bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and localization of major stage-specific polypeptides of infectious Holospora obtusa with monoclonal antibodies
Authors: Wiemann M , Görtz H D
Published: November 2016

With the help of monoclonal antibodies (MAbs) we investigated the occurrence of six polypeptides throughout parts of the life cycle of Holospora obtusa, a bacterium infecting the macronucleus of the ciliate Paramecium caudatum. The polypeptides of interest formed major bands in the protein pattern of the infectious form (IF) of H. obtusa. All MAbs used recognized individual polypeptide bands of the IF proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides were also detected in the reproductive form in trace amounts. Two-dimensional electrophoresis revealed that the 33,000-, 28,000-, and 14,000-Mr polypeptides wre acidic and exhibited multiple isoelectric points under native conditions. Four polypeptides (Mrs of 50,000, 33,000, 28,000, and 20,000) were no longer detected or became drastically reduced within the first 30 min of invasion. Concomitantly, a loss of electron-dense periplasmic material occurred, which is typical for invading IFs (H.-D. Görtz and M. Wiemann, J. Protistol. 24:101-109, 1989). In an attempt to clarify the subcellular localization of the six polypeptides, we performed chloroform extraction studies and identified four of the released polypeptides with MAbs. A 14,000-Mr polypeptide was immunocytochemically localized in the periplasm of the IF. The results showed that the six major polypeptides of the IF were stage specific or stage specifically enriched and are likely to contribute to the electron-dense periplasmic material of the IF.
Primary structure, expression in Escherichia coli, and properties of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from Bacillus megaterium
Authors: Robin C , Blanche F , Cauchois L , Cameron B , Couder M , Crouzet J
Published: November 2016

A Bacillus megaterium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity was subcloned and sequenced. The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron, J. Bacteriol. 171:4222-4231, 1989). The B. megaterium polypeptide was overexpressed in Escherichia coli, partially purified, and shown to exhibit, like P. denitrificans SUMT, substrate inhibition at uroporphyrinogen III concentrations above 0.5 microM, suggesting a common regulation for aerobic cobalamin-producing organisms.
Identification of two porins in Pelobacter venetianus fermenting high-molecular-mass polyethylene glycols
Authors: Schmid A , Benz R , Schink B
Published: November 2016

Porins were purified from cells of the anaerobic gram-negative bacterium Pelobacter venetianus grown with 20-kDa polyethylene glycol. After treatment of the cell envelope fraction with sodium dodecyl sulfate-containing solutions, the murein contained only two major peptidoglycan-associated proteins of 14 and 23 kDa. Both proteins were released from the peptidoglycan by the detergent Triton X-100. Genapol X-80 released only the 23-kDa protein. This protein was purified by chromatography on a hydroxyapatite column. It did not form sodium dodecyl sulfate-resistant oligomers. Reconstituted in lipid bilayer membranes, the 23-kDa protein formed cation-selective channels with a single-channel conductance of 230 pS in 1 M KCl. The channel is not a general-diffusion pore, since its conductance depends only moderately on the salt concentration. The channel conducted ammonium much better than potassium or rubidium ions, suggesting that it is probably involved in ammonium uptake. The outer membrane of P. venetianus contains a further, non-murein-associated pore with an unknown molecular mass. It is also cationically selective and has a single-channel conductance of 1.6 nS in 1 M KCl, which suggests that its effective diameter is similar to that of porins from enteric bacteria.
Saturation mutagenesis of the UASNTR (GATAA) responsible for nitrogen catabolite repression-sensitive transcriptional activation of the allantoin pathway genes in Saccharomyces cerevisiae
Authors: Bysani N , Daugherty J R , Cooper T G
Published: November 2016

Saturation mutagenesis of the UASNTR element responsible for GLN3-dependent, nitrogen catabolite repression-sensitive transcriptional activation of allantoin pathway genes in yeast cells identified the dodecanucleotide sequence 5'-TTNCTGATAAGG-3' as the minimum required for UAS activity. There was significant flexibility in mutant sequences capable of supporting UAS activity, which correlates well with the high variation in UASNTR homologous sequences reported to be upstream of the DAL and DUR genes. Three of nine UASNTR-like sequences 5' of the DAL5 gene supported high-level transcriptional activation. The others, which contained nonpermissive substitutions, were not active.
Identification and initial characterization of the eutF locus of Salmonella typhimurium
Authors: O'Toole G A , Escalante-Semerena J C
Published: November 2016

We report the isolation and initial characterization of mutations in the newly described eutF locus of Salmonella typhimurium LT2. Mutations in eutF render a strain unable to utilize ethanolamine as a source of carbon and/or energy and impair growth on ethanolamine as a sole nitrogen source. Strains carrying eutF mutations exhibit a 2-order-of-magnitude decrease in transcription of the unlinked eutDEABCR operon (50 min), which codes for the enzymes needed to catabolize ethanolamine; have only 10% of the ethanolamine ammonia-lyase activity found in the wild type; and show a marked reduction in the rate of ethanolamine uptake. Deletion mapping and three-factor cross analysis results are consistent with the gene order cobA trp eutF tonB at 34 min on the linkage map. We discuss two possible roles for the EutF protein: (i) as an ethanolamine permease or (ii) as a transcription factor required for the expression of the eutDEABCR operon.
A novel activity in Escherichia coli K-12 that directs restriction of DNA modified at CG dinucleotides
Authors: Kelleher J E , Raleigh E A
Published: November 2016

The restriction systems McrA and McrB of Escherichia coli K-12 are known to attack DNA containing modified cytosine. In strains lacking both activities, however, we observed that DNA methylated at CG dinucleotides (as is mammalian DNA) was still significantly restricted. We show that this substantial barrier to the acceptance of 5-methylcytosine-containing DNA is attributable to a hitherto unknown activity of the Mrr restriction system. Strikingly, the multiple systems used by this gut inhabitant to determine the fate of invading DNA will all limit genetic exchange with its mammalian host(s), reinforcing the idea that one role of DNA methylation is to serve as a "molecular passport" (E. A. Raleigh, R. Trimarchi, and H. Revel, Genetics 122:279-296, 1989).
rbcR [correction of rcbR], a gene coding for a member of the LysR family of transcriptional regulators, is located upstream of the expressed set of ribulose 1,5-bisphosphate carboxylase/oxygenase genes in the photosynthetic bacterium Chromatium vinosum
Authors: Viale A M , Kobayashi H , Akazawa T , Henikoff S
Published: November 2016

An open reading frame, rbcR, was identified 226 bp upstream of rbcAB, i.e., the ribulose 1,5-bisphosphate carboxylase genes expressed in the phototrophic purple bacterium Chromatium vinosum. Several features reveal that rbcR encodes a member of the LysR family of transcriptional regulators, in which an anomalous content of lysine and arginine residues (Lys/Arg anomaly) was found. The expression of rbcR in Escherichia coli as a protein fused to the N-terminal region of beta-galactosidase led to reduced expression of rbcAB. Thus, rbcR is likely to encode a trans-acting transcriptional regulator of rbcAB expression in C. vinosum.
Macromolecular structure and aggregation states of Helicobacter pylori urease
Authors: Austin J W , Doig P , Stewart M , Trust T J
Published: November 2016

Urease purified from Helicobacter pylori by differential ultracentrifugation and fast pressure liquid chromatography was composed of subunits with apparent molecular weights (MrS) of 66,000 and 30,000. Electron microscopy of this purified material demonstrated that it formed disc-shaped macromolecular aggregates that were approximately 13 nm in diameter and 3 nm thick. Images of both negatively stained and shadowed preparations indicated that the discs tended to stack to form pairs and then these pairs further aggregated to form four-disc stacks. This stacking of subunits explains the heterogeneity observed previously in the molecular weight of urease preparations. In some negatively stained preparations there were also some smaller (approximately 8-nm-diameter) annular units present, which may represent individual urease units or possibly an aggregate of one of the two subunits from which urease is constructed.
Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis
Authors: Weinrauch Y , Msadek T , Kunst F , Dubnau D
Published: November 2016

The sequence and properties of the comQ gene are described. comQ was predicted to encode a 34,209-Da protein, and the product of comQ was shown to be required for the development of genetic competence. The apparent transcriptional initiation and termination sites of comQ were mapped, and the location of a likely E sigma A promoter was inferred. The expression of comQ was maximal early in growth and declined as the cells approached the stationary phase. This expression was not dependent on any of the competence regulatory genes tested (comA, comP, sin, abrB, degU, and spo0A). Disruption of comQ in the chromosome prevented the development of competence as well as the transcription of comG, a late competence operon. This disruption also decreased the expression of srfA, a regulatory operon needed for the expression of competence. These and other results suggest a role for ComQ early in the hierarchy of competence regulatory genes, probably as a component of a signal transduction system.
Construction, characterization, and complementation of Rhodospirillum rubrum puf region mutants
Authors: Hessner M J , Wejksnora P J , Collins M L
Published: November 2016

Rhodospirillum rubrum is a facultatively phototrophic bacterium that, under certain growth conditions, forms an intracytoplasmic chromatophore membrane (ICM) housing the photochemical apparatus. The puf operon of R. rubrum encodes protein subunits of the photochemical reaction center and the B880 light-harvesting antenna complex. Mutant strains of R. rubrum were constructed by interposon mutagenesis through which a kanamycin resistance gene cartridge was inserted into restriction sites and in place of restriction fragments of the puf region. Southern blot analysis demonstrated that the defective copies of puf sequences had replaced their normal chromosomal counterparts through homologous recombination. The phenotypes of the mutant strains were evaluated on the basis of puf gene expression, spectral analysis, pigment content of membranes, and electron-microscopic examination of thin sections of cells grown under semi-aerobic and dark anaerobic conditions. Alterations of the puf region affect phototrophic competence and the formation of the ICM. The latter result implies an obligatory role for puf gene products in ICM formation in R. rubrum. One mutant with a deletion in puf structural genes was complemented in trans to the wild-type phenotype. Other mutants could be restored to the wild-type phenotype only by recombination.
Cloning and characterization of a Rhizobium meliloti homolog of the Escherichia coli cell division gene ftsZ
Authors: Margolin W , Corbo J C , Long S R
Published: November 2016

The ftsZ gene is essential for initiation of cell division in Escherichia coli and Bacillus subtilis. To begin our studies of division arrest during differentiation of Rhizobium meliloti bacteroids, we isolated a R. meliloti ftsZ homolog, ftsZRm. Degenerate primers directed towards a conserved region of ftsZ were used to amplify a segment of R. meliloti DNA by polymerase chain reaction, and the product of this reaction was then used to isolate positive clones from a bacteriophage library. The DNA sequence of an open reading frame containing the region of homology indicated that the R. meliloti FtsZ protein (FtsZRm) is 50% homologous to the known E. coli and B. subtilis FtsZ proteins, but at 590 amino acids (63 kDa), it is predicted to be nearly 50% larger. Strong expression of an approximately 70-kDa labeled protein in a coupled in vitro transcription-translation system supports this prediction. The additional 200 amino acids appear to fall in a single internal domain highly enriched for proline and glutamine residues. When we regulated R. meliloti ftsZ (ftsZRm) expression on a high-copy-number plasmid in E. coli with Plac and laclq, cells were smaller than normal in the presence of low FtsZRm levels (with no isopropyl-beta-D-thiogalactopyranoside [IPTG]) and filamentous when FtsZRm was overproduced (with IPTG). These results suggest that low levels of FtsZRm stimulate E. coli cell division, while high levels may be inhibitory.
Mutations that affect activity of the Rhizobium meliloti trpE(G) promoter in Rhizobium meliloti and Escherichia coli
Authors: Bae Y M , Stauffer G V
Published: November 2016

The cloned Rhizobium meliloti trpE(G) gene is not expressed in Escherichia coli. Oligonucleotide-directed mutagenesis was used to introduce base substitution mutations in the promoter region of this gene. Three separate mutations that increased homology of the putative -10 region of this promoter with the E. coli -10 promoter consensus sequence by 1 bp converted this promoter to an active promoter in E. coli. A deletion extending to position -43 from the 5' side had a minor effect on transcription in R. meliloti. However, transcription was nearly eliminated when a deletion extended to position -33, indicating that the crucial domain of the R. meliloti trpE(G) promoter begins in the region downstream of position -43. The R. meliloti trpE(G) promoter has two regions that show homology with the E. coli -35 and -10 promoter consensus sequences. Mutations in these putative -35 and -10 regions, but not in the spacer region, affected promoter strength in R. meliloti. By comparing four known R. meliloti promoter sequences, we identified a highly conserved trimer near position -35 (5'-TTG-3') but no noticeably conserved sequence near position -10.
The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate
Authors: Hryniewicz M M , Kredich N M
Published: November 2016

The cysPTWA operons of Escherichia coli and Salmonella typhimurium encode components of periplasmic transport systems for sulfate and thiosulfate and are regulated as part of the cysteine regulons. In vitro transcription initiation from the cysP promoter was shown to require both CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, which act as inducers, and was inhibited by the anti-inducer sulfide. Thiosulfate was found to be even more potent than sulfide as an anti-inducer. DNase I protection experiments showed two discrete binding sites for CysB protein in the presence of N-acetyl-L-serine. CBS-P1 is located between positions -85 and -41 relative to the major transcription start site, and CBS-P2 is located between positions -19 and +25. Without N-acetyl-L-serine, the CysB protein protected the region between positions -63 and -11, which was designated CBS-P3. In gel mobility shift assays, the mobility of CysB protein-cysP promoter complexes was increased by O-acetyl-L-serine, N-Acetyl-L-serine had no effect in gel shift experiments, presumably because its anionic charge results in its rapid removal from the complex during electrophoresis. Comparison of DNA fragments differing with respect to binding site position indicated that complexes with CysB protein contain DNA that is bent somewhere between CBS-P1 and CBS-P2 and that O-acetyl-L-serine decreases DNA bending. Binding studies with fragments containing either CBS-P2 alone, CBS-P1 alone, or the entire cysP promoter region suggest a model in which the complex of bent DNA observed in the absence of O-acetyl-L-serine contains a single CysB protein molecule bound to CBS-P3. At relatively low CysB protein concentrations, O-acetyl-L-serine would cause a single CysB protein molecule to bind tightly to CBS-P1, rather than to CBS-P3, thereby decreasing DNA bending and increasing complex electrophoretic mobility. At higher CysB protein concentrations, O-acetyl-L-serine would cause a second molecule to bind at CBS-P2, giving a more slowly migrating complex.
Mutations in trans that affect formate dehydrogenase (fdhF) gene expression in Salmonella typhimurium
Authors: Fasciano A , Hallenbeck P C
Published: November 2016

Expression of the fdhF locus of Salmonella typhimurium is shown to be dependent upon ntrA and oxrB. However, the oxrB8 mutation is pleiotropic and also affects the expression of hyd, pepT, and chlC, whereas a mutation in ntrA does not. Insertional inactivation with Tn10 and localized mutagenesis permitted the definition and partial characterization of two new genes, fdhS and fdhR, which appear to be involved in the positive regulation of fdhF expression. Both genes were mapped to the 71- to 72-min region of the S. typhimurium chromosome with the gene order fdhS-crp-fdhR-rpsL. Mutations in fdhS specifically affect fdhF expression without affecting the expression of the other anaerobically induced genes or enzymes that were tested, including hyd, pepT, chlC, nitrite reductase, sulfite reductase, and trimethylamine-N-oxide reductase. Both fdhR and fdhS may be involved in fdhF regulation vis-à-vis oxygen, since localized mutagenesis produced alleles of both genes that permitted the aerobic expression of fdhF. However, fdhR may more directly interact with fdhF, since insertional inactivation of fdhS does not abolish aerobic expression of fdhF in fdhR mutant strains. Taken together, these results suggest that fdhS and fdhR act in concert under anaerobic conditions to activate fdhF transcription.
Factors influencing the specific interaction of Neisseria gonorrhoeae with transforming DNA
Authors: Goodman S D , Scocca J J
Published: November 2016

The specific interaction of transformable Neisseria gonorrhoeae with DNA depends on the recognition of specific 10-residue target sequences. The relative affinity for DNA between 3 and 17 kb in size appears to be linearly related to the frequency of targets on the segment and is unaffected by absolute size. The average frequency of targets in chromosomal DNA of N. gonorrhoeae appears to be approximately one per 1,000 bp.
The Escherichia coli htrP gene product is essential for bacterial growth at high temperatures: mapping, cloning, sequencing, and transcriptional regulation of htrP
Authors: Raina S , Mabey L , Georgopoulos C
Published: November 2016

We identified and characterized a new Escherichia coli gene, htrP. The htrP gene was identified because its insertional inactivation by the Tn10 transposon results in the inability of E. coli to form colonies at temperatures above 37 degrees C and a slow growth phenotype at 30 degrees C. The htrP gene was cloned and mapped to 66.3 min on the E. coli genetic map, 4 kbp clockwise from the tolC gene. The htrP gene was sequenced and shown to code for an acidic, 27,471-Da polypeptide and to be transcribed counterclockwise with respect to the genetic map. The predicted HtrP protein has two potential transmembrane segments and shares an identity of 64.4% over a length of 210 amino acids with the LuxH protein. Despite the fact that the htrP gene is essential for E. coli growth exclusively at high temperatures, the levels of htrP-specific transcripts decrease with increasing temperature.
Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans
Authors: Blanche F , Couder M , Debussche L , Thibaut D , Cameron B , Crouzet J
Published: November 2016

The cobalamin biosynthetic pathway enzyme that catalyzes amidation of 5'-deoxy-5'-adenosyl-cobyrinic acid a,c-diamide was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by a four-column procedure. The purified protein had an isoelectric point of 5.6 and molecular weights of 97,300 as estimated by gel filtration and 57,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. Stepwise Edman degradation provided the sequence of the first 16 amino acid residues at the N terminus. The enzyme catalyzed the four-step amidation sequence from cobyrinic acid a,c-diamide to cobyric acid via the formation of cobyrinic acid triamide, tetraamide, and pentaamide intermediates. The amidations are carried out in a specific order; this order was not determined. The enzyme was specific to coenzyme forms of substrates and did not carry out amidation of the carboxyl group at position f. The amidation reactions were ATP/Mg2+ dependent and exhibited a broad optimum around pH 7.5. L-Glutamine was shown to be the preferred amide group donor (Km congruent to 45 microM) but could be replaced by ammonia (Km = 20 mM). For all of the four partially amidated substrates, the Km values were in the micromolar range and the Vmax values were about 7,000 nmol h-1 mg-1.
Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase
Authors: Hidalgo E , Chen Y M , Lin E C , Aguilar J
Published: November 2016

The gene ald, encoding aldehyde dehydrogenase, has been cloned from a genomic library of Escherichia coli K-12 constructed with plasmid pBR322 by complementing an aldehyde dehydrogenase-deficient mutant. The ald region was sequenced, and a single open reading frame of 479 codons specifying the subunit of the aldehyde dehydrogenase enzyme complex was identified. Determination of the N-terminal amino acid sequence of the enzyme protein unambiguously established the identity and the start codon of the ald gene. Analysis of the 5'- and 3'-flanking sequences indicated that the ald gene is an operon. The deduced amino acid sequence of the ald gene displayed homology with sequences of several aldehyde dehydrogenases of eukaryotic origin but not with microbial glyceraldehyde-3-phosphate dehydrogenase.
Identification and cloning of a fur regulatory gene in Yersinia pestis
Authors: Staggs T M , Perry R D
Published: November 2016

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.
Phosphatidylglyceroylalkylamine, a novel phosphoglycolipid precursor in Deinococcus radiodurans
Authors: Huang Y , Anderson R
Published: November 2016

We report here the structure of a previously uncharacterized phospholipid in the radiation-resistant bacterium Deinococcus radiodurans. This phospholipid, designated lipid 4, was shown by chemical analysis, HF hydrolysis, and nuclear magnetic resonance spectroscopy to be phosphatidylglyceroylalkylamine. Lipid 4 thus contains the unusual lipid constituents glyceric acid and alkylamines, which have previously been identified in two complex phosphoglycolipids from this organism. By [32P]phosphate pulse-chase labeling techniques, lipid 4 was shown to be the precursor of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminylphosphatidylglyceroylalkylamine. While phosphatidylglyceroylalkylamine is rapidly biosynthesized from Pi, its subsequent glycosylation occurs much more slowly. Therefore, we conclude that the final glycosylation step is the rate-limiting event in the biosynthesis of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminyl-phosphatidylglyceroylalkylamine.
Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H' recognition site
Authors: Lee E C , MacWilliams M P , Gumport R I , Gardner J F
Published: November 2016

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.
Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein
Authors: Zuberi A R , Bischoff D S , Ordal G W
Published: November 2016

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.
Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin
Authors: Sen K , Nikaido H
Published: November 2016

Deep rought mutants, which produce very defective lipopolysaccharides, are unable to export normal levels of porins into the outer membrane. In this study, we showed that lipopolysaccharides from such mutants were also unable to facilitate the trimerization, in vitro, of monomeric OmpF porin secreted by spheroplasts of Escherichia coli B/r. In contrast, lipopolysaccharides containing most or all of the core oligosaccharides were able to facilitate trimerization.
Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila
Authors: Raybuck S A , Ramer S E , Abbanat D R , Peters J W , Orme-Johnson W H , Ferry J G , Walsh C T
Published: November 2016

The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.
Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6
Authors: Valdivia R H , Wang L , Winans S C
Published: November 2016

Neoplastic crown gall tumors incited by Agrobacterium tumefaciens release novel amino acid or sugar derivatives known as opines, whose synthesis is directed by genes transferred to plant cells. Agrobacterium cells can transport and catabolize these compounds as sources of carbon and nitrogen. This article describes a region of the pTiA6 plasmid which is required for catabolism of the opine octopine and whose transcription is induced by octopine. This region of the plasmid contains four open reading frames, occQ, occM, occP, and occJ, which show homology to the family of so-called shock-sensitive permeases. TnphoA mutagenesis demonstrated that the OccJ and OccM proteins lie fully or partly in the periplasmic space. The OccJ protein was identified by electrophoresis and found to be fully localized in the periplasmic space. When these proteins were expressed in Escherichia coli, radiolabeled octopine became cell-associated.
Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system
Authors: Vanden Boom T J , Reed K E , Cronan J E
Published: November 2016

We report the isolation and genetic characterization of novel Tn10dTc and Tn1000dKn insertion mutations in and near the lip locus of the Escherichia coli chromosome. The Tn10dTc and Tn1000dKn mutations define two genes, lipA and lipB, involved in lipoic acid biosynthesis. Two representative alleles (lip-2 and lip-9) from the previously reported genetic class of lipoic acid auxotrophic mutants (A. A. Herbert and J. R. Guest, J. Gen. Microbiol. 53:363-381, 1968) were assigned to the lipA complementation group. We have cloned the E. coli lip locus and developed a recombinant plasmid-based genetic system for fine-structure physical-genetic mapping of mutations in this region of the E. coli chromosome. We also report that a recombinant plasmid containing a 5.2-kbp PvuII restriction fragment from the E. coli lip locus produced three proteins of approximately 8, 12, and 36 kDa by using either a maxicell or in vitro transcription translation expression system. The 36-kDa protein was identified as the gene product encoded by the lipA locus. Finally, we have identified a previously unreported lipoylated protein that functions in the glycine cleavage system of E. coli.
Properties of polyphosphate: AMP phosphotransferase of Acinetobacter strain 210A
Authors: Bonting C F , Kortstee G J , Zehnder A J
Published: November 2016

Polyphosphate:AMP phosphotransferase, an enzyme which catalyzes the phosphorylation of AMP to ADP at the expense of polyphosphate, was purified more than 1,500-fold from Acinetobacter strain 210A by streptomycin sulfate precipitation and by Mono-Q, Phenyl Superose, and Superose column chromatography. Streptomycin sulfate precipitation appeared to be an effective step in the purification procedure. During the following chromatographic steps, there was a 29-fold increase in specific activity but the yield was low (0.3%). Kinetic studies showed apparent Km values of 0.26 mM for AMP and 0.8 microM for polyphosphate with an average chain length of 35 phosphate groups. The highest activities were found with polyphosphate molecules of 18 to 44 phosphate residues. The polyphosphate chain was degraded completely to ADP. The mechanism of degradation is processive. No activity was obtained with ortho-, pyro-, tri-, and tetraphosphate. The enzyme was inhibited by pyro-, tri-, and tetraphosphate. The inhibition by tri- and tetraphosphate was mixed with polyphosphate as a substrate. The inhibition constants for the dissociation of the enzyme-inhibitor complex and for the enzyme-inhibitor-substrate complex were 0.9 and 6.5 mM, respectively, for triphosphate and 0.7 and 1.5 mM, respectively, for tetraphosphate.
Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes
Authors: Kozloff L M , Turner M A , Arellano F
Published: November 2016

The preliminary finding that nonprotein additions to the protein product of the ice-nucleating gene of Pseudomonas syringae or Erwinia herbicola are essential for ice nucleation at the warmest temperatures has led to experiments aimed at identifying possible linkages between the ice protein and the other components. It appears that the protein is coupled to various sugars through N- and O-glycan linkages. Mannose residues are apparently bound via an N-glycan bond to the amide nitrogen of one or more of the three essential asparagine residues in the unique amino-terminal portion of the protein. In turn, these mannose residues are involved in the subsequent attachment of phosphatidylinositol to the nucleation structure. This phosphatidylinositol-mannose-protein structure is the critical element in the class A nucleating structure. In addition to sugars attached to the asparagine residues, additional sugar residues appear to be attached by O-glycan linkages to serine and threonine residues in the primary repeating octapeptide, which makes up 70% of the total ice protein. These additional sugar residues include galactose and glucosamine and most likely additional mannose residues. These conclusions were based on (i) the changes in ice-nucleating activity due to the action of N- and O-glycanases, alpha- and beta-mannosidoses, and beta-galactosidase; (ii) immunoblot analyses of ice proteins in cell extracts after enzyme treatments; and (iii) the properties of transformed Ice+ Escherichia coli cells containing plasmids with defined amino-terminal and carboxyl-terminal deletions in the ice gene. Finally, evidence is presented that these sugar residues may play a role in aggregating the ice gene lipoglycoprotein compound into larger aggregates, which are the most effective ice nucleation structures.
Independence of bacteriophage N15 lytic and linear plasmid replication from the heat shock proteins DnaJ, DnaK, and GrpE
Authors: Tilly K
Published: November 2016

The chromosome of the temperate bacteriophage N15 replicates as a linear plasmid with covalently closed ends (or hairpins) when it forms a lysogen. I found that, in contrast to the cases for lambda and the low-copy-number plasmids F and P1, both phage and plasmid replication of N15 are independent of the heat shock proteins DnaJ, DnaK, and GrpE.
Partitioning of a mini-F plasmid into anucleate cells of the mukB null mutant
Authors: Ezaki B , Ogura T , Niki H , Hiraga S
Published: November 2016

The partition-proficient mini-F plasmid pXX325 was stably maintained in the mukB null mutant, which is defective in chromosome partitioning into the two daughter cells. In the null mutant, the plasmid was partitioned into both nucleate and anucleate daughter cells, independently of host chromosomes.
Independent regulation of nifHDK operon transcription and DNA rearrangement during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Golden J W , Whorff L L , Wiest D R
Published: November 2016

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 expresses the genes required for nitrogen fixation in terminally differentiated cells called heterocysts. The nifHDK operon encodes the nitrogenase polypeptides and is expressed at high levels in heterocysts. During heterocyst differentiation, an 11-kb DNA element is excised from the nifD gene by site-specific recombination. The xisA gene, located on the 11-kb element, is required for the excision of the element. Transcription and DNA rearrangement of the nifHDK operon both occur late during heterocyst differentiation, about 18 to 24 h after induction, suggesting that the regulation of these events might be coupled. We show that heterocyst-specific transcription and DNA rearrangement of the nifHDK operon are independent of one another. Northern (RNA) analysis of the xisA mutant strain DW12-2.2, which cannot excise the nifD 11-kb element or fix nitrogen, showed that the nifH and nifD genes are transcribed on unrearranged chromosomes. The nifK gene was not transcribed in DW12-2.2, indicating that its expression is dependent on the nifH promoter and excision of the 11-kb element from the operon. A 1.68-kb DNA fragment containing the nifH promoter was deleted from the chromosome to produce the mutant strain LW1. LW1 formed heterocysts but did not grow on nitrogen-free medium and showed no transcription through nifD. Southern analysis of LW1 showed normal excision of the 11-kb element from the nifHDK operon, indicating that transcription from the nifH promoter is not required for the developmentally regulated DNA rearrangement.
Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus
Authors: Ramachandra M , Seetharam R , Emptage M H , Sariaslani F S
Published: November 2016

We have purified an NADH-dependent ferredoxin reductase from crude extracts of Streptomyces griseus cells grown in soybean flour-enriched medium. The purified protein has a molecular weight of 60,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzyme requires Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing flavoproteins. A relatively large number of hydrophobic amino acid residues are found by amino acid analysis, and beginning with residue 7, a consensus flavin adenine dinucleotide binding sequence, GXGXXGXXXA, is revealed in this protein. In the presence of NADH, the ferredoxin reductase reduces various electron acceptors such as cytochrome c, potassium ferricyanide, dichlorophenolindophenol, and nitroblue tetrazolium. However, only cytochrome c reduction by the ferredoxin reductase is enhanced by the addition of ferredoxin. In the presence of NADH, S. griseus ferredoxin and cytochrome P-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin.
Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides
Authors: Meinke A , Gilkes N R , Kilburn D G , Miller R C , Warren R A
Published: November 2016

Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A. Meinke, C. Braun, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, J. Bacteriol. 173:308-314, 1991). The catalytic domain of 608 amino acids is at the N terminus. The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes. The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families. The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids. Deletion of these 131 amino acids reduces but does not eliminate activity. The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence. The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T. Watanabe, K. Suzuki, K. Oyanagi, K. Ohnishi, and H. Tanaka, J. Biol. Chem. 265:15659-15665, 1990). The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs). It functions as a CBD when fused to a heterologous polypeptide. Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD. The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity.
Upstream induction sequence, the cis-acting element required for response to the allantoin pathway inducer and enhancement of operation of the nitrogen-regulated upstream activation sequence in Saccharomyces cerevisiae
Authors: van Vuuren H J , Daugherty J R , Rai R , Cooper T G
Published: November 2016

Expression of the DAL2, DAL4, DAL7, DUR1,2, and DUR3 genes in Saccharomyces cerevisiae is induced by the presence of allophanate, the last intermediate of the allantoin degradative pathway. Analysis of the DAL7 5'-flanking region identified an element, designated the DAL upstream induction sequence (DAL UIS), required for response to inducer. The operation of this cis-acting element requires functional DAL81 and DAL82 gene products. We determined the DAL UIS structure by using saturation mutagenesis. A specific dodecanucleotide sequence is the minimum required for response of reporter gene transcription to inducer. There are two copies of the sequence in the 5'-flanking region of the DAL7 gene. There are one or more copies of the sequence upstream of each allantoin pathway gene that responds to inducer. The sequence is also found 5' of the allophanate-inducible CAR2 gene as well. No such sequences were detected upstream of allantoin pathway genes that do not respond to the presence of inducer. We also demonstrated that the presence of a UIS element adjacent to the nitrogen-regulated upstream activation sequence significantly enhances its operation.
Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa
Authors: MacGregor C H , Wolff J A , Arora S K , Phibbs P V
Published: November 2016

Mutants which are defective in catabolite repression control (CRC) of multiple independently regulated catabolic pathways have been previously described. The mutations were mapped at 11 min on the Pseudomonas aeruginosa chromosome and designated crc. This report describes the cloning of a gene which restores normal CRC to these Crc- mutants in trans. The gene expressing this CRC activity was subcloned on a 2-kb piece of DNA. When this 2-kb fragment was placed in a plasmid behind a phage T7 promoter and transcribed by T7 RNA polymerase, a soluble protein with a molecular weight (MW) of about 30,000 was produced in Escherichia coli. A soluble protein of identical size was overproduced in a Crc- mutant when it contained the 2-kb fragment on a multicopy plasmid. This protein could not be detected in the mutant containing the vector without the 2-kb insert or with no plasmid. When a 0.3-kb AccI fragment was removed from the crc gene and replaced with a kanamycin resistance cassette, the interrupted crc gene no longer restored CRC to the mutant, and the mutant containing the interrupted gene no longer overproduced the 30,000-MW protein. Pools of intracellular cyclic AMP and the activities of adenylate cyclase and phosphodiesterase were measured in mutant and wild-type strains with and without a plasmid containing the crc gene. No consistent differences between any strains were found in any case. These results provide original evidence for a 30,000-MW protein encoded by crc+ that is required for wild-type CRC in P. aeruginosa and confirms earlier reports that the mode of CRC is cyclic AMP independent in this bacterium.
Purification and characterization of hydroxypyruvate reductase from the facultative methylotroph Methylobacterium extorquens AM1
Authors: Chistoserdova L V , Lidstrom M E
Published: November 2016

Hydroxypyruvate reductase was purified to homogeneity from the facultative methylotroph Methylobacterium extorquens AM1. It has a molecular mass of about 71 kDa, and it consists of two identical subunits with a molecular mass of about 37 kDa. This enzyme uses both NADH (Km = 0.04 mM) and NADPH (Km = 0.06 mM) as cofactors, uses hydroxypyruvate (Km = 0.1 mM) and glyoxylate (Km = 1.5 mM) as the only substrates for the forward reaction, and carries out the reverse reaction with glycerate (Km = 2.6 mM) only. It was not possible to detect the conversion of glycolate to glyoxylate, a proposed role for this enzyme. Kinetics and inhibitory studies of the enzyme from M. extorquens AM1 suggest that hydroxypyruvate reductase is not a site for regulation of the serine cycle at the level of enzyme activity.