bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphoglycerides and phospholipase C in membrane fractions of Escherichia coli B
Authors: Bayer M H , Bayer M E
Published: January 2020

The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions containing sites at which an attachment of inner and outer membranes had been observed in the electron microscope (Int.M). Phosphoglycerides labeled with [14C]palmitic acid and [3H]serine were extracted from membrane fractions and identified by two-dimensional thin-layer chromatography. The amount of phosphatidylethanolamine was highest in the outer membrane, whereas the amounts of phosphatidylglycerol and cardiolipin were highest in the inner membrane. The Int.M fractions were observed to have concentrations of phospholipids intermediate to those of the inner and outer membranes. This result supports the assumption that a concentration gradient of inner membrane-outer membrane lipids might exist at the membrane contact sites. The highest phospholipase C activity was detected in the inner membrane and Int.M fractions. The presence of phospholipase C and other lipolytic enzymes in the Int.M fractions suggests a possible involvement of adhesion sites in lipid metabolism, adding a further set of activities to the function of these domains.
Component A2 of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum
Authors: Rouvière P E , Escalante-Semerena J C , Wolfe R S
Published: January 2020

Component A2 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum has been purified 370-fold by liquid chromatography. Homogeneity was obtained by anaerobic preparative polyacrylamide gel electrophoresis. Component A2 is a colorless, air-stable protein consisting of a single polypeptide as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The relative molecular mass of the native protein was determined by high-performance, size exclusion chromatography to be Mr 52,000; on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a value of Mr 59,000 was obtained. When cell extract was subjected to N6-ATP-agarose affinity chromatography the methylcoenzyme M methylreductase system was resolved into two fractions; one of them was component A2. This work provides a new operational definition for component A2, i.e., its characteristic chromatographic behavior on N6-ATP-agarose. However, its functional definition is its ability to reconstitute the methylreductase activity with components A1, A3, and C. Several attempts to assign a role to component A2 are reported.
Copy mutant of mini-Rts1: lowered binding affinity of mutated RepA protein to direct repeats
Authors: Terawaki Y , Itoh Y
Published: January 2020

Nucleotide sequence analysis of mini-Rts1 and its copy mutant disclosed the presence of two clusters of direct-repeat sequences flanking the coding region for the 33,000-dalton RepA protein and two base substitutions on the mini-Rts1cop1 genome (Kamio et al., J. Bacteriol. 158:307-312, 1984). On subcloning of the left cluster (incI) that is located downstream from repA, the five 24-base-pair repeats expressed a stronger incompatibility toward mini-Rts1 than did the four repeats. The right cluster (incII) that contains three 21-base-pair repeats also exhibited strong incompatibility toward mini-Rts1. By separating the two base substitutions of mini-Rts1cop1, the mutation that is responsible for the copy increase was determined to be a single base change in the RepA coding region. Both clusters of the repeats, cloned separately into the vector plasmid, showed a weaker incompatibility toward mini-Rts1cop1 than to the wild-type mini-Rts1. These findings suggest a lowered binding affinity of the mutated RepA protein to the direct repeats.
Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication
Authors: Chikami G K , Guiney D G , Schmidhauser T J , Helinski D R
Published: January 2020

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.
Frequency among Enterobacteriaceae of the DNA sequences encoding type 1 pili
Authors: Buchanan K , Falkow S , Hull R A , Hull S I
Published: January 2020

Type 1 pili, characterized by mannose-inhibitable agglutination of fowl or guinea pig erythrocytes, have been found throughout the family Enterobacteriaceae. A radiolabeled probe was prepared from a restriction endonuclease-digested fragment of the Escherichia coli pil operon and used to detect homologous DNA sequences in 236 bacteria representing 11 genera of Enterobacteriaceae. Only isolates identified as E. coli or Shigella spp. exhibited homology. In contrast, mannose-sensitive hemagglutination was observed in nine genera. Probe DNA did not hybridize to plasmid DNA, indicating a chromosomal location for the pil operon. Analysis of restriction nuclease-digested whole-cell DNA from 60 E. coli and two Shigella sp. isolates indicated that internal sequences were conserved in most strains, but that changes in flanking sequences in the chromosome were common.
Mutations that define the promoter of ompF, a gene specifying a major outer membrane porin protein
Authors: Taylor R K , Garrett S , Sodergren E , Silhavy T J
Published: January 2020

Expression of the major porin structural genes, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ. To help define the sites required for the expression of both porin genes, we have used a novel selection to identify mutations that decrease ompF transcription. From our assignment of the mRNA start site by the primer extension method, these mutations appear to delineate poorly conserved -10 and -35 consensus promoter regions. In addition, one mutation provides the first genetic evidence that an A residue at position -45 may be important for RNA polymerase recognition.
Structural and functional analyses of the fosfomycin resistance transposon Tn2921
Authors: Navas J , García-Lobo J M , León J , Ortíz J M
Published: January 2020

The fosfomycin resistance transposon Tn2921 is flanked by directly repeated sequences homologous to the Tn10-related insertion sequence IS10. The nonrepeated DNA sequences of Tn2921 can be deleted without affecting the transposition ability of the element, showing that at least one of the direct repeats is an active insertion sequence. Transposition of Tn2921 seems to occur through direct transposition, since cointegrates have not been observed. The evolutionary relatedness of Tn2921 and IS10 is discussed.
Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella sp
Authors: Kato N , Ohta M , Kido N , Ito H , Naito S , Kuno T
Published: January 2020

We extracted an R-form lipopolysaccharide (LPS) by the phenol-water method from Klebsiella sp. strain LEN-111 (O3-:KI-) and followed the changes in ultrastructure of the LPS during the extraction procedure. When the LPS was obtained from the water phase of an extract by addition of 2 volumes of 10 mM MgCI2-ethanol, it consisted of membrane pieces with a hexagonal lattice structure with a lattice constant of 14 to 15 nm. The lattice structure of the LPS was disrupted into short rods with sodium dodecyl sulfate, but the same hexagonal lattice structure was again formed by precipitation with 2 volumes of 10 mM MgCI2-ethanol. The LPS preparation after two cycles of treatment by the phenol-water method, which contained no detectable amounts of proteins, kept an unaltered ability to form the hexagonal lattice structure. Extensive treatment with pronase and extraction with chloroform did not impair the ability of the LPS preparation to form the lattice structure. When the other salts, NaCI, CaCI2 or Zn(CH3COO)2, were used for precipitation of the LPS with ethanol in place of MgCI2, the LPS did not form the hexagonal lattice structure. However, if the LPS precipitated with NaCI-ethanol was converted to the magnesium salt form after it was electrodialyzed, it formed the same hexagonal lattice structure as the LPS precipitated with MgCI2-ethanol. From these results, it was concluded that the R-form LPS has the ability of in vitro self-assembly into a hexagonal lattice structure in the presence of Mg2+ without the help of other components such as proteins and free lipids from outer membrane.
Regulation of the SOS response analyzed by RecA protein amplification
Authors: Calsou P , Salles B
Published: January 2020

A split UV light dose procedure was used in Escherichia coli to induce an SOS function, RecA protein amplification, which was measured by an immunoradiometric assay. The SOS system was partially induced after the first UV irradiation, and the inducing effects of subsequent identical UV doses were quantified. Variations in the inducing effects of successive UV doses were related to modulations of the SOS signal level during SOS induction. A reduction in the level of SOS signal was found after 20 min in the wild-type strain, hypothesized to result from negative control of repair functions. A few DNA repair mutants were tested by the same procedure; the uvrA, recF, and umuC genes were involved in SOS induction control, but we found differences in their respective kinetics of expression. On the contrary, in a recB mutant, only a slight effect was obtained on this control.
Amplification of a chromosomal region in Bacillus subtilis
Authors: Albertini A M , Galizzi A
Published: January 2020

We report on the amplification in Bacillus subtilis of a defined DNA sequence after exposure of the bacteria to increasing levels of antibiotic. The experimental system consisted of transformation of competent cells with a plasmid (pRHA39) unable to replicate in the host and carrying the alpha-amylase gene derived from B. subtilis. Selection of transformants resistant to 5 micrograms of chloramphenicol per ml resulted in the isolation of strains with the plasmid integrated into the chromosome at the site of homology, by a Campbell type mechanism. Starting from such a nontandem duplication, amplification was achieved by growing the bacteria in increasing concentrations of chloramphenicol. By dilution, Southern blotting, and hybridization to a radioactive probe, we estimated a copy number of about 10 for the amplified sequence of samples grown in the presence of 50 micrograms of chloramphenicol per ml. No free plasmid could be detected in the amplified strains. The extent of the amplified region was the same for all transformants, and the endpoints appeared to be the same in all isolates. As a consequence of the amplification, there was a noticeable increase in amylase production, and the amount of enzyme produced correlated with gene dosage. The amplification did not occur in a recE genetic background.
Ultrastructural localization of dipicolinic acid in dormant spores of Bacillus subtilis by immunoelectron microscopy with colloidal gold particles
Authors: Kozuka S , Yasuda Y , Tochikubo K
Published: January 2020

The localization of dipicolinic acid in dormant spores of Bacillus subtilis was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were distributed mainly in the core regions of dormant spores and were not observed in those of germinated or autoclaved spores. This result clearly demonstrates that dipicolinic acid is localized in the cores of dormant spores.
SAL-TOL in vivo recombinant plasmid pKF439
Authors: Furukawa K , Miyazaki T , Tomizuka N
Published: January 2020

SAL-TOL in vivo recombinant plasmid pKF439 was characterized in a strain from a mixed culture of bacteria harboring various degradative plasmids. Analysis of the gene organization of pKF439 revealed that the 57-kilobase TOL fragment, including the 40-kilobase TOL metabolic region, was inserted into the complete SAL replicon at the position of SmaI-C within XhoI-B of SAL. The molecular size of pKF439 was calculated to be 138 kilobases. pKF439 could be transferred to Pseudomonas putida and Pseudomonas aeruginosa at high frequency, and the transconjugants gained the ability to grow with m-xylene, m-toluate, and salicylate as the sole carbon source.
Mutations that convert the four leucine codons of the Salmonella typhimurium leu leader to four threonine codons
Authors: Carter P W , Weiss D L , Weith H L , Calvo J M
Published: January 2020

In Salmonella typhimurium, expression of the leucine operon is regulated by a transcription attenuation mechanism. According to a current model of attenuation, elevated expression of this operon requires that a ribosome stall at one of four adjacent codons for leucine on a leader RNA. We used oligonucleotide-directed mutagenesis to convert the four leucine codons of the S. typhimurium leu leader to four threonine codons. Analysis of the resulting mutant operon showed that almost all regulation by leucine had been abolished. The mutant operon was, instead, partially derepressed by a limitation for charged threonine tRNA. These results provide direct evidence for the function for the four leucine codons postulated by the attenuator model. An unexpected observation made during these studies was that the wild-type leu operon was partially derepressed by starvation for threonine.
Mitomycin-induced lethality of Escherichia coli cells containing the ColE1 Plasmid: involvement of the kil gene
Authors: Zhang S P , Faro A , Zubay G
Published: January 2020

Escherichia coli cells containing the ColE1 plasmid or related plasmids are killed by considerably lower levels of mitomycin C (MTC) than are plasmid-free cells. Since exposure to MTC induces high levels of synthesis of the plasmid-encoded colicin toxin, it was originally thought that the killing effect was due to the increased levels of colicin. This possibility was discounted when it was shown that deletion mutations in the plasmid lacking most of the colicin (cea) gene still sensitized host cells to MTC. Only when the region containing the cea gene promoter was deleted did the killing effect disappear. This led to the suggestion that transcription originating from the cea gene promoter and not the colicin protein itself was required for killing. Transcription-blocking mutations in the cea gene support this suggestion. It was proposed that there is a gene (kil) located downstream from the cea gene in the same operon that is responsible for MTC killing and colicin transport. The precise location of the kil gene in ColE1 can be predicted by piecing together published sequence information. We used available sequence data to construct a number of well-defined plasmid mutants to further examine the relevance of transcription from the cea promoter and the kil gene to drug-induced killing and colicin transport. The most informative mutant had a small insertion in the kil gene. This mutant behaved as predicted; cells containing it had a greatly lowered sensitivity to MTC and were severely inhibited in the transport of colicin.
Effect of growth conditions on peptidoglycan content and cytoplasmic steps of its biosynthesis in Escherichia coli
Authors: Mengin-Lecreulx D , van Heijenoort J
Published: January 2020

In an attempt to bring some insight into how peptidoglycan synthesis is controlled in Escherichia coli, simple parameters, such as cell peptidoglycan content, the pool levels of its seven uridine nucleotide precursors, and the specific activities of five enzymes involved in their formation, were investigated under different growth conditions. When exponential-phase cells with generation times ranging from 25 to 190 min were examined, the peptidoglycan content apparently varied as the cell surface area changed, and no important variations in the pool levels of the nucleotide precursors or in the specific activities of the five enzymes considered were observed. The peptidoglycan of exponential-phase cells accounted for 0.7 to 0.8% of the dry cell weight, whereas that of stationary-phase cells accounted for 1.4 to 1.9%. Depending on the growth conditions, the number of peptidoglycan disaccharide peptide units per cell varied from 2.4 X 10(6) to 5.6 X 10(6). The levels of the nucleotide precursor pools as well as the specific activities of the D-glutamic acid- and D-alanyl-D-alanine-adding enzymes varied little with the growth phase. The specific activities of UDP-N-acetylglucosamine transferase, UDP-N-acetylglucosamine-enolpyruvate reductase, and the diaminopimelic acid-adding enzymes decreased by 20 to 50% at most in the late stationary phase. The results are discussed in terms of the possible importance for cell survival of the maintenance of a high capacity for peptidoglycan synthesis, whatever its rate under various growth conditions, and of a balance between the synthesis and breakdown of peptidoglycan during active growth.
Recycling of murein by Escherichia coli
Authors: Goodell E W
Published: January 2020

The tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid [A2pm]), tetrapeptide (L-Ala-D-Glu-A2pm-D-Ala), and dipeptide (A2pm-D-Ala) which are shed by Escherichia coli from the murein sacculus were found to be reused by the cells to synthesize murein. The tripeptide was used directly, without degradation, to form UDP-N-acetylmuramyl-L-Ala-D-Glu-A2pm. The tetrapeptide lost its carboxy-terminal D-Ala, apparently in the periplasm, before being used. The dipeptide was degraded to D-Ala and A2pm before uptake.
Characterization of the Salmonella typhimurium mgl operon and its gene products
Authors: Müller N , Heine H G , Boos W
Published: January 2020

In Salmonella typhimurium and Escherichia coli the high-affinity galactose transport system, which contains a periplasmic galactose-binding protein as an essential component, is encoded by the mgl genes. The entire mgl region of S. typhimurium is contained on a 6.3-kilobase EcoRI restriction fragment, which has been cloned into plasmid vectors. We determined the extent of the mgl region on this fragment by Tn5 mutagenesis, examination of lacZ fusions to mgl genes, and subcloning smaller restriction fragments. Polyacrylamide gel electrophoresis of protein preparations derived from strains carrying different plasmids was used to identify the mgl gene products. We conclude that the mgl operon consists of four genes that form a single transcription unit: mglB, mglA, mglE, and mglC. The mglB gene codes for galactose-binding protein (33,000 daltons), mglA codes for a membrane-bound protein of 51,000 daltons, and mglC codes for a 29,000-dalton membrane protein. The mglE product was less well characterized. Its existence was inferred from a mglE-lacZ protein fusion located between mglA and mglC. In addition, the coupled transcription-translation in vitro system indicated that mglE codes for a 21,000-dalton protein.
Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO
Authors: Kokjohn T A , Miller R V
Published: January 2020

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.
Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40
Authors: Woolfolk C A
Published: January 2020

The isolation of a xanthine dehydrogenase from Pseudomonas putida 40 which utilizes ferricyanide as an electron acceptor at high efficiency is presented. The new activity is separate from the NAD+ and oxygen-utilizing activities of the same organism but displays a broad pattern for reducing substrates typical of those of previously studied xanthine-oxidizing enzymes. Unlike the previously studied enzymes, the new enzyme appears to lack flavin but possess heme and is resistant to cyanide treatment. However, sensitivity of the purified enzyme to methanol and the selective elimination of the activity when tungstate is added to certain growth media suggest a role for molybdenum. The enzyme is subject to a selective proteolytic action during processing which is not accompanied by denaturation or loss of activity and which is minimized by the continuous exposure of the activity to EDTA and phenylmethylsulfonyl fluoride. Electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate suggests that the enzyme is constructed of subunits with a molecular weight of approximately 72,000. Electrophoresis under native conditions of a purified enzyme previously exposed to magnesium ion reveals a series of major and minor activity bands which display some selectivity toward both electron donors and acceptors. An analysis of the effect of gel concentration on this pattern suggests that the enzyme forms a series of charge and size isomers with a pair of trimeric forms predominating. Comparison of the rate of sedimentation of the enzyme in sucrose gradients with its elution profile from standardized Sepharose 6B columns suggests a molecular weight of 255,000 for the major form of the native enzyme.
Mutations that affect utilization of a promoter in stationary-phase Bacillus subtilis
Authors: Ray C , Hay R E , Carter H L , Moran C P
Published: January 2020

Transcription of the ctc gene in Bacillus subtilis is activated only after exponentially growing cells enter stationary phase. The promoter of the ctc gene is utilized in vitro by two minor forms of RNA polymerase, E sigma 37 and E sigma 32, but not by the most abundant form of RNA polymerase, E sigma 55. We have used the ctc promoter to direct transcription of the xylE gene on plasmid pLC4 and observed that xylE was expressed only in stationary-phase B. subtilis. We also have constructed a series of homologous plasmids that differ only by specific base substitutions in the ctc promoter. We observed that the base substitutions that affected utilization of the ctc promoter in vivo (xylE expression) were the same as those that we had previously shown to affect utilization of the promoter in vitro by E sigma 37 and E sigma 32. We conclude that it is likely that the ctc promoter is utilized in vivo by E sigma 37 or E sigma 32.
Genetic location of genes encoding enterobacterial common antigen
Authors: Meier U , Mayer H
Published: January 2020

A new rff mutation (rff-726) of Escherichia coli is described which affects the biosynthesis of the enterobacterial common antigen. This mutation was detected in an rfe-defective strain. A Tn10 insertion near the rfe locus was isolated to facilitate further mapping. Both mutations rfe and rff were mapped by transduction with bacteriophage P1, giving the gene order ilv rfe rff uvrD metE. The F' factor F14 was able to complement both mutations rfe and rff, whereas the F' factor F16 could complement the rfe but not the rff mutation. The rff mutation did not affect the biosynthesis of N-acetyl-D-mannosaminuronic acid, as the previously described rff mutations in Salmonella typhimurium do (H. C. Lew, H. Nikaido, and P. H. Mäkelä, J. Bacteriol. 136:227-233, 1978), and also did not affect the biosynthesis of other enterobacterial common antigen components; however, the biosynthesis of the complete enterobacterial common antigen molecule was blocked.
Localization of the enantiozymes of 6-hydroxy-nicotine oxidase in Arthrobacter oxidans by electron immunochemistry
Authors: Swafford J R , Reeves H C , Brandsch R
Published: January 2020

During the course of growth of Arthrobacter oxidans, induction of the enantiozymes 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase occurred in the presence of DL-nicotine. Cryoultramicrotomed sections obtained from cells grown to stationary phase were gold immunolabeled. The results obtained demonstrate that both enzymes are localized in the cytoplasm.
Uptake of intact nucleoside monophosphates by Bdellovibrio bacteriovorus 109J
Authors: Ruby E G , McCabe J B , Barke J I
Published: January 2020

The degraded nucleic acids and ribosomes of its prey cell provide Bdellovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 microM in attack phase cells and 35 microM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated.
Suicide vector for transposon mutagenesis in Pseudomonas solanacearum
Authors: Morales V M , Sequeira L
Published: January 2020

A suicide vector was constructed by cloning the transfer genes of the wide-host-range (IncW group) plasmid R388 into the BamHI site of pBR325. This plasmid can deliver Tn5 into Pseudomonas solanacearum at frequencies ranging from 10(-6) to 10(-9) per recipient.
Regions on plasmid pCU1 required for the killing of Klebsiella pneumoniae
Authors: Thatte V , Gill S , Iyer V N
Published: January 2020

Plasmid pCU1 was Kik+ (promotes killing of Klebsiella pneumoniae). All Tn5 insertions within the tra region of pCU1 were Kik-. Two other regions, kikA and kikB, were needed. They may be separated on different plasmids, but both must be mobilized into Klebsiella pneumoniae. Establishment of one kik region in K. pneumoniae followed by receipt of the second did not lead to killing. Kik was therefore intracellular and required concerted and transient action of both regions.
Nitric oxide-dependent proton translocation in various denitrifiers
Authors: Shapleigh J P , Payne W J
Published: January 2020

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.
Effects of the ccd function of the F plasmid on bacterial growth
Authors: Jaffé A , Ogura T , Hiraga S
Published: January 2020

The ccd segment of the mini F plasmid containing the ccdA and ccdB genes controls the coordination between plasmid proliferation and cell physiology and fate. When the DNA replication of a thermosensitive-replication plasmid carrying the ccd segment of mini F is blocked, plasmid DNA molecules are progressively diluted through cell division until the copy number reaches 1 per cell. From this time on, there is little increase in the number of viable cells, although cells continue to divide, resulting in a mixed population of viable cells (mostly plasmid containing), nonviable but residually dividing cells, and nonviable nondividing cells. Results are presented suggesting that plasmid-containing cells are viable and continue to divide, whereas plasmid-free segregants are nonviable and form filaments after a few residual divisions, with DNA synthesis reduced or arrested in the filaments. Although the ccd functions are known to induce the SOS response when plasmid replication is blocked, the production of nonviable plasmid-free segregants is independent of the SOS cell division inhibition mechanism determined by the sfiA and sfiC genes.
Influence of pH, O2, and temperature on the absorption properties of the secondary light-harvesting antenna in members of the family Rhodospirillaceae
Authors: Uffen R L
Published: January 2020

In some Rhodospirillaceae, the primary light-harvesting (LH I) antenna absorbs near-infrared light around 870 nm, whereas LH II (holochrome B800-860) has a major absorption band between 850 and 860 nm (B860) and a minor absorbancy around 800 nm (B800). Results show that, unlike LH I, holochrome B800-860 (LH II) exhibits unstable light absorption properties in whole cells. This was observed in Rhodopseudomonas capsulata grown anaerobically in light in weakly buffered carbohydrate medium; cultures lost both carotenoid-dependent brown-yellow pigmentation and LH II absorbancy. The whole cell spectrophotometric changes were attributed to mild acid conditions generated during sugar metabolism. LH II absorbancy was also destroyed in both R. capsulata and Rhodopseudomonas gelatinosa when cultures growing at neutral pH were acidified to a pH value around 5.0 with HCl. In contrast, during the same time period of exposure to pH 5.0, only a 50% decrease in Rhodopseudomonas sphaeroides LH II B800 absorbancy was measured. At neutral pH, LH II absorbancy in suspensions of nongrowing Rhodopseudomonas spp. was also sensitive to O2 exposure and to incubation at 30 to 40 degrees C. During treatment with O2, the rate of LH II B800 absorption decrease in R. gelatinosa and R. sphaeroides was 60 and 40% per h, respectively, compared with their absorbancy maximum around 860 nm. Both 860-nm absorbancy and the total bacteriochlorophyll content of the cells remained unchanged. On the other hand, no significant decrease in B800 if LH II in R. capsulata occurred during O2 exposure, but a 20% absorption decay rate per h of B800 was observed in cells incubated anaerobically at 40 degrees C. These B800 LH II spectral changes Rhodopseudomonas spp. were prevented by maintaining cells at neutral pH and at 10 degrees C. The near-infrared absorption spectrum of Rhodospirillum rubrum, which does not form LH II, was not significantly influenced by these different pH, aerobic, or temperature conditions.
Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control
Authors: Wu R P , Womble D D , Rownd R H
Published: January 2020

DNA from the replication control region of plasmid NR1 or of the Inc- copy mutant pRR12 was cloned into a pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia coli cells that harbored either NR1 or pRR12. After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further. By this process, 134 plasmids with Inc- mutations in the cloned NR1 or pRR12 DNA were obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertions in the NR1 DNA and were not examined further. Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NR1-derived replicon (Rep+ or Rep-), (ii) the copy numbers of the Rep+ reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.
Comparison of the regulatory regions of ilvGEDA operons from several enteric organisms
Authors: Harms E , Hsu J H , Subrahmanyam C S , Umbarger H E
Published: January 2020

The nucleotide sequence preceding the ilvGEDA operon has been examined and compared in five enteric organisms. The sequence in Escherichia coli B was identical to the earlier-described strain K-12 sequence. The sequences of Salmonella typhimurium and Klebsiella aerogenes were remarkably similar to that of E. coli and identical in that part of the leader region that specified the putative 32-amino-acid peptide. Thus, identical secondary structures could be postulated for the leaders of all three organisms, and regulation of operon expression could be like that postulated earlier for E. coli. Different secondary structures had to be postulated for the leader transcripts of Edwardsiella tarda and Serratia marcescens. Control of attenuation of the operon in these organisms by the level of leucyl tRNA could be explained only if ribosome stalling occurred at a single leucine codon. In both organisms, that single leucine codon is the rarely used CUA rather than the CUG that is in E. coli, S. typhimurium, and K. aerogenes.
Two modes of control of pilA, the gene encoding type 1 pilin in Escherichia coli
Authors: Orndorff P E , Spears P A , Schauer D , Falkow S
Published: January 2020

Type 1 piliation in Escherichia coli is subject to metastable regulation at the transcriptional level (B. I. Eisenstein, Science 214:337-339, 1981). However, the genes controlling in this fashion are not known. We present evidence that the pilA gene, encoding the structural subunit of type 1 pili, is subject to metastable transcriptional regulation. A pilA'-lacZ fusion, constructed in vitro on a recombinant plasmid, was used in conjunction with a recBC sbcB mutant of E. coli K-12 to introduce the fusion into the chromosomal region encoding Pil. This fusion was found to be subject to metastable transcriptional control. The rate of switching from the Lac+ to the Lac- phenotype was 4 X 10(-4) per cell per generation and 6.2 X 10(-4) in the opposite direction. A ca. 10-fold difference in beta-galactosidase activity was observed between phenotypically "ON" (Lac+) and "OFF" (Lac-) populations. P1 transduction experiments showed that the element determining the ON or OFF phenotype was tightly linked to pilA. In addition to the metastable regulation of pilA, a second type of transcriptional regulation was effected by the product of a gene, hyp, adjacent to pilA. By using a recombinant plasmid containing just a pilA'-lacZ fusion and the putative pilA promoter, we found that a lesion in hyp conferred a beta-galactosidase activity about fivefold higher than that of a strain possessing the parental hyp gene. Mutants constructed to have a pilA'-lacZ fusion and a hyp::Tn5-132 mutation in the chromosome exhibited a frequency of switching from Lac+ to Lac- and vice versa indistinguishable from that of the parental strain. However, in the ON mode, hyp::Tn5-132 mutants showed a twofold-higher beta-galactosidase activity. Thus, hyp does not appear to affect metastable variation but does affect the level of transcription of the pilA gene in the ON (transcribed) mode.
Genetic analysis of erythromycin production in Streptomyces erythreus
Authors: Weber J M , Wierman C K , Hutchinson C R
Published: January 2020

Streptomyces erythreus produces the 14-membered macrolide antibiotic erythromycin A. The properties of erythromycin A nonproducing mutants and their genetic linkage to chromosomal markers were used to establish the rudiments of genetic organization of antibiotic production. Thirty-three Ery- mutants, produced by mutagenesis of S. erythreus NRRL 2338 and affecting the formation of the macrolactone and deoxysugar intermediates of erythromycin A biosynthesis, were classified into four phenotypically different groups based on their cosynthesis behavior, the type of biosynthetic intermediate accumulated, and their ability to biotransform known biochemical intermediates of erythromycin A. Demonstration of the occurrence of natural genetic recombination during conjugal mating in S. erythreus enabled comparison of the genetic linkage relationships of three different ery mutations with seven other markers on a simple chromosome map. This established a chromosomal location for the ery mutations, which appear to be located in at least two positions within one interval of the map.
Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations
Authors: Johnson E A , Levine R L , Lin E C
Published: January 2020

Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.
Cell length in a wee dnaA mutant of Escherichia coli
Authors: de la Campa A G , Tormo A , Martínez-Salas E , Aldea M , Vicente M
Published: January 2020

The cell length of the short siblings of dividing pairs formed in the absence of replication by two strains of Escherichia coli, OV-25-9 [dnaA46 wee(Am)] and OV-25-10 [dnaA46 wee(AM) supF] was measured. In the presence of Wee, the length of these cells increased to those values expected for newborn wild-type cells growing under similar conditions. In its absence, cell length remained at values near the minimum unit length possible for newborn cells. Our results show that both cell elongation and the action of Wee are independent of DNA replication, being compatible with the role proposed for Wee in coordination between cell elongation and division.
Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi
Authors: Roeder D L , Collmer A
Published: January 2020

The phytopathogenic enterobacterium Erwinia chrysanthemi contains pel genes encoding several different isozymes of the plant-tissue-disintegrating enzyme pectate lyase (PL). The pelC gene, encoding an isozyme with an approximate isoelectric point of 8.0, was mutagenized by a three-step procedure involving (i) insertional inactivation of the cloned gene by ligation of a kan-containing BamHI fragment from pUC4K with a partial Sau3A digest of E. chrysanthemi pelC DNA in pBR322; (ii) mobilization of the pBR322 derivative from Escherichia coli to E. chrysanthemi by the helper plasmids R64drd11 and pLVC9; and (iii) exchange recombination of the pelC::kan mutation into the E. chrysanthemi chromosome by selection for kanamycin resistance in transconjugants cultured in phosphate-limited medium (which renders pBR322 unstable). The resulting E. chrysanthemi mutant was Kanr Amps, lacked pBR322 sequences, and was deficient in only one of the four major PL isozymes, PLc, as determined by activity-stained isoelectric-focusing polyacrylamide gels. The rates of PL induction and cell growth in a medium containing polygalacturonic acid as the sole carbon source were not significantly reduced in the mutant. No difference was detected in the ability of the mutant to macerate potato tuber tissue. The evidence suggests that this isozyme is not necessary for soft-rot pathogenesis.
Methylation of macromolecules during development in Myxococcus xanthus
Authors: Panasenko S M
Published: January 2020

Covalent modification of macromolecules can serve to alter their biological activities and is therefore frequently involved in regulation. I examined methylation of proteins and carbohydrates during development and vegetative growth in the procaryote Myxococcus xanthus. Striking differences in the patterns of protein methylation occurred when cell development was induced by nutrient deprivation on solid media and when cells were starved in liquid. In addition, a methylated, protease-resistant macromolecule which contained carbohydrate and which may have been an unusual type of lipopolysaccharide was observed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of methylation patterns in various media and an analysis of the time course of methylation indicated that changes in methylation were part of the developmental pathway which includes aggregation. Induction of development in liquid by glycerol produced no changes in methylation.
Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron
Authors: Guthrie E P , Shoemaker N B , Salyers A A
Published: January 2020

We cloned the gene for one of the two chondroitin lyases of Bacteroides thetaiotaomicron into the cosmid vector pHC79 and subcloned it into pBR328. No proteins the size of B. thetaiotaomicron chondroitin lyase I or II (104 to 108 kilodaltons) were detectable in maxicell or in vitro transcription-translation preparations. However, partial purification of the chondroitin lyase activity from the Escherichia coli subclone showed that its properties were similar to those of the B. thetaiotaomicron chondroitin lyases. Antibodies to the chondroitin lyase that was produced in E. coli cross-reacted with the B. thetaiotaomicron chondroitin lyase II but not with chondroitin lyase I. The molecular weight of the enzyme produced in E. coli, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration, was slightly lower than those of the two chondroitin lyases from B. thetaiotaomicron; the enzyme had a higher affinity for bacterial membranes and for heparin-agarose, and cyanogen bromide digestion products of the chondroitin lyase produced in E. coli differed slightly from those of B. thetaiotaomicron chondroitin lyase II. gamma delta mutagenesis was used to locate the chondroitin lyase gene on the subcloned 7.8-kilobase EcoRI fragment. The size of the gene was approximately 3.3 kilobases, as expected for a protein with a molecular weight of 104,000.
Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae
Authors: Willsky G R , Leung J O , Offermann P V , Plotnick E K , Dosch S F
Published: January 2020

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.
Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force
Authors: Murén E M , Randall L L
Published: January 2020

The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with K+, a precursor form of alpha-amylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration.
araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora
Authors: Lei S P , Lin H C , Heffernan L , Wilcox G
Published: January 2020

The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.