bluuebunny/crossref_metadata_2025 huggingface dataset:
The photoactive yellow protein from Ectothiorhodospira halophila as studied with a highly specific polyclonal antiserum: (intra)cellular localization, regulation of expression, and taxonomic distribution of cross-reacting proteins
Authors: Hoff W D , Sprenger W W , Postma P W , Meyer T E , Veenhuis M , Leguijt T , Hellingwerf K J
Published: November 2016

A rabbit antiserum was raised against the photoactive yellow protein (PYP) from Ectothiorhodospira halophila and purified by adsorption experiments to obtain a highly specific polyclonal antiserum. This antiserum was used to obtain the following results. (i) In E. halophila, PYP can be isolated from the fraction of soluble proteins. In the intact cell, however, PYP appeared to be associated with (intra)cytoplasmic membranes, as was concluded from analysis of immunogold-labelled thin sections of the organism. (ii) The regulation of expression of PYP was studied by using dot blot assays, Western blotting (immunoblotting), and rocket immunoelectrophoresis. Under all conditions investigated (light color, salt concentration, and growth phase), PYP was expressed constitutively in E. halophila. However, when Rhodospirillum salexigens was grown aerobically, the expression of PYP was suppressed. (iii) A large number of prokaryotic microorganisms contained a single protein, with an apparent size of approximately 15 kDa, that cross-reacted with the antiserum. Among the positively reacting organisms were both phototrophic and chemotrophic, as well as motile and nonmotile, organisms. After separation of cellular proteins into a membrane fraction and soluble proteins, it was established that organisms adapted to growth at higher salt concentrations tended to have the cross-reacting protein in the soluble fraction. In the cases of R. salexigens and Chromatium salexigens, we have shown that the cross-reacting protein involved is strongly homologous to PYP from E. halophila.
The asiA gene product of bacteriophage T4 is required for middle mode RNA synthesis
Authors: Ouhammouch M , Orsini G , Brody E N
Published: November 2016

The asiA gene of bacteriophage T4 encodes a 10-kDa peptide which binds strongly in vitro to the sigma 70 subunit of Escherichia coli RNA polymerase, thereby weakening sigma 70-core interactions and inhibiting sigma 70-dependent transcription. To assess the physiological role of this protein, we have introduced an amber mutation into the proximal portion of the asiA gene. On suppressor-deficient hosts, this mutant phage (amS22) produces minute plaques and exhibits a pronounced delay in phage production. During these mutant infections, T4 DNA synthesis is strongly delayed, suggesting that the AsiA protein plays an important role during the prereplicative period of phage T4 development. The kinetics of protein synthesis show clearly that while T4 early proteins are synthesized normally, those expressed primarily via the middle mode exhibit a marked inhibition. In fact, the pattern of protein synthesis after amS22 infection resembles greatly that seen after infection by amG1, an amber mutant in motA, a T4 gene whose product is known to control middle mode RNA synthesis. The amber mutations in the motA and asiA genes complement, both for phage growth and for normal kinetics of middle mode protein synthesis. Furthermore, primer extension analyses show that three different MotA-dependent T4 middle promoters are not recognized after infection by the asiA mutant phage. Thus, in conjunction with the MotA protein, the AsiA protein is required for transcription activation at T4 middle mode promoters.
Two different dihydroorotate dehydrogenases in Lactococcus lactis
Authors: Andersen P S , Jansen P J , Hammer K
Published: November 2016

The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic.
Nucleotide sequence and mutational analysis of the gene encoding KpsD, a periplasmic protein involved in transport of polysialic acid in Escherichia coli K1
Authors: Wunder D E , Aaronson W , Hayes S F , Bliss J M , Silver R P
Published: November 2016

The 17-kb kps gene cluster encodes proteins necessary for the synthesis, assembly, and translocation of the polysialic acid capsule of Escherichia coli K1. We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the translocation of the polymer to the cell surface. The nucleotide sequence of the 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determined. Sequence analysis showed an open reading frame for a 558-amino-acid protein with a typical N-terminal prokaryotic signal sequence corresponding to the first 20 amino acids. KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum. A chromosomal insertion mutation was generated in the kpsD gene and results in loss of surface expression of the polysialic acid capsule. Immunodiffusion analysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells. A wild-type copy of kpsD supplied in trans complemented the chromosomal mutation, restoring extracellular expression of the K1 capsule. However, a kpsD deletion derivative (kpsD delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional. Western blot (immunoblot) data from cell fractions expressing KpsD delta C11 suggest that the truncated protein was inefficiently exported into the periplasm and localized primarily to the cytoplasmic membrane.
Mutations affecting lipopolysaccharide enhance ail-mediated entry of Yersinia enterocolitica into mammalian cells
Authors: Pierson D E
Published: November 2016

Two genes of Yersinia enterocolitica, inv and ail, have been identified as having a role in the bacterial adherence to and entry into mammalian cells in vitro. Expression of both genes is regulated by temperature. In stationary phase, ail gene expression is detectable only in bacteria at 37 degrees C, not at lower temperatures. An inv mutant derivative of Y. enterocolitica, which cannot enter mammalian cells when grown at 30 degrees C because of the lack of both inv and ail gene products, was mutagenized with the transposons mini-Tn10 and Tn5B50 to look for an increase in Ail-mediated cell entry. Sixteen mutants that could enter tissue culture cells after growth at 30 degrees C were selected. All of the mutants had increased cell surface Ail levels as detected by an Ail-specific monoclonal antibody. All of the ten Tn5B50 and one of the six mini-Tn10 mutants showed no increase in ail expression, but they had alterations in their lipopolysaccharide (LPS) such that no O side chains were detectable in bacteria grown at 30 degrees C. Thus, these mutants that are increased in their ability to enter cells appear to be so as a result of a change in the LPS on the surface resulting in increased levels of Ail protein able to interact with the mammalian cell surface. In the remaining mini-Tn10 mutants, LPS is normal, and the increase in cell surface Ail levels appears to be due to an increase in ail mRNA present in the cell. These mutants may therefore be affecting a repressor of ail gene expression.
Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a
Authors: Yao Z , Valvano M A
Published: November 2016

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.
Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster
Authors: Stevenson G , Neal B , Liu D , Hobbs M , Packer N H , Batley M , Redmond J W , Lindquist L , Reeves P
Published: November 2016

Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.
Physical map of Mycoplasma gallisepticum
Authors: Tigges E , Minion F C
Published: November 2016

Physical chromosomal maps of two Mycoplasma gallisepticum strains, R and ATCC 19610, were constructed by using field inversion gel electrophoresis. To assist in the ordering of chromosomal fragments and the construction of the chromosomal maps, the gram-positive transposon Tn4001 was modified to serve as a mobile restriction site. The total sizes of the M. gallisepticum R and ATCC 19610 genomes were estimated to be 1,037 and 998 kb, respectively. The restriction enzyme locations for EagI and SmaI were determined along with several transposon insertion sites. The two strain maps were similar except for three small deletions and one additional EagI site in strain ATCC 19610.
Transformation of Actinomyces spp. by a gram-negative broad-host-range plasmid
Authors: Yeung M K , Kozelsky C S
Published: November 2016

The gram-negative broad-host-range vector pJRD215 was transferred by electroporation into strains of Actinomyces viscosus or Actinomyces naeslundii at efficiencies which ranged from 10(2) to 10(7) transformants per microgram of plasmid DNA. The Actinomyces transformants expressed pJRD215-encoded resistance to kanamycin and streptomycin. Moreover, the transforming plasmid DNA had not undergone any deletions or rearrangements, nor had it integrated into the genomes of these strains.
Molecular analysis of the F plasmid traVR region: traV encodes a lipoprotein
Authors: Doran T J , Loh S M , Firth N , Skurray R A
Published: November 2016

The nucleotide sequences of the conjugative F plasmid transfer region genes, traV and traR, have been determined. The deduced amino acid sequence of TraV indicated that it may be a lipoprotein; this was confirmed by examining the effect of globomycin on traV-encoded polypeptides synthesized in minicells. An open reading frame that may represent a previously undetected transfer gene, now designated trbG, was identified immediately upstream of traV. The deduced product of traR was found to share amino acid similarity with proteins from the bacteriophages 186 and P2 and with the dosage-dependent dnaK suppressor DksA.
Identification of flagellar synthesis regulatory and structural genes in a sigma D-dependent operon of Bacillus subtilis
Authors: Mirel D B , Lauer P , Chamberlin M J
Published: November 2016

The sigma D form of RNA polymerase from Bacillus subtilis has been shown previously to direct the synthesis of several transcription units bearing genes for flagellin, motility proteins, and autolysins. In this report, we describe an operon of genes transcribed from the sigma D-dependent promoter PD-1. We have identified three complete open reading frames and one partial one downstream of this promoter; immediately upstream is the previously identified comF locus. The PD-1 operon encodes the presumptive B. subtilis homologs of two Salmonella typhimurium late flagellar genes, flgM and flgK. Also present in this operon are two genes of unknown function, orf139 and orf160, whose products show similarities to the eukaryotic cytoskeletal proteins myosin and vimentin, respectively. orf139 and orf160 may encode proteins that form extended alpha-helical secondary structures and coiled-coil quaternary structures which may be filamentous components of the gram-positive bacterial flagellum. We have characterized the B. subtilis flgM gene further by constructing an in-frame deletion mutation, flgM delta 80, and creating strains of B. subtilis in which this allele has replaced the wild-type copy. By primer extension analysis of cellular RNA, we have shown that the flgM delta 80 mutation relieves the block to transcription of two other sigma D-dependent operons imposed by an unlinked mutation in a gene directing early flagellar synthesis. We conclude that, as in the case of S. typhimurium, early flagellar synthesis in B. subtilis is coupled to late flagellar synthesis through repression of sigma D-dependent transcription by the flgM gene product.
Interaction between the acceptor end of tRNA and the T box stimulates antitermination in the Bacillus subtilis tyrS gene: a new role for the discriminator base
Authors: Grundy F J , Rollins S M , Henkin T M
Published: November 2016

The Bacillus subtilis tyrS gene is a member of a group of gram-positive aminoacyl-tRNA synthetase and amino acid biosynthesis genes which are regulated by transcription antitermination. Each gene in the group is specifically induced by limitation for the appropriate amino acid. This response is mediated by interaction of the cognate tRNA with the mRNA leader region to promote formation of an antiterminator structure. The tRNA interacts with the leader by codon-anticodon pairing at a position designated the specifier sequence which is upstream of the antiterminator. In this study, an additional site of possible contact between the tRNA and the leader was identified through covariation of leader mRNA and tRNA sequences. Mutations in the acceptor end of tRNA(Tyr) could suppress mutations in the side bulge of the antiterminator, in a pattern consistent with base pairing. This base pairing may thereby directly affect the formation and/or function of the antiterminator. The discriminator position of the tRNA, an important identity determinant for a number of tRNAs, including tRNA(Tyr), was shown to act as a second specificity determinant for assuring response to the appropriate tRNA. Furthermore, overproduction of an unchargeable variant of tRNA(Tyr) resulted in antitermination in the absence of limitation for tyrosine, supporting the proposal that uncharged tRNA is the effector in this system.
Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA
Authors: Grundy F J , Turinsky A J , Henkin T M
Published: November 2016

The Bacillus subtilis acsA (acetyl coenzyme A synthetase) and acuABC (acetoin utilization) genes were previously identified in the region downstream from the ccpA gene, which encodes a protein required for catabolite repression of the amyE (alpha-amylase) gene. The acsA and acuABC genes are divergently transcribed, with only 20 bp separating the -35 sequences of their promoters. Expression of these genes was maximal in stationary phase and was repressed by the addition of glucose to the growth medium. Two sites resembling amyO, the cis-acting regulatory target site for amyE, were identified in the acsA and acuABC promoter regions. Glucose repression of acsA and acuABC transcription was dependent on both CcpA and the amyO-like sequences.
Characterization of the operon encoding the YpkA Ser/Thr protein kinase and the YopJ protein of Yersinia pseudotuberculosis
Authors: Galyov E E , Håkansson S , Wolf-Watz H
Published: November 2016

The Ser/Thr protein kinase YpkA, encoded by the virulence plasmid pIB1, is an indispensable virulence determinant of Yersinia pseudotuberculosis [E. E. Galyov, S. Håkansson, A. Forsberg, and H. Wolf-Watz, Nature (London) 361:730-732, 1993]. In this study, the organization of the ypkA-containing operon and the in vitro regulation of this transcriptional unit were characterized. The operon contains two structural genes, ypkA and yopJ, and is regulated by temperature and the extracellular concentration of Ca2+, as are the yop genes. The two proteins were secreted without posttranslational processing, showing that YpkA and YopJ belong to the Yop family. Mutational analysis revealed that, in contrast to all other Yop proteins so far studied, the YopJ protein was dispensable for virulence of Y. pseudotuberculosis.
Requirements for translocation of periplasmic domains in polytopic membrane proteins
Authors: Uhland K , Ehrle R , Zander T , Ehrmann M
Published: November 2016

Periplasmic domains of cytoplasmic membrane proteins require export signals for proper translocation. These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed the isolation of mislocalization mutants. In the original construct, alkaline phosphatase is fused to the second periplasmic domain of the membrane protein, and its activity is thus confined exclusively to the periplasm. Mutants that no longer translocated alkaline phosphatase were selected by complementation of a serB mutation. A total of 11 deletions in the amino terminus were isolated, all of which spanned at least the third transmembrane segment. This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused. Our results obtained in vivo support the model that amino-terminal membrane-spanning segments are required for translocation of large periplasmic domains. In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the secretion apparatus.
Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme
Authors: Ganguli S , Zapata G , Wallis T , Reid C , Boulnois G , Vann W F , Roberts I S
Published: November 2016

The gene encoding for the CMP-NeuNAc synthetase enzyme of Neisseria meningitidis group B was cloned by complementation of a mutant of Escherichia coli defective for this enzyme. The gene (neuA) was isolated on a 4.1-kb fragment of meningococcal chromosomal DNA. Determination of the nucleotide sequence of this fragment revealed the presence of three genes, termed neuA, neuB, and neuC, organized in a single operon. The presence of a truncated ctrA gene at one end of the cloned DNA and a truncated gene encoding for the meningococcal sialyltransferase at the other confirmed that the cloned DNA corresponded to region A and part of region C of the meningococcal capsule gene cluster. The predicted amino acid sequence of the meningococcal NeuA protein was 57% homologous to that of NeuA, the CMP-NeuNAc synthetase encoded by E. coli K1. The predicted molecular mass of meningococcal NeuA protein was 24.8 kDa, which was 6 kDa larger than that formerly predicted (U. Edwards and M. Frosch, FEMS Microbiol. Lett. 96:161-166, 1992). Purification of the recombinant meningococcal NeuA protein together with determination of the N-terminal amino acid sequence confirmed that this 24.8-kDa protein was indeed the meningococcal CMP-NeuNAc synthetase. The predicted amino acid sequences of the two other encoded proteins were homologous to those of the NeuC and NeuB proteins of E. coli K1, two proteins involved in the synthesis of NeuNAc. These results indicate that common steps exist in the biosynthesis of NeuNAc in these two microorganisms.
Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp
Authors: Trust T J , Logan S M , Gustafson C E , Romaniuk P J , Kim N W , Chan V L , Ragan M A , Guerry P , Gutell R R
Published: November 2016

The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria.
Mutation of the putative nucleotide binding site of the Bacillus subtilis membrane protein ComFA abolishes the uptake of DNA during transformation
Authors: Londoño-Vallejo J A , Dubnau D
Published: November 2016

ComFA is a membrane protein required for the uptake of transforming DNA following its binding to the Bacillus subtilis competent-cell surface. ComFA, which resembles members of the DEAD family of ATP-driven helicases, contains sequences similar to those found in many ATP-binding proteins and thought to represent the ATP-binding sites of these proteins. We have suggested that ComFA may function as a DNA translocase and/or helicase, using the energy of ATP hydrolysis to mediate the uptake of DNA. As a partial test of this hypothesis, we have introduced mutations into highly conserved glycyl and lysyl residues of the putative ATP-binding site, located, respectively, at positions 151 and 152, and determined the effects of these alterations on in vivo function. A substitution of the conserved lysyl by a glutamyl residue (K152E) and a double G151R-K152N mutation each resulted in a nearly 1,000-fold decrease in transformability, equivalent to that observed in a ComFA null mutant. A K152N mutation caused a partial loss-of-function phenotype. These effects were manifested at the level of DNA uptake; no marked effects on the final levels of DNA binding were noted. When either the K152E mutant allele or the G151R-K152N double mutant allele was combined in single copy with wild-type comFA, a dominant negative phenotype expressed on the level of DNA uptake was observed, suggesting that ComFA acts in a complex with other proteins, with additional molecules of ComFA, or with both.
The DNA replication fork blocked at the Ter site may be an entrance for the RecBCD enzyme into duplex DNA
Authors: Horiuchi T , Fujimura Y , Nishitani H , Kobayashi T , Hidaka M
Published: November 2016

In Escherichia coli, eight kinds of chromosome-derived DNA fragments (named Hot DNA) were found to exhibit homologous recombinational hotspot activity, with the following properties. (i) The Hot activities of all Hot DNAs were enhanced extensively under RNase H-defective (rnh) conditions. (ii) Seven Hot DNAs were clustered at the DNA replication terminus region on the E. coli chromosome and had Chi activities (H. Nishitani, M. Hidaka, and T. Horiuchi, Mol. Gen. Genet. 240:307-314, 1993). Hot activities of HotA, -B, and -C, the locations of which were close to three DNA replication terminus sites, the TerB, -A, and -C sites, respectively, disappeared when terminus-binding (Tau or Tus) protein was defective, thereby suggesting that their Hot activities are termination event dependent. Other Hot groups showed termination-independent Hot activities. In addition, at least HotA activity proved to be dependent on a Chi sequence, because mutational destruction of the Chi sequence on the HotA DNA fragment resulted in disappearance of the HotA activity. The HotA activity which had disappeared was reactivated by insertion of a new, properly oriented Chi sequence at the position between the HotA DNA and the TerB site. On the basis of these observations and positional and orientational relationships between the Chi and the Ter sequences, we propose a model in which the DNA replication fork blocked at the Ter site provides an entrance for the RecBCD enzyme into duplex DNA.
Why does Escherichia coli have two primary pathways for synthesis of glutamate?
Authors: Helling R B
Published: November 2016

Escherichia coli has two primary pathways for glutamate synthetase-glutamate synthase pathway is known to be essential for synthesis at low ammonium concentrations and for regulation of the glutamine pool, but the necessity for glutamate dehydrogenase (GDH) has been uncertain. The results of competition experiments between the wild type and a GDH-deficient mutant during nutrient-limited growth and of direct enzyme measurements suggest that GDH is used in glutamate synthesis when the cell is limited for energy (and carbon) but ammonium and phosphate are present in excess, while the glutamine synthetase-glutamate synthase pathway is used when the cell is not under energy limitation. The use of alternative routes for glutamate synthesis implies that the energy cost of biosynthesis may be less when energy is limited than when energy is unlimited.
The UGA4 UASNTR site required for GLN3-dependent transcriptional activation also mediates DAL80-responsive regulation and DAL80 protein binding in Saccharomyces cerevisiae
Authors: Cunningham T S , Dorrington R A , Cooper T G
Published: November 2016

Expression of the nitrogen catabolic genes in Saccharomyces cerevisiae, including those of the gamma-aminobutyric acid (UGA) and allantoin (DAL) pathways, is regulated positively by the GLN3 protein and negatively by the DAL80 protein. The deduced sequences of the DAL80 and GLN3 proteins contain a zinc finger motif homologous to those shown to bind GATA sequences. In addition, DAL80 protein has been directly shown to bind to a pair of GATA-containing sequences (URSGATA) in vitro, and a pair of GATA-containing sequences (UASNTR) is required for GLN3-dependent transcriptional activation in a heterologous expression vector. We demonstrate here that the GATA-containing sites upstream of UGA4 required for optimal GLN3-dependent transcriptional activation also mediate DAL80 protein binding in vitro and DAL80-responsive regulation in vivo.
The yeast Saccharomyces kluyveri as a recipient eukaryote in transkingdom conjugation: behavior of transmitted plasmids in transconjugants
Authors: Inomata K , Nishikawa M , Yoshida K
Published: November 2016

The prokaryote Escherichia coli successfully conjugated with the eukaryote Saccharomyces kluyveri, which is relatively distant from the species S. cerevisiae. To achieve this transkingdom conjugation, we constructed three types of conjugative plasmids, namely integrative, replicative, and centromere vectors, for S. cerevisiae. By transfer of any of the three plasmids from E. coli, an S. kluyveri Ura- mutant was converted to the Ura+ phenotype. This phenotype was easily lost under nonselective conditions. Southern analysis of the transconjugants clearly indicated the presence of the plasmids in many different structures and sizes.
A strand-specific endonucleolytic activity with DNA site preference for cleavage in Chlamydia trachomatis
Authors: Mathews S A , Sriprakash K S
Published: November 2016

Endonucleolytic activity was detected in high-salt extracts of Chlamydia trachomatis cell lysates. This nucleolytic activity showed specificity for one strand of a PCR fragment containing the strong chlamydial promoter that regulates synthesis of two short plasmid-specified RNAs. Strand specificity was also observed with supercoiled DNA containing the same region; once again, the same strand was cleaved. The sensitive strand is the nontemplate strand for two short transcripts which initiate at the same nucleotide. Alignment of sequences on either side of the nick sites revealed a consensus which may be required for recognition of the enzymatic activity.
Osmoadaptation in rhizobia: ectoine-induced salt tolerance
Authors: Talibart R , Jebbar M , Gouesbet G , Himdi-Kabbab S , Wróblewski H , Blanco C , Bernard T
Published: November 2016

After having shown that ectoine (a tetrahydropyrimidine) displays osmoprotective properties towards Escherichia coli (M. Jebbar, R. Talibart, K. Gloux, T. Bernard, and Blanco, J. Bacteriol. 174:5027-5035, 1992), we have investigated the involvement of this molecule in the osmotic adaptation of Rhizobium meliloti. Ectoine appeared almost as effective as glycine betaine in improving the growth of R. meliloti under adverse osmotic conditions (0.5 M NaCl). Moreover, improvement of growth of rhizobial strains insensitive to glycine betaine was also observed. Ectoine transport proved inducible, periplasmic protein dependent, and, as shown by competition experiments, distinct from the transport of glycine betaine. Medium osmolarity had little effect on the uptake characteristics, since the rate of influx increased from 12 to only 20 nmol min-1 mg of protein-1 when NaCl concentrations were raised from 0 to 0.3 or 0.5 M, with a constant of transport of 80 microM. Natural-abundance 13C-nuclear magnetic resonance and radiolabelling assays showed that ectoine, unlike glycine betaine, is not intracellularly accumulated and, as a consequence, does not repress the synthesis of endogenous compatible solutes (glutamate, N-acetylglutaminylglutamine amide, and trehalose). Furthermore, the strong rise in glutamate content in cells osmotically stressed in the presence of ectoine suggests that, instead of being involved in osmotic balance restoration, ectoine should play a key role in triggering the synthesis of endogenous osmolytes. Hence, we believe that there are at least two distinct classes of osmoprotectants: those such as glycine betaine or glutamate, which act as genuine osmolytes, and those such as ectoine, which act as chemical mediators.
Identification of the lipopolysaccharide core region as the receptor site for a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa
Authors: Yokota S , Hayashi T , Matsumoto H
Published: November 2016

A temperate phage, phi CTX, is a cytotoxin-converting phage of Pseudomonas aeruginosa. In this study, we characterized the lipopolysaccharide (LPS) structures of phi CTX-resistant mutants derived from phi CTX-sensitive strains. phi CTX infectivity was neutralized by LPS preparations derived from sensitive strains but not by those from resistant strains. phi CTX-resistant mutants had lower-molecular-weight rough (R)-type LPS than the parental strains and lacked the reactivity of some anti-LPS core monoclonal antibodies. Some LPS core components were lacking or significantly decreased in the resistant mutants. These results suggested that a receptor site of the cytotoxin-converting phage phi CTX was the LPS core region and that especially L-rhamnose and D-glucose residues in the outer core were involved in phage binding. The host range of phi CTX was nearly O-serotype dependent, probably because of the diversity of the LPS core structure among P. aeruginosa strains. phi CTX bound to most strains of Homma serotypes A, G, and I but not to strains of serotypes B and E. Furthermore, we found that a genetic locus specifying phi CTX sensitivity (and consequently participating in the biosynthesis of part of the LPS core) existed in or near the locus participating in the determination of O-serotype specificity (somA), which has been mapped between leu-10 and eda-9001. phi CTX, as well as anti-LPS core monoclonal antibodies, will be a good tool for structural characterization of the P. aeruginosa LPS core region.
A third genetic locus required for the formation of heterocysts in Anabaena sp. strain PCC 7120
Authors: Fernández-Piñas F , Leganés F , Wolk C P
Published: November 2016

Mutagenesis of Anabaena sp. strain PCC 7120 with a derivative of transposon Tn5 led to the isolation of a mutant strain, P6, in which heterocysts are not formed (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation of P6 in the wild-type strain reproduced the phenotype of the original mutant. Analysis by pulsed-field gel electrophoresis localized the transposition at ca. 3.44 Mb on the physical map of the chromosome of wild-type Anabaena sp. The transposon was situated within an open reading frame (ORF), which we denote hetP, whose wild-type form was cloned and also sequenced. The predicted HetP protein was not found to show significant sequence similarity to other proteins. The mutation in strain P6 could be complemented by a clone of a fragment of wild-type DNA that includes hetP and at least one additional ORF 3' from hetP, but not by a clone that includes hetP as its only ORF. The latter clone proved highly toxic. The phenotype of the P6 mutant may, therefore, be due to a polar effect of the insertion of the transposon. Filaments of strain P6 and of the wild-type strain, when bearing the complementing fragment on a pDU1-based plasmid, showed an increased frequency of clustered heterocysts compared with that of the wild-type strain.
Isolation of a novel IS3 group insertion element and construction of an integration vector for Lactobacillus spp
Authors: Walker D C , Klaenhammer T R
Published: November 2016

An insertion sequence (IS) element from Lactobacillus johnsonii was isolated, characterized, and exploited to construct an IS-based integration vector. L. johnsonii NCK61, a high-frequency conjugal donor of bacteriocin production (Laf+) and immunity (Lafr), was transformed to erythromycin resistance (Emr) with the shuttle vector pSA3. The NCK61 conjugative functions were used to mobilize pSA3 into a Laf- Lafs EMs recipient. DNA from the Emr transconjugants transformed into Escherichia coli MC1061 yielded a resolution plasmid with the same size as that of pSA3 with a 1.5-kb insertion. The gram-positive replication region of the resolution plasmid was removed to generate a pSA3-based suicide vector (pTRK327) bearing the 1.5-kb insert of Lactobacillus origin. Plasmid pTRK327 inserted randomly into the chromosomes of both Lactobacillus gasseri ATCC 33323 and VPI 11759. No homology was detected between plasmid and total host DNAs, suggesting a Rec-independent insertion. The DNA sequence of the 1.5-kb region revealed the characteristics of an IS element (designated IS1223): a length of 1,492 bp; flanking, 25-bp, imperfect inverted repeats; and two overlapping open reading frames (ORFs). Sequence comparisons revealed 71.1% similarity, including 35.7% identity, between the deduced ORFB protein of the E. coli IS element IS150 and the putative ORFB protein encoded by the Lactobacillus IS element. A putative frameshift site was detected between the overlapping ORFs of the Lactobacillus IS element. It is proposed that, similar to IS150, IS1223 produces an active transposase via translational frameshifting between two tandem, overlapping ORFs.
Secretion of the Serratia marcescens HasA protein by an ABC transporter
Authors: Létoffé S , Ghigo J M , Wandersman C
Published: November 2016

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.
Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli
Authors: Manna D , Gowrishankar J
Published: November 2016

Transcription of the proU operon of Escherichia coli is induced several hundred-fold upon growth at elevated osmolarity, but the underlying mechanisms are incompletely understood. Three cis elements appear to act additively to mediate proU osmoresponsivity: (i) sequences around a promoter, P1, which is situated 250 bp upstream of the first structural gene proV; (ii) sequences around another (sigma 70-dependent) promoter, P2, which is situated 60 bp upstream of proV; and (iii) a negative regulatory element present within the proV coding region. These three cis elements are designated, respectively, P1R, P2R, and NRE. trans-acting mutants with partially derepressed proU expression have been obtained earlier, and a vast majority of the mutations affect the gene encoding the nucleoid protein HNS. In this study we employed a selection for trans-acting mutants with reduced proU+ expression, and we obtained a derivative that had suffered mutations in two separate loci designated dpeA and dpeB. The dpeB mutation caused a marked reduction in promoter P1 expression and was allelic to rpoS, the structural gene for the stationary-phase-specific sigma factor of RNA polymerase. Expression from P1 was markedly induced, in an RpoS-dependent manner, in stationary-phase cultures. In contrast to the behavior of the isolated P1 promoter, transcription from a construct carrying the entire proU cis-regulatory region (P1R plus P2R plus NRE) was not significantly affected by either growth phase or RpoS. The dpeA locus was allelic to hupB, which along with hupA encodes the nucleoid protein HU. hupA hupB double mutants exhibited a pronounced reduction in proU osmotic inducibility. HU appears to affect proU regulation through the P2R mechanism, whereas the effect of HNS is mediated through the NRE.
Regulation of rns, a positive regulatory factor for pili of enterotoxigenic Escherichia coli
Authors: Froehlich B , Husmann L , Caron J , Scott J R
Published: November 2016

Attachment of enterotoxigenic Escherichia coli to the human gut is considered an important early step in infection that leads to diarrhea. This attachment is mediated by pili, which belong to a limited number of serologically distinguishable types. Many of these pili require the product of rns, or a closely related gene, for their expression. We have located the major promoter for rns and found that although its sequence diverges significantly from a sigma-70 promoter consensus sequence, it is very strong. Transcription of rns is negatively regulated both at a region upstream of this promoter and at a region internal to the rns open reading frame. In addition, rns positively regulates its own transcription, probably by counteracting these two negative effects.
Identification of a novel gene, aut, involved in autotrophic growth of Alcaligenes eutrophus
Authors: Freter A , Bowien B
Published: November 2016

The aerobic facultative chemoautotroph Alcaligenes eutrophus was found to possess a novel gene, designated aut, required for both lithoautotrophic (hydrogen plus carbon dioxide) and organoautotrophic (formate) growth (Aut+ phenotype). Insertional mutagenesis by transposon Tn5-Mob localized the gene on a chromosomal 13-kbp EcoRI fragment. Physiological characterization of various Aut- mutants revealed pleiotropic effects caused by the transposon insertion. Heterotrophic growth of the mutants on substrates catabolized via the glycolytic pathway was slower than that of the parent strains, and the colony morphology of the mutants was altered when grown on nutrient agar. The heterotrophic derepression of the cbb operons encoding Calvin cycle enzymes was abolished, although their expression was still inducible in the presence of formate. Apparently, the mutation did not affect the cbb genes directly but impaired the autotrophic growth in a more general manner. The conjugally transferred wild-type EcoRI fragment allowed phenotypic in trans complementation of the mutants. Further subcloning and sequencing identified a single open reading frame (aut) of 495 bp that was sufficient for complementation. The monocistronic aut gene was constitutively transcribed into a 0.65-kb mRNA. However, its expression appeared to be low. Heterologous expression of aut was achieved in Escherichia coli, resulting in overproduction of an 18-kDa protein. Database searches yielded weak partial sequence similarities of the deduced Aut protein sequence to some cytidylyltransferases, but no indication for the exact function of the aut gene was obtained. Hybridizing DNA sequences that might be similar to the aut gene were detected by Southern hybridization in the genome of two other autotrophic bacteria.
Rhizobia catabolize nod gene-inducing flavonoids via C-ring fission mechanisms
Authors: Rao J R , Cooper J E
Published: November 2016

Gas chromatographic and mass spectrometric analyses of derivatized culture medium extracts were used to identify the products of flavonoid metabolism by rhizobia. A number of Rhizobium species and biovars degraded their nod gene-inducing flavonoids by mechanisms which originated in a cleavage of the C-ring of the molecule and which yielded conserved A- and B-ring products among the metabolites. In contrast, Pseudomonas putida degraded quercetin via an initial fission in its A-ring, and Agrobacterium tumefaciens displayed a nonspecific mode of flavonoid degradation which yielded no conserved A- or B-ring products. When incubated with rhizobia, flavonoids with OH substitutions at the 5 and 7 positions yielded phloroglucinol as the conserved A-ring product, and those with a single OH substitution at the 7 position yielded resorcinol. A wider range of structures was found among the B-ring derivatives, including p-coumaric, p-hydroxybenzoic, protocatechuic, phenylacetic, and caffeic acids. The isoflavonoids genistein and daidzein were also degraded via C-ring fission by Rhizobium fredii and Rhizobium sp. strain NGR234, respectively. Partially characterized aromatic metabolites with potential nod gene-inducing activity were detected among the products of naringenin degradation by Rhizobium leguminosarum bv. viciae. The initial structural modification of nod gene-inducing flavonoids by rhizobia can generate chalcones, whose open C-ring system may have implications for the binding of inducers to the nodD gene product.