bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence and transcription mapping of Bacillus subtilis competence genes comB and comA, one of which is related to a family of bacterial regulatory determinants
Authors: Weinrauch Y , Guillen N , Dubnau D A
Published: November 2016

The complete nucleotide sequences of the comA and comB loci of Bacillus subtilis were determined. The products of these genes are required for the development of competence in B. subtilis and for the expression of late-expressing competence genes. The major 5' termini of both the comA and comB transcripts were determined. The inferred promoters of both comA and comB contained sequences that were similar to those found at the -10 and -35 regions of promoters that are used by sigma A-RNA polymerase, the primary form of this enzyme in vegetative cells. The comB gene was located approximately 3 kilobase pairs upstream of the comA gene and encoded a 409-amino-acid protein with a predicted molecular weight of 46,693. The comA locus contained two open reading frames (ORFs) and comB contained one ORF. The predicted amino acid sequence of the comA ORF1 gene product consisted of 214 amino acids, with an aggregate molecular weight of 24,132. The ORF1 product was required for competence, while ORF2, which was cotranscribed with ORF1 and encoded a predicted protein of 126 amino acids, was not. The predicted protein sequence of the comA ORF1 gene product was found to be similar to that of several members of the effector class of procaryotic signal transducers. The C-terminal portion of the predicted comA sequence contained a possible helix-turn-helix motif, which is characteristic of DNA-binding proteins. comA ORF1 was cloned on a multicopy plasmid and was shown to complement the competence-deficient phenotype caused by the comA124 insertion of Tn917lac. Also, the presence of comA ORF1 in multiple copies interfered with sporulation. Anti-peptide antibodies raised to the predicted product of comA ORF1 reacted strongly with a single protein band of about 24,000 daltons in immunoblots. The possible roles of multiple signal transduction systems in triggering the development of competence are discussed.
Nucleotide sequence and genetic organization of the Bacillus subtilis comG operon
Authors: Albano M , Breitling R , Dubnau D A
Published: November 2016

A series of Tn917lac insertions define the comG region of the Bacillus subtilis chromosome. comG mutants are deficient in competence and specifically in the binding of exogenous DNA. The genes included in the comG region are first expressed during the transition from the exponential to the stationary growth phase. From nucleotide sequence information, it was concluded that the comG locus contains seven open reading frames (ORFs), several of which overlap at their termini. High-resolution S1 nuclease mapping and primer extension were used to identify the 5' terminus of the comG mRNA. The sequence upstream from the comG start site closely resembled the consensus recognition sequence for the major B. subtilis vegetative RNA polymerase holoenzyme. Complementation analysis confirmed that the comG ORF1 protein is required for the ability of competent cultures to resolve into two populations with different cell densities on Renografin (E. R. Squibb & Sons, Princeton, N.J.) gradients, as well as for full expression of comE, another late competence locus. The predicted comG ORF1 protein showed significant similarity to the virB ORF11 protein from Agrobacterium tumefaciens, which is probably involved in T-DNA transfer. The N-terminal sequences of comG ORF3 and, to a lesser extent, the comG ORF4 and ORF5 proteins were similar to a class of pilin proteins from members of the genera Bacteroides, Pseudomonas, Neisseria, and Moraxella. All of the comG proteins except comG ORF1 possessed hydrophobic domains that were potentially capable of spanning the bacterial membrane. It is likely that these proteins are membrane associated, and they may comprise part of the DNA transport machinery. When present in multiple copies, a DNA fragment carrying the comG promoter was capable of inhibiting the development of competence as well as the expression of several late com genes, suggesting a role for a transcriptional activator in the expression of those genes.
The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli
Authors: Kuriki Y
Published: November 2016

pBR322 contains the amp gene encoding beta-lactamase. When Escherichia coli carrying this plasmid is exposed to heat shock, beta-lactamase synthesis is repressed transiently at the translational level. To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1. These constructs were introduced into E. coli, and the effect of heat shock of the cells on the synthesis of beta-galactosidase starting from the amp start codon was examined. As is the case for pBR322-encoded synthesis of beta-lactamase, the synthesis of beta-galactosidase encoded by the fused genes also ceased transiently upon heat shock. It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation. As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).
IS861, a group B streptococcal insertion sequence related to IS150 and IS3 of Escherichia coli
Authors: Rubens C E , Heggen L M , Kuypers J M
Published: November 2016

A 1,442-base-pair (bp) insertion sequence (IS861) was identified in the type III group B streptococcal (GBS) strain COH-1. It is flanked by 26-bp imperfect inverted repeats and contains two open reading frames, 1 and 2, encoding 141- and 277-amino-acid proteins, respectively. A 3-bp target sequence, ACA, is duplicated and flanks each inverted repeat. IS861 shares greater than 30% homology with IS3 and IS150 of Escherichia coli, primarily in the region of their putative transposases. Northern (RNA) analysis revealed that RNA is actively transcribed in vivo by IS861 and 17- and 36-kilodalton proteins were synthesized in E. coli maxicell assays. Multiple copies of IS861 were observed throughout the chromosome of COH-1, and one of the copies is located near genes involved in GBS capsule synthesis. IS861 is the first insertion sequence identified in GBS. Its role in GBS and the significance of its relationship to the phylogenetically similar insertion sequences typified by IS150 and IS3 of E. coli are unknown.
(S)-3-hydroxy-3-methylglutaryl coenzyme A reductase, a product of the mva operon of Pseudomonas mevalonii, is regulated at the transcriptional level
Authors: Wang Y L , Beach M J , Rodwell V W
Published: November 2016

We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, the structural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonas mevalonii. The DNA segment that we characterized includes the promoter region for the mva operon. Nuclease S1 mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon. Transcription initiates at -56 bp relative to the first A (+1) of the translation start site. Transcription in vivo was induced by mevalonate. Structural features of the mva promoter region include an 80-bp A + T-rich region, and -12, -24 consensus sequences that resemble sequences of sigma 54 promoters in enteric organisms. The relative amplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation of this operon at the transcriptional level.
Cloning of 18S and 25S rDNAs from the pathogenic fungus Cryptococcus neoformans
Authors: Restrepo B I , Barbour A G
Published: November 2016

Cryptococcus neoformans is an important pathogenic fungus that has been classified as a basidiomycete. Little is known of the molecular genetics of this fungal pathogen. To begin such studies, we devised a procedure for extraction of DNA from cryptococci; this method involved the use of the cell wall-active enzyme NovoZym 234. Using cloned rDNA of Saccharomyces cerevisiae as a probe, we identified homologous restriction fragments in a Southern blot of digested C. neoformans DNA. An 8.6-kilobase HindIII fragment that hybridized with the yeast rDNA probe was ligated with the vector pBR322 and cloned into Escherichia coli. When the fragment was used as a probe, it hybridized to the 18S and 25S rRNAs of C. neoformans in Northern (RNA) blots of native and denatured RNA. It bound at high stringency only weakly to the rRNAs of the ascomycete S. cerevisiae. The locations of the genes for 5/5.8S, 18S, and 25S subunits in the cloned fragment were identified with labeled rRNA of these different types.
Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes
Authors: Yamada M , Makino K , Amemura M , Shinagawa H , Nakata A
Published: November 2016

The phoB gene product of Escherichia coli is the transcriptional activator for the genes in the phosphate regulon as well as for phoB itself, all of which are induced by phosphate starvation. The phoR gene product modulates PhoB function in response to the phosphate concentrations in the medium. We quantitatively compared the levels of expression of the phoA, phoB, phoE, and pstS genes in several phoB mutants with different phenotypes by constructing operon fusions of these genes with the gene for chloramphenicol acetyltransferase. Although all the phoB mutants examined had little activator function for phoA, three among the four mutants showed various levels of the activator function for phoB, pstS, and phoE. To study the functional motifs of the PhoB and PhoR proteins, we cloned and sequenced the four classical phoB and six phoR mutant genes. All of the phoB mutations and one of the phoR mutations were missense mutations, and most of the altered amino acids were in the highly conserved amino acids among the regulatory proteins homologous to PhoB or PhoR protein, such as the OmpR, SfrA, and VirG proteins or the EnvZ, CpxA, and VirA proteins. The other five phoR mutations were nonsense mutations.
Thiol-sensitive genes of Escherichia coli
Authors: Javor G T
Published: November 2016

The effect of 1-thioglycerol on the expression of genes of Escherichia coli was investigated. Pulse-labeled proteins from aerobically growing, 1-thioglycerol-treated E. coli were separated by two-dimensional gel electrophoresis, and their radioactivities were compared with those of identical proteins from nontreated cells. The first 10 min of exposure to thiol stimulated the synthesis of 10% of the observed proteins and inhibited the production of 16% of the proteins. After 30 min of growth with thiol, the synthesis of 44% of the observed proteins was inhibited and synthesis of 18% of the proteins was stimulated. In general, the expression of genes of carbohydrate metabolism, amino acid metabolism, and protein biosynthesis were inhibited, while nucleic acid synthetic and repair gene expressions showed mixed responses. Synthesis of transport proteins was not affected. Transient stimulation of oxidative-stress proteins and sustained stimulation of the expressions of trxB, ompA, and ompB genes and those of several unidentified gene products were also observed. Whether these complex responses merely reflect adjustments by cellular subsystems to a suddenly reducing environment or whether they are manifestations of a reductive-stress regulon will have to await genetic analysis of this phenomenon.
Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein
Authors: Swan D G , Cortes J , Hale R S , Leadlay P F
Published: November 2016

The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ702, CAB was found to be expressed at higher levels than in S. erythraea, with no apparent effects on either growth or sporulation. A more convenient expression system for CAB was obtained by introducing an NdeI site at the initiation codon by using oligonucleotide-directed mutagenesis and placing the gene in the expression vector pT7-7 in Escherichia coli. In this system, CAB was efficiently expressed at levels up to 20 to 30% of total cell protein. When purified to homogeneity from either E. coli or Streptomyces lividans, CAB was found to be identical to the protein previously obtained from S. erythraea.
REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase
Authors: Morrison A , Christensen R B , Alley J , Beck A K , Bernstine E G , Lemontt J F , Lawrence C W
Published: November 2016

We have cloned the REV3 gene of Saccharomyces cerevisiae by complementation of the rev3 defect in UV-induced mutagenesis. The nucleotide sequence of this gene encodes a predicted protein of Mr 172,956 showing significant sequence similarity to Epstein-Barr virus DNA polymerase and to other members of a class of DNA polymerases including human DNA polymerase alpha and yeast DNA polymerase I. REV3 protein shows less sequence identity, and presumably a more distant evolutionary relationship, to the latter two enzymes than they do to each other. Haploids carrying a complete deletion of REV3 are viable. We suggest that induced mutagenesis in S. cerevisiae depends on a specialized DNA polymerase that is not required for other replicative processes. REV3 is located 2.8 centimorgans from CDC60, on chromosome XVI.
Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants
Authors: Hawes M C , Smith L Y
Published: November 2016

Agrobacterium tumefaciens Tn5 mutants deficient in chemotaxis to root exudates were used to study the significance of chemotaxis in crown gall pathogenesis. Mutants deficient in motility and in chemotaxis were fully virulent when inoculated by direct immersion in inoculum, followed by growth for 2 weeks in moist growth pouches. Ability of mutant bacteria to move through soil to infect roots was tested by planting wounded seedlings into air-dried soil or sand that had been infested with inoculum. Mutant bacteria were almost as virulent as the parent on plants grown in sand but were avirulent on soil-grown plants.
Cloning and analysis of an Escherichia coli operon containing the rpmF gene for ribosomal protein L32 and the gene for a 30-kilodalton protein
Authors: Tanaka Y , Tsujimura A , Fujita N , Isono S , Isono K
Published: November 2016

The chromosomal DNA fragments of Escherichia coli K-12 were cloned into a mini-F cosmid, pRE435, after partial digestion with restriction endonuclease Sau3AI. The clones were first screened for PyrC+ and then for other genes, including rpmF encoding ribosomal protein L32 that had been mapped near pyrC (I. Janda, M. Kitakawa, and K. Isono, Mol. Gen. Genet. 201:443-436, 1985). Thus, we obtained a total of five rpmF-containing clones. The rpmF gene was located on the chromosomal segment in one of the clones (pAY2-5) by insertional mutagenesis with transposon gamma delta, followed by analysis of the gene products by the maxicell method. Hybridization analysis of clone pAY2-5 with the ordered clone bank (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) indicates that a gap at the 1,510-kilobase coordinates in the physical map of E. coli can be bridged by this clone. The nucleotide sequence of the region containing rpmF was accordingly established. In addition, the RNA transcripts from the chromosomal region containing rpmF were analyzed, and the transcriptional initiation sites were determined. The results suggest that rpmF forms an operon with the gene termed g30k which codes for a 30-kilodalton protein of unknown function. At least four transcripts were found to code for ribosomal protein L32.
Fine-structure genetic map of the maltose transport operon of Salmonella typhimurium
Authors: Schneider E , Bishop L , Schneider E , Alfandary V , Ames G F
Published: November 2016

We have constructed a fine-structure genetic map of the maltose transport operon in Salmonella typhimurium. We have isolated mal mutants by using indicator plates, penicillin selection, or a proton suicide technique. Mutants were obtained as spontaneous events or were induced by chemical mutagenesis and transposon insertion. Tn10 and Mu d(lac Ap)1 insertion mutations were used to create deletions. Mutations were also obtained in a gene that is equivalent to lamB in Escherichia coli, which codes for the lambda bacteriophage receptor. The gene products in the mutants were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and immunoblotting. Our data indicate that the location of this operon on the Salmonella chromosome as well as the gene order and its orientation are the same as those in E. coli. This map will be useful in studying the mechanism of periplasmic transport in S. typhimurium.
Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli
Authors: Nagasawa H , Sakagami Y , Suzuki A , Suzuki H , Hara H , Hirota Y
Published: November 2016

Chromatographic peptide mapping of lysyl endopeptidase digests of penicillin-binding protein 3 (PBP 3) of Escherichia coli revealed peptides that differed in retention time between the precursor and mature forms. The peptides were purified from a processing-defective (prc) mutant and a wild-type (prc+) strain. These peptides were identified as the C-terminal region of the precursor form and mature PBP 3 by amino acid sequencing. Each of the C-terminal peptides was cleaved into two fragments by trypsin digestion. By sequencing the resultant carboxyl-side fragment derived from the mature form, it was concluded that the C-terminal residue of mature PBP 3 was Val-577, and thus the Val-577-Ile-578 bond is the cleavage site for processing. This conclusion was consistent with the amino acid compositions of the relevant peptides, which suggested that the peptide from the cleavage site to the end of the deduced sequence (Ile-578-Ser-588) was present in the precursor but absent in the mature form. One lysyl peptide bond resisted both lysyl endopeptidase and trypsin and remained uncleaved in the peptide analyzed above.
Genetic organization and sequence of the Pseudomonas cepacia genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase
Authors: Zylstra G J , Olsen R H , Ballou D P
Published: November 2016

The locations of the genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.11.3) on a 9.5-kilobase-pair PstI fragment cloned from the Pseudomonas cepacia DBO1 chromosome were determined. This was accomplished through the construction of several subclones into the broad-host-range cloning vectors pRO2317, pRO2320, and pRO2321. The ability of each subclone to complement mutations in protocatechuate 3,4-dioxygenase (pcaA) was tested in mutant strains derived from P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. These complementation studies also showed that the two subunits were expressed from the same promoter. The nucleotide sequence of the region encoding for protocatechuate 3,4-dioxygenase was determined. The deduced amino acid sequence matched that determined by N-terminal analysis of regions of the isolated enzyme. Although over 400 nucleotides were sequenced before the start of the genes, no homology to known promoters was found. However, a terminator stem-loop structure was found immediately after the genes. The deduced amino acid sequence showed extensive homology with the previously determined amino acid sequence of protocatechuate 3,4-dioxygenase from another Pseudomonas species.
Regulation of expression of the Pasteurella haemolytica leukotoxin determinant
Authors: Strathdee C A , Lo R Y
Published: November 2016

The Pasteurella haemolytica leukotoxin determinant is composed of four contiguous genes encoded on the same DNA strand and denoted lktCABD, in the order of their genetic organization. To gain a better understanding of the expression and regulation of the leukotoxin, the transcripts and promoters of the lkt determinant were mapped. Northern (RNA) blot analysis revealed two sets of transcripts. One set was 3.7 and 3.4 kilobases long, encoded lktCA, and comprised approximately 90% of the transcripts, whereas the other set was 7.4 and 7.1 kilobases long and encoded lktCABD. Two promoters were present, and each had features similar to the Escherichia coli consensus promoter sequences. Both promoters were located upstream from lktC; they were separated by 258 base pairs, as mapped by primer extension analysis. These results suggest a mechanism of expression similar to that of the related E. coli hemolysin. Transcription initiated upstream from lktC at either promoter and continued through lktC and lktA to a rho-independent transcriptional termination signal in the lktA-lktB intercistronic region. This signal attenuated expression by terminating 90% of transcription to generate the 3.7- and 3.4-kilobase lktCA transcripts. The remaining readthrough transcription generated full-length 7.4- and 7.1-kilobase lktCABD transcripts. Expression of the leukotoxin was greatly reduced by growth at 30 degrees C, pH 6.5, and Fe2+ limitation. These conditions also modulated the expression of a number of other secreted proteins, which suggests that all of these secreted proteins are controlled by the same regulatory mechanism.
Patterns of cellular interactions during fruiting-body formation in Myxococcus xanthus
Authors: O'Connor K A , Zusman D R
Published: November 2016

Aggregation and mound formation during development of the myxobacterium Myxococcus xanthus were examined by scanning electron microscopy and light microscopy. Several complex patterns of multicellular associations were observed. These observations imply that complex, organized cell-cell interactions occur during the process of development. Examination of sliced aggregates revealed that, contrary to common perception, the process of sporulation commenced during mound formation rather than after the completion of mound morphogenesis. The morphogenesis of M. xanthus fruiting bodies is compared with the morphogenesis of fruiting bodies of other members of the Myxobacteriales previously described in the literature.
Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication
Authors: Tilly K , Yarmolinsky M
Published: November 2016

Low-copy-number plasmids, such as P1 prophage and the fertility factor F, require a plasmid-encoded replication protein and several host products for replication. Stable maintenance also depends on active partitioning of plasmids into daughter cells. Mini-P1 par+ and par plasmids were found to be destabilized by mutations in the dnaJ, dnaK, and grpE genes of Escherichia coli. The transformation efficiency and stability of mini-F plasmids were also reduced in the mutant strains. These results indicate that heat shock proteins DnaJ, DnaK, and GrpE play roles in the replication of plasmid P1 and probably also in of F.
Organization of the murE-murG region of Escherichia coli: identification of the murD gene encoding the D-glutamic-acid-adding enzyme
Authors: Mengin-Lecreulx D , Parquet C , Desviat L R , Plá J , Flouret B , Ayala J A , van Heijenoort J
Published: November 2016

The 2-min region of the Escherichia coli genome contains a large cluster of genes from pbpB to envA that code for proteins involved in peptidoglycan biosynthesis and cell division. From pLC26-6 of the collection of Clarke and Carbon (L. Clarke and J. Carbon, Cell 9:91-99, 1976) plasmids carrying different fragments from the 8-kilobase-pair region downstream of pbpB were constructed and analyzed for their ability to direct protein synthesis in maxicells, to complement various thermosensitive mutations, and to overproduce enzymatic activities. We report the localization of the previously unidentified murD gene coding for the D-glutamic acid-adding enzyme within this region. Our data show that the genes are in the order pbpB-murE-murF-X-murD-Y-murG, where X and Y represent chromosomal fragments from 1 to 1.5 kilobase pairs, possibly coding for unknown proteins. Furthermore, the murE and murF genes, encoding the meso-diaminopimelic acid and D-alanyl-D-alanine-adding enzymes, respectively, may be translationally coupled when transcription is initiated upstream of murE, within the preceding structural gene pbpB coding for penicillin-binding protein 3.
Is the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive), a copper metalloenzyme?
Authors: Baasov T , Knowles J R
Published: November 2016

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive) was purified from Escherichia coli carrying the plasmid pKB45. Enzyme of high specific catalytic activity (70 mu/mg) was obtained from cells grown only to the early log phase. The purified protein contained Cu(II) and showed an absorption band at 350 nm. Metal-free, catalytically inactive apoenzyme could be produced by dialysis against cyanide ion, and the holoenzyme could be reconstituted in terms of both catalytic activity and A350 by the binding of one Cu(II) ion per enzyme subunit. Zn(II) also reactivated the apoenzyme to about 50% of the level seen with Cu(II), although in this case no band appeared at 350 nm. In contrast to earlier reports that the enzyme contains substoichiometric levels of iron, insignificant amounts of iron were found in the isolated enzyme, and neither Fe(II) nor FE(III) regenerated either an absorption band at 350 nm or any catalytic activity from the apoenzyme. The evident preference of the enzyme as isolated for (Cu)II suggests that the synthase might naturally be a copper metalloenzyme.
Characterization of the cryptic lambdoid prophage DLP12 of Escherichia coli and overlap of the DLP12 integrase gene with the tRNA gene argU
Authors: Lindsey D F , Mullin D A , Walker J R
Published: November 2016

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage lambda. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 alpha 4 beta 4 insertion element. The argU and DLP12 int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of lambda orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo P/ren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.
Environmental regulation of expression of virulence determinants in Bordetella pertussis
Authors: Melton A R , Weiss A A
Published: November 2016

The trans-activator vir is required for expression of all virulence-associated genes in Bordetella pertussis. The nature of the global regulation of these factors by vir and environmental signals was examined by Northern blot analysis and with beta-galactosidase transcriptional fusions in five vir-regulated genes. Northern blots suggested that vir regulates at the level of transcription since Vir- organisms did not exhibit detectable mRNA from vir-regulated loci. Environmental signals such as high levels of salts, nicotinic acid, and 6-chloronicotinic acid or growth at low temperatures were examined. Of all of the cations and anions examined, only SO4 ions eliminated transcription of vir-regulated genes and reduced transcription of vir itself, suggesting that global regulation is obtained by modifying expression of the essential component, vir. Organisms grown on 6-chloronicotinic acid or quinaldic acid did not have detectable transcription from vir-regulated loci. Modulation by nicotinic acid, on the other hand, was strain dependent, acting at the level of transcription in strain 18-323 but not in Tohama I derivatives. Growth at lower temperatures reduced, but did not eliminate, transcription from vir-regulated loci. At 28 degrees C the ratio of pertussis toxin mRNA to recA mRNA (a non-vir-regulated factor) was equivalent to that at 37 degrees C, suggesting that transcription at low temperatures is reduced in a proportional manner and need not involve vir.
S-layer protein gene of Acetogenium kivui: cloning and expression in Escherichia coli and determination of the nucleotide sequence
Authors: Peters J , Peters M , Lottspeich F , Baumeister W
Published: November 2016

Acetogenium kivui is anaerobically growing thermophilic bacterium with a gram-positive type of cell wall structure. The outer surface is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer polypeptide was cloned in Escherichia coli on two overlapping fragments by using the plasmid pUC18 as the vector. It was expressed under control of a cloned Acetogenium promoter or the lacZ gene. We determined the complete sequence of the structural gene. The mature polypeptide comprises 736 amino acids and is preceded by a typical procaryotic signal sequence of 26 amino acids. It i weakly acidic, weakly hydrophilic, and contains a relatively high proportion of hydroxyamino acids, including two clusters of serine and threonine residues. An N-terminal region of about 200 residues is homologous to the N-terminal part of the middle wall protein, one of the two S-layer proteins of Bacillus brevis, and there is also an internal homology within the N-terminal region of the A. kivui polypeptide.
Arsenic oxide-induced thermotolerance in Saccharomyces cerevisiae
Authors: Chang E C , Kosman D J , Willsky G R
Published: November 2016

The growth response of Saccharomyces cerevisiae to arsenite and arsenate and the relationship between the enhancement of heat shock protein (hsp) synthesis caused by these arsenic oxides and thermotolerance are reported. Arsenite and arsenate transiently inhibited cell growth and overall protein synthesis; arsenate enhanced the synthesis of the 42-, 74-, 84-, and 100-kilodalton hsps, whereas arsenite enhanced synthesis of only the 74-kilodalton hsp. The induction of these hsps reached a maximum 45 min following metal oxide treatment and then declined. A delayed thermotolerance peaked 4 h after metal oxide addition, at which time cell growth and protein synthesis were recovering. These data show that the arsenate- and arsenite-induced thermotolerance in S. cerevisiae cells does not appear to be causally related to either hsp synthesis or cell cycle arrest.
Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli
Authors: Kelly-Wintenberg K , Montie T C
Published: November 2016

The flagellin gene was isolated from a Pseudomonas aeruginosa PAO1 genomic bank by conjugation into a PA103 Fla- strain. Flagellin DNA was transferred from motile recipient PA103 Fla+ cells by transformation into Escherichia coli. We show that transformed E. coli expresses flagellin protein. Export of flagellin to the E. coli cell surface was suggested by positive colony blots of unlysed cells and by isolation of flagellin protein from E. coli supernatants.
Trehalose accumulation in vegetative cells and spores of Myxococcus xanthus
Authors: McBride M J , Zusman D R
Published: November 2016

The disaccharide trehalose is found in the spores and cysts of a variety of organisms. We analyzed developing cells of Myxococcus xanthus for trehalose accumulation. Vegetative cells grown in media with low osmotic strengths contained less than 5 micrograms of trehalose per mg of protein. Spores formed in fruiting bodies accumulated up to 1,100 micrograms of trehalose per mg of protein. Spores formed in liquid culture following the addition of glycerol contained up to 300 micrograms of trehalose per mg of protein. The trehalose contents of both spore types decreased rapidly during the early stages of germination. Trehalase activity was not detected in extracts of dormant or germinating spores. Trehalose accumulation in M. xanthus was also associated with elevated osmotic strength. Vegetative cells accumulated up to 214 micrograms of trehalose per mg of protein when grown in media containing elevated levels of solutes.
Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus
Authors: Rua J , De Arriaga D , Busto F , Soler J
Published: November 2016

Repression of the synthesis of isocitrate lyase by glucose and/or induction of the synthesis of isocitrate lyase by acetate in Phycomyces blakesleeanus were demonstrated. Both glycerol and ethanol failed to induce isocitrate lyase activity. Furthermore, glucose appeared to cause an in vivo catabolite inactivation of the derepressed enzyme. Isocitrate lyase was inactivated both reversibly and irreversibly by glucose.
Molybdenum accumulation in chlD mutants of Escherichia coli
Authors: Scott D , Amy N K
Published: November 2016

The content of molybdenum in wild-type and chlD cells was measured under a variety of growth conditions to determine if cells with a defective chlD gene were able to accumulate molybdenum. The chlD cells accumulated less molybdenum than wild-type cells did but concentrated molybdenum to a level at least 20-fold higher than the concentration in the culture medium. Molybdenum was present within spheroplasts of chlD cells and was not dialyzable. The chlD cells accumulated as much molybdenum as wild-type cells did when grown in medium containing 0.1 mM molybdate; thus, the capability of incorporation of molybdenum into cellular component(s) was equivalent to that of the wild type under these conditions.
Identification of toxS, a regulatory gene whose product enhances toxR-mediated activation of the cholera toxin promoter
Authors: Miller V L , DiRita V J , Mekalanos J J
Published: November 2016

We describe the cloning of the toxS gene from Vibrio cholerae E1 Tor strain E7946. This gene lies downstream from the toxR gene, which encodes the transcriptional activator for the cholera toxin (ctx) operon in V. cholerae. We show that ToxS acts in conjunction with ToxR to activate expression of the ctx operon in Escherichia coli. The classical strain 569B, which is attenuated for virulance but which synthesizes high levels of cholera toxin in vitro, carries a deletion of 1.2 kilobase pairs of DNA, downstream from the toxR gene, which removes toxS. We present evidence that toxS is the downstream gene in an operon with toxR.
Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity
Authors: Silen J L , Frank D , Fujishige A , Bone R , Agard D A
Published: November 2016

The alpha-lytic protease of Lysobacter enzymogenes was successfully expressed in Escherichia coli by fusing the promoter and signal sequence of the E. coli phoA gene to the proenzyme portion of the alpha-lytic protease gene. Following induction, active enzyme was found both within cells and in the extracellular medium, where it slowly accumulated to high levels. Use of a similar gene fusion to express the protease domain alone produced inactive enzyme, indicating that the large amino-terminal pro region is necessary for activity. The implications for protein folding are discussed. Furthermore, inactivation of the protease by mutation of the catalytic serine residue resulted in the production of a higher-molecular-weight form of the alpha-lytic protease, suggesting that the enzyme is self-processing in E. coli.
In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen
Authors: Barr K , Nunes-Edwards P , Rick P D
Published: November 2016

The heteropolysaccharide chains of enterobacterial common antigen (ECA) are made up of linear trisaccharide repeat units with the structure----3)-alpha-D-Fuc4NAc-(1----4)- beta-D-ManNAcA-(1----4)-alpha-D-GlcNAc-(1----, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The assembly of these chains involves lipid-linked intermediates, and both GlcNAc-pyrophosphorylundecaprenol (lipid I) and ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) are intermediates in ECA biosynthesis. In this study we demonstrated that lipid II serves as the acceptor of Fuc4NAc residues in the assembly of the trisaccharide repeat unit of ECA chains. Incubation of Escherichia coli membranes with UDP-GlcNAc, UDP-[14C]ManNAcA, and TDP-[3H]Fuc4NAc resulted in the synthesis of a radioactive glycolipid (lipid III) that contained both [14C]ManNAcA and [3H]Fuc4NAc. The oligosaccharide moiety of lipid III was identified as a trisaccharide by gel-permeation chromatography, and the in vitro synthesis of lipid III was dependent on prior synthesis of lipids I and II. Accordingly, the incorporation of [3H]Fuc4NAc into lipid III from the donor TDP-[3H]Fuc4NAc was dependent on the presence of both UDP-GlcNAc and UDP-ManNAcA in the reaction mixtures. In addition, the in vitro synthesis of lipid III was abolished by tunicamycin. Direct conversion of lipid II to lipid III was demonstrated in two-stage reactions in which membranes were initially incubated with UDP-GlcNAc and UDP-[14C]ManNAcA to allow the synthesis of radioactive lipid II. Subsequent addition of TDP-Fuc4Nac to the washed membranes resulted in almost complete conversion of radioactive lipid II to lipid III. The in vitro synthesis of lipid III was also accompanied by the apparent utilization of this lipid intermediate for the assembly of ECA heteropolysaccharide chains. Incubation of membranes with UDP-[3H]GlcNAc, UDP-ManNAcA, and TDP-Fuc4NAc resulted in the apparent incorporation of isotope into ECA polymers, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In addition, the in vitro incorporation of [3H]Fuc4NAc into ECA heteropolysaccharide chains was demonstrated with ether-treated cells that were prepared from delta rfbA mutants of Salmonella typhimurium. These mutants are defective in the synthesis of TDP-Fuc4NAc; as a consequence, they are also defective in the synthesis of lipid III and they accumulate lipid II. Accordingly, incubation of ether-permeabilized cells of delta rfbA mutants with TDP-[3h]Fuc4NAc resulted in the incorporation of isotope into both lipid III and ECA heteropolysaccharide chains.
Two transcribing activities are involved in expression of the Streptomyces galactose operon
Authors: Westpheling J , Brawner M
Published: November 2016

The Streptomyces galactose operon is transcribed from two independently regulated promoters: galP1, located at the 5' end of the operon and responsible for galactose-dependent transcription of the operon, and galP2, an internal constitutive promoter. We identified and partially separated two distinct transcribing activities involved in expression of this operon. Using RNA polymerase from Streptomyces lividans and Streptomyces coelicolor partially purified by chromatography on heparin-agarose and DNA-cellulose, we detected activities capable of initiating transcription in vitro specifically from either galP1 or galP2. Circumstantial evidence suggests that the activity for galP2 transcription is a holoenzyme species associated with the previously described sigma 28 protein (referred to here as sigma C). The galP1-transcribing activity is more difficult to evaluate. This activity may correspond to a holoenzyme species associated with sigma A (formerly sigma 35), although other possibilities are discussed. This would be the second reported example of a catabolite-controlled gene in Streptomyces species expressed from multiple promoters recognized by different holoenzyme forms. This may indicate that the involvement of RNA polymerase heterogeneity in gene expression in Streptomyces species is a more general strategy for regulation than the specialized gene expression seen in Escherichia coli.
Purification and characterization of a low-molecular-weight membrane protein with affinity for the Escherichia coli origin of replication
Authors: Jacq A , Kern R , Tsugita A , Kohiyama M
Published: November 2016

A purification procedure was devised for a low-molecular-mass (about 10-kilodalton) membrane protein from Escherichia coli that was shown to bind specifically to the chromosomal replication origin region (oriC). Nitrocellulose membrane retention assays showed the binding site to be adjacent to the right boundary of the oriC minimal sequence. We determined the amino acid sequence of the N-terminal and C-terminal regions as well as the global amino acid composition of this membrane protein. Specific antibodies against the protein were produced and used to confirm the cell membrane location of the protein. These results demonstrate that this is a new membrane protein, different from the previously described B' protein, with specific binding activity for the oriC region. We propose that this protein be called membrane oriC-binding protein 2 (MOB2 protein).
Occurrence and distribution of gas vesicle genes among cyanobacteria
Authors: Damerval T , Castets A M , Guglielmi G , Houmard J , Tandeau de Marsac N
Published: November 2016

Gas vesicles (GV) are specialized cell inclusions providing many aquatic procaryotes with buoyancy. In the cyanobacterium Calothrix sp. strain PCC 7601, at least four genes are involved in GV formation. One of those, gvpA1, encodes the major structural GV protein (70 amino acids) and belongs to a multigene family (gvpA1, gvpA2, gvpD). The fourth gene, gvpC, encodes a 162-amino-acid protein, the function of which is still unclear. We used the Calothrix gvpA1 and gvpC genes as probes to perform Southern hybridization experiments with DNA extracted from various cyanobacterial strains. The gvpA gene was found in all the strains that synthesize GV, indicating that its product is an obligatory component of GV. Furthermore, it was found to occur as multiple copies in most of the strains tested. The gvpC gene was only detected in some strains able to synthesize a large amount of GV within a short period. This suggests that the gvpC gene product is a dispensable protein for GV formation and is involved in the efficiency of the assembly process. Based on the occurrence of the gvp genes and on DNA-DNA hybridization patterns, genus assignments are discussed.
Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti
Authors: Pleier E , Schmitt R
Published: November 2016

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.
Genetic and biochemical characterization of a mutation (fatA) that allows trans unsaturated fatty acids to replace the essential cis unsaturated fatty acids of Escherichia coli
Authors: DeVeaux L C , Cronan J E , Smith T L
Published: November 2016

Unsaturated fatty acid auxotrophs of Escherichia coli are able to use only unsaturated fatty acids of the cis configuration as the required growth supplement. A mutation in the fatA gene allows such auxotrophs to utilize unsaturated fatty acids with a trans double bond as well as fatty acids having a cis double bond. The fatA gene was mapped to min 69 near argG, and the allele studied (fatA1) was found to be dominant over the wild-type gene. fatA1 mutant strains grew at similar rates when supplemented with elaidate (trans-9-octadecenoate) or oleate (cis-9-octadecenoate). The fat+ strain, however, lysed when supplemented with the trans fatty acid. Physiological characterization of the fatA mutant strain was undertaken. The mutation appeared not to be involved with long-chain fatty acid transport. Introduction of lesions in known fatty acid transport genes abolished trans fatty acid utilization in the fatA mutant strain. Also, growth characteristics of the fat+ and the fatA1 mutant strains on elaidate as the sole carbon source were identical, which indicated comparables rate of fatty acid accumulation. The mutation appeared to be involved with recognition of the trans configuration after uptake into the cell. The levels of trans fatty acid incorporation into the phospholipids of the fat+ and the fatA strains differed considerably, with the mutant incorporating much higher levels. No significant accumulation of elaidate into nonphospholipid cellular components was observed. The fatA mutation did not appear to be involved with the cellular metabolic state, as cyclic AMP had no effect on the ability of the strains to utilize trans fatty acids.
Amplification of bacterial genomic DNA by the polymerase chain reaction and direct sequencing after asymmetric amplification: application to the study of periplasmic permeases
Authors: Shyamala V , Ames G F
Published: November 2016

The polymerase chain reaction (PCR) has been used to amplify DNA fragments by using eucaryotic genomic DNA as a template. We show that bacterial genomic DNA can be used as a template for PCR amplification. We demonstrate that DNA fragments at least as large as 4,400 base pairs can be amplified with fidelity and that the amplified DNA can be used as a substrate for most operations involving DNA. We discuss problems inherent in the direct sequencing of the amplified product, one of the important exploitations of this methodology. We have solved the problems by developing an "asymmetric amplification" method in which one of the oligonucleotide primers is used in limiting amounts, thus allowing the accumulation of single-stranded copies of only one of the DNA strands. As an illustration of the use of PCR in bacteria, we have amplified, sequenced, and subcloned several DNA fragments carrying mutations in genes of the histidine permease operon. These mutations are part of a preliminary approach to studying protein-protein interactions in transport, and their nature is discussed.