bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79(Hly+), RV79(Hly-), and 569B
Authors: Goldberg S L , Murphy J R
Published: January 2020

The Hly region from the chromosome of Vibrio cholerae El Tor strain RV79(Hly-) and the nonhemolytic classical strain 569B were cloned into plasmid vector pBR322. Escherichia coli K-12 transformants possessing these recombinant plasmids were nonhemolytic and were detected with a 32P-labeled hly-specific DNA probe. Restriction endonuclease Sau3AI digestions of the cloned hly loci of two independently obtained RV79(Hly+) convertants, when compared with the digests of cloned RV79(Hly-) loci, revealed that an apparent alteration (10 to 15 base pairs) had occurred. In contrast, an apparent 20-base-pair deletion was present in the cloned hly locus of the classical biotype V. cholerae strain 569B. Maxicell analysis and immunoprecipitation of labeled proteins of E. coli which are encoded by the cloned hly loci of RV79(Hly+) and from nuclease BAL 31-deleted plasmids, as well as immunoprecipitation of [35S]methionine-labeled V. cholerae proteins, suggest that the hemolysin is an 84,000-dalton polypeptide.
Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli
Authors: Sankar P , Lee J H , Shanmugam K T
Published: January 2020

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.
Genetic transfer in Halobacterium volcanii
Authors: Mevarech M , Werczberger R
Published: January 2020

Auxotrophic mutants of Halobacterium volcanii generated by chemical mutagenesis were used to demonstrate a native genetic transfer system in this extremely halophilic member of the class Archaeobacteria.
Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC
Authors: Jacobs T W , Egelhoff T T , Long S R
Published: January 2020

Infection of alfalfa by the soil bacterium Rhizobium meliloti proceeds by deformation of root hairs and bacterial invasion of host tissue by way of an infection thread. We studied an 8.7-kilobase (kb) segment of the R. meliloti megaplasmid, which contains genes required for infection. Site-directed Tn5 mutagenesis was used to examine this fragment for nodulation genes. A total of 81 R. meliloti strains with mapped Tn5 insertions in the 8.7-kb fragment were evaluated for nodulation phenotype on alfalfa plants; 39 of the insertions defined a 3.5-kb segment containing nodulation functions. Of these 39 mutants, 37 were completely nodulation deficient (Nod-), and 2 at the extreme nif-distal end were leaky Nod-. Complementation analysis was performed by inoculating plants with strains carrying a genomic Tn5 at one location and a plasmid-borne Tn5 at another location in the 3.5-kb nodulation segment. Mutations near the right border of the fragment behaved as two distinct complementation groups. The segment in which these mutations are located was analyzed by DNA sequencing. Several open reading frames were found in this region, but the one most likely to function is 1,206 bases long, reading from left to right (nif distal to proximal) and spanning both mutation groups. The genetic behavior of this segment may be due either to the gene product having two functional domains or to a recombinational hot spot between the apparent complementation groups.
Identification of molybdoproteins in Clostridium pasteurianum
Authors: Hinton S M , Mortenson L E
Published: January 2020

Cells of Clostridium pasteurianum whose N source is switched from NH3 to N2 accumulate large amounts of molybdenum beginning 1.5 h before the detection of nitrogenase activity. Anaerobic multiphasic gel electrophoresis and anion-exchange chromatography were used to identify the molybdoproteins and molybdenum-containing components present in N2-fixing cells. In addition to molybdate, six distinct 99Mo-labeled species were detected, i.e., a membrane fragment, the MoFe protein of nitrogenase, formate dehydrogenase, a Mo "binding-storage" protein, a 30-kilodalton molybdoprotein, and a low-molecular-weight molybdenum species. Of these, the MoFe protein, formate dehydrogenase, and the Mo binding-storage protein were present in more than one zone because of complex formation with other proteins, partial denaturation, and variation in the amount of Mo bound to the protein, respectively. In addition to the six proteins, a soluble "free" Mo cofactor in the cytosol was detected by showing that it reconstituted nitrate reductase activity in crude extracts of the Neurospora crassa mutant nit-1.
Secretion of staphylococcal nuclease by Bacillus subtilis
Authors: Kovacevic S , Veal L E , Hsiung H M , Miller J R
Published: January 2020

The staphylococcal nuclease (nuc) gene from Staphylococcus aureus has been cloned and expressed in Bacillus subtilis. The nuclease protein was expressed either from its own promoter and translation start signals, or from a combination of a B. subtilis promoter, ribosome binding site, and a signal peptide sequence. Greater than 80% of the active gene product was secreted into the medium, whereas, when a signal peptide sequence was absent, as little as 4% of the nuclease activity was found in the culture medium. Intracellular (or cell-bound) nuclease, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, was shown to have the molecular weight of the predicted precursor protein with the signal peptide. Levels of nuclease reached 50 mg per liter in the culture medium, depending on the growth medium and the strain used. These findings indicate the prospective use of nuclease as a model system for studying secretion of heterologous proteins in B. subtilis.
Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene
Authors: Lee C H , Hu S T , Swiatek P J , Moseley S L , Allen S D , So M
Published: January 2020

The Escherichia coli heat-stable enterotoxin STII gene in P307 is flanked by inverted repeat sequences, suggesting that the STII gene is part of a transposon. To study the transposability, a DNA fragment containing the putative STII transposon has been cloned. Results of transposition assays indicated that the STII gene can transpose from one plasmid to another. The size of the transposon has been determined to be approximately 9 kilobases. The structure and the location of the STII gene in clinical isolates of Escherichia coli have been investigated by restriction enzyme analyses. The structural genes of STII from different clinical isolates appear to be uniform in size, but the flanking sequences are heterogeneous. This result suggests that the STII genes in different isolates are not on the same transposon as observed in P307.
Enzymatic release of halogens or methanol from some substituted protocatechuic acids
Authors: Kersten P J , Chapman P J , Dagley S
Published: January 2020

Four strains of gram-negative bacteria capable of growing at the expense of 5-chlorovanillate were isolated from soil, and the metabolism of one strain was studied in particular detail. In the presence of alpha, alpha'-bipyridyl, a suspension of 5-chlorovanillate-grown cells accumulated 5-chloroprotocatechuate from 5-chlorovanillate; in the absence of inhibitor these compounds, and various other 5-substituted protocatechuates and vanillates, were oxidized to completion. Cell suspensions of this strain grown on 5-chlorovanillate or vanillate released chloride quantitatively from 5-chlorovanillate and released methanol from syringate. Extracts of cells grown with 4-hydroxybenzoate, vanillate, or syringate possessed high levels of both protocatechuate 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate hydrolase; extracts from acetate-grown cells did not. Protocatechuate 4,5-dioxygenase, purified from strains that could grow with 5-chlorovanillate, oxidized 5-halogeno-protocatechuates and 3-O-methylgallate with the formation of 2-pyrone-4,6-dicarboxylate. A crude extract converted 5-chloroprotocatechuate into pyruvate plus oxaloacetate. On the basis of these observations, a meta-fission reaction sequence is proposed for the bacterial degradation of vanillate and protocatechuate substituted at C-5 of the benzene ring with halogen or methoxyl.
Ion selectivity of gram-negative bacterial porins
Authors: Benz R , Schmid A , Hancock R E
Published: January 2020

Twelve different porins from the gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single-channel conductance between 1.5 and 6 nS in 1 M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free solution. Thus the single-channel conductances of the individual porins could be used to estimate the effective channel diameters of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes containing individual porins gave results that were consistent with the conclusions drawn from the single-channel experiments. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aqueous phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examined, including all of the known constitutive porins of the four gram-negative bacteria studied, were cation selective with a 3- to 40-fold preference for K+ ions over Cl- ions.
Unusual stability of the Methanospirillum hungatei sheath
Authors: Beveridge T J , Stewart M , Doyle R J , Sprott G D
Published: January 2020

The proteinaceous sheath of Methanospirillum hungatei was isolated by lysing cells in 50 mM dithiothreitol, separating the sheath from other cellular material by discontinuous sucrose density centrifugation, and removing the "cell spacers" with dilute NaOH. The isolated sheath material consisted of hollow tubes which had a highly ordered surface array. The stability of the sheath to treatment with denaturants and to enzymatic digestion was examined by a turbidimetric assay in conjunction with electron microscopy and optical or electron diffraction. The sheath was resistant to a range of proteases and also was not digested by peptidoglycan-degrading enzymes, a lipase, a cellulase, a glucosidase, or Rhozyme (a mixture of galactosidases, acetylglucosaminidase, acetylgalactosaminidase, fucosidase, and mannosidases). In addition to being unaffected by common salts, thiol-reducing agents, and EDTA, the layer was resistant to powerful denaturants such as 6 M urea, 6 M guanidinium hydrochloride, 10 M LiSCN, cyanogen bromide, sodium periodate, and 1% sodium dodecyl sulfate. Strong bases, boiling 3 N HCl, and performic acid did attack the sheath; in these cases, the array was systematically disassembled in a progressive manner, which was followed by electron microscopy. The layer was slightly modified by N-bromosuccinimide in urea, but the array remained intact. The stability of the sheath was remarkable, not only as compared to other bacterial surface arrays, but also as compared to proteins generally, and possibly indicated the presence of covalent cross-links between protein subunits.
Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli
Authors: Bremer E , Silhavy T J , Weinstock G M
Published: January 2020

We have constructed several derivatives of bacteriophage lambda that translocate by using the transposition machinery of phage Mu (lambda placMu phages). Each phage carries the c end of Mu, containing the Mu cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S end (beta end). These sequences contain the Mu attachment sites, and their orientation allows the lambda genome to be inserted into other chromosomes, resulting in a lambda prophage flanked by the Mu c and S sequences. These phages provide a means to isolate cells containing fusions of the lac operon to other genes in vivo in a single step. In lambda placMu50, the lacZ and lacY genes, lacking a promoter, were located adjacent to the Mu S sequence. Insertion of lambda placMu50 into a gene in the proper orientation created an operon fusion in which lacZ and lacY were expressed from the promoter of the target gene. We also introduced a gene, kan, which confers kanamycin resistance, into lambda placMu50 and lambda placMu1, an analogous phage for constructing lacZ protein fusions (Bremer et al., J. Bacteriol. 158:1084-1093, 1984). The kan gene, located between the cIII and ssb genes of lambda, permitted cells containing insertions of these phages to be selected independently of their Lac phenotype.
Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport
Authors: Laddaga R A , Bessen R , Silver S
Published: January 2020

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.
Cloning and expression in Escherichia coli of sdhA, the structural gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex
Authors: Magnusson K , Hederstedt L , Rutberg L
Published: January 2020

Bacillus subtilis cytochrome b558 is a transmembrane protein which anchors succinate dehydrogenase (SDH) to the cytoplasmic membrane and is reduced by succinate. The structural gene for this cytochrome was cloned and expressed in Escherichia coli. Random BamHI or BglII fragments of B. subtilis 168 DNA were cloned in the BamHI site of plasmid pHV32. The derived plasmids were used to transform B. subtilis SDH mutants to chloramphenicol resistance by integration of the plasmid via DNA homology. Of some 3,000 transformants tested, 6 were SDH positive and had pHV32 integrated close to the sdh operon. Two plasmids, pKIM2 and pKIM4, with an insert of B. subtilis DNA of 5.7 and 3.4 kilobases, respectively, were generated by transforming E. coli with DNA from the SDH-positive transformants after cleavage with EcoRI or BglII and ligation. In E. coli carrying either of the two plasmids, about 4% of total membrane protein was B. subtilis cytochrome b558. E. coli (pKIM2) also contained antigen which reacted with antibodies specific for the flavoprotein and the iron-sulfur protein subunit of B. subtilis SDH. Enzymatically active, membrane-bound B. subtilis SDH could not be demonstrated in E. coli (pKIM2). The B. subtilis DNA insert in pKIM2 could transform B. subtilis sdhA (cytochrome b558), sdhB (flavoprotein), and sdhC (iron-sulfur protein) mutants to the wild type. The results suggest that pKIM2 carries the whole B. subtilis sdh operon. The data confirm the gene order and the proposed direction of transcription of the B. subtilis sdh operon. Most likely the sdh genes in E. coli(pKIM2) are controlled by their natural promoter.
Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli
Authors: Matsuyama S , Mizushima S
Published: January 2020

A method is presented for the construction of a deletion mutant lacking the micF gene, which has been proposed to negatively regulate expression of the ompF gene. The method includes (i) construction of a temperature-sensitive plasmid containing a chromosomal fragment that carries both flanking regions of the micF gene but does not carry micF itself and (ii) replacement of the corresponding chromosomal domain with the fragment. The method is applicable to construction of a deletion mutant for any Escherichia coli chromosomal gene provided that it is dispensable. The micF deletion was confirmed by genetic and biochemical tests, including nucleotide sequence analysis. ompF expression in the micF deletion mutant thus constructed was normally regulated and was not enhanced. When micF was cloned into a high-copy-number plasmid it repressed ompF gene expression, whereas when cloned into a low-copy-number plasmid it did not. From these results, it is concluded that a single copy of the micF gene on the E. coli chromosome does not play a critical role in ompF gene expression.
Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety
Authors: Le Van Q , Keller P J , Bown D H , Floss H G , Bacher A
Published: January 2020

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.
Starvation for ilvB operon leader amino acids other than leucine or valine does not increase acetohydroxy acid synthase activity in Escherichia coli
Authors: Tsui P , Freundlich M
Published: January 2020

Eleven different amino acids are encoded in the ilvB leader mRNA. Starvation for leucine or valine, but not for any of the other nine amino acids, resulted in high levels of acetohydroxy acid synthase I. These results are discussed in terms of a report (C.A. Hauser and G.W. Hatfield, Proc. Natl. Acad. Sci. U.S.A. 81:76-79, 1984) which suggests that threonine and alanine, in addition to leucine and valine, are involved in the regulation of the ilvB operon.
Regulation of nitrogen fixation in Rhodospirillum rubrum grown under dark, fermentative conditions
Authors: Schultz J E , Gotto J W , Weaver P F , Yoch D C
Published: January 2020

Rhodospirillum rubrum was shown to grow fermentatively on fructose with N2 as a nitrogen source. The nitrogenase activity of these cells was regulated by the NH4+ switch-off/switch-on mechanism in a manner identical to that for photosynthetically grown cells. In vitro, the inactive nitrogenase Fe protein from fermenting cells was reactivated by an endogenous membrane-bound, Mn2+-dependent activating enzyme that was interchangeable with the activating enzyme isolated from photosynthetic membranes.
Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation
Authors: Flores E , Wolk C P
Published: January 2020

Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.
Organization and nucleotide sequence analysis of an rRNA and tRNA gene cluster from Caulobacter crescentus
Authors: Feingold J , Bellofatto V , Shapiro L , Amemiya K
Published: January 2020

rRNA genes of Caulobacter crescentus CB13 were isolated and shown to be present in two gene clusters in the genome. The organization of each rRNA gene cluster was found to be 5'-16S-tRNA spacer-23S-5S-3'. The DNA sequence of 40% of the 16S rRNA gene, the entire 16S/23S intergenic spacer region, and portions of the 23S rRNA gene were determined. Analysis of the nucleotide sequence in the 16S-23S intergenic spacer region revealed the presence of tRNAIle and tRNAAla genes. Large invert repeat sequences were found surrounding the 16S rRNA gene. These inverted repeat sequences are analogous to the RNase III-processing sites in the E. coli rRNA precursor. Small invert repeat sequences were also found flanking the individual tRNA genes. RNA polymerase-binding studies with restriction fragments of the rRNA gene cluster revealed three regions which bound enzyme, and these regions were shown to contain transcription initiation sites. One of these sites was located within the 16S gene near its 3' end, and the other two were found at the 5' end of the 23S gene.
Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae
Authors: Lewis T A , Taylor F R , Parks L W
Published: January 2020

Wild-type Saccharomyces cerevisiae do not accumulate exogenous sterols under aerobic conditions, and a mutant allele conferring sterol auxotrophy (erg7) could be isolated only in strains with a heme deficiency. delta-Aminolevulinic acid (ALA) fed to a hem1 (ALA synthetase-) erg7 (2,3-oxidosqualene cyclase-) sterol-auxotrophic strain of S. cerevisiae inhibited sterol uptake, and growth was negatively affected when intracellular sterol was depleted. The inhibition of sterol uptake (and growth of sterol auxotrophs) by ALA was dependent on the ability to synthesize heme from ALA. A procedure was developed which allowed selection of strains which would take up exogenous sterols but had no apparent defect in heme or ergosterol biosynthesis. One of these sterol uptake control mutants possessed an allele which allowed phenotypic expression of sterol auxotrophy in a heme-competent background.
Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene
Authors: Carlson C A , Stewart G J , Ingraham J L
Published: January 2020

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.
Uncoupling of osmoregulation of the Escherichia coli K-12 ompF gene from ompB-dependent transcription
Authors: Ramakrishnan G , Ikenaka K , Inouye M
Published: January 2020

The expression of the genes for the Escherichia coli K-12 outer membrane proteins, ompF and ompC, is subject to osmoregulation and responds to changes in the osmolarity of the growth medium. The transcription of these genes is dependent on the products of the regulatory locus ompB (comprising the genes ompR and envZ). The native promoter of ompF was replaced with an inducible lpp promoter to eliminate this transcriptional dependence of ompF expression on ompB. As a result, it was possible for the OmpF protein to be produced in an ompB mutant strain that does not normally express ompF. Surprisingly, the expression of ompF under the lpp promoter was still osmoregulated not only in the ompB+ strain but also in two ompB strains tested. These results indicate the involvement of a factor(s) besides the ompR and envZ gene products in the osmoregulation of OmpF production. This factor may interact with a sequence downstream of the ompF promoter. In addition, we show that the expression of ompF under the lpp promoter has no direct effect on ompC expression.
Cloning and sequencing of the beta-lactamase I gene of Bacillus cereus 5/B and its expression in Bacillus subtilis
Authors: Wang W , Mézes P S , Yang Y Q , Blacher R W , Lampen J O
Published: January 2020

The beta-lactamases of Bacillus cereus have attracted interest because they are secreted efficiently, because multiple enzymes are frequently present, and because their regulation has unusual features. beta-Lactamase I of strain 5/B is produced constitutively at a high level, and the exoenzyme appears to be several thousand daltons larger than the corresponding product of strain 569/H. We have cloned the gene for 5/B beta-lactamase I in Escherichia coli and B. subtilis and have sequenced the structural portion and the regulatory regions. The 5/B enzyme is produced at a low level in E. coli RR1(pRWY200) and remains cellbound. In B. subtilis it is formed in large amounts, and over 90% of it is released into the medium. There is a large degree of homology between the promoter and leader peptide regions of the 5/B and 569/H genes; both utilize UUG as the translation initiation codon (P. S. F. Mézes, R. W. Blacher, and J. O. Lampen, (J. Biol. Chem. 260:1218-1223, 1985). Although there are significant differences in the peptide segment where processing would be expected to occur, the NH2 terminus of the major 5/B product from B. subtilis BD170(pRWY215) is His-44, which is the same as the NH2 terminus of the major 569/H product from B. subtilis BD170(pRWM5).
Bacteroides intermedius binds fibrinogen
Authors: Lantz M S , Switalski L M , Kornman K S , Höök M
Published: January 2020

The binding of Bacteroides intermedius VPI 8944 to human fibrinogen has been characterized. The binding is time dependent, at least partially reversible, saturable, and specific. On an average, a maximum of 3,500 fibrinogen molecules bind per bacterial cell, with a dissociation constant of 1.7 X 10(-11) M. These bacteria also exhibit a fibrinogenolytic activity which can be partially inhibited by protease inhibitors. Bacteria release fibrinogenolytic activity into the surrounding medium without loss of binding activity, but more pronounced fibrinogen breakdown occurs when 125I-labeled fibrinogen is associated with the bacteria, suggesting that fibrinogen is degraded at the cell surface. Fibrinogen binding by B. intermedius might represent a mechanism of bacterial tissue adherence.
Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans
Authors: Husain M , Steenkamp D J
Published: January 2020

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.
Evidence for two restriction-modification systems in Halobacterium cutirubrum
Authors: Patterson N H , Pauling C
Published: January 2020

Data from plating experiments indicated that Halobacterium cutirubrum NRC34001 has at least two separate restriction-modification systems. A spontaneous or induced loss of one or both systems resulted in four restriction-modification phenotypes. There was a positive correlation between changes in gas vacuolation phenotypes and either restriction-modification system.
Isolation and expression of the Escherichia coli gene encoding malate dehydrogenase
Authors: Sutherland P , McAlister-Henn L
Published: January 2020

An oligodeoxynucleotide specific for a pentapeptide sequence corresponding to amino acid residues 32 through 36 of Escherichia coli malate dehydrogenase was chemically synthesized and used to identify the mdh gene on plasmid pLC32-38 from the Clarke-Carbon recombinant library. Cells transformed with this plasmid exhibited a 10-fold increase in malate dehydrogenase activity. A 1.2-kilobase PvuII fragment which hybridized with the oligodeoxynucleotide probe was subcloned, and the identity of the mdh structural gene was confirmed by partial nucleotide sequence analysis. The expression of the mdh gene, as measured by both Northern blotting and enzyme assays, was found to vary over a 20-fold range with different culture conditions.
Analysis of outer membrane proteins which are associated with growth of Bacteroides thetaiotaomicron on chondroitin sulfate
Authors: Kotarski S F , Linz J , Braun D M , Salyers A A
Published: January 2020

By analyzing outer membrane proteins of Bacteroides thetaiotaomicron on two-dimensional polyacrylamide gels, we were able to identify 10 protein spots that were associated with growth on chondroitin sulfate but not with growth on glucuronic acid or other monosaccharides. These proteins were distinct from the outer membrane polypeptides that were associated with growth on two other negatively charged polysaccharides, polygalacturonic acid and heparin. Of the 10 protein spots that were associated with growth on chondroitin sulfate, 4 could be detected on immunoblots with antiserum that had been raised against outer membranes from bacteria grown on chondroitin sulfate and then cross-adsorbed with membranes from bacteria grown on glucose. Synthesis of these four proteins appeared to be regulated coordinately with synthesis of the two enzymes that degrade chondroitin sulfate, chondroitin lyase I and II. Although one of the four proteins (Mr 110,000) was similar in molecular weight to the chondroitin lyases, the cross-adsorbed antiserum which detected this outer membrane protein did not cross-react with either of these two enzymes.
Identification of the Escherichia coli deoR and cytR gene products
Authors: Singer J T , Barbier C S , Short S A
Published: January 2020

The protein products encoded by the Escherichia coli deoR and cytR structural genes have been identified based on results obtained from E. coli maxicells harboring (i) recombinant plasmids carrying wild-type deoR and cytR genes, (ii) deletion derivatives of the deoR+ and cytR+ plasmids, (iii) plasmids containing site-specific mutations in the deoR and cytR structural genes, and (iv) plasmids which have transposon Tn1000 inserted into the deoR and cytR structural genes. Analysis of the protein profiles obtained from all the maxicell experiments demonstrated that the deoR gene encodes a protein with a subunit molecular weight of 30,500 and that the product of the cytR gene is a protein with a subunit molecular weight of 37,000.
Ferric iron reductase of Rhodopseudomonas sphaeroides
Authors: Moody M D , Dailey H A
Published: January 2020

Ferric iron reductase activity was examined in the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The specific activities of extracts from cells grown under phototrophic and aerobic conditions were similar and not affected by the concentration of iron in the growth media. The activity was resolved by ion-exchange column chromatography into two fractions, designated iron reductase A and iron reductase B, with molecular weights of 41,000 and 32,000, respectively. Both of these soluble cytoplasmic enzymes required the presence of flavin mononucleotide for activity and utilized NADH to reduce iron supplied as ferric citrate. Iron reductase B was responsible for the majority of activity in crude extracts and was purified 556-fold by conventional protein purification techniques. The apparent Km values of iron reductase B for NADH, Fe3+, and flavin mononucleotide were determined to be 18.2, 8.3, and 3.2 microM, respectively.
Enzymology of the beta-ketoadipate pathway in Trichosporon cutaneum
Authors: Powlowski J B , Ingebrand J , Dagley S
Published: January 2020

Cell extracts were prepared from Trichosporon cutaneum grown with phenol or p-cresol, and activities were assayed for enzymes catalyzing conversion of these two carbon sources into 3-ketoadipate (beta-ketoadipate) and 3-keto-4-methyladipate, respectively. When activities of each enzyme were expressed as a ratio, the rate for methyl-substituted substrate being divided by that for the unsubstituted substrate, it was apparent that p-cresol-grown cells elaborated pairs of enzymes for hydroxylation, dioxygenation, and delactonization. One enzyme of each pair was more active against its methyl-substituted substrate, and the other was more active against its unsubstituted substrate. Column chromatography was used to separate two hydroxylase activities and also 1,2-dioxygenase activities; the catechol 1,2-dioxygenases were further purified to electrophoretic homogeneity. Extracts of phenol-grown cells contained only those enzymes in this group that were more active against unsubstituted substrates. In contrast, whether cells were grown with phenol or p-cresol, only one muconate cycloisomerase (lactonizing enzyme) was elaborated which was more active against 3-methyl-cis,cis-muconate than against cis,cis-muconate; in this respect it differed from a cycloisomerase of another strain of T. cutaneum which has been characterized. The cycloisomerase was purified from both phenol-grown and p-cresol-grown cells, and some characteristics were determined.
Regulation of single and multicopy his operons in Escherichia coli
Authors: Riccio A , Bruni C B , Rosenberg M , Gottesman M , McKenney K , Blasi F
Published: January 2020

We fused segments of the Escherichia coli his regulatory region to galK in single-copy and multicopy vectors. These fusions demonstrated that (i) derepression of his by histidine starvation is due exclusively to attenuation; (ii) the his promoter is metabolically regulated; and (iii) both regulatory systems operate when the his regulatory region is present on a multicopy plasmid. Thus, there is no evidence for titration of his regulatory elements. Deletions of the his anti-attenuator region, carried on multicopy plasmids, cause low-level galK expression. This expression is not stimulated by histidine starvation, but is growth rate dependent. We replaced the his attenuator with the efficient lambda terminator, to. In the context of the his regulatory region, however, lambda to only partially terminates transcription.
Regulation of phenoxazinone synthase expression in Streptomyces antibioticus
Authors: Jones G H
Published: January 2020

The cloned gene for the subunit of phenoxazinone synthase (PHS), an enzyme implicated in the biosynthesis of actinomycin in Streptomyces antibioticus, was used as a probe to study the regulation of the enzyme. The direction of transcription of the PHS gene was determined with end-labeled restriction fragments derived from the gene. Low-resolution S1 mapping revealed that transcription was initiated at a position which may lie within the SphI restriction site, which represents the limit of the cloned sequence. Northern blotting allowed the identification of the putative PHS message. This RNA appeared to be significantly larger than the size required to encode the PHS subunit. RNA dot blotting showed that the increase in PHS specific activity observed in cultures grown on antibiotic production medium, with galactose as a carbon source, was due in part to an increased production of PHS mRNA. PHS was also more stable than most cellular proteins and appeared to be protected against degradation under conditions in which most other proteins are broken down. This protective effect also contributed to the increase in PHS specific activity observed in S. antibioticus cultures grown on production medium. The repression of PHS synthesis by glucose was also reflective of a transcriptional control mechanism. At early time points postinoculation, PHS mRNA levels were lower in cultures grown on glucose as a carbon source than in cultures of the same age grown on galactose. mRNA levels presumably begin to increase only after all the glucose in the medium is utilized. The ability of 5-fluorouracil to stimulate PHS production in young cultures was also due to the synthesis of new mRNA for the enzyme.
Influence of transport energization on the growth yield of Escherichia coli
Authors: Muir M , Williams L , Ferenci T
Published: January 2020

The growth yields of Escherichia coli on glucose, lactose, galactose, maltose, maltotriose, and maltohexaose were estimated under anaerobic conditions in the absence of electron acceptors. The yields on these substrates exhibited significant differences when measured in carbon-limited chemostats at similar growth rates and compared in terms of grams (dry weight) of cells produced per mole of hexose utilized. Maltohexaose was the most efficiently utilized substrate, and galactose was the least efficiently utilized under these conditions. All these sugars were known to be metabolized to glucose 6-phosphate and produced the same pattern of fermentation products. The differences in growth yields were ascribed to differences in energy costs for transport and phosphorylation of these sugars. A formalized treatment of these factors in determining growth yields was established and used to obtain values for the cost of transport and hence the energy-coupling stoichiometries for the transport of substrates via proton symport and binding-protein-dependent mechanisms in vivo. By this approach, the proton-lactose stoichiometry was found to be 1.1 to 1.8 H+ per lactose, equivalent to approximately 0.5 ATP used per lactose transported. The cost of transporting maltose via a binding-protein-dependent mechanism was considerably higher, being over 1 to 1.2 ATP per maltose or maltodextrin transported. The formalized treatment also permitted estimation of the net ATP yield from the metabolism of these sugars; it was calculated that the growth yield data were consistent with the production of 2.8 to 3.2 ATP in the metabolism of glucose 6-phosphate to fermentation products.
Role of protein subunits in Proteus rettgeri penicillin G acylase
Authors: Daumy G O , Danley D , McColl A S
Published: January 2020

Penicillin G acylase from Proteus rettgeri is an 80,000- to 90,000-dalton enzyme composed of two nonidentical subunits. Both subunits were required for enzymatic activity. The 65,000-dalton beta subunit contained a phenylmethylsulfonyl fluoride-sensitive residue required for enzymatic activity, and the 24,500-dalton alpha subunit contained the domain that imparts specificity for the penicillin side chain.
Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus
Authors: Takagi M , Imanaka T , Aiba S
Published: January 2020

The thermostable neutral protease gene nprT of Bacillus stearothermophilus was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1,644 bases and 548 amino acid residues. A Shine-Dalgarno sequence was found 9 bases upstream from the translation start site (ATG), and the deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first 14 amino acids of purified extracellular protease completely matched that deduced from the DNA sequence starting at GTC (Val), 687 bases (229 amino acids) downstream from ATG. This suggests that the protease is translated as a longer polypeptide. The amino acid sequence of the extracellular form of this protease (319 amino acids) was highly homologous to that of the thermostable neutral protease from Bacillus thermoproteolyticus but less homologous to the thermolabile neutral protease from Bacillus subtilis. A promoter region determined by S1 nuclease mapping (TTTTCC for the -35 region and TATTTT for the -10 region) was different from the conserved promoter sequences recognized by the known or factors in bacilli. However, it was very homologous to the promoter sequence of the spo0B gene from B. subtilis. The guanine-plus-cytosine content of the coding region of the nprT gene was 58 mol%, while that of the third letter of the codons was much higher (72 mol%).
Evidence for RecA protein association with the cell membrane and for changes in the levels of major outer membrane proteins in SOS-induced Escherichia coli cells
Authors: Garvey N , St John A C , Witkin E M
Published: January 2020

Membrane fractions from Escherichia coli cells expressing DNA damage-inducible (SOS) functions contain elevated quantities of RecA protein (L. J. Gudas and A. B. Pardee, J. Mol. Biol. 101:459-477, 1976). We used two-dimensional polyacrylamide gel electrophoresis to separate membrane proteins from several strains to determine whether this effect is an artifact due to contamination of membranes during preparation by the large amount of cytoplasmic RecA present in SOS-induced cells. We found that amplification of RecA+ protein without a DNA-damaging treatment does not result in increased RecA-membrane association, whether recA is depressed specifically by an operator-constitutive recA allele or coordinately with other SOS genes by a lexA mutation that inactivates their common repressor. In contrast, large amounts of RecA appear in membrane fractions from undamaged cells of an SOS-constitutive strain carrying recA730, which encodes a spontaneously SOS-activated RecA. We conclude that the increased association of RecA with the membrane fraction requires the presence of the activated form of RecA, and that this association may contribute significantly to the SOS response. We describe also striking effects of SOS expression on the levels of the outer membrane proteins OmpA, OmpC, and OmpF.
Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis
Authors: Belland R J , Trust T J
Published: January 2020

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced into Aeromonas salmonicida 449 which produces a surface protein array known as the A-layer. Kanamycin-resistant exconjugants of 449 with altered ability to produce the A-layer were selected by virtue of their altered colonial morphology and color on medium containing the dye Congo red. Analysis of culture supernatants, periplasmic shock fluid, outer membranes, and whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to A-protein revealed five classes of single-insertion mutations that affected the ability of cells to produce and export A-protein and to assemble the A-layer. These studies suggest that A-protein is produced from a single chromosomal gene. The subunits subsequently pass through the periplasm and across the outer membrane. At least one gene product is required for this export. Assembly of A-layer on the cell surface then requires the presence of O polysaccharide chains on the lipopolysaccharide. In one case, insertion of Tn5 resulted in loss of ability to produce both A-protein and lipopolysaccharide with O polysaccharide chains, suggesting that synthesis of A-protein and synthesis of lipopolysaccharide may involve coordinate regulation.