bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the pAD1-encoded sex pheromone response in Enterococcus faecalis: expression of the positive regulator TraE1
Authors: Tanimoto K , Clewell D B
Published: November 2016

pAD1 is a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis that encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. The response is regulated by a cluster of genes that includes a positive regulatory determinant, traE1, able to activate key structural genes involved in the conjugative process. A negative regulatory determinant, traA, affects the expression of traE1 and is sensitive to the pheromone signal. Between the two determinants is a gene, iad, which encodes a small peptide, iAD1, a competitive inhibitor of cAD1. The determinants (traE1-iad-traA) are oriented such that iad and traE1 are transcribed in the same direction, opposite that of traA. Transcription of iad and traA starts between these determinants and moves outward in each case. A recent report from our laboratory, dealing with transcriptional fusions in the traE1-iad region (L. T. Pontius and D. B. Clewell, J. Bacteriol. 174:3152-3160, 1992), indicated that traE1 expression may be dependent on transcriptional read-through of a terminator(s) between iad and traE1. The present report provides direct analyses of relevant RNA species before and during induction and shows that indeed transcriptional read-through from iad is important in the initial expression of traE1. However, the data show that once traE1 is activated, it can then be expressed independently, probably because of TraE1 activating its own promoter. This view is also supported by genetic complementation studies. In addition, DNA binding studies with TraA showed that the protein binds to the promoter of iad. Binding of TraA to the region between iad and traE1 could not be detected; however, the involvement of TraA in influencing transcription termination in this region is still not ruled out, since additional factors could be involved. A model for the regulation of the pheromone response is presented.
Specific binding of Thiobacillus ferrooxidans RbcR to the intergenic sequence between the rbc operon and the rbcR gene
Authors: Kusano T , Sugawara K
Published: November 2016

The presence of two sets (rbcL1-rbcS1 and rbcL2-rbcS2) of rbc operons has been demonstrated in Thiobacillus ferrooxidans Fe1 (T. Kusano, T. Takeshima, C. Inoue, and K. Sugawara, J. Bacteriol. 173:7313-7323, 1991). A possible regulatory gene, rbcR, 930 bp long and possibly translated into a 309-amino-acid protein, was found upstream from the rbcL1 gene as a single copy. The gene is located divergently to rbcL1 with a 144-bp intergenic sequence. As in the cases of the Chromatium vinosum RbcR and Alcaligenes eutrophus CfxR, T. ferrooxidans RbcR is thought to be a new member of the LysR family, and these proteins share 46.5 and 42.8% identity, respectively. Gel mobility shift assays showed that T. ferrooxidans RbcR, produced in Escherichia coli, binds specifically to the intergenic sequence between rbcL1 and rbcR. Footprinting and site-directed mutagenesis experiments further demonstrated that RbcR binds to overlapping promoter elements of the rbcR and rbcL1 genes. The above data strongly support the participation of RbcR in regulation of the rbcL1-rbcS1 operon and the rbcR gene in T. ferrooxidans.
Molecular cloning, sequencing, and mapping of the gene encoding protease I and characterization of proteinase and proteinase-defective Escherichia coli mutants
Authors: Ichihara S , Matsubara Y , Kato C , Akasaka K , Mizushima S
Published: November 2016

Clones carrying the gene encoding a proteinase were isolated from Clarke and Carbon's collection, using a chromogenic substrate, N-benzyloxycarbonyl-L-phenylalanine beta-naphthyl ester. The three clones isolated, pLC6-33, pLC13-1, and pLC36-46, shared the same chromosomal DNA region. A 0.9-kb Sau3AI fragment within this region was found to be responsible for the overproduction of the proteinase, and the nucleotide sequence of the region was then determined. The proteinase was purified to homogeneity from the soluble fraction of an overproducing strain possessing the cloned gene. N-terminal amino acid sequencing of the purified protein revealed that the cloned gene is the structural gene for the protein, with the protein being synthesized in precursor form with a signal peptide. On the basis of its molecular mass (20 kDa), periplasmic localization, and substrate specificity, we conclude this protein to be protease I. By using the gene cloned on a plasmid, a deletion mutant was constructed in which the gene was replaced by the kanamycin resistance gene (Kmr) on the chromosome. The Kmr gene was mapped at 11.8 min, the gene order being dnaZ-adk-ush-Kmr-purE, which is consistent with the map position of apeA, the gene encoding protease I in Salmonella typhimurium. Therefore, the gene was named apeA. Deletion of the apeA gene, either with or without deletion of other proteinases (protease IV and aminopeptidase N), did not have any effect on cell growth in the various media tested.
Identification of endonucleolytic cleavage sites involved in decay of Escherichia coli trxA mRNA
Authors: Arraiano C , Yancey S D , Kushner S R
Published: November 2016

The degradation of individual mRNAs in Escherichia coli has been studied through the use of a multiple mutant carrying the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and rne-1 (RNase E) alleles. In this triple mutant, discrete mRNA breakdown products are stabilized in vivo at the nonpermissive temperature (Arraiano, C. M., S. D. Yancey, and S. R. Kushner, J. Bacteriol. 170:4625-4633, 1988). In the case of thioredoxin (trxA) mRNA decay, degradation fragments accumulated at early times after a shift to the nonpermissive temperature. Using Northern (RNA) blots, S1 nuclease analysis, and primer extensions, we identified a series of specific endonucleolytic cleavage sites that occur throughout the transcript in both the triple mutant and a wild-type control. The implications of the complex decay patterns observed are discussed.
Mutations affecting the ability of Escherichia coli Lrp to bind DNA, activate transcription, or respond to leucine
Authors: Platko J V , Calvo J M
Published: November 2016

Lrp is a regulatory protein in Escherichia coli that increases expression of some operons and decreases expression of others. Mutations in Lrp were isolated on the basis of their effects on ilvIH, one of the operons regulated positively by Lrp. The ilvIH operon encodes an enzyme involved in the biosynthesis of leucine, valine, and isoleucine, and expression of this operon is repressed when cells are grown in the presence of leucine. Three groups of mutants were isolated. Mutant strains that were resistant to the repressive effects of leucine were termed leucine response mutants. These mutants had changes in the Lrp amino acid sequence between amino acid residues 108 and 149. Mutant strains having low expression of ilvIH in vivo were identified as colonies having reduced expression of a reporter gene. For some of these mutants, called DNA-binding mutants, binding to ilvIH DNA in vitro was markedly reduced. The mutations in these strains caused changes in Lrp between amino acids 16 and 70. Six of ten of these mutations were within a region having a putative helix-turn-helix motif. A third group of mutants had low ilvIH expression in vivo but apparently normal DNA binding in vitro. These mutants were called activation mutants since they affected the ability of Lrp to activate expression. Lrp from these strains had changes in amino acids between residues 76 and 125. This study suggests that Lrp has separate domains responsible for binding DNA, activating transcription, and responding to leucine.
Participation of RAP1 protein in expression of the Saccharomyces cerevisiae arginase (CAR1) gene
Authors: Kovari L Z , Kovari I , Cooper T G
Published: November 2016

Regulated expression of the inducible arginase (CAR1) gene of Saccharomyces cerevisiae has been shown to require three upstream activation sequences (UASs) and an upstream repression sequence, URS1. Two of the UAS elements, UASC1 and UASC2, operate in an inducer-independent manner, while the third, UASI, is inducer dependent. UASC1 and UASC2 were previously shown to contain ABF-1 binding sites that were required for normal transcription. In this work, we demonstrate that UASC1 and UASC2 also contain two and three sites, respectively, that are able to bind RAP1 protein. RAP1 binding to these sites, however, is significantly weaker than that to sites in TEF2 and HMRE. The effects of mutating the sites individually or in combination suggest that at least three of them, two in UASC1 and one in UASC2, probably participate in CAR1 expression.
A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species
Authors: Goldberg J B , Gorman W L , Flynn J L , Ohman D E
Published: November 2016

Conversion of the mucoid phenotype, which results from the production of the exopolysaccharide alginate, is a feature typical of Pseudomonas aeruginosa strains causing chronic pulmonary infections in patients with cystic fibrosis. In this study, we further characterized a recombinant plasmid, called pJF15, that contains DNA from the 65- to 70-min region of the chromosome of mucoid P. aeruginosa FRD1 and has loci involved in alginate conversion. Plasmid pJF15 complements algT mutations in trans and confers the mucoid phenotype in cis following gene replacement. However, the phenotype of nonmucoid P. aeruginosa carrying pJF15 is unchanged. Here we report the identification of a locus immediately downstream of algT, called algN, that may be a negative regulator that blocks algT from activating alginate production. Inactivation of algN by transposon Tn501 insertion allowed algT to stimulate alginate production in trans. The DNA sequence of this region identified an open reading frame that predicts an algN gene product of 33 kDa, but no homology was found to other proteins in a sequence data base. Clones of algT in which algN was deleted caused the activation of alginate biosynthesis in transconjugants of several P. aeruginosa strains. DNA containing algT was shown to hybridize to the genomes of several Pseudomonas species, including P. putida, P. stutzeri, and P. fluorescens. Transconjugants of these species carrying algT DNA (with a deletion of algN) from pJF15 showed a mucoid phenotype and increased production of uronic acid-containing polymers that resembled alginate.
In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs
Authors: Rowley K B , Elford R M , Roberts I , Holmes W M
Published: November 2016

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.
The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression
Authors: Tomoyasu T , Yuki T , Morimura S , Mori H , Yamanaka K , Niki H , Hiraga S , Ogura T
Published: November 2016

The ftsH gene is essential for cell viability in Escherichia coli. We cloned and sequenced the wild-type ftsH gene and the temperature-sensitive ftsH1(Ts) gene. It was suggested that FtsH protein was an integral membrane protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding domain. The ftsH1(Ts) gene was found to have two base substitutions within the coding sequence corresponding to the amino acid substitutions Glu-463 by Lys and Pro-587 by Ala. Homology search revealed that an approximately 200-amino-acid domain, including the putative ATP-binding sequence, is highly homologous (35 to 48% identical) to the domain found in members of a novel, eukaryotic family of putative ATPases, e.g., Sec18p, Pas1p, CDC48p, and TBP-1, which function in protein transport pathways, peroxisome assembly, cell division cycle, and gene expression, respectively. Possible implications of these observations are discussed.
Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species
Authors: Fairhead H , Setlow B , Setlow P
Published: November 2016

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.
The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin
Authors: Moore D , Hamilton C M , Maneewannakul K , Mintz Y , Frost L S , Ippen-Ihler K
Published: November 2016

The Escherichia coli F plasmid gene required for amino-terminal acetylation of F-pilin subunits was identified. Using Western blots (immunoblots), we assayed the reaction of monoclonal antibodies with F-pilin polypeptides in inner membrane preparations from various F mutant strains. It was known that JEL92 recognizes an internal pilin epitope and JEL93 recognizes the acetylated amino-terminal sequence (L.S. Frost, J.S. Lee, D.G. Scraba, and W. Paranchych, J. Bacteriol. 168:192-198, 1986). As expected, neither antibody reacted with inner membranes from F- cells or Flac derivatives that do not synthesize pilin. Mutations that affected the individual activities of F tra genes traA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -P, -R, -U, -V and -W or trb genes trbA, -B, -C, -D, -E, -G, -H, and -I did not prevent JEL92 or JEL93 recognition of membrane pilin. However, Hfr deletion mutants that lacked the most-distal transfer region genes did not express pilin that reacted with JEL93. Nevertheless, all strains that retained traA and traQ did express JEL92-reactive pilin polypeptides. Analysis of strains expressing cloned tra segments showed that traA and traQ suffice for synthesis of JEL92-reactive pilin, but synthesis of JEL93-reactive pilin is additionally dependent on traX. We concluded that the traX product is required for acetylation of F pilin. Interestingly, our data also showed that TraA+ TraQ+ cells synthesize two forms of pilin which migrate at approximately 7 and 8 kDa. In TraX+ cells, both become acetylated and react with JEL93. Preparations of wild-type F-pilus filaments contain both types of subunits.
Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes
Authors: Blomqvist K , Nikkola M , Lehtovaara P , Suihko M L , Airaksinen U , Stråby K B , Knowles J K , Penttilä M E
Published: November 2016

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.
Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIA
Authors: Kessler A C , Haase A , Reeves P R
Published: November 2016

Salmonella enterica and Yersinia pseudotuberculosis are the only examples in nature known to use a variety of 3,6-dideoxyhexose derivatives as O antigen constituents. To allow a comparison of the responsible biosynthetic genes of the two organisms, we have sequenced a section of the Y. pseudotuberculosis serogroup IIA rfb region that contained the genes for the abequose biosynthetic pathway. Comparison of the identified genes with the rfb region of S. enterica LT2 showed that the two dideoxyhexose pathway gene clusters are related. The arrangement of the genes was largely conserved, and the G + C compositions of the two DNA regions were strikingly similar; however, the degree of conservation of nucleotide and protein sequences suggested that the two gene clusters have been evolving independently for considerable time. Hybridization experiments showed that the dideoxyhexose pathway genes are widespread throughout the various serogroups of Y. pseudotuberculosis.
Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis
Authors: Sun D , Setlow P
Published: November 2016

A number of Bacillus subtilis genes involved in NAD biosynthesis have been cloned and sequenced. One of the genes encodes a polypeptide homologous to Escherichia coli L-aspartate oxidase, and its mutation resulted in a nicotinic acid (Nic)-dependent phenotype; this gene was termed nadB. A second open reading frame (orf2) was found downstream of nadB, and an insertional plasmid separating orf2 and nadB also gave a Nic-dependent phenotype. This result suggests that orf2 may also be involved in NAD biosynthesis and that nadB and orf2 are in the same operon. Upstream of nadB was a third gene, transcribed in the opposite direction to that of nadB-orf2. The amino acid sequence derived from the third gene was quite similar to those derived from nifS genes of various nitrogen-fixing bacteria; therefore, the third gene was termed nifS. As with nadB and orf2, mutations in nifS also resulted in a Nic-dependent phenotype. The promoter regions of nadB and nifS overlapped each other and both contained -10 and -35 sequences which resemble those of E sigma A-type promoters. Transcription from both the nifS and nadB promoters, as well as expression of a nadB-lacZ fusion, was repressed by Nic. However, nadB transcription and nadB-lacZ expression were decreased, at most, only slightly by a deletion in nifS. The possible role of the nifS gene product in NAD biosynthesis is discussed.
A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli
Authors: Guilloton M B , Lamblin A F , Kozliak E I , Gerami-Nejad M , Tu C , Silverman D , Anderson P M , Fuchs J A
Published: November 2016

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.
The periplasmic dipeptide permease system transports 5-aminolevulinic acid in Escherichia coli
Authors: Verkamp E , Backman V M , Björnsson J M , Söll D , Eggertsson G
Published: November 2016

In a genetic screen designed to generate Escherichia coli strains completely devoid of the heme precursor 5-aminolevulinic acid (ALA), we isolated a class of mutants which were defective for exogenous ALA uptake. The mutations, designated alu (ALA uptake), mapped to the 80-min region of the E. coli chromosome. They were complemented by a recombinant plasmid containing the dpp operon, which encodes a dipeptide permease transport system. Alu mutants displayed a severe reduction in ALA import, as did a strain with a chromosomal insertion in the first gene of the dpp operon. A recognized substrate of Dpp transport, prolyl-glycine, effectively competed with ALA for uptake. E. coli strains defective in ALA biosynthesis (hemA or hemL) require exogenous ALA to achieve wild-type growth but show limited aerobic and anaerobic growth in the absence of ALA. The presence of an alu or dpp mutation in hemA or hemL strains abolishes growth in the absence of ALA and requires increased levels of ALA for normal growth. We conclude that the alu mutations are within the dpp operon and that the dipeptide transport system mediates uptake of the important metabolite ALA.
Genetic structure and regulation of the cysG gene in Salmonella typhimurium
Authors: Goldman B S , Roth J R
Published: November 2016

Siroheme, a cofactor of both sulfite and nitrite reductase in Salmonella typhimurium, requires the cysG gene for its synthesis. Three steps are required to synthesize siroheme from uroporphyrinogen III, the last common intermediate in the heme and siroheme pathways. All previously characterized cysG mutants were shown to be defective for the synthesis of cobalamin (B12), which shares a common precursor with siroheme. Since few cysG auxotrophs had been previously analyzed and since there is no evidence of siroheme mutants outside of the cysG region, we sought to expand the analysis of the region by isolating more mutations and studying the transcriptional regulation of the cysG gene using lacZ fusions. We isolated and analyzed 66 cysG auxotrophs. All were defective for both siroheme and cobalamin synthesis. Five exceptional mutants were partially defective for the synthesis of both and appear to be leaky. Complementation tests with tandem duplications suggest that the mutations causing the Cys auxotrophy affect only one cistron. The cysG gene is transcribed in a clockwise direction; this was demonstrated by a method that permits determining the orientation of two genes of unknown orientation provided their relative map order is known. The cysG gene was not part of the cysteine regulon, but had a substantial basal level of expression which was induced fivefold when cells were grown anaerobically on nitrite. Finally, we used Mud-generated duplications to genetically determine the organization of the cysG and nirB genes.
Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate
Authors: Karlson U , Dwyer D F , Hooper S W , Moore E R , Timmis K N , Eltis L D
Published: November 2016

A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G + C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P-450: cytochrome P-450RR1 was induced by 2-ethoxyphenol, and cytochrome P-450RR2 was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (KS = 4.2 +/- 0.3 microM) and 2-methoxyphenol (KS = 2.0 +/- 0.1 microM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450RR2 by both 4-methoxybenzoate (KS = 2.1 +/- 0.1 microM) and 4-ethoxybenzoate (KS = 1.6 +/- 0.1 microM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-450RR2, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the O-dealkylation of their respective substrates. The respective products of the O-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers.
A physical map of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius 7 chromosome
Authors: Kondo S , Yamagishi A , Oshima T
Published: November 2016

A chromosomal map of the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius 7 was constructed with four restriction enzymes: NotI, BssHII, RsrII, and EagI. The map indicated that the chromosome is a single circular DNA of 2,760 +/- 20 kb (mean +/- standard error of the mean). rRNA genes were also mapped. They were located at one site in the genome.
Escherichia coli ferredoxin NADP+ reductase: activation of E. coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein
Authors: Bianchi V , Reichard P , Eliasson R , Pontis E , Krook M , Jörnvall H , Haggård-Ljungquist E
Published: November 2016

A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia coli. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dA1, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map of E. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dA1 is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dA1 activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.
Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl
Authors: Kohler H P , Schmid A , van der Maarel M
Published: November 2016

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.
Phase variation in Xenorhabdus luminescens: cloning and sequencing of the lipase gene and analysis of its expression in primary and secondary phases of the bacterium
Authors: Wang H , Dowds B C
Published: November 2016

The phenomenon of phase variation in the insect-pathogenic bacterium Xenorhabdus luminescens was investigated. Differential activity of the lipase enzyme (EC 3.1.1.3) was observed between the two phases of the bacteria. The enzyme was found to be secreted into the culture medium, and about five to six times greater specific activity was secreted by the primary phase than by the secondary form. The lipase gene (lip-1) was cloned and sequenced. The data imply that there is only a single Tween 80-utilizing lipase gene in X. luminescens K122. The sequence revealed a translation product of 645 amino acids, from which a hydrophobic leader sequence of 24 amino acids is removed during processing. The structure of the gene was shown to be the same in the primary and secondary forms of X. luminescens. In addition, transcription was found to start at the same position, 169 bp upstream of the translation initiation codon, in the two forms of the bacteria. Equal amounts of lipase RNA accumulated in the two forms, and at least as much lipase protein was secreted by the secondary form as by the primary. This suggests that the difference in specific activity between the enzymes secreted by the two phases probably arises from a posttranslational type of regulation.
A genetic analysis of various functions of the TyrR protein of Escherichia coli
Authors: Yang J , Ganesan S , Sarsero J , Pittard A J
Published: November 2016

The TyrR protein is involved in both repression and activation of the genes of the TyrR regulon. Correction of an error in a previously published sequence has revealed a Cro-like helix-turn-helix DNA-binding domain near the carboxyl terminus. Site-directed mutagenesis in this region has generated a number of mutants that can no longer repress or activate. Deletions of amino acid residues 5 to 42 produced a protein that could repress but not activate. The central domain of TyrR contains an ATP-binding site and is homologous with the NtrC family of activator proteins. A mutation to site A of the ATP-binding site and other mutations in this region affect tyrosine-mediated repression but do not prevent activation or phenylalanine-mediated repression of aroG.
Transposon mutagenesis in Acinetobacter calcoaceticus RAG-1
Authors: Leahy J G , Jones-Meehan J M , Pullias E L , Colwell R R
Published: November 2016

Molecular genetic studies of Acinetobacter spp. have been greatly limited by the lack of a method for transposon mutagenesis. In this study, a genetically engineered derivative of Tn10, mini-Tn10PttKm, was conjugally transferred in plate matings from Escherichia coli SM10[(lambda pir)(pLOFPttKm)] to Acinetobacter calcoaceticus RAG-1. Transfer frequencies were dependent on mating ratios and varied from 7.9 x 10(-8) to 3.4 x 10(-7) per recipient cell. The 27 lipase-deficient transconjugants which were isolated exhibited several different phenotypes, including gelatinase mutants, esterase mutants, and putative auxotrophs. Southern blot analysis confirmed the insertion of the mini-Tn10PttKm transposon in single, unique sites in five transconjugants. Four of five lipase mutants contained single insertions of mini-Tn10PttKm in the same chromosomal restriction fragments. To our knowledge, this is the first report of the use of a transposon for direct, generalized mutagenesis in Acinetobacter spp.
Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi
Authors: Fice D , Shen Z , Byers D M
Published: November 2016

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.
Axial filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase
Authors: Bylund J E , Haines M A , Piggot P J , Higgins M L
Published: November 2016

When chloramphenicol was added to a culture of Bacillus subtilis in early exponential growth, microscopic observation of cells stained by 4',6-diamidino-2-phenylindole showed nucleoids that had changed in appearance from irregular spheres and dumbbells to large, brightly stained spheres and ovals. In contrast, the addition of chloramphenicol to cultures in mid- and late exponential growth showed cells with elongated nucleoids whose frequency and length increased as the culture approached stationary phase. The kinetics of nucleoid elongation after the addition of chloramphenicol to exponential-phase cultures was complex. Immediately after treatment, the rate of nucleoid elongation was very rapid. The nucleoid then elongated steadily for about 4 min, after which the rate of elongation decreased considerably. Nucleoids of cells treated with 6-(p-hydroxyphenylazo)-uracil (an inhibitor of DNA synthesis) exhibited the immediate rapid elongation upon chloramphenicol treatment but not the subsequent changes. These observations suggest that axial filament formation during stationary phase (stage I of sporulation) in the absence of chloramphenicol results from changes in nucleoid structure that are initiated earlier, during exponential growth.
Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for symbiotic nitrogen fixation is atypical
Authors: Watson R J , Rastogi V K
Published: November 2016

In Rhizobium meliloti, an aspartate aminotransferase (AspAT) encoded within a 7.3-kb HindIII fragment was previously shown to be required for symbiotic nitrogen fixation and aspartate catabolism (V. K. Rastogi and R.J. Watson, J. Bacteriol. 173:2879-2887, 1991). A gene coding for an aromatic aminotransferase located within an 11-kb HindIII fragment was found to complement the AspAT deficiency when overexpressed. The genes encoding these two aminotransferases, designated aatA and tatA, respectively, have been localized by subcloning and transposon Tn5 mutagenesis. Sequencing of the tatA gene revealed that it encodes a protein homologous to an Escherichia coli aromatic aminotransferase and most of the known AspAT enzymes. However, sequencing of the aatA gene region revealed two overlapping open reading frames, neither of which encoded an enzyme with homology to the typical AspATs. Polymerase chain reaction was used to selectively generate one of the candidate sequences for subcloning. The cloned fragment complemented the original nitrogen fixation and aspartate catabolism defects and was shown to encode an AspAT with the expected properties. Sequence analysis showed that the aatA protein has homology to AspATs from two thermophilic bacteria and the eukaryotic tyrosine aminotransferases. These aminotransferases form a distinct class in which only 13 amino acids are conserved in comparison with the well-known AspAT family. DNA homologous to the aatA gene was found to be present in Agrobacterium tumefaciens and other rhizobia but not in Klebsiella pneumoniae or E. coli.
Modulation of the heat shock response by one-carbon metabolism in Escherichia coli
Authors: Gage D J , Neidhardt F C
Published: November 2016

A genetic screen designed to isolate mutants of Escherichia coli W3110 altered in the ability to induce the heat shock response identified a strain unable to induce the heat shock proteins in a rich, defined medium lacking methionine after exposure to 2,4-dinitrophenol. This strain also grew slowly at 28 degrees C and linearly at 42 degrees C in this medium. The abnormal induction of the heat shock proteins and abnormal growth at both high and low temperatures were reversed when methionine was included in the growth medium. The mutation responsible for these phenotypes mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. This reaction is the major source of glycine and one-carbon units in the cell. Because fixed one-carbon units, in the form of methionine, allowed mutant cells to induce the heat shock response after exposure to 2,4-dinitrophenol, a one-carbon restriction may be responsible for the phenotypes described above.
Analysis of features contributing to activity of the single-stranded origin of Bacillus plasmid pBAA1
Authors: Seery L , Devine K M
Published: November 2016

The features which contribute to the activity of the single-stranded origin of the Bacillus plasmid pBAA1 were investigated. This origin is contained on a DNA fragment greater than 116 but less than 191 bases in size. There is the potential to form three stem-loop structures within this fragment. Comparison of the sequence of this origin from pBAA1 with the sequence of a homologous fragment from the Bacillus thuringiensis plasmid pGI2 indicates that both the structure and the relative positioning of the predicted stem-loops are important for origin activity. Deletion analysis suggests that it is the structure of stem-loop III which is important, because it can be replaced by a nonrelated dyad element without significant loss of origin activity. Three sequence motifs are conserved between the origins from pBAA1 and pGI2. Mutation of motif 1 leads to attenuation of single-stranded origin activity. A second motif (motif 3) shares significant homology with a group of single-strand initiation (ssi) sites found on plasmids isolated from Escherichia coli, suggesting that it also contributes to single-stranded origin activity. Our results also indicate that RNA polymerase is utilized to synthesize the RNA primer at the pBAA1 single-stranded origin and that this origin can function in both Bacillus subtilis and Staphylococcus aureus.
Accumulation of bldA-specified tRNA is temporally regulated in Streptomyces coelicolor A3(2)
Authors: Leskiw B K , Mah R , Lawlor E J , Chater K F
Published: November 2016

Deletion of the bldA gene of Streptomyces coelicolor A3(2), which encodes the only tRNA for the rare UUA codon, had no obvious effects on primary growth but interfered with aerial mycelium formation and antibiotic production. To investigate the possible regulatory role of bldA, its transcription start point was identified, and time courses were determined for the appearance of its primary transcript, the processing of the primary transcript to give a mature 5' end, and the apparent efficiency of translation of ampC mRNA, which contains multiple UUA codons. The bldA promoter was active at all times, but processing of the 5' end of the primary transcript was comparatively inefficient in young cultures. This may perhaps involve an antisense RNA, evidence of which was provided by promoter probing and in vitro transcription. The presence of low levels of the processed form of the tRNA in young cultures followed by increased abundance in older cultures contrasted with the pattern observed for accumulation of a different, presumably typical tRNA which was approximately equally abundant throughout growth. The increased accumulation of the 5' processed form of bldA tRNA coincided with more-efficient translation of ampC mRNA in older cultures, supporting the hypothesis that in at least some physiological conditions, bldA may have a regulatory influence on events late in growth, such as morphological differentiation and antibiotic production.
Global regulation of gene expression in Escherichia coli
Authors: Chuang S E , Daniels D L , Blattner F R
Published: November 2016

Global transcription responses of Escherichia coli to various stimuli or genetic defects were studied by measuring mRNA levels in about 400 segments of the genome. Measuring mRNA levels was done by analyzing hybridization to DNA dot blots made with overlapping lambda clones spanning the genome of E. coli K-12. Conditions examined included isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, heat shock, osmotic shock, starvation for various nutrients, entrance of cells into the stationary phase of growth, anaerobic growth in a tube, growth in the gnotobiotic mouse gut, and effects of pleiotropic mutations rpoH, himA, topA, and crp. Most mapped genes known to be regulated by a particular situation were successfully detected. In addition, many chromosomal regions containing no previously known regulated genes were discovered that responded to various stimuli. This new method for studying globally regulated genetic systems in E. coli combines detection, cloning, and physical mapping of a battery of coregulated genes in one step.
Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis
Authors: Kunji E R , Smid E J , Plapp R , Poolman B , Konings W N
Published: November 2016

Lactococcus lactis ML3 possesses two different peptide transport systems of which the substrate size restriction and specificity have been determined. The first system is the earlier-described proton motive force-dependent di-tripeptide carrier (E. J. Smid, A. J. M. Driessen, and W. N. Konings, J. Bacteriol. 171:292-298, 1989). The second system is a metabolic energy-dependent oligopeptide transport system which transports peptides of four to at least six amino acid residues. The involvement of a specific oligopeptide transport system in the utilization of tetra-alanine and penta-alanine was established in a mutant of L. lactis MG1363 that was selected on the basis of resistance to toxic analogs of alanine and alanine-containing di- and tripeptides. This mutant is unable to transport alanine, dialanine, and trialanine but still shows uptake of tetra-alanine and penta-alanine. The oligopeptide transport system has a lower activity than the di-tripeptide transport system. Uptake of oligopeptides occurs in the absence of a proton motive force and is specifically inhibited by vanadate. The oligopeptide transport system is most likely driven by ATP or a related energy-rich, phosphorylated intermediate.