bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aeromonas hydrophila
AH-3 Type III Secretion System Expression and Regulatory Network
Authors: Vilches Silvia , Jimenez Natalia , Tomás Juan M. , Merino Susana 
Published: August 2009

ABSTRACT

The
Aeromonas hydrophila
type III secretion system (T3SS) has been shown to play a crucial role in this pathogen's interactions with its host. We previously described the genetic organization of the T3SS cluster and the existence of at least one effector, called AexT, in
A. hydrophila
strain AH-3. In this study, we analyzed the expression of the T3SS regulon by analyzing the activity of the
aopN-aopD
and
aexT
promoters (T3SS machinery components and effector, respectively) by means of two different techniques: promoterless
gfp
fusions and real-time PCR. The expression of the
A. hydrophila
AH-3 T3SS regulon was induced in response to several environmental factors, of which calcium depletion, a high magnesium concentration, and a high growth temperature were shown to be the major ones. Once the optimal conditions were established, we tested the expression of the T3SS regulon in the background of several virulence determinant knockouts of strain AH-3. The analysis of the data obtained from
axsA
and
aopN
mutants, both of which have been described to be T3SS regulators in other species, allowed us to corroborate their function as the major transcription regulator and valve of the T3SS, respectively, in
Aeromonas hydrophila
. We also demonstrated the existence of a complicated interconnection between the expression of the T3SS and several other different virulence factors, such as the lipopolysaccharide, the PhoPQ two-component system, the
ahyIR
quorum sensing system, and the enzymatic complex pyruvate deshydrogenase. To our knowledge, this is the first study of the
A. hydrophila
T3SS regulatory network.
Serogroup, Virulence, and Genetic Traits of
Vibrio parahaemolyticus
in the Estuarine Ecosystem of Bangladesh
Authors: Alam Munirul , Chowdhury Wasimul B. , Bhuiyan N. A. , Islam Atiqul , Hasan Nur A. , Nair G. Balakrish , Watanabe H. , Siddique A. K. , Huq Anwar , Sack R. Bradley , Akhter M. Z. , Grim Christopher J. , Kam K.-M. , Luey C. K. Y. , Endtz Hubert P. , Cravioto Alejandro , Colwell Rita R. 
Published: August 2009

ABSTRACT

Forty-two strains of
Vibrio parahaemolyticus
were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes
tox
R
and
tlh
were confirmed by PCR in all but two strains, which also lacked
tox
R
. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (
trh
) gene, but not
tdh
. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed
tdh
. Of the 18
tdh
-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single
trh
-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., “clonal cluster,” as did pandemic strains of diverse origin. The demonstrated prevalence of
tdh
-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.
Inhibition of Nitrate Reduction by Chromium(VI) in Anaerobic Soil Microcosms
Authors: Kourtev Peter S. , Nakatsu Cindy H. , Konopka Allan 
Published: August 2009

ABSTRACT

Chromium is often found as a cocontaminant at sites polluted with organic compounds. For nitrate-respiring microbes, Cr(VI) may be not only directly toxic but may also specifically interfere with N reduction. In soil microcosms amended with organic electron donors, Cr(VI), and nitrate, bacteria oxidized added carbon, but relatively low doses of Cr(VI) caused a lag and then lower rates of CO
2
accumulation. Cr(VI) strongly inhibited nitrate reduction; it occurred only after soluble Cr(VI) could not be detected. However, Cr(VI) additions did not eliminate Cr-sensitive populations; after a second dose of Cr(VI), bacterial activity was strongly inhibited. Differences in microbial community composition (assayed by PCR-denaturing gradient gel electrophoresis) driven by different organic substrates (glucose and protein) were smaller than when other electron acceptors had been used. However, the selection of bacterial phylotypes was modified by Cr(VI). Nine isolated clades of facultatively anaerobic Cr(VI)-resistant bacteria were closely related to cultivated members of the phylum
Actinobacteria
or
Firmicutes
. In
Bacillus cereus
GNCR-4, the nature of the electron donor (fermentable or nonfermentable) affected Cr(VI) resistance level and anaerobic nitrate metabolism. Our results indicate that carbon utilization and nitrate reduction in these soils were contingent upon the reduction of added Cr(VI). The amount of Cr(VI) required to inhibit nitrate reduction was 10-fold less than for aerobic catabolism of the same organic substrate. We speculate that the resistance level of a microbial process is directly related to the diversity of microbes capable of conducting it.
Molecular Analysis of Virulence Profiles and Shiga Toxin Genes in Food-Borne Shiga Toxin-Producing
Escherichia coli
Authors: Slanec T. , Fruth A. , Creuzburg K. , Schmidt H. 
Published: August 2009

ABSTRACT

In this study, 75 Shiga toxin (Stx)-producing
Escherichia coli
(STEC) strains originating from foods (
n
= 73) and drinking water (
n
= 2) were analyzed for their
stx
genotype, as well as for further chromosome-, phage-, and plasmid-encoded virulence factors. A broad spectrum of
stx
genes was detected. Fifty-three strains (70.7%) contained
stx
2
or
stx
2
variants, including
stx
2d
, mucus-activatable
stx
2d
,
stx
2e
, and
stx
2g
. Seven strains (9.3%) harbored
stx
1
or
stx
1c
, and 15 strains (20.0%) carried both
stx
2
and/or
stx
2
variants and
stx
1
or
stx
1c
. Beside
stx
, the most abundant accessory virulence markers in STEC food isolates were
iha
(57.3%),
ehxA
(40.0%),
espP
(28.0%), and
subAB
(25.3%). Only four strains were
eae
positive; three of these belonged to the serogroups O26, O103, and O157 and contained a typical enterohemorrhagic
E. coli
virulence spectrum. The results of this study show that a number of STEC strains that occur in foods appear to be pathogenic for humans, based on their virulence profiles. Analysis of
stx
subtypes and detection of additional virulence factors in
eae
-negative strains may help to better assess the risk of such strains for causing human infection.
Neurotoxin Gene Clusters in
Clostridium botulinum
Type Ab Strains
Authors: Lúquez Carolina , Raphael Brian H. , Maslanka Susan E. 
Published: August 2009

ABSTRACT

There is limited knowledge of the neurotoxin gene diversity among
Clostridium botulinum
type Ab strains. Only the sequences of the
bont/A
and
bont/B
genes in
C. botulinum
type Ab strain CDC1436 and the sequence of the
bont/B
gene in
C. botulinum
type Ab strain CDC588 have been reported. In this study, we sequenced the entire
bont/A
- and
bont/B
-associated neurotoxin gene clusters of
C. botulinum
type Ab strain CDC41370 and the
bont/A
gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The
bont/A
nucleotide sequence of strain CDC41370 differed from those of the known
bont/A
subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The
bont/B
nucleotide sequence in strain CDC41370 showed 99.7% identity to the sequence of subtype B1. The
bont/A
nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the
C. botulinum
type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of
p21
-
orfX1
and
orfX2
-
orfX3
varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and
bont/A
and
bont
/B
nucleotide sequences, suggesting that they may have arisen from separate recombination events.
Survival of
Lactobacillus reuteri
DSM 17938 and
Lactobacillus rhamnosus
GG in the Human Gastrointestinal Tract with Daily Consumption of a Low-Fat Probiotic Spread
Authors: Dommels Yvonne E. M. , Kemperman Robèr A. , Zebregs Yvonne E. M. P. , Draaisma René B. , Jol Arne , Wolvers Danielle A. W. , Vaughan Elaine E. , Albers Ruud 
Published: August 2009

ABSTRACT

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Therefore, probiotic strains should be able to survive passage through the human gastrointestinal tract. Human gastrointestinal tract survival of probiotics in a low-fat spread matrix has, however, never been tested. The objective of this randomized, double-blind, placebo-controlled human intervention study was to test the human gastrointestinal tract survival of
Lactobacillus reuteri
DSM 17938 and
Lactobacillus rhamnosus
GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. Forty-two healthy human volunteers were randomly assigned to one of three treatment groups provided with 20 g of placebo spread (
n
= 13), 20 g of spread with a target dose of 1 × 10
9
CFU of
L. reuteri
DSM 17938 (
n
= 13), or 20 g of spread with a target dose of 5 × 10
9
CFU of
L. rhamnosus
GG (
n
= 16) daily for 3 weeks. Fecal samples were obtained before and after the intervention period. A significant increase, compared to the baseline, in the recovery of viable probiotic lactobacilli in fecal samples was demonstrated after 3 weeks of daily consumption of the spread containing either
L. reuteri
DSM 17938 or
L. rhamnosus
GG by selective enumeration. In the placebo group, no increase was detected. The results of selective enumeration were supported by quantitative PCR, detecting a significant increase in DNA resulting from the probiotics after intervention. Overall, our results indicate for the first time that low-fat spread is a suitable carrier for these probiotic strains.
Cytoplasmic Bulk Flow Propels Nuclei in Mature Hyphae of
Neurospora crassa
Authors: Ramos-García Silvia L. , Roberson Robert W. , Freitag Michael , Bartnicki-García Salomón , Mouriño-Pérez Rosa R. 
Published: August 2009

ABSTRACT

We used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of
Neurospora crassa
whose nuclei expressed histone H1-tagged green fluorescent protein (GFP). In addition to the H1-GFP wild-type (WT) strain, we examined nuclear displacement (passive transport) in four mutants deficient in microtubule-related motor proteins (
ro-1
,
ro-3
,
kin-1
, and a
ro-1 kin-1
double mutant). We also treated the WT strain with benomyl and cytochalasin A to disrupt microtubules and actin microfilaments, respectively. We found that the degree of nuclear displacement in the subapical regions of all strains correlated with hyphal elongation rate. The WT strain and that the
ro-1 kin-1
double mutant showed the highest correlation between nuclear movement and hyphal elongation. Although most nuclei seemed to move forward passively, presumably carried by the cytoplasmic bulk flow, a small proportion of the movement detected was either retrograde or accelerated anterograde. The absence of a specific microtubule motor in the mutants
ro-1
,
ro-3
, or
kin-1
did not prevent the anterograde and retrograde migration of nuclei; however, in the
ro-1 kin-1
double mutant retrograde migration was absent. In the WT strain, almost all nuclei were elongated, whereas in all other strains a majority of nuclei were nearly spherical. With only one exception, a sizable exclusion zone was maintained between the apex and the leading nucleus. The
ro-1
mutant showed the largest nucleus exclusion zone; only the treatment with cytochalasin A abolished the exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear to be determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Motor proteins probably play an active role in powering the retrograde or accelerated anterograde migrations of nuclei and may also contribute to passive anterograde displacement by binding nuclei to microtubules.
Dual Acylation Accounts for the Localization of α19-Giardin in the Ventral Flagellum Pair of
Giardia lamblia
Authors: Šarić Mirela , Vahrmann Anke , Niebur Daniela , Kluempers Verena , Hehl Adrian B. , Scholze Henning 
Published: August 2009

ABSTRACT

A
Giardia
-specific protein family denominated as α-giardins, represents the major protein component, besides tubulin, of the cytoskeleton of the human pathogenic parasite
Giardia lamblia
. One of its members, α19-giardin, carries an N-terminal sequence extension of MGCXXS, which in many proteins serves as a target for dual lipid conjugation: myristoylation at the glycine residue after removal of the methionine and palmitoylation at the cysteine residue. As the first experimental evidence of a lipid modification, we found α19-giardin to be associated with the membrane fraction of disrupted trophozoites. After heterologous coexpression of α19-giardin with giardial
N
-myristoyltransferase (NMT) in
E
scherichia coli
, we found the protein in a myristoylated form. Additionally, after heterologous expression together with the palmitoyl transferase Pfa3 in
Saccharomyces cerevisiae
, α19-giardin associates with the membrane of the main vacuole. Immunocytochemical colocalization studies on wild-type
Giardia
trophozoites with tubulin provide evidence that α19-giardin exclusively localizes to the ventral pair of the giardial flagella. A mutant in which the putatively myristoylated N-terminal glycine residue was replaced by alanine lost this specific localization. Our findings suggest that the dual lipidation of α19-giardin is responsible for its specific flagellar localization.
Capsular Polysaccharide Production in
Enterococcus faecalis
and Contribution of CpsF to Capsule Serospecificity
Authors: Thurlow Lance R. , Thomas Vinai Chittezham , Hancock Lynn E. 
Published: August 2009

ABSTRACT

Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen
Enterococcus faecalis
was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of
E. faecalis
were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (
cpsC
to
cpsK
). Using a novel genetic system, we demonstrated that seven of the nine genes in the
cps
operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of
cpsF
in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that
cpsF
is responsible for glucosylation of serotype C capsular polysaccharide in
E. faecalis
.
The Uracil DNA Glycosylase UdgB ofMycobacterium smegmatisProtects the Organism from the Mutagenic Effects of Cytosine and Adenine Deamination
Authors: Wanner Roger M. , Castor Dennis , Güthlein Carolin , Böttger Erik C. , Springer Burkhard , Jiricny Josef 
Published: August 2009

ABSTRACTSpontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB fromPyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB fromMycobacterium smegmatisprotects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase,M. smegmatisUdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil.
tIS
Cpe8
, an IS
1595
-Family Lincomycin Resistance Element Located on a Conjugative Plasmid in
Clostridium perfringens
Authors: Lyras Dena , Adams Vicki , Ballard Susan A. , Teng Wee L. , Howarth Pauline M. , Crellin Paul K. , Bannam Trudi L. , Songer J. Glenn , Rood Julian I. 
Published: August 2009

ABSTRACT

Clostridium perfringens
is a normal gastrointestinal organism that is a reservoir for antibiotic resistance genes and can potentially act as a source from which mobile elements and their associated resistance determinants can be transferred to other bacterial pathogens. Lincomycin resistance in
C. perfringens
is common and is usually encoded by
erm
genes that confer macrolide-lincosamide-streptogramin B resistance. In this study we identified strains that are lincomycin resistant but erythromycin sensitive and showed that the lincomycin resistance determinant was plasmid borne and could be transferred to other
C. perfringens
isolates by conjugation. The plasmid, pJIR2774, is the first conjugative
C. perfringens
R-plasmid to be identified that does not confer tetracycline resistance. Further analysis showed that resistance was encoded by the
lnuP
gene, which encoded a putative lincosamide nucleotidyltransferase and was located on tIS
Cpe8
, a functional transposable genetic element that was a member of the IS
1595
family of transposon-like insertion sequences. This element had significant similarity to the mobilizable lincomycin resistance element tIS
Sag10
from
Streptococcus agalactiae
. Like tIS
Sag10
, tIS
Cpe8
carries a functional origin of transfer within the resistance gene, allowing the element to be mobilized by the conjugative transposon Tn
916
. The similarity of these elements and the finding that they both contain an
oriT
-like region support the hypothesis that conjugation may result in the movement of DNA modules that are not obviously mobile since they are not linked to conjugation or mobilization functions. This process likely plays a significant role in bacterial adaptation and evolution.
Characterization of aMycobacterium tuberculosisESX-3 Conditional Mutant: Essentiality and Rescue by Iron and Zinc
Authors: Serafini Agnese , Boldrin Francesca , Palù Giorgio , Manganelli Riccardo 
Published: August 2009

ABSTRACTRecently, a novel type of secretory pathway, type VII secretion systems (T7SSs), has been characterized in mycobacteria. The chromosomes ofMycobacterium tuberculosisandMycobacterium bovisencode five T7SSs (ESX-1 to ESX-5). The best characterized of them, ESX-1, is involved in host-pathogen interactions, and its deletion is one of the main causes ofM. bovisBCG attenuation. Another T7SS, ESX-3, has been previously shown to be transcriptionally controlled by the zinc uptake repressor (Zur) and by the iron-dependent transcriptional repressor (IdeR), suggesting that it might be involved in zinc and iron homeostasis. In this study, we characterized anM. tuberculosisconditional mutant in which transcription of the ESX-3 gene cluster can be downregulated by anhydrotetracycline. We showed that this T7SS is essential for growth and that this phenotype can be complemented by zinc, iron, or supernatant from a wild-type parental strain culture, demonstrating that the ESX-3 secretion system is responsible for the secretion of some soluble factor(s) required for growth that is probably involved in optimal iron and zinc uptake.
Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of
Escherichia coli
Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis
Authors: Hermes Fatemah A. M. , Cronan John E. 
Published: August 2009

ABSTRACT

The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in
Escherichia coli
, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in
lipB
are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of
lipB
strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene (
lplA
) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced

K
m

values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains.
Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases
Authors: Van de Vijver Pieter , Vondenhoff Gaston H. M. , Kazakov Teymur S. , Semenova Ekaterina , Kuznedelov Konstantin , Metlitskaya Anastasia , Van Aerschot Arthur , Severinov Konstantin 
Published: August 2009

ABSTRACT

Microcin C (McC) is a potent antibacterial agent produced by some strains of
E
scherichia coli
. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the α-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA
Asp
by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.