bluuebunny/crossref_metadata_2025 huggingface dataset:
Compositional biases of bacterial genomes and evolutionary implications
Authors: Karlin S , Mrázek J , Campbell A M
Published: November 2016

We compare and contrast genome-wide compositional biases and distributions of short oligonucleotides across 15 diverse prokaryotes that have substantial genomic sequence collections. These include seven complete genomes (Escherichia coli, Haemophilus influenzae, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis sp. strain PCC6803, Methanococcus jannaschii, and Pyrobaculum aerophilum). A key observation concerns the constancy of the dinucleotide relative abundance profiles over multiple 50-kb disjoint contigs within the same genome. (The profile is rhoXY* = fXY*/fX*fY* for all XY, where fX* denotes the frequency of the nucleotide X and fY* denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complementary sequence.) On the basis of this constancy, we refer to the collection [rhoXY*] as the genome signature. We establish that the differences between [rhoXY*] vectors of 50-kb sample contigs of different genomes virtually always exceed the differences between those of the same genomes. Various di- and tetranucleotide biases are identified. In particular, we find that the dinucleotide CpG=CG is underrepresented in many thermophiles (e.g., M. jannaschii, Sulfolobus sp., and M. thermoautotrophicum) but overrepresented in halobacteria. TA is broadly underrepresented in prokaryotes and eukaryotes, but normal counts appear in Sulfolobus and P. aerophilum sequences. More than for any other bacterial genome, palindromic tetranucleotides are underrepresented in H. influenzae. The M. jannaschii sequence is unprecedented in its extreme underrepresentation of CTAG tetranucleotides and in the anomalous distribution of CTAG sites around the genome. Comparative analysis of numbers of long tetranucleotide microsatellites distinguishes H. influenzae. Dinucleotide relative abundance differences between bacterial sequences are compared. For example, in these assessments of differences, the cyanobacteria Synechocystis, Synechococcus, and Anabaena do not form a coherent group and are as far from each other as general gram-negative sequences are from general gram-positive sequences. The difference of M. jannaschii from low-G+C gram-positive proteobacteria is one-half of the difference from gram-negative proteobacteria. Interpretations and hypotheses center on the role of the genome signature in highlighting similarities and dissimilarities across different classes of prokaryotic species, possible mechanisms underlying the genome signature, the form and level of genome compositional flux, the use of the genome signature as a chronometer of molecular phylogeny, and implications with respect to the three putative eubacterial, archaeal, and eukaryote domains of life and to the origin and early evolution of eukaryotes.
Characterization of transcription organization and analysis of unique expression patterns of an alkyl hydroperoxide reductase C gene (ahpC) and the peroxide regulator operon ahpF-oxyR-orfX from Xanthomonas campestris pv. phaseoli
Authors: Mongkolsuk S , Loprasert S , Whangsuk W , Fuangthong M , Atichartpongkun S
Published: November 2016

We have analyzed the transcription organization of ahpC, ahpF, oxyR, and orfX from Xanthomonas campestris pv. phaseoli. ahpC was transcribed as a monocistronic 0.6-kb mRNA, while ahpF-oxyR-orfX were transcribed as a polycistronic approximately 3.0-kb-long mRNA. The novel transcription organization of these genes has not observed in other bacteria. Western analysis showed that oxidants (peroxides and superoxide anions), a thiol reagent (N-ethylmaleimide), and CdCl2 caused large increases in the steady-state level of AhpC. Growth at alkaline pH also moderately induced AhpC accumulation. Thermal and osmotic stresses did not alter the levels of AhpC. Northern blotting results confirmed that oxidant- and CdCl2-induced AhpC accumulation was due to increased levels of ahpC transcripts. Analysis of oxyR expression revealed a unique pattern. Unlike other bacterial systems, peroxides and a superoxide generator induced accumulation of OxyR. Northern blotting results confirmed that these oxidants induced expression of oxyR operon. This novel regulatory pattern could be generally important. The transcription organization and patterns of chemicals and stress induction of ahpC and oxyR differed from those of other bacteria and are likely to be important for X. campestris pv. phaseoli survival during exposure to oxidants.
Mutations in sdh (succinate dehydrogenase genes) alter the thiamine requirement of Salmonella typhimurium
Authors: Enos-Berlage J L , Downs D M
Published: November 2016

Mutants lacking the first enzyme in de novo purine synthesis (PurF) can synthesize thiamine if increased levels of pantothenate are present in the culture medium (J. L. Enos-Berlage and D. M. Downs, J. Bacteriol. 178:1476-1479, 1996). Derivatives of purF mutants that no longer required pantothenate for thiamine-independent growth were isolated. Analysis of these mutants demonstrated that they were defective in succinate dehydrogenase (Sdh), an enzyme of the tricarboxylic acid cycle. Results of phenotypic analyses suggested that a defect in Sdh decreased the thiamine requirement of Salmonella typhimurium. This reduced requirement correlated with levels of succinyl-coenzyme A (succinyl-CoA), which is synthesized in a thiamine pyrophosphate-dependent reaction. The effect of succinyl-CoA on thiamine metabolism was distinct from the role of pantothenate in thiamine synthesis.
A genetic locus involved in iron utilization unique to some Campylobacter strains
Authors: Guerry P , Perez-Casal J , Yao R , McVeigh A , Trust T J
Published: November 2016

Two genes involved in iron utilization in Campylobacter coli VC167 T1 have been characterized. The cfrA gene encodes a protein with a predicted Mr of 77,653 which, after processing of the leader sequence, has a predicted Mr of 75,635. This protein has significant sequence identity to siderophore receptors of several bacteria, and site-specific mutants defective in cfrA do not synthesize one of two major iron-repressible outer membrane proteins. An adjacent gene encodes a TonB-like protein; a mutant in this gene lost the ability to utilize hemin, ferrichrome, and enterochelin as iron sources. The cfrA and tonB genes of VC167 T1 hybridized to all strains of C. coli and most strains of C. jejuni examined but did not hybridize to several other strains of C. jejuni, suggesting that the thermophilic campylobacters can be separated into two categories based on the presence of these two iron utilization genes.
Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida
Authors: Morawski B , Casy G , Illaszewicz C , Griengl H , Ribbons D W
Published: November 2016

Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies.
Identification of the ahp operon of Salmonella typhimurium as a macrophage-induced locus
Authors: Francis K P , Taylor P D , Inchley C J , Gallagher M P
Published: November 2016

Previously, we tagged a macrophage-induced Salmonella typhimurium locus with Mudlux (K. P. Francis and M. P. Gallagher, Infect. Immun. 61:640-649, 1993). The insertion lies within the OxyR-regulated ahpC locus and conveys alkyl peroxide sensitivity. Plasmid-encoded ahp reverses sensitivity but reduces luminescence. This suggests that OxyR is titrated by the multicopy ahp promoter.
Characterization of the L-malate permease gene (maeP) of Streptococcus bovis ATCC 15352
Authors: Kawai S , Suzuki H , Yamamoto K , Kumagai H
Published: November 2016

A gene which was shown to be cotranscribed with the NAD+-dependent malic enzyme gene (maeE) of Streptococcus bovis ATCC 15352 was revealed to encode L-malate-specific permease (MaeP), which showed high activity at low pHs (pH 5.1 to 5.9). MaeP was strongly inhibited by the ionophores nigericin and valinomycin.
Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease
Authors: Moncrief M B , Hausinger R P
Published: November 2016

In vivo urease metallocenter assembly in Klebsiella aerogenes requires the presence of several accessory proteins (UreD, UreF, and UreG) and is further facilitated by UreE. In this study, UreG was isolated and shown to be a monomer with an Mr of 21,814 +/- 20 based on gel filtration chromatography and mass spectrometric results. Although it contains a P-loop motif typically found in nucleotide-binding proteins, UreG did not bind or hydrolyze ATP or GTP, and it exhibited no affinity for ATP- and GTP-linked agarose resins. Site-directed mutagenesis of ureG allowed the substitution of Ala for Lys-20 or Thr-21 in the P-loop motif and resulted in the production of inactive urease in cells grown in the presence of nickel; hence, an intact P-loop may be essential for UreG to function in vivo. These mutant cells were unable to synthesize the UreD-UreF-UreG-urease apoprotein species that are thought to be the key urease activation complexes in the cell. An insoluble protein species containing UreD, UreF, and UreG (termed the DFG complex) was detected in cells carrying deletions in ureE and the urease structural genes. The DFG complex was solubilized in 0.5% Triton X-100 detergent, shown to bind to an ATP-linked agarose resin, and found to elute from the resin in the presence of Mg-ATP. In cells containing a UreG P-loop variant, the DFG complex was formed but did not bind to the nucleotide-linked resin. These results suggest that the UreG P-loop motif may be essential for nucleotide binding by the DFG complex and support the hypothesis that nucleotide hydrolysis is required for in vivo urease metallocenter assembly.
6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases
Authors: Bouma C L , Reizer J , Reizer A , Robrish S A , Thompson J
Published: November 2016

The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U-14C]maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose. The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively. The 6-phospho-alpha-glucosidase expressed in E. coli (like the enzyme purified from F. mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air. Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E. coli was modulated by addition of various sugars to the growth medium. Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F. mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily. The cloned 2.2-kb Sau3AI DNA fragment from F. mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH. The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F. mortiferum.
Aerobic regulation of the sucABCD genes of Escherichia coli, which encode alpha-ketoglutarate dehydrogenase and succinyl coenzyme A synthetase: roles of ArcA, Fnr, and the upstream sdhCDAB promoter
Authors: Park S J , Chao G , Gunsalus R P
Published: November 2016

The sucABCD genes of Escherichia coli encode subunits for two enzymes of the tricarboxylic acid (TCA) cycle, alpha-ketoglutarate dehydrogenase (sucAB) and succinyl coenzyme A synthetase (sucCD). To examine how these genes are expressed in response to changes in oxygen and carbon availability, a set of sucA-lacZ, sucC-lacZ, sdhCDAB-sucA-lacZ, and sdhC-lacZ fusions were constructed and analyzed in vivo. While the expression of a sucA-lacZ fusion was low under all cell growth conditions tested, the expression of the sucA gene from the upstream sdhC promoter was considerably higher and varied by up to 14-fold depending on the carbon substrate used. Expression of the sdhCDAB-sucA-lacZ fusion varied by fourfold in response to oxygen. In contrast, no expression was seen from a sucC-lacZ reporter fusion, indicating that no promoter immediately precedes the sucCD genes. Taken together, these findings demonstrate that the oxygen and carbon control of sucABCD gene expression occurs by transcriptional regulation of the upstream sdhC promoter. The weaker sucA promoter provides an additional low constitutive level of sucABCD gene expression to supplement transcription from the sdhC promoter. The negative control of sucABCD gene expression seen under anaerobic conditions, like that for the sdhCDAB genes, is provided by the arcA and fnr gene products. These findings establish that the differential expression of eight genes for three of the TCA cycle enzymes in E. coli is controlled from one regulatory element.
Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli
Authors: Purcarea C , Hervé G , Ladjimi M M , Cunin R
Published: November 2016

The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae. Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.
Duplication of the pepF gene and shuffling of DNA fragments on the lactose plasmid of Lactococcus lactis
Authors: Nardi M , Renault P , Monnet V
Published: November 2016

The gene corresponding to the lactococcal oligopeptidase PepF1 (formerly PepF [V. Monnet, M. Nardi, A. Chopin, M.-C. Chopin, and J.-C. Gripon, J. Biol. Chem. 269:32070-32076, 1994]) is located on the lactose-proteinase plasmid of Lactococcus lactis subsp. cremoris NCDO763. Use of the pepF1 gene as a probe with different strains showed that pepF1 is present on the chromosome of Lactococcus lactis subsp. lactis IL1403, whereas there is a second, homologous gene, pepF2, on the chromosome of strain NCDO763. From hybridization, PCR amplification, and sequencing experiments, we deduced that (i) pepF1 and pepF2 exhibit 80% identity and encode two proteins which are 84% identical and (ii) pepF2 is included in an operon composed of three open reading frames and is transcribed from two promoters. The protein, encoded by the gene located downstream of pepF2, shows significant homology with methyltransferases. Analysis of the sequences flanking pepF1 and pepF2 indicates that only a part of the pepF2 operon is present on the plasmid of strain NCDO763, while the operon is intact on the chromosome of strain IL1403. Traces of several recombination events are visible on the lactose-proteinase plasmid. This suggests that the duplication of pepF occurred by recombination from the chromosome of an L. lactis subsp. lactis strain followed by gene transfer. We discuss the possible functions of PepF and the role of its amplification.
Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the beta subclass
Authors: Hurek T , Egener T , Reinhold-Hurek B
Published: November 2016

Nitrogenase is a functionally constant protein catalyzing N2 reduction, which is found in many phylogenetic lineages of Archaea and Bacteria. A phylogenetic analysis of nif genes may provide insights into the evolution of the bacterial genomes. Moreover, it may be used to study diazotrophic communities, when classical isolation techniques may fail to detect all contributing populations. Among six species of the genus Azoarcus, diazotrophic Proteobacteria of the beta subclass, the deduced amino acid sequences of nifH genes of two species were unusually divergent from each other. Nitrogenases of the "authentic" Azoarcus branch formed a monophyletic unit with those of gamma Proteobacteria, thus being in accordance with 16S ribosomal DNA phylogeny. The nitrogenase proteins of the two aberrant strains clustered within the alpha proteobacterial clade with rhizobial nitrogenases. This relationship was supported by bootstrap values of 87 to 98% obtained by various distance and maximum parsimony methods. Phylogenetic distances of NifH proteins indicate a possible lateral gene transfer of nif genes to Azoarcus from a common donor of the alpha subclass at the time of species diversification or several more recent, independent transfers. Application of the phylogenetic analysis to DNA isolated from environmental samples demonstrated novel habitats for Azoarcus: in guts of termites and rice grown in Japan, nifH genes belonging to the authentic Azoarcus branch were detected. This is the first evidence suggesting the occurrence of Azoarcus spp. in a plant other than its originally described host, Kallar grass. Moreover, evidence for expression of nif genes inside grass roots was obtained by in situ hybridization studies with antisense nifH probes.
epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus
Authors: Sugai M , Fujiwara T , Ohta K , Komatsuzawa H , Ohara M , Suginaka H
Published: November 2016

Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide.
Identification and localization of the Tgl protein, which is required for Myxococcus xanthus social motility
Authors: Rodriguez-Soto J P , Kaiser D
Published: November 2016

Tgl protein is required for the production of the type IV pili found at a pole of the Myxococcus xanthus cell. These pili are essential for social motility. Evidence is presented that Tgl is a membrane protein, based on experiments with polyclonal antibody specific for Tgl that was raised against the fusion proteins beta-galactosidase-Tgl and TrpE-Tgl. Immunoaffiity-purified antibody reacted with a protein in M. xanthus having an apparent molecular mass of 27.5 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sequence of the tgl gene translates into a polypeptide of 27 kDa. Although these numbers are close, it is likely that the primary tgl translation product is processed and modified in M. xanthus. The N terminus has a signal peptidase II recognition sequence, cleavage of which is expected to remove 19 amino acid residues. When the tgl gene is expressed in Escherichia coli, the protein product consistently migrates faster in the gel than mature Tgl expressed in M. xanthus, suggesting a second modification by addition which slows migration of the protein from M. xanthus. Tgl, as detected by its specific antibody, sediments with the membrane fraction of cells. It can be extracted with detergents but not with salt or by the addition of chelators for divalent cations. In an equilibrium gradient, Tgl bands at the buoyant density of membranes and with the NADH-oxidase activity. Intact cells failed to bind anti-Tgl antibody, and less than 2% of the total Tgl is released in soluble form from the periplasm. Yet, cells that had been osmotically shocked and treated with paraformaldehyde were able to react with the specific antibody--a reaction absent from cells with a deletion of the tgl transcription unit. Assuming that osmotic shock disrupts the outer membrane, the fractionation and localization data imply that Tgl is attached to the inner or outer membranes, from which it is exposed to the intermembranous space. Tgl is necessary for synthesis of pili in M. xanthus and is the only pilus protein that can be donated by other cells (stimulation). Tgl contains six tandem copies of the tetratrico peptide repeat structural motif. Its membrane localization, capacity for stimulation, and content of tetratrico structural repeats together suggest that Tgl may be necessary for the assembly of pilin subunits into filaments.
Characterization of thiI, a new gene involved in thiazole biosynthesis in Salmonella typhimurium
Authors: Webb E , Claas K , Downs D M
Published: November 2016

Thiamine pyrophosphate (TPP) is a required cofactor in Salmonella typhimurium that is generated de novo by the condensation of 4-amino-5-hydroxymethyl pyrimidine (HMP) pyrophosphate and 4-methyl-5-(beta-hydroxyethyl)-thiazole (THZ) monophosphate. The THZ and HMP moieties are independently synthesized, and labeling studies have demonstrated probable metabolic precursors to both. We present herein the initial characterization of thiI, a gene required for THZ synthesis. We show that thiI is a 1,449-bp open reading frame located at minute 9.65 on the S. typhimurium chromosome and that it encodes a 483-amino-acid protein with a predicted molecular mass of 55 kDa. Unlike genes in the thiamine biosynthetic operon at minute 90, thiI is not transcriptionally regulated by TPP.
Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans
Authors: Tsukioka Y , Yamashita Y , Nakano Y , Oho T , Koga T
Published: November 2016

We had isolated three genes (rmlA, rmlB, and rmlC) involved in dTDP-rhamnose synthesis in Streptococcus mutans and found that three genes were insufficient for dTDP-rhamnose synthesis (Y. Tsukioka, Y. Yamashita, T. Oho, Y. Nakano, and T. Koga, J. Bacteriol. 179:1126-1134, 1997). The rmlD gene of S. mutans, encoding the enzyme which catalyzes the last step of dTDP-rhamnose synthesis, has been cloned and sequenced. The cell extract of Escherichia coli expressing the rmlD gene of S. mutans exhibited enzymatic activity corresponding to its counterpart in Shigella flexneri, a gram-negative bacterium. Rhamnose was not detected in the cell wall preparation purified from the mutant in which the cloned gene was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with autoclaved extracts from the mutant. The rmlD gene product of S. mutans compensated for the incompleteness of dTDP-rhamnose synthesis by the three previously isolated genes. These results indicate that the rmlD gene product is indispensable for the dTDP-rhamnose pathway and subsequently for the synthesis of serotype-specific antigen in S. mutans. Furthermore, conservation of the rmlD gene in Streptococcus species was demonstrated by Southern blot analysis.
Mck1, a member of the glycogen synthase kinase 3 family of protein kinases, is a negative regulator of pyruvate kinase in the yeast Saccharomyces cerevisiae
Authors: Brazill D T , Thorner J , Martin G S
Published: November 2016

An interaction between the Saccharomyces cerevisiae protein kinase Mck1 and pyruvate kinase (Pyk1) was detected by using the two-hybrid method. Purified Mck1 was able to phosphorylate purified Pyk1 on Ser in vitro. Pyruvate kinase activity was elevated in mck1 delta cells. Several of the phenotypes of mck1 delta mutants are similar to those observed in cells overexpressing PYK1. Co-overexpression of MCK1 suppressed all of the phenotypes associated with PYK1 overexpression. These results indicate that Mck1 negatively regulates pyruvate kinase activity, possibly by direct phosphorylation.
Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome
Authors: Pósfai G , Koob M D , Kirkpatrick H A , Blattner F R
Published: November 2016

A system of versatile insertion plasmids was constructed that permits efficient delivery of the target sites of an ultra-rare-cutting endonuclease and the recombinase FLP into preselected sites of the bacterial genome. With the help of this system, the pathogenicity island LEE of the Escherichia coli O157:H7 genome was excised and isolated in vitro, deleted in vivo, rescued as a plasmid, and transferred into another strain.
Evolutionary analysis of the hisCGABdFDEHI gene cluster from the archaeon Sulfolobus solfataricus P2
Authors: Charlebois R L , Sensen C W , Doolittle W F , Brown J R
Published: November 2016

While sequencing the genome of the archaeon Sulfolobus solfataricus P2, we found an 8,313-bp sequence containing a cluster of nine histidine biosynthesis genes in an order different from that of any known his operon. Results of phylogenetic analysis of the coding regions in the putative operon give conflicting evolutionary histories for individual his genes.
Characterization of a second lysine decarboxylase isolated from Escherichia coli
Authors: Kikuchi Y , Kojima H , Tanaka T , Takatsuka Y , Kamio Y
Published: November 2016

We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.
Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis
Authors: Schiött T , Throne-Holst M , Hederstedt L
Published: November 2016

Cytochromes of the c type contain covalently bound heme. In bacteria, they are located on the outside of the cytoplasmic membrane. Cytochrome c synthesis involves export of heme and apocytochrome across the cytoplasmic membrane followed by ligation of heme to the polypeptide. Using radioactive protoheme IX produced in Escherichia coli, we show that Bacillus subtilis can use heme from the growth medium for cytochrome c synthesis. The B. subtilis ccdA gene encodes a 26-kDa integral membrane protein which is required for cytochrome c synthesis (T. Schiött et al., J. Bacteriol. 179:1962-1973, 1997). In this work, we analyzed the stage at which cytochrome c synthesis is blocked in a ccdA deletion mutant. The following steps were found to be normal in the mutant: (i) transcription and translation of cytochrome c structural genes, (ii) translocation of apocytochrome across the cytoplasmic membrane, and (iii) heme transport from the cytoplasm to cytochrome polypeptide on the outer side of the cytoplasmic membrane. It is concluded that CcdA is required for a late step in the cytochrome c synthesis pathway.
Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene
Authors: Mars A E , Kasberg T , Kaschabek S R , van Agteren M H , Janssen D B , Reineke W
Published: November 2016

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.
Occurrence of deletions, associated with genetic instability in Streptomyces ambofaciens, is independent of the linearity of the chromosomal DNA
Authors: Fischer G , Decaris B , Leblond P
Published: November 2016

The chromosomal structures of mutant strains of Streptomyces ambofaciens which have arisen from genetic instability were investigated by using pulsed-field gel electrophoresis and probing with sequences cloned from the unstable region which maps near the ends of the linear chromosomal DNA. The chromosomal structures of seven mutant strains harboring large deletions were classified into three types. (i) Deletions internal to one chromosomal arm were characterized in two of the mutant strains. In these strains, a linear chromosomal structure was retained, as were parts of the terminal inverted repeats sequences (TIRs) and the proteins bound to them. (ii) Four of the mutants presented a deletion including all sequences from the TIRs. A junction fragment homologous to sequences originating from internal region of both arms was characterized. Consequently, the chromosomal DNA of these strains was deduced to be circularized. Furthermore, chromosomal stability was assessed in the progeny of these circular DNA mutants. Additional deletion events were detected in 11 mutants among the 13 strains isolated, demonstrating that circular chromosomes do not correspond to a stabilization of the chromosome structure and that the occurrence of deletion could be independent of the presence of chromosomal ends. (iii) A mutant with DNA amplification was shown to have a linear chromosome with a deletion of all sequences between the amplified region and the end of the chromosome. The other chromosomal arm remained unaffected by deletion and associated with protein.
Cloning, sequencing, and oxygen regulation of the Rhodobacter capsulatus alpha-ketoglutarate dehydrogenase operon
Authors: Dastoor F P , Forrest M E , Beatty J T
Published: November 2016

The Rhodobacter capsulatus sucA, sucB, and lpd genes, which encode the alpha-ketoglutarate dehydrogenase (E1o), the dihydrolipoamide succinyltransferase (E2o), and the dihydrolipoamide dehydrogenase (E3) components of the alpha-ketoglutarate dehydrogenase complex (KGD), respectively, were cloned, sequenced, and used for regulatory analyses. The KGD enzymatic activity was greater in cells grown under aerobic, respiratory growth conditions than under anaerobic, photosynthetic conditions. Similarly, the sucA gene was transcribed differentially, leading to a greater accumulation of sucA mRNAs under respiratory growth conditions than under photosynthetic conditions, although differential rates of mRNA decay could also contribute to the different amounts of sucA mRNAs under these two growth conditions. The sucA promoter was located about 4 kb upstream of the 5' end of the sucA gene, and transcripts greater than 9.5 kb hybridized to a sucA probe, suggesting the presence of an operon that produces a polycistronic mRNA. Thus, these genes seem to be expressed as an unstable primary transcript, and we speculate that posttranscriptional processes control the stoichiometry of KGD proteins.
H-NS and RpoS regulate emergence of Lac Ara+ mutants of Escherichia coli MCS2
Authors: Gómez-Gómez J M , Blázquez J , Baquero F , Martínez J L
Published: November 2016

Two master growth-phase regulatory proteins, H-NS and sigmaS, are involved in the formation of araB-lacZ fusion clones of Escherichia coli MCS2. The stationary-phase sigma factor RpoS is strictly required for the appearance of such mutants, whereas the histone-like protein H-NS represses their emergence. Our results support the idea that genetic changes leading to adaptive mutation in this model system are regulated by physiological signal transduction networks.
Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant
Authors: Kortlüke C , Breese K , Gad'on N , Labahn A , Drews G
Published: November 2016

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.
Redox buffering by melanin and Fe(II) in Cryptococcus neoformans
Authors: Jacobson E S , Hong J D
Published: November 2016

Melanin is a fungal extracellular redox buffer which, in principle, can neutralize antimicrobial oxidants generated by immunologic effector cells, but its source of reducing equivalents is not known. We wondered whether Fe(II) generated by the external ferric reductase of fungi might have the physiologic function of reducing fungal melanin and thereby promoting pathogenesis. We observed that exposure of a melanin film electrode to reductants decreased the open-circuit potential (OCP) and reduced the area of a cyclic voltammetric reduction wave whereas exposure to oxidants produced the opposite effects. Exposure to 10, 100, 1,000 or 10,000 microM Fe(II) decreased the OCP of melanin by 0.015, 0.038, 0.100, and 0.120 V, respectively, relative to a silver-silver chloride standard, and decreased the area of the cyclic voltammetric reduction wave by 27, 35, 50, and 83%, respectively. Moreover, exposure to Fe(II) increased the buffering capacity by 44%, while exposure to millimolar dithionite did not increase the buffering capacity. The ratio of the amount of bound iron to the amount of the incremental increase in the following oxidation wave was approximately 1.0, suggesting that bound iron participates in buffering. Light absorption by melanin suspensions was decreased 14% by treatment with Fe(II), consistent with reduction of melanin. Light absorption by suspensions of melanized Cryptococcus neoformans was decreased 1.3% by treatment with Fe(II) (P < 0.05). Cultures of C. neoformans generated between 2 and 160 microM Fe(II) in culture supernatant, depending upon the strain and the conditions [the higher values were achieved by a constitutive ferric reductase mutant in high concentrations of Fe(III)]. We infer that Fe(II) can reduce melanin under physiologic conditions; moreover, it binds to melanin and cooperatively increases redox buffering. The data support a model for physiologic redox cycling of fungal melanin, whereby electrons exported by the yeast to form extracellular Fe(II) maintain the reducing capacity of the extracellular redox buffer.
Biochemical basis for glucose-induced inhibition of malolactic fermentation in Leuconostoc oenos
Authors: Miranda M , Ramos A , Veiga-da-Cunha M , Loureiro-Dias M C , Santos H
Published: November 2016

The sugar-induced inhibition of malolactic fermentation in cell suspensions of Leuconostoc oenos, recently reclassified as Oenococcus oeni (L. M. T. Dicks, F. Dellaglio, and M. D. Collins, Int. J. Syst. Bacteriol. 45:395-397, 1995) was investigated by in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and manometric techniques. At 2 mM, glucose inhibited malolactic fermentation by 50%, and at 5 mM or higher it caused a maximum inhibitory effect of ca. 70%. Galactose, trehalose, maltose, and mannose caused inhibitory effects similar to that observed with glucose, but ribose and 2-deoxyglucose did not affect the rate of malolactic activity. The addition of fructose or citrate completely relieved the glucose-induced inhibition. Glucose was not catabolized by permeabilized cells, and inhibition of malolactic fermentation was not observed under these conditions. 31P NMR analysis of perchloric acid extracts of cells obtained during glucose-malate cometabolism showed high intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate, and glycerol-3-phosphate. Glucose-6-phosphate, 6-phosphogluconate, and NAD(P)H inhibited the malolactic activity in permeabilized cells or cell extracts, whereas NADP+ had no inhibitory effect. The purified malolactic enzyme was strongly inhibited by NADH, whereas all the other above-mentioned metabolites exerted no inhibitory effect, showing that NADH was responsible for the inhibition of malolactic activity in vivo. The concentration of NADH required to inhibit the activity of the malolactic enzyme by 50% was ca. 25 microM. The data provide a coherent biochemical basis to understand the glucose-induced inhibition of malolactic fermentation in L. oenos.
DNA bending by the TrpI protein of Pseudomonas aeruginosa
Authors: Piñeiro S , Olekhnovich I , Gussin G N
Published: November 2016

TrpI protein, the activator of transcription of the trpBA operon of fluorescent pseudomonads, bends the DNA when it forms either of two well-characterized complexes with the trpBA regulatory region. In complex 1, with TrpI bound only to its strong binding site (site I), the calculated bending angle is 65 to 67 degrees and the center of bending is in the middle of site I. In complex 2, which is required for activation of the trpBA promoter, with TrpI bound both to site I and to the weaker site II, the bending angle is increased to 89 to 90 degrees and the center of bending is at the site I-site II boundary. Indoleglycerol phosphate (InGP), which strongly stimulates formation of complex 2 and is required for activation, does not affect the bending angle of either complex. However, a mutation (-10C/11C) shown previously to affect activation has a small but detectable effect on bending, reducing the calculated bending angle to 83 to 86 degrees. These results suggest a way that DNA bending and InGP may be important for activation.
Differential levels of specific cytochrome c biogenesis proteins in response to oxygen: analysis of the ccl operon in Rhodobacter capsulatus
Authors: Gabbert K K , Goldman B S , Kranz R G
Published: November 2016

The photosynthetic bacterium Rhodobacter capsulatus synthesizes c-type cytochromes under a variety of growth conditions. For example, under aerobic growth, c-type cytochromes are synthesized as part of an electron transport pathway, using oxygen as the terminal electron acceptor. Anaerobically in the light, R. capsulatus requires cytochrome bc1 and other c-type cytochromes for the photosynthetic electron transport pathway. It is shown here that the ccl1 and ccl2 genes of R. capsulatus are required for the synthesis of all c-type cytochromes, including the cytochrome c' protein of unknown function but of structural similarity to cytochrome b562. Polar and nonpolar mutations constructed in each gene demonstrated that the ccl12 genes form an operon. Expression of the ccl12 genes was examined by using lacZ and phoA fusions as translational reporters. Primer extension analysis was used to determine transcriptional control and the start site of the ccl12 promoter. Finally, antiserum to the Ccl2 protein was used to quantitate levels of Ccl2 under six different growth conditions. The Ccl2 protein is present at 20-fold-higher levels under conditions where oxygen is present. In contrast, other cytochromes c biogenesis proteins, HelA and HelX, previously shown to be part of an helABCDX operon, are at relatively similar levels under these six growth conditions. This discovery is discussed in terms of the physiology and evolution of cytochromes c biogenesis, with particular attention to oxidative environments.
Mutation of a single MalK subunit severely impairs maltose transport activity in Escherichia coli
Authors: Davidson A L , Sharma S
Published: November 2016

The maltose transport system of Escherichia coli, a member of the ABC transport superfamily of proteins, consists of a periplasmic maltose binding protein and a membrane-associated translocation complex that contains two copies of the ATP-binding protein MalK. To examine the need for two nucleotide-binding domains in this transport complex, one of the two MalK subunits was inactivated by site-directed mutagenesis. Complexes with mutations in a single subunit were obtained by attaching a polyhistidine tag to the mutagenized version of MalK and by coexpressing both wild-type MalK and mutant (His)6MalK in the same cell. Hybrid complexes containing one mutant (His)6MalK subunit and one wild-type MalK subunit were separated from those containing two mutant (His)6MalK proteins based on differential affinities for a metal chelate column. Purified transport complexes were reconstituted into proteoliposome vesicles and assayed for maltose transport and ATPase activities. When a conserved lysine residue at position 42 that is involved in ATP binding was replaced with asparagine in both MalK subunits, maltose transport and ATPase activities were reduced to 1% of those of the wild type. When the mutation was present in only one of the two subunits, the complex had 6% of the wild-type activities. Replacement of a conserved histidine residue at position 192 in MalK with arginine generated similar results. It is clear from these results that two functional MalK proteins are required for transport activity and that the two nucleotide-binding domains do not function independently to catalyze transport.