bluuebunny/crossref_metadata_2025 huggingface dataset:
Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor
Authors: Gilis A , Khan M A , Cornelis P , Meyer J M , Mergeay M , van der Lelie D
Published: November 2016

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.
Transcriptional regulation of the proton-translocating ATPase (atpIBEFHAGDC) operon of Escherichia coli: control by cell growth rate
Authors: Kasimoglu E , Park S J , Malek J , Tseng C P , Gunsalus R P
Published: November 2016

The F0F1 proton-translocating ATPase complex of Escherichia coli, encoded by the atpIBEFHAGDC operon, catalyzes the synthesis of ATP from ADP and Pi during aerobic and anaerobic growth when respiratory substrates are present. It can also catalyze the reverse reaction to hydrolyze ATP during nonrespiratory conditions (i.e., during fermentation of simple sugars) in order to maintain a electrochemical proton gradient across the cytoplasmic membrane. To examine how the atp genes are expressed under different conditions of cell culture, atpI-lacZ operon fusions were constructed and analyzed in single copy on the bacterial chromosome or on low-copy-number plasmids. Expression varied over a relatively narrow range (about threefold) regardless of the complexity of the cell growth medium, the availability of different electron acceptors or carbon compounds, or the pH of the culture medium. In contrast to prior proposals, atp operon expression was shown to occur from a single promoter located immediately before atpI rather than from within it. The results of continuous-culture experiments suggest that the cell growth rate rather than the type of carbon compound used for growth is the major variable in controlling atp gene expression. Together, these studies establish that synthesis of the F0F1 ATPase is not greatly varied by modulating atp operon transcription.
Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis
Authors: Douglas A L , Hatch T P
Published: November 2016

On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a -35 hexamer region of -42 aaaaaga TATACAaa -28 and an unusual, GC-rich -10 hexamer region of -13 tTATCGCt -6. The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations. The 5' extent of P2 was located at bp -42. Replacement of wild-type sequence with two G's at positions -41 and 40, -35 and 34, and -29 and 28 resulted in severely decreased transcription. Additionally, the spacing between the -35 and -10 hexamers could not be shortened without adversely affecting in vitro activity. Substitution of G at position -13, -10, -7, or -6 had little or no effect on transcription, whereas substitution of G at -11 or -12 significantly decreased promoter strength. Triple point mutations which changed the -10 hexamer from TATCGC to TATTAT,TATATT, or TATAAT had little effect on promoter activity. Unlike the partially purified C. trachomatis sigma66-RNA polymerase used in this study, purified Escherichia coli sigma70-RNA polymerase did not recognize the wild-type P2 promoter. Mutant P2 templates with -10 hexamers that resembled the consensus recognition site were transcribed by E. coli holoenzyme in vitro, suggesting that C. trachomatis sigma66-RNA polymerase has special promoter recognition properties not found in E. coli sigma70-holoenzyme.
Mutations in apbC (mrp) prevent function of the alternative pyrimidine biosynthetic pathway in Salmonella typhimurium
Authors: Petersen L , Downs D M
Published: November 2016

The alternative pyrimidine biosynthetic (APB) pathway can synthesize the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine in Salmonella typhimurium independently of de novo purine biosynthesis. When mutants defective in function of the APB pathway were isolated, the predominant class (40%) were defective in a single locus we have designated apbC. Mutations in apbC block function of the APB pathway since they prevent growth of a purF mutant in the absence of thiamine. Lesions in apbC also cause a thiamine auxotrophy in strains proficient in purine biosynthesis when fructose is provided as the sole carbon and energy source. Results presented here are consistent with ApbC being involved in the conversion of aminoimidazole ribonucleotide to HMP, and we suggest that ApbC performs a redundant step in thiamine synthesis. Sequence analysis demonstrated that apbC mutations were alleles of mrp, a locus previously reported in Escherichia coli as a metG-related protein. We propose that this locus in S. typhimurium be designated apbC to reflect its involvement in thiamine synthesis.
Sulfur-binding protein of flagella of Thiobacillus ferrooxidans
Authors: Ohmura N , Tsugita K , Koizumi J I , Saika H
Published: November 2016

The sulfur-binding protein of Thiobacillus ferrooxidans ATCC 23270 was investigated. The protein composition of the bacterium's cell surface changed according to the culture substrate. Sulfur-grown cells showed greater adhesion to sulfur than iron-grown cells. The sulfur-grown cells synthesized a 40-kDa surface protein which was not synthesized by iron-grown cells. The 40-kDa protein had thiol groups and strongly adhered to elemental sulfur powder. This adhesion was not disturbed by Triton X-100, which can quench hydrophobic interactions. However, adhesion was disturbed by 2-mercaptoethanol, which broke the disulfide bond. The thiol groups of the 40-kDa protein formed a disulfide bond with elemental sulfur and mediated the strong adhesion between T. ferrooxidans cells and elemental sulfur. The 40-kDa protein was located on the flagella. The location of the protein would make it possible for cells to be in closer contact with the surface of elemental sulfur powder.
Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii
Authors: Seyfried M , Daniel R , Gottschalk G
Published: November 2016

The genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli. The B12-free enzyme was purified to homogeneity. It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 (alpha), 21,487 (beta), and 16,121 (gamma) Da, respectively. The enzyme complex has the composition alpha2beta2gamma2. Amino acid alignments with the subunits of the recently sequenced diol dehydratase of Klebsiella oxytoca (T. Tobimatsu, T. Hara, M. Sakaguchi, Y. Kishimoto, Y. Wada, M. Isoda, T. Sakai, and T. Toraya, J. Biol. Chem. 270:7142-7148, 1995) revealed identities between 51.8 and 70.9%.
Identification of LytSR-regulated genes from Staphylococcus aureus
Authors: Brunskill E W , Bayles K W
Published: November 2016

In this report, the characterization of a Staphylococcus aureus operon containing two LytSR-regulated genes, lrgA and lrgB, is described. Sequence and mutagenesis studies of these genes suggest that lrgA encodes a murein hydrolase exporter similar to bacteriophage holin proteins while lrgB may encode a protein having murein hydrolase activity.
Conjugative transposon Tn916: evidence for excision with formation of 5'-protruding termini
Authors: Manganelli R , Ricci S , Pozzi G
Published: November 2016

Conjugative transposons are genetic elements able to promote their own intracellular transposition and intercellular conjugal transfer. They move by an excision-integration system related to that of lambdoid phages, in which the first step is the excision of the transposon from the donor replicon to form a covalently closed circular intermediate which contains a heteroduplex joint. In this work, sequencing both strands of the circular intermediate heteroduplex joint, it was found that, as during lambda phage excision, Tn916 excises from the host DNA by 5'-protruding staggered endonucleolytic cleavages.
Characterization of an OprL null mutant of Pseudomonas putida
Authors: Rodríguez-Herva J J , Ramos J L
Published: November 2016

A Pseudomonas putida oprL null mutant was generated with reverse genetics by using an in vitro-truncated oprL::xylE construct and in vivo allelic exchange. The nature of the mutation introduced in P. putida was confirmed by Southern blotting. Western blots (immunoblots) of peptidoglycan-associated proteins revealed that the OprL protein was not made in the mutant strain, whereas it was detectable as a 19-kDa band in protein preparations of the wild-type strain. The P. putida oprL, mutant exhibited altered cell morphology as revealed by electron microscopy and was more sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA than the wild-type strain. The oprL gene was conserved in a wide variety of the Pseudomonas strains belonging to rRNA group I, which suggests that this gene is important for the maintenance of the cell envelope and cell morphology in this group of microorganisms.
Phospholipid profiles of Clostridium difficile
Authors: Drucker D B , Wardle H M , Boote V
Published: November 2016

Phospholipid molecular species present in 32 isolates of Clostridium difficile were examined by fast atom bombardment-mass spectrometry in negative-ion mode. This revealed major anions consistent with the expected presence of the following phosphatidylglycerol (PG) analogs: PG(31:2), PG(32:1), PG(33:2), PG(33:1), PG(34:2), and PG(34:1). The major phospholipid molecular species are distinct from those of other bacterial groups examined.
Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid/iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila
Authors: Maupin-Furlow J , Ferry J G
Published: November 2016

The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma). The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced. The cdhD gene is upstream of and separated by 3 bp from cdhE. Both genes are preceded by apparent ribosome-binding sites. Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD. The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum. The CdhE sequence had a four-cysteine motif with the potential to bind a 4Fe-4S cluster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy. A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli. The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration. The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom.
slpM, a gene coding for an "S-layer-like array" overexpressed in S-layer mutants of Thermus thermophilus HB8
Authors: Olabarría G , Fernández-Herrero L A , Carrascosa J L , Berenguer J
Published: November 2016

S-layer deletion mutants of Thermus thermophilus HB8 overproduce a regular array which surrounds groups of several cells. Averages of two-dimensional projections revealed a detailed architecture similar in general morphology and unit cell dimensions to that of the S-layer but having a different mass distribution. The structural components of these "S-layer-like arrays" are a group of three proteins of 52 (P52), 50 (P50), and 36 (P36) kDa, which are overexpressed in S-layer mutants. These three proteins specifically bind antibodies against P52, suggesting that the smaller proteins correspond to fragments derived from P52. This hypothesis was demonstrated by the identity of the trypsin digestion products of P52 and P50. The gene slpM, responsible for the synthesis of P52, was cloned by using synthetic oligonucleotides designed from partial amino acid sequences of P52 and P50. When slpM was expressed in Escherichia coli, proteins specifically recognized by anti-P52 antiserum whose electrophoretic mobilities were similar to those of P52 and P36 were detected. The sequence of slpM revealed the existence of an open reading frame in which the amino termini of P52, P50, and P36 were identified. The unprocessed product of slpM is a 469-amino-acid-long polypeptide whose theoretical M(r) (52,131) was in good agreement with the electrophoretic mobility of P52. The properties deduced for the product of slpM are very different from those of any S-layer protein so far sequenced. The possible roles of SlpM in wild-type cells are discussed.
Porins of Vibrio cholerae: purification and characterization of OmpU
Authors: Chakrabarti S R , Chaudhuri K , Sen K , Das J
Published: November 2016

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).
The lipooligosaccharides of Haemophilus ducreyi are highly sialylated
Authors: Melaugh W , Campagnari A A , Gibson B W
Published: November 2016

The major lipooligosaccharides of the sexually transmitted pathogen Haemophilus ducreyi 35000 have been previously found to terminate in N-acetyllactosamine and sialyl-N-acetyllactosamine, Neu5Ac alpha 2-->3Gal beta 1-->4GlcNAc (W. Melaugh, N. J. Phillips, A. A. Campagnari, M. V. Tullius, and B. W. Gibson, Biochemistry 33: 13070-13078, 1994). In this study, mass spectrometry and composition analyses have shown that the lipooligosaccharides from three other H. ducreyi strains also contain N-acetyllactosamine and are highly sialylated (approximately 30 to 50%), although one African strain was found to contain neither of these structural features.
Glucose uptake in Kluyveromyces lactis: role of the HGT1 gene in glucose transport
Authors: Billard P , Ménart S , Blaisonneau J , Bolotin-Fukuhara M , Fukuhara H , Wésolowski-Louvel M
Published: November 2016

A gene for high-affinity glucose transport, HGT1, has been isolated from the lactose-assimilating yeast Kluyveromyces lactis. Disruption strains showed much-reduced uptake of glucose at low concentrations and growth was particularly affected in low-glucose medium. The HGT1 nucleotide sequence implies that it encodes a typical transmembrane protein with 12 hydrophobic domains and with 26 to 31% amino acid identity with the Hxtp family of glucose transport elements in Saccharomyces cerevisiae. Expression is constitutive (in contrast to RAG1, the major gene for low-affinity glucose uptake in K. lactis) and is controlled by several genes also known to affect expression of RAG1. These include RAG5 (which codes for the single hexokinase of K. lactis), which is required for HGT1 transcription, and RAG4, which has a negative effect. The double mutant deltahgt1deltarag1 showed further reduced glucose uptake but still grew quite well on 2% glucose and was not completely impaired even on 0.1% glucose.
Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis
Authors: Mai X , Adams M W
Published: November 2016

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of approximately 5 min) and was purified under anaerobic conditions. It had an M(r) of approximately 210,000 and appeared to be an octomeric enzyme (alpha2beta2gamma2delta2) with four different subunits with M(r)s of 43,000 (alpha), 29,000 (beta), 23,000 (gamma), and 10,000 (delta). The enzyme contained 0.9 mol of thiamine PPi and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent Km values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PPi were approximately 250, 40, 8, and 9 microM, respectively. The enzyme was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.0 (at 80 degrees C). KGOR was quite thermostable, with a half-life at 80 degrees C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.
CodY is required for nutritional repression of Bacillus subtilis genetic competence
Authors: Serror P , Sonenshein A L
Published: November 2016

The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids. This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and was relieved in strains carrying a null mutation in the codY gene. DNase I footprinting experiments showed that purified CodY binds directly to the srfA and comK promoter regions.
Identification of the galE gene and a galE homolog and characterization of their roles in the biosynthesis of lipopolysaccharide in a serotype O:8 strain of Yersinia enterocolitica
Authors: Pierson D E , Carlson S
Published: November 2016

A clone that complements mutations in Yersinia enterocolitica lipopolysaccharide (LPS) core biosynthesis was isolated, and the DNA sequence of the clone was determined. Three complete open reading frames and one partial open reading frame were located on the cloned DNA fragment. The first, partial, open reading frame had homology to the rfbK gene. The remaining reading frames had homology to galE, rol, and gsk. Analysis of the galE homolog indicates that although it can complement an Escherichia coli galE mutant, its primary function in Y. enterocolitica is not in the production of UDP galactose but, instead, some other nucleotide sugar required for LPS biosynthesis. This gene has been renamed lse, for LPS sugar epimerase. The rol homolog has been demonstrated to have a role in Y. enterocolitica serotype 0:8 O-polysaccharide antigen chain length determination. An additional galE homolog has been identified in Y. enterocolitica by homology to the E. coli gene. The product of this gene has UDP galactose 4-epimerase activity in both E. coli and Y. enterocolitica. This gene is linked to the other genes of the galactose utilization pathway, similar to what is seen in other members of the family Enterobacteriaceae. Although Y. enterocolitica 0:8 strains are reported to have galactose as a constituent of LPS, a strain containing a mutation in this galE gene does not exhibit any LPS defects.
Evolutionary relationships among extradiol dioxygenases
Authors: Eltis L D , Bolin J T
Published: November 2016

A structure-validated alignment of 35 extradiol dioxygenase sequences including two-domain and one-domain enzymes was derived. Strictly conserved residues include the metal ion ligands and several catalytically essential active site residues, as well as a number of structurally important residues that are remote from the active site. Phylogenetic analyses based on this alignment indicate that the ancestral extradiol dioxygenase was a one-domain enzyme and that the two-domain enzymes arose from a single genetic duplication event. Subsequent divergence among the two-domain dioxygenases has resulted in several families, two of which are based on substrate preference. In several cases, the two domains of a given enzyme express different phylogenies, suggesting the possibility that such enzymes arose from the recombination of genes encoding different dioxygenases. A phylogeny-based classification system for extradiol dioxygenases is proposed.
Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants
Authors: Francetic O , Kumamoto C A
Published: November 2016

Ribose-binding protein (RBP) is exported to the periplasm of Escherichia coli via the general export pathway. An rbsB-lacZ gene fusion was constructed and used to select mutants defective in RBP export. The spontaneous Lac+ mutants isolated in this selection contained either single-amino-acid substitutions or a deletion of the RBP signal sequence. Intact rbsB genes containing eight different point mutations in the signal sequence were reconstructed, and the effects of the mutations on RBP export were examined. Most of the mutations caused severe defects in RBP export. In addition, different suppressor mutations in SecY/PrlA protein were analyzed for their effects on the export of RBP signal sequence mutants in the presence or absence of SecB. Several RBP signal sequence mutants were efficiently suppressed, but others were not suppressed. Export of an RBP signal sequence mutant in prlA mutant strains was partially dependent on SecB, which is in contrast to the SecB independence of wild-type RBP export. However, the kinetics of export of an RBP signal sequence mutant point to a rapid loss of pre-RBP export competence, which occurs in strains containing or lacking SecB. These results suggest that SecB does not stabilize the export-competent conformation of RBP and may affect translocation by stabilizing the binding of pre-RBP at the translocation site.
Molecular cloning and characterization of Saccharomyces cerevisiae RAD28, the yeast homolog of the human Cockayne syndrome A (CSA) gene
Authors: Bhatia P K , Verhage R A , Brouwer J , Friedberg E C
Published: November 2016

Cockayne syndrome patients exhibit severe developmental and neurological abnormalities. Cells derived from these patients are sensitive to killing by UV radiation and do not support the rapid repair of the transcribed strand of transcriptionally active genes observed in cells from normal individuals. We report the cloning of the Saccharomyces cerevisiae homolog of the Cockayne syndrome A (CSA) gene, which we designate as RAD28. A rad28 null mutant does not manifest increased sensitivity to killing by UV or gamma radiation or to methyl methanesulfonate. Additionally, the rate of repair of the transcribed and nontranscribed strands of the yeast RPB2 gene in the rad28 mutant is identical to that observed in wild-type cells following exposure to UV light. As previously shown for rad7 rad26 and rad16 rad26 double mutants, the rad28 null mutant shows slightly enhanced sensitivity to UV light in the presence of mutations in the RAD7 or RAD16 gene. Both rad28 and rad26 null mutants are hypermutable following exposure to UV light.
Identification and characterization of pbpC, the gene encoding Bacillus subtilis penicillin-binding protein 3
Authors: Murray T , Popham D L , Setlow P
Published: November 2016

Penicillin-binding proteins (PBPs) are enzymes involved in the synthesis of peptidoglycan structures in Bacillus subtilis such as the vegetative cell wall and the spore cortex. The B. subtilis sequencing project has identified a gene (orf16, EMBL accession number D38161) which exhibits significant sequence similarity to genes encoding class B high-molecular-weight PBPs. We have found that orf16 encodes PBP3 and have renamed this locus pbpC. Transcriptional fusions to lacZ were used to demonstrate that pbpC is transcribed primarily during log-phase growth, with lower amounts expressed during sporulation. During spore germination and outgrowth, pbpC expression resumes coincident with an increase in the optical density of the culture. The major promoter for pbpC is located just upstream of the gene; a low level of expression during sporulation appears to originate from much further upstream. Loss of PBP3 does not produce any detectable change in phenotype with respect to cell morphology, growth, sporulation, spore heat resistance, or spore germination and outgrowth. This was also true when the pbpC mutation was combined with mutations affecting other PBP-encoding genes to produce double mutants. These findings are consistent with previous evidence that many PBPs of B. subtilis have redundant functions within the cell.
IS257-mediated cointegration in the evolution of a family of staphylococcal trimethoprim resistance plasmids
Authors: Leelaporn A , Firth N , Paulsen I T , Skurray R A
Published: November 2016

Analyses of the Staphylococcus epidermidis multiresistance plasmids pSK697 and pSK818 have revealed them to be closely related to the trimethoprim resistance plasmid pSK639, also isolated from S. epidermidis. pSK697 and pSK818 were found to contain a cointegrated copy of a second plasmid related to the S. epidermidis multidrug antiseptic and disinfectant resistance plasmid pSK108 and the S. aureus tetracycline resistance plasmid pT181, respectively. In contrast to pSK639, both plasmids were found to contain a third copy of IS257, such that the integrated plasmids in both cases are flanked by a copy of this element. This organization and the presence of duplicated sequences at the extremities of the integrated plasmids implicate IS257 in the formation of these cointegrate plasmids. Sequence analysis of the IS257 elements from these plasmids has provided insights into the probable mechanism of cointegration, viz., nonresolved replicative transposition of IS257.
Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600
Authors: Knobel H R , Egli T , van der Meer J R
Published: November 2016

A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression.
A chimeric disposition of the elongation factor genes in Rickettsia prowazekii
Authors: Syvänen A C , Amiri H , Jamal A , Andersson S G , Kurland C G
Published: November 2016

An exceptional disposition of the elongation factor genes is observed in Rickettsia prowazekii, in which there is only one tuf gene, which is distant from the lone fus gene. In contrast, the closely related bacterium Agrobacterium tumefaciens has the normal bacterial arrangement of two tuf genes, of which one is tightly linked to the fus gene. Analysis of the flanking sequences of the single tuf gene in R. prowazekii shows that it is preceded by two of the four tRNA genes located in the 5' region of the Escherichia coli tufB gene and that it is followed by rpsJ as well as associated ribosomal protein genes, which in E. coli are located downstream of the tufA gene. The fus gene is located within the str operon and is followed by one tRNA gene as well as by the genes secE and nusG, which are located in the 3' region of tufB in E. coli. This atypical disposition of genes suggests that intrachromosomal recombination between duplicated tuf genes has contributed to the evolution of the unique genomic architecture of R. prowazekii.
Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum
Authors: Fox J D , He Y , Shelver D , Roberts G P , Ludden P W
Published: November 2016

In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins. These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. Together these enzymes catalyze the following conversion: CO + H2O --> CO2 + H2. This system enables R. rubrum to grow in the dark on CO as the sole energy source. Expression of this system has been shown previously to be regulated at the transcriptional level by CO. We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R. rubrum genome. These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase. As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3. In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energy-conserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms. We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well. We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins.
Secondary transporters for citrate and the Mg(2+)-citrate complex in Bacillus subtilis are homologous proteins
Authors: Boorsma A , van der Rest M E , Lolkema J S , Konings W N
Published: November 2016

Citrate uptake in Bacillus subtilis is mediated by a secondary transporter that transports the complex of citrate and divalent metal ions. The gene coding for the transporter termed CitM was cloned, sequenced, and functionally expressed in Escherichia coli. Translation of the base sequence to the primary sequence revealed a transporter that is not homologous to any known secondary transporter. However, CitM shares 60% sequence identity with the gene product of open reading frame N15CR that is on the genome of B. subtilis and for which no function is known. The hydropathy profiles of the primary sequences of CitM and the unknown gene product are very similar, and secondary structure prediction algorithms predict 12 transmembrane-spanning segments for both proteins. Open reading frame N15CR was cloned and expressed in E. coli and was shown to be a citrate transporter as well. The transporter is termed CitH. A remarkable difference between the two transporters is that citrate uptake by CitM is stimulated by the presence of Mg2+ ions, while citrate uptake by CitH is inhibited by Mg2+. It is concluded that the substrate of CitM is the Mg(2+)-citrate complex and that CitH transports the free citrate anion. Uptake experiments in right-side-out membrane vesicles derived from E. coli cells expressing either CitM or CitH showed that both transporters catalyze electrogenic proton/substrate symport.
Characterization of a cytoplasmic trehalase of Escherichia coli
Authors: Horlacher R , Uhland K , Klein W , Ehrmann M , Boos W
Published: November 2016

Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source. The pathway of trehalose utilization is different at low and high osmolarity. At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose. Glucose is then taken up by the phosphotransferase system. At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose. treF, the gene encoding this enzyme, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein. The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency. TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA. The nonidentical amino acids of TreF are more polar and more acidic than those of TreA. The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor. Mutants producing 17-fold more TreF than does the wild type were isolated.
Spontaneous tandem sequence duplications reverse the thermal stability of carboxyl-terminal modified 3-isopropylmalate dehydrogenase
Authors: Akanuma S , Yamagishi A , Tanaka N , Oshima T
Published: November 2016

A mutant strain of Thermus thermophilus which contains deletions in the 3'-terminal region of its leuB gene showed a temperature-sensitive growth phenotype in the absence of leucine. Three phenotypically thermostable mutants were isolated from the temperature-sensitive strain by spontaneous evolution. Each pseudorevertant carried a tandem sequence duplication in the 3' region of its leuB gene. The mutated 3-isopropylmalate dehydrogenases encoded by the leuB genes from the pseudorevertants were more thermostable than the enzyme from the temperature-sensitive strain. Structural analyses suggested that the decreased thermostability of the enzyme from the temperature-sensitive strain was caused by reducing hydrophobic and electrostatic interactions in the carboxyl-terminal region and that the recovered stability of the enzymes from the pseudorevertants was due to the restoration of the hydrophobic interaction. Our results indicate that tandem sequence duplications are the general genetic way to alter protein characteristics in evolution.
The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis
Authors: Furuya K , Hutchinson C R
Published: November 2016

DnrN, a protein essential for the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius, was overproduced in Escherichia coli and S. peucetius. The cell-free extract from E. coli was used to conduct DNA-binding assays. The results of gel mobility shift analysis showed that DnrN binds specifically to the dnrI promoter region with a high affinity (Kd = 50 nM). Neither acetyl phosphate nor ATP affected the binding ability, and there was no difference in binding between wild-type DnrN and a mutant form (D-55-->N) lacking the putative phosphorylation site (aspartate 55) of a response regulator protein. Therefore, phosphorylation of DnrN apparently is not necessary for DNA binding. DNase I footprinting analysis indicated binding regions at 37 to 55 bp and 62 to 100 bp upstream of the transcriptional start point of dnrI. Interestingly, the sequence of these regions includes consecutive overlapping triplets [5'-(A/T)GC, 5'-(A/T)CG, 5'-(A/T)C(A/T)] that have been shown to be the preferential binding site of daunorubicin (J. B. Chaires and J. E. Herrera, Biochemistry 29:6145-6153, 1990). This may explain why daunorubicin appeared to inhibit the binding of DnrN to the dnrI promoter, which could result in feedback repression of daunorubicin production. The results of Western blotting (immunoblotting) analysis with His-tagged DnrN antiserum showed that dnrN expression is coincident with daunorubicin production and that the maximum level of DnrN is 0.01% of total protein in the wild-type S. peucetius strain. Since the level of DnrN was lowered in mutant strains that do not produce daunorubicin, we speculate that dnrN and dnrI expression are regulated by daunorubicin.
Structure and function of the Pseudomonas putida integration host factor
Authors: Calb R , Davidovitch A , Koby S , Giladi H , Goldenberg D , Margalit H , Holtel A , Timmis K , Sanchez-Romero J M , de Lorenzo V , Oppenheim A B
Published: November 2016

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.
Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii
Authors: Theisen M
Published: November 2016

Borrelia burgdorferi sensu lato organisms, comprising B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are tick-borne pathogens causing Lyme borreliosis in humans. To identify putative virulence determinants, a B. afzelii DNA library was screened for Congo red dye binding, a property associated with virulence in pathogenic bacteria. One clone was found to carry a 663-nucleotide-long open reading frame encoding a Congo red dye-binding protein with a calculated molecular mass of 25,660 Da. The amino acid sequence deduced from its nucleotide sequence was found to include a consensus bacterial lipidation site present at residues 15 to 18 (Leu-Ser-Gly-Cys). The lipoprotein nature was demonstrated by incorporation of radioactive palmitate; hence, this protein has been termed NlpH, for new lipoprotein H. NlpH is located on the surface of B. afzelii, and the nlpH gene is found on a circular plasmid. The nlpH gene is also found in B. burgdorferi sensu stricto and B. garinii. Immediately upstream of nlpH is located a smaller reading frame encoding a polypeptide containing the casein kinase II phosphorylation recognition sequence, (Ser/Thr)-X-Y-(Glu/Asp), repeated 10 times.
A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis
Authors: Lee A I , Sarsero J P , Yanofsky C
Published: November 2016

In Bacillus subtilis, the tryptophan-activated trp RNA-binding attenuation protein (TRAP) regulates expression of the seven tryptophan biosynthetic genes by binding to specific repeat sequences in the transcripts of the trp operon and of the folate operon, the operon containing trpG. Steinberg observed that strains containing a temperature-sensitive mutant form of tryptophanyl-tRNA synthetase, encoded by the trpS1 allele, produced elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess L-tryptophan (W. Steinberg, J. Bacteriol. 117:1023-1034, 1974). We have confirmed this observation and have shown that expression of two reporter gene fusions, trpE'-'lacZ and trpG'-'lacZ, is also increased under these conditions. Deletion of the terminator or antiterminator RNA secondary structure involved in TRAP regulation of trp operon expression eliminated the trpS1 effect, suggesting that temperature-sensitive expression was mediated by the TRAP protein. Analysis of expression of mtrB, the gene encoding the TRAP subunit, both by examination of a lacZ translational fusion and by measuring the intracellular levels of TRAP by immunoblotting, indicated that the trpS1-induced increase in trp gene expression was not due to inhibition of mtrB expression or to alteration of the amount of TRAP present per cell. Increasing the cellular level of TRAP by overexpressing mtrB partially counteracted the trpS1 effect, demonstrating that active TRAP was limiting in the trpS1 mutant. We also showed that elevated trp operon expression was not due to increased transcription initiation at the upstream aroF promoter, a promoter that also contributes to trp operon expression. We postulate that the increase in trp gene expression observed in the trpS1 mutant is due to the reduced availability of functional TRAP. This could result from inhibition of TRAP function by uncharged tRNA(Trp) molecules or by increased synthesis of some other transcript capable of binding and sequestering the TRAP regulatory protein.
Periodic phenomena in Proteus mirabilis swarm colony development
Authors: Rauprich O , Matsushita M , Weijer C J , Siegert F , Esipov S E , Shapiro J A
Published: November 2016

Proteus mirabilis colonies exhibit striking geometric regularity. Basic microbiological methods and imaging techniques were used to measure periodic macroscopic events in swarm colony morphogenesis. We distinguished three initial phases (lag phase, first swarming phase, and first consolidation phase) followed by repeating cycles of subsequent swarming plus consolidation phases. Each Proteus swarm colony terrace corresponds to one swarming-plus-consolidation cycle. The duration of the lag phase was dependent upon inoculation density in a way that indicated the operation of both cooperative and inhibitory multicellular effects. On our standard medium, the second and subsequent swarm phases displayed structure in the form of internal waves visible with reflected and dark-field illumination. These internal waves resulted from organization of the migrating bacteria into successively thicker cohorts of swarmer cells. Bacterial growth and motility were independently modified by altering the composition of the growth medium. By varying the glucose concentration in the substrate, it was possible to alter biomass production without greatly affecting the kinetics of colony surface area expansion. By varying the agar concentration in the substrate, initial bacterial biomass production was unaffected but colony expansion dynamics were significantly altered. Higher agar concentrations led to slower, shorter swarm phases and longer consolidation phases. Thus, colony growth was restricted by higher agar concentrations but the overall timing of the swarming-plus-consolidation cycles remained constant. None of a variety of factors which had significant effects on colony expansion altered terracing frequencies at 32 degrees C, but the length of the swarming-plus-consolidation cycle was affected by temperature and medium enrichment. Some clinical isolates displayed significant differences in terracing frequencies at 32 degrees C. Our results defined a number of readily quantifiable parameters in swarm colony development. The data showed no connection between nutrient (glucose) depletion and the onset of different phases in swarm colony morphogenesis. Several observations point to the operation of density-dependent thresholds in controlling the transitions between distinct phases.