bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the spoIVB and recN loci of Bacillus subtilis
Authors: Van Hoy B E , Hoch J A
Published: November 2016

Two independent genes, recN and spoIVB, along with their respective promoter and termination regions, were discovered and sequenced in the 3.4-kilobase region between the ahrC and spoOA genes at map position 216 in the Bacillus subtilis chromosome map. The gene encoding a 576-amino-acid protein, which maintains a high homology with the Escherichia coli recN gene product, was adjacent to ahrC. The sequence revealed a 64,472-dalton polypeptide which contained a conserved ATP-binding site and possible lexA-type regulatory binding sequences in its promoter region. A second open reading frame identified as the spoIVB gene was directly downstream of recN. It consisted of 1,275 nucleotides which coded for a 425-amino-acid polypeptide with a molecular weight of 45,976. Phenotypic, genetic, and transcriptional analyses confirmed that this gene was spoIVB. Although no chloroform-resistant spores were produced by spoIVB-inactivated strains, under microscopic examination, phase-gray forespores were visible. The spoIVB165 mutation was localized to a 200-base-pair region in the amino-terminal portion of the polypeptide, spoIVB was not transcribed until hour 2 of sporulation in wild-type B. subtilis cells, as determined by beta-galactosidase activity assays from lacZ transcriptional fusion constructions. We found no amino acid sequence homology between the spoIVB gene product and other known bacterial proteins.
Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli
Authors: Kato N , Ohta M , Kido N , Ito H , Naito S , Hasegawa T , Watabe T , Sasaki K
Published: November 2016

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates (preferential growth along the a axis) or solid columns (preferential growth along the c axis), when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 degrees C for 10 days. Analyses of crystals suggested that they consist of hexagonal lattices with the a axis (a side of the lozenge as a unit cell on the basal plane) of 0.462 nm for all these three kinds of LPSs and the c axes (perpendicular to the basal plane) of 5.85, 8.47, and 8.75 nm for S. minnesota Re and Ra LPSs and E. coli K-12 LPS, respectively, and that hydrocarbon chains of the lipid A portion play the leading part in crystallization, whereas the hydrophilic part of the lipid A (the disaccharide backbone) and R core exhibit a disordered structure or are in a random orientation. The phenomenon of doubling of the a axis to 0.924 nm was observed with crystals of S. minnesota Re LPS when they were incubated in 70% ethanol for an additional 180 days, but not with crystals of S. minnesota Ra LPS or E. coli K-12 LPS. S. minnesota S-form LPS possessing the O-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions.
Genetic analysis of bacteriophage lambda integrase interactions with arm-type attachment site sequences
Authors: Lee E C , Gumport R I , Gardner J F
Published: November 2016

The bacteriophage P22-based challenge phage system was used to study lambda integrase (Int) protein binding to its arm-type recognition sequences in the bacteriophage lambda attachment site. Challenge phages were constructed that carried inserts containing either the contiguous P'123 arm-type sites or the single P'1 site within the P22 phage promoter, Pant, which is required for expression of antirepressor. If Int protein binds to these sequences in vivo, it represses transcription from Pant. We found that Int repressed Pant in phages carrying the P'123 sites more efficiently than those carrying only the P'1 site, suggesting that the protein binds cooperatively at the three adjacent sites. The Int protein from a related lambdoid phage, HK022, also repressed transcription by binding to the same arm-type sites. Mutations in the P'123 or P'1 sites that impair Int binding were isolated by selecting mutant phages that express antirepressor in the presence of Int. DNA sequence analyses showed that most of the mutants in the challenge phages carrying the P'123 sites contained multiple changes and that two mutants contained only single-base-pair changes at positions that are completely conserved among all arm-type sites. Thirty-five mutants were isolated and analyzed from phages containing only the P'1 site. Most mutants contained single-nucleotide changes, and mutations were isolated at 8 of the 10 positions of the site, suggesting that most if not all base pairs in the conserved recognition sequence are involved in Int binding.
Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression
Authors: Forsberg A , Wolf-Watz H
Published: November 2016

The yopE gene of Yersinia pseudotuberculosis was recently sequenced, and YopE was identified as an indispensable virulence determinant when tested in a mouse model (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). In the study described here, the DNA sequences of the yopE genes of Yersinia pestis EV76 and Yersinia enterocolitica 8081 were determined and compared with that of the Y. pseudotuberculosis gene. Only two codons were found to differ, both leading to amino acid replacements, when the gene from Y. pestis was compared. These two replacements were also present in the gene from Y. enterocolitica; in addition, 18 other codons were found to differ. Thirteen of these substitutions led to amino acid replacements. Downstream of the yopE gene, the plasmid partition locus par was found to be conserved in all three species. In Y. enterocolitica 8081, the sequence homology was interrupted by a putative insertion sequence element inserted between the yopE gene and the par region at a position only 5 base pairs downstream of the yopE stop codon. Upstream of the yopE gene, 620 base pairs were conserved in the three species. This region contained a 130-amino-acid-long open reading frame reading in the opposite direction to the yopE gene and expressed a 14-kilodalton protein in minicells. An insertion mutation in this region constructed in Y. pseudotuberculosis expressed significantly lower amounts of YopE protein in vitro than did the corresponding wild type. The expression level could be restored by transcomplementation. This new locus was designated yerA, for yopE-regulating gene A. The yerA mutant was avirulent when mice were challenged by oral infection.
Mechanism of gram variability in select bacteria
Authors: Beveridge T J
Published: November 2016

Gram stains were performed on strains of Actinomyces bovis, Actinomyces viscosus, Arthrobacter globiformis, Bacillus brevis, Butyrivibrio fibrisolvens, Clostridium tetani, Clostridium thermosaccharolyticum, Corynebacterium parvum, Mycobacterium phlei, and Propionibacterium acnes, using a modified Gram regimen that allowed the staining process to be observed by electron microscopy (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Furthermore, since a platinum salt replaced the iodine mordant of the Gram stain, energy-dispersive X-ray spectroscopy could evaluate the stain intensity and location by monitoring the platinum signal. These gram-variable bacteria could be split into two groups on the basis of their staining responses. In the Actinomyces-Arthrobacter-Corynebacterium-Mycobacterium-Propionibacterium group, few cells became gram negative until the exponential growth phase; by mid-exponential phase, 10 to 30% of the cells were gram negative. The cells that became gram negative were a select population of the culture, had initiated septum formation, and were more fragile to the stress of the Gram stain at the division site. As cultures aged to stationary phase, there was a relatively slight increase toward gram negativity (now 15 to 40%) due to the increased lysis of nondividing cells by means of lesions in the side walls; these cells maintained their rod shape but stained gram negative. Those in the Bacillus-Butyrivibrio-Clostridium group also became gram negative as cultures aged but by a separate set of events. These bacteria possessed more complex walls, since they were covered by an S layer. They stained gram positive during lag and the initial exponential growth phases, but as doubling times increased, the wall fabric underlying the S layer became noticeably thinner and diffuse, and the cells became more fragile to the Gram stain. By stationary phase, these cultures were virtually gram negative.
Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens
Authors: Uttaro A D , Cangelosi G A , Geremia R A , Nester E W , Ugalde R A
Published: November 2016

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.
Genetic organization of the hydrogen uptake (hup) cluster from Rhizobium leguminosarum
Authors: Leyva A , Palacios J M , Murillo J , Ruiz-Argüeso T
Published: November 2016

In symbiosis with peas, Rhizobium leguminosarum UPM791 induces the synthesis of a hydrogen uptake (Hup) system that recycles hydrogen generated in nodules by nitrogenase. A cosmid (pAL618) containing hup genes from this strain on a 20-kilobase-pair (kb) DNA insert has previously been isolated in our laboratory (A. Leyva, J. M. Palacios, T. Mozo, and T.Ruiz-Argüeso, J. Bacteriol. 169:4929-4934, 1987). Here we show that cosmid pAL618 contains all of the genetic information required to confer high levels of hydrogenase activity on the naturally Hup- strains R. leguminosarum UML2 and Rhizobium phaseoli CFN42, and we also describe in detail the organization of hup genes on pAL618. To study hup gene organization, site-directed transposon mutagenesis and complementation analysis were carried out. According to the Hup phenotype associated with the transposon insertions, hup genes were found to span a 15-kilobase-pair region within pAL618 insert DNA. Complementation analysis revealed that Hup- mutants fell into six distinct complementation groups that define six transcriptional units, designated regions hupI to hupVI. Region hupI was subcloned and expressed in Escherichia coli cells under the control of a bacteriophage T7 promoter. A polypeptide of ca. 65 kilodaltons that was cross-reactive with antiserum against the large subunit of Bradyrhizobium japonicum hydrogenase was detected both in E. coli cells carrying the cloned hupI region and in pea bacteroids from strain UPM791, indicating that region hupI codes for structural genes of R. leguminosarum hydrogenase.
Cloning and expression of a structural gene from Chlorobium vibrioforme that complements the hemA mutation in Escherichia coli
Authors: Avissar Y J , Beale S I
Published: November 2016

Escherichia coli SASX41B carries the hemA mutation and requires delta-aminolevulinic acid for growth. Strain SASX41B was transformed to prototrophy with pYA1, a plasmid vector carrying a 5.8-kilobase insert of genomic DNA from the green sulfur bacterium Chlorobium vibrioforme. Cell extracts prepared from transformed cells are able to catalyze transfer of label from [1-14C]glutamate or [3,4-3H]glutamyl-tRNA to delta-aminolevullinic acid at rates much higher than extracts of wild-type cells can, whereas extracts prepared from untransformed strain SASX41B cells lack both activities. By comparing the relative abilities of glutamyl-tRNAs derived from several heterologous cell types to function as substrates for the dehydrogenase reaction in extracts of HB101 and SASX41B cells transformed by pYA1, it was determined that the expressed dehydrogenase in the transformed cells resembled that of C. vibrioforme and not that of E. coli. Thus it can be concluded that plasmid pYA1 contains inserted DNA that codes for a structural component of C. vibrioforme glutamyl-tRNA dehydrogenase which confers glutamyl-tRNA substrate specificity.
Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida
Authors: Consevage M W , Phillips A T
Published: November 2016

The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.
Cloning and characterization of the hemA region of the Bacillus subtilis chromosome
Authors: Petricek M , Rutberg L , Schröder I , Hederstedt L
Published: November 2016

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.
Isolation and characterization of Candida albicans morphological mutants derepressed for the formation of filamentous hypha-type structures
Authors: Gil C , Pomés R , Nombela C
Published: November 2016

Several Candida albicans morphological mutants were obtained by a procedure based on a combined treatment with nitrous acid plus UV irradiation and a double-enrichment step to increase the proportion of mutants growing as long filamentous structures. Altered cell morphogenesis in these mutants correlated with an altered colonial phenotype. Two of these mutants, C. albicans NEL102 and NEL103, were selected and characterized. Mutant blastoconidia initiated budding but eventually gave rise to filamentous hypha-type formations. These filaments were long and septate, and they branched very regularly at positions near septa. Calcofluor white (which is known to bind chitin-rich areas) stained septa, branching zones, and filament tips very intensely, as observed under the fluorescence microscope. Wild-type hybrids were obtained by fusing protoplasts of strain NEL102 with B14, another morphological mutant previously described as being permanently pseudomycelial, indicating that genetic determinants responsible for the two altered phenotypes are different. The mutants characterized in this work seemed to sequentially express the morphogenic characteristics of C. albicans, from blastoconidia to hyphae, in the absence of any inducer. Further characterization of these strains could be relevant to gain understanding of the genetic control of dimorphism in this species.
Paracoccus denitrificans cytochrome c1 gene replacement mutants
Authors: Gerhus E , Steinrücke P , Ludwig B
Published: November 2016

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.
The Cpx proteins of Escherichia coli K-12: evidence that cpxA, ecfB, ssd, and eup mutations all identify the same gene
Authors: Rainwater S , Silverman P M
Published: November 2016

An existing cpxA(Ts) mutant was resistant to amikacin at levels that inhibited completely the growth of a cpxA+ and a cpxA deletion strain and failed to grow as efficiently on exogenous proline. These properties are similar to those of mutants altered in a gene mapped to the cpxA locus and variously designated as ecfB, ssd, and eup. The amikacin resistance phenotype of the cpxA mutant was inseparable by recombination from the cpxA mutant phenotype (inability to grow at 41 degrees C without exogenous isoleucine and valine) and was recessive to the cpxA+ allele of a recombinant plasmid. Using methods that ensured independent mutations in the cpxA region of the chromosome, we isolated six new amikacin-resistant mutants following nitrosoguanidine mutagenesis. Three-factor crosses mapped the mutations to the cpxA locus. When transferred by P1 transduction to a cpxB11 Hfr strain, each of the mutations conferred the Tra- and Ilv- phenotypes characteristic of earlier cpxA mutants. Two of the new mutations led to a significantly impaired ability to utilize exogenous proline, and four led to partial resistance to colicin A. Two of the new cpxA alleles were recessive to the cpxA+ allele, and four were dominant, albeit to different degrees. On the basis of these data, we argue that cpxA, ecfB, eup, and ssd are all the same gene. We discuss the cellular function of the cpxA gene product in that light.
Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into the di-chain form
Authors: Dekleva M L , Dasgupta B R
Published: November 2016

A protease that nicks the approximately 150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the approximately 150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an approximately 50-kDa light chain and an approximately 100-kDa heavy chain which are disulfide linked and is indistinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified greater than 1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The approximately 62-kDa amidase (protease) is a complex of 15.5- and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70 degrees C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.
Purification and characterization of a Bacteroides loeschei adhesin that interacts with procaryotic and eucaryotic cells
Authors: London J , Allen J
Published: November 2016

The adhesin of Bacteroides loeschei PK1295 that mediates coaggregation with Streptococcus sanguis 34 and hemagglutination of erythrocytes was purified to electrophoretic homogeneity. The lectinlike protein has an estimated native Mr of 450,000 and consists of six subunits of identical molecular weight (Mr 75,000). The purified adhesin appears to be a basic protein with a pI between 7.4 and 8.0. Amino acid and N-terminal sequence analyses were carried out with the purified protein. These indicated that the protein contains a large number of Asx and Glx residues as well as basic amino acid residues. The binding site of the pure adhesin retained its native configuration during purification. When preincubated with streptococcal partner cells at pH 4.6, the adhesin prevented B. loeschei cells from coaggregating with the streptococci. An adhesin preparation adjusted to a pH of 6.8 rapidly agglutinated both streptococci and neuraminidase-treated erythrocytes. Galactosides inhibited the agglutination reactions.
Identification of the promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli K-12 gene aroG
Authors: Baseggio N , Davies W D , Davidson B E
Published: November 2016

The promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli gene aroG (encoding the phenylalanine-sensitive 3-deoxy-arabinoheptulosonate-7-phosphate synthase) were located. Primer extension analysis and nuclease S1 mapping of in vivo transcripts were used to determine the 5' and 3' ends, respectively, of the mRNA. Both ends exhibited some heterogeneity with respect to length. The 3' end of the major molecular species was located within a region that has structural homology with known rho-independent terminators. The location of the aroG promoter was identified in both strands of the DNA by in vitro DNase I footprinting and methylation protection experiments with RNA polymerase. In these experiments, a region of up to 80 base pairs (bp) was protected by the binding of RNA polymerase. The location of the aroG operator was also identified in both strands of the DNA by in vitro DNase I footprinting with pure TyrR. TyrR protected 26 to 28 bp of DNA containing a 22-bp palindrome (TYR R box) and overlapping the -35 region of the promoter. Mutations in the aroG regulatory DNA were isolated by site-directed mutagenesis and cloned in a low-copy-number plasmid to generate aroG-lac fusions. The effects of the mutations on the regulation of aroG expression were determined by measuring the beta-galactosidase activities of the fusions in strains with tyrR, tyrR+, and multicopy tyrR+ genotypes. The results of this mutant analysis confirmed that the aroG operator contains a single TYR R box.
Cloning and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli
Authors: Russo T A , Thompson J S , Godoy V G , Malamy M H
Published: November 2016

We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type of NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda pR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and Tn1000 mutagenesis, the NANase structural gene and control region that functions in B. fragilis were localized to a 1.5- to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tn1000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.
Constitutive synthesis of a transport function encoded by the Thiobacillus ferrooxidans merC gene cloned in Escherichia coli
Authors: Kusano T , Ji G Y , Inoue C , Silver S
Published: November 2016

Mercuric reductase activity determined by the Thiobacillus ferrooxidans merA gene (cloned and expressed constitutively in Escherichia coli) was measured by volatilization of 203Hg2+. (The absence of a merR regulatory gene in the cloned Thiobacillus mer determinant provides a basis for the constitutive synthesis of this system.) In the absence of the Thiobacillus merC transport gene, the mercury volatilization activity was cryptic and was not seen with whole cells but only with sonication-disrupted cells. The Thiobacillus merC transport function was compared with transport via the merT-merP system of plasmid pDU1358. Both systems, cloned and expressed in E. coli, governed enhanced uptake of 203Hg2+ in a temperature- and concentration-dependent fashion. Uptake via MerT-MerP was greater and conferred greater hypersensitivity to Hg2+ than did uptake with MerC. Mercury uptake was inhibited by N-ethylmaleimide but not by EDTA. Ag+ salts inhibited mercury uptake by the MerT-MerP system but did not inhibit uptake via MerC. Radioactive mercury accumulated by the MerT-MerP and by the MerC systems was exchangeable with nonradioactive Hg2+.
A transferable plasmid associated with AS-48 production in Enterococcus faecalis
Authors: Martínez-Bueno M , Gálvez A , Valdivia E , Maqueda M
Published: November 2016

Enterococcus faecalis S-48 produces a peptide antibiotic, AS-48, and a bacteriocin, Bc-48. We have isolated mutants that lack these inhibitory characteristics. Further analysis of the mutants indicates that a plasmid of 56 kilobases (pMB2) may harbor the genes for AS-48. In conjugation experiments, pMB2 has been transferred into a plasmid-free OG1X strain of E. faecalis. The OG1X(pMB2) transconjugant produces the antibiotic AS-48 in solid medium, and the MIC of AS-48 for this strain is the same as that of the donor strain.
A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene
Authors: Sohail A , Lieb M , Dar M , Bhagwat A S
Published: November 2016

Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing an 11-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.
Osmotic regulation of beta(1-2) glucan synthesis in members of the family Rhizobiaceae
Authors: Zorreguieta A , Cavaignac S , Geremia R A , Ugalde R A
Published: November 2016

High osmolarity in the culture medium of growing Agrobacterium tumefaciens strongly inhibited the accumulation of cellular beta(1-2) glucan. However, the enzymatic system required for the synthesis of this polysaccharide from UDP-glucose was not repressed by high osmolarity. Mutants of A. tumefaciens and Rhizobium meliloti affected in beta(1-2) glucan synthesis were unable to grow normally in low-osmolarity media.
Cloning of a lincosamide resistance determinant from Streptomyces caelestis, the producer of celesticetin, and characterization of the resistance mechanism
Authors: Calcutt M J , Cundliffe E
Published: November 2016

Self-resistance has been investigated in Streptomyces caelestis (producer of the lincosamide antibiotic celesticetin), from which a lincosamide resistance determinant (clr) has been isolated on a 1-kilobase DNA fragment and cloned in Streptomyces lividans. The clr product is a specific methylase which produces a single residue of N6-monomethyladenine in 23S rRNA at position 2058, thereby rendering the 50S ribosmal subunit resistant to the action of lincosamides.
Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601
Authors: Mérida A , Leurentop L , Candau P , Florencio F J
Published: November 2016

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.
Stimulation of glutamine transport by osmotic stress in Escherichia coli K-12
Authors: Gehring K , Hofnung M , Nikaido H
Published: November 2016

Osmotic stress produced by high concentrations of sucrose stimulated the high-affinity transport of glutamine in Escherichia coli cells. Glutamine transport via a low-affinity system was not affected. Osmotic stress produced by NaCl, in contrast, inhibited the transport of glutamine and some other amino acids. Maltose transport was strongly inhibited by osmotic stress.
Cell cycle-independent lysis of Escherichia coli by cefsulodin, an inhibitor of penicillin-binding proteins 1a and 1b
Authors: Jacoby G H , Young K D
Published: November 2016

Cefsulodin lyses actively growing Escherichia coli by binding specifically to penicillin-binding proteins (PBPs) 1a and 1b. Recent findings (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989) have linked cefsulodin-induced lysis to septation during the first division cycle after a nutritional shift-up or chromosome replication realignment. We synchronized cells by membrane filtration to determine whether cefsulodin-induced lysis depended on septation in normally growing cells. Populations of newly divided cells were allowed to grow for variable lengths of time. Cefsulodin was added to these synchronous cultures, which represented points in two to three rounds of the cell cycle. Since the cell numbers were small, a new lysis assay was developed that was based on the release of DNA measured by fluorometry. Lysis occurred at a constant time after addition of the antibiotic, regardless of the time in the cell cycle at which the addition was made. Thus, cefsulodin-induced lysis is not linked to septation or to any other cell cycle-related event.
Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein
Authors: Olson E R , Dunyak D S , Jurss L M , Poorman R A
Published: November 2016

We describe the isolation and analysis of an Escherichia coli gene, dppA, and its role in dipeptide transport. dppA maps near min 79 and encodes a protein (DppA) that has regions of amino acid similarity with a peptide-binding protein from Salmonella typhimurium (OppA). Like OppA, DppA is found in the periplasmic space and thus is most likely a dipeptide-binding protein. Insertional inactivation of dppA results in the inability of a proline auxotroph to utilize Pro-Gly as a proline source. dppA-dependent Pro-Gly utilization does not require any of the three major proline transport systems, demonstrating that DppA is not simply a dipeptidase. An in vivo competition assay was used to show that DppA is probably involved in the transport of dipeptides other than Pro-Gly. Transcription of dppA is repressed by the presence of casamino acids, suggesting that the cell alters its dipeptide transport capabilities in response to an environmental signal.
Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis
Authors: Borchert T V , Nagarajan V
Published: November 2016

A series of alterations in the Bacillus amyloliquefaciens levansucrase signal peptide were made by in vitro mutagenesis, and their effect on the secretion of levansucrase in Bacillus subtilis was studied. Some of the alterations resulted in a completely defective signal peptide. These included the removal of positively charged residues from the N-terminus and disruption of the hydrophobic core of the signal peptide either by introducing a charged residue or by deleting five or more amino acids. Analysis of the signal peptide processing-site alterations revealed that small residues are preferred at the -1 and -3 positions. However, a wide variety of amino acids are tolerated at the +1 position.
Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination
Authors: Sussman M D , Setlow P
Published: November 2016

The gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination, has been cloned from Bacillus megaterium and Bacillus subtilis, and its nucleotide sequence has been determined. Use of a translational gpr-lacZ fusion showed that the B. subtilis gpr gene was expressed primarily, if not exclusively, in the forespore compartment of the sporulating cell, with expression taking place approximately 1 h before expression of glucose dehydrogenase and ssp genes. gpr-lacZ expression was abolished in spoIIAC (sigF) and spoIIIE mutants but was reduced only approximately 50% in a spoIIIG (sigG) mutant. However, the kinetics of the initial approximately 50% of gpr-lacZ expression were unaltered in a spoIIIG mutant. The in vivo transcription start site of gpr has been identified and found to be identical to the in vitro start site on this gene with either E sigma F or E sigma G. Induction of sigma G synthesis in vivo turned on gpr-lacZ expression in parallel with synthesis of glucose dehydrogenase. These data are consistent with gpr transcription during sporulation first by E sigma F and then by E sigma G.
Sequence domains required for the activity of avirulence genes avrB and avrC from Pseudomonas syringae pv. glycinea
Authors: Tamaki S J , Kobayashi D Y , Keen N T
Published: November 2016

avrB and avrC from Pseudomonas syringae pv. glycinea share significant amino acid homology but interact with different soybean resistance genes to elicit the hypersensitive defense reaction. Recombinant genes constructed between avrB and avrC revealed that the central regions were required for avirulence gene activity but the 5' and 3' termini were interchangeable. Recombinants involving the central regions did not yield any detectable avirulence gene activity, and no new avirulence phenotypes were observed from any of the chimeric genes. These results suggest that the protein products of avrB and avrC possess catalytic properties that are required for the avirulence phenotypes.
High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor
Authors: Wu J Y , Siegel L M , Kredich N M
Published: November 2016

The flavoprotein and hemoprotein components of Escherichia coli B NADPH-sulfite reductase are encoded by cysJ and cysI, respectively. Plasmids containing these two genes overexpressed flavoprotein catalytic activity and apohemoprotein by 13- to 35-fold, but NADPH-sulfite reductase holoenzyme activity was increased only 3-fold. Maximum overexpression of holoenzyme activity was achieved by the inclusion in such plasmids of Salmonella typhimurium cysG, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, a cofactor for the hemoprotein. Thus, cofactor deficiency, in this case siroheme, can limit overexpression of a cloned enzyme. Catalytically active holoenzyme accounted for 10% of total soluble protein in a host containing cloned cysJ, cysI, and cysG. A 5.3-kb DNA fragment containing S. typhimurium cysG was sequenced, and the open reading frame corresponding to cysG was identified by subcloning and by identifying plasmid-encoded peptides in maxicells. Comparison with the sequence reported for the E. coli cysG region (J. A. Cole, unpublished data; GenBank sequence ECONIRBC) indicates a gene order of nirB-nirC-cysG in the cloned S. typhimurium fragment. In addition, two open reading frames of unknown identity were found immediately downstream of cysG. One of these contains 11 direct repeats of 33 nucleotides each, which correspond to the consensus amino acid sequence Asp-Asp-Val-Thr-Pro-Pro-Asp-Asp-Ser-Gly-Asp.
Cloning and sequencing of the beta-lactam hydroxylase gene (cefF) from Streptomyces clavuligerus: gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes
Authors: Kovacevic S , Miller J R
Published: November 2016

The deacetylcephalosporin C synthetase (hydroxylase) gene from Streptomyces clavuligerus has been cloned and sequenced. The open reading frame codes for a protein with an Mr of 34,584. The hydroxylase gene (cefF) is closely linked to the epimerase gene (cefD) and the expandase gene (cefE) and is transcribed in the opposite orientation. The hydroxylase and expandase genes are 59 and 71% identical at the amino acid and DNA levels, respectively. cefE and cefF may have arisen from a gene duplication in the actinomycetes.
Effect of the delta subunit on assembly and proton permeability of the F0 proton channel of Escherichia coli F1F0 ATPase
Authors: Angov E , Ng T C , Brusilow W S
Published: November 2016

During the assembly of the Escherichia coli proton-translocating ATPase, the subunits of F1 interact with F0 to increase the proton permeability of the transmembrane proton channel. We tested the involvement of the delta subunit in this process by partially and completely deleting uncH (delta subunit) from a plasmid carrying the genes for the F0 subunits and delta and testing the effects of those F0 plasmids on the growth of unc+ and unc mutant E. coli strains. We found that the delta subunit was required for inhibition of growth of unc+ cells. We also tested membranes isolated from unc-deleted cells containing F0 plasmids for F1-binding ability. In unc-deleted cells, these plasmids produced F0 in amounts comparable to those found in normal unc+ E. coli cells, while having only small effects on cell growth. These studies demonstrate that the delta subunit plays an important role in opening the F0 proton channel but that it does not serve as a temporary plug of F0 during assembly, as had been previously speculated (S. Pati and W. S. A. Brusilow, J. Biol. Chem. 264:2640-2644, 1989).
Temperature sensitivity caused by missense suppressor supH and amber suppressor supP in Escherichia coli
Authors: Thorbjarnardóttir S , Björnsson A , Amundadóttir L , Eggertsson G
Published: November 2016

The temperature-sensitive missense suppressor supH and amber suppressor supP in Escherichia coli are mutations of the serU and leuX genes, respectively. The supH tRNA, tRNA(SerCAA), is expected to recognize UUG codons, which are normally read by tRNA(LeuCAA) and tRNA(LeuUAA), coded for by the leuX gene and the leuZ gene, respectively. We show that supP and supH are incompatible and that strains carrying both supP and a restrictive rpsL allele are temperature sensitive. It is suggested that the temperature sensitivity of both supH and supP strains is caused by deficient reading of UUG codons by tRNA(LeuUAA).
Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis
Authors: Pang A S , Nathoo S , Wong S L
Published: November 2016

Two novel Bacillus subtilis genes that regulate the production of several extracellular enzymes were clones and characterized. These two genes are organized as part of an operon. When cloned in a multicopy plasmid, the first gene (tenA, transcription enhancement) stimulates alkaline protease production at the transcriptional level. The second gene (tenI) exerts an opposite effect to reduce alkaline protease production. The production of neutral protease, levansucrase, and alkaline protease can be stimulated up to 11- to 55-fold. Thus, tenA is a new member of the deg (regulatory genes for degradative enzymes) family in B. subtilis. A functional degS product is required to observe the stimulatory effect from tenA. Between the promoter and the ribosome-binding site of tenA, there exists a terminatorlike structure. Deletion of this structure doubles the expression of tenA. Neither tenA nor tenI is essential for cell growth and the production of extracellular enzymes. However, inactivation of these genes causes a delay in sporulation. This operon is located close to tre on the genetic linkage map. The overall organization of this operon and its relationship with other known regulatory factors in the deg family are discussed.
delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli
Authors: O'Neill G P , Thorbjarnardóttir S , Michelsen U , Pálsson S , Söll D , Eggertsson G
Published: November 2016

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2
Authors: Toone W M , Rudd K E , Friesen J D
Published: November 2016

We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli.