bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum
Authors: Spaink Herman P. , Wijfjes André H. M. , der van Drift Koen M. G. M. , Haverkamp Johan , Thomas‐Oates Jane E. , Lugtenberg Ben J. J. 
Published: October 2006

SummaryThe Rhizobium nodulation genes nodABC are involved in the synthesis of lipo‐chitin oligosac‐charides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three. In vivo radioactive labelling experiments, in which D‐[1‐14C]‐glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo‐chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N‐acetyl group on the non‐reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O‐acetyl substituent on the non‐reducing terminal sugar residue (designated NodBCL metabolites). The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP‐N‐acetyl‐D‐[U‐14C]‐glucosamine was added using thin‐layer chromatography. The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites. The same results were obtained when the NodB and NodC proteins were produced separately in two different strains. On the basis of these and other recent results, we propose that NodB is a chitin oilgosaccharide deacetylase, NodC an N‐acetylglucosaminyltransferase and, by default, NodA is involved in lipie attachment.
Threonine dehydratases of Corynebacterium glutamicum with altered allosteric control: their generation and biochemical and structural analysis
Authors: Möckel Bettina , Eggeling Lothar , Sahm Hermann 
Published: October 2006

SummaryThreonine dehydratase is the key enzyme in L‐isoleucine synthesis, since it is allosterically feedback‐inhibited by L‐isoleucine. With the aim of obtaining regulatorily altered mutants of the threonine dehydratase of Corynebacterium glutamicum, amino acids were specifically exchanged and a new biological system of mutant selection was developed, based on the intoxication of Escherichia coli by ketobutyrate, which is the dehydratase reaction product. A collection of 19 mutant enzymes was generated and genetically and biochemically characterized comprising a whole range of regulatorily and catalytically altered enzymes. Of particular interest is the mutant Val‐323‐Ala, which is characterized by the fact that the L‐isoleucine inhibition is entirely abolished so that the enzyme is always present in a relaxed, high‐activity state. Correspondingly, the Hill coefficient is 1.4, in contrast to the value of 3.4 characteristic of the wild‐type enzyme. Another peculiar mutant generated is the double mutant His‐278‐Arg‐Leu‐351‐Ser. Here, again, L‐isoleucine no longer inhibits catalytic activity, but the effector still promotes major structural changes of the protein, as ascertained from the L‐isoleucine‐dependent loss of pyridoxal‐5 ‐phosphate from this mutant enzyme. Further enzymes obtained are reduced in L‐isoleucine inhibition to a varying degree. Detailed studies on the structure of the enzyme revealed a partially very high similarity of the secondary structure to the mechanistically identical β‐subunit of the tryptophan synthase. This provides further indications concerning the localization of the regulatory and catalytic domain of threonine dehydratase.
DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC
Authors: Barnes Marjorie H. , Tarantino Paul M. , Spacciapoli Peter , Brown Neal C. , Yu Huilan , Dybvig Kevin 
Published: October 2006

SummaryMycoplasmas have originated from Gram‐positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram‐positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)‐positive enzymes to a single exo‐negative species. The objective of this work was to rigorousiy investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram‐positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III‐specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram‐positive bacterial gene encoding Pol III M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram‐positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram‐positive Pol III‐selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra‐resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram‐positive bacteria than previously thought.
The 100 kDa haem:haemopexin‐binding protein of Haemophilus Influenzae: structure and localization
Authors: Cope Leslie D. , Thomas Sharon E. , Latimer Jo L. , Slaughter Clive A. , Müller‐Eberhard Ursula , Hansen Eric J. 
Published: October 2006

SummaryAll Haemophilus influenzae strains have an absolute requirement for exogenously supplied haem for aerobic growth. A majority of strains of H. influenzae type b (Hib) produce a 100 kDa protein which binds haem: haemopexin complexes. This 100 kDa haem:haemopexin binding protein, designated HxuA, was originally detected on the Hib cell surface. Monoclonal antibody (mAb)‐based analyses revealed that the HxuA protein was also present in soluble form in Hib culture supernatants. This soluble HxuA protein exhibited haem:haemopexin‐binding activity in a direct binding assay. Nucleotide sequence analysis of the hxuA gene from Hib strain DL42, together with N‐terminal amino acid analysis of HxuA protein purified from Hib culture supernatant, revealed that this protein was synthesized as a 101 kDa precursor with a leader peptide that was removed to yield a 99kDa protein. Southern blot analysis of chromosomal DNA from four Hib and four non‐typeable H. influenzae (NTHI) strains detected the presence of a single band in each strain that hybridized a Hib hxuA gene probe. Subsequent analysis of these NTHI strains showed that all four strains released into culture supernatant a haem:haemopexin‐binding protein that migrated in SDS‐PAGE at a rate similar or identical to that of the Hib HxuA protein. A Hib hxuA mutant was used to screen an NTHI genomic DNA library and an NTHI gene was cloned that complemented the mutation in this Hib strain. Nucleotide sequence analysis of this NTHI gene revealed that it encoded a protein with 87% identity to the Hib HxuA protein. The expression of HxuA by both Hib and NTHI strains indicates that this particular haem acquisition system is conserved among H. influenzae strains.
Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1
Authors: Hovde Carolyn J. , Marr James C. , Hoffmann Marcy L. , Hackett Sean P. , Chi Young‐in , Crum Kimberlee K. , Stevens Dennis L. , Stauffacher Cynthia V. , Bohach Gregory A. 
Published: October 2006

SummaryThe goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 ‐induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.
Structural analysis and molybdenum‐dependent expression of the pAO1‐encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans
Authors: Grether‐Beck Susanne , Igloi Gabor L. , Pust Stefan , Schilz Emil , Decker Karl , Brandsch Roderich 
Published: October 2006

SummaryThe genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6‐hydroxy‐L‐niotine oxidase (6‐HLNO) gene. NDH is composed of three subunits (A, B and C) of Mr 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe‐S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum‐containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron‐sulphur cluster, that the middle‐sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin‐binding domain of XDH. Expression of both the ndh and the 6‐hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane‐associated form. In the presence of tungsten the fraction of membrane‐associated NDH increased.
The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N‐termini of type 4 prepilin proteins
Authors: Russell Mary A. , Darztns Aldis 
Published: October 2006

SummaryA new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ‐6, was broadly resistant to pili‐specific phages and unable to translocate across solid surfaces by the pilus‐dependent mechanism of twitching motility (Twt−). Immunoblot analysis demonstrated that MJ‐6 was devoid of pili (Pil−) but was unaffected in the production of unassembled pilin pools. Genetic studies aimed at localizing the pilE mutation on the P. aeruginosa PAO chromosome demonstrated a strong co‐linkage between MJ‐6 phage resistance and the proB marker located at 71 min. Cloning of the pilE gene was facilitated by the isolation and identification of a proB+‐containing plasmid from a PAO1 cosmid library. Upon introduction of the PA01 proB+ cosmid clone into MJ‐6, sensitivity to pili‐specific phage, twitching motility and pilus production were restored. The nucleotide sequence of a 1 kb Eco RV‐Clal fragment containing the pilE region revealed a single complete open reading frame with characteristic P. aeruginosa codon bias. PilE, a protein with a molecular weight of 15278, showed significant sequence identity to the pilin precursors of P. aeruginosa and to other type 4 prepilin proteins. The region of highest homology was localized to the N‐terminal 40 amino acid residues. The putative PilE N‐terminus contained a seven‐residue basic leader sequence followed by a consensus cleavage site for prepilin pep‐tidase and a largely hydrophobic region which contained tyrosine residues (Tyr‐24 and Tyr‐27) previously implicated in maintaining pilin subunit‐subunit interactions. The requirement of PilE in pilus biogenesis was confirmed by demonstrating that chromosomal pilE insertion mutants were pilus‐ and twitching‐motility deficient.
TcpA pilin sequences and colonization requirements for O1 and O139 Vibrio cholerae
Authors: Rhine Janet A. , Taylor Ronald K. 
Published: October 2006

SummaryThe distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non‐O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non‐O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non‐O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP‐associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence of tcpA and the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non‐O1 serotypes.
Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase: identification of common target heptamers for both NarP‐ and NarL‐dependent regulation
Authors: Tyson K. L. , Cole J. A. , Busby S. J. W. 
Published: October 2006

SummaryExpression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 691/2 bp upstream of the transcript startpoint. The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of the nrf promoter has been exploited to corroborate the location of the ‐10 hexamer and the FNR‐binding site, and to find the sites essential for nitrite‐dependent activation and nitrate‐dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 741/2 bp upstream from the transcript start. NarL‐dependent repression by nitrate is due to two heptamer sequences that flank the FNR‐binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site.
Transcription of the principal sigma‐factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase
Authors: Fujita Masaya , Tanaka Kan , Takahashi Hideo , Amemural Akinori 
Published: October 2006

SummaryThe rpoS gene encodes the second principal sigma factor of RNA polymerase in stationary‐phase cells in Escherichia coli. We examined the transcription of Pseudomonas aeruginosa rpoS as to the growth of ceils. The results of quantitative S1 nuclease mapping of rpoS and rpoD, encoding the principal sigma factor, indicated that the transcription of rpoS is induced in stationary‐phase cells, whereas that of rpoD is induced in exponential‐phase cells. By high‐resolution S1 nuclease mapping, the 5′‐ and 3′‐ends of rpoS mRNA were determined. The results indicated that rpoS is transcribed as a monocistronic mRNA. The sequence preceding the 5 end of rpoS mRNA showed poor homology to the consensus sequences of the previously known promoters. P. aeruginosa rpoS was not transcribed in E coli. By in vitro transcription assaying, P. aeruginosa rpoS was shown to be transcribed by the RNA polymerase fraction containing the principal sigma (σ70) RNA polymerase of P. aeruginosa
Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis
Authors: Kado Clarence I. 
Published: October 2006

SummaryConjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms. The transfer of DNA between Gram‐negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore. Accumulating evidence suggests that transfer of the T‐DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins. The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity=50.8%) with VirB proteins. Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%). VirB2 protein (12.3 kDa) contains a signal peptidase‐I cleavage sequence that generates a polypeptide of 7.2 kDa. Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase‐I cleavage site that generates the 7.2 kDa pilin structural protein. Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptl operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA‐protein complexes.
A mutation in the receiver domain of the Agrobacterium tumefaciens transcriptional regulator VirG increases its affinity for operator DNA
Authors: Han Dong Cho , Winans Stephen C. 
Published: October 2006

SummaryWe fused the wild‐type Agrobacterium tumefaciens virG gene and the constitutive virGN54D allele to the malE gene of Escherichia coli, and studied the binding of MBP—VirG fusions to the autoregulated virG promoter. MBP—VirGN54D protein bound this promoter with 10‐fold higher affinity than MBP—VirG, and bound to vir box I with eightfold higher affinity than to vir box III. Disruption of vir box III did not alter the affinity for vir box I, suggesting a lack of cooperativity between these sites. We provide evidence that protein bound at a single vir box may have a higher oligomeric state than non‐bound protein, and that a DNA distortion adjacent to vir box I may occur during activation.
Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome‐binding site permits tap‐independent RepA synthesis
Authors: Blomberg Pontus , Engdahl Hilde M. , Malmgren Charlotta , Romby Pascale , Wagner E. Gerhart H. 
Published: October 2006

SummaryThe replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post‐transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem‐loop sequestering the repA ribosome‐binding site blocks tap translation‐independent repA expression. Destabilization of this structure resulted in tap‐independent RepA synthesis, concomitant with a loss of CopA‐mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome‐binding site stem‐loop. An increased spacing between the repA Shine‐Dalgarno region and the start codon permitted even higher rep A expression. In Incα/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.
Structural and functional diversity among bacterial interspersed mosaic elements (BIMEs)
Authors: Bachellier Sophie , Saurin William , Perrin David , Hofnung Maurice , Gilson Eric 
Published: October 2006

SummaryPalindromic units (PU or REP) were defined as 40‐nucleotide DNA sequences which are highly repeated in the genome of several members of the Enterobacteriaceae. They were shown to be a constituent of the bacterial interspersed mosaic element (BIME), in which they are associated with other repetitive sequences. We report here that Escherichia coli PU sequences contain three motifs (Y, Z1 and Z2), leading to the definition of two BIME families. The BIME‐1 family, highly conserved over 145 nucleotides, contains two PUs (motifs Y and Z1). The BIME‐2 family contains a variable number of PUs (motifs Y and Z2). We present evidence, using band shift experiments, that each PU motif binds DNA gyrase with a different affinity. This suggests that the two families are functionally distinct.