bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Characterization of Environmental Bacteria Capable of Extracellular Biosorption of Mercury
Authors: François Fabienne , Lombard Carine , Guigner Jean-Michel , Soreau Paul , Brian-Jaisson Florence , Martino Grégory , Vandervennet Manon , Garcia Daniel , Molinier Anne-Laure , Pignol David , Peduzzi Jean , Zirah Séverine , Rebuffat Sylvie
Published: December 2011

Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl
2
sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl
2
also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.
Establishing Equivalence for Microbial-Growth-Inhibitory Effects (“Iso-Hurdle Rules”) by Analyzing Disparate Listeria monocytogenes Data with a Gamma-Type Predictive Model
Authors: Pujol Laure , Kan-King-Yu Denis , Le Marc Yvan , Johnston Moira D. , Rama-Heuzard Florence , Guillou Sandrine , McClure Peter , Membré Jeanne-Marie
Published: December 2011

Preservative factors act as hurdles against microorganisms by inhibiting their growth; these are essential control measures for particular food-borne pathogens. Different combinations of hurdles can be quantified and compared to each other in terms of their inhibitory effect (“iso-hurdle”). We present here a methodology for establishing microbial iso-hurdle rules in three steps: (i) developing a predictive model based on existing but disparate data sets, (ii) building an experimental design focused on the iso-hurdles using the model output, and (iii) validating the model and the iso-hurdle rules with new data. The methodology is illustrated with
Listeria monocytogenes
. Existing data from industry, a public database, and the literature were collected and analyzed, after which a total of 650 growth rates were retained. A gamma-type model was developed for the factors temperature, pH, a
w
, and acetic, lactic, and sorbic acids. Three iso-hurdle rules were assessed (40 logcount curves generated): salt replacement by addition of organic acids, sorbic acid replacement by addition of acetic and lactic acid, and sorbic acid replacement by addition of lactic/acetic acid and salt. For the three rules, the growth rates were equivalent in the whole experimental domain (γ from 0.1 to 0.5). The lag times were also equivalent in the case of mild inhibitory conditions (γ ≥ 0.2), while they were longer in the presence of salt than acids under stress conditions (γ < 0.2). This methodology allows an assessment of the equivalence of inhibitory effects without intensive data generation; it could be applied to develop milder formulations which guarantee microbial safety and stability.
Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211
Authors: Jiang Chunyan , Wang Hougen , Kang Qianjin , Liu Jing , Bai Linquan
Published: December 2011

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene,
slnC
, was cloned from the salinomycin producer
Streptomyces albus
XM211. The targeted replacement of
slnC
and subsequent
trans
-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing
slnC
was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including
slnC
, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.
Diversity of Five Anaerobic Toluene-Degrading Microbial Communities Investigated Using Stable Isotope Probing
Authors: Sun Weimin , Cupples Alison M.
Published: December 2011

Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [13C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genusThauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the familyComamonadaceae(genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the classClostridia(genusDesulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the classDeltaproteobacteria, within the familySyntrophobacteraceae(digester sludge) orDesulfobulbaceae(contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.
Metatranscriptomic Analysis of Microbes in an Oceanfront Deep-Subsurface Hot Spring Reveals Novel Small RNAs and Type-Specific tRNA Degradation
Authors: Murakami Shinnosuke , Fujishima Kosuke , Tomita Masaru , Kanai Akio
Published: December 2011

Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000 m in Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new
Betaproteobacteria
. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as
Escherichia coli
and
Streptomyces coelicolor
, and the current results suggest that specific tRNA decay occurs more frequently than previously expected.
Carbapenem-Hydrolyzing GES-5-Encoding Gene on Different Plasmid Types Recovered from a Bacterial Community in a Sewage Treatment Plant
Authors: Girlich Delphine , Poirel Laurent , Szczepanowski Rafael , Schlüter Andreas , Nordmann Patrice
Published: December 2011

Plasmids pRSB113 and pRSB115 were recovered from an activated sludge bacterial community of a municipal wastewater treatment plant in Germany. Both plasmids carry the same
bla
GES-5
carbapenemase gene, located within two distinct class 1 integrons. These plasmids have different backbones, belong to different incompatibility groups, and could replicate in both
Pseudomonas aeruginosa
and
Escherichia coli
.
Identification and Characterization of the LysR-Type Transcriptional Regulator HsdR for Steroid-Inducible Expression of the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni
Authors: Gong Wenjie , Xiong Guangming , Maser Edmund
Published: December 2011

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from
Comamonas testosteroni
is a key enzyme in steroid degradation in soil and water. 3α-HSD/CR gene (
hsdA
) expression can be induced by steroids like testosterone and progesterone. Previously, we have shown that the induction of
hsdA
expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking of
hsdA
transcription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem.
276:
9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem.
278
:47400–47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3α-HSD/CR expression in
C. testosteroni
has been identified. The
hsdR
gene is located 2.58 kb downstream from
hsdA
on the
C. testosteroni
ATCC 11996 chromosome with an orientation opposite that of
hsdA
. The
hsdR
gene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of the
hsdA
gene, electrophoresis mobility shift assays showed that HsdR specifically binds to the
hsdA
promoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore,
in vitro
binding assays indicated that HsdR can come into contact with RNA polymerase. As expected, an
hsdR
knockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-type
C. testosteroni
after testosterone induction. In conclusion, HsdR is a positive transcription factor for the
hsdA
gene and promotes the induction of 3α-HSD/CR expression in
C. testosteroni
.
CTX-M-15 Extended-Spectrum β-Lactamase in a Shiga Toxin-Producing Escherichia coli Isolate of Serotype O111:H8
Authors: Valat Charlotte , Haenni Marisa , Saras Estelle , Auvray Frédéric , Forest Karine , Oswald Eric , Madec Jean-Yves
Published: December 2011

We report the discovery of a CTX-M-15-producing
Escherichia coli
(STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic
Escherichia coli
(EHEC) infections. In line with the recent CTX-M-15/O104:H4
E. coli
outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the
E. coli
population, including STEC isolates.
Absence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex CC398 as a Nasal Colonizer of Pigs Raised in an Alternative System
Authors: Cuny Christiane , Friedrich Alexander W. , Witte Wolfgang
Published: December 2011

Livestock-associated methicillin-resistant
Staphylococcus aureus
(LA-MRSA) ST398 isolated from pigs raised in conventional farms was previously reported. Here we report a study on 25 farms adhering to an alternative system. LA-MRSA ST398 was not detected in nasal swabs from 178 pigs or from 89 humans working and living on these farms.
Requirement for RNA Helicase CsdA for Growth of Yersinia pseudotuberculosis IP32953 at Low Temperatures
Authors: Palonen Eveliina , Lindström Miia , Somervuo Panu , Johansson Per , Björkroth Johanna , Korkeala Hannu
Published: December 2011

The expression of
csdA
, encoding an RNA helicase, was induced at 3°C in
Yersinia pseudotuberculosis
. The role of CsdA in
Y. pseudotuberculosis
under cold conditions was confirmed by impaired growth of insertional
csdA
mutants at 3°C. The results suggest that CsdA is crucial for
Y. pseudotuberculosis
survival in the chilled food chain.
Lactic Acid Bacterium and Yeast Microbiotas of 19 Sourdoughs Used for Traditional/Typical Italian Breads: Interactions between Ingredients and Microbial Species Diversity
Authors: Minervini Fabio , Di Cagno Raffaella , Lattanzi Anna , De Angelis Maria , Antonielli Livio , Cardinali Gianluigi , Cappelle Stefan , Gobbetti Marco
Published: December 2011

The study of the microbiotas of 19 Italian sourdoughs used for the manufacture of traditional/typical breads allowed the identification, through a culture-dependent approach, of 20 and 4 species of lactic acid bacteria (LAB) and yeasts, respectively. Numerically, the most frequent LAB isolates were
Lactobacillus sanfranciscensis
(ca. 28% of the total LAB isolates),
Lactobacillus plantarum
(ca. 16%), and
Lactobacillus paralimentarius
(ca. 14%).
Saccharomyces cerevisiae
was identified in 16 sourdoughs.
Candida humilis
,
Kazachstania barnettii
, and
Kazachstania exigua
were also identified. As shown by principal component analysis (PCA), a correlation was found between the ingredients, especially the type of flour, the microbial community, and the biochemical features of sourdoughs.
Triticum durum
flours were characterized by the high level of maltose, glucose, fructose, and free amino acids (FAA) correlated with the sole or main presence of obligately heterofermentative LAB, the lowest number of facultatively heterofermentative strains, and the low cell density of yeasts in the mature sourdoughs. This study highlighted, through a comprehensive and comparative approach, the dominant microbiotas of 19 Italian sourdoughs, which determined some of the peculiarities of the resulting traditional/typical Italian breads.
Enigmatic Presence of Mitochondrial Complex I in Trypanosoma brucei Bloodstream Forms
Authors: Surve Sachin , Heestand Meredith , Panicucci Brian , Schnaufer Achim , Parsons Marilyn
Published: December 2011

The presence of mitochondrial respiratory complex I in the pathogenic bloodstream stages of
Trypanosoma brucei
has been vigorously debated: increased expression of mitochondrially encoded functional complex I mRNAs is countered by low levels of enzymatic activity that show marginal inhibition by the specific inhibitor rotenone. We now show that epitope-tagged versions of multiple complex I subunits assemble into α and β subcomplexes in the bloodstream stage and that these subcomplexes require the mitochondrial genome for their assembly. Despite the presence of these large (740- and 855-kDa) multisubunit complexes, the electron transport activity of complex I is not essential under experimental conditions since null mutants of two core genes (
NUBM
and
NUKM
) showed no growth defect
in vitro
or in mouse infection. Furthermore, the null mutants showed no decrease in NADH:ubiquinone oxidoreductase activity, suggesting that the observed activity is not contributed by complex I. This work conclusively shows that despite the synthesis and assembly of subunit proteins, the enzymatic function of the largest respiratory complex is neither significant nor important in the bloodstream stage. This situation appears to be in striking contrast to that for the other respiratory complexes in this parasite, where physical presence in a life-cycle stage always indicates functional significance.
Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum
Authors: Veiga Tânia , Solis-Escalante Daniel , Romagnoli Gabriele , ten Pierick Angela , Hanemaaijer Mark , Deshmuhk Amit , Wahl Aljoscha , Pronk Jack T. , Daran Jean-Marc
Published: December 2011

The industrial production of penicillin G by
Penicillium chrysogenum
requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of
P. chrysogenum
with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-
13
C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of
P. chrysogenum
cultures grown with phenylalanine and with (NH
4
)
2
SO
4
as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free
Saccharomyces cerevisiae
strain CEN.PK711-7C (
pdc1 pdc5 pdc6Δ aro10Δ thi3Δ
). The introduction of Pc13g09300 restored the growth of this
S. cerevisiae
mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in
P. chrysogenum
. These results provide a basis for the metabolic engineering of
P. chrysogenum
for the production of the penicillin G side chain precursor phenylacetate.
Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity
Authors: Bitsaktsis Constantine , Iglesias Bibiana V. , Li Ying , Colino Jesus , Snapper Clifford M. , Hollingshead Susan K. , Pham Giang , Gosselin Diane R. , Gosselin Edmund J.
Published: December 2011

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen,Francisella tularensis. To determine if a similar strategy could be applied to the important pathogenStreptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevatedS. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethalS. pneumoniaechallenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed againstS. pneumoniaethat appears to be lactoferrin mediated.
Surface-Localized Spermidine Protects the Pseudomonas aeruginosa Outer Membrane from Antibiotic Treatment and Oxidative Stress
Authors: Johnson Lori , Mulcahy Heidi , Kanevets Uliana , Shi Yan , Lewenza Shawn
Published: December 2011

Extracellular DNA acts as a cation chelator and induces the expression of antibiotic resistance genes regulated by Mg2+levels. Here we report the characterization of novel DNA-induced genes inPseudomonas aeruginosathat are annotated as homologs of the spermidine synthesis genesspeD(PA4773) andspeE(PA4774). The addition of sublethal concentrations of DNA and membrane-damaging antibiotics induced expression of the genesPA4773toPA4775, as shown using transcriptionalluxfusions and quantitative RT-PCR. Exogenous polyamine addition prevented DNA- and peptide-mediated gene induction. Mutation ofPA4774resulted in an increased outer membrane (OM) susceptibility phenotype upon polymyxin B, CP10A, and gentamicin treatment. When the membrane-localized fluorescent probe C11-BODIPY581/591was used as an indicator of peroxidation of membrane lipids, thePA4774::luxmutant demonstrated an increased susceptibility to oxidative membrane damage from H2O2treatment. Addition of exogenous polyamines protected the membranes of thePA4774::luxmutant from polymyxin B and H2O2treatment. Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using high-performance liquid chromatography and mass spectrometry. ThePA4774::luxmutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg2+-limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage.
The Translocation Domain in Trimeric Autotransporter Adhesins Is Necessary and Sufficient for Trimerization and Autotransportation
Authors: Mikula Kornelia M. , Leo Jack C. , Łyskowski Andrzej , Kedracka-Krok Sylwia , Pirog Artur , Goldman Adrian
Published: December 2011

Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the β-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the β-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different
Escherichia coli
immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the β-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not.
Genes Involved in Immunity to and Secretion of Aureocin A53, an Atypical Class II Bacteriocin Produced by Staphylococcus aureus A53
Authors: Nascimento Janaína dos Santos , Coelho Marcus Lívio Varella , Ceotto Hilana , Potter Amina , Fleming Luana Rocha , Salehian Zhian , Nes Ingolf F. , Bastos Maria do Carmo de Freire
Published: December 2011

Aureocin A53 is an antimicrobial peptide produced by
Staphylococcus aureus
A53. The genetic determinants involved in aureocin A53 production and immunity to its action are organized in at least four transcriptional units encoded by the 10.4-kb plasmid pRJ9. One transcriptional unit carries only the bacteriocin structural gene,
aucA
. No immunity gene is found downstream of
aucA
, as part of the same transcriptional unit. Further downstream of
aucA
is found an operon which contains the three genes
aucEFG
, whose products seem to associate to form a dedicated ABC transporter. When
aucEFG
were expressed in RN4220, an aureocin A53-sensitive
S. aureus
strain, this strain became partially resistant to the bacteriocin. A gene disruption mutant in
aucE
was defective in aureocin A53 externalization and more sensitive to aureocin A53 than the wild-type strain, showing that
aucEFG
are involved in immunity to aureocin A53 by active extrusion of the bacteriocin. Full resistance to aureocin A53 was exhibited by transformants carrying, besides
aucEFG
, the operon formed by two genes,
aucIB
and
aucIA
, located between
aucA
and
aucEFG
and carried in the opposite strand. AucIA and AucIB share similarities with hypothetical proteins not found in the gene clusters of other bacteriocins. A gene disruption mutant in
orf8
, located upstream of
aucA
and whose product exhibits about 50% similarity to a number of hypothetical membrane proteins found in many Gram-positive bacteria, was strongly affected in aureocin A53 externalization but resistant to aureocin A53, suggesting that Orf8 is also involved in aureocin A53 secretion.
Involvement of T6 Pili in Biofilm Formation by Serotype M6 Streptococcus pyogenes
Authors: Kimura Keiji Richard , Nakata Masanobu , Sumitomo Tomoko , Kreikemeyer Bernd , Podbielski Andreas , Terao Yutaka , Kawabata Shigetada
Published: December 2011

The group A streptococcus (GAS)
Streptococcus pyogenes
is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (
f
ibronectin-binding protein,
c
ollagen-binding protein, and
t
rypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm
in vitro
. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the
tee6
gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the
fctX
and
srtB
genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.
Mutation of the Sensor Kinase
chvG
in Rhizobium leguminosarum Negatively Impacts Cellular Metabolism, Outer Membrane Stability, and Symbiosis
Authors: Vanderlinde Elizabeth M. , Yost Christopher K.
Published: December 2011

Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont
Rhizobium leguminosarum
biovar viciae VF39, mutation of
chvG
, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The
chvG
mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abnormally large amounts of poly-ß-hydroxybutyrate. Mutation of
chvG
caused symbiotic defects on peas, lentils, and vetch; nodules formed by the
chvG
mutant were small and white and contained only a few cells that had failed to differentiate into bacteroids. Mutation of
chvG
also destabilized the outer membrane of
R. leguminosarum
, resulting in increased sensitivity to membrane stressors. Constitutive expression of
ropB
, the outer membrane protein-encoding gene, restored membrane stability and rescued the sensitivity phenotypes described above. Similar phenotypes have been described for mutations in other ChvG-regulated genes encoding a conserved operon of unknown function and in the
fabXL
genes required for synthesis of the lipid A very-long-chain fatty acid, suggesting that ChvG is a key component of the envelope stress response in
Rhizobium leguminosarum
. Collectively, the results of this study demonstrate the important and unique role the ChvG/ChvI TCS plays in the physiology, metabolism, and symbiotic competency of
R. leguminosarum
.
EspD Is Critical for the Virulence-Mediating ESX-1 Secretion System in Mycobacterium tuberculosis
Authors: Chen Jeffrey M. , Boy-Röttger Stefanie , Dhar Neeraj , Sweeney Nathan , Buxton Roger S. , Pojer Florence , Rosenkrands Ida , Cole Stewart T.
Published: December 2011

ESAT-6 system 1 (ESX-1)-mediated secretion in
Mycobacterium tuberculosis
is dependent on proteins encoded by the cotranscribed
espA-espC-espD
gene cluster. While the roles of EspA and EspC with respect to the ESX-1 secretion system have been actively investigated, the function of EspD remains unknown. We show that EspD is secreted by
M. tuberculosis
, but unlike EspA and EsxA, its export does not exclusively require the ESX-1 system. Evidence for stabilization of cellular levels of EspA and EspC by EspD is presented, and depletion of EspD results in loss of EsxA secretion. Site-directed mutagenesis of EspD reveals that its role in the maintenance of cellular levels of EspA in
M. tuberculosis
is distinct from its facilitation of EsxA secretion. The same mutagenesis experiments have also shown that secretion of EspD is not required for the secretion of EsxA. Our findings highlight a critical and complex role for EspD in modulating the ESX-1 secretion system in
M. tuberculosis
.
Isolation and Characterization of the Prochlorococcus Carboxysome Reveal the Presence of the Novel Shell Protein CsoS1D
Authors: Roberts Evan W. , Cai Fei , Kerfeld Cheryl A. , Cannon Gordon C. , Heinhorst Sabine
Published: December 2011

Cyanobacteria, including members of the genus
Prochlorococcus
, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the CO
2
-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is not known. Complicating the study of cyanobacterial carboxysomes has been the inability to obtain homogeneous carboxysome preparations. This study describes the first successful purification and characterization of carboxysomes from the marine cyanobacterium
Prochlorococcus marinus
MED4. Because the isolated
P. marinus
MED4 carboxysomes were free from contaminating membrane proteins, their protein complement could be assessed. In addition to the expected shell proteins, the CsoS1D protein that is not encoded by the canonical
cso
gene clusters of α-cyanobacteria was found to be a low-abundance shell component. This finding and supporting comparative genomic evidence have important implications for carboxysome composition, structure, and function. Our study indicates that carboxysome composition is probably more complex than was previously assumed based on the gene complements of the classical
cso
gene clusters.
Conventional testing methods produce submaximal values of maximum oxygen consumption
Authors: Beltrami Fernando G , Froyd Christian , Mauger Alexis R , Metcalfe Alan J , Marino Frank , Noakes Timothy D
Published: December 2011

Background

This study used a novel protocol to test the hypothesis that a plateau in oxygen consumption (VO2max) during incremental exercise testing to exhaustion represents the maximal capacity of the cardiovascular system to transport oxygen.

Methods

Twenty-six subjects were randomly divided into two groups matched by their initial VO2max. On separate days, the reverse group performed (i) an incremental uphill running test on a treadmill (INC1) plus verification test (VER) at a constant workload 1 km h−1higher than the last completed stage in INC1; (ii) a decremental test (DEC) in which speed started as same as the VER but was reduced progressively and (iii) a final incremental test (INCF). The control group performed only INC on the same days that the reverse group was tested.

Results

VO2maxremained within 0.6 ml kg−1min−1across the three trials for the control group (p=0.93) but was 4.4% higher during DEC compared with INC1(63.9±3.8 vs 61.2±4.8 ml kg−1min−1, respectively, p=0.004) in the reverse group, even though speed at VO2maxwas lower (14.3±1.1 vs 16.2±0.7 km h−1for DEC and INC1, respectively, p=0.0001). VO2maxremained significantly higher during INCF(63.6±3.68 ml kg−1min−1, p=0.01), despite an unchanged exercise time between INC1and INCF.

Conclusion

These findings go against the concept that a plateau in oxygen consumption measured during the classically described INC and VER represents a systemic limitation to oxygen use. The reasons for a higher VO2during INCFfollowing the DEC test are unclear.
Asynchronous Solutions for Nanomagnetic Logic Circuits
Authors: Vacca Marco , Graziano Mariagrazia , Zamboni Maurizio
Published: December 2011

In the years to come new solutions will be required to overcome the limitations of scaled CMOS technology. One approach is to adopt Nano-Magnetic Logic Circuits, highly appealing for their extremely reduced power consumption. Despite the interesting nature of this approach, many problems arise when this technology is considered for real designs. The wire is the most critical of these problems from the circuit implementation point of view. It works as a pipelined interconnection, and its delay in terms of clock cycles depends on its length. Serious complications arise at the design phase, both in terms of synthesis and of physical design.

One possible solution is the use of a delay insensitive asynchronous logic, Null Convention Logic (NCL
TM
). Nevertheless its use has many negative consequences in terms of area occupation and speed loss with respect to a Boolean version. In this article we analyze and compare different solutions: nanomagnetic circuits based on full NCL, mixed Boolean-NCL, and fully Boolean logic. We discuss the advantages of these logics, but also the issues they raise. In particular we analyze feedback signals, which, due to their intrinsic pipelined nature, cause errors that still have not found a solution in the literature. The innovative arrangement we propose solves most of the problems and thus soundly increases the knowledge of this technology. The analysis is performed using a VHDL behavioral model we developed and a microprocessor we designed based on this model, as a sound and realistic test bench.
Comparing ingress and egress detection to secure interdomain routing
Authors: Goebel Christoph , Neumann Dirk , Krishnan Ramayya
Published: December 2011

The global economy and society increasingly depends on computer networks linked together by the Internet. The importance of computer networks reaches far beyond the telecommunications sector since they have become a critical factor for many other crucial infrastructures and markets. With threats mounting and security incidents becoming more frequent, concerns about network security grow.

It is an acknowledged fact that some of the most fundamental network protocols that make the Internet work are exposed to serious threats. One of them is the Border Gateway Protocol (BGP) which determines how Internet traffic is routed through the topology of administratively independent networks that the Internet is comprised of. Despite the existence of a steadily growing number of BGP security proposals, to date none of them has been adopted.

Using a precise definition of BGP robustness we experimentally show that the degree of robustness is distributed unequally across the administrative domains of the Internet, the so-called Autonomous Systems (ASes). The experiments confirm the intuition that the contribution ASes are able to make towards securing the correct working of the inter-domain routing infrastructure by deploying countermeasures against routing attacks differ depending on their position in the AS topology. We also show that the degree of this asymmetry can be controlled by the choice of the security strategy. We compare the strengths and weaknesses of two fundamentally different approaches in increasing BGP's robustness which we termed ingress and egress detection of false route advertisements and indicate their implications. Our quantitative results have important implications for Internet security policy, in particular with respect to the crucial question where to start the deployment of which type of security scheme in order to maximize the Internet's robustness to routing attacks.
Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-β and VEGFR-2 expression
Authors: Chanakira Alice , Dutta Raini , Charboneau Richard , Barke Roderick , Santilli Steven M. , Roy Sabita
Published: December 2011

Despite intensive research studies, theories have yet to focus on the contribution of hypoxia to patency differences observed clinically between arterial vs. venous grafts. This study investigates the differential hypoxic response of smooth muscle cells (SMC) to hypoxia-derived endothelial cell (EC) growth factors. Initiation of SMC proliferation under hypoxia (<5% O2) occurred only after incubation with hypoxic endothelial cell-conditioned media (H-ECM). After the investigation of several possible growth factors in the H-ECM that may be responsible for SMC proliferation, the greatest difference was observed in vascular endothelial growth factor (VEGF-A) and platelet-derived growth factor homodimer B (PDGF-BB) expression. VEGF-A increased (2-fold) significantly ( P < 0.05) in arterial-derived smooth muscle cells (ASMC) under hypoxia compared with venous-derived smooth muscle cells (VSMC), which showed no significant change. VSMC showed significant ( P < 0.05) increase in VEGFR-2 expression under hypoxia compared with ASMC. Incubation with VEGFR-2-neutralizing antibody/PDGFR antagonist in VSMC before addition of H-ECM resulted in decreased proliferation. ASMC proliferation under hypoxia did not decrease during incubation with VEGFR-2-neutralizing antibody but did decrease upon PDGFR antagonist incubation. Current therapies focusing on treating intimal hyperplasia have negated the fact that combinational therapy might be required to combat induction of SMC proliferation. Clinically, therapy with PDGFR antagonists plus anti-VEGFR-2 may prove to be efficacious in managing SMC proliferation in venous-derived grafts.
Effects of elastin degradation and surrounding matrix support on artery stability
Authors: Lee Avione Y. , Han Boyang , Lamm Shawn D. , Fierro Cesar A. , Han Hai-Chao
Published: December 2011

Tortuous arteries are often associated with aging, hypertension, atherosclerosis, and degenerative vascular diseases, but the mechanisms are poorly understood. Our recent theoretical analysis suggested that mechanical instability (buckling) may lead to tortuous blood vessels. The objectives of this study were to determine the critical pressure of artery buckling and the effects of elastin degradation and surrounding matrix support on the mechanical stability of arteries. The mechanical properties and critical buckling pressures, at which arteries become unstable and deform into tortuous shapes, were determined for a group of five normal arteries using pressurized inflation and buckling tests. Another group of nine porcine arteries were treated with elastase (8 U/ml), and the mechanical stiffness and critical pressure were obtained before and after treatment. The effect of surrounding tissue support was simulated using a gelatin gel. The critical pressures of the five normal arteries were 9.52 kPa (SD 1.53) and 17.10 kPa (SD 5.11) at axial stretch ratios of 1.3 and 1.5, respectively, while model predicted critical pressures were 10.11 kPa (SD 3.12) and 17.86 kPa (SD 5.21), respectively. Elastase treatment significantly reduced the critical buckling pressure ( P < 0.01). Arteries with surrounding matrix support buckled into multiple waves at a higher critical pressure. We concluded that artery buckling under luminal pressure can be predicted by a buckling equation. Elastin degradation weakens the arterial wall and reduces the critical pressure, which thus leads to tortuous vessels. These results shed light on the mechanisms of the development of tortuous vessels due to elastin deficiency.
Impairments in the intrinsic contractility of mesenteric collecting lymphatics in a rat model of metabolic syndrome
Authors: Zawieja Scott D. , Wang Wei , Wu Xin , Nepiyushchikh Zhanna V. , Zawieja David C. , Muthuchamy Mariappan
Published: December 2011

Numerous studies on metabolic syndrome (MetSyn), a cluster of metabolic abnormalities, have demonstrated its profound impact on cardiovascular and blood microvascular health; however, the effects of MetSyn on lymphatic function are not well understood. We hypothesized that MetSyn would modulate lymphatic muscle activity and alter muscularized lymphatic function similar to the impairment of blood vessel function associated with MetSyn, particularly given the direct proximity of the lymphatics to the chronically inflamed adipose depots. To test this hypothesis, rats were placed on a high-fructose diet (60%) for 7 wk, and their progression to MetSyn was assessed through serum insulin and triglyceride levels in addition to the expression of metabolic and inflammatory genes in the liver. Mesenteric lymphatic vessels were isolated and subjected to different transmural pressures while lymphatic pumping and contractile parameters were evaluated. Lymphatics from MetSyn rats had significant negative chronotropic effects at all pressures that effectively reduced the intrinsic flow-generating capacity of these vessels by ∼50%. Furthermore, lymphatics were remodeled to a significantly smaller diameter in the animals with MetSyn. Wire myograph experiments demonstrated that permeabilized lymphatics from the MetSyn group exhibited a significant decrease in force generation and were less sensitive to Ca2+, although there were no significant changes in lymphatic muscle cell coverage or morphology. Thus, our data provide the first evidence that MetSyn induces a remodeling of collecting lymphatics, thereby effectively reducing their potential load capabilities and impairing the intrinsic contractility required for proper lymph flow.
Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel
Authors: Sridharan Meera , Bowles Elizabeth A. , Richards Jennifer P. , Krantic Medina , Davis Katie L. , Dietrich Kristine A. , Stephenson Alan H. , Ellsworth Mary L. , Sprague Randy S.
Published: December 2011

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (Gi or Gs), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-xL BH44–23 and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-xL BH44–23 attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.