bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipids and lipopolysaccharide of Aeromonas hydrophila
Authors: Howard S P , Buckley J T
Published: January 2020

The phospholipids and lipopolysaccharide of Aeromonas hydrophila were characterized. Phosphatidylethanolamine and phosphatidylglycerol were the major phospholipid components. The outer membrane contained more phosphatidylethanolamine and less phosphatidylglycerol than the inner membrane, and the phospholipids of the outer membrane contained a higher proportion of saturated fatty acids. Only four fatty acids (C14:0, C16:0, C16:1, and C18:1) were found in the phospholipids. The lipopolysaccharide of A. hydrophila did not contain the eight-carbon sugar 3-deoxyoctulosonic acid nor did it contain C16:0, both of which are typical constituents of the lipopolysaccharide of many other species.
Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images
Authors: Valkenburg J A , Woldringh C L , Brakenhoff G J , van der Voort H T , Nanninga N
Published: January 2020

The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.
Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus
Authors: Yamashita I , Suzuki K , Fukui S
Published: January 2020

The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.
Oxygen regulation in Salmonella typhimurium
Authors: Strauch K L , Lenk J B , Gamble B L , Miller C G
Published: January 2020

Regulation by oxygen of the peptidase T (pepT) locus of Salmonella typhimurium was studied by measuring beta-galactosidase levels in strains containing a pepT::Mu d1(Apr lac) operon fusion. beta-Galactosidase was induced in anaerobic cultures and late-exponential and stationary-phase aerated cultures. Peptidase T activity also was induced under these growth conditions. pepT+ but not pepT strains will utilize as amino acid sources the tripeptides Leu-Leu-Leu and Leu-Gly-Gly only when grown anaerobically. Mutations at two loci, oxrA and oxrB (oxygen regulation) prevent induction of the pepT locus. The oxrA locus is homologous to the fnr locus of Escherichia coli. We have isolated 12 independent Mu d1 insertions (oxd::Mu d1, oxygen dependent) that show induction of beta-galactosidase in anaerobic cultures and stationary-phase aerated cultures. These insertions fall into nine classes based on map location. All of the oxd::Mu d1 insertions are regulated by oxrA and oxrB and therefore define a global regulon that responds to oxygen limitation.
Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli
Authors: Honda H , Kudo T , Horikoshi K
Published: January 2020

The gene for xylanase A of alkalophilic Bacillus sp. strain C-125 was cloned in Escherichia coli with pBR322. The plasmid pCX311 contained 2.6- and 2.0-kilobase-pair HindIII fragments. The characteristics of the purified pCX311-encoded xylanase were the same as those of purified xylanase A from alkalophilic Bacillus sp. strain C-125.
Escherichia coli 6S RNA gene is part of a dual-function transcription unit
Authors: Hsu L M , Zagorski J , Wang Z , Fournier M J
Published: January 2020

The gene coding for the metabolically stable 6S RNA of Escherichia coli has been cloned, sequenced, and partially characterized in expression analyses. The DNA sequence results confirm the accuracy of the previously established RNA sequence and, with genomic hybridization data, reveal that there is only one copy of the 6S DNA in the chromosome. Consistent with its relaxed mode of expression, the promoter region of the 6S RNA gene was found to lack the hypothetical GC-rich discriminator domain common to other stable RNA genes under stringent control. The sequence results also revealed the occurrence of a 540-base-pair open reading frame immediately downstream from the 6S RNA coding region. Results from the expression analyses show that the protein and RNA coding regions are cotranscribed in vitro and that the open reading frame is translated in vivo.
Effect of nitrogen starvation on the morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus
Authors: Stevens S E , Nierzwicki-Bauer S A , Balkwill D L
Published: January 2020

The effects of nitrogen starvation on the morphology and ultrastructure of the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The internal ultrastructural characteristics of vegetative cells changed markedly during nitrogen starvation. Carboxysomes were degraded, while polyphosphate bodies and lipid bodies accumulated. The ultrastructure of mature heterocysts was also affected by nitrogen starvation; their intracytoplasmic membranes vesiculated to form vacuolelike structures and, eventually, large empty regions in the cytoplasm. Nitrogen starvation stimulated extensive heterocyst differentiation in M. laminosus, producing heterocyst frequencies of 17.5% in narrow filaments and 28.3% in wide filaments within 44 h after transfer to N-free conditions. Cells in wide filaments differentiated so extensively that only 16.8% of them failed to initiate the differentiation process within 44 h.
Monovalent cations enable cell wall turnover of the turnover-deficient lyt-15 mutant of Bacillus subtilis
Authors: Cheung H Y , Freese E
Published: January 2020

A lyt-15 mutant reported to be unable to turn over the cell wall exhibited the same rate of wall turnover as the standard strain if the medium contained 0.2 M NaCl, which did not affect growth. Cell wall autolysis was also optimal at 0.2 M NaCl.
Occurrence of diploid strains of Cryptococcus neoformans
Authors: White C W , Jacobson E S
Published: January 2020

A mating between niacin and pantothenate auxotrophs of Cryptococcus neoformans gave a few prototrophic progeny that were self-fertile. These were uninuclear but contained twice as much DNA as the parental strains. Segregation of nutritional markers was observed upon sporulation. We conclude that these self-fertile strains are diploids.
recA-independent recombination between repeated IS50 elements is not caused by an IS50-encoded function
Authors: Phadnis S H , Berg D E
Published: January 2020

Certain pBR322-related plasmids containing direct repeats of the insertion element IS50 appear to be unstable in recA Escherichia coli because smaller recombinant derivatives accumulate rapidly in plasmid DNA populations. We show here that (i) this instability is plasmid specific, but not IS50 specific; (ii) it is due to a detrimental effect exerted by these plasmids on bacterial growth; and (iii) the growth impairment is alleviated in cells harboring the smaller recombinant plasmids. Although a recent report had concluded that accumulation of recombinants reflected an IS50-specific recombination function, when correction is made for the relative growth rates of cells containing the parental and recombinant plasmids the evidence for such a recombination function disappears.
Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum
Authors: Béguin P , Cornet P , Aubert J P
Published: January 2020

The nucleotide sequence of the celA gene, encoding the extracellular endoglucanase A of Clostridium thermocellum, was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 32 amino acids. A segment of 23 amino acids was duplicated at the COOH-terminal end of the protein. The putative GUG initiation codon was preceded by an AGGAGG sequence, typical of procaryotic ribosomal binding sites. The segment of DNA presumably specifying transcriptional initiation contained a high percentage of adenine and thymine residues, including an adenine-thymine tract extending over 54 base pairs.
Physical mapping of a 330 X 10(3)-base-pair region of the Myxococcus xanthus chromosome that is preferentially labeled during spore germination
Authors: Komano T , Inouye S , Inouye M
Published: January 2020

Myxococcus xanthus was pulse-labeled with [3H]thymidine immediately after germination of dimethyl sulfoxide-induced spores. The restriction enzyme digests of the total chromosomal DNA from the pulse-labeled cells were analyzed by one-dimensional as well as two-dimensional agarose gel electrophoresis. Four PstI fragments preferentially labeled at a very early stage of germination were cloned into the unique PstI site of pBR322. By using these clones as probes, a restriction enzyme map was established covering approximately 6% of the total M. xanthus genome (330 X 10(3) base pairs). The distribution of the specific activities of the restriction fragments pulse-labeled after germination suggests a bidirectional mode of DNA replication from a fixed origin.
Cold sensitivity induced by overproduction of UmuDC in Escherichia coli
Authors: Marsh L , Walker G C
Published: January 2020

The UmuD and UmuC proteins of Escherichia coli are essential for mutagenesis by UV and most chemicals. Their mode of action is presently unknown. Strains which lack the LexA repressor [lexA(Def)] and contain a pBR322-derived plasmid carrying the umuDC operon overexpress UmuD and UmuC and become cold sensitive (growth at 42 degrees C but not at 30 degrees C). Deletion mapping showed that the umuDC locus on the plasmid is responsible for conferring cold sensitivity. The conditional lethality appeared due to a rapid and reversible inhibition of DNA synthesis at the nonpermissive temperature. Cold sensitivity was enhanced by the increase of NaCl in the medium to 1% and eliminated by 4% ethanol in the medium. Cold sensitivity was partially suppressed by the lon-100 mutation and completely suppressed by the htpR165 mutation.
Amount and chain length of polyphosphates in Escherichia coli depend on cell growth conditions
Authors: Rao N N , Roberts M F , Torriani A
Published: January 2020

Anaerobiosis induced an accumulation of polyphosphates (poly Pi) in a phosphate-rich medium by an alkaline-phosphatase constitutive mutant of Escherichia coli. The total poly Pi content was maximum at around 6 h of anaerobic growth. Both trichloroacetic acid- and NaOH-soluble poly Pi were found to be present. The acid-soluble fraction consisted mainly of a linear polymer of about 20 +/- 5 phosphate units, whereas the alkali-extractable poly Pi fraction contained at least four molecular species of higher chain length as determined by gel filtration. The majority of poly Pi extracted at 6 h had lower chain lengths than those extracted from cells incubated for 24 h. In vivo 31P nuclear magnetic resonance spectra of E. coli cells as a function of growth conditions were consistent with the in vitro extract results.
Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals
Authors: Mergeay M , Nies D , Schlegel H G , Gerits J , Charles P , Van Gijsegem F
Published: January 2020

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).
7-Methylpterin and 7-methyllumizine: oxidative degradation products of 7-methyl-substituted pteridines in methanogenic bacteria
Authors: White R H
Published: January 2020

7-Methylpterin and 7-methyllumizine were isolated and identified in extracts of methanogenic bacteria which had been extracted in air with ethanol-water. Ethanol-water preparations of cells extracted under nitrogen or hydrogen were devoid of these compounds. Extracts of cells obtained in the presence of air also had an increased amount of a complex arylamine which, on acid hydrolysis, gave 1 mol each of phosphate, 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane, and alpha-hydroxyglutaric acid. Gas chromatography-mass spectrometry was used to identify the 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane as its tetratrimethylsilyl derivative and the alpha-hydroxyglutaric acid as the n-butyl ester derivative of its gamma-lactone. When exposed to air, extracts of cells prepared in the absence of air produced 6-acetyl-7-methylpterin and 7-methylxanthopterin in addition to 7-methylpterin and 7-methyllumizine. It is concluded that these compounds are derived from the oxidative cleavage of the tetrahydromethanopterin, which is present in these bacteria, by a series of reactions analogous to those known to occur in the oxidative cleavage of tetrahydrofolic acid.
Genetic mapping of nth, a gene affecting endonuclease III (thymine glycol-DNA glycosylase) in Escherichia coli K-12
Authors: Weiss B , Cunningham R P
Published: January 2020

The nth gene of Escherichia coli affects the production of endonuclease III, a glycosylase-endonuclease that attacks DNA damaged by oxidizing agents or by ionizing radiation. An nth insertion mutant and a deletion mutant were studied. nth is located between add and tyrS on the linkage map of E. coli K-12 and was 97% linked to tyrS in a transduction with phage P1.
Legume agglutinins that bind to Rhizobium meliloti
Authors: Seegers R , LaRue T A
Published: January 2020

A protein found in seeds and roots of alfalfa (Medicago sativa) was implicated in the specificity of the infection process, based on its binding to the symbiont Rhizobium meliloti. We found an agglutinin with similar properties in seeds and roots of sweet clover (Melilotis alba). The sweet clover differed from alfalfa in nodulation by a mutant strain of R. meliloti, but the agglutinins were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Rhizobium agglutination, and cross-reactivity to antibodies. Similar agglutinins binding R. meliloti were found in seeds of legumes from different cross-inoculation groups, including soybean (Glycine max), cowpea (Vigna unguiculata), pea (Pisum sativum L), and mung bean (Vigna mungo). The agglutinins from these legumes were recognized by antibodies raised against the agglutinins of alfalfa and sweet clover. Seeds of corn (Zea mays) and tomato (Lycopersicon esculentum) contained a protein similar to the legume agglutinin, but it did not react with the antibodies. We conclude that the alfalfa agglutinin is representative of a common legume protein and that there is no evidence for its role in specificity or nodule initiation.
Denitrification by Alcaligenes eutrophus is plasmid dependent
Authors: Römermann D , Friedrich B
Published: January 2020

Curing of the hydrogenase-specifying megaplasmid pHG indigenous to strains of the facultative lithoautotrophic bacterium Alcaligenes eutrophus was correlated with a loss of denitrifying ability (Nitd). The retransfer of plasmid pHG1 reconstituted the Nitd phenotype. Plasmid-free mutants were still capable of converting some nitrate to nitrite, but they did not metabolize nitrite under anaerobic conditions.
Regulation of naphthalene catabolic genes of plasmid NAH7
Authors: Yen K M , Gunsalus I C
Published: January 2020

Tn5 insertion mutations defining a regulatory gene, nahR, of the naphthalene catabolic pathway encoded by the NAH7 plasmid were mapped within a small NAH7 region only a few hundred bases upstream of the nahG gene, the most promoter-proximal gene of the nahGHIJK operon. The nahR mutations blocked the induction of both the nahABCDEF and nahGHIJK operons, and the defect was completely corrected in the presence of the wild-type allele in a trans position. The pleiotropic, recessive, and negative nature of these mutations indicates that the nahR gene specifies a regulatory element which is required to activate both nah operons.
Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn918)
Authors: Clewell D B , An F Y , White B A , Gawron-Burke C
Published: January 2020

Streptococcus faecalis RC73 was found to harbor a conjugative plasmid (pAM373) which confers a mating response to a sex pheromone (cAM373) excreted by plasmid-free members of the same species. The pheromone was also detected in culture filtrates of all of 23 Staphylococcus aureus strains but in only 2 of 22 coagulase negative staphylococcus strains. Streptococcus sanguis Challis and G9B also produced the activity, but 10 other Streptococcus sanguis strains did not. The activity was also produced by Streptococcus faecium 9790. A tetracycline resistance (Tc) determinant present in S. faecalis RC73 was not associated with pAM373 but served as a useful marker in efforts to identify pAM373 among other plasmids present in the strain. Analyses of the Tc determinant showed that it was located on a conjugative transposon very similar to Tn916. Designated Tn918, the transposon could insert into pAM373 as well as into two other hemolysin plasmids. Whereas pAM373 derivatives transferred very well between strains of Streptococcus faecalis, the plasmid would not establish in Staphylococcus aureus or Streptococcus sanguis. However, a derivative of pAM373 carrying Tn918 proved to be a useful delivery vehicle for generating transposon insertions into multiple sites on the staphylococcal chromosome.
Transport of vitamin B12 in Escherichia coli: cloning of the btuCD region
Authors: DeVeaux L C , Kadner R J
Published: January 2020

The transport of vitamin B12 in Escherichia coli requires a specific vitamin B12 receptor protein in the outer membrane and the tonB gene product. In addition, the btuC gene, located at min 38 on the genetic map, has been found to influence vitamin B12 uptake or utilization. The btuC function is required for the growth response to vitamin B12 when the outer membrane transport process (btuB or tonB function) is defective. However, even in a wild-type strain, btuC is required for proper transport of vitamin B12. Additional mutations in the vicinity of btuC were isolated as lac fusions that produced a phenotype similar to that of a btuC mutant. The btuC region was cloned by selection for complementation of a btuC mutation. Complementation testing with plasmids carrying various deletions or transposon Tn1000 insertions demonstrated that the new mutations defined a separate, independently expressed locus, termed btuD. The coding regions for both genes were identified on a 3.4-kilobase HindIII-HincII fragment and were 800 to 1,000 base pairs in length. They were separated by a 600- to 800-base-pair region. The gene order in this portion of the chromosome map was found to be pps-zdh-3::Tn10-btuD-btuC-pheS. Expression of beta-galactosidase in the btuD-lac fusion-bearing strains, whether proficient or defective in vitamin B12 transport, was not regulated by the presence of vitamin B12 in the growth medium.
Kinetic studies on formation of cytochrome oxidase of Rhodopseudomonas capsulata after a shift from phototrophic to chemotrophic growth
Authors: Hüdig H , Drews G
Published: January 2020

Rhodopseudomonas capsulata cells were shifted from phototrophic (anaerobic, light) to chemotrophic (semiaerobic, dark, 10% air saturation) growth conditions. During the adaptation period of 4 h, the bacteriochlorophyll content of cells and membranes decreased, and a newly synthesized 65-kilodalton polypeptide of the cytochrome oxidase was incorporated into the membrane fraction. The enzymatic activity of the cytochrome oxidase increased strongly after a lag time of 2 h. The amount of cytochrome oxidase protein does not follow the same kinetics. The relative amount of a membrane-bound cytochrome c of low molecular weight, which has been proposed to be a donor for the cytochrome oxidase, increased during adaptation.
Effects of light, oxygen, and substrates on steady-state levels of mRNA coding for ribulose-1,5-bisphosphate carboxylase and light-harvesting and reaction center polypeptides in Rhodopseudomonas sphaeroides
Authors: Zhu Y S , Kaplan S
Published: January 2020

The mRNA levels specific for ribulose-1,5-bisphosphate carboxylase, light-harvesting I polypeptides alpha and beta, and reaction center polypeptides L and M were assayed by use of a series of DNA probes specific for each cognate mRNA. Both the steady-state amounts and sizes of the specific mRNAs were measured as a function of the light intensity incident to the culture, the presence or absence of oxygen, and the type of substrate present in the growth medium. Northern hybridization revealed at least two and possibly three transcripts for ribulose-1,5-bisphosphate carboxylase. The cellular level of mRNA specific for ribulose-1,5-bisphosphate carboxylase increased in consort with enzyme activity as a function of both light intensity and reducing state of the substrate. Neither mRNA nor enzyme activity was detectable in aerobically grown cells. For the light-harvesting I and reaction center polypeptides there exist two transcripts, the larger of which appears to be a polycistronic mRNA possessing information for all four polypeptides and a smaller transcript specific for only the alpha and beta polypeptides of the light-harvesting I complex. The regulation of each of these mRNAs was affected by light and oxygen, but was not significantly affected by the oxidation-reduction state of the substrate.
Two homologous genes coding for spore-specific proteins are expressed at different times during development of Myxococcus xanthus
Authors: Teintze M , Thomas R , Furuichi T , Inouye M , Inouye S
Published: January 2020

The ops and tps genes of Myxococcus xanthus have ca. 90% DNA and amino acid sequence homology and are in the same orientation separated by a spacer region of only 1.4 kilobases. The products of the two genes were found to cross-react immunologically, and both were capable of Ca2+-dependent self-assembly on the surface of myxospores. However, the ops and tps genes were expressed very differently during the developmental cycle of M. xanthus. The tps gene is induced early during fruiting body formation on a solid surface, and its product, protein S, is made in large quantities (up to 15% of total protein synthesis). When the cells turn into myxospores, protein S is assembled on the outer surface of the spore. We have now also found it in much smaller quantities inside the spores. The ops gene, on the other hand, appears to be induced later in development, after the cells have sporulated, since the ops gene product was found only inside the spores. When an ops gene under the control of a tps gene promoter was inserted into a wild-type strain, the ops gene product was synthesized at the same time as protein S and assembled onto the spore surface.
Evidence of a common pathway of carbon dioxide reduction to methane in methanogens
Authors: Jones W J , Donnelly M I , Wolfe R S
Published: January 2020

The roles of methanofuran and tetrahydromethanopterin as carriers of C1 moieties in the reduction of carbon dioxide to methane were studied in representatives of diverse groups of methanogens, confirming that these roles, first reported for Methanobacterium thermoautotrophicum, are common for methanogenesis in general. Extracts of the methanogens tested converted formyl-methanofuran and methyl-tetrahydromethanopterin to methane; the extractable cofactors derived from the same methanogens, with one exception, complemented a methanofuran- and tetrahydromethanopterin-deficient enzyme system from M. thermoautotrophicum. The amounts of extractable methanofuran and tetrahydromethanopterin were determined for each representative methanogen.
Slow-growing Rhizobium japonicum comprises two highly divergent symbiotic types
Authors: Stanley J , Brown G G , Verma D P
Published: January 2020

We examined the interrelationships of the genomes of 10 slow-growing strains of Rhizobium japonicum to provide a foundation for molecular genetic studies of these agriculturally important endosymbiotic bacteria of commercial soybeans. The degree of base substitution in and around known symbiotic genes (nif and presumptive nod), constitutively expressed genes (glnA and recA), and two other cloned sequences was estimated from restriction site variation by using cloned DNAs as hybridization probes to genomic Southern blots. Two highly divergent patterns of conservation of nifDH genes and nod-homologous sequences were found. On this basis, we classified the strains as the symbiotic genotypes sTI or sTII. Existing maps of the nif genes of R. japonicum apply only to strains of the sTI genotype. This division was further characterized by four other probes which also distinguished two sublines within sTI. Phenograms were constructed depicting interrelationships according to DNA sequence divergence. sTI and sTII are two highly divergent evolutionary lines consistent with the status of individual species. Neither is related to fast-growing Rhizobium strains (PRC strains) nodulating soybeans.
Unusual organization of the ilvIH promoter of Escherichia coli
Authors: Haughn G W , Squires C H , De Felice M , Largo C T , Calvo J M
Published: January 2020

Analysis of plasmids containing ilvIH-galK fusions indicated that the Escherichia coli ilvIH promoter and sequences sufficient to cause leucine repression lie within 363 base pairs (bp) of ilvI. Experiments designed to locate the promoter and regulatory sequences more precisely gave the following results. The positions of the 5' endpoints of both unlabeled and pulse-labeled ilvIH mRNAs transcribed in vivo lie 30 bp upstream of ilvI. By contrast, the major in vitro RNA endpoints lie at positions further upstream. Several mutations which increase the expression of ilvIH lie 40 to 50 bp upstream of ilvI, within a putative promoter termed P1. Deletion of a 50-bp region immediately upstream of ilvI, which includes P1, resulted in the loss of all ilvIH promoter activity. Deletion of sequences more than 200 bp upstream of ilvI reduced ilvIH promoter activity by more than 80%. These results suggest that transcription of the ilvIH operon is initiated from promoter P1 but that sequences more than 200 bp upstream are required for optimal transcription of the operon.
Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity
Authors: Expert D , Toussaint A
Published: January 2020

A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants. The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions. The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities.
Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport
Authors: Bukau B , Brass J M , Boos W
Published: January 2020

Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. We investigated whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by increasing Ca2+ concentrations up to 400 mM. Plasmolysis was essential for a high efficiency; reconstitution and transformation could be stimulated 5- and 40-fold, respectively, by a high concentration of sucrose (400 mM) in cells incubated with a suboptimal Ca2+ concentration (50 mM). The same divalent cations that promote import of DNA (Ca2+, Ba2+, Sr2+, Mg2+, and Ni2+) also induced import of protein. Ca2+ alone was found to be inefficient in promoting reconstitution; successive treatment with phosphate and Ca2+ ions was essential. Transformation also was observed in the absence of phosphate, but could be stimulated by pretreatment with phosphate. The optimal phosphate concentrations were 100 mM and 1 to 10 mM for reconstitution and transformation, respectively. Heat shock, in which the cells are rapidly transferred from 0 to 42 degrees C, affected the two processes differently. Incubation of cells at 0 degrees C in Ca2+ alone allows rapid entry of protein, but not of DNA. Transformation was observed only when exogenous DNA was still present during the heat shock. Shock fluid containing maltose-binding protein inhibited transformation (with 6 microgram of DNA per ml, half-maximal inhibition occurred at around 300 microgram of shock fluid per ml). DNA inhibited reconstitution (with 5 microgram of shock fluid per ml, half-maximal inhibition occurred at around 3 mg of DNA per ml).
Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography
Authors: Dougherty T J
Published: January 2020

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique.
Nucleotide sequence of an Escherichia coli chromosomal hemolysin
Authors: Felmlee T , Pellett S , Welch R A
Published: January 2020

We determined the DNA sequence of an 8,211-base-pair region encompassing the chromosomal hemolysin, molecularly cloned from an O4 serotype strain of Escherichia coli. All four hemolysin cistrons (transcriptional order, C, A, B, and D) were encoded on the same DNA strand, and their predicted molecular masses were, respectively, 19.7, 109.8, 79.9, and 54.6 kilodaltons. The identification of pSF4000-encoded polypeptides in E. coli minicells corroborated the assignment of the predicted polypeptides for hlyC, hlyA, and hlyD. However, based on the minicell results, two polypeptides appeared to be encoded on the hlyB region, one similar in size to the predicted molecular mass of 79.9 kilodaltons, and the other a smaller 46-kilodalton polypeptide. The four hemolysin gene displayed similar codon usage, which is atypical for E. coli. This reflects the low guanine-plus-cytosine content (40.2%) of the hemolysin DNA sequence and suggests the non-E. coli origin of the hemolysin determinant. In vitro-derived deletions of the hemolysin recombinant plasmid pSF4000 indicated that a region between 433 and 301 base pairs upstream of the putative start of hlyC is necessary for hemolysin synthesis. Based on the DNA sequence, a stem-loop transcription terminator-like structure (a 16-base-pair stem followed by seven uridylates) in the mRNA was predicted distal to the C-terminal end of hlyA. A model for the general transcriptional organization of the E. coli hemolysin determinant is presented.
Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12
Authors: Ballantine S P , Boxer D H
Published: January 2020

Two membrane-bound hydrogenase isoenzymes present in Escherichia coli during anaerobic growth have been resolved. The isoenzymes are immunologically and electrophoretically distinct. The physically more abundant isoenzyme (hydrogenase 1) contains a subunit of Mr 64,000 and is not released from the membrane by exposure to either trypsin or pancreatin. The second isoenzyme (hydrogenase 2) apparently contributes the greater part of the membrane-bound hydrogen:benzyl viologen oxidoreductase activity and exists in two electrophoretic forms revealed by nondenaturing polyacrylamide gel analysis. This isoenzyme is irreversibly inactivated at alkaline pH and gives rise to an active, soluble derivative when the membrane-bound enzyme is exposed to either trypsin or pancreatin. Both hydrogenase isoenzymes contain nickel.
Structural genes for flagellar hook-associated proteins in Salmonella typhimurium
Authors: Homma M , Kutsukake K , Iino T
Published: January 2020

The flaW, flaU, and flaV genes of Salmonella typhimurium LT2 were cloned into pBR322. These genes were mapped on the cloned DNA fragments by restriction endonuclease analysis and construction of the deletion derivatives. Their gene products were identified, by the minicell method, as proteins whose molecular weights were estimated to be 59,000 for the flaW product, 31,000 for the flaU product, and 48,000 for the flaV product. These values are identical to those of three species of hook-associated proteins (HAPs), namely, HAP1, HAP3, and HAP2. Furthermore, antibodies against HAP1, HAP3, and HAP2 specifically reacted with the gene products of flaW, flaU, and flaV, respectively. Therefore, we concluded that they are structural genes for HAPs. The antibodies against HAP1 and HAP3 also specifically reacted with the gene products of flaS and flaT of Escherichia coli, respectively. This indicates that these gene products are HAPs in E. coli. This result is consistent with the demonstration that flaS and flaT of E. coli are functionally homologous with flaW and flaU of S. typhimurium.
Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi)
Authors: van Schie B J , Hellingwerf K J , van Dijken J P , Elferink M G , van Dijl J M , Kuenen J G , Konings W N
Published: January 2020

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.
Transcriptional control of synthesis of acid-soluble proteins in sporulating Bacillus subtilis
Authors: Johnson W C , Mahler I , Phillips K , Tipper D J
Published: January 2020

The major acid-soluble spore proteins (ASSPs) isolated from mature spores of Bacillus subtilis are designated alpha, beta, and gamma (about 60, 60, and 100 amino acids in length, respectively). Alpha and beta are very similar, and gamma is very similar to a less predominant ASSP called delta (about 115 amino acids). A minor and very basic ASSP called epsilon is the same size as alpha and beta but is unrelated antigenically. These and several minor ASSPs comprise at least three related families of sporulation-specific gene products. Expression of the alpha and beta genes, detectable as functional mRNA in vitro, coincides with the time of synthesis of all of the major ASSPs in vivo. This apparently coordinate expression is dependent on at least the spo0A, spoIIA, and spoIIIA loci, but not on the spoIVA or spoVA loci, consistent with the late stage of this expression (initiating at 3.5 h after the start of sporulation and peaking at 5 h after start of sporulation). A few minor ASSPs may be asynchronously expressed.
Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b
Authors: Kuo J S , Doelling V W , Graveline J F , McCoy D W
Published: January 2020

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.
Identification of the merR gene of R100 by using mer-lac gene and operon fusions
Authors: Foster T J , Brown N L
Published: January 2020

Transcriptional (operon) and translational (gene) fusions between the R100 merR gene and lacZ were constructed in vitro in a pBR322 plasmid carrying the mer genes derived from plasmid R100. The translational fusions were oriented in the opposite direction to and divergently from the merTCAD genes. This shows that the reading frame previously thought to be merR was incorrect. Expression of the gene fusion was repressed in trans by a compatible plasmid carrying the R100 merR+ gene, as was a similarly oriented transcriptional fusion. In contrast, expression of beta-galactosidase by the lac fragment located at the same site but in the opposite orientation was at a lower level and was not repressed by merR+.
Asymmetric distribution of charge on the cell wall of Bacillus subtilis
Authors: Sonnenfeld E M , Beveridge T J , Koch A L , Doyle R J
Published: January 2020

The cell wall of Bacillus subtilis is capable of binding different kinds of metal ions. The wall-ion complex appears to be dependent on both phosphoryl from teichoic acid and carboxylate from peptidoglycan. In the present study, cationized ferritin (CF) was used as a probe for charge distribution on the wall of B. subtilis 168. Detergent-extracted cell walls bound CF only on the outer wall face. Completed cell poles bound CF, but septa did not. When the walls were permitted to autolyze briefly, binding of CF occurred on both faces. In contrast, limited hydrolysis of the walls by egg white lysozyme resulted in the penetration of CF into the wall matrix. When walls were made teichoic acid-free, CF-binding asymmetry was preserved, suggesting that carboxyl groups were oriented toward the surface. Walls with carboxylates chemically neutralized also retained charge asymmetry. Phosphate-free and carboxyl-modified walls bound CF only poorly or not at all. These results indicate that negative charges contributed by both phosphate and carboxyl are responsible for the binding of CF and that the observed asymmetry in the distribution of the label is due to the orientation of teichoic acid and muramyl peptides toward the outside of the cell wall, above the plane of the glycan strands.
Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation
Authors: Piret J M , Chater K F
Published: January 2020

Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.6-kilobase fragment of wildtype DNA was cloned which restored sporulation to five independent bldA mutants when lysogenized with the recombinant phage. The cloned gene(s) was dominant over the mutant alleles. Phage integration by recombination of the cloned bldA+ DNA with the bldA region of each mutant produced mainly sporulating colonies, presumably heterozygous bldA+/bldA partial diploids for the insert DNA. However, a minority of these primary transductants were bald and were apparently homozygous bldA/bldA mutant partial diploids, formed by some homogenetization process. The phages released from the bald lysogens carried bldA mutations and were used to show that bldA+ sequences had been cloned and that fine mapping of the region could be performed.
Role for 2-linked-beta-D-glucan in the virulence of Agrobacterium tumefaciens
Authors: Puvanesarajah V , Schell F M , Stacey G , Douglas C J , Nester E W
Published: January 2020

Phenol-water cell extracts of virulent Agrobacterium tumefaciens A348 and several avirulent mutants with a reduced ability to attach to plant surfaces were examined. A low-molecular-weight 2-linked-beta-D-glucan was identified in the cell wall extracts of the virulent wild-type strain. Analyses of phenol-water extracts and culture filtrates of four mutant strains showed that the mutants did not produce any 2-linked-beta-D-glucan. When these mutants were complemented, the ability to produce the glucan described above was restored. These results suggest that there is a role for 2-linked-beta-D-glucans in the attachment of A. tumefaciens to plant cells. One avirulent, attachment-defective mutant retained its ability to produce the low-molecular-weight glucan. This mutation, however, mapped to a different transcriptional unit than the mutants deficient in the glucan described above. Thus, it appears that 2-linked-beta-D-glucan is only one component that may be necessary for attachment of A. tumefaciens to plant cell surfaces.
Further evidence that two unique subunits are essential for expression of hydrogenase activity in Rhizobium japonicum
Authors: Harker A R , Lambert G R , Hanus F J , Evans H J
Published: January 2020

Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G. R. Lambert, M. A. Cantrell, F. J. Hanus, S. A. Russell, K. R. Haddad, and H. J. Evans, Proc. Natl. Acad. Sci. USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides. Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000). Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides. Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein. These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.