bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the lsi region involved in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae
Authors: Petricoin E F , Danaher R J , Stein D C
Published: November 2016

The genetic locus (lsi-1) responsible for the transformation of the lipooligosaccharide (LOS)-defective Neisseria gonorrhoeae mutant FA5100 to LOS expression was studied by deletion mutagenesis and sequence analysis. An open reading frame that was preceded by a leader sequence containing regions with the potential to form hairpin loops was identified. A perfect sigma 70 promoter consensus sequence was found upstream from this open reading frame. Promoter function was screened for functionality by using lac fusion cassettes and in vitro transcription-translation analysis. A frameshift mutation in the lsi-1 gene was constructed by site-directed mutagenesis and introduced into the chromosome of FA19, the LOS-expressing isogenic parent strain of FA5100. The mutant was characterized by Southern blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting) and found to be phenotypically identical to FA5100.
Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene
Authors: van der Ploeg J , van Hall G , Janssen D B
Published: November 2016

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10.
Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis
Authors: Popham D L , Stragier P
Published: November 2016

Mutation of the spoVB gene in Bacillus subtilis causes the production of spores containing a defective cortex and unable to acquire heat resistance. The spoVB locus is highly linked to another spo locus, spoIIIF, characterized by a single mutation (I. L. Lamont and J. Mandelstam, J. Gen. Microbiol. 130:1253-1261, 1984). A 18-kb DNA region overlapping the spoIIIF-spoVB region was cloned in successive steps starting from a Tn917 insertion in the nic locus. The exact location of the spoIIIF and spoVB loci was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spoIIIF and spoVB are a single monocistronic locus encoding a 518-amino-acid polypeptide with features of an integral membrane protein. The precise location of the spoIIIF590 and spoVB91 mutations in that unique open reading frame was determined, and both mutations were sequenced. A null mutation was engineered in the spoIIIF-spoVB locus and led to a typical spoVB phenotype, identical to the phenotype created by either spoIIIF590 or spoVB91, suggesting that the original spoIIIF mutant contained a secondary mutation arresting sporulation at an earlier stage. A transcriptional spoVB-lacZ fusion was constructed, and its expression was found to be directly dependent on RNA polymerase containing sigma E. A null mutation of spoVB had no effect on expression of sspB and cotA, members of the sigma G- and sigma K-controlled regulons respectively, while expression of cotC, a member of the latest known mother cell regulon, was delayed and strongly reduced. These results are consistent with SpoVB being involved in cortex biosynthesis and affecting only indirectly expression of late sporulation genes.
Cloning and sequencing of the gene for a Pseudomonas paucimobilis enzyme that cleaves beta-aryl ether
Authors: Masai E , Katayama Y , Kawai S , Nishikawa S , Yamasaki M , Morohoshi N
Published: November 2016

We isolated Pseudomonas paucimobilis SYK-6, which was able to degrade various dimeric lignin compounds (Y. Katayama, S. Nishikawa, M. Nakamura, K. Yano, M. Yamasaki, N. Morohoshi, and T. Haraguchi, Mokuzai Gakkaishi 33:77-79, 1987). This metabolic process is a distinct characteristic of this bacterium, which is equipped with an enzymatic modification system for various dimeric lignin compounds involved in the tricarboxylic acid cycle. Cleavage of the beta-aryl ether linkage is essential in this process, because this linkage is the most abundant (approximately 50%) in lignin. Here, we report the isolation and characterization of the beta-etherase gene, which contains an open reading frame of 843 bp and which we call ligE. This gene was expressed in Escherichia coli, and the enzyme had the same kinetic properties as the P. paucimobilis SYK-6 enzyme.
Characterization of the heat shock response in Mycobacterium bovis BCG
Authors: Patel B K , Banerjee D K , Butcher P D
Published: November 2016

We have for the first time characterized the heat shock response in mycobacteria both at the level of transcription, by RNA extraction, Northern (RNA) blotting, and hybridization with gene-specific probes for the Mycobacterium tuberculosis 65- and 71-kDa heat shock proteins (HSPs), and at the level of translation, by [35S]methionine labelling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. We observed increased synthesis of 40-, 65-, 71-, and 90-kDa proteins, which appear to be major HSPs in mycobacteria. The 40-, 71-, and 90-kDa HSPs are coordinately regulated in terms of temperature requirements and kinetics of induction but differ in the levels of expression. The 65- and 71-kDa HSPs are differentially regulated in response to temperature, with different kinetics and levels of induction. mRNA transcript sizes for the 71-, 65-, 40-, and 30-kDa proteins were found to be broadly consistent with DNA sequence open reading frames. A maximum increase of about 69-fold in the levels of mRNA for the 71-kDa HSP after 45 min of heat shock at 45 degrees C was observed, whereas the 65-kDa HSP mRNA increased only 5-fold. It was also found that in M. bovis BCG, as in Escherichia coli, a major control mechanism of the heat shock response is operative at the level of transcription. An ability to characterize the heat shock response in mycobacteria provides an experimental model with which to study environmentally regulated gene expression and an opportunity to identify virulence genes, which may coregulate as part of the heat shock regulon.
Release of a cell surface protein during development of Myxococcus xanthus
Authors: Glufka R , Maeba P
Published: November 2016

VGP is a major cell-surface glycoprotein present in vegetative cells of Myxococcus xanthus. Serological assays indicated that this protein was released from cells and accumulated in the medium during development, i.e., aggregation, fruiting body formation, and myxosporulation. Cells induced to form spores in the absence of aggregation retained VGP, indicating that loss of VGP was associated with developmental aggregation rather than myxosporulation. Anti-VGP antibodies inhibited vegetative cell gliding, suggesting the protein may also be required for motility.
Functional analysis of the regulatory region of the yeast phosphatidylserine synthase gene, PSS
Authors: Kodaki T , Nikawa J , Hosaka K , Yamashita S
Published: November 2016

The upstream region of the PSS gene contains three positive cis-acting elements, upstream activation sequences 1 and 2 (UAS1 and UAS2) and a TATA box. The 5' end of UAS1 occurs between positions -239 and -209, and that of UAS2 is between positions -172 and -164. UAS2 contains 5'-TTCACATG-3' as a core sequence at positions -161 to -154. Mutational analysis revealed that this octamer is responsible for the control of PSS expression by inositol and choline. The TATA box is located at positions -112 to -108. In addition, PSS contains a negative cis-acting sequence between UAS2 and the TATA box.
Structural complexity of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli
Authors: Girard M L , Flores M , Brom S , Romero D , Palacios R , Dávila G
Published: November 2016

The complete physical map of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli strain CFN42 was established. The data support the concept that Rhizobium symbiotic genes are part of a complex genomic structure which contains a large amount of reiterated DNA sequences. This plasmid is a circular structure of 390 kb with approximately 10 families of internally reiterated DNA sequences of two to three elements each. One family includes two directly oriented nitrogenase operons situated 120 kb apart. We also found several stretches of pSym that are reiterated in other replicons of the cell. Localization of symbiotic gene sequences by heterologous hybridization revealed that nodABC sequences are separated in two regions, each of which contains a nod boxlike element, and it also suggested the presence of two copies of the nifA and nodD gene sequences. We propose that the complex structure of the symbiotic plasmid allows interactions between repeated DNA sequences which, in turn, might result in frequent rearrangements.
Amplification and deletion of a nod-nif region in the symbiotic plasmid of Rhizobium phaseoli
Authors: Romero D , Brom S , Martínez-Salazar J , Girard M L , Palacios R , Dávila G
Published: November 2016

One remarkable characteristic of the genomes of some Rhizobium species is the frequent occurrence of rearrangements. In some instances these rearrangements alter the symbiotic properties of the strains. However, no detailed molecular mechanisms have been proposed for the generation of these rearrangements. To understand the mechanisms involved in the formation of rearrangements in the genome of Rhizobium phaseoli, we have designed a system which allows the positive selection for amplification and deletion events. We have applied this system to investigate the stability of the symbiotic plasmid of R. phaseoli. High-frequency amplification events were detected which increase the copy number of a 120-kb region carrying nodulation and nitrogen fixation genes two to eight times. Deletion events that affect the same region were also found, albeit at a lower frequency. Both kinds of rearrangements are generated by recombination between reiterated nitrogenase (nifHDK) operons flanking the 120-kb region.
Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23
Authors: Nishiyama M , Horinouchi S , Kobayashi M , Nagasawa T , Yamada H , Beppu T
Published: November 2016

The nitrile hydratase (NHase) of Pseudomonas chlororaphis B23, which is composed of two subunits, alpha and beta, catalyzes the hydration of nitrile compounds to the corresponding amides. The NHase gene of strain B23 was cloned into Escherichia coli by the DNA-probing method with the NHase gene of Rhodococcus sp. strain N-774 as the hybridization probe. Nucleotide sequencing revealed that an amidase showing significant similarity to the amidase of Rhodococcus sp. strain N-774 was also coded by the region just upstream of the subunit alpha-coding sequence. In addition to these three proteins, two open reading frames, P47K and OrfE, were found just downstream of the coding region of subunit beta. The direction and close locations to each other of these open reading frames encoding five proteins (amidase, subunits alpha and beta, P47K, and OrfE, in that order) suggested that these genes were cotranscribed by a single mRNA. Plasmid pPCN4, in which a 6.2-kb sequence covering the region coding for these proteins is placed under control of the lac promoter, directed overproduction of enzymatically active NHase and amidase in response to addition of isopropyl-beta-D-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of subunits alpha and beta of NHase was about 10% of the total cellular proteins and that an additional 38-kDa protein probably encoded by the region upstream of the amidase gene was also produced in a large amount. The 38-kDa protein, as well as P47K and OrfE, appeared to be important for efficient expression of NHase activity in E. coli cells, because plasmids containing the NHase and amidase genes but lacking the region coding for the 38-kDa protein or the region coding for P47K and OrfE failed to express efficient NHase activity.
Negative regulation of Bacillus subtilis sporulation by the spo0E gene product
Authors: Perego M , Hoch J A
Published: November 2016

Transcription of the Bacillus subtilis spo0E gene is controlled by the AbrB transition state regulator. In AbrB+ strains, a single transcript, P1, was observed for the spo0E gene. In an abrB4 mutant strain, a second transcription start site 3 bases upstream from P1 was found to be used for the predominant transcript. P1 transcription was insensitive to the state of the abrB gene. Mutants carrying deletion or antibiotic cassette insertion mutations in the spo0E gene were Spo+. Multiple copies of the spo0E gene, not just the promoter region, were found to render strains incapable of sporulation. Spo+ strains that arose spontaneously from such Spo- strains were found to have deletions in the spo0E coding sequence on the plasmid. Strains carrying a deletion of the spo0E gene segregated Spo- colonies. These colonies were found to have secondary mutations in or near the spo0A, spo0B, or spo0F gene, suggesting that deletion of the spo0E gene results in increased pressure to sporulate that is compensated for by inactivation of one or more of the components of the signal transduction system leading to the initiation of sporulation. spo0E deletions were suppressors of the spo0F221 missense mutation but had no effect on the regulation of the spo0F, kinA, spo0A, or spo0B genes. The results suggest that the spo0E gene product is a negative regulator of the signal transduction pathway leading to sporulation.
Formation of (dA-dT)n cruciforms in Escherichia coli cells under different environmental conditions
Authors: Dayn A , Malkhosyan S , Duzhy D , Lyamichev V , Panchenko Y , Mirkin S
Published: November 2016

We have detected cruciform formation of (dA-dT)n inserts in Escherichia coli cells by analyzing the superhelical density of isolated plasmid DNA samples and by probing intracellular DNA with chloroacetaldehyde. The plasmids we used were pUC19 containing inserts of (dA-dT)n. The cruciforms appeared after cells underwent different stresses: inhibition of protein synthesis, anaerbiosis, and osmotic shock. At the same time, all these stimuli led to an increase in superhelical density of the control pUC19 plasmid DNA. Therefore, we suggest that the increase in plasmid superhelicity in response to different environmental stimuli entails the appearance of cruciform structures. The use of the (dA-dT)n units of various lengths made it possible to estimate the superhelical density of the plasmid DNA in vivo.
Processing and termination of 23S rRNA-5S rRNA-tRNA(Gly) primary transcripts in Thermus thermophilus HB8
Authors: Hartmann R K , Toschka H Y , Erdmann V A
Published: November 2016

The two 23S rRNA-5S rRNA-tRNAGly operons from the extreme thermophilic eubacterium Thermus thermophilus HB8 were used to characterized the in vivo processing and termination of 23S rRNA-5S rRNA-tRNAGly primary transcripts in this organism by nuclease S1 mapping. A processing site in the pre-23S rRNA 3'-flanking region is located approximately 25 nucleotides upstream of 5S rRNA and precedes a putative 23S-5S rRNA spacer antitermination box A. Cleavage at this site and 5S rRNA 5' end formation were shown to be inseparable events. Termination of transcription at the uridine cluster following the termination-associated hairpin was shown to be efficient but leaky. Subsequent to the operon, a functional promoter was detected whose -35 box coincided with the uridine-rich termination region. The promoter directed synthesis of a beta-galactosidase fusion protein in Escherichia coli.
Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo
Authors: Baneyx F , Georgiou G
Published: November 2016

Protease III, the product of the ptr gene, is a 110-kDa periplasmic protease with specificity towards insulin and other low-molecular-weight substrates (less than 7,000 molecular weight) in vitro (Y.-S.E. Cheng and D. Zipser, J. Biol. Chem. 254:4698-4706, 1979). Escherichia coli strains deficient in protease III were constructed by insertional inactivation of the ptr gene. This mutation did not appear to affect the function of the adjoining recB and recC genes. Expression of protein A-beta-lactamase, a protease-sensitive secreted polypeptide, was increased approximately twofold in ptr cells. A comparable increase in the half-life of protein A-beta-lactamase was observed by pulse-chase experiments, suggesting that protease III is involved in the catabolism of high-molecular-weight substrates in vivo, ptr mutants exhibited no detectable phenotypic alterations except for a slight reduction in growth rate. When the ptr mutation was transferred to a strain deficient in the secreted protease DegP, a further decrease in growth rate, as well as an additive increase in the expression of the fusion protein, was observed. A ptr degP ompT mutant strain resulted in a further increase in expression in minimal medium but not in rich medium.
A vector for analysis of promoters in the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Lang J D , Haselkorn R
Published: November 2016

A plasmid vector containing a multiple-cloning site followed by a promoterless chloramphenicol acetyltransferase (cat) gene, protected by transcription terminators and mobilizable by conjugation from Escherichia coli into Anabaena sp. strain PCC 7120, was constructed. The utility of the vector was shown by deletion analysis of the promoter region of the Anabaena psbB gene.
Insertion and excision of Bacteroides conjugative chromosomal elements
Authors: Bedzyk L A , Shoemaker N B , Young K E , Salyers A A
Published: November 2016

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.
Role of nitrogen regulator I (NtrC), the transcriptional activator of glnA in enteric bacteria, in reducing expression of glnA during nitrogen-limited growth
Authors: Shiau S P , Schneider B L , Gu W , Reitzer L J
Published: November 2016

During nitrogen-limited growth, transcription of glnA, which codes for glutamine synthetase, requires sigma 54-RNA polymerase and the phosphorylated from the nitrogen regulator I (NRI; also called NtrC). In cells in which the lac promoter controlled expression of the gene coding for NRI, increasing the intracellular concentration of NRI lowered the level of glutamine synthetase. The reduction in glutamine synthetase does not appear to result from the NRI-dependent sequestering of any protein that affects transcription of glnA. Our results also suggest that the negative effect of a high concentration of NRI on glnA expression is a major determinant of the level of glutamine synthetase activity in nitrogen-limited cells of a wild-type strain. We propose that the inhibition results from an impairment of the interaction between NRI-phosphate and RNA polymerase that stimulates glnA transcription. We discuss a model that can account for this reduction in glutamine synthetase.
Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis
Authors: Köplin R , Arnold W , Hötte B , Simon R , Wang G , Pühler A
Published: November 2016

The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.
Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus
Authors: Averhoff B , Gregg-Jolly L , Elsemore D , Ornston L N
Published: November 2016

DNA within Escherichia coli colonies carrying cloned Acinetobacter calcoaceticus genes transforms mutant A. calocaceticus cells with high efficiency. Therefore, E. coli colonies containing such cloned genes can be identified by replica plating onto a lawn of A. calcoaceticus mutant cells. Transformation of A. calcoaceticus also facilitates gap repair and thus allows recovery of specified chromosomal segments in recombinant plasmids. These procedures were used to demonstrate the clustering of A. calcoaceticus genes required for utilization of p-hydroxybenzoate. Chromosomal linkage of the bacterial genes, contained in different operons separated by about 10 kbp of DNA, may have been selected on the basis of their physiological interdependence.
Phylogenetic analysis of the genus Borrelia: a comparison of North American and European isolates of Borrelia burgdorferi
Authors: Marconi R T , Garon C F
Published: November 2016

We have sequenced the 16S rRNA molecules from four species of Borrelia and from six isolates of Borrelia burgdorferi via the reverse transcriptase primer extension method. The sequences were aligned and evolutionary relationships were determined, including the calculation of evolutionary distances and the construction of a phylogenetic tree. These analyses demonstrate significant divergence among B. burgdorferi isolates, with the European isolates G1 and G2 residing most distant from the main cluster. Signature nucleotides which distinguish B. burgdorferi from all other members of this genus and which distinguish the European isolates G1 and G2 from the North American isolates B31, Sh-2-82, and 1352 were identified. Finally, Southern blot analyses were performed to compare the restriction patterns of the genes coding for rRNA and to relate our data to the grouping scheme of Postic et al. (D. Postic, C. Edlinger, C. Richaud, F. Grimont, J. Dufresne, P. Perolat, G. Baranton, and P. A. D. Grimont, Res. Microbiol. 141:465-475, 1990).
A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon
Authors: Elliott T
Published: November 2016

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.
Evolutionary relationships among sulfur- and iron-oxidizing eubacteria
Authors: Lane D J , Harrison A P , Stahl D , Pace B , Giovannoni S J , Olsen G J , Pace N R
Published: November 2016

Some 37 reverse transcriptase, partial 16S rRNA sequences from sulfur- and/or iron-oxidizing eubacteria, including sequences from species of the genera Thiobacillus, Thiothrix, Thiomicrospira, Acidophilium, "Leptospirillum," Thiovulum, and Chlorobium, have been determined. In addition, 16S sequences from a number of unnamed sulfur- and/or iron-oxidizing bacteria from hydrothermal vent sites, from invertebrate-bacterial endosymbioses, and from various mineral recovery operations also have been determined. The majority of sequences place their bacterial donors in one or another of the subdivisions of the Proteobacteria. However, three unnamed facultatively thermophilic iron-oxidizing isolates, Alv, BC, and TH3, are affiliated with the gram-positive division. One H2S-oxidizer, from the genus Thiovulum, is affiliated with Campylobacter, Wolinella, and other genera in what appears to be a new subdivision of the Proteobacteria. Three "Leptospirillum"-helical vibrioid isolates, BU-1, LfLa, and Z-2, exhibit no clear phylum level affiliation at all, other than their strong relationship to each other. A picture is emerging of an evolutionary widespread capacity for sulfur and/or iron oxidation among the eubacteria.
Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism
Authors: Ochs D , Kaletta C , Entian K D , Beck-Sickinger A , Poralla K
Published: November 2016

The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found.
A directional, high-frequency chromosomal mobilization system for genetic mapping of Rhizobium meliloti
Authors: Klein S , Lohman K , Clover R , Walker G C , Signer E R
Published: November 2016

A system for mapping of the Rhizobium meliloti chromosome that utilizes transposon Tn5-Mob, which carries the mobilization site of IncP plasmid RP4 (R. Simon, Mol. Gen. Genet. 196:413-420, 1984), was developed. Insertions of Tn5-Mob that were located at particular sites on the R. meliloti chromosome were isolated and served as origins of high-frequency chromosomal transfer when IncP tra functions were provided in trans. This approach is, in principle, applicable to any gram-negative bacterium in which Tn5 can transpose and into which IncP plasmids can conjugate.
Synergism between the Trp repressor and Tyr repressor in repression of the aroL promoter of Escherichia coli K-12
Authors: Heatwole V M , Somerville R L
Published: November 2016

Computer analysis identified a potential Trp repressor operator 56 nucleotides downstream of the transcriptional start point of aroL, the gene that encodes shikimate kinase II. Tryptophan-dependent interaction of Trp repressor with this operator was demonstrated in vitro by means of a restriction endonuclease protection assay. Regulation of expression from the aroL promoter was evaluated with several genetically marked Escherichia coli strains by using a single-copy aroL-lacZ transcriptional-translational reporter system. The expression of aroL was repressed 6.9-fold by the Tyr repressor alone and 29-fold when both Tyr and Trp repressors were present. The Trp repressor had no effect on expression from the aroL promoter in the absence of the Tyr repressor. Possible mechanisms for Trp repressor-mediated repression, including cooperative interactions with the Tyr repressor, are discussed.
Evidence for differential regulation of genes in the chondroitin sulfate utilization pathway of Bacteroides thetaiotaomicron
Authors: Hwa V , Salyers A A
Published: November 2016

Expression of the chondroitin sulfate utilization (csu) genes of Bacterioides thetaiotaomicron is regulated by chondroitin sulfate. We have now found, however, that the csu genes are not all regulated in the same way. In particular, the gene encoding beta-glucuronidase (csuE) is expressed under two different conditions that do not lead to expression of other csu genes.
Antigenic diversity of the S-layer proteins from pathogenic strains of Aeromonas hydrophila and Aeromonas veronii biotype sobria
Authors: Kostrzynska M , Dooley J S , Shimojo T , Sakata T , Trust T J
Published: November 2016

The antigenic relatedness of paracrystalline surface array proteins with subunit molecular weights of approximately 52,000 from isolates of Aeromonas hydrophila and Aeromonas veronii biotype sobria belonging to a single heat-stable serogroup was examined. Enzyme-linked immunosorbent assay and immunoblotting with two different polyclonal antisera against surface exposed and non-surface-exposed epitopes of the S-layer protein from A. hydrophila TF7 showed that the S-layer proteins of the mesophilic aeromonads were antigenically diverse. NH2-terminal amino acid sequence analysis of four antigenically different proteins showed that while the proteins were structurally related, they differed in primary sequence. Absorption experiments with heterologous live cells showed that cross-reactive epitopes were in non-surface-exposed regions of the S-layer proteins, while absorption with homologous live cells showed that the immunodominant epitopes of the S-layer protein of strain TF7 were strain specific and exposed on the surface of the native, tetragonal array produced by this strain. Proteolytic digestion of the TF7 S-layer protein with trypsin, chymotrypsin, or endoproteinase Glu-C produced an amino-terminal peptide of approximate Mr 38,000 which was refractile to further proteolytic cleavage under nondenaturing conditions. This peptide carried the immunodominant surface-exposed region of the protein, and chemical cleavage with cyanogen bromide further mapped the portion of these surface-exposed epitopes to a peptide of approximate Mr 26,000, part of which maps within the Mr 38,000 protease-resistant NH2-terminal peptide.
Iron uptake and molecular recognition in Pseudomonas putida: receptor mapping with ferrichrome and its biomimetic analogs
Authors: Jurkevitch E , Hadar Y , Chen Y , Libman J , Shanzer A
Published: November 2016

The presence of an Fe(3+)-ferrichrome uptake system in fluorescent Pseudomonas spp. was demonstrated, and its structural requirements were mapped in Pseudomonas putida with the help of biomimetic ferrichrome analogs. Growth tests, 55Fe3+ uptake, and competition experiments demonstrated that the synthetic L-alanine derivative B5 inhibits the action of ferrichrome but does not facilitate Fe3+ transport, while the enantiomeric D-Ala derivative B6 fails to compete with ferrichrome. Contraction of the molecule's envelope by replacing L-Ala by glycine provided a synthetic carrier, B9, which fully simulates ferrichrome as a growth promoter. Sodium azide inhibited 55Fe3+ uptake of the Gly derivative B9, suggesting an active transport process. These data demonstrate the chiral discrimination of the ferrichrome receptor and its sensitivity to subtle structural changes. They further confirm that receptor binding is a necessary but not sufficient condition for Fe3+ uptake to occur and suggest that binding to the receptor and transport proteins might rely on different recognition patterns.
Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors
Authors: Cornelissen C N , Biswas G D , Tsai J , Paruchuri D K , Thompson S A , Sparling P F
Published: November 2016

The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor.
Regulation of the macrolide-lincosamide-streptogramin B resistance gene ermD
Authors: Hue K K , Bechhofer D H
Published: November 2016

The erythromycin resistance gene ermD, which encodes an rRNA methylase protein, has an unusually long leader region (354 nucleotides). Previously, a single promoter-proximal leader peptide coding sequence was recognized from the nucleotide sequence, and erythromycin-induced ribosome stalling in this sequence was proposed to be required for the induction of methylase translation. We characterized spontaneously occurring and in vitro-constructed leader region mutations in an effort to understand the function of various segments of the long ermD leader region. A second leader peptide coding sequence was identified, and the location of insertion and point mutations that expressed ermD methylase constitutively suggested that translation of the second leader peptide is controlled by ribosome stalling in the first leader peptide. From Northern RNA blot analysis of ermD transcription, it appears that regulation of ermD expression is not by transcriptional attenuation.
Oligopeptidase A is required for normal phage P22 development
Authors: Conlin C A , Vimr E R , Miller C G
Published: November 2016

The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.
Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco
Authors: Hatziloukas E , Panopoulos N J
Published: November 2016

Pseudomonas syringae pv. phaseolicola produces the tripeptide N delta(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginin e (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature, in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicotiana tabacum by Agrobacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.
Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding
Authors: Russell P W , Orndorff P E
Published: November 2016

We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli. Defined, in-frame deletion mutations were generated in vitro in each of these two genes. A double mutation that had deletions identical to both single lesions was also constructed. Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca. 16 kDa and that the fimG gene product was a protein of ca. 14 kDa. Each of the constructions was introduced, via homologous recombination, into the E. coli chromosome. All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes. The latter property was also exhibited by partially purified pili isolated from each of the mutants. Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili. The double mutant displayed the characteristics of both single mutants. However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant. All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation. On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization. In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding.
Determination of the mechanism of retrotransfer by mechanistic mathematical modeling
Authors: Top E , Vanrolleghem P , Mergeay M , Verstraete W
Published: November 2016

Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed. This study deals with retromobilization of nonconjugative plasmids (Tra-Mob+). Plasmid transfer was modeled by two mass action models. The first is based on the hypothesis that retromobilization of the Tra-Mob+ vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host. In the second model, retromobilization is considered to be a two-step process involving two transfer events. The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient. Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population. Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process.
The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate
Authors: Handwerger S , Pucci M J , Volk K J , Liu J , Lee M S
Published: November 2016

Vancomycin resistance plasmids in enterococci carry the genes vanH and vanA, which encode enzymes catalyzing, respectively, the reduction of 2-keto acids to 2-D-hydroxy acids and the addition of D-hydroxy acids to D-alanine. It has therefore been postulated that resistant cells produce peptidoglycan precursors that terminate in the depsipeptide D-alanine-2-D-hydroxy acid rather than the dipeptide D-alanine-D-alanine, thus preventing vancomycin binding (M. Arthur, C. Molinas, T. D. H. Bugg, G. D. Wright, C. T. Walsh, and P. Courvalin, Antimicrob. Agents Chemother. 36:867-869, 1992). In the present work, a cytoplasmic peptidoglycan precursor was isolated from vancomycin-resistant Enterococcus faecalis and analyzed by mass spectrometry, which suggested the structure UDP-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-lactate.
Regulation of narK gene expression in Escherichia coli in response to anaerobiosis, nitrate, iron, and molybdenum
Authors: Kolesnikow T , Schröder I , Gunsalus R P
Published: November 2016

The regulation of the narK gene in Escherichia coli was studied by constructing narK-lacZ gene and operon fusions and analyzing their expression in various mutant strains in response to changes in cell growth conditions. Expression of narK-lacZ was induced 110-fold by a shift to anaerobic growth and a further 8-fold by the presence of nitrate. The fnr gene product mediates this anaerobic response, while nitrate control is mediated by the narL, narX, and narQ gene products. The narX and narQ gene products were shown to sense nitrate independently of one another and could each activate narK expression in a NarL-dependent manner. We provide the first evidence that NarL and FNR interact to ensure optimal expression of narK. IHF and Fis proteins are also required for full activation of narK expression, and their roles in DNA bending are discussed. Finally, the availability of molybdate and iron ions is necessary for optimal narK expression, whereas the availability of nitrite is not. Although the role of the narK gene product in cell metabolism remains uncertain, the pattern of narK gene expression is consistent with a proposed role of NarK in nitrate uptake by the cell for nitrate-linked electron transport.
Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli
Authors: Kälin M , Neujahr H Y , Weissmahr R N , Sejlitz T , Jöhl R , Fiechter A , Reiser J
Published: November 2016

A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.