bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Fungal and Bacterial Plant Pathogens In Vitro and In Planta with Ultrashort Cationic Lipopeptides
Authors: Makovitzki Arik , Viterbo Ada , Brotman Yariv , Chet Ilan , Shai Yechiel 
Published: August 2007

ABSTRACT

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen
Botrytis cinerea
as well as on corn leaves infected with
Cochliobolus heterostrophus
. Similarly, treatment with the lipopeptides of
Arabidopsis
leaves infected with the bacterial leaf pathogen
Pseudomonas syringae
efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.
Seasonal Detection of Human Viruses and Coliphage in Newport Bay, California
Authors: Jiang Sunny C. , Chu Weiping , He Jian-Wen 
Published: August 2007

ABSTRACT
Recent studies have shown that the fecal indicator bacteria (FIB) currently used to indicate water quality in the coastal environment may be inadequate to reflect human viral contamination. Coliphage was suggested as a better indicator of human viral pollution and was proposed by the U.S. EPA as an alternative indicator for fecal pollution in groundwater. In this study, we investigated the occurrence and distribution of FIB, F+ coliphage, and PCR-detectable human adenovirus and enterovirus for an entire year at 15 locations around the Newport Bay watershed, an important southern California estuary for water recreation and an ecological reserve. Peak concentrations and prevalences of FIB and F+ coliphage were associated with winter storms (wet weather). Human adenoviruses and enteroviruses, however, were detected by PCR in ∼5% of samples collected in the summer (dry weather) but only once in wet weather. These results demonstrated that FIB and coliphage have similar seasonal and freshwater-to-saltwater distribution patterns, while the detection of human viruses depends on a distribution pattern that is the opposite of that of FIB and coliphage. This research suggested that coliphage and FIB share similar environmental sources, while sources of human viruses in Newport Bay are perhaps different.
Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers
Authors: Schwarzenbach Kaspar , Widmer Franco , Enkerli Jürg 
Published: August 2007

ABSTRACT

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on
Beauveria brongniartii
, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of
B. brongniartii
. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a
B. brongniartii
BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of
B. brongniartii
based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.
Identification ofN-Acetylhexosamine 1-Kinase in the Complete Lacto-N-Biose I/Galacto-N-Biose Metabolic Pathway inBifidobacterium longum
Authors: Nishimoto Mamoru , Kitaoka Motomitsu 
Published: August 2007

ABSTRACTWe have determined the functions of the enzymes encoded by thelnpB, lnpC, andlnpDgenes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), inBifidobacterium longumJCM1217. ThelnpBgene encodes a novel kinase,N-acetylhexosamine 1-kinase, which producesN-acetylhexosamine 1-phosphate; thelnpCgene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active onN-acetylhexosamine 1-phosphate; and thelnpDgene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operonlnpABCDencodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.
Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in
Sesbania rostrata-Azorhizobium caulinodans
ORS571 Symbiosis
Authors: Suzuki Shino , Aono Toshihiro , Lee Kyung-Bum , Suzuki Tadahiro , Liu Chi-Te , Miwa Hiroki , Wakao Seiji , Iki Taichiro , Oyaizu Hiroshi 
Published: August 2007

ABSTRACT

The molecular and physiological mechanisms behind the maturation and maintenance of N
2
-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood.
Azorhizobium caulinodans
ORS571 is a microsymbiont of the tropical legume
Sesbania rostrata
, forming N
2
-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of
A. caulinodans
ORS571 were individually inoculated onto the stems of
S. rostrata
, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn
5
mutants were selected and categorized into seven nodulation types based on size and N
2
fixation activity. Tn
5
insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as
nod, nif
, and
fix
, respectively, lipopolysaccharide synthesis-related genes, C
4
metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.
mariner
-Based Transposon Mutagenesis of
Rickettsia prowazekii
Authors: Liu Zhi-Mei , Tucker Aimee M. , Driskell Lonnie O. , Wood David O. 
Published: August 2007

ABSTRACT

Rickettsia prowazekii
, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile
mariner
-based,
Himar1
transposon system to generate insertional mutants of
R. prowazekii
. A transposon containing the
R. prowazekii arr-2
rifampin resistance gene and a gene coding for a green fluorescent protein (GFP
UV
) was constructed and placed on a plasmid expressing the
Himar1
transposase. Electroporation of this plasmid into
R. prowazekii
resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques,
R. prowazekii
transformants expressing GFP
UV
were easily visible by fluorescence microscopy. Thus, a
mariner
-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP
UV
fluorescence.
Severe Tryptophan Starvation Blocks Onset of Conventional Persistence and Reduces Reactivation of
Chlamydia trachomatis
Authors: Leonhardt Ralf M. , Lee Seung-Joon , Kavathas Paula B. , Cresswell Peter 
Published: August 2007

ABSTRACT

The intracellular survival of the bacterial pathogen
Chlamydia trachomatis
depends on protein synthesis by the microbe soon after internalization. Pharmacologic inhibition of bacterial translation inhibits early trafficking of the parasitophorous vacuole (inclusion) to the microtubule-organizing center (MTOC) and promotes its fusion with lysosomes, which is normally blocked by
Chlamydia
. Depletion of cellular tryptophan pools by gamma interferon-inducible indoleamine-2,3-dioxygenase (IDO) is believed to be the major innate immune mechanism controlling
C. trachomatis
infection in human cells, an action to which the bacteria can respond by converting into a nonreplicating but highly reactivatable persistent state. However, whether severe IDO-mediated tryptophan starvation can be sufficient to fully arrest the chlamydial life cycle and thereby counteract the onset of persistence is unknown. Here we demonstrate that at low exogenous tryptophan concentrations a substantial fraction of
C. trachomatis
bacteria fail to traffic to the MTOC or to switch into the conventional persistent state in gamma interferon-induced human cells. The organisms stay scattered in the cell periphery, do not retain infectivity, and display only low transcriptional activity. Importantly, the rate at which these aberrant
Chlamydia
bacteria become reactivated upon replenishment of cellular tryptophan pools is substantially lower. Thus, severe tryptophan depletion in cells with high IDO activity affects chlamydial development more rigorously than previously described.
Inhibition of Staphylococcal Biofilm Formation by Nitrite
Authors: Schlag Steffen , Nerz Christiane , Birkenstock Timo A. , Altenberend Florian , Götz Friedrich 
Published: August 2007

ABSTRACT

Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesin (PIA) synthesis and biofilm formation by the human pathogens
Staphylococcus aureus
and
Staphylococcus epidermidis
. In this study we characterized an adaptive response of
S. aureus
SA113 to nitrite-induced stress and show that it involves concomitant impairment of PIA synthesis and biofilm formation. Transcriptional analysis provided evidence that nitrite, either as the endogenous product of respiratory nitrate reduction or after external addition, causes repression of the
icaADBC
gene cluster, mediated likely by IcaR. Comparative microarray analysis revealed a global change in gene expression during growth in the presence of 5 mM sodium nitrite and indicated a response to oxidative and nitrosative stress. Many nitrite-induced genes are involved in DNA repair, detoxification of reactive oxygen and nitrogen species, and iron homeostasis. Moreover, preformed biofilms could be eradicated by the addition of nitrite, likely the result of the formation of toxic acidified nitrite derivatives. Nitrite-mediated inhibition of
S. aureus
biofilm formation was abrogated by the addition of nitric oxide (NO) scavengers, suggesting that NO is directly or indirectly involved. Nitrite also repressed biofilm formation of
S. epidermidis
RP62A.
A TolC-Like Protein Is Required for Heterocyst Development inAnabaenasp. Strain PCC 7120
Authors: Moslavac Suncana , Nicolaisen Kerstin , Mirus Oliver , Al Dehni Fadi , Pernil Rafael , Flores Enrique , Maldener Iris , Schleiff Enrico 
Published: August 2007

ABSTRACTThe filamentous cyanobacteriumAnabaenasp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane β-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 genehgdD(heterocystglycolipiddeposition protein).
Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA
Authors: Uchino Keiichi , Saito Terumi , Gebauer Birgit , Jendrossek Dieter 
Published: August 2007

ABSTRACT

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from
Ralstonia eutropha
catalyzed formation of PHB from
14
C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD
+
, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD
+
/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.
Involvement of the Detoxifying Enzyme Lactoylglutathione Lyase in
Streptococcus mutans
Aciduricity
Authors: Korithoski Bryan , Lévesque Céline M. , Cvitkovitch Dennis G. 
Published: August 2007

ABSTRACT

Streptococcus mutans
, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Its main virulence factors are acidogenicity and aciduricity, the abilities to produce acid and to survive and grow at low pH, respectively. Metabolic processes are finely regulated following acid exposure in
S. mutans
. Proteome analysis of
S. mutans
demonstrated that lactoylglutathione lyase (LGL) was up-regulated during acid challenge. The LGL enzyme catalyzes the conversion of toxic methylglyoxal, derived from glycolysis, to
S
-
d
-lactoylglutathione. Methylglyoxal inhibits the growth of cells in all types of organisms. The current study aimed to investigate the relationship between LGL and aciduricity and acidogenicity in
S. mutans
. An
S. mutans
isogenic mutant defective in
lgl
(LGLKO) was created, and its growth kinetics were characterized. Insertional inactivation of
lgl
resulted in an acid-sensitive phenotype. However, the glycolytic rate at pH 5.0 was greater for LGLKO than for
S. mutans
UA159 wild-type cells. LGL was involved in the detoxification of methylglyoxal, illustrated by the absence of enzyme activity in LGLKO and the hypersensitivity of LGLKO to methylglyoxal, compared with UA159 (MIC of 3.9 and 15.6 mM, respectively). Transcriptional analysis of
lgl
conducted by quantitative real-time PCR revealed that
lgl
was up-regulated (approximately sevenfold) during the exponential growth phase compared with that in the stationary growth phase. Gene expression studies conducted at low pH demonstrated that
lgl
was induced during acidic growth (∼3.5-fold) and following acid adaptation (∼2-fold).This study demonstrates that in
S. mutans
, LGL functions in the detoxification of methylglyoxal, resulting in increased aciduricity.
Epsilon-Toxin Plasmids of
Clostridium perfringens
Type D Are Conjugative
Authors: Hughes Meredith L. , Poon Rachael , Adams Vicki , Sayeed Sameera , Saputo Juliann , Uzal Francisco A. , McClane Bruce A. , Rood Julian I. 
Published: August 2007

ABSTRACT

Isolates of
Clostridium perfringens
type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene,
etx
, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The
etx
gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the
C. perfringens
type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of “unmarked” native ε-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional ε-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the
C. perfringens
typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.
TcpA, an FtsK/SpoIIIE Homolog, Is Essential for Transfer of the Conjugative Plasmid pCW3 inClostridium perfringens
Authors: Parsons Jennifer A. , Bannam Trudi L. , Devenish Rodney J. , Rood Julian I. 
Published: August 2007

ABSTRACTThe conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogenClostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer inC. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that thetcpAgene was essential for the conjugative transfer of pCW3 and that thetcpBgene was not required for transfer. Furthermore, complementation of a pCW3ΔtcpAmutant with divergenttcpAhomologs provided experimental evidence that all of the known conjugative plasmids fromC. perfringensuse a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process inC. perfringens.
Identification and Characterization of TriABC-OpmH, a Triclosan Efflux Pump of
Pseudomonas aeruginosa
Requiring Two Membrane Fusion Proteins
Authors: Mima Takehiko , Joshi Swati , Gomez-Escalada Margarita , Schweizer Herbert P. 
Published: August 2007

ABSTRACT

Pseudomonas aeruginosa
achieves high-level (MIC > 1 mg/ml) triclosan resistance either by constitutive expression of MexAB-OprM, an efflux pump of the resistance nodulation cell division (RND) family, or expression of MexCD-OprJ, MexEF-OprN, and MexJK-OpmH in regulatory mutants. A triclosan-resistant target enzyme and perhaps other mechanisms probably act synergistically with efflux. To probe this notion, we exposed the susceptible Δ(
mexAB-oprM
) Δ(
mexCD-oprJ
) Δ(
mexEF-oprN
) Δ(
mexJK
) Δ(
mexXY
) strain PAO509 to increasing triclosan concentrations and derived a resistant strain, PAO509.5. This mutant overexpressed the PA0156-PA0157-PA0158 pump, which only effluxed triclosan, but not closely related compounds, antibiotics, and divalent cations, and was therefore renamed TriABC. Constitutive expression of the
triABC
operon was due to a single promoter-up mutation. Deletion of two adjacent genes,
pcaR
and
PA0159
, encoding transcriptional regulators had no effect on expression of this operon. TriABC is the only
P. aeruginosa
RND pump which contains two membrane fusion proteins, TriA and TriB, and both are required for efflux pump function. Probably owing to tight transcriptional coupling of the
triABC
genes, complementation of individual mutations was only partially achievable. Full complementation was only observed when a complete
triABC
operon was provided in
trans
, either in single or multiple copies. TriABC associated with OpmH, but not OprM, for assembly of a functional triclosan efflux pump. TriABC is the fifth RND pump in
P. aeruginosa
shown to efficiently efflux triclosan, supporting the notion that efflux is the primary mechanism responsible for this bacterium's high intrinsic and acquired triclosan resistance.