which preceded the mature 33-kDa elastase protein. Three zinc ligands and an active site were predicted for the mature elastase on the basis of its amino acid sequence homology with Bacillus thermoproteolyticus thermolysin.
Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12
Authors: Green B D , Battisti L , Thorne C B
Published: November 2016

The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.
Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR
Authors: Harker A R , Olsen R H , Seidler R J
Published: November 2016

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.
Structure and glycosylation of lipoteichoic acids in Bacillus strains
Authors: Iwasaki H , Shimada A , Yokoyama K , Ito E
Published: November 2016

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.
Cloning and analysis of genes involved in coenzyme B12 biosynthesis in Pseudomonas denitrificans
Authors: Cameron B , Briggs K , Pridmore S , Brefort G , Crouzet J
Published: November 2016

Cobalamin synthesis probably requires 20 to 30 different enzymatic steps. Pseudomonas putida and Agrobacterium tumefaciens mutants deficient in cobalamin synthesis (Cob have been isolated. In P. putida, Cob mutants were identified as being unable to use ethanolamine as a source of nitrogen in the absence of added cobalamin (deamination of ethanolamine requires coenzyme B12 as a cofactor). In A. tumefaciens, Cob mutants were simply screened for their reduced cobalamin synthesis. A genomic library of Pseudomonas denitrificans was constructed on a mobilizable wide-host-range vector. Eleven plasmids from this library were able to complement most of these mutants. By complementation and restriction mapping analysis, four genomic loci of P. denitrificans were found to be responsible for complementation of the Cob mutants. By subcloning fragments from the four genomic loci, we identified at least 14 different genes involved in cobalamin synthesis.
A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli
Authors: Henry M F , Cronan J E
Published: November 2016

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.
Nucleotide sequence of the Salmonella typhimurium mutL gene required for mismatch repair: homology of MutL to HexB of Streptococcus pneumoniae and to PMS1 of the yeast Saccharomyces cerevisiae
Authors: Mankovich J A , McIntyre C A , Walker G C
Published: November 2016

The mutL gene of Salmonella typhimurium LT2 is required for dam-dependent methyl-directed DNA mismatch repair. We have cloned and sequenced the mutL gene of S. typhimurium LT2 and compared its sequence with those of the hexB gene product of the gram-positive bacterium Streptococcus pneumoniae and the PMS1 gene product of the yeast Saccharomyces cerevisiae. MutL was found to be quite similar to the HexB mismatch repair protein of S. pneumoniae and to the mismatch repair protein PMS1 of the yeast S. cerevisiae. The significant similarities among these proteins were confined to their amino-terminal regions and suggest common evolution of the mismatch repair machinery in those organisms. The DNA sequence for mutL predicted a gene encoding a protein of 618 amino acid residues with a molecular weight of 67,761. The assignment of reading frame was confirmed by the construction of a chimeric protein consisting of the first 30 amino acids of LacZ fused to residues 53 through 618 of MutL. Interestingly, the presence of excess amounts of this fusion protein in wild-type mutL+ cells resulted in a trans-dominant effect causing the cell to exhibit a high spontaneous mutation frequency.
Isolation and characterization of mutants with deletions in dnaQ, the gene for the editing subunit of DNA polymerase III in Salmonella typhimurium
Authors: Lancy E D , Lifsics M R , Kehres D G , Maurer R
Published: November 2016

dnaQ (mutD) encodes the editing exonuclease subunit (epsilon) of DNA polymerase III. Previously described mutations in dnaQ include dominant and recessive mutator alleles as well as leaky temperature-sensitive alleles. We describe the properties of strains bearing null mutations (deletion-substitution alleles) of this gene. Null mutants exhibited a growth defect as well as elevated spontaneous mutation. As a consequence of the poor growth of dnaQ mutants and their high mutation rate, these strains were replaced within single colonies by derivatives carrying an extragenic suppressor mutation that compensated the growth defect but apparently not the mutator effect. Sixteen independently derived suppressors mapped in the vicinity of dnaE, the gene for the polymerization subunit (alpha) of DNA polymerase III, and one suppressor that was sequenced encoded an altered alpha polypeptide. Partially purified DNA polymerase III containing this altered alpha subunit was active in polymerization assays. In addition to their dependence on a suppressor mutation affecting alpha, dnaQ mutants strictly required DNA polymerase I for viability. We argue from these data that in the absence of epsilon, DNA replication falters unless secondary mechanisms, including genetically coded alteration in the intrinsic replication capacity of alpha and increased use of DNA polymerase I, come into play. Thus, epsilon plays a role in DNA replication distinct from its known role in controlling spontaneous mutation frequency.
Cloning, expression, and regulation of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase genes
Authors: Zylstra G J , Olsen R H , Ballou D P
Published: November 2016

The genes for the alpha and beta subunits of the enzyme protocatechuate 3,4-dioxygenase (EC 1.13.11.3) were cloned from the Pseudomonas cepacia DBO1 chromosome on a 9.5-kilobase-pair PstI fragment into the broad-host-range cloning vector pRO2317. The resultant clone was able to complement protocatechuate 3,4-dioxugenase mutations in P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. Expression studies showed that the genes were constitutively expressed and subject to catabolite repression in the heterologous host. Since the cloned genes exhibited normal induction patterns when present in P. cepacia DBO1, it was concluded that induction was subject to negative control. Regulatory studies with P. cepacia wild-type and mutant strains showed that protocatechuate 3,4-dioxygenase is induced either by protocatechuate or by beta-carboxymuconate. Further studies of P. cepacia DBO1 showed that p-hydroxybenzoate hydroxylase (EC 1.14.13.2), the preceding enzyme in the pathway, is induced by p-hydroxybenzoate and that beta-carboxymuconate lactonizing enzyme, which catalyzes the reaction following protocatechuate 3,4-dioxygenase, is induced by both p-hydroxybenzoate and beta-ketoadipate.
The 5'-flanking region of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is crucial for growth of the cyanobacterium Synechococcus sp. strain PCC 7942 at the level of CO2 in air
Authors: Friedberg D , Kaplan A , Ariel R , Kessel M , Seijffers J
Published: November 2016

Transformation of the high-CO2-requiring mutants (hcr) O221 and E1 derived from the cyanobacterium Synechococcus sp. strain PCC 7942 by a wild-type DNA library restored their ability to grow at the level of CO2 in air. A plasmid (pE12) containing a 10-kilobase DNA insert was rescued from a O221 heterogenote and proved to transform both O221 and E1 to the wild-type phenotype. The capacity of the pE12 subclones to confer the wild-type phenotype to O221 transformants enabled the mapping of the mutation in O221 (designated hcrO221) within a 232-base-pair PstI-BstXI DNA restriction fragment. Sequence analysis revealed two open reading frames (ORFs) at positions -1745 to -1262 (ORFI) and -1218 to -393 (ORFII) upstream of the rbcL gene. A 3-kilobase PstI fragment of O221 was cloned, and hcrO221 was found to be a point mutation within the PstI-BstXI region -1309 nucleotides upstream of the rbcL gene. The significance of this flanking region for adaptation to air levels of CO2 was further demonstrated by the generation of new hcr mutants following insertional inactivation of wild-type DNA in the BstXI site. Electron microscopy revealed aberrant carboxysome structures in growing cells of the hcr mutants, a defect that was possibly related to the mutation, since transformation with pE12 derivatives restored the carboxysome structure to normal.
Oxidation of Neurospora crassa NADP-specific glutamate dehydrogenase by activated oxygen species
Authors: Aguirre J , Rodríguez R , Hansberg W
Published: November 2016

The glutamine synthetase and the NADP-specific glutamate dehydrogenase activities of Neurospora crassa were lost in a culture without carbon source only when in the presence of air. Glutamine synthetase was previously reported to be liable to in vitro and in vivo inactivation by activated oxygen species. Here we report that NADP-specific glutamate dehydrogenase was remarkably stable in the presence of activated oxygen species but was rendered susceptible to oxidative inactivation when chelated iron was bound to the enzyme and either ascorbate or H2O2 reacted on the bound iron. This reaction gave rise to further modifications of the enzyme monomers by activated oxygen species, to partial dissociation of the oligomeric structure, and to precipitation and fragmentation of the enzyme. The in vitro oxidation reaction was affected by pH, temperature, and binding to the enzyme of NADPH. Heterogeneity in total charge was observed in the purified and immunoprecipitated enzymes, and the relative amounts of enzyme monomers with different isoelectric points changes with time of the oxidizing reaction.
Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa
Authors: Tamaru H , Inoue H
Published: November 2016

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. Production of this enzyme is repressed in vegetative cultures but can be induced by treatment with low concentrations of cycloheximide. Isolation and characterization of a derepressed mutant, the lah-1 mutant, that is capable of producing laccase in vegetative cultures without induction by cycloheximide are described. The lah-1 mutation is mapped between nit-2 and leu-3 on linkage group I, and it behaved as a recessive mutation in a forced heterokaryon. No differences were detected biochemically or immunologically between the laccase protein produced by the lah-1 mutant in the absence of cycloheximide and that induced with cycloheximide in the wild-type strain. This suggests that both laccases (66 kilodaltons) are products of the same structural gene. Relative amounts of laccase in the culture filtrate of the lah-1 mutant were much higher than those induced with cycloheximide in the wild-type strain, demonstrating high efficiency of the lah-1 mutant in production and secretion of laccase. The time course of laccase production by the lah-1 mutant revealed that expression of 66-kilodalton laccase was repressed in conidia and derepressed during vegetative mycelial growth. This suggests that a multiple regulatory mechanism is involved in the production and/or maturation of Neurospora laccase. The lah-1 mutant may be useful for identifying genes that regulate expression of the laccase gene in N. crassa.
Microbial degradation of beta-chlorinated four-carbon aliphatic acids
Authors: Kohler-Staub D , Kohler H P
Published: November 2016

Alcaligenes sp. strain CC1 is able to grow on several alpha-chlorinated aliphatic acids (2-chlorobutyrate, 2-chloropropionate, and chloroacetate), as well as on the beta-chlorinated four-carbon aliphatic acids trans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlorobutyrate as sole carbon and energy sources. Dehalogenation of alpha-chlorinated acids could be measured by using resting cells grown on all the different carbon sources, whereas dehalogenation of beta-chlorinated four-carbon acids could be detected only by using resting cells grown on four-carbon compounds. A constitutive 2-haloacid dehalogenase, which did not show any activity with beta-chlorinated four-carbon acids, was detected in cell extracts. Cell extracts of crotonate-grown cells additionally contained a beta-haloacid dechlorination activity, which acted on trans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlorobutyrate and was strictly dependent on coenzyme A, ATP, and Mg2+. Dechlorination of beta-chlorinated four-carbon acids takes place after activation of the acids to their coenzyme A derivatives and seems to be independent of the constitutive 2-haloacid dehalogenase.
Nucleotide sequence and expression of cheF, an essential gene for chemotaxis in Bacillus subtilis
Authors: Ying C W , Ordal G W
Published: November 2016

The cheF gene, which is involved in chemotaxis in Bacillus subtilis, has been cloned, expressed, and sequenced. This gene is contained in a 0.7-kilobase PstI DNA fragment that was isolated from a lambda Charon 4A B. subtilis chromosomal DNA library. This fragment was subcloned into the expression vector pSI-1 and shown to complement the cheF mutation both for chemotaxis and for methanol production in response to the addition of attractants. Plasmid-encoded DNA expression in B. subtilis maxicells indicated that a membrane-associated polypeptide of 20-kilodaltons was expressed from this 0.7-kilobase DNA. The nucleotide sequence of this DNA fragment was determined, and an open reading frame capable of encoding a putative 175-amino-acid protein (Mr 20,002) was identified. In an effort to understand the function of the cheF protein, the dosage of the cheF gene product was varied by altering the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) during growth. In the presence of high concentrations of IPTG, chemotaxis was inhibited and methanol production was impaired.
Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purification and properties of a sulfur oxygenase reductase from the facultatively anaerobic archaebacterium Desulfurolobus ambivalens
Authors: Kletzin A
Published: November 2016

From aerobically grown cells of the extremely thermophilic, facultatively anaerobic chemolithoautotrophic archaebacterium Desulfurolobus ambivalens (DSM 3772), a soluble oxygenase reductase (SOR) was purified which was not detectable in anaerobically grown cells. In the presence of oxygen but not under a hydrogen atmosphere, the enzyme simultaneously produced sulfite, thiosulfate, and hydrogen sulfide from sulfur. Nonenzymatic control experiments showed that thiosulfate was produced mainly in a chemical reaction between sulfite and sulfur. The maximum specific activity of the purified SOR in sulfite production was 10.6 mumol/mg of protein at pH 7.4 and 85 degrees C. The ratio of sulfite to hydrogen sulfide production was 5:4 in the presence of zinc ions. The temperature range of enzyme activity was 50 to 108 degrees C, with a maximum at 85 degrees C. The molecular mass of the native SOR was 550 kilodaltons, determined by gel filtration. It consisted of identical subunits with an apparent molecular mass of 40 kilodaltons in sodium dodecyl sulfate-gel electrophoresis. The particle diameter in electron micrographs was 15 /+- 1.5 nm. The enzyme activity was inhibited by the thiol-binding reagents p-chloromercuribenzoic acid, N-ethyl maleimide, and 2-iodoacetic acid and by flavin adenine dinucleotide, Fe3+, and Fe2+. It was not affected by CN-, N3-, or reduced glutathione.
Nucleotide sequence of IS492, a novel insertion sequence causing variation in extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica
Authors: Bartlett D H , Silverman M
Published: November 2016

The complete nucleotide sequence of insertion element IS492, which causes reversible inactivation of extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica, is presented. Insertion of IS492 results in the EPS- phenotype, and excision results in restoration of EPS+. DNA sequencing of the site of insertion in the eps locus showed that insertion of IS492 generates a 5-base-pair repeat and that its excision is precise. IS492 is 1,202 nucleotides in length and contains one large open reading frame encoding a protein of 318 amino acids, a candidate for transposition function. No similarity between IS492 and other transposable elements has been found. Unlike the situation with other insertion sequences, no direct or inverted repeats exist at the termini of IS492.
Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process
Authors: Tian G , Wu H C , Ray P H , Tai P C
Published: November 2016

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).
The sigma E subunit of Bacillus subtilis RNA polymerase is present in both forespore and mother cell compartments
Authors: Carlson H C , Haldenwang W G
Published: November 2016

Bacillus subtilis cells harvested 3.5 h after the onset of sporulation (t3.5) were fractionated into extracts enriched in either mother cell or forespore components and were analyzed immunologically for sigma E and its precursor protein, P31. We determined by Western blot (immunoblot) analysis that equivalent amounts of P31 and sigma E were present in both mother cell and forespore extracts. This result implies that, although sigma E is not synthesized until a stage in development when the cell is partitioned into progenitor forespore and mother cell compartments, it probably directs the transcription of genes that are expressed in both of these structures.
Translation of merD in Tn21
Authors: Lee I W , Gambill B D , Summers A O
Published: November 2016

All four sequenced examples of the mercury resistance (mer) operon of gram-negative bacteria have a promoter-distal reading frame, merD, whose removal has little effect on the resistance phenotype and whose translation has not previously been observed. Using merD-lacZ protein fusions, we show that merD is translated. However, Hg(II)-induced merD expression, as measured by beta-galactosidase activity and immunoblotting, is 10- to 15-fold lower than that of fusions to the gene immediately preceding it, merA.
Effects of inhibition of the B subunit of DNA gyrase on conjugation in Escherichia coli
Authors: Hooper D C , Wolfson J S , Tung C , Souza K S , Swartz M N
Published: November 2016

Antagonism of the DNA gyrase B subunit in the donor bacterium by coumermycin or thermal inactivation inhibited transfer of plasmid R64drd-11. Coumermycin also inhibited Hfr transfer, with kinetics after drug removal suggesting that transfer resumed from the point of inhibition, in contrast to inhibition with nalidixic acid, after which transfer reinitiated from the origin of transfer.
Evolution of chemotactic-signal transducers in enteric bacteria
Authors: Dahl M K , Boos W , Manson M D
Published: November 2016

The methyl-accepting chemotactic-signal transducers of the enteric bacteria are transmembrane proteins that consist of a periplasmic receptor domain and a cytoplasmic signaling domain. To study their evolution, transducer genes from Enterobacter aerogenes and Klebsiella pneumoniae were compared with transducer genes from Escherichia coli and Salmonella typhimurium. There are at least two functional transducer genes in the nonmotile species K. pneumoniae, one of which complements the defect in serine taxis of an E. coli tsr mutant. The tse (taxis to serine) gene of E. aerogenes also complements an E. coli tsr mutant; the tas (taxis to aspartate) gene of E. aerogenes complements the defect in aspartate taxis, but not the defect in maltose taxis, of an E. coli tar mutant. The sequence was determined for 5 kilobases of E. aerogenes DNA containing a 3' fragment of the cheA gene, cheW, tse, tas, and a 5' fragment of the cheR gene. The tse and tas genes are in one operon, unlike tsr and tar. The cytoplasmic domains of Tse and Tas are very similar to those of E. coli and S. typhimurium transducers. The periplasmic domain of Tse is homologous to that of Tsr, but Tas and Tar are much less similar in this region. However, several short sequences are conserved in the periplasmic domains of Tsr, Tar, Tse, and Tas but not of Tap and Trg, transducers that do not bind amino acids. These conserved regions include residues implicated in amino-acid binding.
Regulation of glutamine synthetase in Streptomyces coelicolor
Authors: Fisher S H , Wray L V
Published: November 2016

Glutamine synthetase (GS) in Streptomyces coelicolor was shown to be regulated at two levels. First, the S. coelicolor GS protein is subject to a posttranslational covalent modification which is likely to involve adenylylation. Adenylylation is important in regulating GS activity both after sudden changes in ammonium availability and during steady-state growth. Since higher levels of adenylylated GS were seen in S. coelicolor mutants deficient in glutamate synthase than in wild-type cells, glutamine or a metabolite derived from glutamine is likely to be involved in the metabolic signal that regulates GS adenylylation. Second, the GS structural gene (glnA) is transcriptionally regulated in response to nitrogen availability during steady-state growth. Transcription of the glnA gene occurred from the same promoter during vegetative growth, stationary phase, and sporulation. The nucleotide sequence of this promoter has significant homology with the -10, but not the -35, region of the consensus sequence of Streptomyces vegetative promoters. The glnA gene is transcribed as a monocistronic mRNA.
cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression
Authors: Laoide B M , McConnell D J
Published: November 2016

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.
Extracellular secretion of cloned aerolysin and phospholipase by Aeromonas salmonicida
Authors: Wong K R , Green M J , Buckley J T
Published: November 2016

The promoterless structural genes for aerolysin and the extracellular phospholipase of Aeromonas hydrophila were inserted into a multi-host-range expression vector and transferred into Aeromonas salmonicida and Escherichia coli. In both species, gene expression was under the control of the inducible tac promoter of the vector. Neither the phospholipase nor the aerolysin was released by intact E. coli. Instead, both proteins accumulated in the periplasm, leading to reduced growth and eventual cell death. When the aerolysin gene inserted into the vector contained its own promoter, the toxin was expressed constitutively by A. salmonicida but not by E. coli. Production of aerolysin and the phospholipase by A. salmonicida did not affect cell growth, and the proteins were correctly processed and exported by intact cells. Both proteins could also be detected in the periplasm, where their concentrations were considerably higher then they were outside the cells. Periplasmic aerolysin was rapidly released when cells were transferred to fresh medium, indicating that this compartment is part of the normal export pathway and that the protein is not shunted there as a consequence of overproduction. Plasmid-coded aerolysin did not appear to compete with the cell proteins for export components, as even when very large quantities of aerolysin were being exported by A. salmonicida, there was no effect on chromosomal protease release and only a modest reduction in the export of chromosomal phospholipase.
A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope
Authors: Vos P , van Asseldonk M , van Jeveren F , Siezen R , Simons G , de Vos W M
Published: November 2016

The complete nucleotide sequence of a gene located immediately upstream of the Lactococcus lactis subsp. cremoris SK11 prtP gene encoding the cell envelope-attached proteinase was determined. This gene, designated prtM, was found to be transcribed from the same promotor region as was the proteinase gene but in the opposite direction. The prtM gene directed the expression in Escherichia coli of a protein with a size similar to the expected value of 33 kilodaltons, as deduced from the nucleotide sequence data. The derived amino acid sequence of the PrtM protein indicated the presence of a consensus lipoprotein signal sequence at the N terminus, which suggested that PrtM is a lipoprotein. Plasmids containing the prtM gene, the prtP gene, or both were constructed. Expression studies of L. lactis clones containing these plasmids showed that the prtM gene encodes a trans-acting activity involved in the maturation of cell envelope-located and -secreted forms of the SK11 proteinase.
Novel arrangement of rRNA genes in Mycoplasma gallisepticum: separation of the 16S gene of one set from the 23S and 5S genes
Authors: Chen X , Finch L R
Published: November 2016

Large restriction fragments from the DNA of Mycoplasma gallisepticum S6 and PG31, which were prepared by digestion with BglI, BssHII, SmaI, or XhoI and which were separated by pulsed-field electrophoresis, were hybridized with probes containing most, or different parts, of an rRNA operon of Mycoplasma capricolum. The results showed that the genomes contained three widely separated rRNA loci. One locus contained genes for all three rRNA species and another contained 23S and probably 5S rRNA genes, whereas the third appeared to have only a 16S rRNA gene.
Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium
Authors: Rioux C R , Kadner R J
Published: November 2016

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.
Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis
Authors: Finke A , Roberts I , Boulnois G , Pzzani C , Jann K
Published: November 2016

The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes.
Interspecific complementation analysis by protoplast fusion of Candida tropicalis and Candida albicans adenine auxotrophs
Authors: Corner B E , Poulter R T
Published: November 2016

A protocol employing inositol starvation was used to isolate proline and adenine auxotrophs of Candida tropicalis. Interspecific hybrids between red adenine auxotrophs of C. tropicalis and Candida albicans were formed by protoplast fusion. These C. tropicalis red adenine auxotrophs were shown to fall into two complementation groups by crossing them with a known C. albicans ade1 tester strain. It is suggested that these two groups correspond to the ade1 and ade2 mutants of Saccharomyces cerevisiae and C. albicans and that these defined mutants may be useful in attempts to develop transformation systems for C. tropicalis.
Accumulation of 3-(N-morpholino)propanesulfonate by osmotically stressed Escherichia coli K-12
Authors: Cayley S , Record M T , Lewis B A
Published: November 2016

We found that exogenous morpholinopropanesulfonate (MOPS) is concentrated approximately fivefold in the free volume of the cytoplasm of Escherichia coli K-12 (strain MG1665) when grown at high osmolarity (1.1 OsM) in two different media containing 40 mM MOPS. MOPS was not accumulated by E. coli grown in low-osmolarity MOPS-buffered medium or in 1.1 OsM MOPS-buffered medium containing the osmoprotectant glycine betaine. Salmonella typhimurium LT2 did not accumulate MOPS under any condition examined. We infer that accumulation of MOPS by E. coli K-12 is not due to passive equilibration but rather to transport, possibly involving an as yet uncharacterized porter not present in S. typhimurium. Glutamate and MOPS were the only anionic osmolytes we observed by 13C nuclear magnetic resonance in E. coli K-12 grown in MOPS-buffered medium. The increase in positive charge accompanying the increase in the steady-state amount of K+ in cells shifted from low to high external osmolarity appeared to be compensated for by changes in the amounts of putrescine, glutamate, and MOPS. MOPS is not an osmoprotectant, because its accumulation did not increase cell growth rate.