bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of a
trans
-Dichloroethene-Respiring Dehalogenimonas Species in the 1,1,2,2-Tetrachloroethane-Dechlorinating WBC-2 Consortium
Authors: Manchester Marie J. , Hug Laura A. , Zarek Matt , Zila Anna , Edwards Elizabeth A.
Published: May 2012

The WBC-2 consortium is an organohalide-respiring anaerobic microbial enrichment culture capable of dechlorinating 1,1,2,2-tetrachloroethane (TeCA) to ethene. In the WBC-2 culture, TeCA is first transformed to
trans
-dichloroethene (tDCE) by dichloroelimination; tDCE is subsequently transformed to vinyl chloride (VC) and then to ethene by hydrogenolysis. Analysis of 16S rRNA gene clone libraries from culture DNA revealed sequences from three putative dechlorinating organisms belonging to
Dehalococcoides
,
Dehalobacter
, and
Dehalogenimonas
genera. Quantitative PCR primers were designed for each of these sequences, and their abundance was quantified in enrichment cultures over time. These data revealed that complete dechlorination of TeCA to ethene involves all three organisms.
Dehalobacter
spp. grew during the dihaloelimination of TeCA to tDCE, while
Dehalococcoides
and
Dehalogenimonas
spp. grew during hydrogenolysis of tDCE to ethene. This is the first time a genus other than
Dehalococcoides
has been implicated in dechlorination of tDCE to VC.
Ciliates Expel Environmental Legionella-Laden Pellets To Stockpile Food
Authors: Hojo Fuhito , Sato Daisuke , Matsuo Junji , Miyake Masaki , Nakamura Shinji , Kunichika Miyuki , Hayashi Yasuhiro , Yoshida Mitsutaka , Takahashi Kaori , Takemura Hiromu , Kamiya Shigeru , Yamaguchi Hiroyuki
Published: May 2012

When
Tetrahymena
ciliates are cultured with
Legionella pneumophila
, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental
L. pneumophila
, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human
L. pneumophila
isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental
L. pneumophila
for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.
Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy
Authors: Huang Shaoya , Ding Xuezhi , Sun Yunjun , Yang Qi , Xiao Xiuqing , Cao Zhenping , Xia Liqiu
Published: May 2012

The proteome of a newBacillus thuringiensissubsp.kurstakistrain, 4.0718, from the middle vegetative (T1), early sporulation (T2), and late sporulation (T3) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches andB. thuringiensissubspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome ofB. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing theB. thuringiensisproteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.
High-Pressure Inactivation of Human Norovirus Virus-Like Particles Provides Evidence that the Capsid of Human Norovirus Is Highly Pressure Resistant
Authors: Lou Fangfei , Huang Pengwei , Neetoo Hudaa , Gurtler Joshua B. , Niemira Brendan A. , Chen Haiqiang , Jiang Xi , Li Jianrong
Published: May 2012

Human norovirus (NoV) is the leading cause of nonbacterial acute gastroenteritis epidemics worldwide. High-pressure processing (HPP) has been considered a promising nonthermal processing technology to inactivate food- and waterborne viral pathogens. Due to the lack of an effective cell culture method for human NoV, the effectiveness of HPP in inactivating human NoV remains poorly understood. In this study, we evaluated the effectiveness of HPP in disrupting the capsid of human NoV based on the structural and functional integrity of virus-like particles (VLPs) and histo-blood group antigen (HBGA) receptor binding assays. We found that pressurization at 500 to 600 MPa for 2 min, a pressure level that completely inactivates murine norovirus and feline calicivirus, was not sufficient to disrupt the structure and function of human NoV VLPs, even with a holding time of 60 min. Degradation of VLPs increased commensurate with increasing pressure levels more than increasing time. The times required for complete disruption of human NoV VLPs at 700, 800, and 900 MPa were 45, 15, and 2 min, respectively. Human NoV VLPs were more resistant to HPP in their ability to bind type A than type B and O HBGAs. Additionally, the 23-nm VLPs appeared to be much more stable than the 38-nm VLPs. Taken together, our results demonstrated that the human NoV capsid is highly resistant to HPP. While human NoV VLPs may not be fully representative of viable human NoV, destruction of the VLP capsid is highly suggestive of a typical response for viable human NoV.
Oral Spirochetes Implicated in Dental Diseases Are Widespread in Normal Human Subjects and Carry Extremely Diverse Integron Gene Cassettes
Authors: Wu Yu-Wei , Rho Mina , Doak Thomas G. , Ye Yuzhen
Published: May 2012

The NIH Human Microbiome Project (HMP) has produced several hundred metagenomic data sets, allowing studies of the many functional elements in human-associated microbial communities. Here, we survey the distribution of oral spirochetes implicated in dental diseases in normal human individuals, using recombination sites associated with the chromosomal integron in
Treponema
genomes, taking advantage of the multiple copies of the integron recombination sites (repeats) in the genomes, and using a targeted assembly approach that we have developed. We find that integron-containing
Treponema
species are present in ∼80% of the normal human subjects included in the HMP. Further, we are able to
de novo
assemble the integron gene cassettes using our constrained assembly approach, which employs a unique application of the de Bruijn graph assembly information; most of these cassette genes were not assembled in whole-metagenome assemblies and could not be identified by mapping sequencing reads onto the known reference
Treponema
genomes due to the dynamic nature of integron gene cassettes. Our study significantly enriches the gene pool known to be carried by
Treponema
chromosomal integrons, totaling 826 (598 97% nonredundant) genes. We characterize the functions of these gene cassettes: many of these genes have unknown functions. The integron gene cassette arrays found in the human microbiome are extraordinarily dynamic, with different microbial communities sharing only a small number of common genes.
White Mutants of Chloroperoxidase-Secreting Caldariomyces fumago as Superior Production Strains, Revealing an Interaction between Pigmentation and Enzyme Secretion
Authors: Buchhaupt Markus , Hüttmann Sonja , Schrader Jens
Published: May 2012

By mutant-colony screening of
Caldariomyces fumago
, several white mutants were isolated that are superior strains for the production of the valuable enzyme chloroperoxidase (CPO). Their culture supernatant lacks the contaminating dark pigment, which simplifies downstream processing. Furthermore, the CPO content increased significantly faster than the wild-type rate, which uncovers possible interactions between pigmentation and enzyme secretion.
Influence of Artisan Bakery- or Laboratory-Propagated Sourdoughs on the Diversity of Lactic Acid Bacterium and Yeast Microbiotas
Authors: Minervini Fabio , Lattanzi Anna , De Angelis Maria , Di Cagno Raffaella , Gobbetti Marco
Published: May 2012

Seven mature type I sourdoughs were comparatively back-slopped (80 days) at artisan bakery and laboratory levels under constant technology parameters. The cell density of presumptive lactic acid bacteria and related biochemical features were not affected by the environment of propagation. On the contrary, the number of yeasts markedly decreased from artisan bakery to laboratory propagation. During late laboratory propagation, denaturing gradient gel electrophoresis (DGGE) showed that the DNA band corresponding to
Saccharomyces cerevisiae
was no longer detectable in several sourdoughs. Twelve species of lactic acid bacteria were variously identified through a culture-dependent approach. All sourdoughs harbored a certain number of species and strains, which were dominant throughout time and, in several cases, varied depending on the environment of propagation. As shown by statistical permutation analysis, the lactic acid bacterium populations differed among sourdoughs propagated at artisan bakery and laboratory levels.
Lactobacillus plantarum
,
Lactobacillus sakei
, and
Weissella cibaria
dominated in only some sourdoughs back-slopped at artisan bakeries, and
Leuconostoc citreum
seemed to be more persistent under laboratory conditions. Strains of
Lactobacillus sanfranciscensis
were indifferently found in some sourdoughs. Together with the other stable species and strains, other lactic acid bacteria temporarily contaminated the sourdoughs and largely differed between artisan bakery and laboratory levels. The environment of propagation has an undoubted influence on the composition of sourdough yeast and lactic acid bacterium microbiotas.
Intricate Interactions between the Bloom-Forming Cyanobacterium Microcystis aeruginosa and Foreign Genetic Elements, Revealed by Diversified Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Signatures
Authors: Kuno Sotaro , Yoshida Takashi , Kaneko Takakazu , Sako Yoshihiko
Published: May 2012

Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacteriumMicrocystis aeruginosa, we investigated CRISPR in fourM. aeruginosastrains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity toM. aeruginosaphage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided intoM. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure ofM. aeruginosato small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies thatM. aeruginosahas been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.
Identification and Molecular Characterization of Class 1 Integrons in Multiresistant Escherichia coli Isolates from Poultry Litter
Authors: Ponce-Rivas Elizabeth , Muñoz-Márquez María-Enriqueta , Khan Ashraf A.
Published: May 2012

This study describes the prevalence of arrays of class 1 integron cassettes and Qnr determinants (A, B, and S) in 19 fluoroquinolone-resistant
Escherichia coli
isolates from chicken litter.
qnrS
and
qnrA
were the predominant genes in these fluoroquinolone-resistant isolates, and an uncommon array of
aacA4
-
catB3
-
dfrA1
gene cassettes from a class1 integron was found. Additionally,
aadA1
and
dfrA1
gene cassettes, encoding resistance to streptomycin and trimethoprim, constituted the most common genes identified and was located on megaplasmids as well on the chromosome. Antibiotic resistance, pulsed-field gel electrophoresis (PFGE), and plasmid data suggest a genetically diverse origin of poultry
E. coli
isolates.
Reclamation of Ampicillin Sensitivity for the Genetic Manipulation of Legionella pneumophila
Authors: Sutherland Molly C. , Vogel Joseph P.
Published: May 2012

Research on
Legionella pneumophila
, the causative agent of Legionnaires' disease, has been hampered due to the lack of selectable markers for genetic manipulation. We report the construction of a mutant strain of
L. pneumophila
lacking
loxA
, a chromosomally encoded β-lactamase, that has enhanced sensitivity to ampicillin. Also described are a method for converting
Legionella
strains to ampicillin sensitivity and conditions for utilizing
bla
as a selectable marker.
Involvement of a Putative Cyclic AMP Receptor Protein (CRP)-Like Binding Sequence and a CRP-Like Protein in Glucose-Mediated Catabolite Repression of
thn
Genes in Rhodococcus sp. Strain TFB
Authors: Tomás-Gallardo Laura , Santero Eduardo , Floriano Belén
Published: May 2012

Glucose catabolite repression of tetralin catabolic genes in
Rhodococcus
sp. strain TFB was shown to be exerted by a protein homologous to transcriptional regulators of the cyclic AMP receptor (CRP)-FNR family. The protein was detected bound to putative CRP-like boxes localized at the promoters of the
thnA1
and
thnS
genes.
Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker
Authors: Li Baoguang , Chen Jin-Qiang
Published: May 2012

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable
Escherichia coli
O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of
E. coli
O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for
E. coli
O157:H7 strains (
n
= 298). Using this assay, we can detect amounts of genomic DNA of
E. coli
O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)–real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10
7
dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable
E. coli
O157:H7 cells with an 8-h enrichment. In conclusion, this PMA–real-time PCR assay offers a sensitive and specific means to selectively detect viable
E. coli
O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable
E. coli
O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.
A Conserved Two-Component Signal Transduction System Controls the Response to Phosphate Starvation in Bifidobacterium breve UCC2003
Authors: Alvarez-Martin Pablo , Fernández Matilde , O'Connell-Motherway Mary , O'Connell Kerry Joan , Sauvageot Nicolas , Fitzgerald Gerald F. , MacSharry John , Zomer Aldert , van Sinderen Douwe
Published: May 2012

This work reports on the identification and molecular characterization of the two-component regulatory system (2CRS) PhoRP, which controls the response to inorganic phosphate (P
i
) starvation in
Bifidobacterium breve
UCC2003. The response regulator PhoP was shown to bind to the promoter region of
pstSCAB
, specifying a predicted P
i
transporter system, as well as that of
phoU
, which encodes a putative P
i
-responsive regulatory protein. This interaction is assumed to cause transcriptional modulation under conditions of P
i
limitation. Our data suggest that the
phoRP
genes are subject to positive autoregulation and, together with
pstSCAB
and presumably
phoU
, represent the complete regulon controlled by the
phoRP
-encoded 2CRS in
B. breve
UCC2003. Determination of the minimal PhoP binding region combined with bioinformatic analysis revealed the probable recognition sequence of PhoP, designated here as the PHO box, which together with
phoRP
is conserved among many high-GC-content Gram-positive bacteria. The importance of the
phoRP
2CRS in the response of
B. breve
to P
i
starvation conditions was confirmed by analysis of a
B. breve
phoP
insertion mutant which exhibited decreased growth under phosphate-limiting conditions compared to its parent strain UCC2003.
Effects of Subinhibitory Concentrations of Menthol on Adaptation, Morphological, and Gene Expression Changes in Enterohemorrhagic Escherichia coli
Authors: Landau Elad , Shapira Roni
Published: May 2012

Menthol (C
10
H
20
O) possesses antibacterial activity; nevertheless, bacterial adaptation to this compound has never been studied. Here we report that precultivation of enterohemorrhagic
Escherichia coli
(EHEC) strains in increasing subinhibitory (SI) concentrations of menthol significantly elevates (4- to 16-fold) their resistance to menthol. Concomitant morphological alterations included the appearance of mucoid colonies and reduced biofilm production. Scanning electron microscopy (SEM) examination revealed suppressed curli formation in menthol-adapted cells. Expression of the gene
cpsB10
(encoding one of the enzymes responsible for colanic acid production) was elevated in response to SI concentrations of menthol in a laboratory
E. coli
strain, whereas expression in an
rcsC
null mutant was reduced, implicating a partial role for the Rcs phosphorelay system in mediating the menthol signal. Adaptation to menthol also reduced expression of the locus of enterocyte effacement-encoded regulator (Ler). This reduction, together with reduced curli and biofilm formation and elevated mucoidity, suggests a general reduction in bacterial virulence following adaptation to menthol. Our results thus suggest menthol as a potential lead in the recently emerging alternative strategy of targeting bacterial virulence factors to develop new types of anti-infective agents.
Modeling of Phenoxy Acid Herbicide Mineralization and Growth of Microbial Degraders in 15 Soils Monitored by Quantitative Real-Time PCR of the Functional
tfdA
Gene
Authors: Bælum Jacob , Prestat Emmanuel , David Maude M. , Strobel Bjarne W. , Jacobsen Carsten S.
Published: May 2012

Mineralization potentials, rates, and kinetics of the three phenoxy acid (PA) herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), were investigated and compared in 15 soils collected from five continents. The mineralization patterns were fitted by zero/linear or exponential growth forms of the three-half-order models and by logarithmic (log), first-order, or zero-order kinetic models. Prior and subsequent to the mineralization event,
tfdA
genes were quantified using real-time PCR to estimate the genetic potential for degrading PA in the soils. In 25 of the 45 mineralization scenarios, ∼60% mineralization was observed within 118 days. Elevated concentrations of
tfdA
in the range 1 × 10
5
to 5 × 10
7
gene copies g
−1
of soil were observed in soils where mineralization could be described by using growth-linked kinetic models. A clear trend was observed that the mineralization rates of the three PAs occurred in the order 2,4-D > MCPA > MCPP, and a correlation was observed between rapid mineralization and soils exposed to PA previously. Finally, for 2,4-D mineralization, all seven mineralization patterns which were best fitted by the exponential model yielded a higher
tfdA
gene potential after mineralization had occurred than the three mineralization patterns best fitted by the Lin model.
Microbiological and Physicochemical Characterization of Small-Scale Cocoa Fermentations and Screening of Yeast and Bacterial Strains To Develop a Defined Starter Culture
Authors: Pereira Gilberto Vinícius de Melo , Miguel Maria Gabriela da Cruz Pedrozo , Ramos Cíntia Lacerda , Schwan Rosane Freitas
Published: May 2012

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with
Saccharomyces cerevisiae
as the dominant species) and lactic acid bacteria (LAB) (
Lactobacillus fermentum
and
Lactobacillus plantarum
were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (
Acetobacter tropicalis
was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains
L. fermentum
UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol),
S. cerevisiae
UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and
Acetobacter tropicalis
UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.
Upregulation of Plasmid Genes during Stationary Phase in Synechocystis sp. Strain PCC 6803, a Cyanobacterium
Authors: Berla Bertram M. , Pakrasi Himadri B.
Published: May 2012

We analyzed DNA microarrays to identify highly expressed genes during stationary-phase growth ofSynechocystissp. PCC 6803. Many identified genes are on endogenous plasmids, with copy numbers between 0.4 and 7 per chromosome. The promoters of such genes will be useful for synthetic biology applications with this phototrophic host.
O
-Demethylation and Successive Oxidative Dechlorination of Methoxychlor by Bradyrhizobium sp. Strain 17-4, Isolated from River Sediment
Authors: Satsuma Koji , Masuda Minoru , Sato Kiyoshi
Published: May 2012

O
-Demethylation of insecticide methoxychlor is well known as a phase I metabolic reaction in various eukaryotic organisms. Regarding prokaryotic organisms, however, no individual species involved in such reaction have been specified and characterized so far. Here we successfully isolated a bacterium that mediates oxidative transformation of methoxychlor, including
O
-demethylation and dechlorination, from river sediment. The isolate was found to be closely related to
Bradyrhizobium elkanii
at the 16S rRNA gene sequence level (100% identical). However, based on some differences in the physiological properties of this bacterium, we determined that it was actually a different species,
Bradyrhizobium
sp. strain 17-4. The isolate mediated
O
-demethylation of methoxychlor to yield a monophenolic derivative [Mono-OH; 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane] as the primary degradation product. The chiral high-performance liquid chromatography (HPLC) analysis revealed that the isolate possesses high enantioselectivity favoring the formation of (
S
)-Mono-OH (nearly 100%). Accompanied by the sequential
O
-demethylation to form the bis-phenolic derivative Bis-OH [1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane], oxidative dechlorination of the side chain proceeded, and monophenolic carboxylic acid accumulated, followed by the formation of multiple unidentified polar degradation products. The breakdown proceeded more rapidly when reductively dechlorinated (dichloro-form) methoxychlor was applied as the initial substrate. The resultant carboxylic acids and polar degradation products are likely further biodegraded by ubiquitous bacteria. The isolate possibly plays an important role for complete degradation (mineralization) of methoxychlor by providing the readily biodegradable substrates.
Archaeal Tetrathionate Hydrolase Goes Viral: Secretion of a Sulfur Metabolism Enzyme in the Form of Virus-Like Particles
Authors: Krupovic Mart , Peixeiro Nuno , Bettstetter Marcus , Rachel Reinhard , Prangishvili David
Published: May 2012

In the course of screening for virus-host systems in extreme thermal environments, we have isolated a strain of the hyperthermophilic archaeaon
Acidianus hospitalis
producing unusual filamentous particles with a zipper-like appearance. The particles were shown to represent a secreted form of a genuine cellular enzyme, tetrathionate hydrolase, involved in sulfur metabolism.
Genome Sequence and Mutational Analysis of Plant-Growth-Promoting Bacterium Agrobacterium tumefaciens CCNWGS0286 Isolated from a Zinc-Lead Mine Tailing
Authors: Hao Xiuli , Xie Pin , Johnstone Laurel , Miller Susan J. , Rensing Christopher , Wei Gehong
Published: May 2012

The plant-growth-promoting bacterium
Agrobacterium tumefaciens
CCNWGS0286, isolated from the nodules of
Robinia pseudoacacia
growing in zinc-lead mine tailings, both displayed high metal resistance and enhanced the growth of
Robinia
plants in a metal-contaminated environment. Our goal was to determine whether bacterial metal resistance or the capacity to produce phytohormones had a larger impact on the growth of host plants under zinc stress. Eight zinc-sensitive mutants and one zinc-sensitive mutant with reduced indole-3-acetic acid (IAA) production were obtained by transposon mutagenesis. Analysis of the genome sequence and of transcription via reverse transcriptase PCR (RT-PCR) combined with transposon gene disruptions revealed that ZntA-4200 and the transcriptional regulator ZntR1 played important roles in the zinc homeostasis of
A. tumefaciens
CCNWGS0286. In addition, interruption of a putative oligoketide cyclase/lipid transport protein reduced IAA synthesis and also showed reduced zinc and cadmium resistance but had no influence on copper resistance. In greenhouse studies,
R. pseudoacacia
inoculated with
A. tumefaciens
CCNWGS0286 displayed a significant increase in biomass production over that without inoculation, even in a zinc-contaminated environment. Interestingly, the differences in plant biomass improvement among
A. tumefaciens
CCNWGS0286,
A. tumefaciens
C58, and zinc-sensitive mutants 12-2 (
zntA
::Tn
5
) and 15-6 (low IAA production) revealed that phytohormones, rather than genes encoding zinc resistance determinants, were the dominant factor in enhancing plant growth in contaminated soil.
Predominance of a Versatile-Peroxidase-Encoding Gene,
mnp4
, as Demonstrated by Gene Replacement via a Gene Targeting System for Pleurotus ostreatus
Authors: Salame Tomer M. , Knop Doriv , Tal Dana , Levinson Dana , Yarden Oded , Hadar Yitzhak
Published: May 2012

Pleurotus ostreatus
(the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle.
P. ostreatus
is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in
Pleurotus
transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a
P. ostreatus
PC9 homokaryon Δ
ku80
strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δ
ku80
strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δ
ku80
strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by
mnp4
. This enzyme is part of the ligninolytic system of
P. ostreatus
, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn
2+
-deficient media.
mnp4
inactivation provided a direct proof that
mnp4
encodes a key VP responsible for the Mn
2+
-dependent and Mn
2+
-independent peroxidase activity under Mn
2+
-deficient culture conditions.
The Bacterial Surface Layer Provides Protection against Antimicrobial Peptides
Authors: de la Fuente-Núñez César , Mertens Jan , Smit John , Hancock Robert E. W.
Published: May 2012

This report describes a previously unrecognized role for bacterial surface layers as barriers that confer protection against antimicrobial peptides. As antimicrobial peptides exist in natural environments, S-layers may provide a bacterial survival mechanism that has been selected for through evolution.
Characterization of Geographically Distinct Bacterial Communities Associated with Coral Mucus Produced by Acropora spp. and Porites spp
Authors: McKew B. A. , Dumbrell A. J. , Daud S. D. , Hepburn L. , Thorpe E. , Mogensen L. , Whitby C.
Published: May 2012

Acropora
and
Porites
corals are important reef builders in the Indo-Pacific and Caribbean. Bacteria associated with mucus produced by
Porites
spp. and
Acropora
spp. from Caribbean (Punta Maroma, Mexico) and Indo-Pacific (Hoga and Sampela, Indonesia) reefs were determined. Analysis of pyrosequencing libraries showed that bacterial communities from Caribbean corals were significantly more diverse (H′, 3.18 to 4.25) than their Indonesian counterparts (H′, 2.54 to 3.25). Dominant taxa were
Gammaproteobacteria
,
Alphaproteobacteria
,
Firmicutes
, and
Cyanobacteria
, which varied in relative abundance between coral genera and region. Distinct coral host-specific communities were also found; for example,
Clostridiales
were dominant on
Acropora
spp. (at Hoga and the Mexican Caribbean) compared to
Porites
spp. and seawater. Within the
Gammproteobacteria
,
Halomonas
spp. dominated sequence libraries from
Porites
spp. (49%) and
Acropora
spp. (5.6%) from the Mexican Caribbean, compared to the corresponding Indonesian coral libraries (<2%). Interestingly, with the exception of
Porites
spp. from the Mexican Caribbean, there was also a ubiquity of
Psychrobacter
spp., which dominated
Acropora
and
Porites
libraries from Indonesia and
Acropora
libraries from the Caribbean. In conclusion, there was a dominance of
Halomonas
spp. (associated with
Acropora
and
Porites
[Mexican Caribbean]),
Firmicutes
(associated with
Acropora
[Mexican Caribbean] and with
Acropora
and
Porites
[Hoga]), and
Cyanobacteria
(associated with
Acropora
and
Porites
[Hoga] and
Porites
[Sampela]). This is also the first report describing geographically distinct
Psychrobacter
spp. associated with coral mucus. In addition, the predominance of
Clostridiales
associated with
Acropora
spp. provided additional evidence for coral host-specific microorganisms.
Patterns of [FeFe] Hydrogenase Diversity in the Gut Microbial Communities of Lignocellulose-Feeding Higher Termites
Authors: Ballor Nicholas R. , Leadbetter Jared R.
Published: May 2012

Hydrogen is the central free intermediate in the degradation of wood by termite gut microbes and can reach concentrations exceeding those measured for any other biological system. Degenerate primers targeting the largest family of [FeFe] hydrogenases observed in a termite gut metagenome have been used to explore the evolution and representation of these enzymes in termites. Sequences were cloned from the guts of the higher termitesAmitermessp. strain Cost010,Amitermessp. strain JT2,Gnathamitermessp. strain JT5,Microcerotermessp. strain Cost008,Nasutitermessp. strain Cost003, andRhyncotermessp. strain Cost004. Each gut sample harbored a more rich and evenly distributed population of hydrogenase sequences than observed previously in the guts of lower termites andCryptocercus punctulatus.This accentuates the physiological importance of hydrogen for higher termite gut ecosystems and may reflect an increased metabolic burden, or metabolic opportunity, created by a lack of gut protozoa. The sequences were phylogenetically distinct from previously sequenced [FeFe] hydrogenases. Phylogenetic and UniFrac comparisons revealed congruence between host phylogeny and hydrogenase sequence library clustering patterns. This may reflect the combined influences of the stable intimate relationship of gut microbes with their host and environmental alterations in the gut that have occurred over the course of termite evolution. These results accentuate the physiological importance of hydrogen to termite gut ecosystems.
Role of Fig1, a Component of the Low-Affinity Calcium Uptake System, in Growth and Sexual Development of Filamentous Fungi
Authors: Cavinder Brad , Trail Frances
Published: May 2012

The function of Fig1, a transmembrane protein of the low-affinity calcium uptake system (LACS) in fungi, was examined for its role in the growth and development of the plant pathogenFusarium graminearum. The Δfig1mutants failed to produce mature perithecia, and sexual development was halted prior to the formation of perithecium initials. The loss of Fig1 function also resulted in a reduced vegetative growth rate. Macroconidium production was reduced 70-fold in the Δfig1mutants compared to the wild type. The function of the high-affinity calcium uptake system (HACS), comprised of the Ca2+channels Mid1 and Cch1, was previously characterized forF. graminearum. To better understand the roles of the LACS and the HACS, Δfig1Δmid1, Δfig1Δcch1, and Δfig1Δmid1Δcch1double and triple mutants were generated, and the phenotypes of these mutants were more severe than those of the Δfig1mutants. Pathogenicity on wheat was unaffected for the Δfig1mutants, but the Δfig1Δmid1, Δfig1Δcch1, and Δfig1Δmid1Δcch1mutants, lacking both LACS and HACS functions, had reduced pathogenicity. Additionally, Δfig1mutants ofNeurospora crassawere examined and did not affect filamentous growth or female fertility in a Δfig1mating typeAstrain, but the Δfig1mating typeastrain failed to produce fertile fruiting bodies. These results are the first report of Fig1 function in filamentous ascomycetes and expand its role to include complex fruiting body and ascus development.
Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4
Authors: Ocampo J. , McCormack B. , Navarro E. , Moreno S. , Garre V. , Rossi S.
Published: May 2012

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungusMucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. InM. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is thatpkaR4is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. ThepkaR2null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting thatpkaR2participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role inM. circinelloidesphysiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway.
The Heat-Induced Molecular Disaggregase Hsp104 of Candida albicans Plays a Role in Biofilm Formation and Pathogenicity in a Worm Infection Model
Authors: Fiori Alessandro , Kucharíková Soňa , Govaert Gilmer , Cammue Bruno P. A. , Thevissen Karin , Van Dijck Patrick
Published: May 2012

The consequences of deprivation of the molecular chaperone Hsp104 in the fungal pathogen
Candida albicans
were investigated. Mutants lacking
HSP104
became hypersusceptible to lethally high temperatures, similarly to the corresponding mutants of
Saccharomyces cerevisiae
, whereas normal susceptibility was restored upon reintroduction of the gene. By use of a strain whose only copy of
HSP104
is an ectopic gene under the control of a tetracycline-regulated promoter, expression of Hsp104 prior to the administration of heat shock could be demonstrated to be sufficient to confer protection from the subsequent temperature increase. This result points to a key role for Hsp104 in orchestrating the cell response to elevated temperatures. Despite their not showing evident growth or morphological defects, biofilm formation by cells lacking
HSP104
proved to be defective in two established
in vitro
models that use polystyrene and polyurethane as the substrates. Biofilms formed by the wild-type and
HSP104
-reconstituted strains showed patterns of intertwined hyphae in the extracellular matrix. In contrast, biofilm formed by the
hsp104
Δ/
hsp104
Δ mutant showed structural defects and appeared patchy and loose. Decreased virulence of the
hsp104
Δ/
hsp104
Δ mutant was observed in the
Caenorhabditis elegans
infection model, in which high
in vivo
temperature does not play a role. In agreement with the view that stress responses in fungal pathogens may have evolved to provide niche-specific adaptation to environmental conditions, these results provide an indication of a temperature-independent role for Hsp104 in support of
Candida albicans
virulence, in addition to its key role in governing thermotolerance.
Characterization of a Vibrio fischeri Aminopeptidase and Evidence for Its Influence on an Early Stage of Squid Colonization
Authors: Fidopiastis Pat M. , Rader Bethany A. , Gerling David G. , Gutierrez Nestor A. , Watkins Katherine H. , Frey Michelle West , Nyholm Spencer V. , Whistler Cheryl A.
Published: May 2012

Vibrio fischeri
cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid
Euprymna scolopes
. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of
Vibrio fischeri
strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and
k
cat
and

K
m

values that are lower and smaller, respectively, than those of
Escherichia coli
PepN. A
V. fischeri
mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.
Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass
Authors: Blumer-Schuette Sara E. , Giannone Richard J. , Zurawski Jeffrey V. , Ozdemir Inci , Ma Qin , Yin Yanbin , Xu Ying , Kataeva Irina , Poole Farris L. , Adams Michael W. W. , Hamilton-Brehm Scott D. , Elkins James G. , Larimer Frank W. , Land Miriam L. , Hauser Loren J. , Cottingham Robert W. , Hettich Robert L. , Kelly Robert M.
Published: May 2012

Extremely thermophilic bacteria of the genus
Caldicellulosiruptor
utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse
Caldicellulosiruptor
species. The open
Caldicellulosiruptor
pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic
Caldicellulosiruptor
species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral
Caldicellulosiruptor
was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.
Cellular Characterization of the Primosome and Rep Helicase in Processing and Restoration of Replication following Arrest by UV-Induced DNA Damage in Escherichia coli
Authors: Courcelle Charmain T. , Landstrom Allison J. , Anderson Brittany , Courcelle Justin
Published: May 2012

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed.
In vitro
, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments
in vivo
. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both
rep
and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage
in vivo
and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate
de novo
replication downstream of the lesion.
Sinorhizobium meliloti ExoR Is the Target of Periplasmic Proteolysis
Authors: Lu Hai-Yang , Luo Li , Yang Meng-Hua , Cheng Hai-Ping
Published: May 2012

Sinorhizobium meliloti
ExoR regulates the production of succinoglycan and flagella through the ExoS/ChvI two-component regulatory system. ExoR has been proposed to inhibit the ExoS sensor through direct interaction in the periplasm. To understand how ExoR suppression of ExoS is relieved, which is required for the expression of ExoS/ChvI-regulated symbiosis genes, we characterized wild-type ExoR and ExoR95 mutant proteins. In addition to the previously identified precursor and mature forms of ExoR (designated ExoR
p
and ExoR
m
, respectively), we detected a 20-kDa form of ExoR (designated ExoR
c20
) derived from the wild-type ExoR protein, but not from the ExoR95 mutant protein. ExoR
c20
was isolated directly from
S. meliloti
periplasm to identify its N-terminal amino acids and the site of the proteolysis, which is highly conserved among ExoR homologs. ExoR
c20
retains the C terminus of the wild-type ExoR. When expressed directly, ExoR
c20
did not complement the
exoR95
mutation, suggesting that ExoR
c20
does not function directly in the ExoR-ExoS/ChvI regulatory pathway and that ExoR
m
is the functional form of ExoR. A single-amino-acid change (ExoRL81A) at the site of ExoR periplasmic proteolysis resulted in the reduction of the amount of ExoR
m
and the loss of the regulatory function of the ExoR protein. These findings suggest that ExoR
m
is a target of periplasmic proteolysis and that the amount of ExoR
m
could be reduced through effective proteolysis to relieve its suppression of ExoS.