bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence and analysis of the plant-inducible locus pinF from Agrobacterium tumefaciens
Authors: Kanemoto R H , Powell A T , Akiyoshi D E , Regier D A , Kerstetter R A , Nester E W , Hawes M C , Gordon M P
Published: November 2016

Several loci on the tumor-inducing plasmid from Agrobacterium tumefaciens were transcriptionally activated in the presence of wounded plant tissue or extracts. The inducible virulence loci were required for efficient tumor formation. In contrast, the plant-inducible locus pinF was not observed to be absolutely essential for virulence. Mutants in pinF showed an attenuated virulence on a variety of dicotyledonous hosts, and this attenuation became more pronounced with decreasing numbers of bacterial cells in the inoculum. The DNA sequence of a 5.5-kilobase region which included the pinF locus from the octopine-type tumor-inducing plasmid A6 was determined. Four open reading frames consistent with the observed transcription of pinF were observed. Two of the open reading frames, pinF1 and pinF2, coded for polypeptides with relative molecular weights of 47,519 (pinF1) and 46,740 (pinF2). A comparison of the amino acid sequences of pinF1 and pinF2 indicated that they were similar to each other and to known polypeptide sequences for cytochrome P-450 enzymes.
Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage
Authors: Ennis D G , Ossanna N , Mount D W
Published: November 2016

Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein.
Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase
Authors: Saporito S M , Gedenk M , Cunningham R P
Published: November 2016

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.
5-Aminolevulinic acid synthesis in Escherichia coli
Authors: Li J M , Brathwaite O , Cosloy S D , Russell C S
Published: November 2016

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate. tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. S. Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.
Localization of the control region for expression of exotoxin A in Pseudomonas aeruginosa
Authors: Tsaur M L , Clowes R C
Published: November 2016

The 2,760-base-pair (bp) PstI-EcoRI segment of the chromosome of Pseudomonas aeruginosa PA103 which carries the exotoxin A structural gene was expressed from an internal promoter when cloned in a pUC9 derivative and transformed into a nontoxigenic mutant of P. aeruginosa PAO1. The unique terminal EcoRI site was deleted, and a new EcoRI site was substituted for a PvuI site located 107 bp 5' to the transcription initiation site. Following EcoRI cleavage, Bal31 deletions were generated from this site, and an EcoRI linker sequence, GGAATTCC, was inserted in place of the deleted DNA. Mutants with deletions located 73 bp or more upstream of the transcription initiation site retained normal expression, whereas in mutants with deletions extending into the region 69 bp or less upstream of this site, exotoxin synthesis was greatly reduced. From a KpnI site located 473 bp 3' to the transcription initiation site, a similar series of Bal31 deletion mutants were generated in which the inserted EcoRI linker sequence was located within the same 72-bp region. Pairs of mutants from the two deletion series were identified in which the EcoRI linker was located at the same sequence, and these mutant pairs were ligated to derive a series of constructs in which the EcoRI linker sequence GGAATTCC was substituted for an 8-bp sequence within the 72-bp region. Some of these linker-substituted mutants showed greatly reduced exotoxin A synthesis. The results are consistent with a binding site for a positive activator contiguous with the binding site for an RNA polymerase.
Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes
Authors: Baetz A L , Allison M J
Published: November 2016

Oxalyl-coenzyme A (oxalyl-CoA) decarboxylase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography, DEAE anion-exchange chromatography, and gel permeation chromatography. The enzyme is made up of four identical subunits (Mr, 65,000) to give the active enzyme (Mr, 260,000). The enzyme catalyzed the thiamine PPi-dependent decarboxylation of oxalyl-CoA to formate and carbon dioxide. Apparent Km and Vmax values, respectively, were 0.24 mM and 0.25 mumol/min for oxalyl-CoA and 1.1 pM and 0.14 mumol/min for thiamine pyrophosphate. The maximum specific activity was 13.5 microM oxalyl-CoA decarboxylated per min per mg of protein.
Elevated serine catabolism is associated with the heat shock response in Escherichia coli
Authors: Matthews R G , Neidhardt F C
Published: November 2016

The biochemical events associated with the heat shock response are not well understood in any organism, nor have the signals that initiate the induction of heat shock protein synthesis been identified. In this work, we demonstrate that the rate of serine catabolism of Escherichia coli cells grown in glucose minimal medium supplemented with serine is elevated three- to sevenfold when the growth temperature is shifted from 37 to 44 degrees C. Elevations in growth temperature and mutations or treatments that lead to elevated basal rates of serine catabolism at 37 degrees C result in the excretion into the culture medium of acetate derived from exogenous serine. Increases in the basal level of serine catabolism at 37 degrees C do not per se induce a heat shock response but are associated with abnormalities in the pattern of induction of heat shock polypeptides following a temperature shift. We postulate that the events responsible for or resulting from the elevation in serine catabolism associated with a shift-up in temperature modulate the induction of 3 of the 17 heat shock polypeptides identified in E. coli. These observations suggest that heat shock diverts serine away from the production of glycine and C1 units, which are required for initiation of protein synthesis and for nucleotide biosynthesis, and towards acetyl coenzyme A and acetate.
Translational coupling of the two proximal genes in the S10 ribosomal protein operon of Escherichia coli
Authors: Lindahl L , Archer R H , McCormick J R , Freedman L P , Zengel J M
Published: November 2016

We have examined the translational coupling between the first two genes in the S10 ribosomal protein operon. We isolated mutations blocking the translation of the first gene of the operon, coding for S10, and monitored their effects on translation of the downstream gene, coding for L3. All of the mutations inhibiting S10 synthesis also affected the synthesis of L3. However, these experiments were complicated by decreased mRNA synthesis resulting from transcription polarity, which we could only partially eliminate by using a rho-100 strain. To completely eliminate the problem of transcription polarity and obtain a more accurate measurement of the coupling, we replaced the natural S10 promoter with a promoter used by the bacteriophage T7 RNA polymerase. As expected, the T7 RNA polymerase was not subject to transcription polarity. Using this system, we were able to show that a complete abolishment of S10 translation resulted in an 80% inhibition of L3 synthesis. Other experiments show that the synthesis of L3 goes up as a function of increasing S10 synthesis, but the translational coupling does not assure strictly proportional output from the two genes.
Promoter specificity of sigma G-containing RNA polymerase from sporulating cells of Bacillus subtilis: identification of a group of forespore-specific promoters
Authors: Nicholson W L , Sun D X , Setlow B , Setlow P
Published: November 2016

During sporulation in Bacillus subtilis, expression of the genes sspA, sspB, sspC, sspD, and sspE, which encode a family of small, acid-soluble spore proteins, as well as of the spoVA and gdh operons is transcriptionally activated at stage III of sporulation only in the forespore compartment. Transcription of these genes is mediated by RNA polymerase containing sigma G (E sigma G), the product of the sigG gene, which is itself expressed at stage III in the developing forespore. We have determined the 5' ends of transcripts generated both in vivo and in vitro by the action of E sigma G on various genes of B. subtilis and other bacilli. The 5' ends of the in vivo and in vitro mRNAs were found to coincide and were therefore considered to define the transcription initiation sites for the genes examined. We identified highly homologous DNA sequences centered at 35 and 10 base pairs preceding the transcriptional start sites of the genes examined. Consequently, we propose that these sequences define a class of promoters recognized only by E sigma G which allow transcription of genes expressed uniquely at stage III in the developing forespore.
Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102
Authors: Kimbara K , Hashimoto T , Fukuda M , Koana T , Takagi M , Oishi M , Yano K
Published: November 2016

Two genes involved in the degradation of biphenyl were isolated from a gene library of a polychlorinated biphenyl-degrading soil bacterium, Pseudomonas sp. strain KKS102, by using a broad-host-range cosmid vector, pKS13. When a 3.2-kilobase (kb) PstI fragment of a 29-kb cosmid DNA insert was subcloned into pUC18 at the PstI site downstream of the lacZ promoter, Escherichia coli cells carrying this recombinant plasmid expressed 2,3-dihydroxybiphenyl dioxygenase activity. Nucleotide sequencing of the 3.2-kb PstI fragment revealed that there were two open reading frames (ORFI [882 base pairs] and ORFII [834 base pairs], in this gene order). Results of analysis of Tn5 insertion mutants and unidirectional deletion mutants suggested that the ORFI coded for 2,3-dihydroxybiphenyl dioxygenase. When the sequence of ORFI was compared with that of bphC of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa, N. Arima, and T. Miyazaki, J. Bacteriol. 169:427-429, 1987), the homology was 68%, with both strains having the same Shine-Dalgarno sequence. The result of gas chromatography-mass spectrometry analysis of the metabolic product suggested that the ORFII had meta cleavage compound hydrolase activity to produce benzoic acid. DNA sequencing suggested that these two genes were contained in one operon.
Biochemical characterization of avirulent Agrobacterium tumefaciens chvA mutants: synthesis and excretion of beta-(1-2)glucan
Authors: de Iannino N I , Ugalde R A
Published: November 2016

The chvA gene product of Agrobacterium tumefaciens is required for virulence and attachment of bacteria to plant cells. Three chvA mutants were studied. In vivo, they were defective in the synthesis, accumulation, and secretion of beta-(1-2)glucan; however, the 235-kilodalton (kDa) protein known to be involved in the synthesis of beta-(1-2)glucan (A. Zorreguieta and R. Ugalde, J. Bacteriol. 167:947-951, 1986) was present and active in vitro. was present and active in vitro. Two molecular forms of cyclic beta-(1-2)glucan, designated types I and II, were resolved by gel chromatography. Type I beta-(1-2)glucan was substituted with nonglycosidic residues, and type II beta-(1-2)glucan was nonsubstituted. Wild-type cells accumulated type I beta-(1-2)glucan, and chvA mutant cells accumulated mainly type II beta-(1-2)glucan and a small amount of type I beta-(1-2)glucan. Inner membranes of wild-type and chvA mutants formed in vitro type II nonsubstituted beta-(1-2)glucan. A 75-kDa inner membrane protein is proposed to be the chvA gene product. chvA mutant inner membranes had increased levels of 235-kDa protein; partial trypsin digestion patterns suggested that the 235-kDa protein (the gene product of the chvB region) and the gene product of the chvA region form a complex in the inner membrane that is involved in the synthesis, secretion, and modification of beta-(1-2)glucan. All of the defects assigned to the chvA mutation were restored after complementation with plasmid pCD522 containing the entire chvA region.
Differentiation of arcA, arcB, and cpxA mutant phenotypes of Escherichia coli by sex pilus formation and enzyme regulation
Authors: Iuchi S , Furlong D , Lin E C
Published: November 2016

In Escherichia coli, mutations in arcA (dye) or arcB anaerobically derepress the synthesis of a multitude of enzymes of aerobic function, and mutations in arcA or cpxA impair F-pilus formation. It is thought that arcA encodes a promoter-recognizing protein, whereas arcB and cpxA encode sensor proteins which interact with the arcA product. In this study we found that anaerobic growth of a wild-type F' strain decreased the synthesis of both the enzymes and the pilus. Although the two arcA mutants examined were both anaerobically derepressed in the enzymes and impaired in aerobic pilus formation as expected, one mutant hyperproduced the pilus anaerobically. The two arcB mutants examined showed normal pilus formation when grown aerobically. When grown anaerobically they developed more pili than the wild-type strain did when grown aerobically. When a cpxA mutant was examined for synthesis of two aerobic enzymes, normal regulation was found. The available data suggest the following. The arcA product anaerobically represses certain genes of aerobic function and activates certain genes related to F function. It appears that the arcB product senses the redox or energy state; absence of the gene function shifts the arcA product to the nonrepressive form for enzyme synthesis for aerobic pathways. The cpxA product, on the other hand, senses the sexual state; absence of the gene function shifts the arcA product to the inactive form for F-pilus synthesis.
Role of metabolism in the chemotactic response of Rhodobacter sphaeroides to ammonia
Authors: Poole P S , Armitage J P
Published: November 2016

Rhodobacter sphaeroides only showed chemotaxis towards ammonia if grown under nitrogen-limited conditions. This chemotactic response was completely inhibited by the addition of methionine sulfoximine. There was no effect of methionine sulfoximine treatment on motility or taxis towards propionate, demonstrating that the effect is specific to ammonia taxis. It is known that methionine sulfoximine inhibits glutamine synthetase and hence blocks ammonia assimilation. Methionine sulfoximine does not inhibit ammonia transport in R. sphaeroides; therefore, these results suggest that limited metabolism via a specific pathway is required subsequent to transport to elicit a chemotactic response to ammonia. Bacteria grown on high ammonia show transport but no chemotactic response to ammonia, suggesting that the pathway of assimilation is important in eliciting a chemotactic response.
Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit
Authors: Hartmann R K , Erdmann V A
Published: November 2016

A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA.
Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication
Authors: Masai H , Arai K
Published: November 2016

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.
Evidence for posttranslational modification and gene duplication of Campylobacter flagellin
Authors: Logan S M , Trust T J , Guerry P
Published: November 2016

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.
Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation
Authors: LaVallie E R , Stahl M L
Published: November 2016

The flagellin promoter and structural gene from Bacillus subtilis I168 was cloned and sequenced. The amino-terminal protein sequence deduced from the coding sequence of the cloned gene was identical to that of the amino terminus of purified flagellin, indicating that the export of this protein is not directed by a posttranslationally processed N-terminal signal peptide. A sequence that was homologous to that of a consensus sigma 28 RNA polymerase recognition site lay upstream of the proposed translational start site. Amplification of this promoter region on a multicopy plasmid resulted in the formation of long, filamentous cells that accumulated flagellin intracellularly. The chromosomal locus containing the wild-type flagellin allele was replaced with a defective allele of the gene (delta hag-633) that contained a 633-base-pair deletion. Transport analysis of various flagellin gene mutations expressed in the hag deletion strain suggest that the extreme C-terminal portion of flagellin is functionally involved in export of the protein.
Variations in UDP-N-acetylglucosamine and UDP-N-acetylmuramyl-pentapeptide pools in Escherichia coli after inhibition of protein synthesis
Authors: Mengin-Lecreulx D , Siegel E , van Heijenoort J
Published: November 2016

The pool levels of the nucleotide precursors of peptidoglycan were analyzed after inhibition of protein synthesis in various Escherichia coli strains. In all cases UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) cell pools increased upon treatment with chloramphenicol or tetracycline. Similar results were observed after the treatment of K-12 strains with valine. Since the intermediate nucleotide precursors did not accumulate after the arrest of protein synthesis and since a feedback mechanism was unlikely, the increases of the UDP-MurNAc-pentapeptide pool appeared as a consequence of that of the UDP-GlcNAc pool by the unrestricted functioning of the intermediate steps of the pathway. The highest increase (sixfold) of UDP-GlcNAc was observed with strain K-12 HfrH growing in minimal medium and treated with chloramphenicol. When a pair of isogenic Rel+ and Rel- strains were considered, both the UDP-GlcNAc and UDP-MurNAc-pentapeptide pools increased upon treatment with chloramphenicol or valine. However, the UDP-GlcNAc pool of the Rel+ strain was at a high natural level, which increased only moderately (20%) after the addition of valine. The increase of the UDP-GlcNAc pool after the various treatments could be due to an effect on some upstream step by an unknown mechanism. The possible correlations of the variations of the precursor pools with the rate of synthesis and extent of cross-linking of peptidoglycan were also considered.
Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri
Authors: Heiss B , Frunzke K , Zumft W G
Published: November 2016

Nitric oxide (NO) reductase was solubilized by Triton X-100 from the membrane fraction of Pseudomonas stutzeri ZoBell and purified 100-fold to apparent electrophoretic homogeneity. The enzyme consisted of two polypeptides of Mr 38,000 and 17,000 associated with heme b and heme c, respectively. Absorption maxima of the reduced complex were at 420.5, 522.5, and 552.5 nm, with a shoulder at 560 nm. The electron paramagnetic resonance spectrum was characteristic of high- and low-spin ferric heme proteins; no signals typical for iron-sulfur proteins were found. Nitric oxide reductase stoichiometrically transformed NO to nitrous oxide in an ascorbate-phenazine methosulfate-dependent reaction with a specific activity of 11.8 mumols/min per mg of protein. The activity increased to 40 mumols upon the addition of soybean phospholipids, n-octyl-beta-D-glucopyranoside, or its thio derivative to the assay system. Apparent Km values for NO and phenazine methosulfate were 60 and 2 microM, respectively. The pH optimum of the reaction was at 4.8. Cytochrome co was purified from P. stutzeri to permit its distinction from NO reductase. Spectrophotometric binding assays and other criteria also differentiated NO reductase from the respiratory cytochrome bc1 complex.
Multiple control mechanisms for pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12
Authors: Liu C G , Turnbough C L
Published: November 2016

Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated over a several-hundredfold range by pyrimidine availability. This regulation occurs, at least in large part, through a UTP-sensitive attenuation control mechanism in which transcriptional termination at the pyrBI attenuator, a rho-independent transcriptional terminator located immediately upstream of the pyrB structural gene, is regulated by the relative rates of transcription and translation within the pyrBI leader region. There is suggestive evidence that an additional, attenuator-independent control mechanism also contributes to this regulation. To measure the level of regulation that occurs through the attenuation and attenuator-independent control mechanisms, we constructed a mutant strain in which a 9-base-pair deletion was introduced into the attenuator of the chromosomal pyrBI operon. This deletion, which removes the run of thymidine residues at the end of the attenuator, completely abolishes rho-independent transcriptional termination activity. When the mutant strain was grown under conditions of pyrimidine excess, the level of operon expression was 51-fold greater than that of an isogenic pyrBI+ strain. Under conditions of pyrimidine limitation, operon expression was increased an additional 6.5-fold in the mutant. These results demonstrate that the attenuation control mechanism is primarily responsible for pyrimidine-mediated regulation but that there is a significant contribution by an attenuator-independent control mechanism.
Common organization of chromosomal loci for production of different capsular polysaccharides in Haemophilus influenzae
Authors: Kroll J S , Zamze S , Loynds B , Moxon E R
Published: November 2016

Cloned Haemophilus influenzae type b capsulation genes were used as hybridization probes to isolate DNA from the capsulation loci (cap) of other serotypes of H. influenzae. Mapping of the resulting clones and Southern hybridization analysis of chromosomal DNAs from type a, b, c, and d strains showed that in each strain cap was organized in the same way: a central DNA segment specific to each serotype flanked by DNA segments of common structure. We infer that enzymes necessary for the synthesis of specific capsular polysaccharide are encoded in the central segment of cap, while proteins involved in a more general way in the process of capsulation are encoded in the flanking segments. Studies of the function of the DNA in one of these non-serotype-specific flanking segments (J. S. Kroll, I. Hopkins, and E. R. Moxon, Cell 53:347-356, 1988) have previously identified a gene encoding a protein necessary for polysaccharide export, an event now deduced to proceed by a mechanism independent of the nature of the disaccharide subunit in the polysaccharide. The near-total duplication of cap that has been found in most type b strains was not found at the analogous locus in the other serotypes. This reinforces our previous hypothesis, based on study of type b strains alone, that while such a duplication is unnecessary for capsulation, it confers some unexplained survival advantage on the widely prevalent strains with this clinically important serotype.
Replication origins of single-stranded-DNA plasmid pUB110
Authors: Boe L , Gros M F , te Riele H , Ehrlich S D , Gruss A
Published: November 2016

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.
Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures
Authors: Karr D B , Emerich D W
Published: November 2016

Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source.
Stimulation of IS1 excision by bacteriophage P1 ref function
Authors: Lu S D , Lu D , Gottesman M
Published: November 2016

Lysogenization by a c1ts variant of coliphage P1, P1c1.100, markedly increased the frequency of reversion of a galT::IS1 mutation. The formation of Gal+ colonies presumably occurs by microhomologous recombination between the 9-base-pair repeats in galT (CGCCGCTAC) generated by the transposition of IS1. The responsible P1 gene, ref, has been cloned and sequenced. ref encodes a 22.8-kilodalton protein and is located near the P1 site-specific recombination function, cre. Expression of ref was repressed by P1 c+. The absence of a distinctive ribosome-binding site is consistent with a poor translation of ref from an expression vector in vivo. Placement of a ribosome-binding site before ref resulted in the extensive synthesis of the Ref protein. Ref stimulated precise excision in recB or himA cells, but not in recA mutants. Ref was active in lexA3 mutants, suggesting that the recombination activity of RecA was directly involved in the reaction. We have constructed a P1c1.100 ref::Tn10 mutant. The absence of Ref did not appear to restrict dramatically the ability of P1 to grow lytically or to form lysogens. Thus, the role of ref in the physiology of P1 remains to be determined.
Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803
Authors: Labarre J , Chauvat F , Thuriaux P
Published: November 2016

The facultative heterotrophic cyanobacterium Synechocystis sp. strain PCC 6803 was transformed by HaeII Cmr fragments ligated at random to HaeII DNA fragments of the host genome. A similar transformation was done with an AvaII Kmr marker ligated to AvaII host DNA fragments. Integration of the resistance markers into the host genome led to a high frequency of stable Kmr and Cmr transformants. Physical analysis of individual transformants indicated that this result was due to homologous recombination by conversionlike events leading to insertion of the Cmr (or Kmr) gene between two HaeII (or AvaII) sites of the host genome, with precise deletion of the host DNA between these sites. In contrast, integrative crossover of circular DNA molecules with homology to the host DNA is very rare in this cyanobacterium. Strain PCC 6803 was shown to have about 12 genomic copies per cell in standard growth conditions, which complicates the detection of recessive mutations induced by chemical or UV mutagenesis. Random disruption of the host DNA by insertional transformation provides a convenient alternative to transposon mutagenesis in cyanobacteria and may help to overcome the difficulties encountered in generating recessive mutants by classical mutagenesis.
Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli
Authors: Bae Y M , Holmgren E , Crawford I P
Published: November 2016

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzyme of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by S1 nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C----T change at position -9 in the -10 region. The +1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site.
Suppression of recA deficiency in plasmid recombination by bacteriophage lambda beta protein in RecBCD- ExoI- Escherichia coli cells
Authors: Berger I , Cohen A
Published: November 2016

Plasmid recombination, like other homologous recombination in Escherichia coli, requires RecA protein in most conditions. We have found that the plasmid recombination defect in a recA mutant can be efficiently suppressed by the beta protein of bacteriophage lambda. beta protein is required for homologous recombination of lambda chromosomes during lytic phage growth in a recA host and is known to have a strand-annealing activity resembling that of RecA protein. The bioluminescence recombination assay was used for genetic analysis of beta-protein-mediated plasmid recombination. Efficient suppression of the recA mutation by beta protein required the absence of the E. coli nucleases exonuclease I and RecBCD nuclease. These nucleases inhibit a RecA-mediated plasmid recombination pathway that is more efficient than the pathway functioning in wild-type cells. Like RecA-mediated plasmid recombination in RecBCD- ExoI- cells, beta-protein-mediated plasmid recombination depended on concurrent DNA replication and on the activity of the recQ gene. However, unlike RecA-mediated plasmid recombination, beta-protein-mediated recombination in RecBCD- ExoI- cells was independent of recF and recJ activities. We propose that inactivation of exonuclease I and RecBCD nuclease stabilizes a recombination intermediate that is involved in RecA- and beta-protein-catalyzed homologous pairing reactions. We suggest that the intermediate may be linear plasmid DNA with a protruding 3' end, since these nucleases are known to interfere with the synthesis of such linear forms. The different recF and recJ requirements for beta-protein-dependent and RecA-dependent recombinations imply that the mechanisms of formation or processing of the putative intermediate differ in the two cases.
Role of cyclic-AMP-dependent protein kinase in catabolite inactivation of the glucose and galactose transporters in Saccharomyces cerevisiae
Authors: Ramos J , Cirillo V P
Published: November 2016

The derepressed high-affinity glucose transport system and the induced galactose transport system are catabolite inactivated when cells with these transport systems are incubated with glucose. The role of the cyclic AMP cascade in the catabolite inactivation of these transport systems was shown by using mutants affected in the activity of cyclic-AMP-dependent protein kinase (cAPK). In tpk1(w) mutants with reduced cAPK activity, the sugar transport systems were expressed but were not catabolite inactivated. In bcy1 mutants with unbridled cAPK activity resulting from a defective regulatory subunit, the transport systems were absent or present at low levels.
A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site
Authors: Lewis P J , Smith M T , Wake R G
Published: November 2016

The small basic protein encoded by the open reading frame adjacent to the terC site in the Bacillus subtilis chromosome and previously implicated in termination of the replication process was purified. Band retardation assays established that this protein (now called the replication terminator protein, encoded by the rtp gene) binds specifically to a 209-base-pair fragment of DNA within which terC is located.
Glutamine auxotrophs of Bacillus subtilis that overproduce glutamine synthetase antigen have altered conserved amino acids in or near the active site
Authors: Zhang J , Strauch M , Aronson A I
Published: November 2016

A number of mutations within the Bacillus subtilis glutamine synthetase (GS) gene result in altered catalytic properties and overproduction of the GS antigen. The restriction fragments containing mutations from three such mutants were sequenced, and they all had amino acid changes in conserved residues found either within or near sequences contributing to the active site of the Salmonella typhimurium GS.
Overproduced bacteriophage T4 gene 33 protein binds RNA polymerase
Authors: Williams K P , Müller R , Rüger W , Geiduschek E P
Published: November 2016

Bacteriophage T4 gene 33 protein (gp33), which is required for viral late transcription, has been overproduced. The purified gp33 binds to RNA polymerase core from uninfected or T4-infected Escherichia coli, but the major E. coli transcription initiation factor, sigma 70, competed effectively for this binding.
Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli
Authors: Caffrey P , Owen P
Published: November 2016

Antigen 43 has been identified as a unique protein complex in the outer membrane of Escherichia coli. The complex contains two different polypeptides, alpha (Mr, 60,000) and beta (Mr, 53,000), in equal stoichiometry (P. Owen, P. Caffrey, and L.-G. Josefsson, J. Bacteriol. 169:3770-3777, 1987). The alpha subunit was released in a water-soluble form upon heating of outer membranes to 60 degrees C and was purified to apparent homogeneity by gel filtration and ion-exchange chromatography. The purified protein was acidic (pI 4.6) and had a polarity of 49.2%. The N-terminal sequence showed homology with the N termini of certain enterobacterial fimbrial subunits. In addition, antigen 43 underwent a reversible phase variation similar to that of type 1 fimbriae. By use of subunit-specific antisera, it was shown that the purified alpha subunit was capable of reassociating with the beta polypeptide. However, electron microscopic examination indicated that antigen 43 does not form a recognizable surface structure. The available evidence supports the view that antigen 43 is a complex consisting of a peripheral membrane protein (alpha) anchored to a subunit (beta) that is integral to the outer membrane.
Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd
Authors: Tomb J F , Barcak G J , Chandler M S , Redfield R J , Smith H O
Published: November 2016

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.
Segregation of relaxed replicated dimers when DNA ligase and DNA polymerase I are limited during oriC-specific DNA replication
Authors: Munson B R , Maier P G , Greene R S
Published: November 2016

An in vitro Escherichia coli oriC-specific DNA replication system was used to investigate the DNA replication pathways of oriC plasmids. When this system was perturbed by the DNA ligase inhibitor nicotinamide mononucleotide (NMN), alterations occurred in the initiation of DNA synthesis and processing of intermediates and DNA products. Addition of high concentrations of NMN soon after initiation resulted in the accumulation of open circular dimers (OC-OC). These dimers were decatenated to open circular monomers (form II or OC), which were then processed to closed circular supercoiled monomers (form I or CC) products. After a delay, limited ligation of the interlinked dimers (OC-OC to CC-OC and CC-CC) also occurred. Similar results were obtained with replication protein extracts from polA mutants. The presence of NMN before any initiation events took place prolonged the existence of nicked template DNA and promoted, without a lag period, limited incorporation into form II molecules. This DNA synthesis was nonspecific with respect to oriC, as judged by DnaA protein dependence, and presumably occurred at nicks in the template DNA. These results are consistent with oriC-specific initiation requiring closed supercoiled molecules dependent on DNA ligase activity. The results also show that decatenation of dimers occurs readily on nicked dimer and represents an efficient pathway for processing replication intermediates in vitro.
Suppression of a -1 frameshift mutation by a recessive tRNA suppressor which causes doublet decoding
Authors: O'Mahony D J , Hughes D , Thompson S , Atkins J F
Published: November 2016

sufS was found to suppress the only known suppressible-1 frameshift mutation, trpE91, at a site identified as GGA and mapped within the single gene of the only tRNA that can decode GGA in Escherichia coli. It mapped to the same gene in Salmonella typhimurium. sufS alleles were recessive, and dominant alleles could not be isolated. This is in contrast to all other tRNA structural gene mutations identified thus far that cause frameshift suppression. The recessiveness implies that all sufS alleles are poor competitors against their wild-type tRNA(Gly2) counterparts. The base G immediately 5' of the GGA suppression site influenced the level but was not critical for suppression by sufS601. From this result, it is inferred that sufS601 causes frameshifting by doublet decoding.
Reassessment of the role of phospholipids in sexual reproduction by sterol-auxotrophic fungi
Authors: Kerwin J L , Duddles N D
Published: November 2016

Several genera of oomycete fungi which are incapable of de novo sterol synthesis do not require these compounds for vegetative growth. The requirement for an exogenous source of sterols for sexual reproduction by several members of the Pythiaceae has been questioned by reports of apparent induction and maturation of oospores on defined media supplemented with phospholipids in the absence of sterols. A more detailed examination of this phenomenon suggested that trace levels of sterols in the inoculum of some pythiaceous fungi act synergistically with phospholipid medium supplements containing unsaturated fatty acid moieties to induce oosporogenesis. Phospholipid analysis of one species, Pythium ultimum, suggested that only the fatty acid portion of the exogenous phospholipid is taken up by the fungus. Enrichment of the phospholipid fraction of total cell lipid of P. ultimum with unsaturated fatty acids promoted oospore induction, and enhanced levels of unsaturated fatty acids in the neutral lipid fraction increased oospore viability. For some pythiaceous fungi, the levels of sterols required for the maturation of oospores with appropriate phospholipid medium supplementation suggest that these compounds are necessary only for the sparking and critical domain roles previously described in other fungi.
Reconstitution and properties of a coenzyme F420-mediated formate hydrogenlyase system in Methanobacterium formicicum
Authors: Baron S F , Ferry J G
Published: November 2016

Formate hydrogenlyase activity in a cell extract of Methanobacterium formicicum was abolished by removal of coenzyme F420; addition of purified coenzyme F420 restored activity. Formate hydrogenlyase activity was reconstituted with three purified components from M. formicicum: coenzyme F420-reducing hydrogenase, coenzyme F420-reducing formate dehydrogenase, and coenzyme F420. The reconstituted system required added flavin adenine dinucleotide (FAD) for maximal activity. Without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F420-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of formate dehydrogenase or coenzyme F420-reducing hydrogenase from the cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. The formate hydrogenlyase activity was reversible, since both the cell extract and the reconstituted system produced formate from H2 plus CO2 and HCO3-.