bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida
Authors: Parales R E , Harwood C S
Published: November 2016

beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.
Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme
Authors: Heaton M P , Neuhaus F C
Published: November 2016

The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacillus casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound lipoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the dae gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the dae gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% of the total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid.
Identification of the rph (RNase PH) gene of Bacillus subtilis: evidence for suppression of cold-sensitive mutations in Escherichia coli
Authors: Craven M G , Henner D J , Alessi D , Schauer A T , Ost K A , Deutscher M P , Friedman D I
Published: November 2016

A shotgun cloning of Bacillus subtilis DNA into pBR322 yielded a 2-kb fragment that suppresses the cold-sensitive defect of the nusA10(Cs) Escherichia coli mutant. The responsible gene encodes an open reading frame that is greater than 50% identical at the amino acid level to the E. coli rph gene, which was formerly called orfE. This B. subtilis gene is located at 251 degrees adjacent to the gerM gene on the B. subtilis genetic map. It has been named rph because, like its E. coli analog, it encodes a phosphate-dependent exoribonuclease activity, RNase PH, that removes the 3' nucleotides from precursor tRNAs. The cloned B. subtilis rph gene also suppresses the cold-sensitive phenotype of other unrelated cold-sensitive mutants of E. coli, but not the temperature-sensitive phenotype of three temperature-sensitive mutants, including the nusA11(Ts) mutant, that were tested.
In vivo translational start site selection on leaderless mRNA transcribed from the Streptomyces fradiae aph gene
Authors: Jones R L , Jaskula J C , Janssen G R
Published: November 2016

The message of the Streptomyces fradiae aph gene lacks a leader sequence and therefore is translated in the absence of a conventional Shine-Dalgarno interaction between mRNA and 30S ribosomal subunits. Insertion mutations generating short leaders of 2 or 4 nucleotides on the 5' end of the aph transcript reduced translational efficiency. A 4-base leader (5'-AUGC-3') placing a potential out-of-frame start codon immediately upstream of the aph coding sequence prevented detectable translation in the aph reading frame. The upstream AUG in this mutant was able to drive the expression of a reporter gene in a translational fusion vector, implying that this start codon was utilized in favor of the downstream AUG. Additional leaders (5'-AUAUGC-3' or 5'-CAUAUGC-3') placing 2 or 3 nucleotides 5' to the upstream AUG relieved this apparent discrimination, permitting translation of the APH protein from the downstream AUG. These results suggest that the position of a start codon with respect to the 5' terminus of aph mRNA is a determinant of translational efficiency and start site selection.
Regulation of katF and katE in Escherichia coli K-12 by weak acids
Authors: Schellhorn H E , Stones V L
Published: November 2016

Chromosomal transcriptional and translational lacZ fusions to the katE (structural gene for the HPII hydroperoxidase) and katF (putative sigma factor required for katE expression) genes of Escherichia coli were isolated, and the regulation of these fusions was used to identify factors that control the expression of these two important antioxidant factors. While katE was found to be regulated primarily at the level of transcription (since induction patterns were similar for both transcriptional and translational fusions), katF expression was a function of both transcriptional and translational signals. The katE gene was induced 57-fold as cells entered the stationary phase, while katF was induced 23-fold. katF induction was coincident with katE induction and occurred at the onset of the stationary growth phase. Expression of both katE and katF could be induced by resuspending uninduced exponential-phase cells in spent culture supernatant recovered from stationary-phase cells. The component of stationary-phase culture supernatant responsible for induction of the katF regulon appeared to be acetate, since expression of both katE and katF fusions was induced when exponential-phase cells were exposed to this weak acid. Other weak acids, including propionate and benzoate, were also found to be effective inducers of expression of both katF and katE. Induction of katE and katF fusions was unaffected in merodiploid strains containing both mutant and wild-type alleles, indicating that expression of both genes is independent of the wild-type gene product. Examination of catalase zymograms prepared from cells exposed to various levels of acetate revealed that both HPI and HPII catalases are induced by this weak acid, suggesting that there is a common link in the regulation of these two enzymes.
Identification of a novel promoter in the replication control region of plasmid R6K
Authors: Mukerji P , Greener A , Filutowicz M
Published: November 2016

A novel source of transcription has been detected in the replication region of plasmid R6K by using fusions involving the galK reporter gene. The -35 and -10 consensus RNA polymerase binding sites were identified in the region overlapping the binding sites for the R6K-encoded replication protein pi. Transcription from this promoter, designated P2, is repressed in vivo by pi-protein levels that are inhibitory for replication. Promoter-down mutations in P2 induced in vitro by bisulfite mutagenesis result in a reduced copy number of a beta-replicon but not of a gamma-replicon. Implications of the role of P2 in R6K replication are discussed.
Ferric iron uptake in Erwinia chrysanthemi mediated by chrysobactin and related catechol-type compounds
Authors: Persmark M , Expert D , Neilands J B
Published: November 2016

Erwinia chrysanthemi 3937 possesses a saturable, high-affinity transport system for the ferric complex of its native siderophore chrysobactin, [N-alpha-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine]. Uptake of 55Fe-labeled chrysobactin was completely inhibited by respiratory poison or low temperature and was significantly reduced in rich medium. The kinetics of chrysobactin-mediated iron transport were determined to have apparent Km and Vmax values of about 30 nM and of 90 pmol/mg.min, respectively. Isomers of chrysobactin and analogs with progressively shorter side chains mediated ferric iron transport as efficiently as the native siderophore, which indicates that the chrysobactin receptor primarily recognizes the catechol-iron center. Free ligand in excess only moderately reduced the accumulation of 55Fe. Chrysobactin may therefore be regarded as a true siderophore for E. chrysanthemi.
Molecular modelling of the three-dimensional structure and conformational flexibility of bacterial lipopolysaccharide
Authors: Kastowsky M , Gutberlet T , Bradaczek H
Published: November 2016

Molecular modelling techniques have been applied to calculate the three-dimensional architecture and the conformational flexibility of a complete bacterial S-form lipopolysaccharide (LPS) consisting of a hexaacyl lipid A identical to Escherichia coli lipid A, a complete Salmonella typhimurium core oligosaccharide portion, and four repeating units of the Salmonella serogroup B O-specific chain. X-ray powder diffraction experiments on dried samples of LPS were carried out to obtain information on the dimensions of the various LPS partial structures. Up to the Ra-LPS structure, the calculated model dimensions were in good agreement with experimental data and were 2.4 nm for lipid A, 2.8 nm for Re-LPS, 3.5 nm for Rd-LPS, and 4.4 nm for Ra-LPS. The maximum length of a stretched S-form LPS model bearing four repeating units was evaluated to be 9.6 nm; however, energetically favored LPS conformations showed the O-specific chain bent with respect to the Ra-LPS portion and significantly smaller dimensions (about 5.0 to 5.5 nm). According to the calculations, the Ra-LPS moiety has an approximately cylindrical shape and is conformationally well defined, in contrast to the O-specific chain, which was found to be the most flexible portion within the molecule.
Interallelic complementation of dnaE(Ts) mutations
Authors: Bryan S K , Moses R E
Published: November 2016

Some Escherichia coli dnaE(Ts) alleles will functionally complement in trans. The complementation is not due to copy number and is compatible with dimeric interaction.
Characterization of a Bacillus subtilis sporulation operon that includes genes for an RNA polymerase sigma factor and for a putative DD-carboxypeptidase
Authors: Wu J J , Schuch R , Piggot P J
Published: November 2016

At early stages of sporulation, the spoIIA locus is transcribed as a tricistronic (1.7-kb) operon, coding for sigma F and for two proteins that modulate the activity of sigma F. The locus is transcribed as a longer (2.9-kb) transcript at the late stages of sporulation. We show here that the longer transcript contains an additional open reading frame whose product has extensive sequence homology with DD-carboxypeptidases; the corresponding gene is designated dacF. Cotranscription of a morphogene, such as dacF, with the gene for a sigma factor suggests a way to couple transcription regulation with morphogenesis. The predicted N-terminal sequence of the DacF protein and the inhibition of sporulation by a translational dacF-lacZ fusion both suggest that the protein has a signal peptide for transport into or across a membrane. Expression of a dacF-lacZ transcriptional fusion was in the forespore. The 5' end of the 2.9-kb transcript was determined by primer extension analysis. The region 5' to the end showed no homology to promoters recognized by known sigma factors but was homologous to the corresponding region of the forespore-specific 0.3-kb gene of Bacillus subtilis.
Analysis of the Pseudomonas aeruginosa major outer membrane protein OprF by use of truncated OprF derivatives and monoclonal antibodies
Authors: Finnen R L , Martin N L , Siehnel R J , Woodruff W A , Rosok M , Hancock R E
Published: November 2016

TnphoA mutagenesis of the cloned oprF gene was utilized to generate 16 classes of fusions encoding differing lengths of the amino terminus of OprF fused to either alkaline phosphatase or to peptide tags of 1 to 20 amino acids, depending on the orientation and reading frame into which TnphoA was inserted. Representatives of each of the 16 classes were sequenced to determine the precise fusion joint. Four of these 16 representatives which produced in-frame fusions to alkaline phosphatase and another 8 with fusion joints in the amino-terminal half of OprF failed to react with a panel of 10 specific monoclonal antibodies. In contrast, OprF derivatives with predicted fusion joints at amino acids 180, 204, 289, and 299 reacted with one to five of the monoclonal antibodies. Four other immunoreactive OprF derivatives were created by subcloning and encoded amino acids 1 to 187, 188 to 326, 1 to 273 and 1 to 170 plus 301 to 326. On the basis of reactivity with the TnphoA-truncated derivatives and subclones of oprF, the epitopes for all 10 monoclonal antibodies were localized, in part, to specific regions of OprF. Nnie of the 10 monoclonal antibodies, 8 of which recognize surface-exposed epitopes, mapped within the carboxy-terminal region of OprF that is homologous to the Escherichia coli outer membrane protein OmpA. Thus, we concluded that parts of the carboxy terminus of OprF are exposed on the external face of the outer membrane. In addition, a clone containing only the first two cysteine residues of OprF demonstrated reactivity with monoclonal antibodies MA4-4 and MA7-8 that was destroyed by 2-mercaptoethanol treatment, as was reactivity with intact OprF. Thus, we conclude that this first pair of cysteines at residues 176 and 185 of mature OprF form a disulfide bond.
Light and oxygen regulation of the synthesis of bacteriochlorophylls a and c in Chloroflexus aurantiacus
Authors: Oelze J
Published: November 2016

Control of the synthesis of bacteriochlorophylls (Bchls) a and c by light and oxygen was studied in Chloroflexus aurantiacus grown in batch or chemostat culture with serine as the growth-limiting substrate. For comparison, inhibition by gabaculine of the formation of selected tetrapyrroles was studied. The inhibitory effect of gabaculine decreased in the following order of tetrapyrrole formation: coproporphyrin greater than Bchl c greater than Bchl a. Not only did addition of 5-aminolevulinate (ALA) reverse the inhibition by gabaculine, it also caused an increase in Bchl c content when the cultures grew at high concentrations of ALA. Inhibition of Bchl a, Bchl c, and coproporphyrin formation by oxygen was similar to inhibition by gabaculine. Addition of ALA to aerated cultures led to significant accumulation of coproporphyrin. These results suggest that oxygen inhibits tetrapyrrole formation at a site before ALA formation. Control by light was studied with chemostat cultures transferred from 5 klx to 25 klx. This resulted in only a transient increase of the protein level of the culture, while specific contents of Bchls c and a and the ratio Bchl c/Bchl a decreased to lower steady states. However, the specific content of coproporphyrin increased. Addition of ALA to chemostat cultures adapted to 50 klx increased specific coproporphyrin and Bchl c contents by factors of about 20 and 4, respectively, while the specific Bchl a content was only slightly increased and protein levels were unaffected. Increasing the serine concentration caused an initial increase in the specific Bchl c content, which returned to the original value as soon as the protein content had attained its maximal level. These results suggest that light does not control ALA formation as strictly as oxygen and that competition of biomass formation and tetrapyrrole synthesis for common precursors may be influenced by light.
Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1
Authors: Thanabalu T , Hindley J , Berry C
Published: November 2016

The 97-kDa protein Mtx21, derived from the 100-kDa mosquitocidal protein (Mtx) from Bacillus sphaericus SSII-1 by the deletion of the putative signal sequence, was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and the fusion protein was purified by affinity chromatography. The fusion protein bound to glutathione agarose was cleaved with thrombin to release the Mtx21 protein. The 97-kDa Mtx21 protein was found to be toxic to Culex quinquefasciatus larvae with a 50% lethal concentration of 15 ng/ml. Treating Mtx21 with crude mosquito larval gut extracts gave rise to two major peptides of 70 and 27 kDa. Treating the 97-kDa Mtx21 protein with trysin also gave rise to a similar proteolytic cleavage pattern. N-terminal sequencing showed that the 27-kDa peptide was derived from the N-terminal region of the 97-kDa protein and that the 70-kDa protein was from the C-terminal region of the 97-kDa protein. The 27-kDa peptide has all the previously identified regions of homology with the catalytic peptides of the ADP-ribosyltransferase toxins, such as pertussis toxin S1 peptide, while the 70-kDa peptide has three internal regions of homology.
IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions
Authors: Belogurov A A , Delver E P , Rodzevich O V
Published: November 2016

The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.
In vitro activation of the Serratia marcescens hemolysin through modification and complementation
Authors: Ondraczek R , Hobbie S , Braun V
Published: November 2016

The hemolytic activity of Serratia marcescens is determined by two polypeptides, termed ShlA and ShlB. ShlA is synthesized as an inactive precursor (ShlA*) and secreted with the help of ShlB, which is located in the outer membrane. In this study, it is shown that a cell lysate containing ShlB as well as partially purified ShlB converted ShlA* to the active ShlA hemolysin. ShlA remained active after removal of ShlB by column chromatography. In contrast to the stable modification of ShlA* by ShlB, a reversible activation was achieved by adding to ShlA* an N-terminal fragment of ShlA (ShlA16), consisting of 269 amino acid residues of ShlA and 18 residues of the vector. The nonhemolytic ShlA16 complemented ShlA* only when it was synthesized in an ShlB-producing cell. A deletion derivative of ShlA*, lacking residues 4 to 117, was complemented by ShlA16 but not activated by ShlB. Activation of ShlA* by ShlB at 4 degrees C proceeded at a much slower rate than complementation by ShlA16. It is concluded that ShlA* is modified by ShlB. ShlA16 modified by ShlB complements the missing modification of ShlA* in trans. Modification by ShlB occurs in the N-terminal part of ShlA*, which is also the reaction in vivo which results in active ShlA hemolysin in the culture supernatant. The HpmA hemolysin of Proteus mirabilis, which is very similar to ShlA, was also activated in vitro by ShlB and complemented by ShlA16.
Membrane ultrastructure of alkaliphilic Bacillus species studied by rapid-freeze electron microscopy
Authors: Khan S , Ivey D M , Krulwich T A
Published: November 2016

Cells of Bacillus firmus OF4 and Bacillus alcalophilus were examined by rapid-freeze freeze-fracture and freeze-substitution electron microscopy. No special vesicular structures linked to growth at alkaline pH were found, either within or associated with the cytoplasmic membrane. The cytoplasmic membranes of the alkaliphilic bacilli and the neutrophilic Bacillus subtilis BD99 were indistinguishable. Distinctive intramembrane particle rings, presumed to be flagellar structures on the basis of distribution and morphological characteristics, were found in all of these species. These observations indicate that the adaptations required to effect oxidative phosphorylation and flagellar rotation at extreme alkaline pH occur without gross morphological rearrangement.
Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance
Authors: Brom S , García de los Santos A , Stepkowsky T , Flores M , Dávila G , Romero D , Palacios R
Published: November 2016

Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to be the symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used a positive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one (pe), of which only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd, pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharide synthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealed in competition experiments. The strains that were cured (or deleted for pe) were significantly less competitive than the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmids in free-living conditions: the pf- strain was unable to grow on minimal medium, while strains cured of any other plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lacking pb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of the cured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming that the effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobium genome in symbiotic and free-living functions.
Linear- and circular-plasmid copy numbers in Borrelia burgdorferi
Authors: Hinnebusch J , Barbour A G
Published: November 2016

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.
Oxygen-regulated steps in the Rhodobacter capsulatus tetrapyrrole biosynthetic pathway
Authors: Biel A J
Published: November 2016

The effect of exogenous aminolevulinate and porphobilinogen on protoporphyrin accumulation in Rhodobacter capsulatus was measured. Oxygen inhibited protoporphyrin accumulation in strain AJB456, a bchH mutant, even in the presence of exogenous aminolevulinate, suggesting that some step in the formation of protoporphyrin from aminolevulinate is regulated by oxygen. In contrast, in the presence of exogenous porphobilinogen, oxygen did not inhibit protoporphyrin accumulation. The results presented in this study indicate that oxygen regulates the formation of porphobilinogen from aminolevulinate.
Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system
Authors: Kerby R L , Hong S S , Ensign S A , Coppoc L J , Ludden P W , Roberts G P
Published: November 2016

A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif.
The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase
Authors: Sayavedra-Soto L A , Arp D J
Published: November 2016

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.
Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene
Authors: de Vries G E , Arfman N , Terpstra P , Dijkhuizen L
Published: November 2016

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed.
Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin
Authors: Kupke T , Stevanović S , Sahl H G , Götz F
Published: November 2016

The plasmid-encoded epidermin biosynthesis gene, epiD, of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using both the malE fusion system and the T7 RNA polymerase-promoter system. EpiD was identified by Western blotting (immunoblotting) with anti-maltose-binding protein (MBP)-EpiD antiserum. EpiD and the MBP-EpiD fusion protein, which were mainly present in the soluble protein fraction, were purified from the respective E. coli clones. Purified EpiD showed the typical absorption spectrum of an oxidized flavoprotein with maxima at 274, 382, and 453 nm. The coenzyme released from EpiD by heat treatment was identified as flavin mononucleotide. S. epidermidis Tü3298/EMS11, containing a mutation within epiD, was unable to synthesize active epidermin. This mutated gene, epiD*, was cloned in E. coli and expressed as an MBP-EpiD* fusion protein. DNA sequencing of epiD* identified a point mutation that led to replacement of Gly-93 with Asp. Unlike MBP-EpiD, the fusion protein MBP-EpiD* could not bind flavin mononucleotide. We propose that EpiD catalyzes the removal of two reducing equivalents from the cysteine residue of the C-terminal meso-lanthionine to form a --C==C-- double bond and is therefore involved in formation of the unusual S-[(Z)-2-aminovinyl[-D-cysteine structure in epidermin.
Effect of mechanical removal of pili on gliding motility of Myxococcus xanthus
Authors: Rosenbluh A , Eisenbach M
Published: November 2016

Gliding motility of Myxococcus xanthus is governed by both the adventurous (A) and the social (S) motility gene systems. The presence of pili has previously been shown to be correlated with a genetically intact S-motility system (D. Kaiser, Proc. Natl. Acad. Sci. USA 76:5952-5956, 1979). The purpose of the present work was to study the direct effect of mechanical removal of pill on the social motility of M. xanthus. Depiliation resulted in (i) a loss of streaming motility of A- S+ mutants, i.e., strains which are able to move by virtue of the S-motility system only, (ii) no effect on motility in A+ S- mutants, i.e., strains capable of movement by the A-motility system only, and (iii) a retardation of streaming speed in the wild-type strain (A+ S+). Cell-cell cohesion, another characteristic of social behavior, was not affected by mechanical removal of pill. The observation that mechanical depiliation perturbed the motility of strains which rely on the S-motility system strongly supports a role for pili in social motility of M. xanthus.
The 55-kilodalton protein in an oriC complex fraction is glycogen synthase
Authors: Kaidow A , Kataoka T , Wachi M , Takada A , Yamasaki M , Nagai K
Published: November 2016

Three proteins with molecular masses of 35, 55, and 75 kDa were found in an oriC complex fraction after purification through CsCl density gradient centrifugation (W. G. Hendrickson, T. Kusano, H. Yamaki, R. Balakrishnan, M. King, J. Murchie, and M. Schaechter, Cell 30:915-923, 1982). Of these three proteins, the 55-kDa protein was determined to be glycogen synthase on the basis of the N-terminal amino acid sequence and the molecular weight. The oriC complex was formed in glgA mutant cells, which produce no detectable glycogen, as well as in wild-type cells. None of the 35-, 55-, and 75-kDa proteins were detected in the fraction from this mutant. The results indicate that these proteins were not constituents of the oriC complex.
DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein
Authors: Dong Q , Ebright R H
Published: November 2016

The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.
The amount of RepR protein determines the copy number of plasmid pIP501 in Bacillus subtilis
Authors: Brantl S , Behnke D
Published: November 2016

To prove the hypothesis that the amount of RepR protein is the rate-limiting factor for replication of plasmid pIP501 in Bacillus subtilis, the repR gene was placed under control of the inducible promoter pspac. The plasmid copy number of the pIP501 derivative pRS9 could be deliberately adjusted between approximately 1 and 50 to 100 molecules per cell by varying the concentration of the inducer isopropyl-beta-D-thiogalactopyranoside. Construction of a repR-lacZ fusion proved that the increase in copy number was due to a proportional increase in the amount of RepR protein.
Methane from acetate
Authors: Ferry J G
Published: November 2016

The general features are known for the pathway by which most methane is produced in nature. All acetate-utilizing methanogenic microorganisms contain CODH which catalyzes the cleavage of acetyl-CoA; however, the pathway differs from all other acetate-utilizing anaerobes in that the methyl group is reduced to methane with electrons derived from oxidation of the carbonyl group of acetyl-CoA to CO2. The current understanding of the methanogenic fermentation of acetate provides impressions of nature's novel solutions to problems of methyl transfer, electron transport, and energy conservation. The pathway is now at a level of understanding that will permit productive investigations of these and other interesting questions in the near future.
EJ-1, a temperate bacteriophage of Streptococcus pneumoniae with a Myoviridae morphotype
Authors: Díaz E , López R , García J L
Published: November 2016

The first temperate bacteriophage (EJ-1) of Streptococcus pneumoniae with Myoviridae morphotype A1 isolated from a clinical atypical strain has been purified and characterized. This phage has a double-stranded linear genome about 42 kb long, but in contrast to the other pneumococcal temperate phages that have been characterized so far, EJ-1 does not contain any protein covalently linked to it. We have sequenced a fragment of EJ-1 DNA containing the ejl gene, encoding a cell wall lytic enzyme (EJL amidase). This gene has been cloned and expressed in Escherichia coli, and the EJL enzyme was purified and biochemically characterized as an N-acetylmuramyl-L-alanine amidase that shares many similarities with the major pneumococcal autolysin. The EJL amidase is a choline-dependent enzyme that needs the process of conversion to achieve full enzymatic activity, but in contrast to the wild-type pneumococcal LYTA amidase, this process was found to be reversible. Comparisons of the primary structure of this new lytic enzyme with that of the other cell wall lytic enzymes of S. pneumoniae and its bacteriophages characterized so far provided new insights as to the evolutionary relationships between phages and bacteria. The nucleotide sequences of the attachment site (attP) on the phage genome and one of the junctions created by the insertion of the prophage were determined. Interestingly, the attP site was located near the ejl gene, as previously observed for the pneumococcal temperate bacteriophage HB-3 (A. Romero, R. López, and P. García, J. Virol. 66:2860-2864, 1992). A stem-and-loop structure, some adjacent direct and inverted repeats, and two putative integration host factor-binding sites were found in the att sites.
Effect of D-amino acids on structure and synthesis of peptidoglycan in Escherichia coli
Authors: Caparrós M , Pisabarro A G , de Pedro M A
Published: November 2016

Growth of Escherichia coli in the presence of certain D-amino acids, such as D-methionine, results in the incorporation of the D-amino acid into macromolecular peptidoglycan and can be lethal at high concentrations. Previous studies suggested that incorporation was independent of the normal biosynthetic pathway. An enzymatic reaction between the D-amino acid and macromolecular peptidoglycan was proposed as the mechanism of incorporation. The application of more advanced analytical techniques, notably high-pressure liquid chromatography, revealed that the presence of a D-amino acid susceptible to incorporation induced a multiplicity of alterations in peptidoglycan metabolism. Results derived basically from the study of samples treated with D-Met, D-Trp, and D-Phe indicated that the incorporation of a D-amino acid results in the accumulation of two major new muropeptides whose general structures most likely are GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa and GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-Ala-GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa, where D-aa represents a residue of the added D-amino acid. Resting cells are proficient in the incorporation of D-amino acids and can reach peptidoglycan modification levels comparable to those in growing cells. Under our conditions, D-amino acids had no apparent effect on growth or morphology but caused a severe inhibition of peptidoglycan synthesis and cross-linking, possibly leading to a reduction in the amount of peptidoglycan per cell. The properties of the reaction support the involvement of a penicillin-insensitive LD-transpeptidase enzyme in the synthesis of modified muropeptides and a possible inhibitory action of D-amino acids on high-molecular-weight penicillin-binding proteins.
dcd (dCTP deaminase) gene of Escherichia coli: mapping, cloning, sequencing, and identification as a locus of suppressors of lethal dut (dUTPase) mutations
Authors: Wang L , Weiss B
Published: November 2016

In Escherichia coli, most of the dUMP that is used as a substrate for thymidylate synthetase is generated from dCTP through the sequential action of dCTP deaminase and dUTPase. Some mutations of the dut (dUTPase) gene are lethal even when the cells are grown in the presence of thymidine, but their lethality can be suppressed by extragenic mutations that can be produced by transposon insertion. Six suppressor mutations were tested, and all were found to belong to the same complementation group. The affected gene was cloned, it was mapped by hybridization with a library of recombinant DNA, and its nucleotide sequence was determined. The gene is at 2,149 kb on the physical map. Its product, a 21.2-kDa polypeptide, was overproduced 1,000-fold via an expression vector and identified as dCTP deaminase, the enzyme affected in previously described dcd mutants. Null mutations in dcd probably suppress the lethality of dut mutations by reducing the accumulation of dUTP, which would otherwise lead to the excessive incorporation of uracil into DNA.
Cloning and linkage analysis of Neisseria gonorrhoeae DNA methyltransferases
Authors: Gunn J S , Piekarowicz A , Chien R , Stein D C
Published: November 2016

We have cloned DNA methyltransferases (MTases) from various strains of Neisseria gonorrhoeae. Each of these clones represents a single specificity, indicating that the multiple gonococcal MTase specificities are encoded by monospecific MTases. The DNAs of five strains (FA5100, F62, MS11, Pgh3-2, and WR302) were digested with NheI, SpeI, or NheI plus SpeI and subjected to pulsed-field gel electrophoresis. The DNA MTase clones were used to probe Southern blots of these pulsed-field gels to determine whether the MTase genes are linked and whether there are strain-to-strain differences. The results indicate that none of these genes are closely linked, but variable hybridization patterns indicate that there exist restriction fragment length polymorphisms between the strains tested. Most of the chromosomal regions containing these restriction fragment length polymorphisms are clustered in regions containing gonococcal genes known or suspected to antigenically vary via genetic recombination.
Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone
Authors: Fortin C , Nester E W , Dion P
Published: November 2016

Acetosyringone, a phenolic inducer of the virulence (vir) genes of Agrobacterium tumefaciens, inhibited the growth of the nopaline-type strains T37 and C58 incubated under acidic conditions. In the course of a 6-day incubation with acetosyringone, avirulent clones were produced in different proportions by strains T37 and C58 and also by a spontaneous variant of strain C58, denominated C58F. The proportion of avirulent clones in acetosyringone-treated cultures often exceeded 50% for strains T37 and C58F and was of the order of 1% for strain C58. Control cultures not exposed to acetosyringone did not yield avirulent clones. Two other vir inducers, sinapinic acid and syringaldehyde, also inhibited growth and promoted accumulation of avirulent clones in cultures of strains C58F and T37. On the other hand, various acetosyringone analogs reported not to induce the vir genes did not act as growth inhibitors. All of the T37 and most of the C58F avirulent clones examined still carried a Ti plasmid. In all instances examined, avirulent clones still carrying a Ti plasmid were mutated in this plasmid. Mutants of strain C58F lacked the capacity to induce a virB::lacZ fusion in the presence of acetosyringone.
The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae
Authors: Racenis P V , Lai J L , Das A K , Mullick P C , Hajra A K , Greenberg M L
Published: November 2016

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.
Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe
Authors: Gaynor P M , Greenberg M L
Published: November 2016

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.