bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered Profile of Secondary Metabolites in the Root Exudates of Arabidopsis ATP-Binding Cassette Transporter Mutants
Authors: Badri Dayakar V. , Loyola-Vargas Victor M. , Broeckling Corey D. , De-la-Peña Clelia , Jasinski Michal , Santelia Diana , Martinoia Enrico , Sumner Lloyd W. , Banta Lois M. , Stermitz Frank , Vivanco Jorge M.
Published: December 2007

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.
Identification, Biochemical Characterization, and Subcellular Localization of Allantoate Amidohydrolases from Arabidopsis and Soybean
Authors: Werner Andrea K. , Sparkes Imogen A. , Romeis Tina , Witte Claus-Peter
Published: December 2007

Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO2, and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 μ  m manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of l-asparagine and l-aspartate but not d-asparagine. l-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum.
The Contribution of Photosynthesis to the Red Light Response of Stomatal Conductance
Authors: Baroli Irene , Price G. Dean , Badger Murray R. , von Caemmerer Susanne
Published: December 2007

To determine the contribution of photosynthesis on stomatal conductance, we contrasted the stomatal red light response of wild-type tobacco (Nicotiana tabacum ‘W38’) with that of plants impaired in photosynthesis by antisense reductions in the content of either cytochrome b  6  f complex (anti-b/f plants) or Rubisco (anti-SSU plants). Both transgenic genotypes showed a lowered content of the antisense target proteins in guard cells as well as in the mesophyll. In the anti-b/f plants, CO2 assimilation rates were proportional to leaf cytochrome b  6  f content, but there was little effect on stomatal conductance and the rate of stomatal opening. To compare the relationship between photosynthesis and stomatal conductance, wild-type plants and anti-SSU plants were grown at 30 and 300 μmol photon m−2 s−1 irradiance (low light and medium light [ML], respectively). Growth in ML increased CO2 assimilation rates and stomatal conductance in both genotypes. Despite the significantly lower CO2 assimilation rate in the anti-SSU plants, the differences in stomatal conductance between the genotypes were nonsignificant at either growth irradiance. Irrespective of plant genotype, stomatal density in the two leaf surfaces was 2-fold higher in ML-grown plants than in low-light-grown plants and conductance normalized to stomatal density was unaffected by growth irradiance. We conclude that the red light response of stomatal conductance is independent of the concurrent photosynthetic rate of the guard cells or of that of the underlying mesophyll. Furthermore, we suggest that the correlation of photosynthetic capacity and stomatal conductance observed under different light environments is caused by signals largely independent of photosynthesis.
Pectin Methyl Esterase Inhibits Intrusive and Symplastic Cell Growth in Developing Wood Cells ofPopulus
Authors: Siedlecka Anna , Wiklund Susanne , Péronne Marie-Amélie , Micheli Fabienne , Leśniewska Joanna , Sethson Ingmar , Edlund Ulf , Richard Luc , Sundberg Björn , Mellerowicz Ewa J.
Published: December 2007

Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula × tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.
Two Distinct Forms ofM-Locus Protein Kinase Localize to the Plasma Membrane and Interact Directly withS-Locus Receptor Kinase to Transduce Self-Incompatibility Signaling inBrassica rapa
Authors: Kakita Mitsuru , Murase Kohji , Iwano Megumi , Matsumoto Tomohito , Watanabe Masao , Shiba Hiroshi , Isogai Akira , Takayama Seiji
Published: December 2007

Many flowering plants possess systems of self-incompatibility (SI) to prevent inbreeding. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which encodes both the male determinant S-locus protein 11 (SP11/SCR) and the female determinant S-receptor kinase (SRK). Upon self-pollination, the S-haplotype–specific interaction between the pollen-borne SP11 and the cognate stigmatic SRK receptor induces SI signaling in the stigmatic papilla cell and results in rejection of the self-pollen. Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, M-locus protein kinase (MLPK), in the SI signaling, but its exact physiological function remains unknown. In this study, we identified two different MLPK transcripts, MLPKf1 and MLPKf2, which are produced using alternative transcriptional initiation sites and encode two isoforms that differ only at the N termini. While MLPKf1 and MLPKf2 exhibited distinct expression profiles, both were expressed in papilla cells. MLPKf1 localizes to the plasma membrane through its N-terminal myristoylation motif, while MLPKf2 localizes to the plasma membrane through its N-terminal hydrophobic region. Although both MLPKf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, a bimolecular fluorescence complementation assay revealed direct interactions between SRK and the MLPK isoforms in planta. These results suggest that MLPK isoforms localize to the papilla cell membrane and interact directly with SRK to transduce SI signaling.
Interaction between SGT1 and Cytosolic/Nuclear HSC70 Chaperones Regulates Arabidopsis Immune Responses
Authors: Noël Laurent D. , Cagna Giuseppe , Stuttmann Johannes , Wirthmüller Lennart , Betsuyaku Shigeyuki , Witte Claus-Peter , Bhat Riyaz , Pochon Nathalie , Colby Thomas , Parker Jane E.
Published: December 2007

The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene–triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70–SGT1 association.
Cytokinin Regulates Type-A Arabidopsis Response Regulator Activity and Protein Stability via Two-Component Phosphorelay
Authors: To Jennifer P.C. , Deruère Jean , Maxwell Bridey B. , Morris Veronica F. , Hutchison Claire E. , Ferreira Fernando J. , Schaller G. Eric , Kieber Joseph J.
Published: December 2007

The plant hormone cytokinin regulates many aspects of growth and development. Cytokinin signaling involves His kinase receptors that perceive cytokinin and transmit the signal via a multistep phosphorelay similar to bacterial two-component signaling systems. The final targets of this phosphorelay are a set of Arabidopsis thaliana Response Regulator (ARR) proteins containing a receiver domain with a conserved Asp phosphorylation site. One class of these, the type-A ARRs, are negative regulators of cytokinin signaling that are rapidly transcriptionally upregulated in response to cytokinin. In this study, we tested the role of phosphorylation in type-A ARR function. Our results indicate that phosphorylation of the receiver domain is required for type-A ARR function and suggest that negative regulation of cytokinin signaling by the type-A ARRs most likely involves phosphorylation-dependent interactions. Furthermore, we show that a subset of the type-A ARR proteins are stabilized in response to cytokinin in part via phosphorylation. These studies shed light on the mechanism by which type-A ARRs act to negatively regulate cytokinin signaling and reveal a novel mechanism by which cytokinin controls type-A ARR function.
A CRM Domain Protein Functions Dually in Group I and Group II Intron Splicing in Land Plant Chloroplasts
Authors: Asakura Yukari , Barkan Alice
Published: December 2007

The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.
Cytokinins Act Directly on Lateral Root Founder Cells to Inhibit Root Initiation
Authors: Laplaze Laurent , Benkova Eva , Casimiro Ilda , Maes Lies , Vanneste Steffen , Swarup Ranjan , Weijers Dolf , Calvo Vanessa , Parizot Boris , Herrera-Rodriguez Maria Begoña , Offringa Remko , Graham Neil , Doumas Patrick , Friml Jiri , Bogusz Didier , Beeckman Tom , Bennett Malcolm
Published: December 2007

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.
Hydrocarbon fluxes for ionic compound free soldering
Authors: Miyake Toshihiro , Ishida Masaru , Inagaki Satoshi
Published: December 2007

Purpose

The purpose of this paper is to develop a new ionic compound free soldering process.

Design/methodology/approach

The ability of hydrocarbons including 9,10‐dihydroanthracene, eicosane, 2,6,10,14‐tetramethylpentadecane, cyclooctane, and dicyclopentadiene to reduce cuprous or cupric oxide was investigated. The applicability of hydrocarbons as ion free flux reagents was experimentally examined. The hydrocarbons were applied to the soldering of pre‐oxidized copper electrodes under practical conditions.

Findings

A 9,10‐dihydroanthracene was found to be efficient at reducing cuprous or cupric oxide powder under an argon atmosphere at 300°C for 2 min. The high‐reducing ability of the hydrocarbon is in agreement with the low‐homolytical C−H bond dissociation energy calculated based on the density functional theory. A 9,10‐dihydroanthracene was shown, by the highest soldering efficiency and sufficient reliability in the environmental testing, to be applicable as an ion‐free soldering flux.

Originality/value

The findings of this paper suggest a new method for an ionic compound free soldering process.
The effect of supervision style and decision‐making on role stress and satisfaction of senior foreign managers in international joint ventures in China
Authors: Lu Lung‐Tan , Lee Yuan‐Ho
Published: December 2007

Purpose

The aim of this study is to examine the effect of supervision style and decision‐making on role stress (role conflict, role ambiguity, role overload) and satisfaction of senior foreign managers working in international joint ventures (IJVs) in China.

Design/methodology/approach

This paper is a quantitative study using an international mail survey among Sino‐Foreign joint ventures. The final sample size was comprised of 82 Japanese and Taiwanese senior managers working in IJVs in China. Regression analysis was used to assess the relationship among these variables.

Findings

Regression results demonstrated that a tighter supervision style by Japanese managers positively influences role conflict and role overload. Surprisingly, centralized decision‐making by Japanese managers also positively effects role ambiguity in Japanese managers, but centralized decision‐making by Taiwanese managers negatively effects role ambiguity in Taiwanese managers.

Research limitations/implications

While the sample size is relatively small, the findings in this study suggest that the effects of supervision style and decision‐making are different between the Japanese and Taiwanese groups. It is suggested that further research of management styles among eastern and western managers is required.

Originality/value

This study contributes to international business literature and provides insights to expatriates by investigating the relationships among supervision style, decision‐making, role stress, and satisfaction. It also provides some evidence that the impact of decision‐making on role ambiguity is different between national groups, which have been given limited attention in previous literature.
CRE effects on organizational performance: measurement tools for management
Authors: Appel‐Meulenbroek Rianne , Feijts Ben
Published: December 2007

Purpose

The purpose of this paper is to identify potential measures for corporate real estate (CRE) aspects to correlate them with organizational performance. These measures must simplify discussion between corporate real estate management (CREM), architects and users, and help CREM to claim its place at the strategic decision‐making table.

Design/methodology/approach

An analysis is made of literature in relevant fields (e.g. CREM, architecture, ergonomics and installation technology) to identify aspects that influence performance. For the ones that lacked clear measures, a solution was deducted from other fields of study (environmental psychology, geography, urban design). These new measures are applied to two existing office buildings to show their relevance and usability, followed by a discussion on how to use them as a CRE manager.

Findings

In total, 51 CRE aspects affect organizational performance. Particularly, the structural aspects are not quantified or measured in consistent ways, like “open layout” and “visibility”. Methods from the field of spatial network analysis show very promising possibilities for quantifying these configuration related aspects. Also, it does not appear to be very difficult for CREM to start using these methods.

Originality/value

The spatial network analysis methods that are described and applied in this paper, have not been subject of an article in any (corporate) real estate‐related journal yet. Up till now, this field of research appears to take place in a totally different community of academics and practitioners. Hopefully, this paper will make CREM aware of these methods and encourage them to use them.
Comparative In Vitro Activities of Caspofungin and Micafungin, Determined Using the Method of the European Committee on Antimicrobial Susceptibility Testing, against Yeast Isolates Obtained in France in 2005-2006
Authors: Dannaoui E. , Lortholary O. , Raoux D. , Bougnoux M. E. , Galeazzi G. , Lawrence C. , Moissenet D. , Poilane I. , Hoinard D. , Dromer F.
Published: December 2007

The in vitro activities of caspofungin and micafungin against 1,038 yeast isolates have been determined. The caspofungin and micafungin MICs were lower for
Candida albicans
,
Candida glabrata
, and
Candida tropicalis
than for
Candida parapsilosis
,
Candida guilliermondii
, and
Candida krusei
. A clear correlation was seen between the MICs for the two drugs.
First Detection of Mycobacteria in African Rodents and Insectivores, Using Stratified Pool Screening
Authors: Durnez Lies , Eddyani Miriam , Mgode Georgies F. , Katakweba Abdul , Katholi Charles R. , Machang'u Robert R. , Kazwala Rudovik R. , Portaels Françoise , Leirs Herwig
Published: December 2007

With the rising number of patients with human immunodeficiency virus (HIV)/AIDS in developing countries, the control of mycobacteria is of growing importance. Previous studies have shown that rodents and insectivores are carriers of mycobacteria. However, it is not clear how widespread mycobacteria are in these animals and what their role is in spreading them. Therefore, the prevalence of mycobacteria in rodents and insectivores was studied in and around Morogoro, Tanzania. Live rodents were trapped, with three types of live traps, in three habitats. Pieces of organs were pooled per habitat, species, and organ type (stratified pooling); these sample pools were examined for the presence of mycobacteria by PCR, microscopy, and culture methods. The mycobacterial isolates were identified using phenotypic techniques and sequencing. In total, 708 small mammals were collected, 31 of which were shrews. By pool prevalence estimation, 2.65% of the animals were carriers of mycobacteria, with a higher prevalence in the urban areas and in
Cricetomys gambianus
and the insectivore
Crocidura hirta
. Nontuberculous mycobacteria (
Mycobacterium chimaera
,
M. intracellulare
,
M. arupense
,
M. parascrofulaceum
, and
Mycobacterium
spp.) were isolated from
C. gambianus
,
Mastomys natalensis
, and
C. hirta
. This study is the first to report findings of mycobacteria in African rodents and insectivores and the first in mycobacterial ecology to estimate the prevalence of mycobacteria after stratified pool screening. The fact that small mammals in urban areas carry more mycobacteria than those in the fields and that potentially pathogenic mycobacteria were isolated identifies a risk for other animals and humans, especially HIV/AIDS patients, that have a weakened immune system.
Application of Methods for Identifying Broiler Chicken Gut Bacterial Species Linked with Increased Energy Metabolism
Authors: Torok Valeria A. , Ophel-Keller Kathy , Loo Maylene , Hughes Robert J.
Published: December 2007

A high-throughput microbial profiling tool based on terminal restriction fragment length polymorphism was developed to monitor the poultry gut microbiota in response to dietary manipulations. Gut microbial communities from the duodena, jejuna, ilea, and ceca of 48 birds fed either a barley control diet or barley diet supplemented with exogenous enzymes for degrading nonstarch polysaccharide were characterized by using multivariate statistical methods. Analysis of samples showed that gut microbial communities varied significantly among gut sections, except between the duodenum and jejunum. Significant diet-associated differences in gut microbial communities were detected within the ileum and cecum only. The dissimilarity in bacterial community composition between diets was 73 and 66% within the ileum and cecum, respectively. Operational taxonomic units, representing bacterial species or taxonomically related groups, contributing to diet-associated differences were identified. Several bacterial species contributed to differences between diet-related gut microbial community composition, with no individual bacterial species contributing more than 1 to 5% of the total. Using canonical analysis of principal coordinates biplots, we correlated differences in gut microbial community composition within the ileum and cecum to improved performance, as measured by apparent metabolizable energy. This is the first report that directly links differences in the composition of the gut microbial community with improved performance, which implies that the presence of specific beneficial and/or absence of specific detrimental bacterial species may contribute to the improved performance in these birds.
Has Retail Chicken Played a Role in the Decline of Human Campylobacteriosis?
Authors: Gormley Fraser J. , MacRae Marion , Forbes Ken J. , Ogden Iain D. , Dallas John F. , Strachan Norval J. C.
Published: December 2007

Between 2001 and 2006, the incidence of human
Campylobacter
infections decreased by 10 and 27% in Scotland and the Grampian region of Scotland, respectively. Contemporaneous collection and analyses of human and retail-chicken isolates from Grampian were carried out over a 10-week period in 2001 and again in 2006 in order to determine whether the fall in the incidence of human infections was related to the retail-chicken exposure route. Rates of carriage of
Campylobacter
on chicken carcasses from retail outlets in Grampian in 2001 and 2006 were estimated. Chicken-derived
Campylobacter
isolates from 2001 (
n
= 84) and 2006 (
n
= 105) and human-derived isolates from patients with clinical cases of infection in 2001 (
n
= 172) and 2006 (
n
= 119) were typed by multilocus sequence typing. We found no evidence for statistically significant changes in prevalence and counts per carcass. We found by rarefaction that although the degree of diversity in humans tended to be higher than that in chickens, these differences were not significant. The genetic distance between chicken and human isolates from 2001 according to sequence type, clonal complex (CC), or allele composition was not significant, whereas the distances between 2006 isolates at the CC and allele levels were significant. This difference was attributable to a lower proportion of CC-21's being found in retail-chicken isolates from 2006 than in chicken isolates from 2001. We conclude that human exposure to
Campylobacter
via retail chicken is important and that changes in the population structure of campylobacters in this reservoir need to be taken into account in investigating human infection.
High Frequency of Histamine-Producing Bacteria in the Enological Environment and Instability of the Histidine Decarboxylase Production Phenotype
Authors: Lucas Patrick M. , Claisse Olivier , Lonvaud-Funel Aline
Published: December 2007

Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the generaOenococcus,Lactobacillus, andPediococcus. All carry anhdcAgene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targetinghdcAwas developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 107CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 103CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains ofOenococcus oenithat grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover,hdcAwas detected on a large and possibly unstable plasmid in these strains ofO. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 103per ml. Strains ofO. oeniproducing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.
Occurrence and Expression of Luminescence in
Vibrio cholerae
Authors: Grim Christopher J. , Taviani Elisa , Alam Munirul , Huq Anwar , Sack R. Bradley , Colwell Rita R.
Published: December 2007

Several species of the genus
Vibrio
, including
Vibrio cholerae
, are bioluminescent or contain bioluminescent strains. Previous studies have reported that only 10% of
V. cholerae
strains are luminescent. Analysis of 224 isolates of non-O1/non-O139
V. cholerae
collected from Chesapeake Bay, MD, revealed that 52% (116/224) were luminescent when an improved assay method was employed and 58% (130/224) of isolates harbored the
luxA
gene. In contrast, 334 non-O1/non-O139
V. cholerae
strains isolated from two rural provinces in Bangladesh yielded only 21 (6.3%) luminescent and 35 (10.5%)
luxA
+
isolates. An additional 270 clinical and environmental isolates of
V. cholerae
serogroups O1 and O139 were tested, and none were luminescent or harbored
luxA
. These results indicate that bioluminescence may be a trait specific for non-O1/non-O139
V. cholerae
strains that frequently occur in certain environments. Luminescence expression patterns of
V. cholerae
were also investigated, and isolates could be grouped based on expression level. Several strains with defective expression of the
lux
operon, including natural K variants, were identified.
Production of a Monoclonal Antibody Specific for the Major Outer Membrane Protein of
Campylobacter jejuni
and Characterization of the Epitope
Authors: Qian Hongliang , Pang Ervinna , Du Qingyun , Chang Jason , Dong Jin , Toh Say Ling , Ng Fook Kheong , Tan Ai Ling , Kwang Jimmy
Published: December 2007

Campylobacter
species are important enteric pathogens causing disease in humans and animals. There is a lack of a good immunological test that can be used routinely to separate
Campylobacter jejuni
from other
Campylobacter
species. We produced monoclonal antibodies (MAbs) directed against the major outer membrane protein (MOMP) of
C. jejuni
using recombinant MOMP as the antigen. One MAb, designated MAb5C4 and of the immunoglobulin G1 isotype, was found to be potentially specific for
C. jejuni
. Dot blots demonstrated that MAb5C4 reacted with all 29 isolates of
C. jejuni
tested but did not react with 2
C. jejuni
isolates, 26 other
Campylobacter
spp. isolates, and 19 non-
Campylobacter
isolates. Western blotting showed that MAb5C4 bound to a single protein band approximately 43 kDa in size, corresponding to the expected size of
C. jejuni
MOMP. The detection limit of MAb5C4 in a dot blot assay was determined to be about 5 × 10
3
bacteria. The epitope on the MOMP was mapped to a region six amino acids in length with the sequence
216
GGQFNP
221
, which is 97% conserved among
C. jejuni
strains but divergent in other
Campylobacter
spp.; a GenBank search indicated that 95% of
C. jejuni
isolates will be able to be detected from non-
Campylobacter
spp. based on the highly specific and conserved region of the GGQFNP polypeptide. The epitope is predicted to be located in a region that is exposed to the periplasm. MAb5C4 is a potentially specific and sensitive MAb that can be used for the specific detection and identification of
C. jejuni
.
Biochemical and Molecular Characterization ofSaccharomyces cerevisiaeStrains Obtained from Sugar-Cane Juice Fermentations and Their Impact in Cachaça Production
Authors: Oliveira Valdinéia Aparecida , Vicente Maristela Araújo , Fietto Luciano Gomes , de Miranda Castro Ieso , Coutrim Maurício Xavier , Schüller Dorit , Alves Henrique , Casal Margarida , de Oliveira Santos Juliana , Araújo Leandro Dias , da Silva Paulo Henrique Alves , Brandão Rogelio Lopes
Published: December 2007

Saccharomyces cerevisiaestrains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5′,5"-trifluor-d,l-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of theCOX1gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard.
Effects ofADH2Overexpression inSaccharomyces bayanusduring Alcoholic Fermentation
Authors: Maestre Oscar , García-Martínez Teresa , Peinado Rafael A. , Mauricio Juan C.
Published: December 2007

The effect of overexpression of the geneADH2on metabolic and biological activity inSaccharomyces bayanusV5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the geneADH2during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, andl-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.
Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution
Authors: Shanks Orin C. , Atikovic Emina , Blackwood A. Denene , Lu Jingrang , Noble Rachel T. , Domingo Jorge Santo , Seifring Shawn , Sivaganesan Mano , Haugland Richard A.
Published: December 2007

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 106copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing totalBacteroidetes,Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.
Forest Soil Metagenome Gene Cluster Involved in Antifungal Activity Expression in
Escherichia coli
Authors: Chung Eu Jin , Lim He Kyoung , Kim Jin-Cheol , Choi Gyung Ja , Park Eun Jin , Lee Myung Hwan , Chung Young Ryun , Lee Seon-Woo
Published: December 2007

Using two forest soils, we previously constructed two fosmid libraries containing 113,700 members in total. The libraries were screened to select active antifungal clones using
Saccharomyces cerevisiae
as a target fungus. One clone from the Yuseong pine tree rhizosphere soil library, pEAF66, showed
S. cerevisiae
growth inhibition. Despite an intensive effort, active chemicals were not isolated. DNA sequence analysis and transposon mutagenesis of pEAF66 revealed 39 open reading frames (ORFs) and indicated that eight ORFs, probably in one transcriptional unit, might be directly involved in the expression of antifungal activity in
Escherichia coli
. The deduced amino acid sequences of eight ORFs were similar to those of the core genes encoding type II family polyketide synthases, such as the acyl carrier protein (ACP), ACP synthases, aminotransferase, and ACP reductase. The gene cluster involved in antifungal activity was similar in organization to the putative antibiotic production locus of
Pseudomonas putida
KT2440, although we could not select a similar active clone from the KT2440 genomic DNA library in
E. coli
. ORFs encoding ATP binding cassette transporters and membrane proteins were located at both ends of the antifungal gene cluster. Upstream ORFs encoding an IclR family response regulator and a LysR family response regulator were involved in the positive regulation of antifungal gene expression. Our results suggested the metagenomic approach as an alternative to search for novel antifungal antibiotics from unculturable soil bacteria. This is the first report of an antifungal gene cluster obtained from a soil metagenome using
S. cerevisiae
as a target fungus.
Proteomic Analyses of aListeria monocytogenesMutant Lacking σBIdentify New Components of the σBRegulon and Highlight a Role for σBin the Utilization of Glycerol
Authors: Abram F. , Su Wan-Lin , Wiedmann M. , Boor K. J. , Coote P. , Botting C. , Karatzas K. A. G. , O'Byrne C. P.
Published: December 2007

InListeria monocytogenesthe alternative sigma factor σBplays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σBregulon inL. monocytogenes10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigBdeletion strain were compared. Overall, this study identified 38 proteins whose expression was σBdependent; 17 of these proteins were found to require the presence of σBfor full expression, while 21 were expressed at a higher level in the ΔsigBmutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σBin the general stress response and highlighted a probable role for σBin metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σBin glycerol metabolism. Five newly identified members of the σBregulon were shown to be under direct regulation of σBusing reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σBwas shown to play a major role in the stationary phase of growth in complex media.
Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater
Authors: Bae Jinhee , Schwab Kellogg J.
Published: December 2007

Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (
n
= 9) and groundwater samples (
n
= 6). Viral persistence was monitored at 25°C and 4°C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25°C (0.18 and 0.09 log
10
/day for FCV, 0.13 and 0.10 log
10
/day for PV, 0.12 and 0.06 log
10
/day for MS2, and 0.09 and 0.05 log
10
/day for MNV) but not significant at 4°C. According to a multiple linear regression model, the NV NA reduction rates (0.04 ± 0.01 log
10
/day) were not significantly different from the NA reduction rates of MS2 (0.05 ± 0.03 log
10
/day) and MNV (0.04 ± 0.03 log
10
/day) and were significantly different from those of FCV (0.08 ± 0.03 log
10
/day) and PV (0.09 ± 0.03 log
10
/day) at 25°C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.
Characterization of
htrB
and
msbB
Mutants of the Light Organ Symbiont
Vibrio fischeri
Authors: Adin Dawn M. , Phillips Nancy J. , Gibson Bradford W. , Apicella Michael A. , Ruby Edward G. , McFall-Ngai Margaret J. , Hall Daniel B. , Stabb Eric V.
Published: December 2007

Bacterial lipid A is an important mediator of bacterium-host interactions, and secondary acylations added by HtrB and MsbB can be critical for colonization and virulence in pathogenic infections. In contrast,
Vibrio fischeri
lipid A stimulates normal developmental processes in this bacterium's mutualistic host,
Euprymna scolopes
, although the importance of lipid A structure in this symbiosis is unknown. To further examine
V. fischeri
lipid A and its symbiotic function, we identified two paralogs of
htrB
(designated
htrB1
and
htrB2
) and an
msbB
gene in
V. fischeri
ES114 and demonstrated that these genes encode lipid A secondary acyltransferases.
htrB2
and
msbB
are found on the
Vibrio
“housekeeping” chromosome 1 and are conserved in other
Vibrio
species. Mutations in
htrB2
and
msbB
did not impair symbiotic colonization but resulted in phenotypic alterations in culture, including reduced motility and increased luminescence. These mutations also affected sensitivity to sodium dodecyl sulfate, kanamycin, and polymyxin, consistent with changes in membrane permeability. Conversely,
htrB1
is located on the smaller, more variable vibrio chromosome 2, and an
htrB1
mutant was wild-type-like in culture but appeared attenuated in initiating the symbiosis and was outcompeted 2.7-fold during colonization when mixed with the parent. These data suggest that
htrB2
and
msbB
play conserved general roles in vibrio biology, whereas
htrB1
plays a more symbiosis-specific role in
V. fischeri
.
Plasmids of the pRM/pRF Family Occur in DiverseRickettsiaSpecies
Authors: Baldridge Gerald D. , Burkhardt Nicole Y. , Felsheim Roderick F. , Kurtti Timothy J. , Munderloh Ulrike G.
Published: December 2007

The recent discoveries of the pRF and pRM plasmids ofRickettsia felisandR. monacensishave contravened the long-held dogma that plasmids are not present in the bacterial genusRickettsia(Rickettsiales;Rickettsiaceae). We report the existence of plasmids inR. helvetica, R. peacockii, R. amblyommii, andR. massiliaeisolates from ixodid ticks and in anR. hoogstraaliiisolate from an argasid tick.R. peacockiiand four isolates ofR. amblyommiifrom widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. TheR. peacockiiplasmids were lost during long-term serial passage in cultured cells.R. montanensisdid not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that ofR. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. TheR. hoogstraaliiplasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. TheR. hoogstraalii, R. helvetica, andR. amblyommiiplasmids contained homologs of a pRM-carried gene similar to aNitrobactersp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to aBurkholderiasp. transposon resolvase gene.
PneumocystisEncodes a FunctionalS-Adenosylmethionine Synthetase Gene
Authors: Kutty Geetha , Hernandez-Novoa Beatriz , Czapiga Meggan , Kovacs Joseph A.
Published: December 2007

S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6) is the enzyme that catalyzes the synthesis of AdoMet, a molecule important for all cellular organisms. We have cloned and characterized an AdoMet synthetase gene (sam1) fromPneumocystisspp. This gene was transcribed primarily as an ∼1.3-kb mRNA which encodes a protein containing 381 amino acids inP. cariniiorP. murinaand 382 amino acids inP. jirovecii. sam1was also transcribed as part of an apparent polycistronic transcript of ∼5.6 kb, together with a putative chromatin remodeling protein homologous toSaccharomyces cerevisiae, CHD1. Recombinant Sam1, when expressed inEscherichia coli, showed functional enzyme activity. Immunoprecipitation and confocal immunofluorescence analysis using an antipeptide antibody showed that this enzyme is expressed inP. murina. Thus,Pneumocystis, like other organisms, can synthesize its own AdoMet and may not depend on its host for the supply of this important molecule.