bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and partial characterization of the Rhizobium leguminosarum biovar viciae Ca2+-dependent adhesin, which mediates the first step in attachment of cells of the family Rhizobiaceae to plant root hair tips
Authors: Smit G , Logman T J , Boerrigter M E , Kijne J W , Lugtenberg B J
Published: November 2016

The Ca2+-dependent adhesin which mediates the first step in attachment of bacteria of the family Rhizobiaceae to plant root hair tips was isolated from the surface of Rhizobium leguminosarum biovar viciae cells; its ability to inhibit attachment of R. leguminosarum to pea root hair tips was used as a bioassay. Isolated adhesin was found to be able to inhibit attachment of both carbon-limited and manganese-limited R. leguminosarum cells. A multicolumn purification procedure was developed which resulted in pure adhesin, as judged from silver staining of isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electropherograms. The crucial step in purification was the elution of rhizobial proteins by a CaCl2 gradient from a hydroxyapatite matrix. The specific activity increased 1,250 times during purification. The isoelectric point of the adhesin was determined to be 5.1, and the molecular mass was 14 kilodaltons (kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using gel filtration in the presence and absence of Ca2+, the molecular mass of the adhesin was determined to be 15 and 6 kDa, respectively. The adhesin appeared to be a calcium-binding protein. The purified adhesin inhibited attachment of various other rhizobia to pea root hair tips. Also, cell surface preparations of several other rhizobial strains, including Agrobacterium, Bradyrhizobium, and Phyllobacterium spp., showed adhesin activity, suggesting that a common plant receptor is used for attachment of Rhizobiaceae cells and that the adhesin is common among Rhizobiaceae. No attachment-inhibiting activity was detected in cell surface preparations from various other bacterial strains tested. Cell surface preparations from Sym or Ti plasmid-cured Rhizobium and Agrobacterium strains, respectively, also showed adhesin activity, indicating that Sym or Ti plasmid-borne genes are not required for the synthesis and biogenesis of the adhesin. The adhesin was also found to be involved in the attachment of rhizobia to the root hairs of various other legumes and nonlegume plants, including monocotyledonous ones. Since the adhesin appears to be specific for Rhizobiaceae and is Ca2+ dependent, we propose to designate it rhicadhesin. A more detailed model for rhizobial attachment to plant root hairs is discussed.
Expression of a cell surface antigen from Rhizobium leguminosarum 3841 is regulated by oxygen and pH
Authors: Kannenberg E L , Brewin N J
Published: November 2016

Rhizobium leguminosarum bv. viciae 3841 was grown in liquid suspension culture to investigate how culture conditions could affect the expression of a developmentally regulated cell surface antigen associated with lipopolysaccharide. The antigen, which is recognized by monoclonal antibody AFRC MAC 203, was expressed when cultures were grown at neutral pH under low-oxygen conditions (less than 7.5% [vol/vol] O2 in the gas phase). Antigen was also expressed in aerobically grown cultures at pH values below 5.3. The nature of the nitrogen and the carbon sources had no effect on antigen expression except by indirect changes on the pH of the culture medium; similarly, growth in 0.3 M NaCl did not result in antigen expression. The induction of MAC 203 antigen by low-oxygen or low-pH culture conditions is discussed in the context of tissue-specific expression within the legume root nodule.
A methyl-accepting protein is involved in benzoate taxis in Pseudomonas putida
Authors: Harwood C S
Published: November 2016

Pseudomonas putida is attracted to at least two groups of aromatic acids: a benzoate group and a benzoylformate group. Members of the benzoate group of chemoattractants stimulated the methylation of a P. putida polypeptide with an apparent molecular weight of 60,000 in sodium dodecyl sulfate-polyacrylamide gels. This polypeptide is presumed to be a methyl-accepting chemotaxis protein for several reasons: its molecular weight is similar to the molecular weights of Escherichia coli methyl-accepting chemotaxis proteins, the amount of time required to attain maximal methylation correlated with the time needed for behavioral adaptation of P. putida cells to benzoate, and methylation was stimulated by benzoate only in cells induced for chemotaxis to benzoate. Also, a mutant specifically defective in benzoate taxis failed to show any stimulation of methylation upon addition of benzoate. Benzoylformate did not stimulate protein methylation in cells induced for benzoylformate chemotaxis, suggesting that sensory input from this second group of aromatic-acid attractants is processed through a different kind of chemosensory pathway. The chemotactic responses of P. putida cells to benzoate and benzoylformate were not sensitive to external pH over a range (6.2 to 7.7) which would vary the protonated forms of these weak acids by a factor of about 30. This indicates that detection of cytoplasmic pH is not the basis for aromatic-acid taxis in P. putida.
New method for generating deletions and gene replacements in Escherichia coli
Authors: Hamilton C M , Aldea M , Washburn B K , Babitzke P , Kushner S R
Published: November 2016

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C. Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.
The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha
Authors: Leung K Y , Straley S C
Published: November 2016

In Yersinia pestis KIM, there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only Yops M and N are found in easily detectable amounts in the culture medium. In this study, we located and characterized the yopM gene to obtain clues about its role in the virulence of Y. pestis. Rabbit antibody was raised against Yops M and H, copurified from the supernatant of Y. pseudotuberculosis 43(pGW600, pCD1 yopE::Mu dI1[Apr lac]). This antiserum was adsorbed with an Escherichia coli clone that strongly expressed YopH. The resulting YopM-specific antibody was used to screen a HindIII library of pCD1. HindIII-F and several subclones from it expressed YopM in E. coli minicells. A DNA fragment of 1.39 kilobases from HindIII-F was sequenced and found to contain a 367-amino-acid open reading frame capable of encoding a protein with molecular mass (41,566 daltons) and isoelectric point (4.06) similar to those of YopM. The +1 site of the yopM gene was determined by primer extension. The DNA sequence contained repeating structures: 11 pairs of exact direct repeats, two exact inverted repeats, and three palindromes, ranging from 10 to 42 bases in size. One consensus 14-amino-acid sequence was repeated six times in the predicted protein sequence. The YopM sequence shares some significant homology with the von Willebrand factor- and thrombin-binding domain of the alpha chain of human platelet membrane glycoprotein Ib. These findings suggested a testable hypothesis for the function of YopM.
Purification and properties of Myxococcus xanthus cell surface antigen 1604
Authors: Jarvis B W , Dworkin M
Published: November 2016

A cell surface antigen complex from Zwittergent-solubilized Myxococcus xanthus has been purified by immunoaffinity chromatography with monoclonal antibody (MAb) 1604 and by subsequent gel filtration. We propose that the cell surface antigen (CSA) 1604 complex participates in intercellular interactions. The apparent total molecular mass of the CSA 1604 complex is 200 kilodaltons (kDa), as determined by gel filtration and by electrophoresis and Western immunoblot probing with MAb 1604. The antigen epitope recognized by MAb 1604 is on a 51-kDa polypeptide. The CSA complex also contains 14% neutral carbohydrate and a 23-kDa polypeptide that lacks the 1604 epitope. The carbohydrate is most likely part of a lipopolysaccharide (LPS) associated with the CSA, because an MAb recognizing an O antigen epitope from the LPS of M. xanthus also reacted with CSA 1604 on Western immunoblots. Thus, the 200-kDa CSA complex consists of 97 +/- 6 kDa of protein and many associated LPS molecules. The LPS evidently produces the multiplicity of bands observed on Western immunoblots between 100 and 200 kDa. The association with LPS may contribute to the negative charge of the CSA 1604 complex, which has a pI of 4.3. The CSA was clustered on the surface of intact M. xanthus cells after labeling with MAb 1604 and immunogold. Furthermore, fractionation studies indicated that cells grown on a plastic surface had 50% of their total CSA 1604 in the cytosol, 39% in the membrane fraction, and 8% in the periplasm. Saturable binding studies with 125I-MAb 1604 indicated that there were 2,400 CSA 1604 sites per cell. The Kd for MAb 1604 binding to the cell was 9 nM.
Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci
Authors: Hmiel S P , Snavely M D , Florer J B , Maguire M E , Miller C G
Published: November 2016

Salmonella typhimurium strains lacking the CorA Mg2+ transport system retain Mg2+ transport and the ability to grow in medium containing a low concentration of Mg2+. Mutagenesis of a corA strain followed by ampicillin selection allowed isolation of a strain that required Mg2+-supplemented media for growth. This strain contained mutations in at least two loci in addition to corA, designated mgtA and mgtB (for magnesium transport). Strains with mutations at all three loci (corA, mgtA, and mgtB) exhibited no detectable Mg2+ uptake and required 10 mM Mg2+ in the medium for growth at the wild-type rate. A wild-type allele at any one of the three loci was sufficient to restore both Mg2+ transport and growth on 50 microM Mg2+. P22 transduction was used to map the mgt loci. The mgtA mutation was located to approximately 98 map units (cotransducible with pyrB), and mgtB mapped at about 80.5 map units (near gltC). A chromosomal library from S. typhimurium was screened for clones that complemented the Mg2+ requirement of a corA mgtA mgtB mutant. The three classes of plasmids obtained could each independently restore Mg2+ transport to this strain and corresponded to the corA, mgtA, and mgtB loci. Whereas the corA locus of S. typhimurium is analogous to the corA locus previously described for Escherichia coli, neither of the mgt loci described in this report appears analogous to the single mgt locus described in E. coli. Our data in this and the accompanying papers (M. D. Snavely, J. B. Florer, C. G. Miller, and M. E. Maguire, J. Bacteriol. 171:4752-4760, 4761-4766, 1989) indicate that the corA, mgtA, and mgtB loci of S. typhimurium represent three distinct systems that transport Mg2+.
Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg2+ transport systems
Authors: Snavely M D , Florer J B , Miller C G , Maguire M E
Published: November 2016

In Salmonella typhimurium, the corA, mgtA, and mgtB loci are involved in active transport of Mg2+ (S. P. Hmiel, M. D. Snavely, C. G. Miller, and M. E. Maguire, J. Bacteriol. 168:1444-1450, 1988; S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). In this study, the gene products coded for by the corA, mgtA, and mgtB genes were identified by using plasmid expression in Escherichia coli maxicells. Complementation was assessed by introducing plasmids into a Mg2+-dependent corA mgtA mgtB strain and determining the ability of the plasmid to restore growth on medium without a Mg2+ supplement. Complementing plasmids containing corA expressed a 42-kilodalton (kDa) protein. This protein was not expressed by plasmids containing insertions or deletions that eliminated complementation. A plasmid containing mgtA expressed 37- and 91-kDa gene products. Data obtained with subclones and insertions in this plasmid indicated that plasmids expressing only the 91-kDa polypeptide complemented; plasmids that did not express this protein did not complement regardless of whether they expressed the 37-kDa protein. Plasmids carrying mgtB expressed a single protein of 102 kDa whose presence or absence correlated with the ability of the plasmid to complement the Mg2+-dependent triple mutant. Fractionation of labeled maxicells demonstrated that the 42-kDa corA, the 91-kDa mgtA, and the 102-kDa mgtB gene products are all tightly associated with the membrane, a location consistent with involvement in a transport process. These data provide further support the for existence of three distinct systems for Mg2+ transport in S. typhimurium.
Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12
Authors: Roa B B , Connolly D M , Winkler M E
Published: November 2016

We report that pdxA, which is required for de novo biosynthesis of pyridoxine (vitamin B6) and pyridoxal phosphate, belongs to an unusual, multifunctional operon. The pdxA gene was cloned in the same 3.5-kilobase BamHI-EcoRI restriction fragment that contains ksgA, which encodes the 16S rRNA modification enzyme m6(2)A methyltransferase, and apaH, which encodes diadenosine tetraphosphatase (ApppA hydrolase). Previously, Blanchin-Roland et al. showed that ksgA and apaH form a complex operon (Mol. Gen. Genet. 205:515-522, 1986). The pdxA gene was located on recombinant plasmids by subcloning, complementation, and insertion mutagenesis, and chromosomal insertions at five positions upstream from ksgA inactivated pdxA function. DNA sequence analysis and minicell translation experiments demonstrated that pdxA encoded a 35.1-kilodalton polypeptide and that the stop codon of pdxA overlapped the start codon of ksgA by 2 nucleotides. The translational start codon of pdxA was tentatively assigned based on polypeptide size and on the presence of a unique sequence that was also found near the translational start of PdxB. This conserved sequence may play a role in translational control of certain pyridoxine biosynthetic genes. RNase T2 mapping of chromosomal transcripts confirmed that pdxA and ksgA were members of the same complex operon, yet about half of ksgA transcripts arose in vivo under some culture conditions from an internal promoter mapped near the end of pdxA. Transcript analysis further suggested that pdxA is not the first gene in the operon. These structural features support the idea that pyridoxine-biosynthetic genes are members of complex operons, perhaps to interweave coenzyme biosynthesis genetically with other metabolic processes. The results are also considered in terms of ksgA expression.
Cell-cycle-dependent polar morphogenesis in Caulobacter crescentus: roles of phospholipid, DNA, and protein syntheses
Authors: O'Neill E A , Bender R A
Published: November 2016

During swarmer cell differentiation in Caulobacter crescentus, morphogenesis at the swarmer pole is characterized by the loss of the flagellum, by the loss of phage receptor activity (PRA) (the ability of the cell to adsorb phage phi CbK), and finally by the initiation of stalk outgrowth at the site formerly occupied by the flagellum and the PRA. We show here that each of these events is a cell cycle-dependent event requiring continuous protein synthesis for its execution but occurring normally in the absence of DNA synthesis or phospholipid synthesis. During stalked-cell differentiation, the flagellum and PRA reappear and the stalk elongates considerably. We show here that these events are also cell cycle dependent, requiring not only de novo protein synthesis but also DNA and phospholipid syntheses. When synchronous cells dividing 160 min after collection were used, PRA reappearance occurred at 110 min. This PRA reappearance was dependent on a phospholipid synthesis-requiring event occurring at 70 min, a DNA synthesis-requiring event occurring at 95 min, and a protein synthesis-requiring event occurring at 108 min. In the absence of net phospholipid synthesis, stalk elongation appeared more or less normal, but the stalks eventually became fragile, and by 240 min, most of the stalks had broken off, leaving only stubs attached to the cell body.
Staphylococcus aureus chromosomal mutation plaC1 amplifies plasmid pT181 by depressing synthesis of its negative-effector countertranscripts
Authors: Iordanescu S
Published: November 2016

A Staphylococcus aureus chromosomal mutation, plaC1, which leads specifically to the amplification of plasmid pT181 has previously been described (S. Iordanescu, Plasmid 10:130-137, 1983). The mechanism by which plaC1 amplifies plasmid pT181 has been approached in two ways: determination of the plasmid region required for the specific response to the plaC1 mutation and evaluation of different parameters of pT181 replication control by using transcriptional and translational fusions with the beta-lactamase gene as an indicator gene. The results obtained indicate that the control region of plasmid pT181 represents the target of the plaC1 effect, which acts primarily by depressing the synthesis of plasmid pT181 countertranscripts, those small, untranslated RNA molecules playing the roles of negative effectors in the replication control mechanism of the plasmid. In turn, the reduction in countertranscript synthesis leads to an increase in the production of the initiator protein RepC, which is limiting for plasmid replication, and a higher plasmid copy number.
MalI, a novel protein involved in regulation of the maltose system of Escherichia coli, is highly homologous to the repressor proteins GalR, CytR, and LacI
Authors: Reidl J , Römisch K , Ehrmann M , Boos W
Published: November 2016

The maltose regulon of Escherichia coli comprises several operons that are under common regulatory control of the MalT activator protein. Five mal genes, organized in two divergent operons, code for a binding-protein-dependent transport system specific for maltose and maltodextrins. MalK, one of the subunits of this transport system, not only is essential for transport but also plays a role in regulation. Mutations abolishing MalK function not only result in inability to transport maltose but also cause constitutive expression of the maltose regulon. For this constitutivity to be exerted, the function of an additional gene product, MalI, is necessary. Using the constitutive expression of a malK-lacZ fusion as a signal, we cloned the malI gene, expressed it in minicells, and determined its DNA sequence. The sequence predicted a protein of 34,729 molecular weight, in agreement with the apparent molecular weight of the protein (35,000) when expressed in minicells and analyzed by polyacrylamide gel electrophoresis and autoradiography. MalI exhibited high homology to the repressor proteins GalR, CytR, and LacI. When the amino acid sequences were appropriately aligned, MalI showed 28% identity to GalR, 21% to CytR, and 24% to LacI. Including conservative amino acid exchanges, these numbers increased to 69, 56, and 58%, respectively. The regions of high homology were clustered in particular at the N-terminal portion of the protein that includes the helix-turn-helix motif thought to be involved in DNA binding. The protein contained a short stretch of 30 amino acids that was surprisingly homologous to a sequence in MalT. The amino-terminal half of the protein exhibited significant homology with MalK. The transcriptional start of malI was determined by reverse transcriptase and by S1 nuclease mapping. We found a possible binding site for cyclic AMP receptor protein in the promoter region of malI as well as two perfect direct repeats of 14 base pairs with twofold symmetry indicating their possible role as operator sites. Upstream to malI we observed a divergent open reading frame that extended to the end of the sequenced DNA.
Transformation of the archaebacterium Halobacterium volcanii with genomic DNA
Authors: Cline S W , Schalkwyk L C , Doolittle W F
Published: November 2016

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.
Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli
Authors: Conley M P , Wolfe A J , Blair D F , Berg H C
Published: November 2016

If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.
L-arabinose metabolism in Herbaspirillum seropedicae
Authors: Mathias A L , Rigo L U , Funayama S , Pedrosa F O
Published: November 2016

The pathway for L-arabinose metabolism in Herbaspirillum seropedicae was shown to involve nonphosphorylated intermediates and to produce alpha-ketoglutarate. The activities of the enzymes and the natures of several intermediates were determined. The pathway was inducible by L-arabinose, and two key enzymes, L-arabinose dehydrogenase and 2-keto-glutarate semialdehyde dehydrogenase, were present in all strains of H. seropedicae tested.
A procaryotic regulatory factor with a histone H1-like carboxy-terminal domain: clonal variation of repeats within algP, a gene involved in regulation of mucoidy in Pseudomonas aeruginosa
Authors: Deretic V , Konyecsni W M
Published: November 2016

A novel procaryotic transcriptional regulatory element, AlgP, with a histone H1-like carboxy-terminal domain was identified in Pseudomonas aeruginosa. AlgP is required for transcription of the key biosynthetic gene algD, which is necessary for production of the exopolysaccharide alginate causing mucoidy in P. aeruginosa. Mucoidy is a critical virulence determinant of P. aeruginosa invariably associated with the respiratory infections causing high mortality in cystic fibrosis. Here we show that AlgP and histones H1 both have repeated units of the Lys-Pro-Ala-Ala motif (KPAA) and its variations within their long (over 100 amino acids) carboxy-terminal domains. This region of histone H1 tails has been shown to bind to the linker DNA in eucaryotic chromatin fibers. A synthetic 50-mer peptide consisting of repeats from the AlgP carboxy-terminal domain was found to bind DNA in a mobility shift DNA-binding assay. AlgP is encoded by a gene that contains multiple direct repeats organized as tandem, head-to-tail, 12-base-pair (bp) units overlapping with six highly conserved 75-bp units. The repetitive structure of the algP gene appears to participate in the processes underlying the metastable character of mucoidy in P. aeruginosa. Relatively large DNA rearrangements spanning the region with tandem direct repeats encoding the carboxy-terminal histone H1-like structure of AlgP were detected in several strains upon conversion from the mucoid to the nonmucoid phenotype. The frequency of the detectable algP rearrangements associated with the transition into the nonmucoid state varied from strain to strain and ranged from 0 to 50%. The nonmucoid derivatives with the clearly rearranged chromosomal copy of algP were complemented to mucoidy with plasmids containing algP from P. aeruginosa PAO. When a random collection of mucoid strains, isolated from different cystic fibrosis patients, was analyzed by using polymerase chain reaction, an additional level of strain-dependent sequence variation in algP was observed. Variations in the number of the 12-bp repeats were found; however, they did not appear to influence the mucoid status of the strains examined. Thus, the repeated region of algP appears to be a hot spot for DNA rearrangements and strain-dependent variability.
Similar organization of the sigB and spoIIA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase
Authors: Kalman S , Duncan M L , Thomas S M , Price C W
Published: November 2016

Bacillus subtilis sigma-B is an alternate sigma factor implicated in controlling stationary-phase gene expression. We characterized the genetic organization and regulation of the region containing the sigma-B structural gene (sigB) to learn which metabolic signals and protein factors govern sigma-B function. sigB lay in an operon with four open reading frames (orfs) in the order orfV-orfW-sigB-orfX, and lacZ gene fusions showed that all four frames were translated in vivo. Experiments with primer extension, S1 nuclease mapping, and lacZ transcriptional fusions found that sigB operon transcription initiated early in stationary phase from a site 32 nucleotides upstream of orfV and terminated 34 nucleotides downstream of orfX. Fusion expression was abolished in a strain carrying an in-frame deletion in sigB, suggesting that sigma-B positively regulated its own synthesis, and deletions in the sigB promoter region showed that sequences identical to the sigma-B-dependent ctc promoter were essential for promoter activity. Fusion expression was greatly enhanced in a strain carrying an insertion mutation in orfX, suggesting that the 22-kilodalton (kDa) orfX product was a negative effector of sigma-B expression or activity. Notably, the genetic organization of the sigB operon was strikingly similar to that of the B. subtilis spoIIA operon, which has the gene order spoIIAA-spoIIAB-spoIIAC, with spoIIAC encoding the sporulation-essential sigma-F. The predicted sequence of the 12-kDa orfV product was 32% identical to that of the 13-kDa SpoIIAA protein, and the 18-kDa orfW product was 27% identical to the 16-kDa SpoIIAB protein. On the basis of this clear evolutionary conservation, we speculate these protein pairs regulate their respective sigma factors by a similar molecular mechanism and that the spoIIA and sigB operons might control divergent branches of stationary-phase gene expression.
Localization of a second SigH promoter in the Bacillus subtilis sigA operon and regulation of dnaE expression by the promoter
Authors: Qi F X , Doi R H
Published: November 2016

The presence of a second SigH promoter in the sigA operon of Bacillus subtilis was demonstrated by use of a promoter probe plasmid, a sigH deletion mutant, primer extension studies, and in vitro transcription with E sigma H holoenzyme. Both SigH promoters were expressed at low levels even during the growth phase but were expressed at higher levels during the early stationary phase. Expression from the upstream SigH promoter allowed the expression of both dnaE and sigA genes; however, expression from the downstream SigH promoter, which was located in the ribosome-binding site of the dnaE gene, resulted only in the expression of the sigA gene, since the truncated dnaE ribosome-binding site could not be used for initiating translation. Thus, promoter switching during the early stationary phase resulted not only in expression from SigH promoters but also in differential expression of the genes in the sigA operon.
Purification of the Escherichia coli purine regulon repressor and identification of corepressors
Authors: Rolfes R J , Zalkin H
Published: November 2016

The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 microM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K.F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.
Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli
Authors: Sambasivarao D , Scraba D G , Trieber C , Weiner J H
Published: November 2016

Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.
Sequence and expression of the Escherichia coli recR locus
Authors: Yeung T , Mullin D A , Chen K S , Craig E A , Bardwell J C , Walker J R
Published: November 2016

The Escherichia coli RecR protein participates in a recombinational DNA repair process. Its gene is located in a region of chromosome that extends from 502 to 509 kilobases on the physical map and that contains apt, dnaX, orf12-recR, htpG, and adk. Most, if not all, of these are involved in nucleic acid metabolism. The orf12-recR reading frames consist of 935 base pairs and overlap by one nucleotide, with the 3' A of the orf12 termination codon forming the 5' nucleotide of the recR initiation codon. The orf12-recR promoter was located upstream of orf12 by sequence analysis, promoter cloning, and S1 nuclease protection analysis. The start point of transcription was determined by primer extension. The transcript 5' end contained a long, apparently untranslated region of 199 nucleotides. Absence of a detectable promoter specific for recR and the overlap of the orf12 and recR reading frames suggest that translation of recR is coupled to that of orf12. By maxicell analysis, it was determined that both orf12 and recR are translated.
Identification of a Caulobacter basal body structural gene and a cis-acting site required for activation of transcription
Authors: Dingwall A , Gober J W , Shapiro L
Published: November 2016

The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle. One of these genes, flbN, is required early in the flagellar assembly process. The flbN gene was cloned and sequenced, and the time of transcription activation was determined. The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide. The flbN-encoded protein has 30.8% identity with the protein encoded by the Salmonella typhimurium basal body L-ring gene, flgH. Site-directed mutagenesis and gel mobility shift assays identified a binding site at -100 from the transcription start site for a trans-acting protein, RF-2, that functions to partially activate flbN transcription at a defined time in the cell cycle. The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria. Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation. Transcription activation of flbN in C. crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation.
Relationship of cellulosomal and noncellulosomal xylanases of Clostridium thermocellum to cellulose-degrading enzymes
Authors: Morag E , Bayer E A , Lamed R
Published: November 2016

Xylanase activity of Clostridium thermocellum, an anaerobic thermophilic cellulolytic bacterium, was characterized. The activity was localized both in the cellulosome (the principal multienzyme, cellulose-solubilizing protein complex) and in noncellulosomal fractions. Each of these fractions contained at least four major polypeptide bands which contributed to the xylanolytic activity. In both cases, pH and temperature optima, product pattern, and other features of the xylanase activity were almost identical. The main difference was in the average molecular weights of the respective polypeptides which appeared responsible for the activity. In the noncellulosomal fraction, xylanases with Mrs ranging from 30,000 to 65,000 were detected. Distinct from these were the cellulosomal xylanases, which exhibited much larger Mrs (up to 170,000). The cellulosome-associated xylanases corresponded to known cellulosomal subunits, some of which also exhibited endoglucanase activity, and others which coincided with subunits which appeared to express exoglucanaselike activity. In contrast, the noncellulosomal xylanases hydrolyzed xylan exclusively. beta-Glucosidase and beta-xylosidase activities were shown to be the action of different enzymes; both were associated exclusively with the cell and were not components of the cellulosome. Despite the lack of growth on and utilization of xylan or its degradation products, C. thermocellum produces a highly developed xylanolytic apparatus which is interlinked with its cellulase system.
Phylogenetic distribution of branched RNA-linked multicopy single-stranded DNA among natural isolates of Escherichia coli
Authors: Herzer P J , Inouye S , Inouye M , Whittam T S
Published: November 2016

Multicopy single-stranded DNA (msDNA), a branched DNA-RNA molecule, has been shown in Escherichia coli B and clinical strain Cl-1 to be synthesized by reverse transcriptase. We report that 13% of the strains of the ECOR collection, a sample of 72 E. coli isolates representing the breadth of genetic variation of the species, produce msDNA. Three of the four major subspecific groups include msDNA-producing strains. Screening of 25 isolates that are genetically related to msDNA-producing clinical strains uncovered 22 additional msDNA-producing strains. A phylogenetic tree based on allelic variation detected electrophoretically at 20 enzyme-encoding loci revealed two major clusters and several deep branches composed of strains that synthesize msDNA. Although E. coli K-12 does not harbor msDNA, other closely related strains of the K-12 family do. The results support the hypothesis that msDNA-synthesizing systems, including reverse transcriptase genes, were acquired recently and independently in different lineages of E. coli.
Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product
Authors: Cotter P A , Chepuri V , Gennis R B , Gunsalus R P
Published: November 2016

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases that catalyze the oxidation of ubiquinol-8 and the reduction of oxygen to water. They are the cytochrome o oxidase complex encoded by cyoABCDE and the cytochrome d oxidase complex encoded by cydAB. To determine how these genes are regulated in response to a variety of environmental stimuli, including oxygen, we examined their expression by using lacZ protein fusions in wild-type and fnr mutant strains of E. coli. Anaerobic growth resulted in a 140-fold repression of cyoA'-'lacZ expression relative to aerobic growth and a 3-fold increase in cydA'-'lacZ expression. Anaerobic repression of both fusions was mediated in part by the fnr gene product, as evidenced by a 30-fold derepression of cyoA'-'lacZ expression and a 4-fold derepression of cydA'-'lacZ expression in an fnr deletion strain. Supplying wild-type fnr in trans restored wild-type repression for both fusions. Fnr thus functions as an anaerobic repressor of both cyoABCDE and cydAB expression. Reduced-minus-oxidized difference spectrum analyses of cell membranes confirmed the effect of the fnr gene product on the production of cytochrome d oxidase in the cell. Based on the pattern of anaerobic cydAB expression observed, we propose the existence of a second, as yet unidentified, regulatory element that must function either to activate cydAB expression as oxygen becomes limiting or to repress cydAB expression aerobically. Whereas cytochrome o oxidase encoded by cyoABCDE appears to be produced only under oxygen-rich growth conditions, in keeping with its biochemical properties, cytochrome d oxidase is expressed moderately aerobically and is elevated yet further when oxygen becomes limiting so that the organism can cope better under oxygen starvation conditions. We also examined cyoABCDE and cydAB expression in response to growth on alternative carbon compounds and to changes in the culture medium pH and osmolarity.
Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO
Authors: Temple L , Cuskey S M , Perkins R E , Bass R C , Morales N M , Christie G E , Olsen R H , Phibbs P V
Published: November 2016

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.
Unusual septum formation in Streptococcus pneumoniae mutants with an alteration in the D,D-carboxypeptidase penicillin-binding protein 3
Authors: Schuster C , Dobrinski B , Hakenbeck R
Published: November 2016

An internal 630-bp DNA fragment of the gene encoding penicillin-binding protein 3 (PBP 3) (dacA) of Streptococcus pneumoniae was identified in a lambda gt11 gene bank screened with anti-PBP 3 antiserum. The deduced 210-amino-acid sequence showed a high degree of homology to the low-molecular-weight PBPs 5 and 6 of Escherichia coli and Bacillus subtilis PBP 5. Viable mutants lacking a C-terminal part of PBP 3 were obtained after a plasmid containing the dacA fragment was integrated into the PBP 3 gene by homologous recombination. The truncated PBP 3* was still active in terms of beta-lactam binding. Most PBP 3 was found in the growth medium, indicating that membrane anchoring of PBP 3 is provided by the C terminus, as has been shown for other D,D-carboxypeptidases. The mutant cells grew with a slower generation time than the wild type in the shape of irregular enlarged spheres. In addition, as revealed by electron microscopy, cell separation was severely affected, septa were found unevenly distributed at multiple sites within the cells, and the murein layer appeared variable in thickness.
Metabolic relationships between pyridoxine (vitamin B6) and serine biosynthesis in Escherichia coli K-12
Authors: Lam H M , Winkler M E
Published: November 2016

We propose a pathway leading from erythrose-4-phosphate and glutamate to nitrogen 1 and carbons 5,5', and 6 of the pyridoxine ring. This pathway, which parallels the phosphorylated pathway of serine biosynthesis, is predicted on the homology between PdxB and SerA, the structural similarity between serine and 4-hydroxythreonine, and the possible involvement of SerC in pyridoxine biosynthesis. Several predictions of this hypothetical scheme were tested. Consistent with the proposed pathway, supplement inhibition patterns strongly suggest that SerA enzyme acts in a an alternate pathway of pyridoxine biosynthesis in pdxB mutants. Direct enzyme assays detected erythrose-4-phosphate dehydrogenase activity in crude extracts, which again supports the proposed pathway. Chromosomal insertions in serC caused a requirement for pyridoxine, serine, and aromatic compounds, which directly verified that SerC functions in the pyridoxine biosynthetic pathway. Complementation analysis showed that pdxF and pdxC mutations reported previously are most likely alleles of serC. Growth of serC chromosomal insertion mutants on glycoalaldehyde was found to occur without acquisition of second-site mutations and confirmed that pdxB and serC, but not pdxA, function in the same branch of the pyridoxine pathway. In addition, serC::mini-Mu d insertions revealed that the complex serC-aroA operon lacks internal promoters, that the amino terminus of SerC is not strictly essential for activity, and that antisense transcription occurs in the serC-aroA operon. Growth responses of pdxA, pdxB, and serC mutants to beta-hydroxypyruvate, D-alanine, and glycolate could also be reconciled with the proposed pathway. Finally, the proposed scheme is consistent with previous isotope labeling data and accounts for several other observations about pyridoxine biosynthesis. Together, these physiological and biochemical analyses support the proposed pathway and an evolutionary scenario in which this branch of the pyridoxine pathway evolved from the serine pathway by gene recruitment.
Biosynthesis of a membrane adhesion zone fraction throughout the cell cycle of Escherichia coli
Authors: Joseleau-Petit D , Kepes F , Peutat L , D'Ari R , Rothfield L I
Published: November 2016

Synchronized cells of Escherichia coli were pulse-labeled with [3H]leucine and subjected to membrane fractionation to determine whether a fraction that is enriched for membrane-murein adhesion zones (fraction OML) was preferentially generated at specific times during the cell cycle, as previously suggested from studies of lkyD and cha mutants. Contrary to this prediction, the experiments showed that OML was formed continuously during the division cycle.
Cloning of an aroF allele encoding a tyrosine-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase
Authors: Weaver L M , Herrmann K M
Published: November 2016

In Escherichia coli, genes aroF+, aroG+, and aroH+ encode isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. A single base pair change in aroF causes a Pro-148-to-Leu-148 substitution and results in a tyrosine-insensitive enzyme.
Microcin H47, a chromosome-encoded microcin antibiotic of Escherichia coli
Authors: Laviña M , Gaggero C , Moreno F
Published: November 2016

Microcin H47 (MccH47) is a novel microcin antibiotic produced by a natural Escherichia coli isolate. In contrast to all the other colicins and microcins examined to date, which are plasmid encoded, the genes for MccH47 synthesis and immunity are located on the chromosome. These genetic determinants were cloned and shown to extend over a continuous DNA region of ca. 10 kb.
Molecular analysis of an IS200 insertion in the gpt gene of Salmonella typhimurium LT2
Authors: O'Reilly C , Black G W , Laffey R , McConnell D J
Published: November 2016

A strain of Salmonella typimurium LT2 has been isolated which carries an insertion of approximately 700 bp in the gpt gene. The insertion in the gpt gene was shown to be the Salmonella-specific element IS200. The mutation in strain CR1 arose without selection during storage and is only the second phenotypically identified mutation caused by the insertion of IS200.