bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis
Authors: Jacques Marie-Agnès , Durand Karine , Orgeur Geoffrey , Balidas Samuel , Fricot Céline , Bonneau Sophie , Quillévéré Anne , Audusseau Corinne , Olivier Valérie , Grimault Valérie , Mathis René
Published: September 2012

The genus
Clavibacter
comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose.
Clavibacter michiganensis
subsp.
michiganensis
is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of
C. michiganensis
subsp.
michiganensis
strains, relatives (strains from the four other
C. michiganensis
subspecies), and nonpathogenic
Clavibacter
-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (
atpD
,
dnaK
,
gyrB
,
ppK
,
recA
, and
rpoB
). We compared this “framework” with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main
C. michiganensis
subsp.
michiganensis
pathogenicity determinants. We showed that
C. michiganensis
subsp.
michiganensis
is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic
Clavibacter
-like strains were identified as
C. michiganensis
using 16S rRNA gene sequencing. These strains, while cross-reacting with
C. michiganensis
subsp.
michiganensis
identification tools, are phylogenetically distinct from the pathogenic strains but belong to the
C. michiganensis
clade.
C. michiganensis
subsp.
michiganensis
clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced.
Preferential Utilization of
d
-Lactate by Shewanella oneidensis
Authors: Brutinel Evan D. , Gralnick Jeffrey A.
Published: September 2012

Shewanella oneidensis
couples oxidation of lactate to respiration of many substrates. Here we report that
llpR
(

l

-
l
actate-
p
ositive
r
egulator, SO_3460) encodes a positive regulator of
l
-lactate utilization distinct from previously studied regulators. We also demonstrate
d
-lactate inhibition of
l
-lactate utilization in
S. oneidensis
, resulting in preferential utilization of the
d
isomer.
Different Antibiotic Resistance and Sporulation Properties within Multiclonal Clostridium difficile PCR Ribotypes 078, 126, and 033 in a Single Calf Farm
Authors: Zidaric Valerija , Pardon Bart , dos Vultos Tiago , Deprez Piet , Brouwer Michael Sebastiaan Maria , Roberts Adam P. , Henriques Adriano O. , Rupnik Maja
Published: September 2012

Clostridium difficile
strains were sampled periodically from 50 animals at a single veal calf farm over a period of 6 months. At arrival, 10% of animals were
C. difficile
positive, and the peak incidence was determined to occur at the age of 18 days (16%). The prevalence then decreased, and at slaughter,
C. difficile
could not be isolated. Six different PCR ribotypes were detected, and strains within a single PCR ribotype could be differentiated further by pulsed-field gel electrophoresis (PFGE). The PCR ribotype diversity was high up to the animal age of 18 days, but at later sampling points, PCR ribotype 078 and the highly related PCR ribotype 126 predominated. Resistance to tetracycline, doxycycline, and erythromycin was detected, while all strains were susceptible to amoxicillin and metronidazole. Multiple variations of the resistance gene
tet
(M) were present at the same sampling point, and these changed over time. We have shown that PCR ribotypes often associated with cattle (ribotypes 078, 126, and 033) were not clonal but differed in PFGE type, sporulation properties, antibiotic sensitivities, and tetracycline resistance determinants, suggesting that multiple strains of the same PCR ribotype infected the calves and that calves were likely to be infected prior to arrival at the farm. Importantly, strains isolated at later time points were more likely to be resistant to tetracycline and erythromycin and showed higher early sporulation efficiencies
in vitro
, suggesting that these two properties converge to promote the persistence of
C. difficile
in the environment or in hosts.
“Methanoplasmatales,” Thermoplasmatales-Related Archaea in Termite Guts and Other Environments, Are the Seventh Order of Methanogens
Authors: Paul Kristina , Nonoh James O. , Mikulski Lena , Brune Andreas
Published: September 2012

The
Euryarchaeota
comprise both methanogenic and nonmethanogenic orders and many lineages of uncultivated archaea with unknown properties. One of these deep-branching lineages, distantly related to the
Thermoplasmatales
, has been discovered in various environments, including marine habitats, soil, and also the intestinal tracts of termites and mammals. By comparative phylogenetic analysis, we connected this lineage of 16S rRNA genes to a large clade of unknown
mcrA
gene sequences, a functional marker for methanogenesis, obtained from the same habitats. The identical topologies of 16S rRNA and
mcrA
gene trees and the perfect congruence of all branches, including several novel groups that we obtained from the guts of termites and cockroaches, strongly suggested that they stem from the same microorganisms. This was further corroborated by two highly enriched cultures of closely related methanogens from the guts of a higher termite (
Cubitermes ugandensis
) and a millipede (
Anadenobolus
sp.), which represented one of the arthropod-specific clusters in the respective trees. Numerous other pairs of habitat-specific sequence clusters were obtained from the guts of other termites and cockroaches but were also found in previously published data sets from the intestinal tracts of mammals (e.g., rumen cluster C) and other environments. Together with the recently described
Methanomassiliicoccus luminyensis
isolated from human feces, which falls into rice cluster III, the results of our study strongly support the idea that the entire clade of “uncultured
Thermoplasmatales
” in fact represents the seventh order of methanogenic archaea, for which the provisional name “
Methanoplasmatales
” is proposed.
Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-Electrospray Ionization Mass Spectrometry: Isolate Testing and Analysis of Food Samples
Authors: Pierce Sarah E. , Bell Rebecca L. , Hellberg Rosalee S. , Cheng Chorng-Ming , Chen Kai-Shun , Williams-Hill Donna M. , Martin William B. , Allard Marc W.
Published: September 2012

An assay to identify the common food-borne pathogens
Salmonella
,
Escherichia coli
,
Shigella
, and
Listeria monocytogenes
was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140
Salmonella enterica
, 139
E. coli
, 11
Shigella
, and 36
Listeria
patterns and 18 other
Enterobacteriaceae
organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of
S. enterica
,
E. coli
, and
Shigella
species were analyzed. Overall, 100% of
S. enterica
, 99% of
E. coli
, and 73% of
Shigella
spp. were detected using this assay. The assay was also able to identify 30% of the
S. enterica
serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of
S. enterica
from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.
Biotin-Binding Proteins in the Defense of Mushrooms against Predators and Parasites
Authors: Bleuler-Martinez Silvia , Schmieder Stefanie , Aebi Markus , Künzler Markus
Published: September 2012

Tamavidins are fungal biotin-binding proteins (BBPs) displaying antifungal activity against phytopathogens. Here we show high toxicity of tamavidins toward nematodes, insects, and amoebae. As these organisms represent important phyla of fungal predators and parasites, we propose that BBPs are part of the chemical defense system of fungi.
Chemotactic Behavior of Pathogenic and Nonpathogenic Leptospira Species
Authors: Lambert Ambroise , Takahashi Naoko , Charon Nyles W. , Picardeau Mathieu
Published: September 2012

We have developed a capillary tube assay in combination with real-time PCR to quantitate the number of chemoattracted
Leptospira
cells. We identified Tween 80, glucose, sucrose, and pyruvate as attractants for
Leptospira
cells; amino acids and vitamin B
12
were found to be nonchemotactic or weakly chemotactic. This assay has the general applicability to further our understanding of leptospiral chemotaxis.
Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient, Nitrogen-Limited Enological Conditions
Authors: Aceituno Felipe F. , Orellana Marcelo , Torres Jorge , Mendoza Sebastián , Slater Alex W. , Melo Francisco , Agosin Eduardo
Published: September 2012

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.
Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores
Authors: den Besten Heidy M. W. , van Melis Clint C. J. , Sanders Jan Willem , Nierop Groot Masja N. , Abee Tjakko
Published: September 2012

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.
Methicillin-Resistant Staphylococcus aureus Phage Plaque Size Enhancement Using Sublethal Concentrations of Antibiotics
Authors: Kaur Sandeep , Harjai Kusum , Chhibber Sanjay
Published: September 2012

Phage therapy presents an alternative approach against the emerging methicillin-resistantStaphylococcus aureus(MRSA) threat. Some of the problems encountered during isolation of MRSA phages include the high prevalence of enteric phages in natural sources, nonspecific absorption of viable phage, and the formation of pinpoint or tiny plaques. The phage isolated in this study, MR-5, also formed tiny plaques against its hostS. aureusATCC 43300 (MRSA), making its detection and enumeration difficult. An improved method of increasing the plaque size of MRSA phage by incorporating sublethal concentrations of three different classes of antibiotics (inhibitors of protein synthesis) in the classical double-layer agar (DLA) method was investigated. The β-lactam and quinolone antibiotics commonly employed in earlier studies for increasing the plaque size did not show any significant effect on the plaque size of isolated MR-5 phage. Linezolid (oxazolidinone class), tetracycline, and ketolide antibiotics brought significant enhancements (3 times the original size) in the plaque size of MR-5 phage. Prior treatment with these antibiotics resulted in significant reductions in the time of adsorption and the latent period of MR-5 phage. To rule out whether the action of linezolid (which brought the maximum increase in plaque size) was specific for a single phage only, its effect on the plaque size of seven otherS. aureus-specific phages was also assessed. Significant enhancements in the plaque size of these phages were observed. These results indicate that this modification can therefore safely be incorporated in the traditional DLA overlay method to search for new MRSA-virulent phages.
Electrosynthesis of Commodity Chemicals by an Autotrophic Microbial Community
Authors: Marshall Christopher W. , Ross Daniel E. , Fichot Erin B. , Norman R. Sean , May Harold D.
Published: September 2012

A microbial community originating from brewery waste produced methane, acetate, and hydrogen when selected on a granular graphite cathode poised at −590 mV versus the standard hydrogen electrode (SHE) with CO
2
as the only carbon source. This is the first report on the simultaneous electrosynthesis of these commodity chemicals and the first description of electroacetogenesis by a microbial community. Deep sequencing of the active community 16S rRNA revealed a dynamic microbial community composed of an invariant
Archaea
population of
Methanobacterium
spp. and a shifting
Bacteria
population.
Acetobacterium
spp. were the most abundant
Bacteria
on the cathode when acetogenesis dominated. Methane was generally the dominant product with rates increasing from <1 to 7 mM day
−1
(per cathode liquid volume) and was concomitantly produced with acetate and hydrogen. Acetogenesis increased to >4 mM day
−1
(accumulated to 28.5 mM over 12 days), and methanogenesis ceased following the addition of 2-bromoethanesulfonic acid. Traces of hydrogen accumulated during initial selection and subsequently accelerated to >11 mM day
−1
(versus 0.045 mM day
−1
abiotic production). The hypothesis of electrosynthetic biocatalysis occurring at the microbe-electrode interface was supported by a catalytic wave (midpoint potential of −460 mV versus SHE) in cyclic voltammetry scans of the biocathode, the lack of redox active components in the medium, and the generation of comparatively high amounts of products (even after medium exchange). In addition, the volumetric production rates of these three commodity chemicals are marked improvements for electrosynthesis, advancing the process toward economic feasibility.
Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1
Authors: Weiss Michael , Denger Karin , Huhn Thomas , Schleheck David
Published: September 2012

Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C
4
-SPC). Second, these SPCs are mineralized. 3-C
4
-SPC is mineralized by
Comamonas testosteroni
KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C
4
-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria.
Anaerobic Benzene Oxidation by Geobacter Species
Authors: Zhang Tian , Bain Timothy S. , Nevin Kelly P. , Barlett Melissa A. , Lovley Derek R.
Published: September 2012

The abundance ofGeobacterspecies in contaminated aquifers in which benzene is anaerobically degraded has led to the suggestion that someGeobacterspecies might be capable of anaerobic benzene degradation, but this has never been documented. A strain ofGeobacter, designated strain Ben, was isolated from sediments from the Fe(III)-reducing zone of a petroleum-contaminated aquifer in which there was significant capacity for anaerobic benzene oxidation. Strain Ben grew in a medium with benzene as the sole electron donor and Fe(III) oxide as the sole electron acceptor. Furthermore, additional evaluation ofGeobacter metallireducensdemonstrated that it could also grow in benzene-Fe(III) medium. In both strain Ben andG. metallireducensthe stoichiometry of benzene metabolism and Fe(III) reduction was consistent with the oxidation of benzene to carbon dioxide with Fe(III) serving as the sole electron acceptor. With benzene as the electron donor, and Fe(III) oxide (strain Ben) or Fe(III) citrate (G. metallireducens) as the electron acceptor, the cell yields of strain Ben andG. metallireducenswere 3.2 × 109and 8.4 × 109cells/mmol of Fe(III) reduced, respectively. Strain Ben also oxidized benzene with anthraquinone-2,6-disulfonate (AQDS) as the sole electron acceptor with cell yields of 5.9 × 109cells/mmol of AQDS reduced. Strain Ben serves as model organism for the study of anaerobic benzene metabolism in petroleum-contaminated aquifers, andG. metallireducensis the first anaerobic benzene-degrading organism that can be genetically manipulated.
Exposure to Solute Stress Affects Genome-Wide Expression but Not the Polycyclic Aromatic Hydrocarbon-Degrading Activity of Sphingomonas sp. Strain LH128 in Biofilms
Authors: Fida Tekle Tafese , Breugelmans Philip , Lavigne Rob , Coronado Edith , Johnson David R. , van der Meer Jan Roelof , Mayer Antonia P. , Heipieper Hermann J. , Hofkens Johan , Springael Dirk
Published: September 2012

Members of the genusSphingomonasare important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degradingSphingomonassp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity.
Evaluation of the Liaison Automated Testing System for Diagnosis of Congenital Toxoplasmosis
Authors: Prusa Andrea-Romana , Hayde Michael , Pollak Arnold , Herkner Kurt R. , Kasper David C.
Published: September 2012

Congenital toxoplasmosis is a worldwide health problem, and different screening strategies exist. Testing of toxoplasma-specific antibodies in infants identifies congenital toxoplasmosis during the first year of life. However, experience with commercial available immunoassays is limited. The aim of this study was to evaluate both the performance and analytical characteristics of the Liaison diagnostic system in infants. In a retrospective study, serumToxoplasma gondiiantibodies were measured in samples from 333 infants, including 212 noninfected infants and 121 infants with congenital toxoplasmosis. A total of 1,157 umbilical cord blood and peripheral serum samples were analyzed. Liaison toxoplasma-specific IgG and IgM antibodies and the IgG avidity index were compared to the infection status of the infant, determined by the Sabin-Feldman dye test and immunosorbent agglutination assay—IgM. All noninfected infants were seronegative by Liaison IgG within the first year of life. The Liaison system showed a sensitivity of 81.8%, a specificity of 100.0%, a positive predictive value of 100.0%, a negative predictive value of 90.6%, and overall agreement of 84.4% by comparison with the dye test. Overall agreement of both IgM test systems was 96.0%. In this study cohort, avidity did not show a potential diagnostic benefit for the detection of congenital infection. In conclusion, the Liaison system is a valuable tool to monitor the serologic course of infants at risk. A final serologic confirmatory test is recommended to improve the rate of detection of congenital toxoplasmosis at 1 year of life. Protocols of routine follow-up testing in infants and accurate diagnostic tools after acute gestational infections are needed to improve medical care.
Sudden Oak Death: Interactions of the Exotic Oomycete Phytophthora ramorum with Naïve North American Hosts
Authors: Garbelotto Matteo , Hayden Katherine J.
Published: September 2012

Ten years after a threatening and previously unknown disease of oaks and tanoaks appeared in coastal California, a significant amount of progress has been made toward the understanding of its causal agent
Phytophthora ramorum
and of the novel pathosystems associated with this exotic organism. However, a complete understanding of the ecology and epidemiology of this species still eludes us. In part, our inability to fully understand this organism is due to its phylogenetic, phylogeographic, phenotypic, and epidemiological complexities, all reviewed in this paper. Most lines of evidence suggest that the high degree of disease severity reported in California is not simply due to a generalized lack of resistance or tolerance in naïve hosts but also to an innate ability of the pathogen to survive in unfavorable climatic conditions and to reproduce rapidly when conditions become once again favorable.
The SWI/SNF KlSnf2 Subunit Controls the Glucose Signaling Pathway To Coordinate Glycolysis and Glucose Transport in Kluyveromyces lactis
Authors: Cotton Pascale , Soulard Alexandre , Wésolowski-Louvel Micheline , Lemaire Marc
Published: September 2012

In
Kluyveromyces lactis
, the expression of the major glucose permease gene
RAG1
is controlled by extracellular glucose through a signaling cascade similar to the
Saccharomyces cerevisiae
Snf3/Rgt2/Rgt1 pathway. We have identified a key component of the
K. lactis
glucose signaling pathway by characterizing a new mutation,
rag20-1
, which impairs the regulation of
RAG1
and hexokinase
RAG5
genes by glucose. Functional complementation of the
rag20-1
mutation identified the
KlSNF2
gene, which encodes a protein 59% identical to
S. cerevisiae
Snf2, the major subunit of the SWI/SNF chromatin remodeling complex. Reverse transcription-quantitative PCR and chromatin immunoprecipitation analyses confirmed that the KlSnf2 protein binds to
RAG1
and
RAG5
promoters and promotes the recruitment of the basic helix-loop-helix Sck1 activator. Besides this transcriptional effect, KlSnf2 is also implicated in the glucose signaling pathway by controlling Sms1 and KlRgt1 posttranscriptional modifications. When KlSnf2 is absent, Sms1 is not degraded in the presence of glucose, leading to constitutive
RAG1
gene repression by KlRgt1. Our work points out the crucial role played by KlSnf2 in the regulation of glucose transport and metabolism in
K. lactis
, notably, by suggesting a link between chromatin remodeling and the glucose signaling pathway.
Pseudohyphal Growth of Cryptococcus neoformans Is a Reversible Dimorphic Transition in Response to Ammonium That Requires Amt1 and Amt2 Ammonium Permeases
Authors: Lee Soo Chan , Phadke Sujal , Sun Sheng , Heitman Joseph
Published: September 2012

Cryptococcus neoformansis a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19).C. neoformansgrows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth ofC. neoformanshas been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found thatC. neoformansdisplays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed withCryptococcus neoformansserotypes A and D andCryptococcus gattii. C. neoformanspseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by theAMT1andAMT2genes are required for pseudohyphal growth. Bothamt1andamt2mutants are capable of forming pseudohyphae; however,amt1 amt2double mutants do not form pseudohyphae. Interestingly,C. gattiipseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern inC. neoformansbut a mutational genetic event inC. gattiiand provide new insights into understanding pseudohyphal growth ofCryptococcus.
The Putative Guanine Nucleotide Exchange Factor RicA Mediates Upstream Signaling for Growth and Development in Aspergillus
Authors: Kwon Nak-Jung , Park Hee-Soo , Jung Seunho , Kim Sun Chang , Yu Jae-Hyuk
Published: September 2012

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized
ricA
, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus
Aspergillus nidulans
and the opportunistic human pathogen
Aspergillus fumigatus
. In both species,
ricA
mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of
ricA
results in severely impaired colony growth and the total (for
A. nidulans
) or near (for
A. fumigatus
) absence of asexual sporulation (conidiation). The overexpression (OE) of the
A. fumigatus ricA
gene (Af
ricA
) restores growth and conidiation in the ΔAn
ricA
mutant to some extent, indicating partial conservation of RicA function in
Aspergillus
. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not
sfgA
,
flbA
,
rgsB
, or
rgsC
, restored vegetative growth and conidiation in ΔAn
ricA
. Furthermore, we found that RicA can physically interact with GanB in yeast and
in vitro
. Moreover, the presence of two copies or OE of
pkaA
suppresses the profound defects caused by ΔAn
ricA
, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in
A. nidulans
. Despite the lack of conidiation,
brlA
and
vosA
mRNAs accumulated to normal levels in the Δ
ricA
mutant. In addition, mutants overexpressing
fluG
or
brlA
(OE
fluG
or OE
brlA
) failed to restore development in the ΔAn
ricA
mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in
A. nidulans
.
Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan
Authors: Eugster Marcel R. , Loessner Martin J.
Published: September 2012

The C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. TheListeria monocytogenesPly118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure ofListeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding usingL. monocytogenesserovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundanttagOhomologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of eithertagO1(EGDelmo0959) ortagO2(EGDelmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficientListeriacells. Substitution oftagOfrom an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from otherListeriaphage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.
Construction and Characterization of a Derivative of Neisseria gonorrhoeae Strain MS11 Devoid of All
opa
Genes
Authors: LeVan Adriana , Zimmerman Lindsey I. , Mahle Amanda C. , Swanson Karen V. , DeShong Philip , Park Juhee , Edwards Vonetta L. , Song Wenxia , Stein Daniel C.
Published: September 2012

To better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11Δopa) that had the coding sequence for all 11 Opa proteins deleted. The MS11Δopa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11Δopa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11Δopa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11Δopa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ∼85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11Δopa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11Δopa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.
Mycobacterium tuberculosis CwsA Interacts with CrgA and Wag31, and the CrgA-CwsA Complex Is Involved in Peptidoglycan Synthesis and Cell Shape Determination
Authors: Plocinski P. , Arora N. , Sarva K. , Blaszczyk E. , Qin H. , Das N. , Plocinska R. , Ziolkiewicz M. , Dziadek J. , Kiran M. , Gorla P. , Cross T. A. , Madiraju M. , Rajagopalan M.
Published: September 2012

Bacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from
Mycobacterium tuberculosis
, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in
M. smegmatis
, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [
14
C]
N
-acetylglucosamine cell wall precursor. The
M. smegmatis
MSMEG_0023
crgA
double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for
c
ell
w
all synthesis and cell
s
hape protein
A
, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.
A MarR-Type Regulator Directly Activates Transcription from the Brucella abortus virB Promoter by Sharing a Redundant Role with HutC
Authors: Sieira Rodrigo , Arocena Gastón M. , Zorreguieta Angeles , Comerci Diego J. , Ugalde Rodolfo A.
Published: September 2012

Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus
Brucella
essentially depends on the integrity of the T4SS-encoding
virB
operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the
virB
genes that were isolated from total protein extracts of
Brucella
. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the
virB
promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA
in vitro
. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated
virB
expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the
virB
promoter and is probably involved in coordinating gene expression in response to specific environmental signals.
Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis
Authors: Bisson Gregory P. , Mehaffy Carolina , Broeckling Corey , Prenni Jessica , Rifat Dalin , Lun Desmond S. , Burgos Marcos , Weissman Drew , Karakousis Petros C. , Dobos Karen
Published: September 2012

Multidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominantMycobacterium tuberculosismutations in therpoBgene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains ofM. tuberculosis(wild type) to those of their respective rifampin-resistant,rpoBmutant progeny strains with confirmed rifampin monoresistance following antitubercular therapy. Each of these strains was also used to infect gamma interferon- and lipopolysaccharide-activated murine J774A.1 macrophages to analyze transcriptional responses in a physiologically relevant model. BothrpoBmutants showed significant upregulation of the polyketide synthase genesppsA-ppsEanddrrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids inM. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins.ppsA(Rv2931),ppsB(Rv2932), andppsC(Rv2933) were also found to be upregulated more than 10-fold in the BeijingrpoBmutant strain relative to its wild-type parent strain during infection of activated murine macrophages. In addition, metabolomics identified precursors of phthiocerol dimycocerosate, but not the intact molecule itself, in greater abundance in bothrpoBmutant isolates. These data suggest thatrpoBmutation inM. tuberculosismay trigger compensatory transcriptional changes in secondary metabolism genes analogous to those observed in related actinobacteria. These findings may assist in developing novel methods to diagnose and treat drug-resistantM. tuberculosisinfections.
Genetic and Biochemical Characterizations of Enzymes Involved in Streptococcus pneumoniae Serotype 2 Capsule Synthesis Demonstrate that Cps2T (WchF) Catalyzes the Committed Step by Addition of β1-4 Rhamnose, the Second Sugar Residue in the Repeat Unit
Authors: James David B. A. , Yother Janet
Published: September 2012

Five genes (
cps2E
,
cps2T
,
cps2F
,
cps2G
, and
cps2I
) are predicted to encode the glycosyltransferases responsible for synthesis of the
Streptococcus pneumoniae
serotype 2 capsule repeat unit, which is polymerized to yield a branched surface structure containing glucose-glucuronic acid linked to a glucose-rhamnose-rhamnose-rhamnose backbone. Cps2E is the initiating glycosyltransferase, but experimental evidence supporting the functions of the remaining glycosyltransferases is lacking. To biochemically characterize the glycosyltransferases, the donor substrate dTDP-rhamnose was first synthesized using recombinant
S. pneumoniae
enzymes Cps2L, Cps2M, Cps2N, and Cps2O. In
in vitro
assays with each of the glycosyltransferases, only reaction mixtures containing recombinant Cps2T, dTDP-rhamnose, and the Cps2E product (undecaprenyl pyrophosphate glucose) generated a new product, which was consistent with lipid-linked glucose-rhamnose.
cps2T
,
cps2F
, and
cps2I
deletion mutants produced no detectable capsule, but trace amounts of capsule were detectable in Δ
cps2G
mutants, suggesting that Cps2G adds a nonbackbone sugar. All Δ
cps2F
, Δ
cps2G
, and Δ
cps2I
mutants contained different secondary suppressor mutations in
cps2E
, indicating that the initial mutations were lethal in the absence of reduced repeat unit synthesis. Δ
cps2T
mutants did not contain secondary mutations affecting capsule synthesis. The requirement for secondary mutations in mutants lacking Cps2F, Cps2G, and Cps2I indicates that these activities occur downstream of the committed step in capsule synthesis and reveal that Cps2T catalyzes this step. Therefore, Cps2T is the β1-4 rhamnosyltransferase that adds the second sugar to the repeat unit and, as the committed step in type 2 repeat unit synthesis, is predicted to be an important point of capsule regulation.
Iron-Regulated Surface Determinant (Isd) Proteins of Staphylococcus lugdunensis
Authors: Zapotoczna Marta , Heilbronner Simon , Speziale Pietro , Foster Timothy J.
Published: September 2012

Staphylococcus lugdunensis
is the only coagulase-negative
Staphylococcus
species with a locus encoding iron-regulated surface determinant (Isd) proteins. In
Staphylococcus aureus
, the Isd proteins capture heme from hemoglobin and transfer it across the wall to a membrane-bound transporter, which delivers it into the cytoplasm, where heme oxygenases release iron. The Isd proteins of
S. lugdunensis
are expressed under iron-restricted conditions. We propose that
S. lugdunensis
IsdB and IsdC proteins perform the same functions as those of
S. aureus
.
S. lugdunensis
IsdB is the only hemoglobin receptor within the
isd
locus. It specifically binds human hemoglobin with a dissociation constant (

K
d

) of 23 nM and transfers heme on IsdC. IsdB expression promotes bacterial growth in an iron-limited medium containing human hemoglobin but not mouse hemoglobin. This correlates with weak binding of IsdB to mouse hemoglobin
in vitro
. Unlike IsdB and IsdC, the proteins IsdJ and IsdK are not sorted to the cell wall in
S. lugdunensis
. In contrast, IsdJ expressed in
S. aureus
and
Lactococcus lactis
is anchored to peptidoglycan, suggesting that
S. lugdunensis
sortases may differ in signal recognition or could be defective. IsdJ and IsdK are present in the culture supernatant, suggesting that they could acquire heme from the external milieu. The IsdA protein of
S. aureus
protects bacteria from bactericidal lipids due to its hydrophilic C-terminal domain. IsdJ has a similar region and protected
S. aureus
and
L. lactis
as efficiently as IsdA but, possibly due to its location, was less effective in its natural host.
Fur Activates Expression of the 2-Oxoglutarate Oxidoreductase Genes (
oorDABC
) in Helicobacter pylori
Authors: Gilbreath Jeremy J. , West Abby L. , Pich Oscar Q. , Carpenter Beth M. , Michel Sarah , Merrell D. Scott
Published: September 2012

Helicobacter pylori
is a highly successful pathogen that colonizes the gastric mucosa of ∼50% of the world's population. Within this colonization niche, the bacteria encounter large fluctuations in nutrient availability. As such, it is critical that this organism regulate expression of key metabolic enzymes so that they are present when environmental conditions are optimal for growth. One such enzyme is the 2-oxoglutarate (α-ketoglutarate) oxidoreductase (OOR), which catalyzes the conversion of α-ketoglutarate to succinyl coenzyme A (succinyl-CoA) and CO
2
. Previous studies from our group suggested that the genes that encode the OOR are activated by iron-bound Fur (Fe-Fur); microarray analysis showed that expression of
oorD
,
oorA
, and
oorC
was altered in a
fur
mutant strain of
H. pylori.
The goal of the present work was to more thoroughly characterize expression of the
oorDABC
genes in
H. pylori
as well as to define the role of Fe-Fur in this process. Here we show that these four genes are cotranscribed as an operon and that expression of the operon is decreased in a
fur
mutant strain. Transcriptional start site mapping and promoter analysis revealed the presence of a canonical extended −10 element but a poorly conserved −35 element upstream of the +1. Additionally, we identified a conserved Fur binding sequence ∼130 bp upstream of the transcriptional start site. Transcriptional analysis using promoter fusions revealed that this binding sequence was required for Fe-Fur-mediated activation. Finally, fluorescence anisotropy assays indicate that Fe-Fur specifically bound this Fur box with a relatively high affinity (dissociation constant [

K
d

] = 200 nM). These findings provide novel insight into the genetic regulation of a key metabolic enzyme and add to our understanding of the diverse roles Fur plays in gene regulation in
H. pylori
.
Identification of Proteins Likely To Be Involved in Morphogenesis, Cell Division, and Signal Transduction in Planctomycetes by Comparative Genomics
Authors: Jogler Christian , Waldmann Jost , Huang Xiaoluo , Jogler Mareike , Glöckner Frank Oliver , Mascher Thorsten , Kolter Roberto
Published: September 2012

Members of the
Planctomycetes
clade share many unusual features for bacteria. Their cytoplasm contains membrane-bound compartments, they lack peptidoglycan and FtsZ, they divide by polar budding, and they are capable of endocytosis. Planctomycete genomes have remained enigmatic, generally being quite large (up to 9 Mb), and on average, 55% of their predicted proteins are of unknown function. Importantly, proteins related to the unusual traits of
Planctomycetes
remain largely unknown. Thus, we embarked on bioinformatic analyses of these genomes in an effort to predict proteins that are likely to be involved in compartmentalization, cell division, and signal transduction. We used three complementary strategies. First, we defined the
Planctomycetes
core genome and subtracted genes of well-studied model organisms. Second, we analyzed the gene content and synteny of morphogenesis and cell division genes and combined both methods using a “guilt-by-association” approach. Third, we identified signal transduction systems as well as sigma factors. These analyses provide a manageable list of candidate genes for future genetic studies and provide evidence for complex signaling in the
Planctomycetes
akin to that observed for bacteria with complex life-styles, such as
Myxococcus xanthus
.