bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis
Authors: Nakano M M , Marahiel M A , Zuber P
Published: November 2016

Surfactin is a lipopeptide antibiotic produced by the cells of Bacillus subtilis ATCC 21332. A genetic locus responsible for surfactin production (sfp) was transferred from ATCC 21332 to JH642, a derivative of the standard B. subtilis 168. To study the sfp locus at the molecular level, a Tn917 insertion mutant that was blocked in surfactin production (srf) was isolated. The srf::Tn917 mutation was found to be closely linked to sfp, and both loci mapped by PBS1 phage transduction to the chromosomal region between aroI and mtlB. These studies suggest that JH642, a strain which is not a producer of surfactin (genotypically sfp0), contains at least some of the genes encoding surfactin production. Expression of the srf gene(s) was examined in both sfp and sfp0 cells by assaying beta-galactosidase activity encoded by a promoterless lacZ gene that was fused to the srf::Tn917 insertion. In cells of both strains, srf-directed beta-galactosidase activity increased when cells entered the stationary phase of the growth curve, but the activity in sfp cells was higher than that in sfp0 cells. srf-lacZ expression was partially impaired by a mutation in spo0A. In sfp0 cells, this dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation. In the sfp cells, the abrB mutation could not restore the defect conferred by the spo0A mutation. These data suggest that the sfp locus, which is responsible for surfactin production, alters the transcriptional regulation of srf in JH642 cells.
Primary structure and mapping of the hupA gene of Salmonella typhimurium
Authors: Higgins N P , Hillyard D
Published: November 2016

In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. Comparison of hupA of E. coli and S. typhimurium revealed that the HU-2 proteins were identical and that there was high conservation of nucleotide sequences outside the coding frames of the genes. A 300-member genomic library of S. typhimurium was constructed by using random transposition of MudP, a specialized chimeric P22-Mu phage that packages chromosomal DNA unidirectionally from its insertion point. Oligonucleotide hybridization against the library identified one MudP insertion that lies within 28 kilobases of hupA; the MudP was 12% linked to purH at 90.5 min on the standard map. Plasmids expressing HU-2 had a surprising phenotype; they caused growth arrest when they were introduced into E. coli strains bearing a himA or hip mutation. These results suggest that IHF and HU have interactive roles in bacteria.
Analysis of the recE locus of Escherichia coli K-12 by use of polyclonal antibodies to exonuclease VIII
Authors: Luisi-DeLuca C , Clark A J , Kolodner R D
Published: November 2016

Exonuclease VIII (exoVIII) of Escherichia coli has been purified from a strain carrying a plasmid-encoded recE gene by using a new procedure. This procedure yielded 30 times more protein per gram of cells, and the protein had a twofold higher specific activity than the enzyme purified by the previously published procedure (J. W. Joseph and R. Kolodner, J. Biol. Chem. 258:10411-10417, 1983). The sequence of the 12 N-terminal amino acids was also obtained and found to correspond to one of the open reading frames predicted from the nucleic acid sequence of the recE region of Rac (C. Chu, A. Templin, and A. J. Clark, manuscript in preparation). Polyclonal antibodies directed against purified exoVIII were also prepared. Cell-free extracts prepared from strains containing a wide range of chromosomal- or plasmid-encoded point, insertion, and deletion mutations which result in expression of exoVIII were examined by Western blot (immunoblot) analysis. This analysis showed that two point sbcA mutations (sbcA5 and sbcA23) and the sbc insertion mutations led to the synthesis of the 140-kilodalton (kDa) polypeptide of wild-type exoVIII. Plasmid-encoded partial deletion mutations of recE reduced the size of the cross-reacting protein(s) in direct proportion to the size of the deletion, even though exonuclease activity was still present. The analysis suggests that 39 kDa of the 140-kDa exoVIII subunit is all that is essential for exonuclease activity. One of the truncated but functional exonucleases (the pRAC3 exonuclease) has been purified and confirmed to be a 41-kDa polypeptide. The first 18 amino acids from the N terminus of the 41-kDa pRAC3 exonuclease were sequenced and fond to correspond to one of the translational start signals predicted from the nucleotide sequence of radC (Chu et al., in preparation).
Cysteine biosynthesis in Saccharomyces cerevisiae: mutation that confers cystathionine beta-synthase deficiency
Authors: Ono B , Shirahige Y , Nanjoh A , Andou N , Ohue H , Ishino-Arao Y
Published: November 2016

The cys2-1 mutation of Saccharomyces cerevisiae was originally thought to confer cysteine dependence through a serine O-acetyltransferase deficiency. In this study, we show that cys2-1 strains lack not only serine O-acetyltransferase but also cystathionine beta-synthase. However, a prototrophic strain was found to be serine O-acetyltransferase deficient because of a mutation allelic to cys2-1. Moreover, revertants obtained from cys2-1 strains had serine O-acetyltransferase but not cystathionine beta-synthase, whereas transformants obtained by treating a cys2-1 strain with an S. cerevisiae genomic library had cystathionine beta-synthase but not serine O-acetyltransferase. From these observations, we conclude that cys2-1 (serine O-acetyltransferase deficiency) accompanies a very closely linked mutation that causes cystathionine beta-synthase deficiency and that these mutations together confer cysteine dependence. This newly identified mutation is named cys4-1. These results not only support our previous hypothesis that S. cerevisiae has two functional cysteine biosynthetic pathways but also reveal an interesting gene arrangement of the cysteine biosynthetic system.
Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide
Authors: Gofshtein-Gandman L V , Keynan A , Milner Y
Published: November 2016

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. cereus T was inhibited by p-chloromercuribenzene sulfonic acid. The D-BAPAases of B. subtilis and B. cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively. D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm). The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.
Two cellular components, PrlA and SecB, that recognize different sequence determinants are required for efficient protein export
Authors: Trun N J , Stader J , Lupas A , Kumamoto C , Silhavy T J
Published: November 2016

We exploited the conditional-lethal phenotype of secB null mutations to demonstrate that SecB function was required for PrlA-mediated suppression of signal sequence mutations. The results of these experiments provide information about the functions performed and the sequence determinants recognized by each of these components of the protein export machinery of Escherichia coli.
Factors affecting transfer frequency of pAM beta 1 from Streptococcus faecalis to Lactobacillus plantarum
Authors: Sasaki Y , Taketomo N , Sasaki T
Published: November 2016

Conjugal transfer of streptococcal plasmid pAM beta 1 to Lactobacillus plantarum by a filter mating method was examined under various conditions. The transfer frequency depended greatly on the type, pore size, and side (front or back) of the membrane filter. The passage of sterilized water through the membrane under reduced pressure after donor and recipient cells were trapped on it increased the transfer frequency about 10-fold.
EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphorylated in vitro
Authors: Igo M M , Silhavy T J
Published: November 2016

By fusing the transcriptional and translational start signals of lacZ to envZ, we have obtained high-level synthesis of a truncated EnvZ protein (EnvZ115) in which the first 38 amino acids of EnvZ are replaced with the first 8 amino acids of LacZ. Using this construct, we have partially purified the EnvZ115 protein and demonstrated that this protein can be phosphorylated in vitro. We suggest that phosphorylation may be an important feature of EnvZ function.
Evidence for movement of the alpha-amylase gene into two phylogenetically distant Bacillus stearothermophilus strains
Authors: Satoh H , Nishida H , Isono K
Published: November 2016

The gene for an alpha-amylase cloned from strain DY-5 of Bacillus stearothermophilus was used to examine to what extent the corresponding genes are structurally similar in other B. stearothermophilus strains. The structure of the gene itself was almost identical in DY-5 and a group of strains represented by strain 799. The gene was not detected at all in strain DSM2334, which was phenotypically amylase deficient. Comparison of the structure of 5S rRNA and electrophoretic pattern of the ribosomal proteins indicates that strains DY-5 and DSM2334 are closely related to each other, whereas strain 799 is phylogenetically very distant from the two. We estimate that strain 799 separated from DY-5 and DSM2334 some 420 million years ago. Nucleotide sequencing of the region containing the amylase gene from strains DY-5 and 799 revealed the presence of a 3.4-kilobase stretch that was highly similar in the two strains. Furthermore, comparison of the restriction map surrounding the amylase gene of DY-5 with that of a corresponding region in DSM2334 indicated that the former strain contained an extra segment 5.5 kilobases in length, which included the 3.4-kilobase stretch mentioned above. This segment was missing in DSM2334. It thus appears that the alpha-amylase gene was brought into strains DY-5 and 799 from outside despite a large phylogenetic distance.
Structure and expression of the Bacillus subtilis sin operon
Authors: Gaur N K , Cabane K , Smith I
Published: November 2016

The newly identified sin gene affects late growth processes in Bacillus subtilis when it is overexpressed or inactivated in the chromosome. S1 nuclease mapping of the sin gene transcripts in vivo reveals the existence of three transcripts (RNAI, RNAII, and RNAIII). By correlating 5' ends of sin gene transcripts with DNA sequence, we have identified three different promoterlike sequences (P1, P2, and P3) for these transcripts. 3'-End mapping of these transcripts identified three prominent termination sites at the end of the sin gene. These termination sites are localized on two hairpin structures previously identified from the DNA sequence. The most abundant transcript, RNAIII, coded only for the sin gene, while the polycistronic transcripts RNAII and RNAI coded for the sin gene and ORF1 that precedes the sin gene. S1 mapping and translational lacZ fusion studies indicated that ORF1 and the sin gene are regulated differently. ORF1 expression is under developmental control, increasing at the end of vegetative growth, and requires functional spo0A and spo0H gene products. The sin gene is expressed at an almost constant and relatively low level throughout growth and remains largely unaffected by spo0A and spo0H mutations.
Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis
Authors: Thiel T
Published: November 2016

Cells of the cyanobacterium Anabaena variabilis starved for phosphate for 3 days took up phosphate at about 100 times the rate of unstarved cells. Kinetic data suggested that a new transport system had been induced by starvation for phosphate. The inducible phosphate transport system was quickly repressed by addition of Pi. Phosphate-starved cells were more sensitive to the toxic effects of arsenate than were unstarved cells, but phosphate could alleviate some of the toxicity. Arsenate was a noncompetitive inhibitor of phosphate transport; however, the apparent Ki values were high, particularly for phosphate-replete cells. Preincubation of phosphate-starved cells with arsenate caused subsequent inhibition of phosphate transport, suggesting that intracellular arsenate inhibited phosphate transport. This effect was not seen in phosphate-replete cells.
A plasmid of Rhizobium meliloti 41 encodes catabolism of two compounds from root exudate of Calystegium sepium
Authors: Tepfer D , Goldmann A , Pamboukdjian N , Maille M , Lepingle A , Chevalier D , Dénarié J , Rosenberg C
Published: November 2016

Our objectives were to identify substances produced by plant roots that might act as nutritional mediators of specific plant-bacterium relationships and to delineate the bacterial genes responsible for catabolizing these substances. We discovered new compounds, which we call calystegins, that have the characteristics of nutritional mediators. They were detected in only 3 of 105 species of higher plants examined: Calystegia sepium, Convolvulus arvensis (both of the Convolvulaceae family), and Atropa belladonna. Calystegins are abundant in organs in contact with the rhizosphere and are not found, or are observed only in small quantities, in aerial plant parts. Just as the synthesis of calystegins is infrequent in the plant kingdom, their catabolism is rare among rhizosphere bacteria that associate with plants and influence their growth. Of 42 such bacteria tested, only one (Rhizobium meliloti 41) was able to catabolize calystegins and use them as a sole source of carbon and nitrogen. The calystegin catabolism gene(s) (cac) in this strain is located on a self-transmissible plasmid (pRme41a), which is not essential to nitrogen-fixing symbiosis with legumes. We suggest that under natural conditions calystegins provide an exclusive carbon and nitrogen source to rhizosphere bacteria which are able to catabolize these compounds. Calystegins (and the corresponding microbial catabolic genes) might be used to analyze and possibly modify rhizosphere ecology.
Partial purification and characterization of an inactive precursor of Pseudomonas aeruginosa elastase
Authors: Kessler E , Safrin M
Published: November 2016

An inactive precursor of the extracellular elastase of Pseudomonas aeruginosa was extensively purified by immunoadsorption chromatography of the soluble bacterial cell fraction on a column of Sepharose coupled to antielastase antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified precursor fraction revealed two major protein bands with molecular weights of about 36,000 (P36) and 20,000 (P20) that in the absence of sodium dodecyl sulfate were associated with each other. The following findings identify P36 as the elastase precursor and indicate that proteolytic processing of this molecule is required for activation: (i) P36 is larger than the elastase, and it binds antielastase antibodies; (ii) trypsin activation is associated with the disappearance of P36 and the appearance of a new protein band migrating identically with the elastase and reacting with antibodies against the elastase; (iii) peptide maps generated from P36 and the elastase are similar although not identical. P20 by itself was not recognized by antielastase antibodies. Its association with P36 accounts for its adsorption to the immunoaffinity column and suggests that it may serve in elastase secretion.
Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12
Authors: Stoker K , Oltmann L F , Stouthamer A H
Published: November 2016

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.
ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification
Authors: Grodberg J , Dunn J J
Published: November 2016

Bacteriophage T7 RNA polymerase is stable in Escherichia coli but very susceptible to cleavage by at least one endoprotease after cell lysis. The major source of this endoprotease activity was found to be localized to the outer membrane of the cell. A rapid whole-cell assay was developed to screen different strains for the presence of this proteolytic activity. Using this assay, we identified some common laboratory strains that totally lack the protease. Genetic and Southern analyses of these null strains allowed us to conclude that the protease that cleaves T7 RNA polymerase is OmpT (formerly termed protein a), a known outer membrane endoprotease, and that the null phenotype results from deletion of the OmpT structural gene. A recombinant plasmid carrying the ompT gene enables these deletion strains to synthesize OmpT and converts them to a protease-positive phenotype. The plasmid led to overproduction of OmpT protein and protease activity in the E. coli K-12 and B strains we used, but only weak expression in the E. coli C strain, C1757. This strain-dependent difference in ompT expression was investigated with respect to the known influence of envZ on OmpT synthesis. A small deletion in the ompT region of the plasmid greatly diminishes the amount of OmpT protein and plasmid-encoded protease present in outer membranes. Use of ompT deletion strains for production of T7 RNA polymerase from the cloned gene has made purification of intact T7 RNA polymerase routine. Such strains may be useful for purification of other proteins expressed in E. coli.
Genetic analyses of the mannitol permease of Escherichia coli: isolation and characterization of a transport-deficient mutant which retains phosphorylation activity
Authors: Manayan R , Tenn G , Yee H B , Desai J D , Yamada M , Saier M H
Published: November 2016

Three positive selection procedures were developed for the isolation of plasmid-encoded mutants which were defective in the mannitol enzyme II (IIMtl) of the phosphotransferase system (mtlA mutants). The mutants were characterized with respect to the following properties: (i) fermentation, (ii) transport, (iii) phosphoenolpyruvate(PEP)-dependent phosphorylation, and (iv) mannitol-1-phosphate-dependent transphosphorylation of mannitol. Cell lysis in response to indole acrylic acid, which causes the lethal overexpression of the plasmid-encoded mtlA gene, was also scored. No correlation was noted between residual IIMtl activity in the mutants and sensitivity to the toxic effect of indole acrylic acid. Plasmid-encoded mutants were isolated with (i) total or partial loss of all activities assayed, (ii) nearly normal rates of transphosphorylation but reduced rates of PEP-dependent phosphorylation, (iii) nearly normal rates of PEP-dependent phosphorylation but reduced rates of transphosphorylation, and (iv) total loss of transport activity but substantial retention of both phosphorylation activities in vitro. A mutant of this fourth class was extensively characterized. The mutant IIMtl was shown to be more thermolabile than the wild-type enzyme, it exhibited altered kinetic behavior, and it was shown to arise by a single nucleotide substitution (G-895----A) in the mtlA gene, causing a single amino acyl substitution (Gly-253----Glu) in the permease. The results show that a single amino acyl substitution can abolish transport function without abolishing phosphorylation activity. This work serves to identify a site which is crucial to the transport function of the enzyme.
Mapping of Agrobacterium tumefaciens chromosomal genes affecting cellulose synthesis and bacterial attachment to host cells
Authors: Robertson J L , Holliday T , Matthysse A G
Published: November 2016

Six chromosomal transposon mutations in Agrobacterium tumefaciens which result in an inability to synthesize cellulose fibrils were mapped to the vicinity of trp-2 and met-6. Mutations which result in an inability to attach to plant cells, attC43 and attC69, also mapped in this region, as did one Tn5 mutation which caused overproduction of cellulose. Another cellulose overproduction mutation mapped at a distance and was closely linked to ilv-13. The results suggest that there is a region of the A. tumefaciens chromosome located near met-6 which is concerned with the interaction of the bacterium with the host cell surface.
Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype
Authors: Flynn J L , Ohman D E
Published: November 2016

Strains of Pseudomonas aeruginosa causing chronic pulmonary infections in patients with cystic fibrosis are known to convert to a mucoid form in vivo characterized by the production of the exopolysaccharide alginate. The alginate production trait is not stable, and mucoid strains frequently convert back to the nonmucoid form in vitro. The DNA involved in these spontaneous alginate conversions, referred to as algS, was shown here to map near hisI and pru markers on the chromosome of strain FRD, an isolate from a cystic fibrosis patient. Although cloning algS+ by trans-complementation was not possible, a clone (pJF5) was isolated that caused algS mutants to convert to the Alg+ phenotype at detectable frequencies (approximately 0.1%) in vitro. Gene replacement with transposon-marked pJF5 followed by mapping studies showed that pJF5 contained DNA transducibly close to algS in the chromosome. Another clone was identified called pJF15 which did contain algS+ from mucoid P. aeruginosa. The plasmid-borne algS+ locus could not complement spontaneous algS mutations in trans, but its cis-acting activity was readily observed after gene replacement with the algS mutant chromosome by using an adjacent transposon as the selectable marker. pJF15 also contained a trans-active gene called algT+ in addition to the cis-active gene algS+. The algT gene was localized on pJF15 by using deletion mapping and transposon mutagenesis. By using gene replacement, algT::Tn501 mutants of P. aeruginosa were constructed which were shown to be complemented in trans by pJF15. Both algS and algT were located on a DNA fragment approximately 3 kilobases in size. The algS gene may be a genetic switch which regulates the process of alginate conversion.
Molecular cloning and expression of the Escherichia coli dimethyl sulfoxide reductase operon
Authors: Bilous P T , Weiner J H
Published: November 2016

The dimethyl sulfoxide (DMSO) reductase operon coding for a membrane-bound iron-sulfur, molybdoenzyme, which functions as a terminal reductase in Escherichia coli, has been isolated and cloned from an E. coli gene bank. Two clones, MV12(pLC19-36) and MV12(pLC43-43), overexpressed both DMSO and trimethylamine N-oxide (TMAO) reductase activities 13- to 15-fold compared with wild-type cells. Amplification was highest in cells grown anaerobically on fumarate, while cells grown on DMSO or TMAO displayed reduced levels of enzyme amplification. Growth on nitrate or aerobic growth repressed expression of the enzyme. A 6.5-kilobase-pair DNA restriction endonuclease fragment was subcloned from pLC19-36 into the vector pBR322, yielding a recombinant DMSO reductase plasmid, pDMS159. Two polypeptides were amplified and identified on sodium dodecyl sulfate-polyacrylamide gels of proteins from E. coli HB101 harboring pDMS159: a membrane-bound protein with molecular weight 82,600 and a soluble polypeptide with molecular weight 23,600. Three plasmid-encoded polypeptides with molecular weights of 87,500, 23,300, and 22,600 were detected by in vivo transcription/translation studies. The smallest subunit was poorly defined and not detectable by Coomassie blue staining. The DMSO reductase operon was localized to the 20.0-min position on the E. coli linkage map.
New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis
Authors: Kuriki T , Okada S , Imanaka T
Published: November 2016

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.
Evidence for natural gene transfer from gram-positive cocci to Escherichia coli
Authors: Brisson-Noël A , Arthur M , Courvalin P
Published: November 2016

High-level resistance to macrolide-lincosamide-streptogramin type B (MLS) antibiotics in Escherichia coli BM2570 is due to the presence on the conjugative plasmid pIP1527 of the MLS resistance determinant ermBC, which is almost identical to the erm genes previously described in plasmid pAM77 from Streptococcus sanguis (ermAM) and in transposon Tn917 from Enterococcus faecalis (ermB). This gene and its regulatory region are located downstream from the insertion sequence IS1. The 23S rRNA methylase encoded by pIP1527 differs by three and six amino acids from those encoded by Tn917 and pAM77, respectively. Unlike the streptococcal elements which confer the inducible MLS phenotype, the ermBC gene is expressed constitutively in E. coli and Bacillus subtilis, due to several mutations in the regulatory region. Transcription of the ermBC gene starts from three different sites following three overlapping promoters which function in both E. coli and B. subtilis. Promoters P2 and P3 are located within the region homologous to pAM77 and Tn917, and P1 is a hybrid promoter constituted by -35 and -10 sequences located at the end of IS15 and in the streptococcal region, respectively. These results constitute evidence for the recent in vivo transfer from Streptococcus spp. to E. coli. This transfer could have been mediated by transposons such as Tn917 or Tn1545 from Streptococcus pneumoniae, which also bears an MLS determinant that is homologous to ermB. We speculate that the insertion sequences IS15 and IS1 could have played a role in the expression and dissemination of ermBC, which has been found in numerous strains of enterobacteria.
Characterization and mapping of the agrocinopine-agrocin 84 locus on the nopaline Ti plasmid pTiC58
Authors: Hayman G T , Farrand S K
Published: November 2016

Overlapping segments of pTiC58 inserted into cosmid vectors were used to characterize the agrocinopine-agrocin 84 locus from the nopaline/agrocinopine A and B Agrobacterium tumefaciens strain C58. All of the clones conferring agrocin 84 sensitivity on agrobacteria also conferred uptake of agrocin 84 and agrocinopines A and B. Transposon Tn3-HoHo1 insertion mutations of one such clone were generated that simultaneously abolished agrocin 84 sensitivity and transport of agrocinopines A and B and agrocin 84. Such insertions were found to cluster within a 4.4-kilobase region. Analysis of beta-galactosidase activity in these insertion mutants suggested a single transcriptional unit regulated at the transcriptional level by agrocinopines A and B. The smallest DNA fragment subcloned from the region to confer all three activities was 8.5 kilobases long. This subclone was still properly regulated, indicating that the regulatory gene is closely linked to the locus. The data are consistent with a single operon encoding catabolism of agrocinopines A and B and conferring sensitivity to agrocin 84. Based on these results, we support the locus name acc, for agrocinopine catabolism.
A progenitor of the outer membrane LamB trimer
Authors: Stader J , Silhavy T J
Published: November 2016

During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane. Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB).
Plasmids of Azotobacter vinelandii
Authors: Maia M , Sanchez J M , Vela G R
Published: November 2016

Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, and each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures.
Conditional impairment of cell division and altered lethality in hipA mutants of Escherichia coli K-12
Authors: Scherrer R , Moyed H S
Published: November 2016

Mutations in hipA, a gene of Escherichia coli K-12, greatly reduce the lethality of selective inhibition of peptidoglycan synthesis. These mutations have also been found to reduce the lethality that accompanies either selective inhibition of DNA synthesis or heat shock of strains defective in htpR. In addition, the mutant alleles of hipA are responsible for a reversible cold-sensitive block in cell division and synthesis of macromolecules, particularly peptidoglycan. Recombination between the chromosome of hipA mutants and plasmids containing noncomplementing fragments of hipA+ revealed that the mutations responsible for both cold sensitivity and reduced lethality were probably identical and, in any case, lay within the first 360 base pairs of the coding region of hipA, probably within the first 50 base pairs. We suggest that the pleiotropic effects of mutations in hipA reflect the involvement of this gene in cell division.
Induction of base substitution mutations by aflatoxin B1 is mucAB dependent in Escherichia coli
Authors: Foster P L , Groopman J D , Eisenstadt E
Published: November 2016

Recovery of aflatoxin B1-induced base substitution mutations in Escherichia coli was almost completely dependent on the presence of the SOS-mutagenesis-enhancing operon mucAB+; the normal E. coli analog, umuDC+, was not sufficient. Yet aflatoxin B1 induced the SOS response, including the umuDC operon, as well as did UV light. Neither preinduction of the SOS response nor the presence of additional copies of umuDC+ allowed the recovery of aflatoxin B1-induced base substitutions. Thus, the premutagenic DNA lesions induced by aflatoxin B1 reveal a functional difference between UmuDC and MucAB. We estimate that in the presence of MucAB the probability that aflatoxin B1-induced DNA lesions will be converted into mutations is increased at least 10-fold.
Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici
Authors: Thomashow L S , Weller D M
Published: November 2016

Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G. graminis var. tritici and other fungal root pathogens. Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots. Six independent, prototrophic Phz- mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings. Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library. These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear. These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.
Factors influencing cell shape in the mutans group of streptococci
Authors: Tao L , MacAlister T J , Tanzer J M
Published: November 2016

Electron and light microscopic and growth studies of representatives of the diverse species of mutans streptococci revealed the cells to be either bacillary or coccoid in shape. Some strains changed from bacillary to coccoid if the HCO3-/K+ ratio of the media was increased and from coccoid to bacillary if the ratio was decreased. Doubling times of rods and cocci were the same despite an HCO3-/K+ ratio change between 0.008 and 2.84. For strain 10449S, no tested anions or cations substituted for HCO3- or K+ to produce this effect, except for B4O7(2-). Strain 10449S grown at a high B4O7(2-)/K+ ratio became ellipsoid, and this phenomenon was associated with slower doubling times. Up to three incomplete septa could be observed in one rod, but no more than one incomplete septum could be observed in either ellipsoid or spherical cells. Interseptal distances were greatest in rods, shorter in spheres, and shortest in ellipses. All of the above differences were statistically significant (P less than 0.001).
Transduction in Bacillus stearothermophilus
Authors: Welker N E
Published: November 2016

Temperate and virulent bacteriophages isolated from soil were shown to carry out generalized transduction of Bacillus stearothermophilus NUB36. A transducing frequency of 1 X 10(-5) to 7 X 10(-4) was obtained for temperate phages TP-42 and TP-56. The transducing frequency for virulent phage TP-68 was two to three orders of magnitude lower. Cotransfer analysis with the three phages showed that hom-1 is linked to thr-1 and that gly-1 is linked to his-1.
Recognition of individual strains of fast-growing rhizobia by using profiles of membrane proteins and lipopolysaccharides
Authors: de Maagd R , van Rossum C , Lugtenberg B J
Published: November 2016

Membrane protein and lipopolysaccharide profiles of Rhizobium leguminosarum (biovars viciae, trifolii, and phaseoli), R. meliloti, and Agrobacterium tumefaciens strains were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Differences in one or both profiles allowed us to distinguish all 18 R. leguminosarum strains tested in this study from each other.
Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid
Authors: Innes R W , Hirose M A , Kuempel P L
Published: November 2016

Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods. Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R. trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover. One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability. This subclone included all known nodulation genes of R. trifolii ANU843 and the nitrogenase structural genes nifHDK. In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization. Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover. In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules. No homology to known nif and fix genes could be detected in this latter region.
Formation of the chlorophyll precursor delta-aminolevulinic acid in cyanobacteria requires aminoacylation of a tRNAGlu species
Authors: O'Neill G P , Peterson D M , Schön A , Chen M W , Söll D
Published: November 2016

In the chloroplasts of higher plants and algae, the biosynthesis of the chlorophyll precursor delta-aminolevulinic acid (ALA) involves at least three enzymes and a tRNA species. Here we demonstrate that in cell extracts of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 ALA was formed from glutamate in a series of reactions in which activation of glutamate by glutamyl-tRNAGlu formation was the first step. The activated glutamate was reduced by a dehydrogenase which displayed tRNA sequence specificity. Fractionation of strain 6803 tRNA by reverse-phase chromatography and polyacrylamide gel electrophoresis yielded two pure tRNAGlu species which stimulated ALA synthesis in vitro. These tRNAs had identical primary sequences but differed in the nucleotide modification of their anticodon. The 6803 tRNAGlu was similar to the sequences of tRNAGlu species or tRNAGlu genes from Escherichia coli and from chloroplasts of Euglena gracilis and higher plants. Southern blot analysis revealed at least two tRNAGlu gene copies in the 6803 chromosome. A glutamate-1-semialdehyde aminotransferase, the terminal enzyme in the conversion of glutamate to ALA in chloroplasts, was detected in 6803 cell extracts by the conversion of glutamate-1-semialdehyde to ALA and by the inhibition of this reaction by gabaculin.
Selection of in vivo deletions in Saccharomyces cerevisiae
Authors: Bitoun R , Zamir A
Published: November 2016

A general screening procedure has been devised for the selection of in vivo-generated deletions in haploid Saccharomyces cerevisiae cells. It is based on the introduction into a cyh2 host (resistant to the drug cycloheximide) of a tandemly duplicated CYH2 gene (a dominant allele, conferring sensitivity to cycloheximide), and subsequent selection for Cyhr derivatives. The duplicated CYH2 gene has been introduced on CEN ARS plasmids or integrated into chromosome II. A variable but significant proportion of the Cyhr derivatives of such transformants were deletion mutants in which both CYH2 copies had suffered deletions. Some of the deletions extended into sequences outside the tandemly duplicated CYH2 gene. A total of 61 independently selected deletions ranged in length from 3.1 to over 20 kilobases and had no obvious preferred endpoints. Restriction analysis showed that other frequently isolated Cyhr derivatives appeared to retain one of the two CYH2 copies. Such single-copy derivatives of CEN ARS plasmids did not contain a functional CYH2 gene. The frequency of true deletions in CEN ARS plasmids, of approximately 10(-7) per viable cell, was comparable in RAD52 and rad52 strains. Chromosomal deletions, which occurred at a frequency of approximately 10(-8) per viable cell, were observed only in rad52 hosts. Derivatives exhibiting an additional altered phenotype, such as the inactivation of a neighboring gene or, less frequently, the transcriptional activation of a previously silent gene, were isolated by screening deletion mutants. These results show that the method described can be used for in vivo deletion mapping or for the generation of gene fusions.
Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli
Authors: Schultz J E , Latter G I , Matin A
Published: November 2016

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).
Molecular analysis of a composite chromosomal conjugative element (Tn3701) of Streptococcus pyogenes
Authors: Le Bouguénec C , de Cespédès G , Horaud T
Published: November 2016

The plasmid-free Streptococcus pyogenes A454 contains a conjugative element, Tn3701, encoding resistance to erythromycin (Emr), tetracycline (Tcr), and minocycline (Mnr). We have mapped a 50-kilobase (kb) chromosomal region of A454 corresponding to the internal part of Tn3701. Tn3701 includes a 19.7-kb structure, designated Tn3703, on which the Emr Tcr Mnr determinants were localized. Tn3703 was very similar in structure to Tn916. Translocation of the Emr Tcr Mnr markers from A454 onto pIP964, an Enterococcus faecalis hemolysin plasmid, yielded different pIP964 derivatives. When the inserts of four of these derivatives were aligned with the 50-kb region of Tn3701, three of them were found to result from the transposition of Tn3703 and one resulted from the insertion of a 44.0-kb portion of Tn3701, including Tn3703. Tn3701 inserted, apparently without changing its structure, in the chromosomes of various streptococcal transconjugants, as well as in one of the 12 E. faecalis transconjugants studied. Tn3703 inserted at different chromosomal sites in four E. faecalis transconjugants, and one copy of Tn3701 plus an additional copy of Tn3703 were detected in the chromosomes of seven transconjugants.
Regulation of carbon and electron flow in Propionispira arboris: relationship of catabolic enzyme levels to carbon substrates fermented during propionate formation via the methylmalonyl coenzyme A pathway
Authors: Thompson T E , Zeikus J G
Published: November 2016

A detailed study of the glucose fermentation pathway and the modulation of catabolic oxidoreductase activities by energy sources (i.e., glucose versus lactate or fumarate) in Propionispira arboris was performed. 14C radiotracer data show the CO2 produced from pyruvate oxidation comes exclusively from the C-3 and C-4 positions of glucose. Significant specific activities of glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase were detected, which substantiates the utilization of the Embden-Meyerhoff-Parnas path for glucose metabolism. The methylmalonyl coenzyme A pathway for pyruvate reduction to propionate was established by detection of significant activities (greater than 16 nmol/min per mg of protein) of methylmalonyl coenzyme A transcarboxylase, malate dehydrogenase, and fumarate reductase in cell-free extracts and by 13C nuclear magnetic resonance spectroscopic demonstration of randomization of label from [2-13C]pyruvate into positions 2 and 3 of propionate. The specific activity of pyruvate-ferredoxin oxidoreductase, malate dehydrogenase, fumarate reductase, and transcarboxylase varied significantly in cells grown on different energy sources. D-Lactate dehydrogenase (non-NADH linked) was present in cells of P. arboris grown on lactate but not in cells grown on glucose or fumarate. These results indicate that growth substrates regulate synthesis of enzymes specific for the methylmalonyl coenzyme A path and initial substrate transformation.
Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens
Authors: Winans S C , Kerstetter R A , Nester E W
Published: November 2016

We have used transcriptional and translational fusions between various vir gene promoters and the lacZ gene to study the regulation of vir genes. Like other vir promoters, the virA promoter was induced by acetosyringone in a virA virG-dependent fashion. In addition to being induced by acetosyringone, the virG promoter was partially induced by acidic growth conditions and by starvation for inorganic phosphate. These two conditions appeared to act synergistically. The response to low pH and to phosphate starvation occurred in the absence of the Ti plasmid and must therefore have been mediated by chromosomal genes. Two transposon-generated mutations were obtained which attenuated induction by low pH. One of these transposons was cloned along with flanking DNA; the flanking DNA was sequenced (858 base pairs total), and the predicted amino acid sequence showed homology with a family of proteins including the Rhizobium leguminosarum nodI gene, many of whose members bind ATP and have been implicated in active transport systems. These results are discussed as possible explanations for previous observations that the induction of the octopine vir regulon (i) occurs only in acidic media and (ii) shows hyperbolic kinetics after a long lag phase.