Nature of the hisD3018 Frameshift Mutation in Salmonella typhimurium

Histidinol dehydrogenase from three differing revertants of ICR-191A-induced frameshift hisD3018 has been purified and examined for amino acid replacements. The enzyme from one spontaneously arising revertant, R7, contains an extra proline residue, whereas that from another, R5, contains an extensive frameshifted sequence, four amino acid residues of which have been identified to date. The amino acid replacement data are in agreement with the in vitro code word assignments and allow the characterization of the hisD3018 frameshift as an addition of one nucleotide pair, most likely guanine plus cytosine. Enzymatic data for those ICR-191A-induced revertants of hisD3018 arising within the hisD gene indicate that the enzyme is wild type and, therefore, that ICR-191A can cause deletions as well as additions of single base pairs. The wild-type amino acid sequence is restored in enzyme from an N-methyl-N′-nitro-N-nitrosoguanidine (NG)-induced revertant, R29, suggesting that NG is a base-deleting as well as a base-substituting mutagen. The unusual response of hisD3018 to external suppressors is considered in terms of reinitiation of protein synthesis out of phase, coupled with suppression of a nonpermissive missense codon so generated, and of an alternative hypothesis invoking a true frameshift suppressor transfer ribonucleic acid with an extended or deleted anticodon.     

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.     

Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.     

Bilophila wadsworthia in Ear Infections: A Report of Three Cases

During a 3-year period routine anaerobic cultures were examined for the presence of Bilophila wadsworthia. Using taurine-supplemented Bacteroides–Bile–Esculin agar for isolation, we observed three cases of complicated polybacterial ear infections where B. wadsworthia was involved. The first case involved a 69-year-old female patient presenting an otitis externa following stapedectomy where B. wadsworthia was isolated. The second patient, 30-year-old woman, with a 2-decade history of otitis and otorrhoea presented with a cholesteatoma, complicated by brain abscess formation and B. wadsworthia was isolated from the purulent ear secretion as well as from the abscess material. The third case, a 39-year-old male patient suffering from cholesteatoma presented with otorrhoea and otalgia, B. wadsworthia was isolated from purulent ear secretion. In all cases, B. wadsworthia was part of mixed aerobic–anaerobic infections. Because this species was not found in 200 ear swabs from 100 healthy volunteers and was not detectable in throat swabs or saliva from of these patients, an exogenous origin of these outer and middle ear infections as well as an infection by fecal contamination seems more probable than ascending infections from the pharynx or the ear canal.     

Differentiating Coptis chinensis from Coptis japonica and other Coptis species used in Coptidis Rhizoma based on partial trnL-F intergenic spacer sequences

Dried rhizomes of Coptis species are utilized as “Coptidis Rhizoma” (CR), an important herbal medicinal material in traditional Chinese medicine. Almost all CRs traded in the Korean herbal medicine market originate from Coptis chinensis (“Chun Hwang-Lyun” in Korean medical terminology). Other minor CRs originate from Coptis japonica (“Il Hwang-Lyun”). Although there is an obvious discrepancy in the price of traded CRs in the herbal market depending on the Coptis species, CRs originating from C. chinensis and C. japonica are often confused. Furthermore, the CR traded as “Chun Hwang-Lyun” is occasionally mixed with rhizomes of Coptis deltoidea and/or Coptis omeiensis. Therefore, we sought to discriminate C. chinensis from C. japonica, as well as C. deltoidea and C. omeiensis, by using nucleotide sequence differences in the partial trnL-F intergenic spacer. We developed an efficient real-time polymerase chain reaction (PCR)-based discrimination assay to separate samples of C. chinensis from those of C. japonica without the need to separate the DNA markers by using gel electrophoresis. In addition, we developed a multiplex PCR method with which we were able to discriminate samples of C. chinensis from those of C. deltoidea and C. omeiensis by amplifying the 153-bp DNA marker in C. chinensis in a single PCR process.     

Mineralization of Paraoxon and Its Use as a Sole C and P Source by a Rationally Designed Catabolic Pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into β-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

Early Carboniferous spiriferoid brachiopods from the Qaidam Basin,Northwest China: Taxonomy,biostratigraphy and biogeography

Six Early Carboniferous brachiopod species in four genera of the Superfamily Spiriferoidea are described from the Qaidam Basin, northwestern China, including a new genus, Qaidamospirifer, and two new species: Grandispirifer qaidamensis and Qaidamospirifer elongatus. Additionally, a new genus, Triangulospirifer, is also proposed to replace Triangularia Poletaev, 2001 that was preoccupied by a Devonian molluscan genus.On the basis of the new material as well as published information, we have reviewed the taxonomic composition and the stratigraphic and palaeobiogeographic distributions of the three previously established genera from the viewpoint of palaeobiogeography. The study reveals that Grandispirifer has a relatively long stratigraphic range from the late Tournaisian to Serpukhovian. During this interval, the genus attained a wide geographical distribution, reaching Northwest China, western Yunnan of West China, Japan, as well as Iran and North Africa. Angiospirifer first occurred in western Europe in the Viséan, and later migrated to North Africa during the late Viséan. In the Serpukhovian, it migrated eastward, reaching the Donets Basin of Ukraine and the Qaidam Basin in Northwest China. Anthracothyrina evolved from Brachythyrina in North Africa in late Viséan, then dispersed north-westward to western and eastern Europe and, further eastward to the Qaidam Basin during the Serpukhovian.     

Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

Pyocyanin: production,applications, challenges and new insights

Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium and is one of the most commercially and biotechnologically valuable microorganisms. Strains of P. aeruginosa secrete a variety of redox-active phenazine compounds, the most well studied being pyocyanin. Pyocyanin is responsible for the blue-green colour characteristic of Pseudomonas spp. It is considered both as a virulence factor and a quorum sensing signalling molecule for P. aeruginosa. Pyocyanin is an electrochemically active metabolite, involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognised as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. This review summarises recent advances of pyocyanin production from P. aeruginosa with special attention to antagonistic property and bio-control activity. The review also covers the challenges and new insights into pyocyanin from P. aeruginosa.     

Cordyceps cuncunae (Ascomycota, Hypocreales), a new pleoanamorphic species from temperate rainforest in southern Chile

Cordyceps cuncunae Palfner sp. nov. is reported from Valdivian rainforest in southern Chile, parasiting larvae of an unidentified ghost moth species (Lepidoptera, Hepialidae) which probably feed on roots of Laureliopsis philippiana. Morphology and anatomy of stromata as well as morphological and molecular characteristics of mycelium in pure culture which produces two anamorphs, one of them Lecanicillium-like, are described. The systematic position of the new taxon within the most recent generic concept is discussed. This is the first record of an endemic Cordyceps species from Chile.     

Phosphate Starvation-Inducible Gene ushA Encodes a 5′ Nucleotidase Required for Growth of Corynebacterium glutamicum on Media with Nucleotides as the Phosphorus Source

Phosphorus is an essential component of macromolecules, like DNA, and central metabolic intermediates, such as sugar phosphates, and bacteria possess enzymes and control mechanisms that provide an optimal supply of phosphorus from the environment. UDP-sugar hydrolases and 5′ nucleotidases may play roles in signal transduction, as they do in mammals, in nucleotide salvage, as demonstrated for UshA of Escherichia coli, or in phosphorus metabolism. The Corynebacterium glutamicum gene ushA was found to encode a secreted enzyme which is active as a 5′ nucleotidase and a UDP-sugar hydrolase. This enzyme was synthesized and secreted into the medium when C. glutamicum was starved for inorganic phosphate. UshA was required for growth of C. glutamicum on AMP and UDP-glucose as sole sources of phosphorus. Thus, in contrast to UshA from E. coli, C. glutamicum UshA is an important component of the phosphate starvation response of this species and is necessary to access nucleotides and related compounds as sources of phosphorus.     

Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.     

Propionate-Degrading Bacterium, Syntrophobacter wolinii sp. nov. gen. nov., from Methanogenic Ecosystems

A new genus and species of a nonmotile gram-negative rod, Syntrophobacter wolinii, is the first bacterium described which degrades propionate only in coculture with an H₂-using organism and in the absence of light or exogenous electron acceptors such as O₂, sulfate, or nitrate. It was isolated from methanogenic enrichments from an anaerobic municipal sewage digestor, using anaerobic roll tubes containing a medium with propionate as the energy source in association with an H₂-using, sulfate-reducing Desulfovibrio sp. which cannot utilize fatty acids other than formate. S. wolinii produced acetate and, presumably, CO₂ and H₂ (or formate) from propionate. In media without sulfate and with Methanospirillum hungatei, a methanogen that uses only H₂-CO₂ or formate as an energy source, acetate, methane, and, presumably, CO₂ were produced from propionate and only small amounts of Desulfovibrio sp. were present. Isolation in coculture with the methanogen was not successful. S. wolinii does not use other saturated fatty acids as energy sources.     

Identification of the UUG codon as a translational initiation codon in vivo

The lac repressor protein was purified from an Escherichia coli strain carrying an amber mutation in the lacI gene and the tyrosine-inserting amber suppressor, Su3. Protein sequencing showed a change at position 62 in the repressor polypeptide chain from leucine to tyrosine, proving that the amber was derived from a UUG codon at this point in the message. This establishes UUG as an initiation codon in vivo, since it has been previously shown that translational reinitiation can occur at position 62.     

Edge lengths of trees from sequence data

Frequently there is a need to determine lengths associated with each edge of a phylogenetic tree, as these are often used as an indication of relative time intervals. Where this tree has been constructed from sequence data of r characters for n taxa, using the maximum parsimony model, an edge length can be determined from the differences between the inferred sequences of the end vertices of that edge. These inferred sequences are often not uniquely defined; a range of possible sequences are possible at a given internal vertex. In this paper we introduce an efficient [O(r×n)] algorithm which calculates the range of lengths on any edge over all the minimal labelings and significantly reduces the number of potential cases to be considered to obtain an objective measure of edge length.     

SU3, an oxocycloartane diglucoside from Sutherlandia humilis

The structure of a new cycloartane-type triterpene glycoside was determined as 24,25-O-β-d-diglucopyranosyl-6α-hydroxycycloart-3-one (SU3) by spectroscopic methods. This is the first cycloartane diglycoside reported from the genus Sutherlandia (an important South African traditional medicine and general tonic known as cancer bush). It was isolated from a dwarf form of S. frutescens, currently known as Sutherlandia humilis.     

Cambrian stem-group annelids and a metameric origin of the annelid head

The oldest fossil annelids come from the Early Cambrian Sirius Passet and Guanshan biotas and Middle Cambrian Burgess Shale. While these are among the best preserved polychaete fossils, their relationship to living taxa is contentious, having been interpreted either as members of extant clades or as a grade outside the crown group. New morphological observations from five Cambrian species include the oldest polychaete with head appendages, a new specimen of Pygocirrus from Sirius Passet, and an undescribed form from the Burgess Shale. We propose that the palps of Canadia are on an anterior segment bearing neuropodia and that the head of Phragmochaeta is formed of a segment bearing biramous parapodia and chaetae. The unusual anatomy of these taxa suggests that the head is not differentiated into a prostomium and peristomium, that palps are derived from a modified parapodium and that the annelid head was originally a parapodium-bearing segment. Canadia, Phragmochaeta and the Marble Canyon annelid share the presence of protective notochaetae, interpreted as a primitive character state subsequently lost in Pygocirrus and Burgessochaeta, in which the head is clearly differentiated from the trunk.     

Isolation and characterisation of λtra transducing phages from EDFL223 (Flac traB::EDλ4)

A series of λ transducing phages carrying transfer (tra) genes have been isolated from an abnormal lysogen where the λ prophage was inserted into the traB gene of Flac. These have been characterised genetically by complementation analysis with Flac tra point mutants, and by studying tra gene expression during λ repression. Taken together, the phages cover the entire transfer region except for oriT and traI. The tra genes were expressed from the λ promoter pl in all cases, as well as from the tra promoters when these were present. Evidence was obtained suggesting that in F there is a separate promoter for traI. DNA isolated from the λtra phages was characterised physico-chemically by determination of the endonuclease R.EcoRI fragmentation pattern, and by electron microscope heteroduplex analysis. These data were used to construct an improved physical map of the transfer region.     

Expression of the xylan-degrading genes of Bacillus pumilus IPO in Saccharomyces cerevisiae

Xylanase (xynA) and β-xylosidase (xynB) genes of Bacillus pumilus were expressed in Saccharomyces cerevisiae by using the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae. Yeast cells harboring a plasmid pNAX2 containing xynA produced xylanase in the cytoplasm of the cell to an extent as much as 5% of the total soluble protein in the cell extract. Xylanase produced in yeast had an extra methionine at the N-terminus, but had the same specific activity as that produced by B. pumilus IPO. The xylanase in the yeast was not glycosylated and was immunologically identical to that of B. pumilus IPO. Yeast cells harboring a plasmid pYXB containing xynB produced β-xylosidase in the cytoplasm of the cell (3% of the total soluble protein). β-Xylosidase purified from the yeast strain exhibited specific activity nearly equal to the value of enzyme purified from B. pumilus, and had an N-terminal sequence identical to the sequence of the enzyme from B. pumilus.