bluuebunny/crossref_metadata_2025 huggingface dataset:
Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1
Authors: Watt S R , Clarke A J
Published: November 2016

Two extracellular autolysins have been detected in the spent culture supernatants of Pseudomonas aeruginosa PAO1 by using renaturing polyacrylamide gel electrophoresis. The two autolysins were isolated from the culture supernatant by trichloroacetic acid precipitation and were shown to have apparent molecular masses of 26 and 29 kDa. The 26-kDa autolysin first appears during the early exponential phase of growth and then declines sharply, while the 29-kDa autolysin first appears in the late exponential phase of growth and continues well into the stationary phase. Fractionation of whole cells indicated that the 26-kDa enzyme was also localized within the periplasm, with a lesser amount of activity associated with the cytoplasmic membrane. The 29-kDa autolytic activity was distributed within the cell equally between the periplasm and the cytoplasmic membrane. The pH optima of the isolated 26- and 29-kDa autolysins are 6.0 and 5.0, respectively. Further evidence from both protease susceptibility and inhibition studies confirms that these two extracellular autolysins isolated from P. aeruginosa PAO1 are separate and distinct.
In vivo footprinting analysis of Lrp binding to the ilvIH promoter region of Escherichia coli
Authors: Marasco R , Varcamonti M , La Cara F , Ricca E , De Felice M , Sacco M
Published: November 2016

An in vivo footprinting analysis of the ilvIH regulatory region of Escherichia coli showed that the transcription activator Lrp binds to six sites, scattered over 250 bp upstream of the transcriptional start point. When Lrp-mediated activation was impaired by the presence of exogenous leucine, only one promoter-distal site (site 2) was partially protected by Lrp binding. Equilibrium dialysis experiments showed the formation of an Lrp-leucine complex in vitro. These results suggest that leucine negatively affects ilvIH transcription because its interaction with Lrp reduces the efficiency of binding of the regulatory protein to the promoter region.
Phylogeny of mycoplasmalike organisms (phytoplasmas): a basis for their classification
Authors: Gundersen D E , Lee I M , Rehner S A , Davis R E , Kingsbury D T
Published: November 2016

A global phylogenetic analysis using parsimony of 16S rRNA gene sequences from 46 mollicutes, 19 mycoplasmalike organisms (MLOs) (new trivial name, phytoplasmas), and several related bacteria placed the MLOs definitively among the members of the class Mollicutes and revealed that MLOs form a large discrete monophyletic clade, paraphyletic to the Acholeplasma species, within the Anaeroplasma clade. Within the MLO clade resolved in the global mollicutes phylogeny and a comprehensive MLO phylogeny derived by parsimony analyses of 16S rRNA gene sequences from 30 diverse MLOs representative of nearly all known distinct MLO groups, five major phylogenetic groups with a total of 11 distinct subclades (monophyletic groups or taxa) could be recognized. These MLO subclades (roman numerals) and designated type strains were as follows: i, Maryland aster yellows AY1; ii, apple proliferation AP-A; iii, peanut witches'-broom PnWB; iv, Canada peach X CX; v, rice yellow dwarf RYD; vi, pigeon pea witches'-broom PPWB; vii, palm lethal yellowing LY; viii, ash yellows AshY; ix, clover proliferation CP; x, elm yellows EY; and xi, loofah witches'-broom LfWB. The designations of subclades and their phylogenetic positions within the MLO clade were supported by a congruent phylogeny derived by parsimony analyses of ribosomal protein L22 gene sequences from most representative MLOs. On the basis of the phylogenies inferred in the present study, we propose that MLOs should be represented taxonomically at the minimal level of genus and that each phylogenetically distinct MLO subclade identified should represent at least a distinct species under this new genus.
Properties of Bacillus subtilis small, acid-soluble spore proteins with changes in the sequence recognized by their specific protease
Authors: Carrillo-Martinez Y , Setlow P
Published: November 2016

Alpha/beta-type small, acid-soluble proteins (SASP) of dormant spores of Bacillus subtilis bind to DNA and increase its resistance to a variety of damaging agents both in vivo and in vitro. When spores germinate, degradation of alpha/beta-type SASP is rapidly initiated by a sequence-specific protease, which is termed GPR. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the wild-type alpha/beta-type SASP SspCwt; all three mutations change residues in the highly conserved sequence recognized by GPR. In one mutant protein (SspCV), residue 33 (Ser) was changed to Val; in the second (SspCDL), residues 30 and 31 (Glu and Ile) were changed to Asp and Leu, respectively; and in the third mutant protein (SspCDLV), residues 30, 31, and 33 were changed to Asp, Leu, and Val. All three mutant proteins were rapidly degraded by GPR during spore germination, and SspCDL and SspCDLV were degraded by GPR in vitro at rates 8 to 9% of that for SspCwt, although not exclusively at the single site cleaved by GPR in SspCwt. These results indicate (i) that the sequence specificity of GPR is broader than originally imagined and (ii) that GPR can cleave the sequence in SspCDLV. Since the latter sequence is identical to that cleaved during the proteolytic activation of GPR, this result further supports an autoprocessing model for GPR activation during sporulation. The properties of these mutant proteins were also examined, both in vivo in B. subtilis spores and in Escherichia coli and in vitro with purified protein. SspC(v) interacted with DNA similarly to SspC(wt) in vivo, resorting UV and heat resistance to spores lacking major alpha/beta-type SASP to the same extent as SspC(wt). In contrasst, SspC(DL) had much less effect on DNA properties in vivo and bound strongly only to poly(dG) . poly(dC) in vitro; SspC(DLV) exhibited only weak binding to poly(dG).poly(dC) in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the binding of these proteins to spore DNA and alteration of DNA properties and show further that the GRP recognition region in alpha/beta-type SASP plays some role in DNA binding.
Anaerobic regulation of the hydrogenase 1 (hya) operon of Escherichia coli
Authors: Brøndsted L , Atlung T
Published: November 2016

Using a transcriptional fusion to the lacZ gene, we have analyzed the anaerobic regulation of the hydrogenase 1 (hya) operon in response to different anaerobic growth conditions and to mutations in regulatory genes. We found that the transcription of the hya operon was induced when the growth condition was changed from aerobic to anaerobic and that this induction was independent of Fnr but dependent on regulators AppY and ArcA. Furthermore, we found that the transcription of the hya operon was not regulated by the cyclic AMP-cyclic AMP receptor protein complex. Investigation of the effects of different anaerobic growth conditions on the expression of the hya operon showed that expression was induced by formate and repressed by nitrate. Formate induction was not mediated by the fhlA gene product, and nitrate repression was not mediated by the narL gene product. We found a high level of anaerobic expression of the hya operon in glucose medium supplemented with formate and in glycerol medium supplemented with fumarate, suggesting that hydrogenase isoenzyme 1 has a function during both fermentative growth and anaerobic respiration.
Analysis of Bacillus subtilis hut operon expression indicates that histidine-dependent induction is mediated primarily by transcriptional antitermination and that amino acid repression is mediated by two mechanisms: regulation of transcription initiation and inhibition of histidine transport
Authors: Wray L V , Fisher S H
Published: November 2016

Expression of the Bacillus subtilis hut operon is induced by histidine and subject to regulation by carbon catabolite repression and amino acid repression. A set of hut-lacZ transcriptional fusions was constructed and used to identify the cis-acting sites required for histidine induction and amino acid repression. Histidine induction was found to be primarily mediated by transcriptional antitermination at a palindromic sequence located immediately downstream of the first structural gene in the hut operon, hutP. High levels of histidine induction were observed only in hut-lacZ fusions which contained this palindromic sequence. The hutC1 mutation, which results in constitutive expression of the hut operon, was sequenced and found to contain a GC to TA transversion located within the stem-loop structure. Transcription of hut DNA in vitro revealed that the palindromic structure functions as a transcriptional terminator with wild-type hut DNA but not with hutC1 DNA. Two sites were found to be involved in amino acid repression of hut expression: (i) an operator, hutOA, which lies downstream of the hut promoter, and (ii) the hut terminator. The rate of [14C]histidine uptake in amino acid-grown cells was sixfold lower than that seen in cells grown without amino acids. Thus, inhibition of histidine transport in amino acid-grown cells indirectly regulates hut expression by interfering with histidine induction at the hut terminator.
Studies of the biosynthesis of 3,6-dideoxyhexoses: molecular cloning and characterization of the asc (ascarylose) region from Yersinia pseudotuberculosis serogroup VA
Authors: Thorson J S , Lo S F , Ploux O , He X , Liu H W
Published: November 2016

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to occur naturally, four have been found in various strains of Salmonella enterica (abequose, tyvelose, paratose, and colitose) and all five, including ascarylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region harboring the abequose biosynthetic genes from Y. pseudotuberculosis serogroup HA, the detailed genetic principles underlying a 3,6-dideoxyhexose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dideoxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (O antigen) cluster of Salmonella spp., we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a detailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In addition, the functional cloning of this region has allowed the expression of Ep (alpha-D-glucose cytidylyltransferase), Eod (CDP-D-glucose 4,6-dehydratase), E1 (CDP-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase), E3 (CDP-6-deoxy-delta 3,4-glucoseen reductase), Eep (CDP-3,6-dideoxy-D-glycero-D- glycero-4-hexulose-5-epimerase), and Ered (CDP-3,6-dideoxy-L-glycero-D-glycero-4-hexulose-4-reductase), facilitating future mechanistic studies of this intriguing biosynthetic pathway.
The H-NS protein is involved in the biogenesis of flagella in Escherichia coli
Authors: Bertin P , Terao E , Lee E H , Lejeune P , Colson C , Danchin A , Collatz E
Published: November 2016

The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. In this paper, we show that motile behavior was affected in Escherichia coli hns mutants. The loss of motility resulted from a complete lack of flagella. A decrease in the level of transcription of the flhD and fliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation. Furthermore, the Fla- phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex. These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway.
Time-resolved detection of three intracellular signals controlling photomorphogenesis in Physarum polycephalum
Authors: Starostzik C , Marwan W
Published: November 2016

Incompetent plasmodia of Physarum polycephalum exposed to a light pulse sporulated after reaching the competent stage. Fusion of irradiated plasmodia with dark-incubated plasmodia and analysis of sporulation indicated the presence of a morphogenetic signal. It is concluded that a logic AND gate integrates the photoreceptor signal and the competence signal and controls the formation of the morphogenetic signal.
Sequencing analysis reveals a unique gene organization in the gyrB region of Mycoplasma hominis
Authors: Ladefoged S A , Christiansen G
Published: November 2016

The homolog of the gyrB gene, which has been reported to be present in the vicinity of the initiation site of replication in bacteria, was mapped on the Mycoplasma hominis genome, and the region was subsequently sequenced. Five open reading frames were identified flanking the gyrB gene, one of which showed similarity to that which encodes the LicA protein of Haemophilus influenzae. The organization of the genes in the region showed no resemblance to that in the corresponding regions of other bacteria sequenced so far. The gyrA gene was mapped 35 kb downstream from the gyrB gene.
Molecular cloning and characterization of the pgm gene encoding phosphoglucomutase of Escherichia coli
Authors: Lu M , Kleckner N
Published: November 2016

We report here the identification and characterization of pgm, a gene in Escherichia coli that encodes the enzyme phosphoglucomutase, specifically required for the catalysis of the interconversion of glucose 1-phosphate and glucose 6-phosphate. The predicted amino acid sequence of the pgm gene is highly conserved in E. coli, Acetobacter xylinum, Saccharomyces cerevisiae, rabbits, and humans. pgm deletion mutant strains are deficient in phosphoglucomutase activity.
Iron-responsive genetic regulation in Campylobacter jejuni: cloning and characterization of a fur homolog
Authors: Wooldridge K G , Williams P H , Ketley J M
Published: November 2016

The Fur protein of Escherichia coli represses transcription from Fur-responsive genes in an iron-dependent manner. We have demonstrated a Fur-like iron-responsive genetic regulatory activity operating in Campylobacter jejuni by using a chloramphenicol acetyl transferase reporter gene separated from its promoter by a synthetic Fur-responsive operator. A fur-like gene has been cloned from C. jejuni by partial functional complementation of an E. coli fur mutation. Sequence analysis has shown that, at the amino acid level, the C. jejuni Fur protein is 35% identical with its E. coli counterpart.
Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo
Authors: Gaughran J P , Lai M H , Kirsch D R , Silverman S J
Published: November 2016

Nikkomycin Z inhibits chitin synthase in vitro but does not exhibit antifungal activity against many pathogens. Assays of chitin synthase isozymes and growth assays with isozyme mutants were used to demonstrate that nikkomycin Z is a selective inhibitor of chitin synthase 3. The resistance of chitin synthase 2 to nikkomycin Z in vitro is likely responsible for the poor activity of this antibiotic against Saccharomyces cerevisiae.
Expression of both Bacillus subtilis threonyl-tRNA synthetase genes is autogenously regulated
Authors: Gendron N , Putzer H , Grunberg-Manago M
Published: November 2016

The "housekeeping" threonyl-tRNA synthetase gene (thrS) of Bacillus subtilis is shown to be transcribed in vivo and in vitro from a single promoter. In vitro, 85% of all messages transcribed from the thrS promoter are terminated at a strong factor-independent terminator localized upstream of the thrS Shine-Dalgarno sequence, within the 305-nucleotide-long leader region. Overexpression of thrS represses transcriptional and translational thrS-lacZ fusions to a similar extent, suggesting that thrS is autoregulated at the transcriptional level. We show that autogenous control does not act at the level of transcription initiation but involves antitermination of the transcription mechanism. thrZ, the second threonyl-tRNA synthetase gene, is also autogenously regulated. However, the ability of the ThrS synthetase to repress thrS as well as thrZ expression is much greater than that of the ThrZ synthetase.
Strain identification and 5S rRNA gene characterization of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius
Authors: Durovic P , Kutay U , Schleper C , Dennis P P
Published: November 2016

A commonly used laboratory Sulfolobus strain has been unambiguously identified as Sulfolobus acidocaldarius DSM639. The 5S rRNA gene from this strain was cloned and sequenced. It differs at 17 of 124 positions from the identical 5S rRNA sequences from Sulfolobus solfataricus and a strain apparently misidentified as S. acidocaldarius. Analysis of the transcripts from the 5S rRNA gene failed to identify any precursor extending a significant distance beyond the 5' or 3' boundary of the 5S rRNA-coding sequence. This result suggests that the primary transcript of the 5S rRNA gene corresponds in length (within 1 or 2 nucleotides) to the mature 5S rRNA sequence found in 50S ribosomal subunits.
Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis
Authors: Martinussen J , Hammer K
Published: November 2016

Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced, and the putative amino acid sequence was deduced. The promoter was mapped by both primer extension and analysis of beta-galactosidase expressed from strains carrying fusion between upp promoter fragments and the lacLM gene. The results showed that the upp gene was expressed from its own promoter. After in vitro construction of an internal deletion, a upp mutant was constructed by a double-crossover event. This implicated the utilization of a plasmid with a thermosensitive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial strains. The phenotype of the uracil phosphoribosyltransferase-deficient strain was established. Surprisingly, the upp strain is resistant only to very low concentrations of 5-fluorouracil. Secondary mutants in thymidine phosphorylase and thymidine kinase were isolated by selection for resistance to high concentrations of 5-fluorouracil.
Structural studies of the side chain of outer membrane lipopolysaccharide from Pseudomonas syringae pv. coriandricola W-43
Authors: Das S , Ramm M , Kochanowski H , Basu S
Published: November 2016

The lipopolysaccharide (LPS) was isolated from Pseudomonas syringae pv. coriandricola W-43 by hot phenol-water extraction. Rhamnose and 3-N-acetyl-3-deoxyfucose were found to be the major sugar constituents of the LPS together with N-acetylglucosamine, N-acetylgalactosamine, heptose, and 3-deoxy-D-manno-octulosonic acid (Kdo). The main fatty acids of lipid A of the LPS were 3-OH-C:10, C12:0, 2-OH-C12:0, and 3-OH-C12:0. The O-specific polysaccharide liberated from the LPS by mild-acid hydrolysis was purified by gel permeation chromatography. The compositional analysis of the O-specific polysaccharide revealed the presence of L-rhamnose and 3-N-acetyl-3-deoxy-D-fucose in a molar ratio of 4:1. The primary structure of the O-specific polysaccharide was established by methylation analysis together with 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional shift-correlated and one-dimensional nuclear Overhauser effect spectroscopy. The polysaccharide moiety was found to consist of a tetrasaccharide rhamnan backbone, and 3-N-acetyl-3-deoxy-D-fucose constitutes the side chain of the branched pentasaccharide repeating unit of the polysaccharide.
Genetics of the serine cycle in Methylobacterium extorquens AM1: cloning, sequence, mutation, and physiological effect of glyA, the gene for serine hydroxymethyltransferase
Authors: Chistoserdova L V , Lidstrom M E
Published: November 2016

The gene (glyA) of Methylobacterium extorquens AM1 encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the serine cycle for C1 assimilation, was isolated by using a synthetic oligonucleotide with a sequence based on amino acid sequence conserved in SHMTs from different sources. The amino acid sequence deduced from the gene revealed high similarity to those of known SHMTs. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced an SHMT null mutant. Surprisingly, this mutant had lost its ability to grow on C1 as well as on C2 compounds but was still able to grow on succinate. The DNA fragment containing glyA was shown not to be linked with fragments carrying serine cycle genes identified earlier, making it the fourth chromosomal region of M. extorquens AM1 to be indicated as being involved in C1 assimilation.
Efficient gene transfer in Synechococcus sp. strains PCC 7942 and PCC 6301 by interspecies conjugation and chromosomal recombination
Authors: Tsinoremas N F , Kutach A K , Strayer C A , Golden S S
Published: November 2016

We developed a versatile, efficient genetic transfer method for Synechococcus sp. strains PCC 7942 and PCC 6301 that exceeds natural transformation efficiencies by orders of magnitude. As a test case, we complemented a histidine auxotroph and identified a hisS homolog of PCC 7942 as the complementing gene.
Plasmids bearing hfq and the hns-like gene stpA complement hns mutants in modulating arginine decarboxylase gene expression in Escherichia coli
Authors: Shi X , Bennett G N
Published: November 2016

Biodegradative arginine decarboxylase is inducible by acid and is derepressed in an hns mutant. Several plasmids from an Escherichia coli library that could complement the hns phenotype were characterized and placed into groups. One group includes plasmids that contain the hns gene and are considered true complements. Another group was found to carry the hfq gene, which encodes the host factor HF-1 for bacteriophage Q beta replication. Plasmids of the third group contain inserts that mapped at 60.2 min on the E. coli chromosome. We identified an open reading frame (stpA) with a deduced amino acid sequence showing more than 60% identity with the sequences of H-NS proteins from several species as being responsible for the hns complementing phenotype of the third group.
Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107
Authors: Sugano Y , Kodama H , Terada I , Yamazaki Y , Noma M
Published: November 2016

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.
A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli
Authors: Zhang H Z , Lory S , Donnenberg M S
Published: November 2016

Enteropathogenic Escherichia coli, a leading agent of infantile diarrhea worldwide, adheres to tissue culture cells in a pattern called "localized adherence." Localized adherence is associated with bundle-forming pili encoded by the plasmid bfpA gene, the product of which is homologous with the major structural subunit proteins of type IV fimbriae in other bacteria. Several of these proteins have been shown to be processed from a precursor by a specific prepilin peptidase. We cloned restriction fragments downstream of the bfpA gene into an E. coli-Pseudomonas aeruginosa shuttle vector and mobilized them into a P. aeruginosa prepilin peptidase (pilD) mutant. A plasmid containing a 1.3-kb PstI-BamHI fragment was able to complement the pilD mutation, as demonstrated by restoration of sensitivity to the pilus-specific bacteriophage PO4. The DNA sequence of this fragment revealed an open reading frame, designated bfpP, the predicted product of which is homologous to other prepilin peptidases, including TcpJ of Vibrio cholerae (30% identical amino acids), PulO of Klebsiella oxytoca (29%), and PilD of P. aeruginosa (28%). A bfpA::TnphoA mutant complemented with a bfpA-containing DNA fragment only partially processes the BfpA protein. When complemented with a larger fragment containing bfpP as well as bfpA, the mutant expresses the fully processed BfpA protein. P. aeruginosa PAK, but not a pilD mutant of PAK, expresses mature BfpA protein when the bfpA gene is mobilized into this strain. Thus, as in other type IV fimbria systems, enteropathogenic E. coli utilizes a specific prepilin peptidase to process the major subunit of the bundle-forming pilus. This prepilin petidase contains sequence and reciprocal functional homologies with the PilD protein of P. aeruginosa.
Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure
Authors: Dalton H M , Poulsen L K , Halasz P , Angles M L , Goodman A E , Marshall K C
Published: November 2016

The effects of surfaces on the physiology of bacteria adhering to surfaces or immobilized within biofilms are receiving more interest. A study of the effects of hydrophobic and hydrophilic substrata on the colonization behavior of a marine bacterium, SW5, revealed major differences in the morphology of SW5 on these surfaces. Using epifluorescence, scanning confocal laser, and on-line visualization (time-lapse video) microscopy, the organisms at hydrophobic surfaces were characterized by the formation of tightly packed biofilms, consisting of single and paired cells, whereas those at hydrophilic surfaces exhibited sparse colonization and the formation of chains more than 100 microns long, anchored at the surface by the terminal (colonizing) cell. The results are discussed in terms of the possible factors inducing the observed morphological differences and the significance of these differences in terms of biofilm structure and plasmid transfer when SW5 is the recipient organism.
Carboxyl-terminal amino acid residues in elongation factor G essential for ribosome association and translocation
Authors: Hou Y , Yaskowiak E S , March P E
Published: November 2016

The translocation of ribosomes on mRNA is carried out by cellular machinery that has been extremely well conserved across the entire spectrum of living species. This process requires elongation factor G (EF-G, or EF-2 in archaebacteria and eukaryotes), which is a member of the GTPase superfamily. Using genetic techniques, we have identified a series of mutated alleles of fusA (the Escherichia coli gene that encodes EF-G) that were unable to support protein synthesis in vivo. These alleles encode proteins with point mutations at codons 495 (a variant with a Q-to-P change at codon 495 [Q495P]), 502 (G502D), and 563 (G563D) and a nonsense mutation at codon 608. Biochemical analyses demonstrated that EF-G Q495P, G502D, and delta 608-703 were not disrupted in guanine nucleotide binding but were deficient in ribosome-dependent GTP hydrolysis and guanine nucleotide-dependent ribosome association. We propose that all of these mutations are present in a domain that is essential for ribosome association and that GTP hydrolysis was deficient as a secondary consequence of impaired binding to 70S ribosomes.
Atomic composition of the hydrophobic and hydrophilic membrane sides of self-assembled SC3p hydrophobin
Authors: Wösten H A , de Vries O M , van der Mei H C , Busscher H J , Wessels J G
Published: November 2016

The hydrophobin SC3p of Schizophyllum commune self-assembles into a 10-nm-thick amphipathic membrane at hydrophilic-hydrophobic interfaces. X-ray photoelectron spectroscopy of the hydrophobic membrane side of SC3p, assembled in vitro, showed an atomic composition similar to the calculated composition of SC3p when glycosylation was taken into account. The atomic composition measured at the hydrophilic membrane side deviated from that at the hydrophobic side and indicated the presence of a lower number of peptide bonds. High levels of S and N were detected only on mycelia carrying hydrophobic aerial hyphae, as expected with assembled SC3p present at the surface of these hyphae.
Biochemical characterization of the essential GTP-binding protein Obg of Bacillus subtilis
Authors: Welsh K M , Trach K A , Folger C , Hoch J A
Published: November 2016

An essential guanine nucleotide-binding protein, Obg, of Bacillus subtilis has been characterized with respect to its enzymatic activity for GTP. The protein was seen to hydrolyze GTP with a Km of 5.4 microM and a kcat of 0.0061 min-1 at 37 degrees C. GDP was a competitive inhibitor of this hydrolysis, with an inhibition constant of 1.7 microM at 37 degrees C. The dissociation constant for GDP from the Obg protein was 0.5 microM at 4 degrees C and was estimated to be 1.3 microM at 37 degrees C. Approximately 80% of the purified protein was capable of binding GDP. In addition to hydrolysis of GTP, Obg was seen to autophosphorylate with this substrate. Subsequent release of the covalent phosphate proceeds at too slow a rate to account for the overall rate of GTP hydrolysis, indicating that in vitro hydrolysis does not proceed via the observed phosphoamidate intermediate. It was speculated that the phosphorylated form of the enzyme may represent either a switched-on or a switched-off configuration, either of which may be normally induced by an effector molecule. This enzyme from a temperature-sensitive mutant of Obg did not show significantly altered GTPase activity at the nonpermissive temperature.
Molecular cloning of the Escherichia coli B L-fucose-D-arabinose gene cluster
Authors: Elsinghorst E A , Mortlock R P
Published: November 2016

To metabolize the uncommon pentose D-arabinose, enteric bacteria often recruit the enzymes of the L-fucose pathway by a regulatory mutation. However, Escherichia coli B can grow on D-arabinose without the requirement of a mutation, using some of the L-fucose enzymes and a D-ribulokinase that is distinct from the L-fuculokinase of the L-fucose pathway. To study this naturally occurring D-arabinose pathway, we cloned and partially characterized the E. coli B L-fucose-D-arabinose gene cluster and compared it with the L-fucose gene cluster of E. coli K-12. The order of the fucA, -P, -I, and -K genes was the same in the two E. coli strains. However, the E. coli B gene cluster contained a 5.2-kb segment located between the fucA and fucP genes that was not present in E. coli K-12. This segment carried the darK gene, which encodes the D-ribulokinase needed for growth on D-arabinose by E. coli B. The darK gene was not homologous with any of the L-fucose genes or with chromosomal DNA from other D-arabinose-utilizing bacteria. D-Ribulokinase and L-fuculokinase were purified to apparent homogeneity and partially characterized. The molecular weights, substrate specificities, and kinetic parameters of these two enzymes were very dissimilar, which together with DNA hybridization analysis, suggested that these enzymes are not related. D-Arabinose metabolism by E. coli B appears to be the result of acquisitive evolution, but the source of the darK gene has not been determined.
Hfr mapping of mutations in Bordetella pertussis that define a genetic locus involved in virulence gene regulation
Authors: Stibitz S , Carbonetti N H
Published: November 2016

We report the development of techniques for the genetic mapping of point mutations in the bacterial pathogen Bordetella pertussis. A plasmid vector which is self-transmissible by conjugation and which, by insertion into the B. pertussis chromosome, can mobilize chromosomal sequences during conjugation with a recipient B. pertussis bacterium has been constructed. This vector is used in conjunction with a set of strains containing kanamycin resistance gene insertions at defined physical locations in the B. pertussis genome. In crosses between these donor strains and a mutant recipient strain, transfer of a chromosomal segment flanking the kanamycin resistance gene insertion is selected for, and the percentage of exconjugants which reacquire the wild-type trait is scored. In this way the linkage of the mutant allele to these markers, and thus the approximate chromosomal position of the mutant allele, is determined. We have used this genetic system to map a newly described locus in B. pertussis involved in the regulation of the virulence genes ptx (pertussis toxin) and cya (adenylate cyclase toxin).
Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum
Authors: Mateos L M , Pisabarro A , Pátek M , Malumbres M , Guerrero C , Eikmanns B J , Sahm H , Martín J F
Published: November 2016

Two genes, hom (encoding homoserine dehydrogenase) and thrB (encoding homoserine kinase), of the threonine biosynthetic pathway are clustered in the chromosome of Brevibacterium lactofermentum in the order 5' hom-thrB 3', separated by only 10 bp. The Brevibacterium thrB gene is expressed in Escherichia coli, in Brevibacterium lactofermentum, and in Corynebacterium glutamicum and complements auxotrophs of all three organisms deficient in homoserine kinase, whereas the Brevibacterium hom gene did not complement two different E. coli auxotrophs lacking homoserine dehydrogenase. However, complementation was obtained when the homoserine dehydrogenase was expressed as a fusion protein in E. coli. Northern (RNA) analysis showed that the hom-thrB cluster is transcribed, giving two different transcripts of 2.5 and 1.1 kb. The 2.5-kb transcript corresponds to the entire cluster hom-thrB (i.e., they form a bicistronic operon), and the short transcript (1.1 kb) originates from the thrB gene. The promoter in front of hom and the hom-internal promoter in front of thrB were subcloned in promoter-probe vectors of E. coli and corynebacteria. The thrB promoter is efficiently recognized both in E. coli and corynebacteria, whereas the hom promoter is functional in corynebacteria but not in E. coli. The transcription start points of both promoters have been identified by primer extension and S1 mapping analysis. The thrB promoter was located in an 87-bp fragment that overlaps with the end of the hom gene. A functional transcriptional terminator located downstream from the cluster was subcloned in terminator-probe vectors.
Phosphorylation of cytadherence-accessory proteins in Mycoplasma pneumoniae
Authors: Dirksen L B , Krebes K A , Krause D C
Published: November 2016

Attachment to host cells of the respiratory epithelium by Mycoplasma pneumoniae is a complex, multicomponent process, requiring a number of accessory proteins in addition to adhesins directly involved in receptor binding. In this study, protein phosphorylation of the cytadherence-accessory proteins HMW1, HMW2, and HMW4 of M. pneumoniae was examined using biochemical and immunological techniques. The initial indication of protein modification came from Western immunoblot analysis of the two-dimensional polyacrylamide gel electrophoresis (PAGE) profile of M. pneumoniae proteins, revealing multiple spots for both HMW1 and HMW4 that varied in pI but not in size. M. pneumoniae cultured in the presence of H3(32)PO4 exhibited numerous phosphorylated proteins as detected by sodium dodecyl sulfate-PAGE and autoradiography. These included proteins corresponding to HMW1, HMW2, and HMW4 in electrophoretic mobility. The Triton X-100 partitioning characteristics of these phosphorylated proteins was identical to that described previously for HMW1, -2, and -4. Furthermore, these protein bands were absent when a noncytadhering variant deficient in HMW1-5 was examined in the same manner. Finally, the availability of antiserum to HMW1 and -4 enabled us to confirm by radioimmunoprecipitation that HMW1 and HMW4 are phosphoproteins. Phosphoamino acid analysis of acid-hydrolyzed HMW1 and HMW2 identified primarily phosphothreonine and, to a lesser extent, phosphoserine in HMW1 and predominantly phosphoserine, with a trace of phosphothreonine, in HMW2. Neither protein contained phosphotyrosine. HMW1-HMW5 are components of a cytoskeleton-like structure in M. pneumoniae that is thought to function in cell division, changes in cell morphology, gliding motility, and the localization of adhesins in the mycoplasma membrane. Phosphorylation may regulate cytoskeleton dynamics involving these cytadherence-accessory proteins.
The incN plasmid replicon: two pathways of DNA polymerase I-independent replication
Authors: Kim H Y , Banerjee S K , Iyer V N
Published: November 2016

The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons. Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts. The region also specifies a protein called RepA. We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA. oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected. The requirements for oriB usage can thus be distinguished from those for oriS usage. The oriB region can be recovered as a plasmid only if RepA is provided in trans. These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids. Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range.
Isolation of a Bacillus subtilis spoIIGA allele that suppresses processing-negative mutations in the Pro-sigma E gene (sigE)
Authors: Peters H K , Haldenwang W G
Published: November 2016

sigma E, a sporulation-essential sigma factor of Bacillus subtilis, is formed by a developmentally regulated proteolysis which removes 27 to 29 amino acids from the amino terminus of an inactive precursor protein (Pro-sigma E). A mutation which facilitates the conversion of inefficiently processed Pro-sigma E variants into mature sigma E was identified and mapped to spoIIGA. The isolation of such a mutation argues that SpoIIGA is directly involved in the Pro-sigma E processing reaction.
Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome
Authors: Zuerner R L , Stanton T B
Published: November 2016

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.
The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis
Authors: Cohen G , Argaman A , Schreiber R , Mislovati M , Aharonowitz Y
Published: November 2016

Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene.
Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa
Authors: Schweizer H P , Po C
Published: November 2016

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.
The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation
Authors: Magill N G , Cowan A E , Koppel D E , Setlow P
Published: November 2016

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.
Inducibility of the TOL catabolic pathway in Pseudomonas putida (pWW0) growing on succinate in continuous culture: evidence of carbon catabolite repression control
Authors: Duetz W A , Marqués S , de Jong C , Ramos J L , van Andel J G
Published: November 2016

The TOL catabolic genes in Pseudomonas putida (pWW0) are clustered in the upper operon, encoding enzymes for the conversion of toluene and xylenes to benzoate and toluates, and the meta-cleavage operon, encoding enzymes for the conversion of the benzoate and toluates to tricarboxylic acid cycle intermediates. In this study, it was shown that cells growing in a chemostat under succinate growth-limiting conditions express both the upper and meta-cleavage pathways in response to o-xylene, a nonmetabolizable effector of the XylR regulatory protein. The dilution rate maintained in the succinate-limited chemostat cultures influenced the synthesis levels of TOL pathway enzymes, their steady-state levels, and their turnover rates. Cells growing in the presence of nonlimiting concentrations of succinate in continuous culture did not express pathway enzymes in response to the addition of o-xylene, which was due to a blockage at the transcriptional level. Expression of the meta-cleavage pathway in response to 2,3-dimethylbenzoate, a nonmetabolizable effector of the XylS regulatory protein, was 93% lower in cultures exposed to succinate at nonlimiting concentrations than in the succinate-limited chemostats. The mRNA level of xylS during nonlimited growth on succinate was very low compared with that in succinate-limited cultures, suggesting that suppression of expression of the meta-cleavage pathway is regulated mainly by the level of the XylS regulator.