bluuebunny/crossref_metadata_2025 huggingface dataset:
Residual-layer-free printing by selective filling of self-assembled monolayer-treated mold
Authors: Hong Franklin Chau-Nan , Kao Yu-Chih
Published: June 2011

In this study, the authors achieved residual-layer-free printing by reversal imprint with the grooves or cavities of the mold selectively filled with polymer. By treating the protrusion surface and groove surface of the mold with alkylsilane and fluoroalkylsilane self-assembled-monolayers (SAMs), respectively, the polymer solution could selectively fill the grooves or cavities of the mold by spin coating, with no deposition on the protrusion surface. By stamping the selectively filled mold on the substrate, residual-layer-free patterns of polymer could be obtained. The residual-layer-free printing could simplify the fabrication of micron-scale three-dimensional structures with smooth edges and low distortion. The solvent in the polymer solution for spin coating of the mold plays an important role in the selective filling. The mechanism of selective filling was found to be further induced by the dynamic adhesion of the polymer solution to the SAM-treated surfaces.
Increased Transfer of a Multidrug Resistance Plasmid in Escherichia coli Biofilms at the Air-Liquid Interface
Authors: Król Jaroslaw E. , Nguyen Hung Duc , Rogers Linda M. , Beyenal Haluk , Krone Stephen M. , Top Eva M.
Published: June 2011

Although biofilms represent a common bacterial lifestyle in clinically and environmentally important habitats, there is scant information on the extent of gene transfer in these spatially structured populations. The objective of this study was to gain insight into factors that affect transfer of the promiscuous multidrug resistance plasmid pB10 in
Escherichia coli
biofilms. Biofilms were grown in different experimental settings, and plasmid transfer was monitored using laser scanning confocal microscopy and plate counting. In closed flow cells, plasmid transfer in surface-attached submerged biofilms was negligible. In contrast, a high plasmid transfer efficiency was observed in a biofilm floating at the air-liquid interface in an open flow cell with low flow rates. A vertical flow cell and a batch culture biofilm reactor were then used to detect plasmid transfer at different depths away from the air-liquid interface. Extensive plasmid transfer occurred only in a narrow zone near that interface. The much lower transfer frequencies in the lower zones coincided with rapidly decreasing oxygen concentrations. However, when an
E. coli
csrA
mutant was used as the recipient, a thick biofilm was obtained at all depths, and plasmid transfer occurred at similar frequencies throughout. These results and data from separate aerobic and anaerobic matings suggest that oxygen can affect IncP-1 plasmid transfer efficiency, not only directly but also indirectly, through influencing population densities and therefore colocalization of donors and recipients. In conclusion, the air-liquid interface can be a hot spot for plasmid-mediated gene transfer due to high densities of juxtaposed donor and recipient cells.
Large-Scale Comparative Phenotypic and Genomic Analyses Reveal Ecological Preferences of Shewanella Species and Identify Metabolic Pathways Conserved at the Genus Level
Authors: Rodrigues Jorge L. M. , Serres Margrethe H. , Tiedje James M.
Published: June 2011

The use of comparative genomics for the study of different microbiological species has increased substantially as sequence technologies become more affordable. However, efforts to fully link a genotype to its phenotype remain limited to the development of one mutant at a time. In this study, we provided a high-throughput alternative to this limiting step by coupling comparative genomics to the use of phenotype arrays for five sequencedShewanellastrains. Positive phenotypes were obtained for 441 nutrients (C, N, P, and S sources), with N-based compounds being the most utilized for all strains. Many genes and pathways predicted by genome analyses were confirmed with the comparative phenotype assay, and three degradation pathways believed to be missing inShewanellawere confirmed as missing. A number of previously unknown gene products were predicted to be parts of pathways or to have a function, expanding the number of gene targets for future genetic analyses. Ecologically, the comparative high-throughput phenotype analysis provided insights into niche specialization among the five different strains. For example,Shewanella amazonensisstrain SB2B, isolated from the Amazon River delta, was capable of utilizing 60 C compounds, whereasShewanellasp. strain W3-18-1, isolated from deep marine sediment, utilized only 25 of them. In spite of the large number of nutrient sources yielding positive results, our study indicated that except for the N sources, they were not sufficiently informative to predict growth phenotypes from increasing evolutionary distances. Our results indicate the importance of phenotypic evaluation for confirming genome predictions. This strategy will accelerate the functional discovery of genes and provide an ecological framework for microbial genome sequencing projects.
Discovery of a Gene Involved in a Third Bacterial Protoporphyrinogen Oxidase Activity through Comparative Genomic Analysis and Functional Complementation
Authors: Boynton Tye O. , Gerdes Svetlana , Craven Sarah H. , Neidle Ellen L. , Phillips John D. , Dailey Harry A.
Published: June 2011

Tetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained “missing” in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizing
Acinetobacter baylyi
ADP1, to identify one of the missing enzymes, a new protoporphyrinogen oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known protoporphyrinogen oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome of
A. baylyi
, a bacterium that lacks both
hemG
and
hemY
, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked protoporphyrinogen oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne protoporphyrinogen oxidase gene from a number of different organisms, such as
hemG
from
Escherichia coli
,
hemY
from
Myxococcus xanthus
, or the human gene for protoporphyrinogen oxidase.
Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks
Authors: Sekse C. , Sunde M. , Lindstedt B.-A. , Hopp P. , Bruheim T. , Cudjoe K. S. , Kvitle B. , Urdahl A. M.
Published: June 2011

A national survey of
Escherichia coli
O26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, and
E. coli
O26 was detected in 17.9% of the flocks. One hundred forty-two
E. coli
O26 isolates were examined for flagellar antigens (H typing) and four virulence genes, including
stx
and
eae
, to identify possible Shiga toxin-producing
E. coli
(STEC) and enteropathogenic
E. coli
(EPEC). Most isolates (129 out of 142) were identified as
E. coli
O26:H11. They possessed
eae
and may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between the
E. coli
O26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all the
E. coli
O26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing on
E. coli
O26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.
Global Gene Expression Profile for Swarming Bacillus cereus Bacteria
Authors: Salvetti Sara , Faegri Karoline , Ghelardi Emilia , Kolstø Anne-Brit , Senesi Sonia
Published: June 2011

Bacillus cereus
can use swarming to move over and colonize solid surfaces in different environments. This kind of motility is a collective behavior accompanied by the production of long and hyperflagellate swarm cells. In this study, the genome-wide transcriptional response of
B. cereus
ATCC 14579 during swarming was analyzed. Swarming was shown to trigger the differential expression (>2-fold change) of 118 genes. Downregulated genes included those required for basic cellular metabolism. In accordance with the hyperflagellate phenotype of the swarm cell, genes encoding flagellin were overexpressed. Some genes associated with K
+
transport, phBC6A51 phage genes, and the binding component of the enterotoxin hemolysin BL (HBL) were also induced. Quantitative reverse transcription-PCR (qRT-PCR) experiments indicated an almost 2-fold upregulation of the entire
hbl
operon during swarming. Finally, BC1435 and BC1436, orthologs of
liaI-liaH
that are known to be involved in the resistance of
Bacillus subtilis
to daptomycin, were upregulated under swarming conditions. Accordingly, phenotypic assays showed reduced susceptibility of swarming
B. cereus
cells to daptomycin, and P

spac

-induced hyper-expression of these genes in liquid medium highlighted the role of BC1435 and BC1436 in the response of
B. cereus
to daptomycin.
Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol
Authors: Trinh Cong T. , Li Johnny , Blanch Harvey W. , Clark Douglas S.
Published: June 2011

Fermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design an
Escherichia coli
strain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism of
E. coli
was decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growth
E. coli
cannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesigned
E. coli
strain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producing
E. coli
strain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.
Electroactivity of Phototrophic River Biofilms and Constitutive Cultivable Bacteria
Authors: Lyautey Emilie , Cournet Amandine , Morin Soizic , Boulêtreau Stéphanie , Etcheverry Luc , Charcosset Jean-Yves , Delmas François , Bergel Alain , Garabetian Frédéric
Published: June 2011

Electroactivity is a property of microorganisms assembled in biofilms that has been highlighted in a variety of environments. This characteristic was assessed for phototrophic river biofilms at the community scale and at the bacterial population scale. At the community scale, electroactivity was evaluated on stainless steel and copper alloy coupons used both as biofilm colonization supports and as working electrodes. At the population scale, the ability of environmental bacterial strains to catalyze oxygen reduction was assessed by cyclic voltammetry. Our data demonstrate that phototrophic river biofilm development on the electrodes, measured by dry mass and chlorophyll
a
content, resulted in significant increases of the recorded potentials, with potentials of up to +120 mV/saturated calomel electrode (SCE) on stainless steel electrodes and +60 mV/SCE on copper electrodes. Thirty-two bacterial strains isolated from natural phototrophic river biofilms were tested by cyclic voltammetry. Twenty-five were able to catalyze oxygen reduction, with shifts of potential ranging from 0.06 to 0.23 V, cathodic peak potentials ranging from −0.36 to −0.76 V/SCE, and peak amplitudes ranging from −9.5 to −19.4 μA. These isolates were diversified phylogenetically (
Actinobacteria
,
Firmicutes
,
Bacteroidetes
, and
Alpha
-,
Beta
-, and
Gammaproteobacteria
) and exhibited various phenotypic properties (Gram stain, oxidase, and catalase characteristics). These data suggest that phototrophic river biofilm communities and/or most of their constitutive bacterial populations present the ability to promote electronic exchange with a metallic electrode, supporting the following possibilities: (i) development of electrochemistry-based sensors allowing
in situ
phototrophic river biofilm detection and (ii) production of microbial fuel cell inocula under oligotrophic conditions.
Statistical Analysis of Long- and Short-Range Forces Involved in Bacterial Adhesion to Substratum Surfaces as Measured Using Atomic Force Microscopy
Authors: Chen Yun , Busscher Henk J. , van der Mei Henny C. , Norde Willem
Published: June 2011

Surface thermodynamic analyses of microbial adhesion using measured contact angles on solid substrata and microbial cell surfaces are widely employed to determine the nature of the adhesion forces, i.e., the interplay between Lifshitz-van der Waals and acid-base forces. While surface thermodynamic analyses are often viewed critically, atomic force microscopy (AFM) can also provide information on the nature of the adhesion forces by means of Poisson analysis of the measured forces. This review first presents a description of Poisson analysis and its underlying assumptions. The data available from the literature for different combinations of bacterial strains and substrata are then summarized, leading to the conclusion that bacterial adhesion to surfaces is generally dominated by short-range, attractive acid-base interactions, in combination with long-range, weaker Lifshitz-van der Waals forces. This is in line with the findings of surface thermodynamic analyses of bacterial adhesion. Comparison with single-molecule ligand-receptor forces from the literature suggests that the short-range-force contribution from Poisson analysis involves a discrete adhesive bacterial cell surface site rather than a single molecular force. The adhesion force arising from these cell surface sites and the number of sites available may differ from strain to strain. Force spectroscopy, however, involves the tedious task of identifying the minor peaks in the AFM retraction force-distance curve. This step can be avoided by carrying out Poisson analysis on the work of adhesion, which can also be derived from retraction force-distance curves. This newly proposed way of performing Poisson analysis confirms that multiple molecular bonds, rather than a single molecular bond, contribute to a discrete adhesive bacterial cell surface site.
Identification of Loci and Functional Characterization of Trichothecene Biosynthesis Genes in Filamentous Fungi of the Genus Trichoderma
Authors: Cardoza R. E. , Malmierca M. G. , Hermosa M. R. , Alexander N. J. , McCormick S. P. , Proctor R. H. , Tijerino A. M. , Rumbero A. , Monte E. , Gutiérrez S.
Published: June 2011

Trichothecenes are mycotoxins produced by
Trichoderma
,
Fusarium
, and at least four other genera in the fungal order
Hypocreales
.
Fusarium
has a trichothecene biosynthetic gene (
TRI
) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize
TRI
gene orthologues (
tri
) in
Trichoderma arundinaceum
and
Trichoderma brevicompactum
. Our results indicate that both
Trichoderma
species have a
tri
cluster that consists of orthologues of seven genes present in the
Fusarium
TRI
cluster. Organization of genes in the cluster is the same in the two
Trichoderma
species but differs from the organization in
Fusarium
. Sequence and functional analysis revealed that the gene (
tri5
) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both
Trichoderma
species rather than inside the cluster as it is in
Fusarium
. Heterologous expression analysis revealed that two
T. arundinaceum
cluster genes (
tri4
and
tri11
) differ in function from their
Fusarium
orthologues. The
Tatri4
-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in
Fusarium
. The
Tatri11
-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the
Fusarium
orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the
tri/TRI
cluster have been conserved during evolution of
Trichoderma
and
Fusarium
, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.
Role of Nitric Oxide and Flavohemoglobin Homolog Genes in Aspergillus nidulans Sexual Development and Mycotoxin Production
Authors: Baidya Sachin , Cary Jeffrey W. , Grayburn W. Scott , Calvo A. M.
Published: June 2011

Flavohemoglobins are widely distributed in both prokaryotes and eukaryotes. These proteins are involved in reducing nitric oxide levels. Deletion of the
Aspergillus nidulans
flavohemoglobin gene
fhbA
induced sexual development and decreased sterigmatocystin production. Supplementation with a nitric oxide-releasing compound promoted cleistothecial formation and increased
nsdD
and
steA
expression, indicating that nitric oxide induces sexual development. This is the first study on the effect of nitric oxide on morphogenesis and secondary metabolism in fungi.
Determination of Microbial Diversity of Aeromonas Strains on the Basis of Multilocus Sequence Typing, Phenotype, and Presence of Putative Virulence Genes
Authors: Martino Maria Elena , Fasolato Luca , Montemurro Filomena , Rosteghin Marina , Manfrin Amedeo , Patarnello Tomaso , Novelli Enrico , Cardazzo Barbara
Published: June 2011

The genus
Aeromonas
has been described as comprising several species associated with the aquatic environment, which represents their principal reservoir.
Aeromonas
spp. are commonly isolated from diseased and healthy fish, but the involvement of such bacteria in human infection and gastroenteritis has frequently been reported. The primary challenge in establishing an unequivocal link between the
Aeromonas
genus and pathogenesis in humans is the extremely complicated taxonomy. With the aim of clarifying taxonomic relationships among the strains and phenotypes, a multilocus sequencing approach was developed and applied to characterize 23 type and reference strains of
Aeromonas
spp. and a collection of 77 field strains isolated from fish, crustaceans, and mollusks. All strains were also screened for putative determinants of virulence by PCR (
ast
,
ahh1
,
act
,
asa1
,
eno
,
ascV
, and
aexT
) and the production of acylated homoserine lactones (AHLs). In addition, the phenotypic fingerprinting obtained from 29 biochemical tests was submitted to the nonparametric combination (NPC) test methodology to define the statistical differences among the identified genetic clusters. Multilocus sequence typing (MLST) achieved precise strain genotyping, and the phylogenetic analysis of concatenated sequences delineated the relationship among the taxa belonging to the genus
Aeromonas
, providing a powerful tool for outbreak traceability, host range diffusion, and ecological studies. The NPC test showed the feasibility of phenotypic differentiation among the majority of the MLST clusters by using a selection of tests or the entire biochemical fingerprinting. A Web-based MLST sequence database (
http://pubmlst.org/aeromonas
) specific for the
Aeromonas
genus was developed and implemented with all the results.
Exceptional Production of both Prodigiosin and Cycloprodigiosin as Major Metabolic Constituents by a Novel Marine Bacterium, Zooshikella rubidus S1-1
Authors: Lee Jong Suk , Kim Yong-Sook , Park Sooyeon , Kim Jihoon , Kang So-Jung , Lee Mi-Hwa , Ryu Sangryeol , Choi Jong Myoung , Oh Tae-Kwang , Yoon Jung-Hoon
Published: June 2011

A Gram-negative, red-pigment-producing marine bacterial strain, designated S1-1, was isolated from the tidal flat sediment of the Yellow Sea, Korea. On the basis of phenotypic, phylogenetic, and genetic data, strain S1-1 (KCTC 11448BP) represented a new species of the genus
Zooshikella
. Thus, we propose the name
Zooshikella rubidus
sp. nov. Liquid chromatography and mass spectrometry of the red pigments produced by strain S1-1 revealed that the major metabolic compounds were prodigiosin and cycloprodigiosin. In addition, this organism produced six minor prodigiosin analogues, including two new structures that were previously unknown. To our knowledge, this is the first description of a microorganism that simultaneously produces prodigiosin and cycloprodigiosin as two major metabolites. Both prodigiosin and cycloprodigiosin showed antimicrobial activity against several microbial species. These bacteria were approximately 1.5-fold more sensitive to cycloprodigiosin than to prodigiosin. The metabolites also showed anticancer activity against human melanoma cells, which showed significantly more sensitivity to prodigiosin than to cycloprodigiosin. The secondary metabolite profiles of strain S1-1 and two reference bacterial strains were compared by liquid chromatography-mass spectrometry. Multivariate statistical analyses based on secondary metabolite profiles by liquid chromatography-mass spectrometry indicated that the metabolite profile of strain S1-1 could clearly be distinguished from those of two phylogenetically related, prodigiosin-producing bacterial strains.
Decay of Bacterial Pathogens, Fecal Indicators, and Real-Time Quantitative PCR Genetic Markers in Manure-Amended Soils
Authors: Rogers Shane W. , Donnelly Matthew , Peed Lindsay , Kelty Catherine A. , Mondal Sumona , Zhong Zirong , Shanks Orin C.
Published: June 2011

This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green fluorescent protein-expressing
Escherichia coli
O157:H7/pZs and red fluorescent protein-expressing
Salmonella enterica
serovar Typhimurium/pDs were added to laboratory-scale manure-amended soil microcosms with moisture contents of 60% or 80% field capacity and incubated at temperatures of −20°C, 10°C, or 25°C for 120 days. A two-stage first-order decay model was used to determine stage 1 and stage 2 first-order decay rate coefficients and transition times for each organism and qPCR genetic marker in each treatment. Genetic markers for FIB (
Enterococcus
spp.,
E. coli
, and
Bacteroidales
) exhibited decay rate coefficients similar to that of
E. coli
O157:H7/pZs but not of
S. enterica
serovar Typhimurium/pDs and persisted at detectable levels longer than both pathogens. Concentrations of these two bacterial pathogens, their counterpart qPCR genetic markers (
stx1
and
ttrRSBCA
, respectively), and FIB genetic markers were also correlated (
r
= 0.528 to 0.745). This suggests that these qPCR genetic markers may be reliable conservative surrogates for monitoring fecal pollution from manure-amended land. Host-associated qPCR genetic markers for microbial source tracking decayed rapidly to nondetectable concentrations, long before FIB,
Salmonella enterica
serovar Typhimurium/pDs, and
E. coli
O157:H7/pZs. Although good indicators of point source or recent nonpoint source fecal contamination events, these host-associated qPCR genetic markers may not be reliable indicators of nonpoint source fecal contamination events that occur weeks following manure application on land.
Temporal and Spatial Variability in Culturable Pathogenic Vibrio spp. in Lake Pontchartrain, Louisiana, following Hurricanes Katrina and Rita
Authors: Nigro Olivia D. , Hou Aixin , Vithanage Gayatri , Fujioka Roger S. , Steward Grieg F.
Published: June 2011

We investigated the abundance, distribution, and virulence gene content ofVibrio cholerae,V. parahaemolyticus, andV. vulnificusin the waters of southern Lake Pontchartrain in Louisiana on four occasions from October 2005 to September 2006, using selective cultivation and molecular assays. The three targeted pathogenic vibrios were generally below the detection level in January 2006, when the water was cold (13°C), and most abundant in September 2006, when the lake water was warmest (30°C). The maximum values for these species were higher than reported previously for the lake by severalfold to orders of magnitude. The only variable consistently correlated with total vibrio abundance within a single sampling was distance from shore (P= 0.000). Multiple linear regression of the entire data set revealed that distance from shore, temperature, and turbidity together explained 82.1% of the variability in total vibrio CFU. The log-transformed mean abundance ofV. vulnificusCFU in the lake was significantly correlated with temperature (P= 0.014), but not salinity (P= 0.625). Virulence-associated genes ofV. cholerae(ctx) andV. parahaemolyticus(trhandtdh) were not detected in any isolates of these species (n= 128 andn= 20, respectively). In contrast, 16S rRNA typing ofV. vulnificus(n= 298) revealed the presence of both environmental (type A) and clinical (type B) strains. The percentage of the B-typeV. vulnificuswas significantly higher in the lake in October 2005 (35.8% of the total) than at other sampling times (P≤ 0.004), consistent with the view that these strains represent distinct ecotypes.
Synthesis and Characterization of Chimeric Proteins Based on Cellulase and Xylanase from an Insect Gut Bacterium
Authors: Adlakha Nidhi , Rajagopal Raman , Kumar Saravanan , Reddy Vanga Siva , Yazdani Syed Shams
Published: June 2011

Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a
Paenibacillus
strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of
Paenibacillus
. Endo5A and Xyl11D were overexpressed in
Escherichia coli
, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.
Investigating Antibacterial Effects of Garlic (Allium sativum) Concentrate and Garlic-Derived Organosulfur Compounds on Campylobacter jejuni by Using Fourier Transform Infrared Spectroscopy, Raman Spectroscopy, and Electron Microscopy
Authors: Lu Xiaonan , Rasco Barbara A. , Jabal Jamie M. F. , Aston D. Eric , Lin Mengshi , Konkel Michael E.
Published: June 2011

Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy were used to study the cell injury and inactivation of
Campylobacter jejuni
from exposure to antioxidants from garlic.
C. jejuni
was treated with various concentrations of garlic concentrate and garlic-derived organosulfur compounds in growth media and saline at 4, 22, and 35°C. The antimicrobial activities of the diallyl sulfides increased with the number of sulfur atoms (diallyl sulfide < diallyl disulfide < diallyl trisulfide). FT-IR spectroscopy confirmed that organosulfur compounds are responsible for the substantial antimicrobial activity of garlic, much greater than those of garlic phenolic compounds, as indicated by changes in the spectral features of proteins, lipids, and polysaccharides in the bacterial cell membranes. Confocal Raman microscopy (532-nm-gold-particle substrate) and Raman mapping of a single bacterium confirmed the intracellular uptake of sulfur and phenolic components. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to verify cell damage. Principal-component analysis (PCA), discriminant function analysis (DFA), and soft independent modeling of class analogs (SIMCA) were performed, and results were cross validated to differentiate bacteria based upon the degree of cell injury. Partial least-squares regression (PLSR) was employed to quantify and predict actual numbers of healthy and injured bacterial cells remaining following treatment. PLSR-based loading plots were investigated to further verify the changes in the cell membrane of
C. jejuni
treated with organosulfur compounds. We demonstrated that bacterial injury and inactivation could be accurately investigated by complementary infrared and Raman spectroscopies using a chemical-based, “whole-organism fingerprint” with the aid of chemometrics and electron microscopy.
Temporal Variability of Coastal Planctomycetes Clades at Kabeltonne Station, North Sea
Authors: Pizzetti Ilaria , Fuchs Bernhard M. , Gerdts Gunnar , Wichels Antje , Wiltshire Karen H. , Amann Rudolf
Published: June 2011

Members of the bacterial phylum
Planctomycetes
are reported in marine water samples worldwide, but quantitative information is scarce. Here we investigated the phylogenetic diversity, abundance, and distribution of
Planctomycetes
in surface waters off the German North Sea island Helgoland during different seasons by 16S rRNA gene analysis and catalyzed reporter deposition fluorescence
in situ
hybridization (CARD-FISH). Generally
Planctomycetes
are more abundant in samples collected in summer and autumn than in samples collected in winter and spring. Statistical analysis revealed that
Planctomycetes
abundance was correlated to the
Centrales
diatom bloom in spring 2007. The analysis of size-fractionated seawater samples and of macroaggregates showed that ∼90% of the
Planctomycetes
reside in the >3-μm size fraction. Comparative sequence analysis of 184 almost full-length 16S rRNA genes revealed three dominant clades. The clades, named
Planctomyces
-related group A, uncultured
Planctomycetes
group B, and
Pirellula
-related group D, were monitored by CARD-FISH using newly developed oligonucleotide probes. All three clades showed recurrent abundance patterns during two annual sampling campaigns. Uncultured
Planctomycetes
group B was most abundant in autumn samples, while
Planctomyces
-related group A was present in high numbers only during late autumn and winter. The levels of
Pirellula
-related group D were more constant throughout the year, with elevated counts in summer. Our analyses suggest that the seasonal succession of the
Planctomycetes
is correlated with algal blooms. We hypothesize that the niche partitioning of the different clades might be caused by their algal substrates.
Differential Proteomic Analysis of Rickettsia prowazekii Propagated in Diverse Host Backgrounds
Authors: Tucker Aimee M. , Driskell Lonnie O. , Pannell Lewis K. , Wood David O.
Published: June 2011

The obligate intracellular growth of
Rickettsia prowazekii
places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of
R. prowazekii
grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.
A Pmk1-Interacting Gene Is Involved in Appressorium Differentiation and Plant Infection in
Magnaporthe oryzae
Authors: Zhang Haifeng , Xue Chaoyang , Kong Lingan , Li Guotian , Xu Jin-Rong
Published: June 2011

In the rice blast fungus
Magnaporthe oryzae
, the
PMK1
mitogen-activated protein (MAP) kinase gene regulates appressorium formation and infectious growth. Its homologs in many other fungi also play critical roles in fungal development and pathogenicity. However, the targets of this important MAP kinase and its interacting genes are not well characterized. In this study, we constructed two yeast two-hybrid libraries of
M. oryzae
and screened for Pmk1-interacting proteins. Among the nine Pmk1-interacting clones (PICs) identified, two of them,
PIC1
and
PIC5
, were selected for further characterization. Pic1 has one putative nuclear localization signal and one putative MAP kinase phosphorylation site. Pic5 contains one transmembrane domain and two functionally unknown CTNS (cystinosin/ERS1p repeat) motifs. The interaction of Pmk1 with Pic1 or Pic5 was confirmed by coimmunoprecipitation assays. Targeted gene deletion of
PIC1
had no apparent effects on vegetative growth and pathogenicity but resulted in a significant reduction in conidiation and abnormal germ tube differentiation on onion epidermal cells. Deletion of
PIC5
led to a reduction in conidiation and hyphal growth. Autolysis of aerial hyphae became visible in cultures older than 4 days. The
pic5
mutant was defective in germ tube growth and appressorium differentiation. It was reduced in appressorial penetration and virulence on the plant. Both
PIC1
and
PIC5
are conserved in filamentous ascomycetes, but none of their orthologs have been functionally characterized. Our data indicate that
PIC5
is a novel virulence factor involved in appressorium differentiation and pathogenesis in
M. oryzae
.
Clustering of Nuclei in Multinucleated Hyphae Is Prevented by Dynein-Driven Bidirectional Nuclear Movements and Microtubule Growth Control in Ashbya gossypii
Authors: Grava Sandrine , Keller Miyako , Voegeli Sylvia , Seger Shanon , Lang Claudia , Philippsen Peter
Published: June 2011

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with
Ashbya gossypii
revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain
DYN1
promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in
A. gossypii
and thus reveal a novel cellular role for dynein.
Rab11 Function in Trypanosoma brucei: Identification of Conserved and Novel Interaction Partners
Authors: Gabernet-Castello Carme , DuBois Kelly N. , Nimmo Camus , Field Mark C.
Published: June 2011

The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in
Trypanosoma brucei
, where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in
Homo sapiens
, interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.
Multiple Roles of Ypd1 Phosphotransfer Protein in Viability, Stress Response, and Virulence Factor Regulation in Cryptococcus neoformans
Authors: Lee Jang-Won , Ko Young-Joon , Kim Seo-Young , Bahn Yong-Sun
Published: June 2011

Ypd1 is a key phosphorelay protein that controls eukaryotic two-component systems, but its function in
Cryptococcus neoformans
is not known. Here, we report that Ypd1 is required for the viability of
C. neoformans
via the Hog1 mitogen-activated protein kinase (MAPK) pathway but plays multiple cellular roles in both a Hog1-dependent and -independent manner.
Helicobacter pyloriPossesses Four Coiled-Coil-Rich Proteins That Form Extended Filamentous Structures and Control Cell Shape and Motility
Authors: Specht Mara , Schätzle Sarah , Graumann Peter L. , Waidner Barbara
Published: June 2011

We identified two additional genes ofHelicobacter pyloriencoding Ccrp proteins. All four Ccrps have different multimerization and filamentation properties and different types of smallest subunits and do not copurify, suggesting a system of individual Ccrp filaments. Despite the presence of morphologically unaltered flagella, allccrpmutants displayed significantly reduced motility.
Identification of Amino Acid Residues Critical for Catalysis of Holliday Junction Resolution by Mycoplasma genitalium RecU
Authors: Sluijter Marcel , Aslam Mohammad , Hartwig Nico G. , van Rossum Annemarie M. C. , Vink Cornelis
Published: June 2011

The RecU protein from
Mycoplasma genitalium
, RecU

Mge

, is a 19.4-kDa Holliday junction (HJ) resolvase that binds in a nonspecific fashion to HJ substrates and, in the presence of Mn
2+
, cleaves these substrates at a specific sequence (5′-G/TC↓C/TTA/GG-3′). To identify amino acid residues that are crucial for HJ binding and/or cleavage, we generated a series of 16 deletion mutants (9 N- and 7 C-terminal deletion mutants) and 31 point mutants of RecU

Mge

. The point mutations were introduced at amino acid positions that are highly conserved among bacterial RecU-like sequences. All mutants were purified and tested for the ability to bind to, and cleave, HJ substrates. We found the five N-terminal and three C-terminal amino acid residues of RecU

Mge

to be dispensable for its catalytic activities. Among the 31 point mutants, 7 mutants were found to be inactive in both HJ binding and cleavage. Interestingly, in 12 other mutants, these two activities were uncoupled; while these proteins displayed HJ-binding characteristics similar to those of wild-type RecU

Mge

, they were unable to cleave HJ substrates. Thus, 12 amino acid residues were identified (E11, K31, D57, Y58, Y66, D68, E70, K72, T74, K76, Q88, and L92) that may play either a direct or indirect role in the catalysis of HJ resolution.
Multiplicity of 3-Ketosteroid-9α-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids
Authors: Petrusma Mirjan , Hessels Gerda , Dijkhuizen Lubbert , van der Geize Robert
Published: June 2011

The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of
kshA
, encoding the oxygenase component of 3-ketosteroid 9α-hydroxylase, a key enzyme in steroid catabolism. Five
kshA
homologues (
kshA1
to
kshA5
) were previously identified in
Rhodococcus rhodochrous
DSM43269. These KshA
DSM43269
homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in the catabolism of different classes of steroids, i.e., sterols, pregnanes, androstenes, and bile acids, was investigated. Enzyme activity assays showed that all KSH enzymes with KshA
DSM43269
homologues are C-9 α-hydroxylases acting on a wide range of 3-ketosteroids, but not on 3-hydroxysteroids. KshA5 appeared to be the most versatile enzyme, with the broadest substrate range but without a clear substrate preference. In contrast, KshA1 was found to be dedicated to cholic acid catabolism. Transcriptional analysis and functional complementation studies revealed that
kshA5
supported growth on any of the different classes of steroids tested, consistent with its broad expression induction pattern. The presence of multiple
kshA
genes in the
R. rhodochrous
DSM43269 genome, each displaying unique steroid induction patterns and substrate ranges, appears to facilitate a dynamic and fine-tuned steroid catabolism, with C-9 α-hydroxylation occurring at different levels during microbial steroid degradation.
Physiological Roles of the cyAbrB Transcriptional Regulator Pair Sll0822 and Sll0359 in Synechocystis sp. strain PCC 6803
Authors: Yamauchi Yuki , Kaniya Yuki , Kaneko Yasuko , Hihara Yukako
Published: June 2011

All known cyanobacterial genomes possess multiple copies of genes encoding AbrB-like transcriptional regulators, known as cyAbrBs, which are distinct from those conserved among other bacterial species. In this study, we addressed the physiological roles of Sll0822 and Sll0359, the two cyAbrBs in
Synechocystis
sp. strain PCC 6803, under nonstress conditions (20 μmol of photons m
−2
s
−1
in ambient CO
2
). When the sll0822 gene was disrupted, the expression levels of nitrogen-related genes such as
urtA
,
amt1
, and
glnB
significantly decreased compared with those in the wild-type cells. Possibly due to the increase of the cellular carbon/nitrogen ratio in the sll0822-disrupted cells, a decrease in pigment contents, downregulation of carbon-uptake related genes, and aberrant accumulation of glycogen took place. Moreover, the mutant exhibited the decrease in the expression level of cytokinesis-related genes such as
ftsZ
and
ftsQ
, resulting in the defect in cell division and significant increase in cell size. The pleiotrophic phenotype of the mutant was efficiently suppressed by the introduction of Sll0822 and also partially suppressed by the introduction of Sll0359. When His-tagged cyAbrBs were purified from overexpression strains, Sll0359 and Sll0822 were copurified with each other. The cyAbrBs in
Synechocystis
sp. strain PCC 6803 seem to interact with each other and regulate carbon and nitrogen metabolism as well as the cell division process under nonstress conditions.
Novel Insights into the Regulation of LexA in the Cyanobacterium Synechocystis sp. Strain PCC 6803
Authors: Oliveira Paulo , Lindblad Peter
Published: June 2011

The transcription factor LexA in the cyanobacterium
Synechocystis
sp. strain PCC 6803 has been shown to regulate genes that are not directly involved in DNA repair but instead in several different metabolic pathways. However, the signal transduction pathways remain largely uncharacterized. The present work gives novel insights into the regulation of LexA in this unicellular cyanobacterium. A combination of Northern and Western blotting, using specific antibodies against the cyanobacterial LexA, was employed to show that this transcription regulator is under posttranscriptional control, in addition to the classical and already-described transcriptional regulation. Moreover, detailed two-dimensional (2D) electrophoresis analyses of the protein revealed that LexA undergoes posttranslational modifications. Finally, a fully segregated LexA::GFP (green fluorescent protein) fusion-modified strain was produced to image LexA's spatial distribution in live cells. The fusion protein retains DNA binding capabilities, and the GFP fluorescence indicates that LexA is localized in the innermost region of the cytoplasm, decorating the DNA in an evenly distributed pattern. The implications of these findings for the overall role of LexA in
Synechocystis
sp. strain PCC 6803 are further discussed.