bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species
Authors: Sung M H , Tanizawa K , Tanaka H , Kuramitsu S , Kagamiyama H , Soda K
Published: November 2016

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.
Purified Escherichia coli F-factor TraY protein binds oriT
Authors: Lahue E E , Matson S W
Published: November 2016

The traY gene of the Escherichia coli F plasmid has been shown by genetic studies (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980) to be involved in the site-specific nicking reaction at oriT required for the initiation of DNA transfer during bacterial conjugation. In order to assign a biochemical function to TraY protein, the traY gene was cloned in a plasmid vector which utilizes the strong T7 phi 10 promoter to overproduce the protein. The plasmid-encoded TraY protein was specifically labeled with [35S]methionine, and purification of the polypeptide was accomplished by monitoring the radioactive label. Purified TraY protein had a relative molecular mass of approximately 17,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino terminus of the purified protein was sequenced to confirm that the protein was encoded by the traY gene. The protein sequence revealed that the start codon for the TraY protein was a UUG codon 36 base pairs upstream of the AUG start site originally deduced from the DNA sequence (T. Fowler, L. Taylor, and R. Thompson, Gene 26:79-89, 1983). This start sequence confirmed the premise of Inamoto et al. that the F-plasmid TraY polypeptide-coding sequence would begin with UUG, creating a reading frame which renders a large degree of amino acid sequence identity with the TraY polypeptide from R100 (S. Inamoto, Y. Yoshioka, and E. Ohtsubo, J. Bacteriol. 170:2749-2757, 1988). The purified TraY protein from F bound specifically to the origin of transfer region of the F plasmid. However, no nicking activity was detected at oriT by using TraY protein or TraY protein in conjunction with helicase I.
Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids
Authors: François V , Conter A , Louarn J M
Published: November 2016

Conjugative temperature-sensitive plasmids were derived from pSC101. These plasmids are useful in genetic analysis for two reasons: (i) they render possible the construction of new Hfr lines by plasmid integration at predetermined chromosomal loci via Tn10 inverse transposition, and (ii) the Hfr characters are transducible via bacteriophage P1. We also showed that replication from pSC101 origin is deleterious for the plasmid-chromosome fusion.
Isolation of an Lc-specific Escherichia coli bacteriophage
Authors: Fralick J A , Diedrich D L , Casey-Wood S
Published: November 2016

We isolated an OmpF-specific bacteriophage whose host range mutant, SQ108h2, requires the presence of the Lc porin for its attachment and which can be used to screen or select for Lc-defective mutants among Escherichia coli K-12 strains lysogenic for the PA-2 converting phage.
Overproduction of acetate kinase activates the phosphate regulon in the absence of the phoR and phoM functions in Escherichia coli
Authors: Lee T Y , Makino K , Shinagawa H , Nakata A
Published: November 2016

A DNA fragment of Escherichia coli cloned on pBR322 elevated the production of alkaline phosphatase and phosphate-binding protein in a phoR phoM strain. Nucleotide sequence analysis and enzyme assays revealed that the DNA fragment contained the ackA gene, which codes for acetate kinase. A high gene dosage of ackA was needed to induce the production of alkaline phosphatase and phosphate-binding protein in this strain. Overexpression of ackA elevated the intracellular ATP concentration, an effect that might be related to activation of the phosphate regulon in the phoR phoM strain.
Regulation of the Salmonella typhimurium aroF gene in Escherichia coli
Authors: Muday G K , Herrmann K M
Published: November 2016

The Salmonella typhimurium aroF gene, encoding the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, was localized to a chromosomal PstI fragment by Southern blotting with an Escherichia coli aroF probe. This fragment was cloned by screening a plasmid library for complementation of an E. coli aroF mutant. The nucleotide sequence of S. typhimurium aroF was determined and compared with its E. coli homolog. The nucleotide sequences are 85.1% identical, and the corresponding amino acid sequences are 96.1% identical. The E. coli genes encoding the three DAHP synthase isoenzymes are evolutionarily more distant from one another than are the homologous aroF genes of E. coli and S. typhimurium. The S. typhimurium aroF regulatory region contains three imperfect palindromes, two upstream of the promoter and one overlapping the promoter, that are nearly identical to operators aroFo1, aroFo2, and TyrR box 1 of E. coli. The aroFo1 and aroFo2 sequences of the two organisms are each separated by three turns of the DNA helix with no sequence similarity. The 5' ends of the aroF transcripts for both organisms contain untranslated regions with potential stem-loop structures. Translational fusions of the aroF regulatory regions to lacZ were constructed and then introduced in single copy into the E. coli chromosome. beta-Galactosidase assays for tyrR-mediated regulation of aroF-lacZ expression revealed that the E. coli TyrR repressor apparently recognizes the operators of both organisms with about equal efficiency.
Identification and characterization of a new replication region in the Neisseria gonorrhoeae beta-lactamase plasmid pFA3
Authors: Gilbride K A , Brunton J L
Published: November 2016

The 7.1-kilobase-pair (kbp) plasmid pFA3 specifies TEM beta-lactamase production in Neisseria gonorrhoeae. We studied the minimal region required for replication of this plasmid in Escherichia coli by constructing a set of nested deletions of the 3.4-kbp PstI-HindIII fragment. The smallest fragment capable of maintenance in E. coli when ligated to a streptomycin-spectinomycin resistance cassette was 2.0 kbp in size and was different from another autonomously replicating fragment of pFA3 reported by K. H. Yeung and J. Dillon (Plasmid 20:232-240, 1988). The fragment contained single BamHI and XbaI sites and specified a 39-K protein. Fragments subcloned from the minimal region or constructed by deletion from the 3' or 5' ends were not capable of autonomous replication. Mutants constructed by end filling and religating DNA cleaved at the BamHI or XbaI sites were not capable of autonomous replication and no longer produced the 39K protein. These results suggest that replication is dependent on the 39K protein. DNA sequence analysis of the region showed an A-T-rich region followed by four 22-bp direct repeats followed by an open reading frame encoding a 39K basic protein.
Sequence analysis and comparison of int and xis genes from staphylococcal bacteriophages L54a and phi 11
Authors: Ye Z H , Buranen S L , Lee C Y
Published: November 2016

The DNA fragment encoding the integrase and excisionase genes involved in site-specific recombination of staphylococcal bacteriophage phi 11 was cloned and sequenced. The int and xis genes and the recombination site, attP, were highly clustered in a 1.7-kilobase DNA fragment with the gene order attP-int-xis. The int and xis genes were transcribed divergently, with the int gene transcribed toward the attp site and the xis gene transcribed away from the attP site. The deduced Int is a basic protein of 348 residues with an estimated molecular weight of 41,357. In contrast, the deduced Xis is an acidic protein containing 66 amino acids with an estimated molecular weight of 7,621. The site-specific recombination system of phi 11 was compared with that of a closely related bacteriophage, L54a.
Second-site revertants of the P1 copN22 copy mutant
Authors: Froehlich B J , Scott J R
Published: November 2016

Miniplasmids with the P1 copN22 mutation have a copy number about seven times that of the wild type. Selection for reduced copy number from this plasmid led to the isolation of second-site pseudorevertants, called poc mutants. DNA sequence analysis showed that all six independent poc mutants have a single base change in the same codon of the repA gene. This implicates the amino acid at this location, either directly or indirectly, in interactions important for copy number control.
Effects of alfalfa nod gene-inducing flavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes
Authors: Hartwig U A , Maxwell C A , Joseph C M , Phillips D A
Published: November 2016

Transcription of the nodulation genes nodABC in Rhizobium meliloti requires a plant flavonoid signal and nodD, a family of bacterial regulatory genes (nodD1, nodD2, and nodD3). Results from this study show that all previously identified nod gene inducers released by alfalfa seeds and roots induced nodABC-lacZ transcription in R. meliloti containing extra copies of nodD1, but only 4,4'-dihydroxy-2'-methoxychalcone gave high levels of induction with extra copies of nodD2. While mixtures of the methoxychalcone and luteolin showed a positive synergism with extra NodD1 protein, they apparently competed for binding to the NodD2 protein.
Starvation-induced cross protection against osmotic challenge in Escherichia coli
Authors: Jenkins D E , Chaisson S A , Matin A
Published: November 2016

Stationary-phase Escherichia coli cultures showed enhanced osmotic resistance as compared with cultures in mid-logarithmic growth or preadapted to osmotic stress. The osmotolerance that developed during starvation or osmotic adaptation required de novo protein synthesis. Of the 22 polypeptides induced during osmotic shock, five were also starvation proteins.
Occurrence of poly-beta-hydroxybutyrate in Spirulina species
Authors: Vincenzini M , Sili C , de Philippis R , Ena A , Materassi R
Published: November 2016

Several strains of photoautotrophically grown Spirulina spp. contained poly-beta-hydroxybutyrate (PHB) at concentrations never exceeding a few milligrams per gram of dry weight. Under mixotrophic growth conditions in the presence of acetate, PHB reached values greater than 2.5% of dry weight. With pyruvate, no significant effect on PHB accumulation was obtained.
Development of thermotolerance in Neurospora crassa by heat shock and other stresses eliciting peroxidase induction
Authors: Kapoor M , Sreenivasan G M , Goel N , Lewis J
Published: November 2016

Hyperthermia, CdCl2, sodium arsenite, and H2O2 led to the rapid appearance of high levels of peroxidase in Neurospora crassa cultures and induced tolerance toward normally lethal temperatures in 60-h-old colonies. Intracellular superoxide dismutase levels did not correlate with the development of thermotolerance.
Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens
Authors: Mannarelli B M , Evans S , Lee D
Published: November 2016

A gene coding for xylanase activity, xynA, from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens 49 was cloned into Escherichia coli JM83 by using plasmid pUC19. The gene was located on a 2.3-kilobase (kb) DNA insert composed of two adjacent EcoRI fragments of 1.65 and 0.65 kb. Expression of xylanase activity required parts of both EcoRI segments. In E. coli, the cloned xylanase enzyme was not secreted and remained cell associated. The enzyme exhibited no arabinosidase, cellulase, alpha-glucosidase, or xylosidase activity. The isoelectric point of the cloned protein was approximately 9.8, and optimal xylanase activity was obtained at pH 5.4. The nucleotide sequence of the 1,535-base-pair EcoRV-EcoRI segment from the B. fibrisolvens chromosome that included the xynA gene was determined. An open reading frame was found that encoded a 411-amino-acid-residue polypeptide of 46,664 daltons. A putative ribosome-binding site, promoter, and leader sequence were identified. Comparison of the XynA protein sequence with that of the XynA protein from alkalophilic Bacillus sp. strain C-125 revealed considerable homology, with 37% identical residues or conservative changes. The presence of the cloned xylanase gene in other strains of Butyrivibrio was examined by Southern hybridization. The cloned xylanase gene hybridized strongly to chromosomal sequences in only two of five closely related strains.
Phospholipid biosynthesis in Candida albicans: regulation by the precursors inositol and choline
Authors: Klig L S , Friedli L , Schmid E
Published: November 2016

Phospholipid metabolism in the pathogenic fungus Candida albicans was examined. The phospholipid biosynthetic pathways of C. albicans were elucidated and were shown to be similar to those of Saccharomyces cerevisiae. However, marked differences were seen between these two fungi in the regulation of the pathways in response to exogenously provided precursors inositol and choline. In S. cerevisiae, the biosynthesis of phosphatidylcholine via methylation of phosphatidylethanolamine appears to be regulated in response to inositol and choline; provision of choline alone does not repress the activity of this pathway (G. M. Carman and S. A. Henry, Annu. Rev. Biochem. 58:636-669, 1989). The same pathway in C. albicans responds to the exogenous provision of choline. Possible explanations for the observed differences in regulation are discussed.
Relationship between changes in buoyant density and formation of new sites of cell wall growth in cultures of streptococci (Enterococcus hirae ATCC 9790) undergoing a nutritional shift-up
Authors: Higgins M L , Haines M , Whalen M , Glaser D , Bylund J
Published: November 2016

When the glutamate concentration of cultures of Enterococcus hirae was raised from 20 to 300 micrograms/ml, the mass doubling time decreased from ca. 85 to 45 min in 9 min, but balanced growth was not reestablished for 30 to 40 min. During the unbalanced period of growth, RNA and protein synthesis proceeded more rapidly than did peptidoglycan synthesis, buoyant density increased from ca. 1.1024 to 1.1075 g/ml, and the rate of formation of new cell wall growth sites transitorily accelerated above the new growth rate. When studied as a function of cell size, all cultures showed buoyant density to decrease around cell separation, increase as cells increased in size, and then plateau when cells reached large volumes. Greater increases in buoyant density as a function of cell size were seen after shift-up, with the greatest increases observed at 15 to 20 min after shift-up, when the rate of formation of new sites was also maximal. In a population of cells examined by electron microscopy 15 min after shift-up, buoyant density and the frequency of cells with new sites increased as old sites approached the size of two poles. These data were consistent with a model whereby buoyant density increases in the terminal stages of the cell cycle when the surface grows slower than the cytoplasm. The greater the difference in the rates of inside to outside growth, the greater the increase in buoyant density and the more frequently new sites will be initiated.
Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli
Authors: MacInnes J I , Kim J E , Lian C J , Soltes G A
Published: November 2016

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.
High-level expression of the FtsA protein inhibits cell septation in Escherichia coli K-12
Authors: Wang H C , Gayda R C
Published: November 2016

DNA fragments encoding the ftsA gene were subcloned into plasmids downstream of a lac promoter or a tac promoter. These plasmid constructs, when transformed into wild-type and mutant strains, inhibited normal cell septation, causing the formation of long nonseptate filaments. This phenotype is due to overproduction of the FtsA protein.
Isolation of temperature-sensitive McrA and McrB mutations and complementation analysis of the McrBC region of Escherichia coli K-12
Authors: Piekarowicz A , Yuan R , Stein D C
Published: November 2016

We isolated temperature-sensitive mcrA and mcrBC mutants of Escherichia coli. At 42 degrees C, they were unable to restrict the T-even bacteriophages T6gt and T4gt or plasmids encoding cloned DNA methylase genes whose specificities confer sensitivity to the McrA and McrBC nucleases. Complementation analysis of the McrBC region (mcrB251) with the complete cloned McrBC system or a derivative with mcrB alone indicated that the mutation shows an absolute defect for the restriction of DNA containing hydroxymethylcytosine and a thermosensitive defect for the restriction of DNA containing methylcytosine. The properties of the McrA temperature-sensitive mutants suggest that some of these mutations can also influence the restriction of DNA containing hydroxymethylcytosine or methylcytosine residues.
Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX)
Authors: Stoorvogel J , van Bussel M J , van de Klundert J A
Published: November 2016

We have described a gene coding for an Enterobacter cloacae protein, provisionally called OmpX (J. Stoorvogel, M. J. A. W. M. van Bussel, J. Tommassen, and J. A. M. van de Klundert, J. Bacteriol. 173:156-160, 1991). In the work reported here, OmpX was localized in the cell envelope by means of sucrose gradient fractionation of membrane vesicles. Overproduction of OmpX in Escherichia coli from a multicopy plasmid resulted in a reduction in the amount of OmpF. No accumulation of OmpF, of its uncleft precursor, or of its degradation products could be detected in various cell fractions by Western immunoblot analysis using monoclonal antibodies produced in response to OmpF. A decrease in the rate of synthesis of ompF mRNA was indicated by a beta-galactosidase assay in an ompF-lacZ fusion strain containing the cloned ompX gene and by Northern (RNA) blot analysis. These results indicate that the inhibition is at the level of transcription. Colony hybridization, using an internal ompX fragment as a probe, showed a widespread distribution of the ompX gene among clinical isolates of members of the family Enterobacteriaceae. To study the function of the OmpX protein and its role in the regulation of porin protein synthesis, the ompX gene was deleted from the Enterobacter cloacae chromosome and replaced by the aphA gene. The absence of the ompX gene had no apparent effect on cell growth or on the regulation of the porin proteins.
Permissive sites and topology of an outer membrane protein with a reporter epitope
Authors: Charbit A , Ronco J , Michel V , Werts C , Hofnung M
Published: November 2016

We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12. This approach consists of inserting the same reporter foreign antigenic determinant (the C3 epitope from poliovirus) at different sites of LamB so that the resulting hybrid proteins have essentially kept the in vivo biological properties of LamB and therefore its cellular location and structure; the corresponding sites are called permissive sites. A specific monoclonal antibody can then be used to examine the position of the reporter epitope with respect to the protein and the membrane. We present an improved and efficient procedure that led us to identify eight new permissive sites in LamB. These sites appear to be distributed on both sides of the membrane. At one of them (after residue 253), the C3 epitope was detected on intact bacteria, providing the first direct argument for exposure of the corresponding LamB region at the cell surface. At this site as well as at four others (after residues 183, 219, 236, and 352), the C3 epitope could be detected with the C3 monoclonal antibody at the surface of the extracted trimeric LamB-C3 hybrid proteins. We provide a number of convergent arguments showing that the hybrid proteins are not strongly distorted with respect to the wild-type protein so that the conclusions drawn are also valid for this protein. These conclusions are essentially in agreement with the proposed folding model for the LamB protein. They agree, in particular, with the idea that regions 183 and 352 are exposed to the periplasm. In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.
Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi
Authors: Meinke A , Braun C , Gilkes N R , Kilburn D G , Miller R C , Warren R A
Published: November 2016

The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonas fimi. CenB comprises 1,012 amino acids and has a molecular weight of 105,905. The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are greater than 60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C. fimi cellulases Cex and CenA. A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB. The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulose-binding site. The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E. CenB releases mostly cellobiose and cellotetraose from cellohexaose. Like CenA, CenB hydrolyzes the beta-1,4-glucosidic bond with inversion of the anomeric configuration. The pH optimum for CenB is 8.5, and that for CenA is 7.5.
Genetic evidence for interaction of sigma A with two promoters in Bacillus subtilis
Authors: Kenney T J , Moran C P
Published: November 2016

The specificity of promoter binding by RNA polymerase is governed by the sigma subunit. Recent studies, in which single-amino-acid substitutions in sigma factors have been found to suppress the effects of specific base pair substitutions in promoters, support the model that these sigma factors make sequence-specific contacts with nucleotides at the -10 and -35 regions of promoters. We found that single-amino-acid substitutions in the putative -35 region and -10 region recognition domains of sigma A specifically suppressed the effects of mutations in the -35 and -10 regions, respectively, of two promoters that are expressed in exponentially growing Bacillus subtilis. These mutations change the specificity of sigma A, the primary sigma factor in growing B. subtilis, and demonstrate that this sigma factor interacts with promoters in a manner similar to that of its homolog in Escherichia coli, sigma 70. These mutant derivatives of sigma A also provide a tool that may be useful for determining whether sigma A uses specific promoters in vivo.
Protein synthesis in vitro by Micrococcus luteus
Authors: Farwell M A , Rabinowitz J C
Published: November 2016

Bacillus subtilis and related gram-positive bacteria which have low to moderate genomic G + C contents are unable to efficiently translate mRNA derived from gram-negative bacteria, whereas Escherichia coli and other gram-negative bacteria are able to translate mRNA from both types of organisms. This phenomenon has been termed translational species specificity. Ribosomes from the low-G + C-content group (low-G + C group) of gram-positive organisms (B. subtilis and relatives) lack an equivalent to Escherichia ribosomal protein S1. The requirement for S1 for translation in E. coli (G. van Dieijen, P. H. van Knippenberg, J. van Duin, B. Koekman, and P. H. Pouwels, Mol. Gen. Genet. 153:75-80, 1977) and its specific role (A.R. Subramanian, Trends Biochem. Sci. 9:491-494, 1984) have been proposed. The group of gram-positive bacteria characterized by high genomic G + C content (formerly Actinomyces species and relatives) contain S1, in contrast to the low-G + C group (K. Mikulik, J. Smardova, A. Jiranova, and P. Branny, Eur. J. Biochem. 155:557-563, 1986). It is not known whether members of the high-G + C group are translationally specific, although there is evidence that one genus, Streptomyces, can express Escherichia genes in vivo (M. J. Bibb and S. N. Cohen, Mol. Gen. Genet. 187:265-277, 1985; J. L. Schottel, M. J. Bibb, and S. N. Cohen, J. Bacteriol. 146:360-368, 1981). In order to determine whether the organisms of this group are translationally specific, we examined the in vitro translational characteristics of a member of the high-G + C group, Micrococcus luteus, whose genomic G + C content is 73%. A semipurified coupled transcription-translation system of M. luteus translates Escherichia mRNA as well as Bacillus and Micrococcus mRNA. Therefore, M. luteus is translationally nonspecific and resembles E. coli rather than B. subtilis in its translational characteristics.
Protein U, a late-developmental spore coat protein of Myxococcus xanthus, is a secretory protein
Authors: Gollop R , Inouye M , Inouye S
Published: November 2016

Protein U is a spore coat protein produced at the late stage of development of Myxococcus xanthus. This protein was isolated from developmental cells, and its amino-terminal sequence was determined. On the basis of this sequence, the gene for protein U (pru) was cloned and its DNA sequence was determined, revealing an open reading frame of 179 codons. The product from this open reading frame has a typical signal peptide of 25 amino acid residues at the amino terminal end, followed by protein U of 154 residues. This result indicates that protein U is produced as a secretory precursor, pro-protein U, which is then secreted across the membrane to assemble on the spore surface. This is in sharp contrast to protein S, a major spore coat protein produced early in development, which has no signal peptide, indicating that there are two distinct pathways for trafficking of spore coat proteins during the differentiation of M. xanthus.
Further characterizations of bleomycin-sensitive (blm) mutants of Saccharomyces cerevisiae with implications for a radiomimetic model
Authors: Moore C W
Published: November 2016

Direct selection for 12 mutations (blm) conferring hypersensitivities to lethal effects of bleomycins in Saccharomyces cerevisiae resulted in mutants exhibiting cross-hypersensitivity to ionizing radiation and hydrogen peroxide. Remaining mutations did not confer cross-hypersensitivity to radiation. All blm mutations were recessive, except codominant blm3-1, and were assigned to seven complementation groups.
Rapid site-specific DNA inversion in Escherichia coli mutants lacking the histonelike protein H-NS
Authors: Kawula T H , Orndorff P E
Published: November 2016

Escherichia coli pilG mutants are thought to have a dramatically higher DNA inversion rate as measured by the site-specific DNA inversion of the type 1 pili pilA promoter. DNA sequence of the pilG gene confirmed its identity to the gene encoding the bacterial histonelike protein H-NS. Unlike other histonelike protein complexes that enhance site-specific DNA recombination, the H-NS protein inhibited this process. This inhibition was indicated by the increased inversion rate of the pilA promoter region effected by two different mutant pilG alleles. One of these alleles, pilG1, conferred a mutant phenotype only at low temperature attributable to a T-to-G transversion in the -35 sequence of the pilG promoter. The other allele, pilG2-tetR, was an insertion mutation in the pilG coding region that conferred the mutant phenotype independent of temperature. We measured an approximately 100-fold-increased pilA promoter inversion rate in the mutant by exploiting the temperature-dependent expression of pilG1 and using a novel rapid-population-sampling method. Contrary to one current view on how the H-NS protein might act to increase DNA inversion rate, we found no evidence to support the hypothesis that DNA supercoiling affected pilA promoter inversion.
Salmonella typhimurium prfA mutants defective in release factor 1
Authors: Elliott T , Wang X
Published: November 2016

Mutations have been characterized that map in the prfA gene of Salmonella typhimurium. These weak amber suppressors show increased readthrough of UAG but not UAA or UGA codons. Some hemA mutants exhibit a similar suppressor activity due to transcriptional polarity on prfA. All of the suppressors mapping in prfA are recessive to the wild type. Two mutant prfA genes were cloned onto plasmids, and their DNA sequences were determined. A method was devised for transferring the sequenced mutant alleles back to their original location in S. typhimurium via an Escherichia coli recD strain that carries the entire S. typhimurium hemA-prfA operon as a chromosomal insertion in trp. This reconstruction experiment showed that the mutations sequenced are sufficient to confer the suppressor phenotype.
The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli
Authors: McCann M P , Kidwell J P , Matin A
Published: November 2016

KatF is required for the expression of some 32 carbon starvation proteins in Escherichia coli including 6 previously identified as Pex. Mutants with the katF gene survive carbon and nitrogen starvation poorly. Many of the KatF-regulated starvation proteins are common to those induced by other stresses, and the mutant failed to develop starvation-mediated cross protection to osmotic, oxidative, and heat stresses. Furthermore, thermal resistance was not induced in the mutant by heat preadaptation, and it exhibited an altered pattern of protein synthesis at elevated temperature. Thus, KatF is a major switch that controls the starvation-mediated resistant state in E. coli.
Molecular cloning of two linked loci that increase the transformability of transformation-deficient mutants of Haemophilus influenzae
Authors: Larson T G , Roszczyk E , Goodgal S H
Published: November 2016

A plasmid containing a 13.3-kb insert (pER194) was isolated from an EcoRI genomic library of Haemophilus influenzae on the basis of its ability to increase the transformability of the transformation-deficient mutants Com-78 and Com-101. The plasmid failed to increase the transformability of the Rec-1 and Rec-2 mutants, indicating that the mutations producing the Com-78 and Com-101 phenotypes are distinct from those giving rise to the Rec-1 and Rec-2 phenotypes. The physical mapping of the cloned fragment on the H. influenzae chromosome was found to be consistent with the genetic mapping of the Com-101 trait. A 2.8-kb EcoRI-BglII subfragment, representing one end of the 13.3-kb clone, was found to increase the transformation frequency of the Com-78 and Com-101 mutants when supplied in trans, indicating that the subfragment carries one or more loci required for chromosomal transformation. The corresponding region of the Com-101 chromosome was determined by hybridization analysis to contain a 0.3-kb insertion, suggesting that the Com-101 strain may contain an insertion mutation at this locus. A 3.0-kb EcoRI-MluI subfragment, representing the other end of the 13.3-kb EcoRI fragment, was found to increase the transformation frequency of the Com-101 mutant but not of the Com-78 mutant, suggesting that the Com-101 phenotype results from a complex genotype involving mutations at two or more transformation-related loci. This conclusion is consistent with data indicating that the Com-101 trait can be genetically separated into at least two components.
Expression of the bop gene cluster of Halobacterium halobium is induced by low oxygen tension and by light
Authors: Shand R F , Betlach M C
Published: November 2016

The bop gene cluster consists of at least three genes: bop (bacterio-opsin), brp (bacterio-opsin-related protein), and bat (bacterio-opsin activator). We have quantitated transcript levels from these genes in a wild-type and bacterioruberin-deficient mutant of Halobacterium halobium under conditions which affect purple membrane synthesis. In wild-type cultures grown under high oxygen tension in the dark, bop and bat transcript levels were low during steady-state growth and then increased approximately 29- and approximately 45-fold, respectively, upon entry into stationary phase. brp gene transcription remained very low and essentially unchanged under these conditions. In addition, exposure of wild-type cultures growing under high oxygen tension to 30,000 lx of light stimulated expression of all three genes, especially brp. In contrast to the wild-type, transcription from all three genes in the bacterioruberin mutant was very high during steady-state growth under high oxygen tension in the dark. Cultures of the bacterioruberin mutant were shifted at early stationary phase to low oxygen tension to determine whether oxygen concentrations lower than those present in stationary phase would induce transcription of the bop gene cluster in this strain. Indeed, transcription was induced, suggesting that the bop gene cluster is not completely uncoupled from regulation by oxygen tension in the bacterioruberin mutant. From these data, we propose a regulatory model involving two different mechanisms: (i) bat gene expression is induced under conditions of low oxygen tension and the bat gene product activates bop gene expression and (ii) light induces brp transcription, which stimulates or modulates bat transcription.
Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting
Authors: Rothmel R K , Shinabarger D L , Parsek M R , Aldrich T L , Chakrabarty A M
Published: November 2016

CatR, a LysR family protein, positively regulates the Pseudomonas putida catBC operon, which is required for growth on benzoate as a sole carbon source. Transcriptional studies show that the catR and catBC promoters are divergent and overlapping by 2 bp. A beta-galactosidase promoter probe vector was constructed to analyze expression from the catR and catBC promoters under induced and uninduced conditions. As predicted, the catBC promoter is expressed only under induced conditions, while the catR promoter is constitutive. CatR has been shown to specifically bind the catRBC promoter region, and this property was used to devise a purification protocol for CatR. Linear M13 DNA containing the catRBC control region was covalently bound to cyanogen bromide-activated Sepharose in order to construct a DNA affinity column. Crude extracts containing hyperproduced CatR protein were then incubated with the affinity resin under binding conditions, and the CatR protein was eluted with 1 M NaCl. CatR was also purified by heparin-agarose chromatography. This highly purified protein was used for gel retardation and hydroxyl-radical footprinting studies. From this analysis, it was shown that CatR binds upstream of the catBC promoter within the transcribed region of catR.
Type 1 fimbriae of Salmonella enteritidis
Authors: Müller K H , Collinson S K , Trust T J , Kay W W
Published: November 2016

Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21.
Temperature-induced protein synthesis in Bacillus stearothermophilus NUB36
Authors: Wu L , Welker N E
Published: November 2016

Cultures of Bacillus stearothermophilus subjected to a temperature shift-up or shift-down of 15 degrees C within the normal temperature range of growth (45 to 65 degrees C) enter a transient adaptation period before exponential growth at the new temperature. The de novo synthesis of some proteins coincides with the adaptation period.
Purification of glucose-inducible outer membrane protein OprB of Pseudomonas putida and reconstitution of glucose-specific pores
Authors: Saravolac E G , Taylor N F , Benz R , Hancock R E
Published: November 2016

A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida. OprB was surface expressed in whole cells, had a high beta-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation. Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (KS = 110 mM) and other carbohydrates. However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.
Resuscitation of Vibrio vulnificus from the viable but nonculturable state
Authors: Nilsson L , Oliver J D , Kjelleberg S
Published: November 2016

Stationary-phase-grown cells of the estuarine bacterium Vibrio vulnificus became nonculturable in nutrient-limited artificial seawater microcosms after 27 days at 5 degrees C. When the nonculturable cells were subjected to temperature upshift by being placed at room temperature, the original bacterial numbers were detectable by plate counts after 3 days, with a corresponding increase in the direct viable counts from 3% to over 80% of the total cell count. No increase in the total cell count was observed during resuscitation, indicating that the plate count increases were not due to growth of a few culturable cells. Chloramphenicol and ampicillin totally inhibited resuscitation of the nonculturable cells when added to samples that had been at room temperature for up to 24 h. After 72 h of resuscitation, the inhibitors had an easily detectable but reduced effect on the resuscitated cells, indicating that protein and peptidoglycan synthesis were still ongoing. Major changes in the morphology of the cells were discovered. Nonculturable cells of V. vulnificus were small cocci (approximately 1.0 micron in diameter). Upon resuscitation, the cells became large rods with a size of mid-log-phase cells (3.0 microns in length). Four days after the cells had become fully resuscitated, the cell size had decreased to approximately 1.5 micron in length and 0.7 micron in width. The cells were able to go through at least two cycles of nonculturability and subsequent resuscitation without changes in the total cell count. This is the first report of resuscitation, without the addition of nutrient, of nonculturable cells, and it is suggested that temperature may be the determining factor in the resuscitation from this survival, or adaptation, state of certain species in estuarine environments.
The Actinobacillus pleuropneumoniae hemolysin determinant: unlinked appCA and appBD loci flanked by pseudogenes
Authors: Chang Y F , Young R , Struck D K
Published: November 2016

The appBD genes encoding the secretion functions for the 110-kDa RTX hemolysin of Actinobacillus pleuropneumoniae have been cloned and sequenced. Unlike analogous genes from other RTX determinants, the appBD genes do not lie immediately downstream from the hemolysin structural gene, appA. Although isolated from a diverse group of gram-negative organisms, the appBD genes and the characterized RTX BD genes from other organisms all exhibit a high degree of homology at both the DNA and predicted amino acid sequence levels. Analysis of the DNA sequences 3' to appA and 5' to appB suggests that these regions harbor remnant RTX B and A pseudogenes, respectively. Although the appA gene is most similar to the lktA gene from Pasteurella haemolytica (Y. F. Chang, R. Young, and D. K. Struck, DNA 8:635-647, 1989), the RTX A pseudogene upstream from appB most closely resembles the hlyB gene from Escherichia coli, suggesting that the appCA and appBD operons were derived from different ancestral RTX determinants.
Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Alam J , Vrba J M , Cai Y , Martin J A , Weislo L J , Curtis S E
Published: November 2016

A family of repetitive elements from the cyanobacterium Anabaena sp. strain PCC 7120 was identified through the proximity of one element to the psbAI gene. Four members of this seven-member family were isolated and shown to have structures characteristic of bacterial insertion sequences. Each element is approximately 1,200 bp in length, is delimited by a 30-bp inverted repeat, and contains two open reading frames in tandem on the same DNA strand. The four copies differ from each other by small insertions or deletions, some of which alter the open reading frames. By using a system designed to trap insertion elements, one of the elements, denoted IS895, was shown to be mobile. The target site was not duplicated upon insertion of the element. Two other filamentous cyanobacterial strains were also found to contain sequences homologous to IS895.
Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(beta-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli
Authors: Pries A , Priefert H , Krüger N , Steinbüchel A
Published: November 2016

From genomic libraries of Alcaligenes eutrophus H16 in lambda L47 and in pVK100, we cloned DNA fragments which restored the wild-type phenotype to poly(beta-hydroxybutyric acid) (PHB)-leaky mutants derived from strains H16 and JMP222. The nucleotide sequence analysis of a 4.5-kb region of one of these fragments revealed two adjacent open reading frames (ORF) which are relevant for the expression of the PHB-leaky phenotype. The 1,799-bp ORF1 represented a gene which was referred to as phbI. The amino acid sequence of the putative protein I (Mr, 65,167), which was deduced from phbI, exhibited 38.9% identity with the primary structure of enzyme I of the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system (PEP-PTS). The upstream 579-bp ORF2 was separated by 50 bp from ORF1. It included the 270-bp phbH gene which encoded protein H (Mr, 9,469). This protein exhibited 34.9% identity to the HPr protein of the E. coli PEP-PTS. Insertions of Tn5 in different PHB-leaky mutants were mapped at eight different positions in phbI and at one position in phbH. Mutants defective in phbH or phbI exhibited no pleiotropic effects and were not altered with respect to the utilization of fructose. However, PHB was degraded at a higher rate in the stationary growth phase. The functions of these HPr- and enzyme I-like proteins in the metabolism of PHB are still unknown. Evidence for the involvement of these proteins in regulation of the metabolism of intracellular PHB was obtained, and a hypothetical model is proposed.
Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes
Authors: Cameron B , Guilhot C , Blanche F , Cauchois L , Rouyez M C , Rigault S , Levy-Schil S , Crouzet J
Published: November 2016

A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis. The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT). Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertion-mediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid.