bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and functional properties of the folate transport protein from a methotrexate-resistant subline of Lactobacillus casei
Authors: Ananthanarayanan M , Kojima J M , Henderson G B
Published: January 2020

A methotrexate-resistant subline of Lactobacillus casei has been isolated which transports folate at a reduced rate and contains a binding protein whose affinity for folate (Kd = 280 nM) is considerably lower than that of the corresponding protein of wild-type cells (Kd = 0.6 nM). After the addition of mercaptoethanol, however, this same protein exhibits a high affinity for folate (Kd = 1.2 nM) and transports the substrate at a normal rate. Subsequent removal of mercaptoethanol causes a rapid reversal of the activation process. Binding protein labeled covalently with carbodiimide-activated [3H]folate, solubilized with Triton X-100, and subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate had an apparent molecular weight which was approximately twofold higher than that of the corresponding protein of wild-type cells, but it could be reduced to the parental size (Mr = 20,000) by prior treatment with mercaptoethanol. Purified binding protein also exhibited a similarly elevated molecular weight, and its amino acid composition was indistinguishable from that of the wild-type counterpart, except for the presence of a single cysteine residue. These findings indicate that the mutant binding protein exists in a low-affinity form due to disulfide bridge formation between two homologous protein subunits and that cleavage of this bond by mercaptoethanol generates the high-affinity state. The rapid and specific interconversion of these binding forms suggests further that the high-affinity form of the binding protein also resides in the membrane as a dimer, held together by noncovalent interactions.
Intermediary carbon metabolism of Azospirillum brasilense
Authors: Loh W H , Randles C I , Sharp W R , Miller R H
Published: January 2020

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.
Direct evidence for specific binding of the replicative origin of the Escherichia coli chromosome to the membrane
Authors: Kusano T , Steinmetz D , Hendrickson W G , Murchie J , King M , Benson A , Schaechter M
Published: January 2020

The origin of replication of the Escherichia coli chromosomal DNA binds with high affinity to outer membrane preparations. This specific binding requires a 463-base-pair region of origin DNA between positions -45 and +417 of the oriC map. We show that binding does not require the presence of adjacent regions. From further analysis, we conclude that more than one binding site resides within the 325-base-pair fragment between positions +38 (BamHI) and +417 (XhoI). When this fragment is cut, two pieces bind with high affinity and one binds with lesser affinity. The binding ability of one of the high affinity sites is abolished by cutting it at position +92 with BamHI.
Activation of trehalase by membrane-depolarizing agents in yeast vegetative cells and ascospores
Authors: Thevelein J M
Published: January 2020

The membrane-depolarizing agents 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazone, and nystatin are known to cause a rapid increase in the cyclic AMP level in fungal cells. Addition of these proton ionophores to yeast stationary-phase cells or ascospores causes an immediate 10-fold increase in trehalase activity. This observation is in agreement with a role for cyclic AMP-induced phosphorylation in the activation process of trehalase. It also provides an explanation for previous results on the induction of trehalose breakdown by 2,4-dinitrophenol in resting yeast cells.
Conversion of D-mannitol to D-ribose: a newly discovered pathway in Escherichia coli
Authors: Rosenberg H , Hardy C M
Published: January 2020

A mutant (mtlD) strain of Escherichia coli unable to oxidize mannitol-1-phosphate to fructose-6-phosphate was used to study the fate of mannitol-1-phosphate. D-[1-14C]mannitol entered the cells via the phosphotransferase system and was phosphorylated equally at carbon 1 or 6. The label disappeared gradually from the mannitol-1-phosphate pool, and some 60% of the 14C was recovered in nucleic acids. Ribose was isolated from the purified RNA. The 14C label distribution in the isolated ribose precluded a simple hexose-to-pentose conversion by elimination of one terminal carbon from mannitol-1-phosphate. The 14C from mannitol-1-phosphate that did not enter macromolecules was found in CO2 and in some organic, non-phosphorylated compounds that were not identified. We suggest that the de novo synthesis of mannitol-1-phosphate in E. coli may be a reaction specifically dedicated to the biosynthesis of ribose.
Isolation and characterization of two distinct fractions from the inner membrane of dormant Bacillus megaterium spores
Authors: Swerdlow R D , Setlow P
Published: January 2020

Two distinct membrane bands were obtained after sucrose velocity gradient centrifugation of crude inner membranes from dormant Bacillus megaterium spores disrupted under conditions which minimized endogenous enzyme action. These two inner membrane fractions (termed LD and HD) contained similar amounts of total and individual phospholipid species. However, LD and HD differed significantly in phospholipid/protein ratios (4.3 and 0.47 mg/mg, respectively), equilibrium densities (1.12 and 1.18 g/cm3), NADH oxidase specific activity (less than 0.01 and 0.13 mumol/min X mg), and content of specific proteins. In contrast, crude membranes prepared in identical fashion from germinated spores gave only a single inner membrane band (termed G) on sucrose velocity gradients. G had a phospholipid/protein ratio of 0.98 mg/mg, an equilibrium density of 1.16 g/cm3, and an NADH oxidase specific activity of 2.1 mumol/min X mg. Essentially all of the proteins present in LD or HD or both were found in G, consistent with the latter membrane being derived from a mixture of LD and HD. No evidence was found suggesting that there is significant degradation of dormant spore inner membrane protein upon spore germination.
Identification of a glutaminyl-tRNA synthetase mutation Saccharomyces cerevisiae
Authors: Mitchell A P , Ludmerer S W
Published: January 2020

Saccharomyces cerevisiae glutaminyl-tRNA synthetase mutants were isolated through systematic screening of tight Gln- derivatives of a leaky glutamine auxotroph. These mutations define a single nuclear gene, GLN4. The gln4-1 mutation is specific for Gln-tRNA synthetase and shows a dosage effect in heterozygous diploids. The wild-type Gln-tRNA synthetase exhibits a Km for glutamine of 25 microM; the gln4-1 mutation increases this value 20-fold. These observations strongly suggest that GLN4 encodes the Gln-tRNA synthetase.
Post-transcriptional regulation of chloramphenicol acetyl transferase
Authors: Byeon W H , Weisblum B
Published: January 2020

The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) mRNA encoded by plasmid pC194 was determined experimentally by using [alpha-32P]ATP-labeled runoff transcripts partially digested with T1 RNase. By partial digestion of the in vitro transcripts with S1, T1, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions, which contain the complementary sequences CCUCC and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12-base-pair inverted complementary repeat sequence (-19.5 kcal [ca. -81.7 kJ] per mol). The CAT mRNA was stable to digestion by T1 RNase at the four guanosine residues in the Shine and Dalgarno sequence GGAGG , even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194 , induced with chloramphenicol, contain about the same concentration of pulse-labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the non- acetylatable chloramphenicol analog fluorothiamphenicol was tested by using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro with BAL 31 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by chloramphenicol required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role in regulating its own synthesis.
Positive control of the L-rhamnose genetic system in Salmonella typhimurium LT2
Authors: Al-Zarban S , Heffernan L , Nishitani J , Ransone L , Wilcox G
Published: January 2020

A total of 28 L-rhamnose-negative mutants in Salmonella typhimurium LT2 were all linked by P22 transduction and were classified into five groups on the basis of genetic and biochemical experiments. Deletion mapping showed that the gene order was rhaD rhaA rhaB rhaC rhaT . rhaA mutants lacked an inducible L-rhamnose isomerase, rhaB mutants lacked an inducible L- rhamnulokinase , and rhaD mutants were probably defective in L- rhamnulose -1-phosphate aldolase. Mutants that were unable to accumulate L-[14C]rhamnose but could grow on 1% L-rhamnose were designated rhaT to indicate a defect in L-rhamnose transport. Genetic evidence supports the hypothesis that the rhaC gene is a positive regulator of rha gene expression. (i) Pleiotropically negative mutants in the rhaC gene were isolated at a high frequency. (ii) Mutants containing an insertion or deletion within the rhaC gene had a pleiotropically negative phenotype. (iii) Complementation tests indicated that rhaC + was dominant to rhaC -. (iv) Rha+ revertants of deletion and Tn10 insertion mutations in the rhaC gene were isolated.
Oxygen-dependent proton efflux in cyanobacteria (blue-green algae)
Authors: Scherer S , Stürzl E , Böger P
Published: January 2020

The oxygen-dependent proton efflux (in the dark) of intact cells of Anabaena variabilis and four other cyanobacteria (blue-green algae) was investigated. In contrast to bacteria and isolated mitochondria, an H+/e ratio (= protons translocated per electron transported) of only 0.23 to 0.35 and a P/e ratio of 0.8 to 1.5 were observed, indicative of respiratory electron transport being localized essentially on the thylakoids, not on the cytoplasmic membrane. Oxygen-induced acidification of the medium was sensitive to cyanide and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Inhibitors such as 2,6-dinitrophenol and vanadate exhibited a significant decrease in the H+/e ratio. After the oxygen pulse, electron transport started immediately, but proton efflux lagged 40 to 60 s behind, a period also needed before maximum ATP pool levels were attained. We suggest that proton efflux in A. variabilis is due to a proton-translocating ATP hydrolase (ATP-consuming ATPase) rather than to respiratory electron transport located on the cytoplasmic membrane.
Mapping of the lipoprotein signal peptidase gene (lsp)
Authors: Regue M , Remenick J , Tokunaga M , Mackie G A , Wu H C
Published: January 2020

A pBR322 plasmid which contains a fragment of Escherichia coli DNA encoding the lipoprotein signal peptidase gene was used to transform Hfr polA1 strains. Ampr transformants were used as donors in conjugation experiments, and the location of the plasmid amp gene adjacent to the chromosomal lsp gene was determined to be near the thr ara loci of the E. coli chromosome. P1 transduction experiments established that the location of the lsp gene is closely linked to that of dapB , at 0.5 to 0.6 min on the E. coli genetic map. The position of the lsp gene was further determined to be between ileS and dapB by complementation analysis of an E. coli mutant showing temperature-sensitive prolipoprotein signal peptidase activity.
Mannityl opine analogs allow isolation of catabolic pathway regulatory mutants
Authors: Chilton W S , Chilton M D
Published: January 2020

Five virulent Agrobacterium spp. strains that can catabolize the mannityl opines mannopine (MOP), mannopinic acid ( MOA ), and agropinic acid (AGA) were tested for their ability to grow on analogs of these compounds. Analogs containing alternative amino acids replacing glutamic acid or glutamine were generally refused by these bacteria, but mutants were obtained that catabolized the entire family of analogs. In the case of strain C58C1 (pRi 8196), we demonstrated that typical mutants were constitutive for MOP uptake, whereas the wild-type parent was inducible by MOP. Analogs of MOA prepared from a variety of sugars instead of mannose were generally refused, except for a strain carrying pTi B6-806, which grew well on all such analogs. The analogs allowed selection of mutants of all strains. Although most wild-type strains were inducible for AGA uptake, typical mutants selected from strain C58C1 (pRi 8196) were found to be constitutive for uptake of AGA, as was the wild-type strain carrying pTi B6-806. Such constitutive mutants grew on all sugar analogs of MOP, MOA , and AGA tested. The pTi B6-806-containing strain was tested for growth on a more extended series of analogs, including tetrose , triose, diose , and disaccharide analogs, all of which were accepted. Only ketose analogs were refused. Selection of promiscuous regulatory mutants by the two types of opine analogs suggests that the repressor proteins of MOP and AGA permease/ catabolase systems are chiefly responsible for the specificity of the pathways.
Cloning genes for proline biosynthesis from Neisseria gonorrhoeae: identification by interspecific complementation of Escherichia coli mutants
Authors: Stein D C , Silver L E , Clark V L , Young F E
Published: January 2020

DNA from Neisseria gonorrhoeae KH45 was partially digested with Sau3A and inserted into the BamHI site of the cloning vector pLES2 . After introduction into Escherichia coli JM83 by transformation, two different size classes of plasmids were isolated that could complement the proAB deletion of JM83 . These plasmids ( pLES4 and pLES7 ) were characterized by restriction endonuclease digestion. Southern hybridization demonstrated that the inserts had sequence homology. Various deletions of these plasmids were constructed that had lost the ability to complement the proA lesion of chi 463, the proB lesion of chi 340, or both (plasmids pLES9 , pLES8 , and pLES10 , respectively). These deleted plasmids were introduced into a proline-requiring strain of N. gonorrhoeae, F62, with plasmids pLES4 , pLES7 , and pLES8 possessing the ability to correct the proline requirement of F62. Further analysis indicated that the hybrid plasmids were stably maintained as plasmids in N. gonorrhoeae.
Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis
Authors: Dauenhauer S A , Hull R A , Williams R P
Published: January 2020

Prodigiosin, the bright red pigment produced by many strains of Serratia marcescens, is synthesized by a bifurcated pathway that terminates in the enzymatic condensation of the two final products, a monopyrrole and a bipyrrole . Sau3A fragments of S. marcescens ( Nima ) DNA were introduced into a strain of Escherichia coli K-12 by use of the cosmid vector pHC79 , and transformed clones were selected based on resistance to ampicillin. Among 879 transformants screened, 2 could be induced to synthesize prodigiosin when supplied with either one or both terminal products of the bifurcated pathway. Data are presented to support the idea that production of prodigiosin is not usually mediated by a plasmid.
Immunological analysis of the heme proteins present in aerobically grown Escherichia coli
Authors: Kranz R G , Barassi C A , Gennis R B
Published: January 2020

Immunological methods were used to obtain information about Escherichia coli heme proteins. There is a membrane-bound catalase which consists of a single subunit (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis) which is also present in the soluble fraction. Antibodies raised against purified, soluble cytochrome b562 showed that this cytochrome is not related to any of the membrane-bound cytochromes, including the b562 component of the cytochrome o complex. Cytochrome b556 is immunologically unrelated to the cytochrome b556 NR associated with the nitrate reductase system. Cytochrome b556 and cytochrome o are not present in a constant ratio in the membrane.
Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins
Authors: Wretlind B , Pavlovskis O R
Published: January 2020

We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme.
Transposon mutagenesis of marine Vibrio spp
Authors: Belas R , Mileham A , Simon M , Silverman M
Published: January 2020

Coliphage P1 was used to transduce derivatives of transposons Tn5 and mini-Mu into marine Vibrio spp. Transposon Tn5 encoding tetracycline resistance (Tn5-132) was used to isolate mutants of Vibrio harveyi defective in genes for bioluminescence (lux). Insertion of transposon Tn5-132 into the lux gene region was demonstrated by intraspecific transduction with phage hv-1 and by Southern blot hybridization. Transposon mini-Mu, modified to specify tetracycline resistance, was employed to mutagenize genes for lateral flagella synthesis of Vibrio parahaemolyticus. Mini-Mu contains the lacZ structural gene, and transposition results in transcriptional fusion of Vibrio genes with the transposon lacZ gene. Thus, in these fusions, lacZ expression was proportional to the level of transcription of the target gene. Regulation of lateral flagella gene expression was studied in vivo by measuring beta-galactosidase activity, and conditions which activate transcription of these genes were identified. A method for gene cloning with transposon-induced mutations is discussed.
Physical mapping and complementation analysis of transposon Tn5 mutations in Caulobacter crescentus: organization of transcriptional units in the hook gene cluster
Authors: Ohta N , Swanson E , Ely B , Newton A
Published: January 2020

Using the cloned DNA from the hook protein gene region of Caulobacter crescentus ( Ohta et al., Proc. Natl. Acad. Sci. U.S.A. 79:4863-4867, 1982), we have identified and physically mapped 19 Tn5-induced and 2 spontaneous insertion mutations to this region of the chromosome. These nonmotile mutants define a major cluster of fla genes that covers approximately 17 kilobases on the chromosome (hook gene cluster). Complementation analysis of the mutants using DNA fragments from the region subcloned in the broad host range plasmid pRK290 has shown that these fla genes are organized into at least five transcriptional units (I to V). Transcriptional unit II contains at least one gene in addition to the hook protein gene, which makes this the first operon described in C. crescentus. Expression of the hook protein gene and the genetically unlinked flagellin A and B genes by this set of mutants also furnishes additional insights into the hierarchial regulation of flagellar genes. We have found that the spontaneous insertion mutant ( SC511 ) of the hook protein gene ( flaK ) makes no flagellin A or B and that genes downstream from the hook protein gene are required in trans for expression of the hook protein operon and the flagellin A and B genes. Recombination and complementation results thus place flaK , flaJ , flaN , and flaO (R. C. Johnson and B. Ely , J. Bacteriol . 137:627-634, 1979) in the hook gene cluster, identify at least three new genes ( flbD , flbG , and flbF ), and suggest that this cluster may contain several additional, as yet unidentified, fla genes.
Cloning and characterization of the Salmonella typhimurium metE gene
Authors: Schulte L L , Stauffer L T , Stauffer G V
Published: January 2020

The metE gene of Salmonella typhimurium was cloned into plasmid pACYC184 by using a lambda gt7 -metE transducing phage as a source of metE DNA. The recombinant plasmid, designated pGS41 , carries a 12.8-kilobase-pair EcoRI insert fragment. The metE gene was subcloned from pGS41 into plasmid pBR322 on a 4.2-kilobase-pair EcoRI-HindIII fragment (plasmid pGS47 ) and a 4.5-kilobase-pair PstI fragment (plasmid pGS69 ). The location of metE in these plasmids was determined by transposon Tn5 insertional inactivation experiments. A metE-encoded polypeptide of 92,500 Mr was detected in a minicell system by using metE+ plasmids as templates. Truncated polypeptides replaced the 92,500-Mr polypeptide when plasmid derivatives containing Tn5 insertions that inactivate metE were used in the system. A comparison of the site of each Tn5 insertion and the size of the polypeptide made in the minicell system allowed us to determine the direction of transcription and translation. The location of the metE promoter was determined by using an in vitro transcription system and by RNA polymerase protection of restriction endonuclease sites.
Characterization of transposon Tn5-facilitated donor strains and development of a chromosomal linkage map for Agrobacterium tumefaciens
Authors: Pischl D L , Farrand S K
Published: January 2020

We have further characterized the transposon Tn5-facilitated chromosomal gene transfer system developed for Agrobacterium tumefaciens 15955. Using a strain whose chromosome contained Tn5, we compared the chromosome-mobilizing ability of plasmid pDP37 (containing Tn5) with that of its parent plasmid R68.45. For R68.45, we observed nonpolar transmission from multiple origins. For pDP37 we found polarized transmission from a single origin near ilv. When we examined the transmission gradients of a number of pDP37-containing donor strains each differing at the site of the chromosomal insertion we found just two classes. One set of donors transmitted markers with a gradient of Ilv+ greater than Rifr greater than His+ greater than Met+, whereas the second set transferred His+ greater than Rifr greater than Ilv+ greater than Met+. Using representatives from each transmission class of donor strains, we conducted matings to measure the degree of linkage between pairs of adjacent donor markers. From this information we developed a map of the A. tumefaciens 15955 chromosome. Attempts to isolate R-prime plasmids or Hfr-like donors were unsuccessful.
General transduction in Rhizobium meliloti
Authors: Finan T M , Hartweig E , LeMieux K , Bergman K , Walker G C , Signer E R
Published: January 2020

General transduction by phage phi M12 in Rhizobium meliloti SU47 and its derivatives is described. Cotransduction and selection for Tn5 insertions which are closely linked to specific loci were demonstrated. A derivative of SU47 carrying the recA::Tn5 allele of R. meliloti 102F34 could be transduced for plasmid R68.45 but not for chromosomally located alleles. Phage phi M12 is morphologically similar to Escherichia coli phage T4, and restriction endonuclease analysis indicated that the phage DNA was ca. 160 kilobases in size.
Characterization of the cell wall and outer membrane of Rhodopseudomonas capsulata
Authors: Flammann H T , Weckesser J
Published: January 2020

Sucrose density gradient centrifugation of cell envelopes of chemotrophically grown cells of Rhodopseudomonas capsulata St. Louis (= ATCC 23782) resulted in the separation of a cytoplasmic membrane from a cell wall fraction (buoyant densities, 1.139 and 1.215 g/cm3, respectively). The cell wall fractions (untreated or Triton extracted) contained peptidoglycan- and lipopolysaccharide-specific components. Their neutral sugar content, mainly rhamnose and galactose, was high (250 and 100 micrograms/mg [dry weight] of material) due to a non-lipopolysaccharide polymer. The fatty acid content was low (less than or equal to 60 micrograms/mg [dry weight] of material), and half of it was contributed by lipopolysaccharide (3-OH-C10:0, C12:1, and 3-oxo-C14:0). The predominant other fatty acid was C18:1. An outer membrane fraction, obtained by lysozyme treatment of the Triton-extracted cell wall, showed essentially the same chemical composition except for almost complete removal of peptidoglycan. Saline extraction (0.9% NaCl, 37 degrees C, 2 h) removed a lipopolysaccharide-protein(-phospholipid?) complex from whole cells of R. capsulata St. Louis. The polypeptide patterns of the cell wall and outer membrane as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comprised 20 to 25 different polypeptides (most of them very faint) and were dominated by a single, heat-modifiable major protein (Mr 69,000 after solubilization below 60 degrees C; Mr 33,000 at temperatures above 70 degrees C).
Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp. strain 109D
Authors: Diedrich D L , Duran C P , Conti S F
Published: January 2020

The ability of Bdellovibrio sp. to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired. Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp. could acquire the OmpC protein in the absence of the OmpF protein. However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane. The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp. exhibited a kinetic and temporal preference for the OmpF protein. Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency.
Chemosensory and thermosensory excitation in adaptation-deficient mutants of Escherichia coli
Authors: Imae Y , Mizuno T , Maeda K
Published: January 2020

Methyl-accepting chemotaxis protein-methyltransferase-deficient mutants, cheR mutants, of Escherichia coli showed a tumble response to repellents only at low temperatures, and the resultant tumbling lasted unless the condition was changed. The swimming pattern of the repellent-treated cells was different at different temperatures, indicating that the absolute temperature is a determinant of the tumbling frequency of those cells. The tumbling of those cells was also suppressed by the addition of attractants. Under a suitable repellent concentration, the tumbling frequency of the cells was found to be simply determined by the ligand occupancy of chemoreceptors for many attractants. In a methyl-accepting chemotaxis protein-methylesterase-deficient mutant, a cheB deletion mutant, the tumbling frequency was also determined by receptor occupancy of some attractants. These results indicate that in the adaptation-deficient mutants, sensory signals are produced in proportion to the amount of ligand-bound or of thermally altered receptors and transmitted to the flagellar motors without any modification. Thus, it is concluded that the adaptation system, namely, the methylation-demethylation system of methyl-accepting chemotaxis proteins, is not concerned with the step of chemosensory or thermosensory excitation. A simple model is proposed to explain how the swimming pattern of the adaptation-deficient mutants is determined.
Transposon Tn5 encodes streptomycin resistance in nonenteric bacteria
Authors: O'Neill E A , Kiely G M , Bender R A
Published: January 2020

Strains of Caulobacter crescentus, Pseudomonas putida, Acinetobacter calcoaceticus, Rhizobium meliloti, and Rhodopseudomonas sphaeroides carrying the kanamycin resistance-encoding transposon Tn5 were 15 to 500 times more resistant to streptomycin than transposon-free strains. The streptomycin resistance determinant, which is separable from the kanamycin resistance determinant of Tn5, was not expressed in Escherichia coli or Klebsiella aerogenes.
Cloning of the iron superoxide dismutase gene (sodB) in Escherichia coli K-12
Authors: Sakamoto H , Touati D
Published: January 2020

A clone overproducing iron superoxide dismutase has been isolated from an Escherichia coli cosmid bank. Subcloning located the gene responsible for iron superoxide dismutase overproduction on a 6.6-kilobase PstI restriction endonuclease fragment. Maxicell analysis, followed by immunological identification of iron superoxide dismutase protein, demonstrated that the structural gene, sodB, of iron superoxide dismutase has been cloned.
Kinetics of methylation in Escherichia coli K-12
Authors: Lyons S M , Schendel P F
Published: January 2020

Newly synthesized DNA is undermethylated in E. coli K-12. The amount of N6-methyl deoxyadenylic acid in labeled DNA varied from 0.3 mol% of total adenine for a 2-min pulse to 1.7 mol% for DNA that was labeled for more than two generations.
Physical mapping of hemolysin plasmid pCW2, which codes for virulence of a nephropathogenic Escherichia coli strain
Authors: Waalwijk C , de Graaff J , MacLaren D M
Published: January 2020

The hemolysin plasmid pCW2, which enhances the virulence of Escherichia coli, has been mapped. Comparison of the region coding for hemolysin with other hemolysin determinants reveals differences that could explain differences in the ability to confer virulence.
Aspartate-specific peptidases in Salmonella typhimurium: mutants deficient in peptidase E
Authors: Carter T H , Miller C G
Published: January 2020

The only dipeptide found to serve as a leucine source for a Salmonella strain lacking peptidases N, A, B, D, P, and Q was alpha-L-aspartyl-L-leucine. A peptidase (peptidase E) that specifically hydrolyzes Asp-X peptides was identified and partially purified from cell extracts. The enzyme (molecular weight, 35,000) is inactive toward dipeptides with N-terminal asparagine or glutamic acid. Mutants (pepE) lacking this enzyme were isolated by screening extracts for loss of the activity. Genetic mapping placed the pepE locus at 91.5 map units and established the gene order metA pepE zja-861::Tn5 malB. Duplications of the pepE locus showed a gene dosage effect on levels of peptidase E, suggesting that pepE is the structural gene for this enzyme. Mutations in pepE resulted in the loss of the ability to grow on Asp-Pro as a proline source but did not affect utilization of other dipeptides with N-terminal aspartic acid. Loss of peptidase E did not cause a detectable impairment in protein degradation. Two other peptidases present in cell extracts of mutants lacking peptidases N, A, B, D, P, Q, and E also hydrolyze many Asp-X dipeptides.
Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins
Authors: Morona R , Klose M , Henning U
Published: January 2020

The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.
Positive control of expression of the argECBH gene cluster in vitro by guanosine 5'-diphosphate 3'-diphosphate
Authors: Zidwick M J , Korshus J , Rogers P
Published: January 2020

By using a cell-free system derived from Escherichia coli, it was found that guanosine 5'-diphosphate 3'-diphosphate (ppGpp) was a positive effector for expression of both wings of bidirectionally transcribed argECBH gene cluster. A 7- to 20-fold increase in the synthesis of both argininosuccinase (the argH enzyme) and N-acetylornithinase (the argE enzyme) resulted with added ppGpp (0.2 mM optimum). Synthesis of hybridizable argECBH mRNA was enhanced only 30 to 100% by added ppGpp. Of the various guanosine nucleotides tested, only pppGpp mimicked ppGpp. Added ppGpp had no important effect upon (i) measurable argE or argH enzyme activity, (ii) total protein synthesis in the cell-free system, or (iii) the rate of decay of hybridizable argECBH mRNA. With extracts of an argR+ strain, added ppGpp had no effect on the repression of enzyme or mRNA synthesis by L-arginine. By using a two-stage system in which the bulk of argECBH mRNA was synthesized while protein synthesis was delayed, we showed that ppGpp acted at some point during transcription.
Organization and expression of genes responsible for type 1 piliation in Escherichia coli
Authors: Orndorff P E , Falkow S
Published: January 2020

The genetic organization of a segment of recombinant DNA conferring the capacity of synthesize E. coli type 1 pili was examined. This 11.2-kilobase (kb) segment of DNA, derived from a clinical isolate, conferred a piliated phenotype (Pil+) on a nonpiliated (Pil-) strain of E. coli K-12 that lacked DNA homologous to the 11.2-kb region. Insertional mutagenesis, deletion mutagenesis, and subcloning of various regions of the 11.2-kb fragment allowed the localization of five genes, each encoding a polypeptide, that were associated with pilus expression. Three gene products, 17, 86, and 30 kilodaltons (kd) in size, were involved in pilus assembly; assembly of the 17-kd structural (pilin) protein into pili was not seen in mutants lacking either the 86- or 30-kd proteins, but pilin synthesis and proteolytic processing were not affected. The fourth polypeptide, 23 kd in size, appeared to be involved in the regulation of pilus expression because mutants lacking this protein exhibited a 40-fold increase in the amount of pilin antigen per cell. The last protein, 14 kd in size, was not associated with piliation by genetic criteria; however, the 14-kd protein was immunoprecipitated with pili, suggesting an association with pili or immunological cross-reactivity with pilin. Immunoprecipitates of minicell transcription translation products revealed that pilus polymerization was taking place in minicells. This may facilitate the study of the molecular steps in pilus biosynthesis and, as a consequence, provide clues to the assembly of supramolecular structures in general.
Oxygen toxicity in Streptococcus mutans: manganese, iron, and superoxide dismutase
Authors: Martin M E , Strachan R C , Aranha H , Evans S L , Salin M L , Welch B , Arceneaux J E , Byers B R
Published: January 2020

When cultured anaerobically in a chemically defined medium that was treated with Chelex-100 to lower its trace metal content, Streptococcus mutans OMZ176 had no apparent requirement for manganese or iron. Manganese or iron was necessary for aerobic cultivation in deep static cultures. During continuous aerobic cultivation in a stirred chemostat, iron did not support the growth rate achieved with manganese. Since the dissolved oxygen level in the chemostat cultures was higher than the final level in the static cultures, manganese may be required for growth at elevated oxygen levels. In medium supplemented with manganese, cells grown anaerobically contained a low level of superoxide dismutase (SOD) activity; aerobic cultivation increased SOD activity at least threefold. In iron-supplemented medium, cells grown anaerobically also had low SOD activity; aerobic incubation resulted in little increase in SOD activity. Polyacrylamide gel electrophoresis of the cell extracts revealed a major band and a minor band of SOD activity in the cells grown with manganese; however, cells grown with iron contained a single band of SOD activity with an Rf value similar to that of the major band found in cells grown with manganese. None of the SOD activity bands were abolished by the inclusion of 2 mM hydrogen peroxide in the SOD activity strain. S. mutans may not produce a separate iron-containing SOD but may insert either iron or manganese into an apo-SOD protein. Alternatively, iron may function in another activity (not SOD) that augments the defense against oxygen toxicity at low SOD levels.
Isolation of new white collar mutants of Neurospora crassa and studies on their behavior in the blue light-induced formation of protoperithecia
Authors: Degli-Innocenti F , Russo V E
Published: January 2020

White collar (wc) mutants of Neurospora crassa are thought to be regulatory mutants blocked in the photoinduction of carotenogenesis. Eight new wc mutants have been isolated after UV mutagenesis; their morphology and linear growth rate are not altered, although blue light-induced carotenogenesis is completely blocked. All of the wc mutations fall into two complementation groups corresponding to the already-known wc-1 and wc-2 loci. It is shown that the wc mutations impair another blue light effect, the photoinduction of protoperithecia formation, as well as the low constitutive production of protoperithecia in the dark. These effects are not due to the lack of carotenoids since the albino mutants show a normal sexual development. The pleiotropic effects of the mutations in the wc genes indicate that they play a key role in the mechanisms of regulation of the blue light-induced responses of N. crassa.
Insertion mutagenesis of the metJBLF gene cluster of Escherichia coli K-12: evidence for an metBL operon
Authors: Greene R C , Smith A A
Published: January 2020

The effects of Mu or transposon 5 insertions on the expression of genes of the metJBLF cluster show that metB and metL form an operon, transcribed from metB to metL, and that metF and metJ are independently transcribed.
Changes in cell surface hydrophobicity of Myxococcus xanthus are correlated with sporulation-related events in the developmental program
Authors: Kupfer D , Zusman D R
Published: January 2020

Cell surface hydrophobicity was measured in the bacterium Myxococcus xanthus during vegetative growth, fruiting body formation, and glycerol-induced spore formation by the method of Rosenberg et al. (FEMS Microbiol. Lett. 9:29-33, 1980). A significant decrease in cell surface hydrophobicity was observed 12 to 36 h after fruiting body formation and 60 to 120 min after glycerol-induced sporulation. The hydrophilic shift was correlated with the ability of the cells to sporulate but not with their ability to aggregate. Sucrose gradient purification removed the hydrophilic substance from the fruiting body spores but not from the glycerol-induced spores. The change in cell surface hydrophobicity in M. xanthus should be a useful developmental marker.
In vitro polymerization of flagellin excreted by a short-flagellum Salmonella typhimurium mutant
Authors: Ikeda T , Kamiya R , Yamaguchi S
Published: January 2020

The culture medium of a short-flagellum mutant of Salmonella typhimurium contained a large amount of mutant flagellin and a small amount of strong inhibitors of flagellin polymerization. After being freed of the inhibitors, the mutant flagellin could be polymerized in vitro, although under nonphysiological conditions.
Regulation of transcription of the Escherichia coli phosphoenolpyruvate carboxykinase locus: studies with pck-lacZ operon fusions
Authors: Goldie H
Published: January 2020

Mutants of Escherichia coli containing genetic fusions of lacZ to the pck (phosphoenolpyruvate carboxykinase) locus were isolated by using Mu d(lacZ Ampr) bacteriophage. Synthesis of beta-galactosidase in these strains is regulated by cyclic AMP and glucose (catabolite repression). Synthesis of beta-galactosidase by pck-lacZ fusions was induced in log-phase cells growing on gluconeogenic media, was repressed by glucose, and was also induced up to 100-fold at the onset of stationary phase in LB medium. This stationary-phase induction required cyclic AMP and some other unknown regulatory signal.
Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation
Authors: Yang M Y , Ferrari E , Henner D J
Published: January 2020

The neutral protease gene of Bacillus subtilis has been cloned, and its nucleotide sequence has been determined. The cloned gene was used to create an in vitro-derived deletion mutation, which was used to replace the wild-type copy of the gene. This deletion, in combination with a deletion of the alkaline protease gene, completely abolished protease production. The loss of the proteases had no detectable effect on growth, morphology, or sporulation.
Regulation of the F-factor pif operon: pifO, a site required in cis for autoregulation, titrates the pifC product in trans
Authors: Miller J F , Malamy M H
Published: January 2020

F factor pifC, pifA, and pifB gene expression is subject to negative regulation by the product of the pifC locus (J.F. Miller and M. H. Malamy, J. Bacteriol. 156:338-347, 1983). In this paper, we describe the properties of a new regulatory site in the pif region, pifO, which is required in cis for autoregulation of pif gene expression. Spontaneous pifO mutations were isolated that allow expression of a pifC-lacZ protein fusion in the presence of pifC product in trans. Recombination of these pifO mutations onto F'lac results in increased pifA and pifB activity. Thus, a single regulatory element, pifO, regulates pifC, pifA, and pifB expression in cis. The presence of multiple copies of a fragment from the pif region carrying wild-type pifO sequences (F coordinates 42.9 to 42.43 kilobases) in trans to F'lac results in an increase in pifA and pifB activity as measured by inhibition of T7 plating. When the pifO mutations are recombined onto a plasmid carrying pifO, the resulting recombinants are greatly decreased in the ability to increase F'lac pif expression. These results suggest that increased F'lac pifA and pifB expression caused by pifO sequences in trans is a consequence of titration of pifC product and derepression of the pif operon.
Legionella pneumophila surface antigens cloned and expressed in Escherichia coli are translocated to the host cell surface and interact with specific anti-Legionella antibodies
Authors: Engleberg N C , Pearlman E , Eisenstein B I
Published: January 2020

Escherichia coli clones that express Legionella pneumophila antigens were isolated from a plasmid genomic library, and their antigens were characterized by immunoblotting with rabbit anti-L. pneumophila sera. Because previous studies of L. pneumophila antigens by whole-cell radioimmunoprecipitation suggested that comigrating native antigens were surface localized, we conducted experiments to determine if the cloned antigens were surface expressed in E. coli. Aliquots of antisera were absorbed by intact cells of three representative antigen-producing E. coli clones, and surface-bound antibodies were acid eluted from the intact cells. Immunoblots made with selectively absorbed antisera and eluted antibodies confirmed that reactivity to the homologous cloned antigens could be specifically absorbed from the antisera and then eluted from the cells, demonstrating a surface (antibody-accessible) localization in the cloned state. Antibodies eluted from the surface of an E. coli clone that expressed a 19-kilodalton antigen reacted with the surface of L. pneumophila in a liquid-phase, whole-cell enzyme-linked immunosorbent assay. In addition, intact cells of this clone were used in an enzyme-linked immunosorbent assay to detect serum antibody. E. coli cells that express foreign antigens on their surfaces can be used to develop antigen-specific immunoassays and to affinity purify monospecific antibodies.