bluuebunny/crossref_metadata_2025 huggingface dataset:
Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II
Authors: LeBlanc H N , Beatty J T
Published: November 2016

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.
Metabolic activities of metronidazole-sensitive and -resistant strains of Helicobacter pylori: repression of pyruvate oxidoreductase and expression of isocitrate lyase activity correlate with resistance
Authors: Hoffman P S , Goodwin A , Johnsen J , Magee K , Veldhuyzen van Zanten S J
Published: November 2016

In this study, we compared metronidazole (Mtz)-sensitive and -resistant strains of Helicobacter pylori for metabolic differences that might correlate with drug resistance. Included in this study was an isogenic Mtz(r) strain, HP1107, that was constructed by transforming genomic DNA from Mtz(r) strain HP439 into Mtz(s) strain HP500. Enzyme activities were also measured for Mtz(r) strains grown in the presence or absence of 18 micrograms of metronidazole per ml (ca. one-half of the MIC). These studies confirmed the presence of the Embden-Meyerhof-Parnas, Entner-Doudoroff, and pentose pathways. H. pylori strains expressed enzymatic activities indicative of a complete and active Krebs cycle. All strains expressed pyruvate oxidoreductase (POR) and alpha-ketoglutarate oxidoreductase (KOR) as measured with the redox-active dye benzyl viologen (30 to 96 nmol/min/mg of protein for POR and 30 nmol/min/mg of protein for KOR). When grown in the presence of Mtz at > or = 3.5 micrograms/ml, Mtz(r) strains expressed no detectable POR or KOR activity. The apparent repression of POR and KOR activities by Mtz affected bacterial growth as manifest by extended lag periods and growth yield reductions of > 30%. A dose-dependent relationship was demonstrated between the metronidazole concentration in the growth medium and the specific activity of POR measured in bacterial cell extracts. The observed repression was not due to inactivation of POR by Mtz. In addition to repression of POR and KOR activities, growth in the presence of Mtz also led to decreases in the activities of various Krebs cycle enzymes, including aconitase, isocitrate dehydrogenase and succinate dehydrogenase. All of the Mtz(r) strains examined expressed isocitrate lyase and malate synthase activities indicative of the glyoxylate bypass. No isocitrate lyase activity was detected in Mtz(s) strain HP500. Isocitrate lyase activity was expressed by HP500 following transformation to Mtz resistance (Mtz(r) strain HP1107) with DNA from an Mtz(r) strain. The results of this study suggest that Mtz resistance may be a recessive trait, possibly involving inactivation of a regulatory gene, that results in constitutive expression of isocitrate lyase. Repression of POR and KOR activities in response to low levels of Mtz may be a general response of H. pylori strains to Mtz, but only resistant strains manage to survive via activation of compensatory metabolic pathways.
Modulation of NifA activity by PII in Azospirillum brasilense: evidence for a regulatory role of the NifA N-terminal domain
Authors: Arsene F , Kaminski P A , Elmerich C
Published: November 2016

Azospirillum brasilense NifA, which is synthesized under all physiological conditions, exists in an active or inactive from depending on the availability of ammonia. The activity also depends on the presence of PII, as NifA is inactive in a glnB mutant. To investigate further the mechanism that regulates NifA activity, several deletions of the nifA coding sequence covering the amino-terminal domain of NifA were constructed. The ability of these truncated NifA proteins to activate the nifH promoter in the absence or presence of ammonia was assayed in A. brasilense wild-type and mutant strains. Our results suggest that the N-terminal domain is not essential for NifA activity. This domain plays an inhibitory role which prevents NifA activity in the presence of ammonia. The truncated proteins were also able to restore nif gene expression to a glnB mutant, suggesting that PII is required to activate NifA by preventing the inhibitory effect of its N-terminal domain under conditions of nitrogen fixation. Low levels of nitrogenase activity in the presence of ammonia were also observed when the truncated gene was introduced into a strain devoid of the ADP-ribosylation control of nitrogenase. We propose a model for the regulation of NifA activity in A. brasilense.
Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli
Authors: Berry A M , Lock R A , Paton J C
Published: November 2016

Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence of more than one neuraminidase-encoding gene. Only one stable pneumococcal neuraminidase gene (designated nanA) has been described. In the present study, we isolated and characterized a second neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5kb downstream). nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames. NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide. There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purified from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa. The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.
Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3
Authors: Tosques I E , Shi J , Shapleigh J P
Published: November 2016

During denitrification, the production and consumption of nitric oxide (NO), an obligatory and freely diffusible intermediate, must be tightly regulated in order to prevent accumulation of this highly reactive nitrogen oxide. Sequencing upstream of norCB, the structural genes for NO reductase, in the denitrifying bacterium Rhodobacter sphaeroides 2.4.3, we have identified a gene, designated nnrR, which encodes a protein that is a member of the cyclic AMP receptor family of transcriptional regulators. Insertional inactivation of nnrR prevents growth on nitrite, as well as the reduction of nitrite and NO, but has no effect on reduction of nitrate or photosynthetic growth. By using nirK-lacZ and norB-lacZ fusions, we have shown that NnrR is a positive transcriptional regulator of these genes. nnrR is expressed at a low constitutive level throughout the growth of R. sphaeroides 2.4.3. These results show that NnrR is not a global regulator but is instead a regulator of genes whose products are directly responsible for production and reduction of NO. Evidence is also presented suggesting that an NnrR homolog may be present in the nondenitrifying bacterium R. sphaeroides 2.4.1. The likely effector of NnrR activity, as determined on the basis of work detailed in this paper and other studies, is discussed.
Expression of the structural gene, laf1, encoding the flagellin of the lateral flagella in Azospirillum brasilense Sp7
Authors: Moens S , Schloter M , Vanderleyden J
Published: November 2016

The induction of the lateral flagella of Azospirillum brasilense Sp7 was studied by using a translational fusion between the laf1 promoter and gusA. The fusion was induced when cells were grown on solid media but not when they were grown in broth. The fusion was also induced by incubation of liquid-grown cells with an anti-polar flagellum polyclonal antiserum. Hindrance of polar-flagellum rotation is suggested to be the signal for this induction.
Effect of viscosity on swimming by the lateral and polar flagella of Vibrio alginolyticus
Authors: Atsumi T , Maekawa Y , Yamada T , Kawagishi I , Imae Y , Homma M
Published: November 2016

By using mutants of Vibrio alginolyticus with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+), we examined the relationship between swimming speed and the viscosity of the medium for each flagellar system. Pof+ Laf- cells could not swim in the high-viscosity environment (ca. 200 cP) in which Pof- Laf+ cells swam at 20 microns/s. The Pof- Laf+ cells swam at about 20 microns/s at normal viscosity (1 cP) without the viscous agent, and the speed increased to 40 microns/s at about 5 cP and then decreased gradually as the viscosity was increased further. These results show the functional difference between polar and lateral flagella in viscous environments.
Isolation and characterization of the structural gene for OmpL, a pressure-regulated porin-like protein from the deep-sea bacterium Photobacterium species strain SS9
Authors: Welch T J , Bartlett D H
Published: November 2016

Transposon-directed cloning was used to isolate the ompL gene from the deep-sea bacterium Photobacterium species strain SS9. The deduced amino acid sequence of OmpL displays sequence homology to porin proteins from enteric bacteria. Gene fusion and primer extension analyses indicate that ompL is transcriptionally regulated by pressure.
Single-stranded DNA-binding protein enhances the stability of CTG triplet repeats in Escherichia coli
Authors: Rosche W A , Jaworski A , Kang S , Kramer S F , Larson J E , Geidroc D P , Wells R D , Sinden R R
Published: November 2016

The stability of CTG triplet repeats was analyzed in Escherichia coli to identify processes responsible for their genetic instability. Using a biochemical assay for stability, we show that the absence of single-stranded-DNA-binding protein leads to an increase in the frequency of large deletions within the triplet repeats.
Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis
Authors: Liu L , Nakano M M , Lee O H , Zuber P
Published: November 2016

The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.
Identification and characterization of additional flagellin genes from Vibrio anguillarum
Authors: McGee K , Hörstedt P , Milton D L
Published: November 2016

Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE. The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between both organisms. For each gene, in-frame deletions of the entire gene, the 5' end, and the 3' end were made. Mutant analysis showed that each mutation, except those in flaE, caused a loss of flagellin from the filament. However, no obvious structural loss in the filament, as determined by electron microscopy, and only slight decreases in motility were seen. Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype. The 5'-end deletions of flaD and flaE decreased virulence significantly (>10(4)-fold) of infections via both the intraperitoneal and immersion routes. These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence.
An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181
Authors: Zhao A C , Khan S A
Published: November 2016

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.
The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis
Authors: Cordes C , Meima R , Twiest B , Kazemier B , Venema G , van Dijl J M , Bron S
Published: November 2016

The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the Escherichia coli beta-galactosidase (lacZ) gene. Two processes affected the accumulation of pGP1 variants with deletions in the penP-lacZ region. First, divergent transcription from genes upstream of penP-lacZ increased pGP1 deletion frequencies up to about 10-fold. Second, the removal of the PenP signal peptide resulted in completely stable plasmids, indicating that the entry of the PenP fragment into the protein export pathway is an important factor in the instability of pGP1. On the basis of these results, we propose a model in which the temporary anchoring of the plasmid to the membrane through the cotranscriptional and cotranslational entry of PenP into the protein export pathway creates domains of local hypersupercoiling, which we assume to be targets for deletion formation.
Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases
Authors: Spence E L , Kawamukai M , Sanvoisin J , Braven H , Bugg T D
Published: November 2016

The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.
Rhodobacter capsulatus CycH: a bipartite gene product with pleiotropic effects on the biogenesis of structurally different c-type cytochromes
Authors: Lang S E , Jenney F E , Daldal F
Published: November 2016

While searching for components of the soluble electron carrier (cytochrome c2)-independent photosynthetic (Ps) growth pathway in Rhodobacter capsulatus, a Ps- mutant (FJM13) was isolated from a Ps+ cytochrome c2-strain. This mutant could be complemented to Ps+ growth by cycA encoding the soluble cytochrome c2 but was unable to produce several c-type cytochromes. Only cytochrome c1 of the cytochrome bc1 complex was present in FJM13 cells grown on enriched medium, while cells grown on minimal medium contained at various levels all c-type cytochromes, including the membrane-bound electron carrier cytochrome cy. Complementation of FJM13 by a chromosomal library lacking cycA yielded a DNA fragment which also complemented a previously described Ps- mutant, MT113, known to lack all c-type cytochromes. Deletion and DNA sequence analyses revealed an open reading frame homologous to cycH, involved in cytochrome c biogenesis. The cycH gene product (CycH) is predicted to be a bipartite protein with membrane-associated amino-terminal (CycH1) and periplasmic carboxyl-terminal (CycH2) subdomains. Mutations eliminating CyCH drastically decrease the production or all known c-type cytochromes. However, mutations truncating only its CycH2 subdomain always produce cytochrome c1 and affect the presence of other cytochromes to different degrees in a growth medium-dependent manner. Thus, the subdomain CycH1 is sufficient for the proper maturation of cytochrome c1 which is the only known c-type cytochrome anchored to the cytoplasmic membrane by its carboxyl terminus, while CycH2 is required for efficient biogenesis of other c-type cytochromes. These findings demonstrate that the two subdomains of CycH play different roles in the biogenesis of topologically distinct c-type cytochromes and reconcile the apparently conflicting data previously obtained for other species.
Demonstration of a folded monomeric form of porin PhoE of Escherichia coli in vivo
Authors: Van Gelder P , Tommassen J
Published: November 2016

The porins in the outer membranes of gram-negative bacteria are trimeric proteins. A folded monomeric form of the Escherichia coli porin PhoE, with a higher electrophoretic mobility than that of the denatured protein, has recently been detected in in vitro folding studies. To investigate the possible biological significance of the folded monomer, we attempted to detect this form in vivo. After pulse-labeling, folded monomers could be detected by immunoprecipitation. Furthermore, folded monomers were detected in a preparation of mutant PhoE porins, in which the subunit interactions were weakened by a E-66-->R substitution. Together, these results show that the folded monomer is not an in vitro folding artifact but an integral part of the native trimer.
Salmonella typhi contains identical intervening sequences in all seven rrl genes
Authors: Mattatall N R , Daines D A , Liu S L , Sanderson K E
Published: November 2016

Salmonella typhi Ty2 rrl genes contain intervening sequences (IVSs) in helix-25 but not in helix-45 on the basis of observed 23S rRNA fragmentation caused by IVS excision. We have confirmed this and shown all seven IVSs to be identical by isolating genomic DNA fragments containing each of the seven rrl genes from S. typhi Ty2 by use of pulsed-field gel electrophoresis; each rrl gene was amplified by PCR in the helix-25 and helix-45 regions and cycle sequenced. Thirty independent wild-type S. typhi strains, tested by genomic PCR and DraI restriction, also have seven rrl genes with helix-25 IVSs and no helix-45 IVSs. We propose that IVS homogeneity in S. typhi occurs because gene conversion drives IVS sequence maintenance and because adaptation to human hosts results in limited clonal diversity.
Insertional inactivation of the gene for collagen-binding protein has a pleiotropic effect on the phenotype of Staphylococcus aureus
Authors: Hienz S A , Palma M , Flock J I
Published: November 2016

This report describes phenotypical changes caused by the insertional inactivation of the gene for the collagen-binding protein in Staphylococcus aureus PH100. Insertional inactivation resulted in reductions in the amount of fibronectin-binding protein in PH100 and the ability of intact cells to aggregate in the presence of fibronectin. However, the capacity of PH100 to adhere to immobilized fibronectin remained the same.
A germination-specific spore cortex-lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterization of the enzyme
Authors: Moriyama R , Kudoh S , Miyata S , Nonobe S , Hattori A , Makino S
Published: November 2016

A gene (sleB) encoding a 24-kDa germination-specific spore cortex-lytic enzyme, probably an N-acetylmuramyl-L-alanine amidase, was cloned from Bacillus cereus, and its nucleotide sequence was determined. It was indicated that the enzyme is produced as a 259-residue protein with a signal sequence of 32 residues and is present in dormant spores in its active form. Sulfhydryl reagents inactivated the enzyme, but mutation of a single cysteine of the protein, Cys-258, to Gly did not cause complete inactivation of the enzyme, suggesting that the residue does not function as the catalytic center of enzyme.
PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa
Authors: Dean C R , Neshat S , Poole K
Published: November 2016

PfeR (Regulator) and PfeS (Sensor), members of the superfamily of so-called two-component regulatory protein pairs, are required for the enterobactin-inducible production of the ferric enterobactin receptor (PfeA) in Pseudomonas aeruginosa. A pfeR knockout mutant failed to demonstrate enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that PfeR acts at the level of pfeA gene expression. Consistent with this, PfeR overexpressed in P. aeruginosa bound, in bandshift assays, the promoter region of pfeA. Such binding was enhanced when PfeR-containing extracts were prepared from cells cultured in the presence of enterobactin, consistent with a model of PfeR as an enterobactin-responsive activator of pfeA expression. A region showing homology to the consensus binding sequence for the global iron repressor Fur was identified upstream of pfeR, suggesting that the pfeRS operon is iron regulated. As expected, expression of a pfeR-lacZ fusion in P. aeruginosa was increased under conditions of iron limitation. Enterobactin failed, however, to provide any enhancement of pfeR-lacZ expression under iron-limiting conditions, indicating that PfeR does not positively regulate pfeRS expression. A pfeA knockout mutant demonstrated enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that the receptor is not required for the enterobactin inducibility of pfeA gene expression. Such mutants show growth, albeit reduced, in enterobactin-supplemented iron-limiting minimal medium, indicating that a second route of uptake across the outer membrane exists for ferric enterobactin in P. aeruginosa and may be important for the initial induction of pfeA in response to enterobactin.
Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus
Authors: Brayman T G , Hausinger R P
Published: November 2016

Klebsiella aerogenes UreE, one of four accessory proteins involved in urease metallocenter assembly, contains a histidine-rich C terminus (10 of the last 15 residues) that is likely to participate in metal ion coordination by this nickel-binding protein. To study the function of the histidine-rich region in urease activation, ureE in the urease gene cluster was mutated to result in synthesis of a truncated peptide, H144* UreE, lacking the final 15 residues. Urease activity in cells containing H144* UreE approached the activities for cells possessing the wild-type protein at nickel ion concentrations ranging from 0 to 1 mM in both nutrient-rich and minimal media. In contrast, clear reductions in urease activities were observed when two ureE deletion mutant strains were examined, especially at lower nickel ion concentrations. Surprisingly, the H144* UreE, like the wild-type protein, was readily purified with a nickel-nitrilotriacetic acid resin. Denaturing polyacrylamide gel electrophoretic analysis and N-terminal sequencing confirmed that the protein was a truncated UreE. Size exclusion chromatography indicated that the H144* UreE peptide associated into a homodimer, as known for the wild-type protein. The truncated protein was shown to cooperatively bind 1.9 +/- 0.2 Ni(II) ions as assessed by equilibrium dialysis measurements, compared with the 6.05 +/- 0.25 Ni ions per dimer reported previously for the native protein. These results demonstrate that the histidine-rich motif is not essential to UreE function and is not solely responsible for UreE nickel-binding ability. Rather, we propose that internal nickel binding sites of UreE participate in urease metallocenter assembly.
Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr
Authors: Reizer J , Bergstedt U , Galinier A , Küster E , Saier M H , Hillen W , Steinmetz M , Deutscher J
Published: November 2016

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.
Translational autoregulation of the sgm gene from Micromonospora zionensis
Authors: Kojic M , Topisirovic L , Vasiljevic B
Published: November 2016

The sisomicin-gentamicin resistance methylase gene (sgm) from Micromonospora zionensis (the producer of antibiotic G-52 [6-N-methyl-sisomicin]) encodes an enzyme that modifies 16S rRNA and thereby confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Here, we report that this gene is regulated on the translational level. The Escherichia coli lacZ gene and operon fusion system was used, and it was shown that an extra copy of the sgm gene decreases the activity of the fusion protein. These results suggested that expression of the sgm gene is regulated by the translational autorepression because of binding of the methylase to its own mRNA. It was shown by computer analysis that the same hexanucleotide (CCGCCC) is present 14 bp before the ribosome-binding site and in the C-1400 region of 16S rRNA, i.e., the region in which most of the aminoglycosides act. A deletion that removes the hexanucleotide before the gene fusion is not prone to negative autoregulation. This mode of regulation of the sgm gene ensures that enough methylase molecules protect the cell from the action of its own antibiotic. On the other hand, if all of the ribosomes are modified, Sgm methylase binds to its own mRNA in an autorepressive manner.
The uvrB gene of Pseudomonas aeruginosa is not DNA damage inducible
Authors: Rivera E , Vila L , Barbé J
Published: November 2016

The uvrB gene of Pseudomonas aeruginosa has been isolated from a genomic library by complementation of an Escherichia coli uvrB mutant. The complete nucleotide sequence of P. aeruginosa uvrB consists of 2,013 bp, encoding a polypeptide of 670 amino acids. A P. aeruginosa SOS consensus region, which functions as a binding site for the LexA repressor molecule, is not present in the upstream region of the uvrB gene isolated. By transcriptional fusions with a reporter gene, it has been demonstrated that, contrary to what happens with the homologous gene of E. coli, the P. aeruginosa uvrB gene is not DNA damage inducible. Nevertheless, the UvrB protein must be functional in P. aeruginosa cells because a uvrB-defective mutant is extremely sensitive to UV radiation.
Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima
Authors: Lee P J , Stock A M
Published: November 2016

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli. Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins. In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP. Thus, the N-terminal hydrophobic region is not required for kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.
Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus
Authors: Henstra S A , Tolner B , ten Hoeve Duurkens R H , Konings W N , Robillard G T
Published: November 2016

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mt1) were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB,a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtl4 gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB(Mtl) of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60 degrees C of B. stearothermophilus HCB(Mt1) expressed in E. coli was two times lower than that of E. coli IICB(Mtl). IICB(Mtl) in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.
Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980
Authors: Egelseer E M , Schocher I , Sleytr U B , Sára M
Published: November 2016

During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half could function as a binding site for the hmwA.
Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2
Authors: Hu S T , Lee L C , Lei G S
Published: November 2016

The genome of the transposable element IS2 contains five open reading frames that are capable of encoding proteins greater than 50 amino acids; however, only one IS2 protein of 14 kDa had been detected. By replacing the major IS2 promoter located in the right terminal repeat of IS2 with the T7 promoter to express IS2 genes, we have detected another IS2 protein of 46 kDa. This 46-kDa protein was designated InsAB'. Analyses of the InsAB' sequence revealed motifs that are characteristic of transposases of other transposable elements. InsAB' has the ability to bind both terminal repeat sequences of IS2. It was shown to bind a 27-bp sequence (5'-GTTAAGTGATAACAGATGTCTGGAAAT-3', positions 1316 to 1290 by our numbering system [16 to 42 by the previous numbering system]) located at the inner end of the right terminal repeat and a 31-bp sequence (5'-TTATTTAAGTGATATTGGTTGTCTGGAGATT-3', positions 46 to 16 [1286 to 1316]), including the last 27 bp of the inner end and the adjacent 4 bp of the left terminal repeat of IS2. This result suggests that InsAB' is a transposase of IS2. Since there is no open reading frame capable of encoding a 46-kDa protein in the entire IS2 genome, this 46-kDa protein is probably produced by a translational frameshifting mechanism.
Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein
Authors: Revill W P , Bibb M J , Hopwood D A
Published: November 2016

We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.
Global changes in protein composition of N2-fixing-Azoarcus sp. strain BH72 upon diazosome formation
Authors: Karg T , Reinhold-Hurek B
Published: November 2016

The strictly respiratory, diazotrophic bacterium Azoarcus sp. strain BH72 fixes nitrogen under microaerobic conditions. In empirically optimized batch cultures at nanomolar O2 concentrations in the presence of proline, cells can shift into a state of higher activity and respiratory efficiency of N2 fixation in which intracytoplasmic membrane stacks (diazosomes) related to N2 fixation are formed. Induction of intracytoplasmic membranes is most pronounced in coculture of Azoarcus sp. strain BH72 with an ascomycete originating from the same host plant, Kallar grass. To initiate studies on function of diazosomes and regulation of their formation, diazosome-containing bacteria were compared with respect to composition or total cellular and membrane proteins with diazosome-free cells fixing nitrogen under standard conditions. In two-dimensional protein gels, we detected striking differences in protein patterns upon diazosome formation: (i) 7.3% of major proteins disappeared, and only 73% of the total proteins of control cells were detectable, indicating that diazosome-containing cells have a more specialized metabolism; (ii) nine new proteins appeared and five proteins increased in concentration, designated DP1 to DP 15; and (iii) five new major membrane proteins (MP1 to MP6) were detected, indicating that membranes might have specialized functions. N-terminal amino acid sequence analysis of DP1 to DP4 allowed us to preliminarily identify DP4 as the glnB gene product P(II), an intracellular signal transmitter known to be involved in the regulation of nitrogen metabolism. According to its electrophoretic mobility, it might be uridylylated in diazosome-free cells but not in diazosome-containing cells, or it may represent a second, not identical P(II) protein. Oligonucleotides deduced from N-terminal sequences of DP1 and DP4 specifically hybridized to chromosomal DNA of Azoarcus sp. strain BH72 in Southern hybridizations.
Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation
Authors: Mateos L M , Schäfer A , Kalinowski J , Martin J F , Pühler A
Published: November 2016

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure.
Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme
Authors: Sakurada K , Ohta T , Fujishiro K , Hasegawa M , Aisaka K
Published: November 2016

We have cloned a gene (aphA) encoding acetylpolyamine amidohydrolase from Mycoplana ramosa ATCC 49678, (previously named Mycoplana bullata). A genomic library of M. ramosa was screened with an oligonucleotide probe designed from a N-terminal amino acid sequence of the enzyme purified from M. ramosa. Nucleotide sequence analysis revealed an open reading frame of 1,023 bp which encodes a polypeptide with a molecular mass of 36,337 Da. This is the first report of the structure of acetylpolyamine amidohydrolase. The aphA gene was subcloned under the control of the trc promoter and was expressed in Escherichia coli MM294. The recombinant enzyme was purified, and the enzymatic properties were characterized. Substrate specificities, Km values, and Vmax values were identical to those of the native enzyme purified from M. ramosa. In the analysis of the metal-substituted enzymes, we found that the acid limb of pH rate profiles shifts from 7.2 for the original zinc enzyme to 6.6 for the cobalt enzyme. This change suggests that the zinc atom is essential for the catalytic activity of the enzyme similarly to the zinc atom in carboxypeptidase A.
Markerless deletions of pil genes in Myxococcus xanthus generated by counterselection with the Bacillus subtilis sacB gene
Authors: Wu S S , Kaiser D
Published: November 2016

In-frame deletions of pilA and pilS were constructed in Myxococcus xanthus with a plasmid integration-excision strategy facilitated by sacB. sacB conferred sucrose sensitivity upon its M. xanthus host only when it lay in the same orientation as adjacent M. xanthus genes. Gene orientation also affected the efficiency of sucrose counterselection in the sucrose-sensitive strains. The deltapilA mutant lacked pili and social motility, while the deltapilS mutant showed no defect in either phenotype.
Positive regulation by nitrite of the nitrate assimilation operon in the cyanobacteria Synechococcus sp. strain PCC 7942 and Plectonema boryanum
Authors: Kikuchi H , Aichi M , Suzuki I , Omato T
Published: November 2016

In the absence of fixation of ammonium to glutamine, nitrate and nitrite activated transcription of the nitrate assimilation (nirA-nrtABCD-narB) operon of Synechococcus sp. strain PCC 7942. In a nitrate reductase-deficient mutant, only nitrite activated transcription, indicating that nitrite is the actual activator of the operon. Nitrate and nitrite were also found to activate the transcription of a nitrate assimilation operon in the filamentous nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum.
Rgg is a positive transcriptional regulator of the Streptococcus gordonii gtfG gene
Authors: Sulavik M C , Clewell D B
Published: November 2016

The Streptococcus gordonii (Challis) glucosyltransferase-encoding determinant gtfG is regulated by the product of the adjacent gene rgg. Results of analyses described here showed that in both S. gordonii and Escherichia coli Rgg is a positive transcriptional regulator of glucosyltransferase expression. In addition, the transcriptional start sites of both gtfG and rgg were determined.