bluuebunny/crossref_metadata_2025 huggingface dataset:
An observational study of the microbiome of the maternal pouch and saliva of the tammar wallaby, Macropus eugenii, and of the gastrointestinal tract of the pouch young
Authors: Chhour Kim-Ly , Hinds Lyn A. , Jacques Nicholas A. , Deane Elizabeth M.
Published: November 2009

Marsupial mammals, born in an extremely atricial state with no functional immune system, offer a unique opportunity to investigate both the developing microbiome and its relationship to that of the mother and the potential influence of this microbiome upon the development of the immune system. In this study we used a well-established marsupial model animal, Macropus eugenii, the tammar wallaby, to document the microbiome of three related sites: the maternal pouch and saliva, and the gastrointestinal tract (GIT) of the young animal. We used molecular-based methods, targeting the 16S rDNA gene to determine the bacterial diversity at these study sites. In the maternal pouch, 41 unique phylotypes, predominantly belonging to the phylum Actinobacteria, were detected, while in the saliva, 48 unique phylotypes were found that predominantly belonged to the phylum Proteobacteria. The GIT of the pouch young had a complex microbiome of 53 unique phylotypes, even though the pouch young were still permanently attached to the teat and had only been exposed to the external environment for a few minutes immediately after birth while making their way from the birth canal to the maternal pouch. Of these 53 phylotypes, only nine were detected at maternal sites. Overall, the majority of bacteria isolated were novel species (<97 % identity to known 16S rDNA sequences), and each study site (i.e. maternal pouch and saliva, and the GIT of the pouch young) possessed its own unique microbiome.
Null mutation analysis of an afsA-family gene, barX, that is involved in biosynthesis of the γ-butyrolactone autoregulator in Streptomyces virginiae
Authors: Lee Yong Jik , Kitani Shigeru , Nihira Takuya
Published: November 2009

Virginiae butanolide (VB) is a γ-butyrolactone autoregulator that triggers production of the streptogramin antibiotic virginiamycin in Streptomyces virginiae. Our previous studies suggested that the barX gene, an afsA-family gene, is likely to participate in the regulatory pathway for the production of VB, rather than in the biosynthetic pathway of VB itself, in contrast to the function of other afsA-family genes. Mutation analysis now shows that BarX at least plays an enzymic role in the VB biosynthetic pathway. Heterologous expression of the afsA gene from Streptomyces griseus into the barX mutant partially restored the deficiency of virginiamycin production, suggesting that afsA-family genes have a common ability to synthesize the γ-butyrolactone autoregulators. Taken together with previous works relating to the function of an afsA-family gene, these results support the idea that streptomycetes have two biosynthetic pathways for the γ-butyrolactone autoregulators.
Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome
Authors: Kunze Markus , Zerlin Kay F. , Retzlaff Alexander , Pohl Jens O. , Schmidt Eberhard , Janssen Dick B. , Vilchez-Vargas Ramiro , Pieper Dietmar H. , Reineke Walter
Published: November 2009

Pseudomonas putidaGJ31 has been reported to grow on chlorobenzene using ameta-cleavage pathway with chlorocatechol 2,3-dioxygenase (CbzE) as a key enzyme. The CbzE-encoding gene was found to be localized on the 180 kb plasmid pKW1 in acbzTEXGScluster, which is flanked by transposases and encodes only a partial (chloro)catecholmeta-cleavage pathway comprising ferredoxin reductase, chlorocatechol 2,3-dioxygenase, an unknown protein, 2-hydroxymuconic semialdehyde dehydrogenase and glutathioneS-transferase. Downstream ofcbzTEXGSare locatedcbzJ, encoding a novel type of 2-hydroxypent-2,4-dienoate hydratase, and a transposon region highly similar to Tn5501. Upstream ofcbzTEXGS,traNEOFGtransfer genes were found. The search for gene clusters possibly completing the (chloro)catechol metabolic pathway of GJ31 revealed the presence of two additional catabolic gene clusters on pKW1. ThemhpRBCDFETPcluster encodes enzymes for the dissimilation of 2,3-dihydroxyphenylpropionate in a novel arrangement characterized by the absence of a gene encoding 3-(3-hydroxyphenyl)propionate monooxygenase and the presence of a GntR-type regulator, whereas thenahINLOMKJcluster encodes part of the naphthalene metabolic pathway. Transcription studies supported their possible involvement in chlorobenzene degradation. The upper pathway cluster, comprising genes encoding a chlorobenzene dioxygenase and a chlorobenzene dihydrodiol dehydrogenase, was localized on the chromosome. A high level of transcription in response to chlorobenzene revealed it to be crucial for chlorobenzene degradation. The chlorobenzene degradation pathway in strain GJ31 is thus a mosaic encoded by four gene clusters.
myo-Inositol transport by Salmonella enterica serovar Typhimurium
Authors: Kröger Carsten , Stolz Jürgen , Fuchs Thilo M.
Published: November 2009

InSalmonella entericaserovar Typhimurium, the genomic island GEI4417/4436 has recently been identified to be responsible formyo-inositol (MI) utilization. Here, two of the four island-encoded permeases are identified as the MI transporters of this pathogen. In-frame deletion ofiolT1(STM4418) led to a severe growth defect, and deletion ofiolT1(STM4419) to a slight growth defect in the presence of MI. These phenotypes could be complemented by providing the putative transporter genesin trans.Bioluminescence-based reporter assays demonstrated a strong induction of their promoters PiolT1and PiolT2in the presence of MI but not of glucose. Deletion ofiolR, which encodes the negative regulator of most genes involved in MI degradation, resulted in upregulation of PiolT1and PiolT2, indicating that the expression of IolT1 and IolT2 is repressed by IolR. This finding was supported by bandshift assays using purified IolR. Both transporters are located in the membrane when expressed inEscherichia coli. Heterologously expressed IolT1 had its optimal activity at pH 5.5. Together with the strongly reduced MI uptake in the presence of protonophores, this indicates that IolT1 operates as a proton symporter. Usingmyo-[1,2-[3H](N)]inositol, a saturable uptake activity of IolT1 with aKmvalue between 0.49 and 0.79 mM was determined in DH5αexpressing IolT1, inS. entericaserovar Typhimurium strain 14028, and in mutant 14028 ΔiolT2. Phylogenetic analysis of IolT1 identified putative MI transporters in Gram-negative bacteria also able to utilize MI.
Transcriptional profiling and functional analysis of heterokaryon incompatibility in Neurospora crassa reveals that reactive oxygen species, but not metacaspases, are associated with programmed cell death
Authors: Hutchison Elizabeth , Brown Sarah , Tian Chaoguang , Glass N. Louise
Published: November 2009

Heterokaryon incompatibility (HI) is a nonself recognition phenomenon occurring in filamentous fungi that is important for limiting resource plundering and restricting viral transfer between strains. Nonself recognition and HI occurs during hyphal fusion between strains that differ athetloci. If two strains undergo hyphal fusion, but differ in allelic specificity at ahetlocus, the fusion cell is compartmentalized and undergoes a rapid programmed cell death (PCD). Incompatible heterokaryons show a macroscopic phenotype of slow growth and diminished conidiation, and a microscopic phenotype of hyphal compartmentation and cell death. To understand processes associated with HI and PCD, we used whole-genome microarrays forNeurospora crassato assess transcriptional differences associated with induction of HI mediated by differences inhet-c pin-chaplotype. Our data show that HI is a dynamic and transcriptionally active process. The production of reactive oxygen species is implicated in the execution of HI and PCD inN. crassa, as are several genes involved in phosphatidylinositol and calcium signalling pathways. However, genes encoding mammalian homologues of caspases or apoptosis-inducing factor (AIF) are not required for HI or programmed cell death. These data indicate that PCD during HI occurs via a novel and possibly fungal-specific mechanism, making this pathway an attractive drug target for control of fungal infections.
Escherichia coli heat-shock proteins IbpA and IbpB affect biofilm formation by influencing the level of extracellular indole
Authors: Kuczyńska-Wiśnik Dorota , Matuszewska Ewelina , Laskowska Ewa
Published: November 2009

The development of Escherichia coli biofilm requires the differential expression of various genes implicated in cell signalling, stress responses, motility and the synthesis of structures responsible for cell attachment. The ibpAB operon is among the stress-response genes most induced during growth of the E. coli biofilm. In this study we demonstrated, to our knowledge for the first time, that the lack of IbpAB proteins in E. coli cells inhibited the formation of biofilm at the air–liquid interface, although it allowed normal planktonic growth. We showed that ibpAB mutant cells experienced endogenous oxidative stress, which might result from a decreased catalase activity. The endogenous oxidative stress in ibpAB cells led to increased expression of tryptophanase, an enzyme which catalyses the synthesis of indole. We demonstrated that the formation of biofilm by the ibpAB mutant was delayed due to the increase in the extracellular concentration of indole, which is known to play the role of a signal molecule, inhibiting biofilm growth.
Molecular evidence favouring step-wise evolution of Mozambique Vibrio cholerae O1 El Tor hybrid strain
Authors: Halder Kalpataru , Das Bhabatosh , Nair G. Balakrish , Bhadra Rupak K.
Published: November 2009

The ctxAB operon, encoding cholera toxin (CT) in Vibrio cholerae, is carried by the genome of a filamentous phage, CTXΦ. Usually, specific CTXΦ infect each of the two important biotypes, classical and El Tor, of epidemic V. cholerae strains belonging to serogroup O1, and are called CTXclassΦ and CTXETΦ, respectively. However, an unusual hybrid El Tor strain carrying CTXclassΦ caused the cholera epidemic in Mozambique in 2004. To understand the evolution of that strain, we have further analysed some representative hybrid El Tor strains isolated in Kolkata, India, in 1992, and the results indicate that both the Mozambique and the Indian strains are infected with a unique CTXclassΦ having only four copies of the tandem heptamer repeat sequence 5′-TTTTGAT-3′ present in the ctxAB promoter (P
ctxAB
) region, like in CTXETΦ. Usually, the P
ctxAB
of the classical biotype contains seven to eight copies of such sequences. However, sequence analyses of the P
ctxAB
regions of several classical strains indicated that the copy number of heptamer repeat sequences might vary from four to eight copies, which was previously unknown. Since the hybrid strains analysed in this study carry four copies of the heptamer sequences, it may thus serve as a marker to trace the strain in future. Interestingly, while the Mozambique strain is devoid of an El Tor-specific free RS1 element or pre-CTX prophage, the Indian hybrid strains carry such elements. The free RS1 has been mapped, cloned and sequenced. As in pre-CTX and CTX prophages, multiple copies of free RS1 elements were found to be integrated in tandem in the large chromosomal dif site. Since Indian hybrid El Tor strains carry either free RS1 or pre-CTX prophage in their large chromosomes, it is possible that the Mozambique hybrid El Tor strain has evolved from these progenitor strains by step-wise deletion of CTX genetic elements from their large chromosomes.
Carbonic anhydrases in fungi
Authors: Elleuche Skander , Pöggeler Stefanie
Published: November 2009

Carbonic anhydrases (CAs) are metalloenzymes that catalyse the interconversion of carbon dioxide and bicarbonate with high efficiency. This reaction is fundamental to biological processes such as respiration, photosynthesis, pH homeostasis, CO2 transport and electrolyte secretion. CAs are distributed among all three domains of life, and are currently divided into five evolutionarily unrelated classes (α, β, γ, δ and ζ). Fungal CAs have only recently been identified and characterized in detail. While Saccharomyces cerevisiae and Candida albicans each have only one β-CA, multiple copies of β-CA- and α-CA-encoding genes are found in other fungi. Recent work demonstrates that CAs play an important role in the CO2-sensing system of fungal pathogens and in the regulation of sexual development. This review focuses on CA functions in S. cerevisiae, the fungal pathogens C. albicans and Cryptococcus neoformans, and the filamentous ascomycete Sordaria macrospora.
Transcriptional regulation of the novobiocin biosynthetic gene cluster
Authors: Dangel Volker , Härle Johannes , Goerke Christiane , Wolz Christiane , Gust Bertolt , Pernodet Jean-Luc , Heide Lutz
Published: November 2009

The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes novE and novG. We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of novH, but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene novG and insertion of a terminator cassette into the biosynthetic gene novH led to a strong reduction of the number of transcripts of the genes located between novH and novW. This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of novG was found to depend on the presence of NovE, suggesting that the two regulatory genes, novE and novG, act in a cascade-like mechanism. The resistance gene gyrBR
, encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from novH to novW. However, when the gyrase inhibitor novobiocin accumulates in the cultures, gyrBR
is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.
Characterization of a broad-host-range flagellum-dependent phage that mediates high-efficiency generalized transduction in, and between, Serratia and Pantoea
Authors: Evans T. J. , Crow M. A. , Williamson N. R. , Orme W. , Thomson N. R. , Komitopoulou E. , Salmond G. P. C.
Published: November 2009

A phage (ΦOT8) isolated onSerratiasp. ATCC 39006 was shown to be flagellum-dependent, and to mediate generalized transduction with high efficiency (up to 10−4transductants per p.f.u.). ΦOT8 was shown to have a broad host range because it also infected a strain ofPantoea agglomeransisolated from the rhizosphere. Transduction of plasmid-borne antibiotic resistance between the two bacterial genera was demonstrated, consistent with purported ecological roles of phages in dissemination of genes between bacterial genera.Serratiasp. ATCC 39006 andP. agglomeransproduce a number of interesting secondary metabolites that have potential applications in cancer therapy and biocontrol of fungal infections. ΦOT8 has utility as a powerful functional genomics tool in these bacteria.
Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions
Authors: Avanzo Petra , Sabotič Jerica , Anžlovar Sabina , Popovič Tatjana , Leonardi Adrijana , Pain Roger H. , Kos Janko , Brzin Jože
Published: November 2009

We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K
i in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family.
Two type IV secretion systems with different functions in Burkholderia cenocepacia K56-2
Authors: Zhang Ruifu , LiPuma John J. , Gonzalez Carlos F.
Published: November 2009

Bacterial type IV secretion systems (T4SS) perform two fundamental functions related to pathogenesis: the delivery of effector molecules to eukaryotic target cells, and genetic exchange. Two T4SSs have been identified inBurkholderia cenocepaciaK56-2, a representative of the ET12 lineage of theB. cepaciacomplex (Bcc). The plant tissue watersoaking (Ptw) T4SS encoded on a resident 92 kb plasmid is a chimera composed of VirB/D4 and F-specific subunits, and is responsible for the translocation of effector(s) that have been linked to the Ptw phenotype. The bc-VirB/D4 system located on chromosome II displays homology to the VirB/D4 T4SS ofAgrobacterium tumefaciens. In contrast to the Ptw T4SS, the bc-VirB/D4 T4SS was found to be dispensable for Ptw effector(s) secretion, but was found to be involved in plasmid mobilization. The fertility inhibitor Osa did not affect the secretion of Ptw effector(s) via the Ptw system, but did disrupt the mobilization of a RSF1010 derivative plasmid.
Development of a non-invasive murine infection model for acute otitis media
Authors: Stol K. , van Selm S. , van den Berg S. , Bootsma H. J. , Blokx W. A. M. , Graamans K. , Tonnaer E. L. G. M. , Hermans P. W. M.
Published: November 2009

Otitis media (OM) is one of the most frequent diseases in childhood, andStreptococcus pneumoniaeis among the main causative bacterial agents. Since current experimental models used to study the bacterial pathogenesis of OM have several limitations, such as the invasiveness of the experimental procedures, we developed a non-invasive murine OM model. In our model, adapted from a previously developed rat OM model, a pressure cabin is used in which a 40 kPa pressure increase is applied to translocate pneumococci from the nasopharyngeal cavity into both mouse middle ears. Wild-type pneumococci were found to persist in the middle ear cavity for 144 h after infection, with a maximum bacterial load at 96 h. Inflammation was confirmed at 96 and 144 h post-infection by IL-1βand TNF-αcytokine analysis and histopathology. Subsequently, we investigated the contribution of two surface-associated pneumococcal proteins, the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA), to experimental OM in our model. Pneumococci lacking theslrAgene, but not those lacking theppmAgene, were significantly reduced in virulence in the OM model. Importantly, pneumococci lacking both genes were significantly more attenuated than the ΔslrAsingle mutant. This additive effect suggests that SlrA and PpmA exert complementary functions during experimental OM. In conclusion, we have developed a highly reproducible and non-invasive murine infection model for pneumococcal OM using a pressure cabin, which is very suitable to study pneumococcal pathogenesis and virulencein vivo.
Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis
Authors: Chen Jiemin , Kriakov Jordan , Singh Albel , Jacobs William R. , Besra Gurdyal S. , Bhatt Apoorva
Published: November 2009

Mycobacteriophages have played an important role in the development of genetic tools and diagnostics for pathogenic mycobacteria, including Mycobacterium tuberculosis. However, despite the isolation of numerous phages that infect mycobacteria, the mechanisms of mycobacteriophage infection remain poorly understood, and knowledge about phage receptors is minimal. In an effort to identify the receptor for phage I3, we screened a library of Mycobacterium smegmatis transposon mutants for phage-resistant strains. All four phage I3-resistant mutants isolated were found to have transposon insertions in genes located in a cluster involved in the biosynthesis of the cell-wall-associated glycopeptidolipid (GPL), and consequently the mutants did not synthesize GPLs. The loss of GPLs correlated specifically with phage I3 resistance, as all mutants retained sensitivity to two other mycobacteriophages: D29 and Bxz1. In order to define the minimal receptor for phage I3, we then tested the phage sensitivity of previously described GPL-deficient mutants of M. smegmatis that accumulate biosynthesis intermediates of GPLs. The results indicated that, while the removal of most sugar residues from the fatty acyl tetrapeptide (FATP) core of GPL did not affect sensitivity to phage I3, a single methylated rhamnose, transferred by the rhamnosyltransferase Gtf2 to the FATP core, was critical for phage binding.
SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae
Authors: Gale Cheryl A. , Leonard Michelle D. , Finley Kenneth R. , Christensen Leah , McClellan Mark , Abbey Darren , Kurischko Cornelia , Bensen Eric , Tzafrir Iris , Kauffman Sarah , Becker Jeff , Berman Judith
Published: November 2009

The early endocytic patch protein Sla2 is important for morphogenesis and growth rates inSaccharomyces cerevisiaeandCandida albicans,but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2or ScSLA2are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Onlysla2strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis ofC. albicansinfections.
Processing, assembly and localization of a Bacillus anthracis spore protein
Authors: Moody K. L. , Driks A. , Rother G. L. , Cote C. K. , Brueggemann E. E. , Hines H. B. , Friedlander A. M. , Bozue J.
Published: November 2009

All Bacillus spores are encased in macromolecular shells. One of these is a proteinacious shell called the coat that, in Bacillus subtilis, provides critical protective functions. The Bacillus anthracis spore is the infectious particle for the disease anthrax. Therefore, the coat is of particular interest because it may provide essential protective functions required for the appearance of anthrax. Here, we analyse a protein component of the spore outer layers that was previously designated BxpA. Our data indicate that a significant amount of BxpA is located below the spore coat and associated with the cortex. By SDS-PAGE, BxpA migrates as a 9 kDa species when extracted from Sterne strain spores, and as 11 and 14 kDa species from Ames strain spores, even though it has predicted masses of 27 and 29 kDa, respectively, in these two strains. We investigated the possibility that BxpA is subject to post-translational processing as previously suggested. In B. subtilis, a subset of coat proteins is proteolysed or cross-linked by the spore proteins YabG or Tgl, respectively. To investigate the possibility that similar processing occurs in B. anthracis, we generated mutations in the yabG or tgl genes in the Sterne and Ames strains and analysed the consequences for BxpA assembly by SDS-PAGE. We found that in a tgl mutant of B. anthracis, the apparent mass of BxpA increased. This is consistent with the possibility that Tgl directs the cross-linking of BxpA into a form that normally does not enter the gel. Unexpectedly, the apparent mass of BxpA also increased in a yabG mutant, suggesting a relatively complex role for proteolysis in spore protein maturation in B. anthracis. These data reveal a previously unobserved event in spore protein maturation in B. anthracis. We speculate that proteolysis and cross-linking are ubiquitous spore assembly mechanisms throughout the genus Bacillus.
A distinct physiological role of MutY in mutation prevention in mycobacteria
Authors: Kurthkoti Krishna , Srinath Thiruneelakantan , Kumar Pradeep , Malshetty Vidyasagar S. , Sang Pau Biak , Jain Ruchi , Manjunath Ramanathapuram , Varshney Umesh
Published: November 2009

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain ofMycobacterium smegmatisto investigate the role of this enzyme in DNA repair. The MutY deficiency inM. smegmatisdid not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin-resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region ofrpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatisandM. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG : A, 8-oxoG : G or 8-oxoG : T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts ofM. smegmatissuggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria.
Remnant signal peptides on non-exported enzymes: implications for the evolution of prokaryotic respiratory chains
Authors: Ize Bérengère , Coulthurst Sarah J. , Hatzixanthis Kostas , Caldelari Isabelle , Buchanan Grant , Barclay Elaine C. , Richardson David J. , Palmer Tracy , Sargent Frank
Published: November 2009

The twin-arginine translocation (Tat) pathway is a prokaryotic protein targeting system dedicated to the transmembrane translocation of folded proteins. Substrate proteins are directed to the Tat translocase by signal peptides bearing a conserved SRRxFLK ‘twin-arginine’ motif. InEscherichia coli, most of the 27 periplasmically located Tat substrates are cofactor-containing respiratory enzymes, and many of these harbour a molybdenum cofactor at their active site. Molybdenum cofactor-containing proteins are not exclusively located in the periplasm, however, with the major respiratory nitrate reductase (NarG) and the biotin sulfoxide reductase (BisC), for example, being located at the cytoplasmic side of the membrane. Interestingly, both NarG and BisC contain ‘N-tail’ regions that bear some sequence similarity to twin-arginine signal peptides. In this work, we have examined the relationship between the non-exported N-tails and the Tat system. Using a sensitive genetic screen for Tat transport, variant N-tails were identified that displayed Tat transport activity. For the NarG 36-residue N-tail, six amino acid changes were needed to induce transport activity. However, these changes interfered with binding by the NarJ biosynthetic chaperone and impaired biosynthesis of the native enzyme. For the BisC 36-residue N-tail, only five amino acid substitutions were needed to restore Tat transport activity. These modifications also impairedin vivoBisC activity, but it was not possible to identify a biosynthetic chaperone for this enzyme. These data highlight an intimate genetic and evolutionary link between some non-exported redox enzymes and those transported across membranes by the Tat translocation system.
Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002
Authors: Audy Julie , Labrie Steve , Roy Denis , LaPointe Gisèle
Published: November 2009

The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080±120 mg EPS l−1 (P<0.01) after 24 h of incubation. For fructose, galactose and glucose, EPS production was similar, at 512±63, 564±165 and 616±93 mg EPS l−1, respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production (galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase (wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.
A tyrosine O-prenyltransferase catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL
Authors: Kremer Anika , Li Shu-Ming
Published: November 2009

A putative prenyltransferase gene sirD has been identified in the gene cluster encoding the biosynthesis of the phytotoxin sirodesmin PL in Leptosphaeria maculans. The gene product was found to comprise 449 aa, with a molecular mass of 51 kDa. In this study, the coding region of sirD was amplified by PCR from cDNA, cloned into pQE70, and overexpressed in Escherichia coli. The overproduced protein was purified to apparent homogeneity, and characterized biochemically. The dimeric recombinant SirD was found to catalyse the O-prenylation of l-Tyr in the presence of dimethylallyl diphosphate; this was demonstrated unequivocally by isolation and structural elucidation of the enzymic product. Therefore, SirD catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL. K
m values for l-Tyr and dimethylallyl diphosphate were determined as 0.13 and 0.17 mM, respectively. Interestingly, SirD was found to share significant sequence similarity with indole prenyltransferases, which catalyse prenyl transfer reactions onto different positions of indole rings. In contrast to indole prenyltransferases, which accept indole derivatives, but not Tyr or structures derived thereof, as substrates, SirD also prenylated l-Trp, resulting in the formation of 7-dimethylallyltryptophan. A K
m value of 0.23 mM was determined for l-Trp. Turnover numbers of 1.0 and 0.06 S−1 were calculated for l-Tyr and l-Trp, respectively.
Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota
Authors: Berg Ivan A. , Ramos-Vera W. Hugo , Petri Anna , Huber Harald , Fuchs Georg
Published: November 2009

Two new autotrophic carbon fixation cycles have been recently described in Crenarchaeota. The 3-hydroxypropionate/4-hydroxybutyrate cycle using acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme has been identified for (micro)aerobic members of the Sulfolobales. The dicarboxylate/4-hydroxybutyrate cycle using oxygen-sensitive pyruvate synthase and phosphoenolpyruvate carboxylase as carboxylating enzymes has been found in members of the anaerobic Desulfurococcales and Thermoproteales. However, Sulfolobales include anaerobic and Desulfurococcales aerobic autotrophic representatives, raising the question of which of the two cycles they use. We studied the mechanisms of autotrophic CO2 fixation in the strictly anaerobic Stygiolobus azoricus (Sulfolobales) and in the facultatively aerobic Pyrolobus fumarii (Desulfurococcales). The activities of all enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle were found in the anaerobic S. azoricus. In contrast, the aerobic or denitrifying P. fumarii possesses all enzyme activities of the dicarboxylate/4-hydroxybutyrate cycle. We conclude that autotrophic Crenarchaeota use one of the two cycles, and that their distribution correlates with the 16S rRNA-based phylogeny of this group, rather than with the aerobic or anaerobic lifestyle.
The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease
Authors: Sauri Ana , Soprova Zora , Wickström David , de Gier Jan-Willem , Van der Schors Roel C. , Smit August B. , Jong Wouter S. P. , Luirink Joen
Published: November 2009

Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms aβ-barrel pore in the outer membrane implicated in translocation of the upstream ‘passenger’ domain across the outer membrane. However, recent structural data suggest that the diameter of theβ-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage theβ-domain was not inserted and folded as an integralβ-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Bam (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane.
Fungal physiology and the origins of molecular biology
Authors: Brambl Robert
Published: November 2009

Molecular biology has several distinct origins, but especially important are those contributed by fungal and yeast physiology, biochemistry and genetics. From the first gene action studies that became the basis of our understanding of the relationship between genes and proteins, through chromosome structure, mitochondrial genetics and membrane biogenesis, gene silencing and circadian clocks, studies with these organisms have yielded basic insight into these processes applicable to all eukaryotes. Examples are cited of pioneering studies with fungi that have stimulated new research in clinical medicine and agriculture; these studies include sexual interactions, cell stress responses, the cytoskeleton and pathogenesis. Studies with the yeasts and fungi have been effective in applying the techniques and insights gained from other types of experimental systems to research in fungal cell signalling, cell development and hyphal morphogenesis.
Origin and distribution of tick-borne encephalitis virus strains of the Siberian subtype in the Middle Urals, the north-west of Russia and the Baltic countries
Authors: Kovalev S. Yu. , Chernykh D. N. , Kokorev V. S. , Snitkovskaya T. E. , Romanenko V. V.
Published: November 2009

Tick-borne encephalitis virus (TBEV) plays an important role in infectious human morbidity, particularly in Russia and the Middle Urals. The Siberian subtype of TBEV (S-TBEV) is dominant in the Middle Urals. Determining the origin of S-TBEV strains in this territory and also in the European part of Russia and the Baltic countries is very important for understanding the cause of its distribution. The surface glycoprotein E gene was partially sequenced in 165 S-TBEV isolates collected in the Middle Urals between 1966 and 2008. Nucleotide and amino acid sequence identity of the studied isolates is 94 and 97.4 %, respectively. Eighty per cent of them are represented by six clusters with identical amino acid sequences in the glycoprotein E fragment analysed. We have determined four types of isolate distribution in the explored territory: local, split, corridor and diffuse. The average rate of nucleotide substitutions per site year−1 is estimated to be 1.56×10−4. The age of the S-TBEV population was evaluated to be slightly less than 400 years. Phylogenetic analysis of the data and comparison with historical events indicate that the distribution of S-TBEV strains in the Middle Urals and the European part of Russia originated twice from different foci in western Siberia. This is related to the first land road into Siberia and the Trans-Siberian Way, which functioned at different times. The main reason for such rapid distribution of S-TBEV strains is the anthropogenic factor, i.e. human economic activity during the colonization of new territories in Siberia in the recent past.
Novel virus-associated proteins encoded by UL112–113 of human cytomegalovirus
Authors: Wang Shang-Kwei , Hu Cheng-Hui , Lu Miao-Chan , Duh Chang-Yih , Liao Pao-Chi , Tyan Yu-Chang
Published: November 2009

Evidence suggests that the products of the human cytomegalovirus (HCMV) UL112–113 genes are involved in viral DNA replication during lytic infection. A polyclonal antibody was raised against the UL112 open reading frame (ORF) to characterize its function in detail. Immunoblots utilizing the UL112 antibody identified seven distinct protein bands (p20, p26, p28, p34, p43, p50 and p84) expressed during the HCMV infectious cycle. After screening a cDNA library constructed from cells 72 h after infection with HCMV, only four different cDNA protein-producing constructs were obtained, and their ORFs corresponded to p34, p43, p50 and p84. The proteins p20, p26 and p28 were further shown to be selectively included within mature HCMV particles, virions, non-infectious enveloped particles and dense bodies. Immunoaffinity protein purification was used to prepare the samples for liquid chromatography coupled to tandem mass spectrometry. This analysis revealed that p20, p26 and p28 were derived from the UL112 ORF, most likely through post-translational proteolytic cleavage.
Transmissions of variant Creutzfeldt–Jakob disease from brain and lymphoreticular tissue show uniform and conserved bovine spongiform encephalopathy-related phenotypic properties on primary and secondary passage in wild-type mice
Authors: Ritchie Diane L. , Boyle Aileen , McConnell Irene , Head Mark W. , Ironside James W. , Bruce Moira E.
Published: November 2009

Prion strains are defined by their biological properties after transmission to wild-type mice, specifically by their incubation periods and patterns of vacuolar pathology (‘lesion profiles’). Preliminary results from transmissions of variant Creutzfeldt–Jakob disease (vCJD) to wild-type mice provided the first compelling evidence for the close similarity of the vCJD agent to the agent causing bovine spongiform encephalopathy (BSE). Complete results from this investigation, including the transmission characteristics of vCJD from brain and peripheral tissues of 10 cases (after primary transmission and subsequent mouse-to-mouse passage), have now been analysed. All 10 vCJD sources resulted in consistent incubation periods and lesion profiles, suggesting that all 10 patients were infected with the same strain of agent. Incubation periods suggested that infectious titres may be subject to regional variation within the brain. Comparison of incubation periods and lesion profiles from transmission of brain and peripheral tissues showed no evidence of tissue-specific modification in the biological properties of the agent. Analysis of the protease-resistant prion protein (PrPres) by Western blotting from primary and subsequent passages in mice showed a glycosylation pattern closely resembling that of vCJD in humans, the so-called BSE ‘glycoform signature’. Minor variations in PrPres fragment size were evident between mouse strains carrying different alleles of the gene encoding PrP both in primary transmissions and on further passages of vCJD brain. Overall, the results closely resembled those of previously reported transmissions of BSE in the same mouse strains, consistent with BSE being the origin of all of these vCJD cases.
Prevalence and genetic variability of tick-borne encephalitis virus in host-seeking Ixodes ricinus in northern Italy
Authors: Carpi Giovanna , Bertolotti Luigi , Rosati Sergio , Rizzoli Annapaola
Published: November 2009

Tick-borne encephalitis (TBE) is a severe disease that has been endemic in north-east Italy since 1992. Over the past two decades, there has been an increase in the number of human cases reported in many European countries, including Italy. To assess the current TBE infection risk, questing ticks were collected from known TBE foci, as well as from a site in northern Italy where no human infections have been reported previously. A total of 1739Ixodes ricinus(1485 nymphs and 254 adults) was collected and analysed for TBEV prevalence by a real-time RT-PCR targeting the 3′ untranslated region. Phylogenetic analyses of the partial envelope gene were conducted on two newly sequenced TBE virus (TBEV) strains and 28 previously published sequences to investigate the genealogical relationships of the circulating TBEV strains. These phylogenetic analyses confirmed a previous report that the European TBEV subtype is the only subtype circulating within the TBE foci in north-east Italy. Interestingly, nucleotide sequence analysis revealed a high degree of divergence (mean 2.54 %) between the TBEV strains recovered in the Italian province of Trento, despite the circulation of a single TBEV subtype. This elevated genetic variability within a single TBE focus may reflect local differences in the long-standing evolutionary dynamics of TBEV at this site relative to previously characterized sites, or more recent and continuous reintroduction of various TBEV strains.
Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells
Authors: Agis-Juárez Raúl Azael , Galván Iván , Medina Fernando , Daikoku Takiko , Padmanabhan R. , Ludert Juan E. , del Angel Rosa M.
Published: November 2009

The 3′ untranslated region (3′UTR) of the dengue virus (DENV) genome contain several sequences required for translation, replication and cyclization processes. This region also binds cellular proteins such as La, polypyrimidine tract-binding protein (PTB), Y box-binding protein 1, poly(A)-binding protein and the translation initiation factor eEF-1α. PTB is a cellular protein that interacts with the regulatory sequences of positive-strand RNA viruses such as several picornaviruses and hepatitis C virus. In the present report, it was demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection. At 48 h post-infection, PTB, as well as the DENV proteins NS1 and NS3, were found to co-localize with the endoplasmic reticulum marker calnexin. Silencing of PTB expression inhibited virus translation and replication, whilst overexpression of PTB augmented these processes. Thus, these results provide evidence that, during infection, PTB moves from the nucleus to the cytoplasm and plays an important role in the DENV replicative cycle.
Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment
Authors: Olveira J. G. , Souto S. , Dopazo C. P. , Thiéry R. , Barja J. L. , Bandín I.
Published: November 2009

Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV.
Sequence characteristics of potato virus Y recombinants
Authors: Hu Xiaojun , Karasev Alexander V. , Brown Celeste J. , Lorenzen Jim H.
Published: November 2009

Potato virus Y (PVY) is one of the most economically important plant pathogens. The PVY genome has a high degree of genetic variability and is also subject to recombination. New recombinants have been reported in many countries since the 1980s, but the origin of these recombinant strains and the physical and evolutionary mechanisms driving their emergence are not clear at the moment. The replicase-mediated template-switching model is considered the most likely mechanism for forming new RNA virus recombinants. Two factors, RNA secondary structure (especially stem–loop structures) and AU-rich regions, have been reported to affect recombination in this model. In this study, we investigated the influence of these two factors on PVY recombination from two perspectives: their distribution along the whole genome and differences between regions flanking the recombination junctions (RJs). Based on their distributions, only a few identified RJs in PVY genomes were located in lower negative FORS-D, i.e. having greater secondary-structure potential and higher AU-content regions, but most RJs had more negative FORS-D values upstream and/or higher AU content downstream. Our whole-genome analyses showed that RNA secondary structures and/or AU-rich regions at some sites may have affected PVY recombination, but in general they were not the main forces driving PVY recombination.