bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase
Authors: Okada M , Matsuzaki H , Shibuya I , Matsumoto K
Published: November 2016

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.
Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae
Authors: Rich J J , Kinscherf T G , Kitten T , Willis D K
Published: November 2016

Mutational analysis of the bean-pathogenic Pseudomonas syringae pv. syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase. The analysis of a collection of spontaneous mutants of P. syringae pv. syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin. The gacA gene originally was identified as a regulator of extracellular antibiotic production by Pseudomonas fluorescens, and the predicted GacA protein is a member of the FixJ family of bacterial response regulators. The sequence of the putative B728a GacA protein revealed 92% identity with the P. fluorescens GacA protein. An insertional mutation within the P. syringae pv. syringae gacA gene abrogated lesion formation on beans, production of extracellular protease, and production of the toxin syringomycin, the same phenotypes affected by a lemA mutation. DNA sequence analysis identified the P. syringae pv. syringae uvrC gene immediately downstream of the gacA gene, an arrangement conserved in P. fluorescens and Escherichia coli. The gacA insertional mutant was sensitive to UV, presumably because of polarity on transcription of the downstream uvrC gene. Southwestern (DNA-protein) analysis revealed that the lemA and gacA genes were required for the full expression of a DNA binding activity.
Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism
Authors: Chattopadhyay S , Mukherjee A , Ghosh S
Published: November 2016

A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis. The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S. Chattopadhyay, A. Mukherjee, and S. Ghosh, J. Bacteriol. 175:3240-3243, 1993) has now been completely sequenced. The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively. The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus. A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene. Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source. The reason for this is not clear. A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A. brasilense.
Physical characterization of the flagella and flagellins from Methanospirillum hungatei
Authors: Faguy D M , Koval S F , Jarrell K F
Published: November 2016

Flagellar filaments from Methanospirillum hungatei GP1 and JF1 were isolated and subjected to a variety of physical and chemical treatments. The filaments were stable to temperatures up to 80 degrees C and over the pH range of 4 to 10. The flagellar filaments were dissociated in the detergents (final concentration of 0.5%) Triton X-100, Tween 20, Tween 80, Brij 58, N-octylglucoside, cetyltrimethylammonium bromide, and Zwittergent 3-14, remaining intact in only two of the detergents tested, sodium deoxycholate and 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS). Spheroplasting techniques were used to separate the internal cells from the complex sheath, S-layer (cell wall), and end plugs of M. hungatei. The flagellar basal structure was visualized after solubilization of membranes by CHAPS or deoxycholate. The basal structure appeared to be a simple knob with no apparent ring or hook structures. The multiple, glycosylated flagellins constituting the flagellar filaments were cleaved by proteases and cyanogen bromide. The cyanogen bromide-generated fragments of M. hungatei GP1 flagellins were partially sequenced to provide internal sequence information. In addition, the amino acid composition of each flagellin was determined and indicated that the flagellins are distinct gene products, rather than differentially glycosylated forms of the same gene product.
Hyperrecombination in the terminus region of the Escherichia coli chromosome: possible relation to nucleoid organization
Authors: Louarn J , Cornet F , François V , Patte J , Louarn J M
Published: November 2016

The terminus region of the Escherichia coli chromosome is the scene of frequent homologous recombination. This can be demonstrated by formation of deletions between directly repeated sequences which flank a genetic marker whose loss can be easily detected. We report here that terminal recombination events are restricted to a relatively large terminal recombination zone (TRZ). On one side of the TRZ, the transition from the region with a high excision rate to the normal (low) excision rates of the rest of the chromosome occurs along a DNA stretch of less than 1 min. No specific border of this domain has been defined. To identify factors inducing terminal recombination, we examined its relation to two other phenomena affecting the same region, site-specific recombination at the dif locus and site-specific replication pausing. Both the location and the efficiency of terminal recombination remained unchanged after inactivation of the dif-specific recombination system. Similarly, inactivation of site-specific replication pausing or displacement of the replication fork trap so that termination occurs about 200 kb away from the normal region had no clear effect on this phenomenon. Therefore, terminal recombination is not a direct consequence of either dif-specific recombination or replication termination. Furthermore, deletions encompassing the wild-type TRZ do not eliminate hyperrecombination. Terminal recombination therefore cannot be attributed to the activity of some unique sequence of the region. A possible explanation of terminal hyperrecombination involves nucleoid organization and its remodeling after replication: we propose that post replicative reconstruction of the nucleoid organization results in a displacement of the catenation links between sister chromosomes to the last chromosomal domain to be rebuilt. Unrelated to replication termination, this process would facilitate interactions between the catenated molecules and would make the domain highly susceptible to recombination between sister chromosomes.
Characterization of devA, a gene required for the maturation of proheterocysts in the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Maldener I , Fiedler G , Ernst A , Fernández-Piñas F , Wolk C P
Published: November 2016

Mutant M7, obtained by transposon mutagenesis of the cyanobacterium Anabaena sp. strain PCC 7120, is impaired in the development of mature heterocysts. Under aerobic conditions, the mutant is unable to fix N2 because of a deficiency of at least two components of the oxygen-protective mechanisms: a hemoprotein-coupled oxidative reaction and heterocyst-specific glycolipids. DNA contiguous with the inserted transposon was recovered from the mutant and sequenced. The transposon had inserted itself within a 732-bp open reading frame designated devA. The wild-type form of devA, obtained from a lambda-EMBL3 library of Anabaena sp. DNA, had the identical sequence. Directed mutagenesis of devA in the wild-type strain showed that the phenotype of the mutant was caused by insertion of the transposon. The wild-type form of devA on a shuttle vector complemented the mutation in M7. Expression of devA by whole filaments, monitored following nitrogen stepdown by using luxAB as the reporter, increased ca. eightfold during differentiation; the increase within differentiating cells was much greater. The deduced sequence of the DevA protein shows strong similarity to the ATP-binding subunit of binding protein-dependent transport systems. The product of devA may, therefore, be a component of a periplasmic permease that is required for the transition from a proheterocyst to a mature, nitrogen-fixing heterocyst.
Nucleotide sequence of the afimbrial-adhesin-encoding afa-3 gene cluster and its translocation via flanking IS1 insertion sequences
Authors: Garcia M I , Labigne A , Le Bouguenec C
Published: November 2016

The afa gene clusters encode afimbrial adhesins (AFAs) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains. The plasmid-borne afa-3 gene cluster is responsible for the biosynthesis of the AFA-III adhesin that belongs to the Dr family of hemagglutinins. Reported in this work is the nucleotide sequence of the 9.2-kb insert of the recombinant plasmid pILL61, which contains the afa-3 gene cluster cloned from a cystitis-associated E. coli strain (A30). The afa-3 gene cluster was shown to contain six open reading frames, designated afaA to afaF. It was organized in two divergent transcriptional units. Five of the six Afa products showed marked homologies with proteins encoded by previously described adhesion systems that allowed us to attribute to each of them a putative function in the biogenesis of the AFA-III adhesin. AfaE was identified as the structural adhesin product, whereas AfaB and AfaC were recognized as periplasmic chaperone and outer membrane anchor proteins, respectively. The AfaA and AfaF products were shown to be homologous to the PapI-PapB transcriptional regulatory proteins. No function could be attributed to the AfaD product, the gene of which was previously shown to be dispensable for the synthesis of a functional adhesin. Upstream of the afa-3 gene cluster, a 1.2-kb region was found to be 96% identical to the RepFIB sequence of one of the enterotoxigenic E. coli plasmids (P307), suggesting a common ancestor plasmid. This region contains an integrase-like gene (int). Sequence analysis revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster. Two other IS1 elements were detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments. The afa-3 gene cluster was therefore found to be flanked by two IS1 elements in direct orientation and two in opposite orientations. The afa-3 gene cluster, flanked by two directly oriented IS1 elements, was shown to translocate from a recombinant plasmid to the E. coli chromosome. This translocation event occurred via IS1-specific recombination mediated by a recA-independent mechanism.
Molecular characterization of the Salmonella typhimurium flhB operon and its protein products
Authors: Minamino T , Iino T , Kutuskake K
Published: November 2016

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, may constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.
Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100
Authors: DasSarma S , Arora P , Lin F , Molinari E , Yin L R
Published: November 2016

To study the functions of the 13 gvp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster. We constructed a 24.5-kb Escherichia coli-H. halobium shuttle plasmid, pFL2, containing the gvp gene cluster and introduced a kanamycin resistance (kappa) cassette into each gene (except for gvpA). Transformation of H. halobium SD109, which had the entire gvp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the kappa cassette in any of the gvp genes, except gvpM, did not lead to production of normal gas vesicles. Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis. In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the kappa cassette was deleted and only small (15 to 54-bp) insertions remained. The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the kappa insertions resulted in phenotypic reversion. Electron microscopic analysis of the kappa mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicle than in the wild type, while interruptions of gvpF, -G, -H, -J, -K, and -L produce no discernible vesicle intermediates. These results indicate the gvpA, -C, and -N, which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL, -K, -J, -I, -H, -G, and -F, which have the leftward transcriptional orientation encode proteins involved in early steps in the assembly of gas vesicles.
Aerobic catabolism of phenylacetic acid in Pseudomonas putida U: biochemical characterization of a specific phenylacetic acid transport system and formal demonstration that phenylacetyl-coenzyme A is a catabolic intermediate
Authors: Schleissner C , Olivera E R , Fernández-Valverde M , Luengo J M
Published: November 2016

The phenylacetic acid transport system (PATS) of Pseudomonas putida U was studied after this bacterium was cultured in a chemically defined medium containing phenylacetic acid (PA) as the sole carbon source. Kinetic measurement was carried out, in vivo, at 30 degrees C in 50 mM phosphate buffer (pH 7.0). Under these conditions, the uptake rate was linear for at least 3 min and the value of Km was 13 microM. The PATS is an active transport system that is strongly inhibited by 2,4-dinitrophenol, 4-nitrophenol (100%), KCN (97%), 2-nitrophenol (90%), or NaN3 (80%) added at a 1 mM final concentration (each). Glucose or D-lactate (10 mM each) increases the PATS in starved cells (140%), whereas arsenate (20 mM), NaF, or N,N'-dicyclohexylcarbodiimide (1 mM) did not cause any effect. Furthermore, the PATS is insensitive to osmotic shock. These data strongly suggest that the energy for the PATS is derived only from an electron transport system which causes an energy-rich membrane state. The thiol-containing compounds mercaptoethanol, glutathione, and dithiothreitol have no significant effect on the PATS, whereas thiol-modifying reagents such as N-ethylmaleimide and iodoacetate strongly inhibit uptake (100 and 93%, respectively). Molecular analogs of PA with a substitution (i) on the ring or (ii) on the acetyl moiety or those containing (iii) a different ring but keeping the acetyl moiety constant inhibit uptake to different extents. None of the compounds tested significantly increase the PA uptake rate except adipic acid, which greatly stimulates it (163%). The PATS is induced by PA and also, gratuitously, by some phenyl derivatives containing an even number of carbon atoms on the aliphatic moiety (4-phenyl-butyric, 6-phenylhexanoic, and 8-phenyloctanoic acids). However, similar compounds with an odd number of carbon atoms (benzoic, 3-phenylpropionic, 5-phenylvaleric, 7-phenylheptanoic, and 9-phenylnonanoic acids) as well as many other PA derivatives do not induce the system, suggesting that the true inducer molecule is phenylacetyl-coenzyme A (PA-CoA). Furthermore, after P. putida U is cultured in the same medium containing other carbon sources (glucose or octanoic, benzoic, or 4-hydroxyphenylacetic acid) in the place of PA, the PATS and PA-CoA are not detected; neither the PATS nor PA-CoA is found in cases in which mutants (PA- and PCL-) lacking the enzyme which catalyzed the initial step of the PA degradation (phenylacetyl-CoA ligase) are used. PA-CoA has been extracted from bacteria and identified as a true PA catabolite by high-performance liquid chromatography and also enzymatically with pure acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum.
Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg
Authors: Braks I J , Hoppert M , Roge S , Mayer F
Published: November 2016

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.
Analysis of the genetic requirements for inducible multiple-antibiotic resistance associated with the mar locus in Escherichia coli
Authors: Sulavik M C , Gambino L F , Miller P F
Published: November 2016

A series of novel genetic constructs derived from the marRAB operon was used to determine the role of this gene cluster in salicylate-inducible multiple-antibiotic resistance in Escherichia coli. Our findings indicate that regulated antibiotic resistance associated with this locus requires only the products of marR and marA, without any neighboring genes.
The XbaI-BlnI-CeuI genomic cleavage map of Salmonella paratyphi B
Authors: Liu S L , Hessel A , Cheng H Y , Sanderson K E
Published: November 2016

The genomic cleavage map of Salmonella paratyphi B was determined through digestion with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 19 XbaI sites, 10 BlnI sites, and 7 CeuI sites. The fragments were arranged in order through excision of fragments from the gel, redigestion with a second enzyme, end labelling with 32P, and reelectrophoresis. Tn10 transposons inserted in 61 different genes of S. typhimurium LT2 were transduced by use of bacteriophage P22 into S. paratyphi B. The locations of Tn10 insertions on the chromosome of S. paratyphi B were determined by use of XbaI and BlnI sites in Tn10, revealing the positions of genes with Tn10 insertions in S. paratyphi B. All seven CeuI sites (in rrl genes for 23S rRNA) and most of the XbaI and BlnI sites in rrn genes for Glt-tRNA are conserved, but only about half of the XbaI and BlnI sites outside rrn genes are conserved. Gene order is identical in the 68 genes that we could compare between S. paratyphi B and S. typhimurium LT2, and the lengths of intervals between the genes are often the same, but there are several instances of differences in interval lengths, indicating that insertions or deletions of DNA have occurred during the evolutionary divergence of these bacteria.
Characterization of the genome region encoding an fdxH-type ferredoxin and a new 2[4Fe-4S] ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110
Authors: Schrautemeier B , Cassing A , Böhme H
Published: November 2016

A genomic DNA region with four consecutive open reading frames, including an fdxH-type gene, has been sequenced and initially characterized for the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110. The fdxH gene encodes a [2Fe-2S]-type ferredoxin, 98 amino acids in length, with a deduced molecular mass of 10.9 kDa. Conserved residues include two characteristic lysines at positions 10 and 11, shown recently to be important for interaction with nitrogenase reductase (S. Schmitz, B. Schrautermeier, and H. Böhme, Mol. Gen. Genet. 240:455-460, 1993). The gene is transcribed only under anaerobic nitrogenase-inducing conditions, whereas the Plectonema petF gene, encoding a different (type 1) [2Fe-2S] ferredoxin, is only transcribed in cultures growing with combined nitrogen. The fdxH gene was expressed in Escherichia coli as a holoprotein. The purified protein was able to effectively donate electrons to cyanobacterial nitrogenase, whereas PetF from the same organism was not. The occurrence of FdxH in the nonheterocystous genus Plectonema demonstrates for the first time that FdxH-type ferredoxins are not exclusively expressed within heterocysts, as is true for cyanobacteria differentiating these cells for nitrogen fixation under aerobic growth conditions. Two open reading frames that precede fdxH have high similarity to those found at a corresponding location in Anabaena sp. strain PCC 7120. In the latter organism, they are transcribed only under nitrogen-fixing conditions, but the functions of their gene products remain unclear (D. Borthakur, M. Basche, W. J. Buikema, P. B. Borthakur, and R. Haselkorn, Mol. Gen. Genet. 221:227-234, 1990). An fdxB-type gene encoding a 2[4Fe-4S] ferredoxin not previously identified in cyanobacteria is located immediately downstream of fdxH in P. boryanum.
Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants
Authors: Breedveld M W , Yoo J S , Reinhold V N , Miller K J
Published: November 2016

The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp. are believed to provide functions during hypoosmotic adaptation and legume nodulation. In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties. Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear. In the present study, we show that R. meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile. In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R. meliloti ndvA mutant.
Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron
Authors: Schmitt M P , Holmes R K
Published: November 2016

DtxR is an iron-dependent sequence-specific DNA-binding protein that binds to the tox operator, an inverted-repeat nucleotide sequence located upstream from the diphtheria toxin gene. In this study, two additional iron-regulated promoter/operator sequences (IRP1 and IRP2) that are controlled by DtxR were cloned from the chromosome of Corynebacterium diphtheriae and characterized. Operon fusions to lacZ were used to analyze expression from IRP1 and IRP2 in Escherichia coli. Transcription from both promoters was strongly repressed in high-iron medium in the presence of the cloned dtxR gene; however, transcription in the absence of dtxR was 50- to 100-fold greater, regardless of the iron concentration. Purified DtxR altered the electrophoretic mobility of DNA fragments carrying IRP1 or IRP2, and the nucleotide sequences of the two promoter/operator regions indicated that they are both homologous with the tox operator. DtxR protected an approximately 30-bp region on both IRP1 and IRP2 from DNase I digestion. A 19-bp consensus DtxR-binding site was derived from a comparison of the various DtxR-regulated operator/promoter sequences. Footprinting experiments using hydroxyl radicals and dimethyl sulfate demonstrated that DtxR interacted with these operators in a symmetrical manner, probably as a dimer or multimer. The deduced amino acid sequence of an open reading frame (ORF1) located downstream from IRP1 was homologous with a family of periplasmic proteins involved in iron transport in gram-negative bacteria and with the ferrichrome receptor, FhuD, from Bacillus subtilis. These findings suggest that ORF1 encodes a membrane-associated lipoprotein that may serve as the receptor for a ferric-siderophore complex in C. diphtheriae.
Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ
Authors: Baumgartner J W , Kim C , Brissette R E , Inouye M , Park C , Hazelbauer G L
Published: November 2016

Chemoreceptor Trg and osmosensor EnvZ of Escherichia coli share a common transmembrane organization but have essentially unrelated primary structures. We created a hybrid gene coding for a protein in which Trg contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ contributed its cytoplasmic kinase/phosphatase domain. Trz1 transduced recognition of sugar-occupied, ribose-binding protein by its periplasmic domain into activation of its cytoplasmic kinase/phosphatase domain as assessed in vivo by using an ompC-lacZ fusion gene. Functional coupling of sugar-binding protein recognition to kinase/phosphatase activity indicates shared features of intramolecular signalling in the two parent proteins. In combination with previous documentation of transduction of aspartate recognition by an analogous fusion protein created from chemoreceptor Tar and EnvZ, the data indicate a common mechanism of transmembrane signal transduction by chemoreceptors and EnvZ. Signalling through the fusion proteins implies functional interaction between heterologous domains, but the minimal sequence identity among relevant segments of EnvZ, Tar, and Trg indicates that the link does not require extensive, specific interactions among side chains. The few positions of identity in those three sequences cluster in transmembrane segment 1 and the short chemoreceptor sequence in the cytoplasmic part of the hybrid proteins. These regions may be particularly important in physical and functional coupling. The specific cellular conditions necessary to observe ligand-dependent activation of Trz1 can be understood in the context of the importance of phosphatase control in EnvZ signalling and limitations on maximal receptor occupancy in binding protein-mediated recognition.
Physical map of the genome of Acholeplasma oculi ISM1499 and construction of a Tn4001 derivative for macrorestriction chromosomal mapping
Authors: Tigges E , Minion F C
Published: November 2016

A physical chromosomal map of Acholeplasma oculi ISM1499 was constructed by using field inversion gel electrophoresis. To assist in the ordering of the chromosomal fragments, a modified transposon, Tn4001.1064, was constructed. It was also used to rescue mycoplasmal chromosomal sequences adjacent to transposon insertion sites in a one-step cloning procedure. The total size of the A. oculi ISM1499 genome was estimated to be 1,633 kb. The restriction enzyme sites for ApaI, BssHII, EagI, and SmaI were positioned on the map along with several transposon insertion sites.
Molecular cloning of bacterial DNA in vivo using a transposable R6K ori and a P1vir phage
Authors: Stojiljkovic I , Bozja J , Salaj-Smic E
Published: November 2016

A new method of cloning in vivo using the P1vir phage and transposon Tn5-rpsL oriR6K was developed. The method relies upon recircularization of transducing DNA containing a transposon insertion in a recombination-deficient strain of Escherichia coli K-12 and subsequent stable replication of the recircularized DNA. Using this method, we were able to clone in vivo the chromosomal region located between approximately 7.1 and 9.2 min on the E. coli K-12 map in a 95-kb plasmid.
Streptomyces lividans glycosylates the linker region of a beta-1,4-glycanase from Cellulomonas fimi
Authors: Ong E , Kilburn D G , Miller R C , Warren R A
Published: November 2016

The beta-1,4-glycanase Cex of the gram-positive bacterium Cellulomonas fimi is a glycoprotein comprising a C-terminal cellulose-binding domain connected to an N-terminal catalytic domain by a linker containing only prolyl and threonyl (PT) residues. Cex is also glycosylated by Streptomyces lividans. The glycosylation of Cex produced in both C. fimi and S. lividans protects the enzyme from proteolysis. When the gene fragments encoding the cellulose-binding domain of Cex (CBDCex), the PT linker plus CBDCex (PT-CBDCex), and the catalytic domain plus CBDCex of Cex were expressed in S. lividans, only PT-CBDCex was glycosylated. Therefore, all the glycans must be O linked because only the PT linker was glycosylated. A glycosylated form and a nonglycosylated form of PT-CBDCex were produced by S. lividans. The glycosylated form of PT-CBDCex was heterogeneous; its average carbohydrate content was approximately 10 mol of D-mannose equivalents per mol of protein, but the glycans contained from 4 to 12 alpha-D-mannosyl and alpha-D-galactosyl residues. Glycosylated Cex from S. lividans was also heterogeneous. The presence of glycans on PT-CBDCex increased its affinity for bacterial microcrystalline cellulose. The location of glycosylation only on the linker region of Cex correlates with the properties conferred on the enzyme by the glycans.
Cloning, sequencing, and disruption of a levanase gene of Bacillus polymyxa CF43
Authors: Bezzate S , Steinmetz M , Aymerich S
Published: November 2016

The Bacillus polymyxa CF43 lelA gene, expressing both sucrose and fructan hydrolase activities, was isolated from a genomic library of B. polymyxa screened in Bacillus subtilis. The gene was detected as expressing sucrose hydrolase activity; B. subtilis transformants did not secrete the lelA gene product (LelA) into the extracellular medium. A 1.7-kb DNA fragment sufficient for lelA expression in Escherichia coli was sequenced. It contains a 548-codon open reading frame. The deduced amino acid sequence shows 54% identity with mature B. subtilis levanase and is similar to other fructanases and sucrases (beta-D-fructosyltransferases). Multiple-sequence alignment of 14 of these proteins revealed several previously unreported features. LelA appears to be a 512-amino-acid polypeptide containing no canonical signal peptide. The hydrolytic activities of LelA on sucrose, levan, and inulin were compared with those of B. subtilis levanase and sucrase, confirming that LelA is indeed a fructanase. The lelA gene in the chromosome of B. polymyxa was disrupted with a chloramphenicol resistance gene (cat) by "inter-gramic" conjugation: the lelA::cat insertion on a mobilizable plasmid was transferred from an E. coli transformant to B. polymyxa CF43, and B. polymyxa transconjugants containing the lelA::cat construct replacing the wild-type lelA gene in their chromosomes were selected directly. The growth of the mutant strain on levan, inulin, and sucrose was not affected.
The sphR product, a two-component system response regulator protein, regulates phosphate assimilation in Synechococcus sp. strain PCC 7942 by binding to two sites upstream from the phoA promoter
Authors: Nagaya M , Aiba H , Mizuno T
Published: November 2016

In the photosynthetic cyanobacterium Synechococcus sp. strain PCC 7942, the sphS and sphR genes were previously suggested to encode a typical pair of two-component signal transduction proteins. A deletion mutant strain lacking these genes failed to exhibit induction of alkaline phosphatase, the phoA gene product, in response to phosphate limitation in the medium. The SphR protein was overexpressed in Escherichia coli and then purified to near homogeneity. A truncated form of the SphS polypeptide (named SphS*) was also isolated. Here, we demonstrate that purified SphR is phosphorylated by phosphotransfer from SphS and binds to two distinct sites upstream from the phoA promoter. From these results, we conclude that the SphS and SphR proteins are directly involved in the regulation of phoA transcription in response to phosphate limitation in Synechococcus species.
Expansion of bacteriocin activity and host range upon complementation of two peptides encoded within the lactacin F operon
Authors: Allison G E , Fremaux C , Klaenhammer T R
Published: November 2016

Lactacin F is a membrane-active bacteriocin produced by Lactobacillus johnsonii VPI11088 (Laf+). The genetic determinants encoding lactacin F are organized in a 1-kb polycistronic operon composed of a promoter (P(laf)), three genes (lafA, lafX, and ORFZ), and a functional rho-independent transcription terminator. Two Laf- derivatives of VPI11088, designated NCK64 and NCK65, were characterized. NCK64 contained a frameshift mutation in the lafA gene causing premature termination of translation. NCK65 harbored a 10-kb chromosomal deletion covering the laf operon. When the lafA gene was cloned independently and expressed in NCK65, bacteriocin activity was limited to L. helveticus 87, only one of the six known lactacin F-sensitive (Lafs) indicators. When lafX was introduced into NCK65, no bacteriocin activity against any of the sensitive strains was detected. Genetic combination of lafA and lafX, in cis or in trans, restored bacteriocin activity against all Lafs indicators. When two NCK65 clones containing either lafA or lafX were plated slightly apart on agar plates, fully active lactacin F was present in the intervening area where the two excreted gene products, LafA and LafX, diffused together. The genetic analysis revealed that the interaction of two bacteriocinogenic peptides encoded within the laf operon is likely to participate in the formation of poration complexes in the membranes of susceptible bacteria.
Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli
Authors: González-Flecha B , Demple B
Published: November 2016

Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a reporter of gene expression.
Characterization of the fliL gene in the flagellar regulon of Escherichia coli and Salmonella typhimurium
Authors: Raha M , Sockett H , Macnab R M
Published: November 2016

filL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli. A spontaneous fliL mutant of S. typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch. However, in-frame deletions of the E. coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis. Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the effect on motility caused by truncation of the gene is probably an indirect one.
A gene (ccmA) required for carboxysome formation in the cyanobacterium Synechocystis sp. strain PCC6803
Authors: Ogawa T , Marco E , Orus M I
Published: November 2016

A high-CO2-requiring mutant, G7, of Synechocystis sp. strain PCC6803 capable of inorganic carbon transport but unable to utilize the intracellular inorganic carbon pool for photosynthesis was isolated. Transmission electron micrographs of the mutant indicated that the mutant does not have any carboxysomes. A clone (pHPG7) with a 7.5-kbp DNA insert that transforms the G7 mutant to the wild-type phenotype was isolated from a genomic library of wild-type Synechocystis sp. strain PCC6803. Complementation tests with subclones identified the mutation site in G7 within 208 bp. Sequencing of nucleotides in this region elucidated an open reading frame, designated ccmA, encoding a protein of 302 amino acids. Cloning and sequence analysis of the respective G7 gene revealed an A-to-G substitution that results in an Asp-to-Gly substitution in the deduced amino acid. The result indicated that the ccmA gene encodes a protein essential for the formation of carboxysomes. An open reading frame encoding a proline-rich protein of 271 amino acids was found downstream of the ccmA gene, but no ccm-like genes or rbc operon was found in this region.
Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium
Authors: Sirisena D M , MacLachlan P R , Liu S L , Hessel A , Sanderson K E
Published: November 2016

We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified as rfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoA ligase. A plasmid carrying rfaF complements an rfaF mutant of S. typhimurium; rfaD and kbl are homologous to and in the same location as the equivalent genes in Escherichia coli K-12. The RfaF (heptosyl transferase II) protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to be heptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function in heptose binding. E. coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing from S. typhimurium. This DNA includes yibB, which is an open reading frame of unknown function, and two promoters upstream of rfaD (P3, a heat-shock promoter, and P2). Both S. typhimurium and E. coli rfaD genes share a normal consensus promoter (P1). We postulate that the yibB segment is an insertion into the line leading to E. coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S. typhimurium. The G+C content of the rfaLKZYJI genes of both S. typhimurium LT2 and E. coli K-12 is about 35%, much lower than the average of enteric bacteria; if this low G+C content is due to lateral transfer from a source of low G+C content, it must have occurred prior to evolutionary divergence of the two genera.
Regulation of sigma D expression and activity by spo0, abrB, and sin gene products in Bacillus subtilis
Authors: Márquez-Magaña L M , Mirel D B , Chamberlin M J
Published: November 2016

Expression of sigma D protein and of the hag gene, which is transcribed by the sigma D holoenzyme, is not dependent on spo0, abrB, or sin gene products in Bacillus subtilis. Preliminary results, however, suggest that a signal mediated by the spo0K locus may be responsible for the inhibition of sigma D activity during the stationary phase.
Plasmids of the pT181 family show replication-specific initiator protein modification
Authors: Rasooly A , Projan S J , Novick R P
Published: November 2016

The rolling circle plasmids of Staphylococcus aureus regulate their replication by controlling initiator (Rep) protein synthesis. It was demonstrated recently that the pT181 initiator protein RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC* (A. Rasooly and R. P. Novick, Science, 262:1048-1050). We establish here that this initiator modification occurs with four other members of the pT181 family and that it occurs in Bacillus subtilis as well as S. aureus. These results suggest that Rep conversion to Rep* is probably universal among plasmids of the pT181 family and is not host dependent.
Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis
Authors: Borchert S , Stachelhaus T , Marahiel M A
Published: November 2016

The deduced amino acid sequence of the gsp gene, located upstream of the 5' end of the gramicidin S operon (grs operon) in Bacillus brevis, showed a high degree of similarity to the sfp gene product, which is located downstream of the srfA operon in B. subtilis. The gsp gene complemented in trans a defect in the sfp gene (sfpO) and promoted production of the lipopeptide antibiotic surfactin. The functional homology of Gsp and Sfp and the sequence similarity of these two proteins to EntD suggest that the three proteins represent a new class of proteins involved in peptide secretion, in support of a hypothesis published previously (T. H. Grossman, M. Tuckman, S. Ellestad, and M. S. Osburne, J. Bacteriol. 175:6203-6211, 1993).
Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes
Authors: Lung H Y , Baetz A L , Peck A B
Published: November 2016

Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species by an activation-decarboxylation reaction which yields formate and CO2. oxc, the gene encoding the oxalic acid-degrading enzyme oxalyl-coenzyme A decarboxylase, was cloned from the bacterium Oxalobacter formigenes. The DNA sequence revealed a single open reading frame of 1,704 bp capable of encoding a 568-amino-acid protein with a molecular weight of 60,691. The identification of a presumed promoter region and a rho-independent termination sequence indicates that this gene is not part of a polycistronic operon. A PCR fragment encoding the open reading frame, when overexpressed in Escherichia coli, produced a product which cross-reacted antigenically with native enzyme on Western blots (immunoblots), appeared to form homodimers spontaneously, and exhibited enzymatic activity similar to that of the purified native enzyme.