bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and biochemical characterization of a hydroxyneurosporene desaturase involved in the biosynthetic pathway of the carotenoid spheroidene in Rhodobacter sphaeroides
Authors: Albrecht M , Ruther A , Sandmann G
Published: November 2016

Hydroxyneurosporene desaturase is involved in the carotenoid biosynthetic pathway of Rhodobacter species. The gene encoding this enzyme was expressed in Escherichia coli, purified, and biochemically characterized. The resulting protein contained an N-terminal six-histidine extension which derived from the cloning vector; this allowed for a one-step purification of the enzyme to homogeneity after solubilization with Nonidet P-40. The hydrogen acceptor in the C-3,4 desaturation reaction was molecular oxygen. NAD+, NADP+, and flavin adenine dinucleotide had no influence on enzymatic activity. Different acyclic 1-hydroxycarotenoids were tested as substrates. Very good conversion was achieved with 1-hydroxyneurosporene and 1-hydroxylycopene, whereas 1-hydroxy-gamma-carotene and 1,1'-dihydroxylycopene were much less effective. From 1'-hydroxy-3,4-didehydrolycopene only trace amounts of product were obtained, and 1-methoxyneurosporene was not converted by purified hydroxyneurosporene desaturase. A Km of 13.4 microM was determined for 1-hydroxyneurosporene.
The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus
Authors: Ahn Y T , Wu X L , Biswal S , Velmurugan S , Volkert F C , Jayaram M
Published: November 2016

The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain. Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.
Methylmalonyl coenzyme A selectivity of cloned and expressed acyltransferase and beta-ketoacyl synthase domains of mycocerosic acid synthase from Mycobacterium bovis BCG
Authors: Fernandes N D , Kolattukudy P E
Published: November 2016

Methyl-branched fatty acids and polyketides occur in a variety of living organisms. Previous studies have established that multifunctional enzymes use methylmalonyl coenzyme A (CoA) as the substrate to generate methyl-branched products such as mycocerosic acids and polyketides. However, we do not know which of the component activities show selectivity for methylmalonyl-CoA in any biological system. A comparison of homologies of the domains of the multifunctional synthases that selectively use malonyl-CoA or methylmalonyl-CoA suggested that the acyltransferase (AT) and beta-ketoacyl synthase (KS) domains might be responsible for the substrate selectivity. To test this hypothesis, we expressed the AT and KS domains of the mycocerosic acid synthase (MAS) gene from Mycobacterium bovis BCG in Escherichia coli and examined whether they confer to synthases that normally do not use methylmalonyl-CoA the ability to incorporate methylmalonyl-CoA into fatty acids. Both the AT and the KS domains of MAS showed selectivity for methylmalonyl-CoA over malonyl-CoA. Acyl carrier protein (ACP)-dependent elongation of the n-C12 acyl primer mainly by one methylmalonyl-CoA unit was catalyzed by an E. coli fatty acid synthase preparation only in the presence of the expressed MAS domains. An ACP-dependent elongation of the n-C20 acyl primer by one methylmalonyl-CoA extender unit was catalyzed by fatty acid synthase from Mycobacterium smegmatis only in the presence of the expressed MAS domains. These results show methylmalonyl-CoA selectivity for the AT and KS domains of MAS. These domains may be useful in producing novel polyketides by genetic engineering.
Specificities of FemA and FemB for different glycine residues: FemB cannot substitute for FemA in staphylococcal peptidoglycan pentaglycine side chain formation
Authors: Ehlert K , Schröder W , Labischinski H
Published: November 2016

The femAB operon codes for two nearly identical approximately 50-kDa proteins involved in the formation of the staphylococcal pentaglycine interpeptide bridge. Sequencing and analysis of the femA region of mutants isolated by chemical mutagenesis and selection for lysostaphin resistance revealed point mutations leading to the expression of truncated FemA proteins. These femA mutants, although still producing an intact FemB, exhibited a phenotype identical as that described for femAB double mutants. Thus, FemA seems to be essential for the addition of glycine residues 2 and 3 only, whereas FemB is involved in the attachment of exclusively glycine residues 4 and 5. Although FemB has 39% identity with FemA, it cannot substitute for FemA. The FemA and FemB proteins seem to be highly specific in regard to the position of the glycine residues that they attach.
Essential role of the consensus nucleotide-binding site of PtlH in secretion of pertussis toxin from Bordetella pertussis
Authors: Kotob S I , Burns D L
Published: November 2016

PtlH is a member of a specialized set of transport proteins that is essential for secretion of pertussis toxin (PT) from Bordetella pertussis. Previously, PtlH was shown to contain a consensus nucleotide-binding motif. Here, we demonstrate that introduction of plasmids containing mutant forms of ptlH, altered in the putative nucleotide-binding region, into a wild-type strain of B. pertussis resulted in inhibition of PT secretion. Thus, this region of PtlH appears to be essential for protein function. Moreover, the observed dominant negative phenotype suggests that PtlH either functions as a multimer or interacts with another component necessary for secretion of PT.
Role of NifS in maturation of glutamine phosphoribosylpyrophosphate amidotransferase
Authors: Chen S , Zheng L , Dean D R , Zalkin H
Published: November 2016

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is synthesized as an inactive precursor that requires two maturation steps: incorporation of a [4Fe-4S] center and cleavage of an 11-residue NH2-terminal propeptide. Overproduction from a multicopy plasmid in Escherichia coli leads to the formation of soluble proenzyme and mature enzyme forms as well as a small fraction of insoluble proenzyme. Heterologous expression of Azotobacter vinelandii nifS from a compatible plasmid increased the maturation of the soluble proenzyme three- to fourfold without influencing the content of the insoluble fraction. These results support a role for NifS in heterologous Fe-S cluster assembly and enzyme maturation.
Temperature-sensitive lesions in the Francisella novicida valA gene cloned into an Escherichia coli msbA lpxK mutant affecting deoxycholate resistance and lipopolysaccharide assembly at the restrictive temperature
Authors: McDonald M K , Cowley S C , Nano F E
Published: November 2016

The valAB locus of Francisella novicida has previously been found to be highly similar at the deduced amino acid level to msbA lpxK of Escherichia coli. Both ValA and MsbA are members of the superfamily of ABC transporters, and they appear to have similar functions. In this study we describe the isolation of a temperature-sensitive valAB locus. DNA sequence analysis indicates that the only changes to the ValAB deduced amino acid sequence are changes of S453 to an F and T458 to an I in ValA. E. coli strains defective in msbA and expressing temperature-sensitive ValA rapidly ceased growth when shifted from a permissive temperature to a restrictive temperature. After 1 h at the restrictive temperature, cells were much more sensitive to deoxycholate treatment. To test the hypothesis that ValA is responsible for the transport or assembly of lipopolysaccharide, we introduced gseA, a Kdo (3-deoxy-D-manno-octulosonic acid) transferase from Chlamydia trachomatis, into a strain with a temperature-sensitive valA allele and a nonfunctional msbA locus. These recombinants were defective in cell surface expression of the chlamydial genus-specific epitope within 15 min of a shift to the nonpermissive temperature. Also, there was enhanced association of the epitope with the inner membrane after a shift to the nonpermissive temperature. Thus, we propose that ValA is involved in the transport of lipopolysaccharide to the outer membrane.
N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere
Authors: Wood D W , Gong F , Daykin M M , Williams P , Pierson L S
Published: November 2016

Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which suppress take-all disease of wheat by inhibition of the causative agent Gaeumannomyces graminis var. tritici. Phenazines also enhance survival of 30-84 within the wheat rhizosphere in competition with other organisms. Expression of the phenazine biosynthetic operon is controlled by the phzR/phzI N-acyl-homoserine lactone (AHL) response system (L. S. Pierson III et al., J. Bacterial 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). By using high-pressure liquid chromatography coupled with high-resolution mass spectrometry, the AHL produced by PhzI has now been identified as N-hexanoyl-homoserine lactone (HHL). In addition, the ability of HHL to serve as an interpopulation signal molecule in the wheat rhizosphere has been examined by using isogenic reporter strains. Disruption of phzI reduced expression of the phenazine biosynthetic operon 1,000-fold in the wheat rhizosphere. Coinoculation of an isogenic strain which produced the endogenous HHL signal restored phenazine gene expression in the phzI mutant to wild-type levels in situ. These results demonstrate that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere.
Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity
Authors: Peist R , Koch A , Bolek P , Sewitz S , Kolbus T , Boos W
Published: November 2016

malQ mutants of Escherichia coli lacking amylomaltase cannot grow on maltose. They express the maltose system constitutively and are sensitive to maltose when grown on another carbon source. In an attempt to isolate a multicopy suppressor that would result in growth on maltose, we transformed a malQ mutant with a gene bank of E. coli DNA which had been digested with Sau3a and cloned in pBR322. We screened the transformants on MacConkey maltose plates. A colony was isolated that appeared to be resistant to maltose and was pink on these plates, but it was still unable to grow on minimal medium with maltose as the carbon source. The plasmid was isolated, and the gene causing this phenotype was characterized. The deduced amino acid sequence of the encoded protein shows homology to that of lipases and esterases. We termed the gene aes, for acetyl esterase. Extracts of cells harboring plasmid-encoded aes under its own promoter exhibit a fivefold higher capacity to hydrolyze p-nitrophenyl acetate than do extracts of cells of plasmid-free strains. Similarly, strains harboring plasmid-encoded aes are able to grow on triacetyl glycerol (triacetin) whereas the plasmid-free strains are not. The expression of plasmid-encoded aes resulted in strong repression of the maltose transport genes in malT+ strains (10-fold reduction), but not in a malT(Con) strain which is independent of the inducer. Also, overproduction of MalT counteracted the Aes-dependent repression, indicating a direct interaction between MalT and Aes.
Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis
Authors: Kondoh O , Tachibana Y , Ohya Y , Arisawa M , Watanabe T
Published: November 2016

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.
Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B
Authors: Kim S W , Joo S , Choi G , Cho H S , Oh B H , Choi K Y
Published: November 2016

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.
Regulation of expression of the pilA gene in Myxococcus xanthus
Authors: Wu S S , Kaiser D
Published: November 2016

Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.
bldA-dependent expression of the Streptomyces exfoliatus M11 lipase gene (lipA) is mediated by the product of a contiguous gene, lipR, encoding a putative transcriptional activator
Authors: Servín-González L , Castro C , Pérez C , Rubio M , Valdez F
Published: November 2016

Extracellular lipase synthesis by Streptomyces lividans 66 carrying the cloned lipase gene (lipA) from Streptomyces exfoliatus M11 was found to be growth phase dependent, since lipase was secreted into the medium mainly during the stationary phase; S1 nuclease protection experiments revealed abundant lipA transcripts in RNA preparations obtained during the stationary phase but not in those obtained during exponential growth. Transcription from the lipA promoter was dependent on the presence of lipR, a contiguous downstream gene with a very high guanine-plus-cytosine content (80.2%). The deduced lipR product consists of a protein of 934 amino acids that shows similarity to known transcriptional activators and has a strong helix-turn-helix motif at its C terminus; this motif is part of a domain homologous to DNA-binding domains of bacterial regulators of the UhpA/LuxR superfamily. The lipR sequence revealed the presence of a leucine residue, encoded by the rare TTA codon, which caused bldA dependence of lipA transcription in Streptomyces coelicolor A3(2); replacement of the TTA codon by the alternate CTC leucine codon alleviated bidA dependence but not the apparent growth phase-dependent regulation of lipA transcription. When lipR expression was induced in a controlled fashion during the exponential growth phase, by placing it under the inducible tipA promoter, lipase synthesis was shifted to the exponential growth phase, indicating that the timing of lipR expression, and not its bldA dependence, is the main cause for stationary-phase transcription of lipA.
A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability
Authors: Parish T , Liu J , Nikaido H , Stoker N G
Published: November 2016

A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes.
msDNA-Ec48, the smallest multicopy single-stranded DNA from Escherichia coli
Authors: Mao J R , Inouye S , Inouye M
Published: November 2016

Previously we have reported a novel bacterial reverse transcriptase (RT) from Escherichia coli ECOR58 strains in which the YXDD box was replaced with LVDD (J.-R. Mao, S. Inouye, and M. Inouye, Biochem. Biophys. Res. Commun. 227:489-493, 1996). Here we determined the structure of the multicopy single-stranded DNA (msDNA) produced by the RT. The msDNA was found to consist of a single-stranded DNA of 48 nucleotides in length, the shortest msDNA thus far identified from natural sources. The msDNA, the RT, and the retron are designated msDNA-Ec48, RT-Ec48, and retron-Ec48, respectively. On the basis of the structure of the msr gene, the RNA molecule of msDNA-Ec48 is predicted to be composed of 119 ribonucleotides; it is the longest RNA among the known msDNAs. Analysis of the DNA sequences flanking the retron indicates that retron-Ec48 is associated with a prophage related to phages P2 and P4.
Comparison of the bacterial HelA protein to the F508 region of the cystic fibrosis transmembrane regulator
Authors: Goldman B S , Sherman D A , Kranz R G
Published: November 2016

The HelA protein of Rhodobacter capsulatus is the ATP-binding-cassette subunit of an exporter complex required for cytochrome c biogenesis. By primary sequence comparisons the F88 residue of HelA is similar to the F508 residue of the cystic fibrosis transmembrane regulator (CFTR) protein. Previous studies have established that CFTR F508delta or F508R proteins are defective but F508C is functional. Our results demonstrate that the HelA F88 mutants functionally mimic the phenotypes of known CFTR F508 mutants. The phenotypes of additional HelA mutants and the in vivo steady-state levels of these proteins are also reported.
Dinitrogenase reductase-activating glycohydrolase can be released from chromatophores of Rhodospirillum rubrum by treatment with MgGDP
Authors: Norén A , Nordlund S
Published: November 2016

Dinitrogenase reductase-activating glycohydrolase (DRAG), involved in the regulation of nitrogenase activity in Rhodospirillum rubrum, is associated with chromatophore membranes in cell extracts. We show that DRAG can be specifically released by treatment with MgGDP; other nucleotides studied had no effect. The DRAG activity released corresponds to the release of DRAG protein.
Bordetella bronchiseptica expresses the fimbrial structural subunit gene fimA
Authors: Boschwitz J S , van der Heide H G , Mooi F R , Relman D A
Published: November 2016

The differential host species specificities of Bordetella pertussis, B. parapertussis, and B. bronchiseptica might be explained by polymorphisms in adherence factor genes. We have found that B. parapertussis and B. bronchiseptica, unlike B. pertussis, contain a full-length gene for the fimbrial subunit FimA. B. bronchiseptica expresses fimA in a BvgAS-dependent fashion.
Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase
Authors: Rashkova S , Spudich G M , Christie P J
Published: November 2016

The VirB11 ATPase is a putative component of the transport machinery responsible for directing the export of nucleoprotein particles (T complexes) across the Agrobacterium tumefaciens envelope to susceptible plant cells. Fractionation and membrane treatment studies showed that approximately 30% of VirB11 partitioned as soluble protein, whereas the remaining protein was only partially solubilized with urea from cytoplasmic membranes of wild-type strain A348 as well as a Ti-plasmidless strain expressing virB11 from an IncP replicon. Mutations in virB11 affecting protein function were mapped near the amino terminus (Q6L, P13L, and E25G), just upstream of a region encoding a Walker A nucleotide-binding site (F154H;L155M), and within the Walker A motif (P170L, K175Q, and delta GKT174-176). The K175Q and delta GKT174-176 mutant proteins partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. The virB11F154H;L155M allele was transdominant over wild-type virB11 in a merodiploid assay, providing strong evidence that at least one form of VirB11 functions as a homo- or heteromultimer. An allele with a deletion of the first half of the gene, virB11 delta1-156, was transdominant in a merodiploid assay, indicating that the C-terminal half of VirB11 contains a protein interaction domain. Products of both virB11 delta1-156 and virB11 delta158-343, which synthesizes the N-terminal half of VirB11, associated tightly with the A. tumefaciens membrane, suggesting that both halves of VirB11 contain membrane interaction determinants.
4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases
Authors: Gibson J , Dispensa M , Harwood C S
Published: November 2016

The anaerobic degradation of 4-hydroxybenzoate is initiated by the formation of 4-hydroxybenzoyl coenzyme A, with the next step proposed to be a dehydroxylation to benzoyl coenzyme A, the starting compound for a central pathway of aromatic compound ring reduction and cleavage. Three open reading frames, divergently transcribed from the 4-hydroxybenzoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris. These genes, named hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respectively. The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nicotine metabolism that have conserved binding sites for [2Fe-2S] clusters and a molybdenum cofactor. Cassette disruption of the hbaB gene yielded a mutant that was unable to grow anaerobically on 4-hydroxybenzoate but grew normally on benzoate. The hbaB mutant cells did not accumulate [14C]benzoyl coenzyme A during short-term uptake of [14C]4-hydroxybenzoate, but benzoyl coenzyme A was the major radioactive metabolite formed by the wild type. In addition, crude extracts of the mutant failed to convert 4-hydroxybenzoyl coenzyme A to benzoyl coenzyme A. This evidence indicates that the hbaBCD genes encode the subunits of a 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating). The sizes of the specified polypeptides are similar to those reported for 4-hydroxybenzoyl coenzyme A reductase isolated from the denitrifying bacterium Thauera aromatica. The amino acid consensus sequence for a molybdenum cofactor binding site is in HbaC. This cofactor appears to be an essential component because anaerobic growth of R. palustris on 4-hydroxybenzoate, but not on benzoate, was retarded unless 0.1 microM molybdate was added to the medium. Neither tungstate nor vanadate replaced molybdate, and tungstate competitively inhibited growth stimulation by molybdate.
Mutations that increase expression of the rpoS gene and decrease its dependence on hfq function in Salmonella typhimurium
Authors: Brown L , Elliott T
Published: November 2016

The RpoS transcription factor (also called sigmaS or sigma38) is required for the expression of a number of stationary-phase and osmotically inducible genes in enteric bacteria. RpoS is also a virulence factor for several pathogenic species, including Salmonella typhimurium. The activity of RpoS is regulated in response to many different signals, at the levels of both synthesis and proteolysis. Previous work with rpoS-lac protein fusions has suggested that translation of rpoS requires hfq function. The product of the hfq gene, host factor I (HF-I), is a ribosome-associated, site-specific RNA-binding protein originally characterized for its role in replication of the RNA bacteriophage Qbeta of Escherichia coli. In this study, the role of HF-I was explored by isolating suppressor mutations that map to the region directly upstream of rpoS. These mutations increase rpoS-lac expression in the absence of HF-I and also confer substantial independence from HF-I. DNA sequence analysis of the mutants suggests a model in which the RNA secondary structure near the ribosome binding site of the rpoS mRNA plays an important role in limiting expression in the wild type. Genetic tests of the model confirm its predictions, at least in part. It seems likely that the mutations analyzed here activate a suppression pathway that bypasses the normal HF-I-dependent route of rpoS expression; however, it is also possible that some of them identify a sequence element with an inhibitory function that is directly counteracted by HF-I.
Analysis of the cbbXYZ operon in Rhodobacter sphaeroides
Authors: Gibson J L , Tabita F R
Published: November 2016

Three genes, cbbX, cbbY, and cbbZ were found downstream from the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes of Rhodobacter sphaeroides. As in chemoautotrophic bacteria, cbbZ was shown to encode phosphoglycolate phosphatase (PGP), whereas the identities of cbbX and cbbY are not known. To determine the physiological function of the cbbXYZ gene products, we constructed R. sphaeroides strains in which the genes were inactivated and characterized the resultant mutant strains according to growth phenotype and levels of RubisCO and PGP. Only a mutation in cbbX resulted in a discernible phenotype, namely, impaired photoautotrophic growth. No PGP activity was observed in any of the mutants, suggesting that the three genes are transcriptionally linked. Studies with a spontaneous chemoautotrophic competent derivative of the CBBX mutant suggested that the cbbXYZ gene products are not essential for chemoautotrophic growth. PGP activity determined in the wild-type strain grown under a variety of growth conditions, and in various strains containing mutations in Calvin-Benson-Bassham cycle structural and regulatory genes, indicated that transcription of the cbb(I) operon influenced expression of the downstream cbbXYZ operon.
Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes
Authors: de Crécy-Lagard V , Blanc V , Gil P , Naudin L , Lorenzon S , Famechon A , Bamas-Jacques N , Crouzet J , Thibaut D
Published: November 2016

Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed.
Apparent and real recombination frequencies in multicopy plasmids: the need for a novel approach in frequency determination
Authors: Chédin F , Dervyn R , Ehrlich S D , Noirot P
Published: November 2016

Recombination studies of bacteria are often carried out with multicopy plasmids, and recombination frequencies are often deduced from the proportion of cells in the population that express a recombinant phenotype. These frequencies should however be called apparent frequencies, since detection of the recombinant cells requires not only the formation of a rearranged plasmid but also its establishment in the cell. The establishment of the recombinant plasmid can possibly be affected by its interaction with the parental plasmids. To test this hypothesis, we have used a plasmid system enabling the study of deletion formation between short direct repeats (18 bp) in Bacillus subtilis and developed a method by which deletion frequencies are measured under conditions under which interaction is abolished. Real deletion frequencies were thus determined and compared with apparent deletion frequencies. Real frequencies were underestimated by a factor ranging from 4- to 500-fold, depending upon the plasmid under study. This implies that a large majority of the recombinant molecules that are formed are generally not detected. We show that apparent deletion frequencies strongly depend upon (i) the parental plasmid copy number, (ii) the ability of the recombinant molecules to form heterodimeric plasmids, and (iii) the fitness of the recombinant molecules relative to that of parental molecules. Finally, we show that under conditions under which all recombinant molecules are scored, transcription can inhibit the deletion process 10-fold.
Determinants for overproduction of the Pseudomonas oleovorans cytoplasmic membrane protein alkane hydroxylase in alk+ Escherichia coli W3110
Authors: Nieboer M , Gunnewijk M , van Beilen J B , Witholt B
Published: November 2016

The Pseudomonas oleovorans alkB gene is expressed in alk+ Escherichia coli W3110 to 10 to 15% of the total cell protein, which is exceptional for a (foreign) cytoplasmic membrane protein. In other E. coli recombinants such as alk+ HB101, AlkB constitutes 2 to 3% of the total protein. In this study, we have investigated which factors determine the expression level of alkB in alk+ W3110. In particular, we have investigated the role of AlkB-induced stimulation of phospholipid synthesis. Blocking phospholipid synthesis in alk+ W3110 did not specifically alter the expression of alkB, and we conclude that stimulation of phospholipid synthesis is not a prerequisite for high-level expression of the membrane protein. W3110 is able to produce exceptionally high levels of alkane monooxygenase, because the rate of alkB mRNA synthesis in W3110 is an order of magnitude higher than that in HB101. This may be due in part to the higher copy number of pGEc47 in W3110 in comparison with HB101.
Lack of functional complementation between Bordetella pertussis filamentous hemagglutinin and Proteus mirabilis HpmA hemolysin secretion machineries
Authors: Jacob-Dubuisson F , Buisine C , Willery E , Renauld-Mongénie G , Locht C
Published: November 2016

The gram-negative bacterium Bordetella pertussis has adapted specific secretion machineries for each of its major secretory proteins. In particular, the highly efficient secretion of filamentous hemagglutinin (FHA) is mediated by the accessory protein FhaC. FhaC belongs to a family of outer membrane proteins which are involved in the secretion of large adhesins or in the activation and secretion of Ca2+-independent hemolysins by several gram-negative bacteria. FHA shares with these hemolysins a 115-residue-long amino-proximal region essential for its secretion. To compare the secretory pathways of these hemolysins and FHA, we attempted functional transcomplementation between FhaC and the Proteus mirabilis hemolysin accessory protein HpmB. HpmB could not promote the secretion of FHA derivatives. Likewise, FhaC proved to be unable to mediate secretion and activation of HpmA, the cognate secretory partner of HpmB. In contrast, ShlB, the accessory protein of the closely related Serratia marcescens hemolysin, was able to activate and secrete HpmA. Two invariant asparagine residues lying in the region of homology shared by secretory proteins and shown to be essential for the secretion and activation of the hemolysins were replaced in FHA by site-directed mutagenesis. Replacements of these residues indicated that both are involved in, but only the first one is crucial to, FHA secretion. This slight discrepancy together with the lack of functional complementation demonstrates major differences between the hemolysins and FHA secretion machineries.
Two polypeptide products of the Escherichia coli cell division gene ftsW and a possible role for FtsW in FtsZ function
Authors: Khattar M M , Addinall S G , Stedul K H , Boyle D S , Lutkenhaus J , Donachie W D
Published: November 2016

Two new mutations in the cell division gene ftsW have been isolated and characterized. The ftsW263(Ts) mutation results in a block to division at the initiation stage, similar to that previously observed with the ftsW201(Ts) mutation. The ftsW1640(Ts) mutation, however, causes a block to division at a later stage. The ftsW201 and ftsW263 mutants were shown to be phenotypically sensitive to the genetic background and growth conditions and are possibly relA dependent. Immunofluorescence microscopy showed that the FtsZ protein can localize to presumptive division sites in strains carrying ftsW(Ts) mutations at the nonpermissive temperature, suggesting that FtsW is unlikely to be specifically required for the localization of FtsZ to the division site. Examination of the localization of FtsZ in an ftsW rodA double mutant (lemon-shaped cells) revealed several classes of cells ranging from a common class where an FtsZ ring structure is absent to a class where FtsZ forms a complete ring at the midpoint of a lemon-shaped cell, suggesting a role for FtsW in the establishment of a stable FtsZ-based septal structure. We further demonstrate that two FtsW peptides, FtsWL (large) and FtsWS (small), can be identified and that the expression of ftsWS is sufficient for complementation of ftsW(Ts) mutations.
Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids
Authors: Montesinos M L , Herrero A , Flores E
Published: November 2016

The activities of uptake of thirteen 14C-labeled amino acids were determined in nine cyanobacteria, including the unicellular strains Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803; the filamentous strain Pseudanabaena sp. strain PCC 6903, and the filamentous, heterocyst-forming strains Anabaena sp. strains PCC 7120 and PCC 7937; Nostoc sp. strains PCC 7413 and PCC 7107; Calothrix sp. strain PCC 7601 (which is a mutant unable to develop heterocysts); and Fischerella muscicola UTEX 1829. Amino acid transport mutants, selected as mutants resistant to some amino acid analogs, were isolated from the Anabaena, Nostoc, Calothrix, and Pseudanabaena strains. All of the tested cyanobacteria bear at least a neutral amino acid transport system, and some strains also bear transport systems specific for basic or acidic amino acids. Two genes, natA and natB, encoding elements (conserved component, NatA, and periplasmic binding protein, NatB) of an ABC-type permease for neutral amino acids were identified by insertional mutagenesis of strain PCC 6803 open reading frames from the recently published genomic DNA sequence of this cyanobacterium. DNA sequences homologous to natA and natB from strain PCC 6803 were detected by hybridization in eight cyanobacterial strains tested. Mutants unable to transport neutral amino acids, including natA and natB insertional mutants, accumulated in the extracellular medium a set of amino acids that always included Ala, Val, Phe, Ile, and Leu. A general role for a cyanobacterial neutral amino acid permease in recapture of hydrophobic amino acids leaked from the cells is suggested.
Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation
Authors: Kuzminov A , Stahl F W
Published: November 2016

To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair. Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease. We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities. Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme. We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells. Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease. A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E. coli is discussed.
Properties and gene structure of the Thermotoga maritima alpha-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium
Authors: Liebl W , Stemplinger I , Ruile P
Published: November 2016

Thermotoga maritima MSB8 has a chromosomal alpha-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-alpha-glucanotransferase of the same strain and to alpha-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can be translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima alpha-amylase appears to be the first known example of a lipoprotein alpha-amylase. This is in agreement with observations pointing to the membrane localization of this enzyme in T. maritima. Following the signal peptide, a 25-residue putative linker sequence rich in serine and threonine was found. The amylase gene was expressed in E. coli, and the recombinant enzyme was purified and characterized. The molecular mass of the recombinant enzyme was estimated at 61 kDa by denaturing gel electrophoresis (63 kDa by gel permeation chromatography). In a 10-min assay at the optimum pH of 7.0, the optimum temperature of amylase activity was 85 to 90 degrees C. Like the alpha-amylases of many other organisms, the activity of the T. maritima alpha-amylase was dependent on Ca2+. The final products of hydrolysis of soluble starch and amylose were mainly glucose and maltose. The extraordinarily high specific activity of the T. maritima alpha-amylase (about 5.6 x 10(3) U/mg of protein at 80 degrees C, pH 7, with amylose as the substrate) together with its extreme thermal stability makes this enzyme an interesting candidate for biotechnological applications in the starch processing industry.
Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression
Authors: Haussmann C , Rohdich F , Lottspeich F , Eberhardt S , Scheuring J , Mackamul S , Bacher A
Published: November 2016

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome.
Genetic characterization of the pdu operon: use of 1,2-propanediol in Salmonella typhimurium
Authors: Walter D , Ailion M , Roth J
Published: November 2016

Salmonella typhimurium is able to catabolize 1,2-propanediol for use as the sole carbon and energy source; the first enzyme of this pathway requires the cofactor adenosyl cobalamin (Ado-B12). Surprisingly, Salmonella can use propanediol as the sole carbon source only in the presence of oxygen but can synthesize Ado-B12 only anaerobically. To understand this situation, we have studied the pdu operon, which encodes proteins for propanediol degradation. A set of pdu mutants defective in aerobic degradation of propanediol (with exogenous vitamin B12) defines four distinct complementation groups. Mutations in two of these groups (pduC and pduD) eliminate propanediol dehydratase activity. Based on mutant phenotypes, a third complementation group (pduG) appears to encode a cobalamin adenosyl transferase activity. No function has been assigned to the pduJ complementation group. Propionaldehyde dehydrogenase activity is eliminated by mutations in any of the four identified complementation groups, suggesting that this activity may require a complex of proteins encoded by the operon. None of the mutations analyzed affects either of the first two genes of the operon (pduA and pduB), which were identified by DNA sequence analysis. Available data suggest that the pdu operon includes enough DNA for about 15 genes and that the four genetically identified genes are the only ones required for aerobic use of propanediol.
AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae
Authors: Schlumpberger M , Schaeffeler E , Straub M , Bredschneider M , Wolf D H , Thumm M
Published: November 2016

Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.