bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of sugar transport via the multiple sugar metabolism operon of Streptococcus mutans by the phosphoenolpyruvate phosphotransferase system
Authors: Cvitkovitch D G , Boyd D A , Hamilton I R
Published: November 2016

In this report, we provide evidence that the transport of sugars in Streptococcus mutans via the multiple sugar metabolism system is regulated by the phosphoenolpyruvate phosphotransferase system. A ptsI-defective mutant (DC10), when grown on the multiple sugar metabolism system substrate raffinose, exhibited reduced growth, transport, and glycolytic activity with raffinose relative to the parent strain BM71. Inhibition of [3H]raffinose uptake was also observed in both BM71 and DC10 with increasing concentrations of glucose and the glucose analogs alpha-methyl glucoside and 2-deoxyglucose.
Replication through the terminus region of the Bacillus subtilis chromosome is not essential for the formation of a division septum that partitions the DNA
Authors: Wu L J , Franks A H , Wake R G
Published: November 2016

Germinated and outgrowing spores of a temperature-sensitive DNA initiation mutant of Bacillus subtilis were allowed to initiate a single round of replication by being shifted from 34 to 47 degrees C at the appropriate time. The DNA replication inhibitor 6-(parahydroxyphenylazo)-uracil was added to separate portions of the culture at various times during the round. Samples were collected from each around the time of the first division septation for measurements of the extent of the round completed, the level of division septation, the position of the septum within the outgrown cell, and the distribution of DNA (nucleoid) in relation to the septum. The extent of replication was measured directly through a hybridization approach. The results show clearly that a central division septum can close down onto a chromosome that is only partially replicated (to a minimum extent of about 60% of the round) such that DNA appears on both sides of the septum and frequently very close to it. It is concluded, as claimed previously on the basis of a less direct approach (T. McGinness and R.G. Wake, J. Mol. Biol. 134:251-264, 1979), that replication through the terminus region of the chromosome is not essential for the formation of a division septum that partitions the DNA.
Identification of channel-forming activity in the cell wall of Corynebacterium glutamicum
Authors: Niederweis M , Maier E , Lichtinger T , Benz R , Krämer R
Published: November 2016

The cell wall of the gram-positive Corynebacterium glutamicum was prepared. It contained an ion-permeable channel with a single-channel conductance of about 6 nS in 1 M KCl. The mobility sequence of the ions in the channel is similar to that in the aqueous phase, suggesting that it is a water-filled channel wide enough to allow unhindered diffusion of ions. The results indicate that we have identified the hydrophilic pathway through the mycolic acid layer of C. glutamicum.
Metabolism of polychlorinated phenols by Pseudomonas cepacia AC1100: determination of the first two steps and specific inhibitory effect of methimazole
Authors: Tomasi I , Artaud I , Bertheau Y , Mansuy D
Published: November 2016

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 metabolize both dichlorophenols, such as 2,4-dichlorophenol, 2,6-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol, and more highly substituted phenols, such as 2,4,6-trichlorophenol and pentachlorophenol, to the corresponding chlorohydroquinones. The first hydroxylation occurs in the para position of the phenol regardless of whether this position is replaced by a chlorine substituent. The first evidence leading to the characterization of para-hydroxylase as a flavin-containing enzyme is provided by the inhibitory effect of methimazole, an alternate substrate for this monooxygenase, on the degradative ability of the strain. In a second step, with tetrachlorohydroquinone, trichlorohydroxyquinone was isolated and completely characterized. Trichlorohydroxyquinone was also obtained from tetrachloroquinone. Incubation of the cells in the presence of an external source of NADPH prevents the further degradation of tetrachlorohydroquinone, suggesting that the quinone derived from the two-electron oxidation of the hydroquinone is more likely the substrate for the second hydroxylation.
Caulobacter FliQ and FliR membrane proteins, required for flagellar biogenesis and cell division, belong to a family of virulence factor export proteins
Authors: Zhuang W Y , Shapiro L
Published: November 2016

The Caulobacter crescentus fliQ and fliR genes encode membrane proteins that have a role in an early step of flagellar biogenesis and belong to a family of proteins implicated in the export of virulence factors. These include the MopD and MopE proteins from Erwinia carotovora, the Spa9 and Spa29 proteins from Shigella flexneri, and the YscS protein from Yersinia pestis. Inclusion in this family of proteins suggests that FliQ and FliR may participate in an export pathway required for flagellum assembly. In addition, mutations in either fliQ or fliR exhibit defects in cell division and thus may participate directly or indirectly in the division process. fliQ and fliR are class II flagellar genes residing near the top of the regulatory hierarchy that determines the order of flagellar gene transcription. The promoter sequence of the fliQR operon differs from most known bacterial promoter sequences but is similar to other Caulobacter class II flagellar gene promoter sequences. The conserved nucleotides in the promoter region are clustered in the -10, -20 to -30, and -35 regions. The importance of the conserved bases for promoter activity was demonstrated by mutational analysis. Transcription of the fliQR operon is initiated at a specific time in the cell cycle, and deletion analysis revealed that the minimal sequence required for transcriptional activation resides within 59 bp of the start site.
Dual mode of energy coupling by the oxyanion-translocating ArsB protein
Authors: Dey S , Rosen B P
Published: November 2016

The arsA and arsB genes of the ars operon of R-factor R773 confer arsenite resistance in Escherichia coli by coding for an anion-translocating ATPase. Arsenite resistance and the in vivo energetics of arsenite transport were compared in cells expressing the arsA and arsB genes and those expressing just the arsB gene. Cells expressing the arsB gene exhibited intermediate arsenite resistance compared with cells expressing both the arsA and arsB genes. Both types of cells exhibited energy-dependent arsenite exclusion. Exclusion of 73AsO2- from cells expressing only the arsB gene was coupled to electrochemical energy, while in cells expressing both genes, transport was coupled to chemical energy, most likely ATP. These results suggest that the Ars anion transport system can be either an obligatory ATP-coupled primary pump or a secondary carrier coupled to the proton motive force, depending on the subunit composition of the transport complex.
UDP-glucose is a potential intracellular signal molecule in the control of expression of sigma S and sigma S-dependent genes in Escherichia coli
Authors: Böhringer J , Fischer D , Mosler G , Hengge-Aronis R
Published: November 2016

The sigma S subunit of RNA polymerase is the master regulator of a regulatory network that controls stationary-phase induction as well as osmotic regulation of many genes in Escherichia coli. In an attempt to identify additional regulatory components in this network, we have isolated Tn10 insertion mutations that in trans alter the expression of osmY and other sigma S-dependent genes. One of these mutations conferred glucose sensitivity and was localized in pgi (encoding phosphoglucose isomerase). pgi::Tn10 strains exhibit increased basal levels of expression of osmY and otsBA in exponentially growing cells and reduced osmotic inducibility of these genes. A similar phenotype was also observed for pgm and galU mutants, which are deficient in phosphoglucomutase and UDP-glucose pyrophosphorylase, respectively. This indicates that the observed effects on gene expression are related to the lack of UDP-glucose (or a derivative thereof), which is common to all three mutants. Mutants deficient in UDP-galactose epimerase (galE mutants) and trehalose-6-phosphate synthase (otsA mutants) do not exhibit such an effect on gene expression, and an mdoA mutant that is deficient in the first step of the synthesis of membrane-derived oligosaccharides, shows only a partial increase in the expression of osmY. We therefore propose that the cellular content of UDP-glucose serves as an internal signal that controls expression of osmY and other sigma S-dependent genes. In addition, we demonstrate that pgi, pgm, and galU mutants contain increased levels of sigma S during steady-state growth, indicating that UDP-glucose interferes with the expression of sigma S itself.
Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa
Authors: Cunningham L , Williams H D
Published: November 2016

The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.
Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR homolog from Brevibacterium lactofermentum
Authors: Oguiza J A , Tao X , Marcos A T , Martín J F , Murphy J R
Published: November 2016

A homolog of the Corynebacterium diphtheriae dtxR gene was isolated from Brevibacterium lactofermentum. The product of the B. lactofermentum dtxR gene was immunoreactive with polyclonal anti-DtxR antibodies and functioned as an iron-activated repressor capable of regulating the expression of beta-galactosidase from a diphtheria tox promoter/operator transcriptional fusion in recombinant Escherichia coli. The extents of induction by increasing concentrations of the chelator 2,2'-dipyridyl were identical in cells expressing DtxR from either C. diphtheriae or B. lactofermentum.
Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2)
Authors: van Wezel G P , Buttner M J , Vijgenboom E , Bosch L , Hopwood D A , Kieser H M
Published: November 2016

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.
Heterogeneity of genome sizes among natural isolates of Escherichia coli
Authors: Bergthorsson U , Ochman H
Published: November 2016

Comparisons of the genetic maps of Escherichia coli K-12 and Salmonella typhimurium LT2 suggest that the size and organization of bacterial chromosomes are highly conserved. Employing pulsed-field gel electrophoresis, we have estimated the extent of variation in genome size among 14 natural isolates of E. coli. The BlnI and NotI restriction fragment patterns were highly variable among isolates, and genome sizes ranged from 4,660 to 5,300 kb, which is several hundred kilobases larger than the variation detected between enteric species. Genome size differences increase with the evolutionary genetic distance between lineages of E. coli, and there are differences in genome size among the major subgroups of E. coli. In general, the genomes of natural isolates are larger than those of laboratory strains, largely because of the fact that laboratory strains were derived from the subgroup of E. coli with the smallest genomes.
Molecular analysis of a metalloprotease from Proteus mirabilis
Authors: Wassif C , Cheek D , Belas R
Published: November 2016

Proteus mirabilis is known for its ability to differentiate from swimmer to swarmer cells, a process crucial for the pathogenesis of these bacteria during urinary tract infections. Among the many virulence factors produced during swarmer cell differentiation is an extracellular metalloprotease. A cosmid containing a large fragment of P. mirabilis chromosomal DNA was obtained by measuring protease expression in recombinant Escherichia coli. The recombinant and native enzymes were purified to over 95% homogeneity from culture supernatants by use of phenyl-Sepharose affinity chromatography and found to be identical. The activity of the 55-kDa enzyme was stimulated by divalent cations (Ca2+ > Mg2+) and inhibited by a chelator of these cations. The enzyme possesses substrate specificity for both serum and secretory forms of immunoglobulin A1 (IgA1) and IgA2 as well as IgG and, unlike classic IgA proteases, digested to completion both human and mouse IgA. Following subcloning, a 5-kb DNA fragment encoding recombinant protease activity was identified by insertional mutagenesis with Tn5. Four open reading frames were identified within this 5-kb region by limited nucleotide sequence analysis of DNA flanking the transposon. The nucleotide and deduced amino acid sequences of the metalloprotease structural gene (zapA) were obtained. Computerized homology studies revealed that the P. mirabilis metalloprotein is a member of the serralysin family of proteases and may be part of an operon comprising genes encoding an ATP-dependent ABC transporter in addition to the metalloprotease. The relevance of the metalloprotease to swarmer cell differentiation and pathogenicity is discussed.
Phosphorylating enzymes involved in glucose fermentation of Actinomyces naeslundii
Authors: Takahashi N , Kalfas S , Yamada T
Published: November 2016

Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phosphoglycerate kinase, pyruvate kinase, and acetate kinase coupled GDP as well as ADP, but P(i) compounds were not their phosphoryl acceptor. Cell extracts showed GDP-dependent activity of phosphoenolpyruvate carboxykinase, which assimilates bicarbonate and phosphoenolpyruvate into oxaloacetate, a precursor of succinate. Considerable amounts of GTP, polyphosphate, and PPi were found in glucose-fermenting cells, indicating that these compounds may serve as phosphoryl donors or acceptors in Actinomyces cells. PPi could be generated from UTP and glucose 1-phosphate through catalysis of UDP-glucose synthase, which provides UDP-glucose, a precursor of glycogen.
Electron transport-dependent taxis in Rhodobacter sphaeroides
Authors: Gauden D E , Armitage J P
Published: November 2016

Rhodobacter sphaeroides showed chemotaxis to the terminal electron acceptors oxygen and dimethyl sulfoxide, and the responses to these effectors were shown to be influenced by the relative activities of the different electron transport pathways. R. sphaeroides cells tethered by their flagella showed a step-down response to a decrease in the oxygen or dimethyl sulfoxide concentration when using them as terminal acceptors. Bacteria using photosynthetic electron transport, however, showed a step-down response to oxygen addition. Addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone did not cause a transient behavioral response, although it decreased the electrochemical proton gradient (delta p) and increased the rate of electron transport. However, removal of the ionophore, which caused an increase in delta p and a decrease in the electron transport rate, resulted in a step-down response. Together, these data suggest that behavioral responses of R. sphaeroides to electron transport effectors are caused by changes in the rate of electron transport rather than changes in delta p.
Cloning, sequencing, and characterization of a membrane-associated Prevotella ruminicola B(1)4 beta-glucosidase with cellodextrinase and cyanoglycosidase activities
Authors: Wulff-Strobel C R , Wilson D B
Published: November 2016

Prevotella ruminicola B(1)4 is a gram-negative, anaerobic gastrointestinal bacterium. A 2.4-kbp chromosomal fragment from P. ruminicola encoding an 87-kDa aryl-glucosidase (CdxA) with cellodextrinase activity was cloned into Escherichia coli DH5 alpha and sequenced. CdxA activity was found predominantly in the membrane fraction of both P. ruminicola and E. coli, but P. ruminicola localized the protein extracellularly while E. coli did not. The hydrolase had the highest activity on cellodextrins (3.43 to 4.13 mumol of glucose released min-1 mg of protein-1) and p-nitrophenyl-beta-D-glucoside (3.54 mumol min-1 mg of protein-1). Significant activity (70% of p-nitrophenyl-beta-D-glucoside activity) was also detected on arbutin and prunasin. Less activity was obtained with cellobiose, amygdalin, or gentiobiose. CdxA attacks cellodextrins from the nonreducing end, releasing glucose units, and appears to be an exo-1,4-beta-glucosidase (EC 3.2.1.74) which also is able to attack beta-1,6 linkages. Comparison of the deduced amino acid sequence with other glycosyl-hydrolases suggests that this enzyme belongs to family 3 (B. Henrissat, Biochem. J. 280:309-316, 1991). On the basis of this sequence alignment, the catalytic residues are believed to be Asp-275 and Glu-265. This is the first report of a cloned ruminal bacterial enzyme which can cleave cyanogenic plant compounds and which may therefore contribute to cyanide toxicity in ruminants.
Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus
Authors: Schnell S , Steinman H M
Published: November 2016

Although cytosolic superoxide dismutases (SODs) are widely distributed among bacteria, only a small number of species contain a periplasmic SOD. One of these is Caulobacter crescentus, which has a copper-zinc SOD (CuZnSOD) in the periplasm and an iron SOD (FeSOD) in the cytosol. The function of periplasmic CuZnSOD was studied by characterizing a mutant of C. crescentus with an insertionally inactivated CuZnSOD gene. Wild-type and mutant strains showed identical tolerance to intracellular superoxide. However, in response to extracellular superoxide, the presence of periplasmic CuZnSOD increased survival by as much as 20-fold. This is the first demonstration that periplasmic SOD defends against external superoxide of environmental origin. This result has implications for those bacterial pathogens that contain a CuZnSOD. C. crescentus was shown to contain a single catalase/peroxidase which, like Escherichia coli KatG catalase/peroxidase, is present in both the periplasmic and cytoplasmic fractions. The growth stage dependence of C. crescentus catalase/peroxidase and SOD activity was studied. Although FeSOD activity was identical in exponential- and stationary-phase cultures, CuZnSOD was induced nearly 4-fold in stationary phase and the catalase/peroxidase was induced nearly 100-fold. Induction of antioxidant enzymes in the periplasm of C. crescentus appears to be an important attribute of the stationary-phase response and may be a useful tool for studying its regulation.
Inactivation of Serpulina hyodysenteriae flaA1 and flaB1 periplasmic flagellar genes by electroporation-mediated allelic exchange
Authors: Rosey E L , Kennedy M J , Petrella D K , Ulrich R G , Yancey R J
Published: November 2016

Serpulina hyodysenteriae, the etiologic agent of swine dysentery, contains complex periplasmic flagella which are composed of multiple class A and class B polypeptides. To examine the role these proteins play in flagellar synthesis, structure, and function and to develop strains which may provide insight into the importance of motility in the etiology of this pathogen, we constructed specific periplasmic flagellar mutations in S. hyodysenteriae B204. The cloned flaA1 and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and/or kanamycin gene cassettes. Following delivery of these suicide plasmids into S. hyodysenteriae, homologous recombination and allelic exchange at the targeted chromosomal flaA1 and flaB1 genes was verified by PCR, sequence, and Southern analysis. The utility of a chloramphenicol resistance gene cassette for targeted gene disruption was demonstrated and found more amenable than kanamycin as a selective marker in S. hyodysenteriae. Immunoblots of cell lysates of the flagellar mutants with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath or core protein. Both mutations selectively abolished expression of the targeted gene without affecting synthesis of the other flagellar polypeptide. flaA1 and flaB1 mutant strains exhibited altered motility in vitro and were less efficient in movement through a liquid medium. Paradoxically, isogenic strains containing specifically disrupted flaA1 or flaB1 alleles were capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. This is the first report of specific inactivation of a motility-associated gene in spirochetes.
Unusual ancestry of dehydratases associated with quinate catabolism in Acinetobacter calcoaceticus
Authors: Elsemore D A , Ornston L N
Published: November 2016

Catabolism of quinate to protocatechuate requires the consecutive action of quinate dehydrogenase (QuiA), dehydroquinate dehydratase (QuiB), and dehydroshikimate dehyratase (QuiC), Genes for catabolism of protocatechuate are encoded by the pca operon in the Acinetobacter calcoaceticus chromosome. Observations reported here demonstrate that A. calcoaceticus qui genes are clustered in the order quiBCXA directly downstream from the pca operon. Sequence comparisons indicate that quiX encodes a porin, but the specific function of this protein has not been clearly established. Properties of mutants created by insertion of omega elements show that quiBC is expressed as part of a single transcript, but there is also an independent transcriptional initiation site directly upstream of quiA. The deduced amino acid sequence of QuiC does not resemble any other known sequence. A. calcoaceticus QuiB is most directly related to a family of enzymes with identical catalytic activity and biosynthetic AroD function in coliform bacteria. Evolution of A. calcoaceticus quiB appears to have been accompanied by fusion of a leader sequence for transport of the encoded protein into the inner membrane, and the location of reactions catalyzed by the mature enzyme may account for the failure of A. calcoaceticus aroD to achieve effective complementation of null mutations in quiB. Analysis of a genetic site where a DNA segment encoding a leader sequence was transposed adds to evidence suggesting horizontal transfer of nucleotide sequences within genes during evolution.
Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain
Authors: Fijalkowska I J , Schaaper R M
Published: November 2016

We have previously isolated seven mutants of Escherichia coli which replicate their DNA with increased fidelity. These mutants were isolated as suppressors of the elevated mutability of a mismatch-repair-defective mutL strain. Each mutant was shown to contain a single amino acid substitution in the dnaE gene product, the alpha (i.e., polymerase) subunit of DNA polymerase III holoenzyme responsible for replicating the E. coli chromosome. The mechanism(s) by which these antimutators exert their effect is of interest. Here, we have examined the effects of the antimutator alleles in a mutD5 mutator strain. This strain carries a mutation in the dnaQ gene, which results in defective exonucleolytic proofreading. Our results show that dnaE mutations also confer a strong antimutator phenotype in this background, the effects being generally much greater than those observed previously in the mutL background. The results suggest that the dnaE antimutator alleles can exert their effect independently of exonucleolytic proofreading activity. The large magnitude of the antimutator effects in the mutD5 background can be ascribed, at least in part, to the (additional) restoration of DNA mismatch repair, which is generally impaired in mutD5 strains because of error saturation. The high mutability of mutD5 strains was exploited to isolate a strong new dnaE antimutator allele on the basis of its ability to suppress the high reversion rate of an A.T-->T.A transversion in this background. A model suggesting how the dnaE antimutator alleles might exert their effects in proofreading-proficient and -deficient backgrounds is presented.
Derived structure of the putative sialic acid transporter from Escherichia coli predicts a novel sugar permease domain
Authors: Martinez J , Steenbergen S , Vimr E
Published: November 2016

Catabolism of sialic acids by Escherichia coli requires the genes nanA and nanT, which were previously mapped between argG and rpoN (E.R. Vimr and F.A. Troy, J. Bacteriol. 164:845-853, 1985). This organization is confirmed and extended by physical mapping techniques. An open reading frame beginning 135 bp from the nanA translational stop codon could code for a 53,547-Da hydrophobic polypeptide predicted to contain 14 transmembrane segments. Complementation analysis confirmed that nanT is required for sialic acid uptake when expressed in trans. NanT is homologous to a putative permease encoded by open reading frame 425, which maps between leuX and fecE in the E. coli chromosome. However, unlike this hypothetical permease or previously reported monosaccharide transporters, NanT contains a centrally located domain with two additional potential membrane-spanning segments plus one amphiphilic alpha-helix that may be important for the structure and function of sialic acid-permease.
Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation
Authors: Linkerhägner K , Oelze J
Published: November 2016

The presumed beneficial effect of hydrogenase on growth of diazotrophic bacteria was reinvestigated with carbon-limited chemostat cultures of the hydrogenase-deficient mutant hoxKG of Azotobacter vinelandii and its parent. The results revealed that hydrogen recycling was too low to benefit the cellular energy metabolism or activities of nitrogenase and respiration.
A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti
Authors: Breedveld M W , Hadley J A , Miller K J
Published: November 2016

The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection. In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions. In the present study, we have used a thin-layer chromatographic screening method to identify a novel R. meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone. Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents. As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells. Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.
The physical map of the chromosome of a serogroup A strain of Neisseria meningitidis shows complex rearrangements relative to the chromosomes of the two mapped strains of the closely related species N. gonorrhoeae
Authors: Dempsey J A , Wallace A B , Cannon J G
Published: November 2016

A physical map of the chromosome of N. meningitidis Z2491 (serogroup A, subgroup IV-1) has been constructed. Z2491 DNA was digested with NheI, SpeI, SgfI, PacI, BglII, or PmeI, resulting in a limited number of fragments that were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis. The estimated genome size for this strain was 2,226 kb. To construct the map, probes corresponding to single-copy genes or sequences were used on Southern blots of chromosomal DNA digested with the different mapping enzymes and subjected to CHEF electrophoresis. By determining which fragments from different digests hybridized to each specific probe, it was possible to walk back and forth between digests to form a circular macrorestriction map. The intervals between mapped restriction sites range from 10 to 143 kb in size. A total of 117 markers have been placed on the map; 75 represent identified genes, with the remaining markers defined by anonymous cloned fragments of neisserial DNA. Comparison of the arrangement of genetic loci in Z2491 with that in gonococcal strain FA1090, for which a physical map was previously constructed, revealed complex genomic rearrangements between the two strains. Although gene order is generally conserved over much of the chromosome, a region of approximately 500 kb shows translocation and/or inversion of multiple blocks of markers between the two strains. Even within the relatively conserved portions of the maps, several genetic markers are in different positions in Z2491 and FA1090.
Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK
Authors: Yuan G , Wong S L
Published: November 2016

An inverted repeat sequence known as CIRCE (controlling inverted repeat of chaperone expression) in the Bacillus subtilis groE operon has been suggested to function as an operator. To identify the regulatory gene directly or indirectly involved in CIRCE-mediated heat-inducible groE expression, B. subtilis WBG2, carrying an integrated groE-bgaB transcription fusion in the amyE locus, was mutagenized. Dark blue colonies formed at 37 degrees C represent mutants which constitutively produce BgaB (a thermostable beta-galactosidase) at high levels. Seven mutants (WBG101 to WBG107) were selected for further characterization. They all overproduced BgaB, GroEL, and DnaK simultaneously at 37 degrees C. These mutants could be restored to normal by introducing a plasmid carrying a functional copy of orf39, the first gene in the B. subtilis dnaK operon. Genomic sequencing of these mutants demonstrated that they all carried a single mutation in orf39. These mutations can be divided into three groups: (i) Gly-307 to Asp, (ii) Ser-122 to Phe, and (iii) Gly-63 to Glu. By using a binary vector system in E. coli, production of ORF39 was found to negatively regulate the expression of groE-bgaB in a CIRCE-specific manner. Under the heat shock condition, the negative regulation mediated by ORF39 was abolished. Mobility shift of the CIRCE-containing probe was also observed with the crude extract prepared from the E. coli strain that overproduced ORF39. Therefore, ORF39 is the negative regulatory factor which regulates both groE and dnaK expression in B. subtilis. It is likely to function as a CIRCE-specific repressor.
Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis
Authors: Boucher P E , Stibitz S
Published: November 2016

Regulation of virulence factor expression in Bordetella pertussis is mediated by the BvgAS two-component regulatory system. Although previous studies have demonstrated that the transcriptional regulation of the filamentous hemagglutinin gene (fhaB) involves binding of the BvgA activator directly to the fhaB promoter region, the mechanism of pertussis toxin operon (ptx) regulation by BvgA has remained unclear. We demonstrate in vitro the specific binding of BvgA to a region upstream of the ptx promoter that encompasses a 20-bp directly repeated sequence (positions -157 to -117) previously shown to be critical for BvgA-dependent activation. This binding is strictly dependent on the phosphorylation of BvgA, which can be obtained by incubation of BvgA with acetyl phosphate. By DNase I protection studies, we demonstrate the synergistic binding of BvgA-phosphate and purified Escherichia coli RNA polymerase to the ptx promoter. In the presence of the polymerase holoenzyme, a greatly extended footprint encompassing the region between -163 and the putative polymerase binding site was observed. The implications of these observations for pertussis toxin expression and regulation are discussed.
Isolation and cloning of a protein-serine/threonine phosphatase from an archaeon
Authors: Leng J , Cameron A J , Buckel S , Kennelly P J
Published: November 2016

A divalent metal ion-stimulated protein-serine/threonine phosphatase, PP1-arch, was purified approximately 1,000-fold from the extreme acidothermophilic archaeon Sulfolobus solfataricus (ATCC 35091). Purified preparations contained 40 to 70% of total protein as PP1-arch, as determined by assay-ing sodium dodecyl sulfate-polyacrylamide gels for protein phosphatase activity. The first 25 amino acids of the protein's sequence were identified, as well as an internal sequence spanning some 20 amino acids. Using this information, we cloned the gene for PP1-arch via the application of PCR and conventional cloning techniques. The gene for PP1-arch predicted a protein of 293 amino acids that bore striking resemblance to the members of the major family of protein-serine/threonine phosphatases from members of the domain Eucarya, the PP1/2A/2B superfamily. The core of the protein, spanning residues 4 to 275, possessed 29 to 31% identity with these eucaryal protein phosphatases. Of the 42 residues found to be absolutely conserved among the known eucaryal members of the PP1/2A/2B superfamily, 33 were present in PP1-arch. If highly conservative substitutions are included, this total reached 37. The great degree of sequence conservation between molecules from two distinct phylogenetic domains implies that the members of this enzyme superfamily had evolved as specialized, dedicated protein phosphatases prior to the divergence of members of the Archaea and Eucarya from one another.
Mycobacterium smegmatis dnaA region and autonomous replication activity
Authors: Rajagopalan M , Qin M H , Nash D R , Madiraju M V
Published: November 2016

Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element. As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene. Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5' end of the dnaN gene, which codes for the beta subunit of DNA polymerase III. Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M. smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M. smegmatis. We suggest that the M. smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.
Flagellar structure and hyperthermophily: analysis of a single flagellin gene and its product in Aquifex pyrophilus
Authors: Behammer W , Shao Z , Mages W , Rachel R , Stetter K O , Schmitt R
Published: November 2016

The polytrichously inserted flagella of Aquifex pyrophilus, a marine hyperthermophilic bacterium growing at 85 degrees C, were isolated and purified. Electron micrographs of the 19-nm-diameter flagellar filaments show prominent helical arrays of subunits. The primary structure of these 54-kDa flagellin monomers determining the helical shape and heat stability of filaments was of particular interest. The genomic region encoding the flagellin subunit (flaA gene) and an upstream open reading frame (orf1) were cloned and sequenced. The 1,503-bp flaA and 696-bp orf1 are preceded by separate sigma 28-like promoters and ribosome-binding motifs and succeeded by palindromic transcription terminators. Both genes are actively transcribed, but the nature and function of the orf1-encoded 231-residue polypeptide remain unknown. The deduced primary structure of the 501-amino-acid flagellin encoded by flaA consists of conserved N- and C-terminal regions and a variable 246-residue central domain. In comparison to mesophilic flagellins, the thermostable A. pyrophilus flagellin is characterized by increases in aromatic residues and prolines as well as by a 7.9% +/- 3.2% increase in all hydrophobic residues that is balanced by a respective decrease in hydrophilic residues. This composition is thought to form more compact flagellin monomers and stable interface contacts between neighboring subunits in the polymer.
The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae
Authors: ter Schure E G , Silljé H H , Verkleij A J , Boonstra J , Verrips C T
Published: November 2016

Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation. The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant. Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4. The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux.
Cloning and overexpression of the Rhodobacter capsulatus hemH gene
Authors: Kanazireva E , Biel A J
Published: November 2016

In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of the hemH gene, ferrochelatase. We have cloned the R. capsulatus hemH gene by complementation of an Escherichia coli hemH mutant. When a plasmid carrying the hemH gene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase in R. capsulatus, thereby reducing porphyrin synthesis.
Isolation of NAD cycle mutants defective in nicotinamide mononucleotide deamidase in Salmonella typhimurium
Authors: Cheng W , Roth J
Published: November 2016

The NAD or pyridine nucleotide cycle is the sequence of reactions involved in the breakdown of NAD to nicotinamide mononucleotide (NMN) and regeneration of NAD. This cycle is fivefold more active during aerobic growth of Salmonella typhimurium and under this condition breaks down half of the NAD pool every 90 min. DNA ligase is known to convert NAD to NMN but is only a minor contributor to the NAD cycle during aerobic growth. The dominant aerobic route of NMN formation is otherwise uncharacterized. Accumulated NMN generated by either of these routes is potentially dangerous in that it can inhibit the essential enzyme DNA ligase. The reactions which recycle NMN to NAD may serve to minimize the inhibition of ligase and other enzymes by accumulated NMN. The predominant recycling reaction in S. typhimurium appears to be NMN deamidase, which converts NMN directly to the biosynthetic intermediate nicotinic acid mononucleotide. Mutants defective in this recycling step were isolated and characterized. By starting with a ligase-deficient (lig mutant) parent strain that requires deamidase to assimilate exogenous NMN, two classes of mutants that are unable to grow on minimal NMN media were isolated. One class (pncC) maps at 83.7 min and shows only 2% of the wild-type levels of NMN deamidase. Under aerobic conditions, a lig+ allele allows a pncC mutant to grow on NMN and restores some deamidase activity. This growth ability and enzyme activity are not found in lig+ strains grown without oxygen. This suggests that the existence of a second NMN deamidase (pncL) dependent on ligase and stimulated during aerobic growth. The second class of mutants (pncD) gains a requirement for isoleucine plus valine with growth in the presence of exogenous NMN. We propose that pncD mutations reduce the activity of an ilv biosynthetic enzyme that is naturally sensitive to inhibition by NMN.
Structure-function relationship of bacterial prolipoprotein diacylglyceryl transferase: functionally significant conserved regions
Authors: Qi H Y , Sankaran K , Gan K , Wu H C
Published: November 2016

The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.
Construction and characterization of a fimZ mutant of Salmonella typhimurium
Authors: Yeh K S , Hancox L S , Clegg S
Published: November 2016

The Salmonella typhimurium fimA gene is controlled by several ancillary fim genes. One of these genes, fimZ, appears to be involved in increasing the expression of fimA. A fimZ mutant of S. typhimurium was constructed by allelic exchange, and this mutant was found to be nonfimbriate. The fimZ mutant demonstrated decreased levels of fimA expression compared with the parental strain when both were grown under conditions favoring fimbrial expression. An examination of the predicted amino acid sequence, deduced from the nucleotide sequence of fimZ, indicated that the FimZ polypeptide possessed a DNA binding motif. Bacterial lysates, derived from strains transformed with recombinant plasmids possessing a fimZ gene, demonstrated DNA binding activity with a fragment containing the fimA promoter. Lysates without a FimZ polypeptide did not exhibit any binding activity. These data are consistent with FimZ being a transcriptional activator of fimA, and FimZ acts by binding to the promoter region.
Fragmentations of the large-subunit rRNA in the family Rhizobiaceae
Authors: Selenska-Pobell S , Evguenieva-Hackenberg E
Published: November 2016

A 130-nucleotide-long rRNA species corresponding to the 5' end of the 23S rRNA gene was found in 96 strains belonging to different Rhizobium, Bradyrhizobium, and Agrobacterium species. Additional fragmentation in the central region of the large-subunit rRNA occurred in all agrobacteria, except Agrobacterium vitis, and in most Rhizobium leguminosarum and Rhizobium etli strains but did not occur in any of the other rhizobia and bradyrhizobia studied.
Delineation of AbrB-binding sites on the Bacillus subtilis spo0H, kinB, ftsAZ, and pbpE promoters and use of a derived homology to identify a previously unsuspected binding site in the bsuB1 methylase promote
Authors: Strauch M A
Published: November 2016

DNase I footprinting experiments showed that AbrB binds to the regulatory regions of the spo0H, kinB, ftsAZ, and pbpE genes. A conserved motif was found in these and other AbrB-binding sites. A search for Bacillus subtilis DNA sequences containing this motif led to the prediction that AbrB would bind to the promoter controlling the bsuB1 methylase gene. DNase I footprinting experiments confirmed this prediction.