bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli
Authors: Borst D W , Blumenthal R M , Matthews R G
Published: November 2016

Most studies of global regulatory proteins are performed in vitro or involve phenotypic comparisons between wild-type and mutant strains. We report the use of strains in which the gene for the leucine-responsive regulatory protein (lrp) is transcribed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters for the purpose of continuously varying the in vivo concentration of Lrp. To obtain a broad range of Lrp concentrations, strains were employed that contained the lrp fusion either in the chromosome (I. C. Blomfield, P. J. Calie, K. J. Eberhardt, M. S. McClain, and B. I. Eisenstein, J. Bacteriol. 175:27-36, 1993) or on a multicopy plasmid. Western blot (immunoblot) analysis with polyclonal antiserum to Lrp confirmed that Lrp levels could be varied more than 70-fold by growing the strains in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium containing different amounts of IPTG. Expression of an Lrp-regulated gltB::lacZ operon fusion was measured over this range of Lrp concentrations. beta-Galactosidase activity rose with increasing Lrp levels up to the level of Lrp found in wild-type strains, at which point expression is maximal. The presence of leucine in the medium increased the level of Lrp necessary to achieve half-maximal expression of the gltB::lacZ fusion, as predicted by earlier in vitro studies (B. R. Ernsting, J. W. Denninger, R. M. Blumenthal, and R. G. Matthews, J. Bacteriol. 175:7160-7169, 1993). Interestingly, levels of Lrp greater than those in wild-type cells interfered with activation of gltB::lacZ expression. The growth rate of cultures correlated with the intracellular Lrp concentration: levels of Lrp either lower or higher than wild-type levels resulted in significantly slower growth rates. Thus, the level of Lrp in the cell appears to be optimal for rapid growth in minimal medium, and the gltBDF control region is designed to give maximal expression at this Lrp level.
Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro
Authors: Wassenaar R W , Daas P J , Geerts W J , Keltjens J T , van der Drift C
Published: November 2016

The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.
Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter
Authors: Tan M , Engel J N
Published: November 2016

Chlamydia trachomatis RNA polymerase was partially purified by heparin-agarose chromatography and used in conjunction with a plasmid-borne G-less cassette template to characterize the C. trachomatis rRNA P1 promoter in vitro. Stepwise mutational analysis revealed that sequences in the -10, -25, and -35 regions are necessary for promoter activity, but no sequence upstream of position -40 is required. Partially purified C. trachomatis RNA polymerase and purified Escherichia coli holoenzyme exhibited some differences in promoter specificity.
FlgM is a primary regulator of sigmaD activity, and its absence restores motility to a sinR mutant
Authors: Fredrick K , Helmann J D
Published: November 2016

We have used mini-Tn1O mutagenesis to identify negative regulators of sigmaD activity. Nine independent insertions were mapped to five genes: flgM, flgK, fliD, fliS, and fliT, suggesting that FlgM export is regulated similarly in Bacillus subtilis and Salmonella typhimurium. We show that a deletion of flgM can restore sigmaD activity to a sinR null mutant of B. subtilis, although fla/che operon expression is affected by neither SinR nor FlgM.
Identification and characterization of the tolQRA genes of Pseudomonas aeruginosa
Authors: Dennis J J , Lafontaine E R , Sokol P A
Published: November 2016

The tolQ, tolR, and tolA genes from Pseudomonas aeruginosa PAO were cloned using degenerate oligonucleotide PCR primers designed based on conserved transmembrane regions of Escherichia coli TolQ and TolR and E. coli and Pseudomonas putida ExbB and ExbD. The resulting PCR product was used as a probe to isolate a 6.5-kb DNA fragment containing P. aeruginosa tolQ, tolR, and tolA. The nucleotide sequence of a 2.9-kb DNA fragment containing the tolQ, tolR, and tolA genes was determined. The DNA sequence predicts TolQ to be a 25,250-Da protein exhibiting 53% identity to E. coli TolQ. TolR is predicted to be a 15,788-Da protein, sharing 38% identity with the E. coli TolR protein. The P. aeruginosa tolA sequence predicts a 37,813-Da protein with 27% identity to the E. coli TolA. The P. aeruginosa TolQRA proteins were expressed in E. coli minicells. Analysis of plasmid-encoded tolQ::lacZ and tolA::lacZ promoter fusions in E. coli indicated that these genes are expressed at different levels, suggesting transcription from different promoters. Transcriptional analysis of the tol genes in P. aeruginosa revealed that the tolQ and tolR genes are cotranscribed as an approximately 1.5-kb transcript and that tolA is transcribed from its own promoter as an approximately 1.2-kb transcript. The P. aeruginosa Tol proteins were functionally unable to complement E. coli tol mutants, although P. aeruginosa TolQ was able to complement the iron-limited growth of an E. coli exbB mutant. Introduction of the tolQRA genes in the tol-like mutant PAO 1652 restored pyocin AR41 killing, indicating that the Tol proteins are involved in the uptake of pyocin AR41 in P. aeruginosa. Attempts to inactivate the chromosomal copy of the tolA or tolQ gene in the parent strain PAO proved to be unsuccessful, and we propose that inactivation of these genes in P. aeruginosa results in a lethal phenotype.
FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin
Authors: Lo W S , Dranginis A M
Published: November 2016

We report the characterization of a gene encoding a novel flocculin related to the STA genes of yeast, which encode secreted glucoamylase. The STA genes comprise sequences that are homologous to the sporulation-specific glucoamylase SGA and to two other sequences, S2 and S1. We find that S2 and S1 are part of a single gene which we have named FLO11. The sequence of FLO11 reveals a 4,104-bp open reading frame on chromosome IX whose predicted product is similar in overall structure to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. An amino-terminal domain containing a signal sequence and a carboxy-terminal domain with homology to GPI (glycosyl-phosphatidyl-inositol) anchor-containing proteins are separated by a central domain containing a highly repeated threonine- and serine-rich sequence. Yeast cells that express FLO11 aggregate in the calcium-dependent process of flocculation. Flocculation is abolished when FLO11 is disrupted. The product of STA1 also is shown to have flocculating activity. When a green fluorescent protein fusion of FLO11 was expressed from the FLO11 promoter on a single-copy plasmid, fluorescence was observed in vivo at the periphery of cells. We propose that FLO11 encodes a flocculin because of its demonstrated role in flocculation, its structural similarity to other members of the FLO gene family, and the cell surface location of its product. FLO11 gene sequences are present in all yeast strains tested, including all standard laboratory strains, unlike the STA genes which are present only in the variant strain Saccharomyces cerevisiae var. diastaticus. FLO11 differs from all other yeast flocculins in that it is located near a centromere rather than a telomere, and its expression is regulated by mating type. Repression of FLO11-dependent flocculation in diploids is conferred by the mating-type repressor al/alpha2.
Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro
Authors: Lu Y , Switzer R L
Published: November 2016

Transcriptional attenuation of the pyrimidine biosynthetic (pyr) operon from Bacillus subtilis was reconstituted with an in vitro system that consisted of pyr DNA templates, B. subtilis RNA polymerase, four ribonucleoside triphosphates, and the purified B. subtilis PyrR regulatory protein. The templates used each specified one of the three known attenuation regions of the pyr operon. Runoff (read-though) and terminated transcripts of the predicted lengths were the only major products synthesized. Transcription of the template that specifies the 5' leader attenuation region of the operon was examined in detail. Termination of transcription at the attenuator was strongly promoted by the combination of PyrR plus UMP. The concentration of UMP required for half-maximal effect was 2.5 microM. UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UDP was only effective at 100 times the concentration of UMP. Other pyrimidine and purine metabolites tested did not affect termination. PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferase activity of PyrR, antagonized UMP-dependent transcriptional termination, but uracil did not. Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr attenuation regions. The results strongly support the model for transcriptional regulation of the B. subtilis pyr operon previously proposed by R. J. Turner, Y. Lu, and R. L. Switzer (J. Bacteriol. 176:3708-3722, 1994).
Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway
Authors: Cánovas D , Vargas C , Csonka L N , Ventosa A , Nieto J J
Published: November 2016

The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.
Enhanced antibiotic production by manipulation of the Streptomyces peucetius dnrH and dnmT genes involved in doxorubicin (adriamycin) biosynthesis
Authors: Scotti C , Hutchinson C R
Published: November 2016

Sequence analysis of a 3.4-kb region Streptomyces peucetius daunorubicin (DNR) gene cluster established the presence of the dnrH and dnmT genes. In dnrH mutants, DNR production increased 8.5-fold, compared with that in the wild-type strain, while dnmT mutants accumulated epsilon-rhodomycinone (RHO), which normally becomes glycosylated in daunorubicin biosynthesis. Hence, dnmT may be involved in the biosynthesis or attachment of daunosamine to RHO or in the regulation of this process. Since the DnrH protein is similar to known glycosyl transferases, this protein may catalyze the conversion of DNR to its polyglycosylated forms, known as baumycins. Overexpression of dnmT in the wild-type and dnrH mutant strains resulted in a major decrease in RHO accumulation and increase in DNR production.
Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen
Authors: Hassett D J
Published: November 2016

Pseudomonas aeruginosa produced alginate and elevated algD (encoding GDPmannose 6-dehydrogenase) transcription under strict anaerobic conditions, especially when using nitrate as a terminal electron acceptor. Purified alginate added to bacterial suspensions caused a decrease in growth, suggesting that alginate contributes to oxygen limitation for the organism and likely for patients afflicted with the inherited autosomal disease cystic fibrosis.
Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon
Authors: Stein J L , Marsh T L , Wu K Y , Shizuya H , DeLong E F
Published: November 2016

One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated it close relationship to that of previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea. Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea. Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.
Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation
Authors: Mattimore V , Battista J R
Published: November 2016

Forty-one ionizing radiation-sensitive strains of Deinococcus radiodurans were evaluated for their ability to survive 6 weeks of desiccation. All exhibited a substantial loss of viability upon rehydration compared with wild-type D. radiodurans. Examination of chromosomal DNA from desiccated cultures revealed a time-dependent increase in DNA damage, as measured by an increase in DNA double-strand breaks. The evidence presented suggests that D. radiodurans' ionizing radiation resistance is incidental, a consequence of this organism's adaptation to a common physiological stress, dehydration.
Flagellar assembly in Caulobacter crescentus: a basal body P-ring null mutation affects stability of the L-ring protein
Authors: Mohr C D , Jenal U , Shapiro L
Published: November 2016

The P- and L-rings are structural components of the flagellar basal body that are positioned in the periplasmic space and outer membrane, respectively. In order to explore the mechanism of P- and L-ring assembly, we examined the effect of a null mutation in the gene encoding the P-ring subunit, FlgI, on the expression, stability, and subcellular localization of the L-ring subunit, FlgH, in Caulobacter crescentus. Transcription of the L-ring gene and synthesis of the L-ring protein were both increased in the P-ring null mutant. However, steady-state L-ring protein levels were dramatically reduced compared with those of wild type. This reduction, which was not observed in flagellar hook mutants, was due to a decreased stability of the L-ring protein. The instability of the L-ring protein was apparent throughout the cell cycle of the P-ring mutant and contrasted with the fairly constant level of L-ring protein during the cell cycle of wild-type cells. Low levels of the L-ring protein were detected exclusively in the cell envelope of cells lacking the P-ring, suggesting that, in the absence of P-ring assembly, L-ring monomers are unable to form multimeric rings and are thus subject to proteolysis in the periplasm.
Sequencing, distribution, and inactivation of the dipeptidase A gene (pepDA) from Lactobacillus helveticus CNRZ32
Authors: Dudley E G , Husgen A C , He W , Steele J L
Published: November 2016

Previously, the gene for a general dipeptidase (pepDA) was isolated from a gene bank of Lactobacillus helveticus CNRZ32. The pepDA gene consists of a 1,422-bp open reading frame which could encode a polypeptide of 53.5 kDa. No significant identity was found between the deduced amino acid sequence of the pepDA product and the sequence for other polypeptides reported in GenBank. Southern hybridization studies with a pepDA probe indicated that the nucleotide sequence for pepDA is not well conserved among a variety of lactic acid bacteria. Growth studies indicated that a pepDA deletion had no detectable effect on growth rate or acid production by L. helveticus CNRZ32 in milk. Furthermore, no difference in total cellular dipeptidase activity was detected between the mutant and wild-type strains during logarithmic growth in MRS medium.
Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants
Authors: Hove-Jensen B
Published: November 2016

Phosphoribosyl diphosphate-lacking (delta prs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene of the Pho regulon because the ability of pst or phoU mutations to suppress the NAD requirement requires PhoB, the transcriptional activator of the Pho regulon.
Genetic analysis of Rhizobium meliloti bacA-phoA fusion results in identification of degP: two loci required for symbiosis are closely linked to degP
Authors: Glazebrook J , Ichige A , Walker G C
Published: November 2016

The function of the Rhizobium meliloti bacA gene, which is a homolog of the Escherichia coli sbmA gene, is required for an intermediate step in nodule development. A strain carrying the bacA386::TnphoA fusion was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and three mutants that had higher levels of alkaline phosphatase activity were identified. The mutations in these strains were recessive and mapped to the same genetic locus. The gene affected by these mutations was identified and sequenced and was found to be a homolog of the E. coli degP gene, which encodes a periplasmic endopeptidase. Although degP function is important for the virulence of certain intracellular pathogens of mammals, it is not required for the R. meliloti-alfalfa symbiosis. The genetic analyses involving degP were complicated by the presence of a locus immediately upstream of depP that was lethal when present in multiple copies in a DegP- background. R. meliloti derivatives carrying insertion mutations in this locus displayed an N,N,N',N'-tetramethyl-p-phenylenediamine oxidase-negative phenotype, elicited the formation of white cylindrical nodules that did not fix nitrogen, and grew slowly in rich medium, suggesting that the locus was a cyc gene encoding a protein involved in the biosynthesis of a component or components of a respiratory chain. The previously identified fix-382::TnphoA, which similarly causes the formation of white cylindrical nodules that do not fix nitrogen, was shown to affect a gene that is separate from this cyc gene but extremely closely linked to it.
Synthesis of immediate upshift (Iup) proteins during recovery of marine Vibrio sp. strain S14 subjected to long-term carbon starvation
Authors: Marouga R , Kjelleberg S
Published: November 2016

Proteins induced during the initial phase of recovery after long-term carbon starvation in the marine Vibrio sp. strain S14 were identified by two-dimensional gel electrophoresis analysis. Nutritional upshift experiments with pulse-labeled cells were performed after addition of glucose to cells starved for 48 h. Eighteen proteins synthesized during the first 3 min after substrate addition were identified and designated immediate upshift proteins (Iup proteins). They were induced at least 10-fold compared with the rate of synthesis during starvation. Of the Iup proteins, five are not found in exponentially growing cells. Subsequent to the first 3 min of glucose addition, a complex pattern of sequential synthesis of proteins made during a transient phase as well as proteins made during 60 min of the outgrowth response was monitored. To resolve whether the Iup proteins were synthesized from stable transcripts, the initiation of transcription was inhibited by rifampin (Rif). Addition of Rif 5 min prior to glucose promoted upshift resulted in the synthesis of 12 Iup proteins. Furthermore, three Iup proteins were still induced by cells that were Rif treated 20 min prior to the upshift. These results suggest that stable but silent transcripts exist during starvation and that the translation of these mRNA species is initiated by substrate addition. This regulatory mechanism may be essential for an immediate initiation of the recovery program by the nongrowing cell.
Genetic and topological analyses of the bop promoter of Halobacterium halobium: stimulation by DNA supercoiling and non-B-DNA structure
Authors: Yang C F , Kim J M , Molinari E , DasSarma S
Published: November 2016

The bop gene of wild-type Halobacterium halobium NRC-1 is transcriptionally induced more than 20-fold under microaerobic conditions. bop transcription is inhibited by novobiocin, a DNA gyrase inhibitor, at concentrations subinhibitory for growth. The exposure of NRC-1 cultures to novobiocin concentrations inhibiting bop transcription was found to partially relax plasmid DNA supercoiling, indicating the requirement of high DNA supercoiling for bop transcription. Next, the bop promoter region was cloned on an H. halobium plasmid vector and introduced into NRC-1 and S9, a bop overproducer strain. The cloned promoter was active in both H. halobium strains, but at a higher level in the overproducer than in the wild type. Transcription from the bop promoter on the plasmid was found to be inhibited by novobiocin to a similar extent as was transcription from the chromosome. When the cloned promoter was introduced into S9 mutant strains with insertions in either of two putative regulatory genes, brp and bat, no transcription was detectable, indicating that these genes serve to activate transcription from the bop promoter in trans. Deletion analysis of the cloned bop promoter from a site approximately 480 bp upstream of bop showed that a 53-bp region 5' to the transcription start site is sufficient for transcription, but a 28-bp region is not. An 11-bp alternating purine-pyrimidine sequence within the functional promoter region, centered 23 bp 5' to the transcription start point, was found to display DNA supercoiling-dependent sensitivity to S1 nuclease and OsO4, which is consistent with a non-B-DNA conformation similar to that of left-handed Z-DNA and suggests the involvement of unusual DNA structure in supercoiling-stimulated bop gene transcription.
Transcription of the Anabaena sp. strain PCC 7120 ntcA gene: multiple transcripts and NtcA binding
Authors: Ramasubramanian T S , Wei T F , Oldham A K , Golden J W
Published: November 2016

The Anabaena sp. strain PCC 7120 ntcA gene showed multiple transcripts with different 5' ends. The relative abundance of transcripts varied in response to nitrogen availability. The ntcA product, NtcA, showed binding to the promoter region of its own gene. The binding site mapped to a region between the transcription start site used under nitrogen-replete conditions and the start sites used under nitrogen-limiting conditions, suggesting that NtcA regulates its own expression.
Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath)
Authors: Zahn J A , DiSpirito A A
Published: November 2016

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide.
Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease
Authors: Waller P R , Sauer R T
Published: November 2016

The degQ and degS genes of Escherichia coli encode proteins of 455 and 355 residues, respectively, which are homologs of the DegP protease. The purified DegQ protein has the properties of a serine endoprotease and is processed by the removal of a 27-residue amino-terminal signal sequence. A plasmid expressing degQ rescues the temperature-sensitive phenotype of a strain bearing the degP41 deletion, implying that DegQ, like DegP, functions as a periplasmic protease in vivo. Deletions in the degQ gene cause no obvious growth defect, while those in the degS gene result in a small-colony phenotype. The latter phenotype is rescued by a plasmid expressing the degS gene but not by plasmids expressing the degQ or degP genes. This result and the inability of a plasmid expressing degS to rescue the temperature-sensitive degP41 phenotype indicate that the DegS protein is functionally different from the DegQ and DegP proteins.
Identification of a membrane protein involved in activation of the KinB pathway to sporulation in Bacillus subtilis
Authors: Dartois V , Djavakhishvili T , Hoch J A
Published: November 2016

The initiation of sporulation in Bacillus subtilis is dependent on the phosphorylation of the Spo0A transcription factor mediated by the phosphorelay and by two major kinases, KinA and KinB. Temporal expression of these kinases was analyzed, and an assessment of their respective contributions to the production of Spo0A-P was undertaken. The results show that KinB is expressed and activated prior to KinA; i.e., the two kinases are solicited sequentially in the sporulation process and are thought to be activated by different signaling pathways. A strategy was developed to isolate mutations specifically affecting the KinB pathway, using the newly improved mini-Tn10 delivery vector pIC333. Several mutants were obtained, one of which carried a transposon in a gene coding for a small integral membrane protein, named KbaA. Inactivation of the kbaA gene appeared to affect KinB activity but not transcription of kinB. A Spo+ suppressor (kinB45) of the kbaA null mutation was isolated in the promoter region of kinB. An eightfold increase of kinB expression levels over wild-type levels was observed in the kinB45 mutant. Thus, overexpression of the kinB-kapB operon was sufficient to overcome the sporulation defect caused by inactivation of kbaA in a KinA- strain. Transcription of kinB was found to be repressed by SinR, while the kinB45 mutant was no longer sensitive to SinR regulation. Implications of these observations on the transcriptional regulation of kinB and the role of KbaA in KinB activation are discussed.
Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens
Authors: Doty S L , Yu M C , Lundin J I , Heath J D , Nester E W
Published: November 2016

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.
Modulation of luminescence operon expression by N-octanoyl-L-homoserine lactone in ainS mutants of Vibrio fischeri
Authors: Kuo A , Callahan S M , Dunlap P V
Published: November 2016

Population density-dependent expression of luminescence in Vibrio fischeri is controlled by the autoinducer N-3-oxohexanoyl-L-homoserine lactone (autoinducer 1 [AI-1]), which via LuxR activates transcription of the lux operon (luxICDABEG, encoding the putative autoinducer synthase [LuxI] and the luminescence enzymes). We recently identified a novel V. fischeri locus, ainS, necessary for the synthesis of a second autoinducer, N-octanoyl-L-homoserine lactone (AI-2), which via LuxR can activate lux operon transcription in the absence of AI-1. To define the regulatory role of AI-2, a luxI ainS double mutant was constructed; in contrast to the parental strain and a luxI mutant, the luxI ainS mutant exhibited no induction of luminescence and produced no detectable luminescence autoinducer, demonstrating that V. fischeri makes no luminescence autoinducers other than those whose synthesis is directed by luxI and ainS. A mutant defective only in ainS exhibited accelerated luminescence induction compared with that of the parental strain, indicating that AI-2 functions in V. fischeri to delay luminescence induction. Consistent with that observation, the exogenous addition of AI-2 inhibited induction in a dose-dependent manner in V. fischeri and Escherichia coli carrying the lux genes. AI-2 did not mediate luxR negative autoregulation, alone or in the presence of AI-1, and inhibited luminescence induction in E. coli regardless of whether luxR was under the control of its native promoter or a foreign one. Increasing amounts of AI-1 overcame the inhibitory effect of AI-2, and equal activation of luminescence required 25- to 45-fold-more AI-2 than AI-1. We conclude that AI-2 inhibits lux operon transcription. The data are consistent with a model in which AI-2 competitively inhibits the association of AI-1 with LuxR, forming a complex with LuxR which has a markedly lower lux operon-inducing specific activity than that of AI-1-LuxR. AI-2 apparently functions in V. fischeri to suppress or delay induction at low and intermediate population densities.
Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state
Authors: Zeilstra-Ryalls J H , Kaplan S
Published: November 2016

Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome 1. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings.
Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK)
Authors: Kahler C M , Carlson R W , Rahman M M , Martin L E , Stephens D S
Published: November 2016

A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.
Topological characterization of the essential Escherichia coli cell division protein FtsN
Authors: Dai K , Xu Y , Lutkenhaus J
Published: November 2016

Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function. These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed.
Surface display of a functional single-chain Fv antibody on staphylococci
Authors: Gunneriusson E , Samuelson P , Uhlen M , Nygren P A , Stähl S
Published: November 2016

Two different host-vector expression systems designed for cell surface display of chimeric receptors on Staphylococcus xylosus and Staphylococcus carnosus have been evaluated for surface display of a mouse immunoglobulin G1(kappa) [IgG1(kappa)] anti-human IgE single-chain Fv (scFv) antibody fragment. To achieve surface anchoring of the chimeric receptors containing the scFv, the cell surface attachment regions from Staphylococcus aureus protein A were used in both expression systems. The different chimeric receptors could be recovered from cell wall extracts of both S. xylosus and S. carnosus, and surface localization was demonstrated by taking advantage of a serum albumin-binding reporter region present within the two types of receptors. In addition, the two different recombinant staphylococci carrying hybrid receptors containing the scFv were demonstrated to react with the antigen, which was human IgE, in whole-cell enzyme-linked immunosorbent assays. This is the first report of an antibody fragment expressed in a functional form anchored to the surface of gram-positive bacteria. The potential use of recombinant gram-positive bacteria as whole-cell diagnostic devices or alternatives to filamentous phages for surface display of scFv libraries is discussed.
Characterization of a glutathione-dependent formaldehyde dehydrogenase from Rhodobacter sphaeroides
Authors: Barber R D , Rott M A , Donohue T J
Published: November 2016

Glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) represent a ubiquitous class of enzymes, found in both prokaryotes and eukaryotes. During the course of studying energy-generating pathways in the photosynthetic bacterium Rhodobacter sphaeroides, a gene (adhI) encoding a GSH-FDH homolog has been identified as part of an operon (adhI-cycI) that also encodes an isoform of the cytochrome c2 family of electron transport proteins (isocytochrome c2). Enzyme assays with crude Escherichia coli extracts expressing AdhI show that this protein has the characteristic substrate preference of a GSH-FDH. Ferguson plot analysis with zymograms suggests that the functional form of AdhI is a homodimer of approximately40-kDa subunits, analogous to other GSH-FDH enzymes. These properties of AdhI were used to show that mutations which increase or decrease adhI expression change the specific activity of GSH-FDH in R. sphaeroides extracts. In addition, expression of the presumed adhI-cycI operon appears to be transcriptionally regulated, since the abundance of the major adhI-specific primer extension product is increased by the trans-acting spd-7 mutation, which increases the level of both isocytochrome c2 and AdhI activity. While transcriptional linkage of adhI and cycI could suggest a function in a common metabolic pathway, isocytochrome c2 (periplasm) and AdhI (cytoplasm) are localized in separate compartments of R. sphaeroides. Potential roles for AdhI in carbon and energy generation and the possible relationship of GSH-FDH activity to isocytochrome c2 will be discussed based on the commonly accepted physiological functions of GSH-FDH enzymes in prokaryotes and eukaryotes.
Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum
Authors: Davis R , Lehman L , Petrovich R , Shah V K , Roberts G P , Ludden P W
Published: November 2016

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.
Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region
Authors: Furuya N , Komano T
Published: November 2016

A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.
Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens
Authors: Fullner K J , Nester E W
Published: November 2016

Early studies on Agrobacterium tumefaciens showed that development of tumors on plants following infection by A. tumefaciens was optimal at temperatures around 22 degrees C and did not occur at temperatures above 29 degrees C. To assess whether this inability to induce tumors is due to a defect in the T-DNA transfer machinery, mobilization of an incompatibility group Q (IncQ) plasmid by the T-DNA transfer machinery of A. tumefaciens was tested at various temperatures. Optimal transfer occurred when matings were performed at 19 degrees C, and transfer was not seen when matings were incubated above 28 degrees C. Transfer of the IncQ plasmid was dependent upon induction of the virB and virD operons by acetosyringone but was not dependent upon induction of the tra genes by octopine. However, alterations in the level of vir gene induction could not account for the decrease in transfer with increasing temperature. A. tumefaciens did successfully mobilize IncQ plasmids at higher temperatures when alternative transfer machineries were provided. Thus, the defect in transfer at high temperature is apparently in the T-DNA transfer machinery itself. As these data correlate with earlier tumorigenesis studies, we propose that tumor suppression at higher temperatures results from a T-DNA transfer machinery which does not function properly.