bluuebunny/crossref_metadata_2025 huggingface dataset:
Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei
Authors: Qi Feifei , Zhang Weixin , Zhang Fengjie , Chen Guanjun , Liu Weifeng
Published: April 2014

N-linked glycosylation modulates and diversifies the structures and functions of the eukaryotic proteome through both intrinsic and extrinsic effects on proteins. We investigated the significance of the three N-linked glycans on the catalytic domain of cellobiohydrolase I (CBH1) from the filamentous fungus
Trichoderma reesei
in its secretion and activity. While the removal of one or two N-glycosylation sites hardly affected the extracellular secretion of CBH1, eliminating all of the glycosylation sites did induce expression of the unfolded protein response (UPR) target genes, and secretion of this CBH1 variant was severely compromised in a calnexin gene deletion strain. Further characterization of the purified CBH1 variants showed that, compared to Asn270, the thermal reactivity of CBH1 was significantly decreased by removal of either Asn45 or Asn384 glycosylation site during the catalyzed hydrolysis of soluble substrate. Combinatorial loss of these two N-linked glycans further exacerbated the temperature-dependent inactivation. In contrast, this thermal labile property was less severe when hydrolyzing insoluble cellulose. Analysis of the structural integrity of CBH1 variants revealed that removal of N-glycosylation at Asn384 had a more pronounced effect on the integrity of regular secondary structure compared to the loss of Asn45 or Asn270. These data implicate differential roles of N-glycosylation modifications in contributing to the stability of specific functional regions of CBH1 and highlight the potential of improving the thermostability of CBH1 by tuning proper interactions between glycans and functional residues.
HrcT Is a Key Component of the Type III Secretion System in Xanthomonas spp. and Also Regulates the Expression of the Key
hrp
Transcriptional Activator HrpX
Authors: Liu Zhi-Yang , Zou Li-Fang , Xue Xiao-Bo , Cai Lu-Lu , Ma Wen-Xiu , Xiong Li , Ji Zhi-Yuan , Chen Gong-You
Published: April 2014

The type III secretion system (T3SS), encoded by
hrp
(hypersensitive response and pathogenicity) genes in Gram-negative phytopathogenic bacteria, delivers repertoires of T3SS effectors (T3SEs) into plant cells to trigger the hypersensitive response (HR) in nonhost or resistant-host plants and promote pathogenicity in susceptible plants. The expression of
hrp
genes in
Xanthomonas
is regulated by two key regulatory proteins, HrpG and HrpX. However, the interactions between
hrp
gene products in directing T3SE secretion are largely unknown. Here we demonstrated that HrcT of
X. oryzae
pv. oryzicola functions as a T3SS component and positively regulates the expression of
hrpX
. Transcription of
hrcT
occurs via two distinct promoters; one (T1) is with the
hrpB
operon and the second (T3) within
hrpB7
Via either promoter T1 or T3, the defect in Hrp phenotype by
hrcT
deletion was corrected in the presence of
hrcT
only from
Xanthomonas
species but not from other phytopathogenic bacteria. An N-terminally truncated HrcT was able to bind the
hrpX
promoter and activate the expression of
hrpX
, supporting that HrcT is a positive regulator of
hrpX
. A revised model showing the regulatory interactions between HrcT, HrpX, and HrpG is proposed.
High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage
Authors: Yang Junjie , Sun Bingbing , Huang He , Jiang Yu , Diao Liuyang , Chen Biao , Xu Chongmao , Wang Xin , Liu Jinle , Jiang Weihong , Yang Sheng
Published: April 2014

Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the
Escherichia coli
chromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used for
cadA
knockout,
gdhA
knock-in, seamless deletion of
pepD
, site-directed mutagenesis of the essential
metK
gene, and replacement of
metK
with the
Rickettsia
S
-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.
Genomic Features of a Bumble Bee Symbiont Reflect Its Host Environment
Authors: Martinson Vincent G. , Magoc Tanja , Koch Hauke , Salzberg Steven L. , Moran Nancy A.
Published: April 2014

Here, we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name “
Candidatus
Schmidhempelia bombi,” that was inadvertently sequenced alongside the genome of its host, the bumble bee,
Bombus impatiens
. This symbiont is a member of the recently described bacterial order
Orbales
, which has been collected from the guts of diverse insect species; however, “
Ca
. Schmidhempelia” has been identified exclusively with bumble bees. Metabolic reconstruction reveals that “
Ca
. Schmidhempelia” lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochrome
o
, and most genes in the tricarboxylic acid (TCA) cycle. “
Ca
. Schmidhempelia” has retained NADH dehydrogenase II, the low-oxygen specific cytochrome
bd
, anaerobic nitrate respiration, mixed-acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type 6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratory
B. impatiens
revealed that “
Ca
. Schmidhempelia” is present in 90% of individuals and, therefore, may provide benefits to its host.
Distributions of Salmonella Subtypes Differ between Two U.S. Produce-Growing Regions
Authors: Strawn Laura K. , Danyluk Michelle D. , Worobo Randy W. , Wiedmann Martin
Published: April 2014

Salmonella
accounts for approximately 50% of produce-associated outbreaks in the United States, several of which have been traced back to contamination in the produce production environment. To quantify
Salmonella
diversity and aid in identification of
Salmonella
contamination sources, we characterized
Salmonella
isolates from two geographically diverse produce-growing regions in the United States. Initially, we characterized the
Salmonella
serotype and subtype diversity associated with 1,677 samples collected from 33 produce farms in New York State (NYS). Among these 1,677 samples, 74 were
Salmonella
positive, yielding 80 unique isolates (from 147 total isolates), which represented 14 serovars and 23 different pulsed-field gel electrophoresis (PFGE) types. To explore regional
Salmonella
diversity associated with production environments, we collected a smaller set of samples (
n
= 65) from South Florida (SFL) production environments and compared the
Salmonella
diversity associated with these samples with the diversity found among NYS production environments. Among these 65 samples, 23 were
Salmonella
positive, yielding 32 unique isolates (from 81 total isolates), which represented 11 serovars and 17 different PFGE types. The most common serovars isolated in NYS were
Salmonella enterica
serovars Newport, Cerro, and Thompson, while common serovars isolated in SFL were
Salmonella
serovars Saphra and Newport and
S. enterica
subsp.
diarizonae
serovar 50:r:z. High PFGE type diversity (Simpson's diversity index, 0.90 ± 0.02) was observed among
Salmonella
isolates across both regions; only three PFGE types were shared between the two regions. The probability of three or fewer shared PFGE types was <0.000001; therefore,
Salmonella
isolates were considerably different between the two sampled regions. These findings suggest the potential for PFGE-based source tracking of
Salmonella
in production environments.
Stress Response and Adaptation of Listeria monocytogenes 08-5923 Exposed to a Sublethal Dose of Carnocyclin A
Authors: Liu Xiaoji , Basu Urmila , Miller Petr , McMullen Lynn M.
Published: April 2014

Carnocyclin A (CCLA) is an antimicrobial peptide produced by
Carnobacterium maltaromaticum
ATCC PTA-5313, which can be used to control the growth of
Listeria monocytogenes
in ready-to-eat meat products. The aim of this research was to elucidate the cellular responses of
L. monocytogenes
08-5923 exposed to a sublethal dose of CCLA. Microarray, quantitative reverse transcription-PCR, tandem mass spectrometry, and electron microscopy were used to investigate the alteration in gene expression, protein production, and morphological changes in cells of
Listeria
following treatment with CCLA. The genes involved in metabolism (
baiE
,
trn
, and
pykA
), cell wall synthesis (
murZ
and
dacB2
), and cell division (
clpE
and
divIVA
) were upregulated following a 15-min exposure to CCLA as a result of stress responses. Genes involved in cell division, cell wall synthesis, flagellar synthesis, and metabolism were downregulated after 4 h as a result of adaptation. Analysis of total soluble proteins confirmed the downregulation of
pykA
and
gnd
after 4 h of exposure to CCLA. The absence of flagella was observed in
L. monocytogenes
following 30 h of exposure to CCLA. A sublethal dose of CCLA induced adaptation in
L. monocytogenes
08-5923 by inhibition of expression of genes and proteins critical for synthesis of cell wall structures and maintaining metabolic functions. Both the mannose- and cellobiose-specific phosphotransferase systems could be targets for CCLA.
Biotic Interactions and Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the Upper Mississippi River
Authors: Korajkic Asja , McMinn Brian R. , Shanks Orin C. , Sivaganesan Mano , Fout G. Shay , Ashbolt Nicholas J.
Published: April 2014

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (
Escherichia coli
and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An
in situ
mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (
r
2
, 0.96 to 0.98;
P
< 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.
Promotion of Endodontic Lesions in Rats by a Novel Extraradicular Biofilm Model Using Obturation Materials
Authors: Kuremoto Katsutaka , Noiri Yuichiro , Ishimoto Takuya , Yoneda Naomichi , Yamamoto Reiko , Maezono Hazuki , Nakano Takayoshi , Hayashi Mikako , Ebisu Shigeyuki
Published: April 2014

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an
in vivo
extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (
P
< 0.05 by Welch's
t
test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.
From Metagenomics to Pure Culture: Isolation and Characterization of the Moderately Halophilic Bacterium Spiribacter salinus gen. nov., sp. nov
Authors: León María José , Fernández Ana B. , Ghai Rohit , Sánchez-Porro Cristina , Rodriguez-Valera Francisco , Ventosa Antonio
Published: April 2014

Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both
Euryarchaeota
and halophilic bacteria, with a gammaproteobacterium closely related to the genera
Alkalilimnicola
and
Arhodomonas
being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40
T
, isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40
T
has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera
Alkalilimnicola
(94.9 to 94.5%),
Alkalispirillum
(94.3%), and
Arhodomonas
(93.9%) within the family
Ectothiorhodospiraceae
. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name
Spiribacter salinus
gen. nov., sp. nov., is proposed, with strain M19-40
T
(= CECT 8282
T
= IBRC-M 10768
T
= LMG 27464
T
) being the type strain.
Posttranscriptional Regulation of 2,4-Diacetylphloroglucinol Production by GidA and TrmE in Pseudomonas fluorescens 2P24
Authors: Zhang Wei , Zhao Zhao , Zhang Bo , Wu Xiao-Gang , Ren Zheng-Guang , Zhang Li-Qun
Published: April 2014

Pseudomonas fluorescens
2P24 is a soilborne bacterium that synthesizes and excretes multiple antimicrobial metabolites. The polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG), synthesized by the
phlACBD
locus, is its major biocontrol determinant. This study investigated two mutants defective in antagonistic activity against
Rhizoctonia solani
. Deletion of the
gidA
(PM701) or
trmE
(PM702) gene from strain 2P24 completely inhibited the production of 2,4-DAPG and its precursors, monoacetylphloroglucinol (MAPG) and phloroglucinol (PG). The transcription of the
phlA
gene was not affected, but the translation of the
phlA
and
phlD
genes was reduced significantly. Two components of the Gac/Rsm pathway, RsmA and RsmE, were found to be regulated by
gidA
and
trmE
, whereas the other components, RsmX, RsmY, and RsmZ, were not. The regulation of 2,4-DAPG production by
gidA
and
trmE
, however, was independent of the Gac/Rsm pathway. Both the
gidA
and
trmE
mutants were unable to produce PG but could convert PG to MAPG and MAPG to 2,4-DAPG. Overexpression of PhlD in the
gidA
and
trmE
mutants could restore the production of PG and 2,4-DAPG. Taken together, these findings suggest that GidA and TrmE are positive regulatory elements that influence the biosynthesis of 2,4-DAPG posttranscriptionally.
Insights into the Microbial Degradation of Rubber and Gutta-Percha by Analysis of the Complete Genome of Nocardia nova SH22a
Authors: Luo Quan , Hiessl Sebastian , Poehlein Anja , Daniel Rolf , Steinbüchel Alexander
Published: April 2014

The complete genome sequence ofNocardia novaSH22a was determined in light of the remarkable ability of rubber and gutta-percha (GP) degradation of this strain. The genome consists of a circular chromosome of 8,348,532 bp with a G+C content of 67.77% and 7,583 predicted protein-encoding genes. Functions were assigned to 72.45% of the coding sequences. Among them, a large number of genes probably involved in the metabolism of xenobiotics and hardly degradable compounds, as well as genes that participate in the synthesis of polyketide- and/or nonribosomal peptide-type secondary metabolites, were detected. Based onin silicoanalyses and experimental studies, such as transposon mutagenesis and directed gene deletion studies, the pathways of rubber and GP degradation were proposed and the relationship between both pathways was unraveled. The genes involved include,inter alia, genes participating in cell envelope synthesis (long-chain-fatty-acid–AMP ligase and arabinofuranosyltransferase), β-oxidation (α-methylacyl-coenzyme A [α-methylacyl-CoA] racemase), propionate catabolism (acyl-CoA carboxylase), gluconeogenesis (phosphoenolpyruvate carboxykinase), and transmembrane substrate uptake (Mce [mammalian cell entry] transporter). This study not only improves our insights into the mechanism of microbial degradation of rubber and GP but also expands our knowledge of the genusNocardiaregarding metabolic diversity.
Use of Silicate Minerals for pH Control during Reductive Dechlorination of Chloroethenes in Batch Cultures of Different Microbial Consortia
Authors: Lacroix Elsa , Brovelli Alessandro , Barry D. A. , Holliger Christof
Published: April 2014

In chloroethene-contaminated sites undergoing
in situ
bioremediation, groundwater acidification is a frequent problem in the source zone, and buffering strategies have to be implemented to maintain the pH in the neutral range. An alternative to conventional soluble buffers is silicate mineral particles as a long-term source of alkalinity. In previous studies, the buffering potentials of these minerals have been evaluated based on abiotic dissolution tests and geochemical modeling. In the present study, the buffering potentials of four silicate minerals (andradite, diopside, fayalite, and forsterite) were tested in batch cultures amended with tetrachloroethene (PCE) and inoculated with different organohalide-respiring consortia. Another objective of this study was to determine the influence of pH on the different steps of PCE dechlorination. The consortia showed significant differences in sensitivities toward acidic pH for the different dechlorination steps. Molecular analysis indicated that
Dehalococcoides
spp. that were present in all consortia were the most pH-sensitive organohalide-respiring guild members compared to
Sulfurospirillum
spp. and
Dehalobacter
spp. In batch cultures with silicate mineral particles as pH-buffering agents, all four minerals tested were able to maintain the pH in the appropriate range for reductive dechlorination of chloroethenes. However, complete dechlorination to ethene was observed only with forsterite, diopside, and fayalite. Dissolution of andradite increased the redox potential and did not allow dechlorination. With forsterite, diopside, and fayalite, dechlorination to ethene was observed but at much lower rates for the last two dechlorination steps than with the positive control. This indicated an inhibition effect of silicate minerals and/or their dissolution products on reductive dechlorination of
cis
-dichloroethene and vinyl chloride. Hence, despite the proven pH-buffering potential of silicate minerals, compatibility with the bacterial community involved in
in situ
bioremediation has to be carefully evaluated prior to their use for pH control at a specific site.
Microscopy, Culture, and Quantitative Real-Time PCR Examination Confirm Internalization of Mycobacteria in Plants
Authors: Kaevska M. , Lvoncik S. , Slana I. , Kulich P. , Kralik P.
Published: April 2014

The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown
in vitro
and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of
Mycobacterium avium
(10
8
to 10
10
cells/g of soil) and feces from animals with paratuberculosis. We detected
M. avium
subsp.
avium
,
hominissuis
, and
paratuberculosis
in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10
4
cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.
Diversity and Transport of Microorganisms in Intertidal Sands of the California Coast
Authors: Boehm Alexandria B. , Yamahara Kevan M. , Sassoubre Lauren M.
Published: April 2014

Forced by tides and waves, large volumes of seawater are flushed through the beach daily. Organic material and nutrients in seawater are remineralized and cycled as they pass through the beach. Microorganisms are responsible for most of the biogeochemical cycling in the beach; however, few studies have characterized their diversity in intertidal sands, and little work has characterized the extent to which microbes are transported between different compartments of the beach. The present study uses next-generation massively parallel sequencing to characterize the microbial community present at 49 beaches along the coast of California. In addition, we characterize the transport of microorganisms within intertidal sands using laboratory column experiments. We identified extensive diversity in the beach sands. Nearly 1,000 unique taxa were identified in sands from 10 or more unique beaches, suggesting the existence of a group of “cosmopolitan” sand microorganisms. A biogeographical analysis identified a taxon-distance relationship among the beaches. In addition, sands with similar grain size, organic carbon content, exposed to a similar wave climate, and having the same degree of anthropogenic influence tended to have similar microbial communities. Column experiments identified microbes readily mobilized by seawater infiltrating through unsaturated intertidal sands. The ease with which microbes were mobilized suggests that intertidal sands may represent a reservoir of bacteria that seed the beach aquifer where they may partake in biogeochemical cycling.
High Genetic Diversity among Strains of the Unindustrialized Lactic Acid Bacterium Carnobacterium maltaromaticum in Dairy Products as Revealed by Multilocus Sequence Typing
Authors: Rahman Abdur , Cailliez-Grimal Catherine , Bontemps Cyril , Payot Sophie , Chaillou Stéphane , Revol-Junelles Anne-Marie , Borges Frédéric
Published: April 2014

Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter.
Carnobacterium maltaromaticum
is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes
C. maltaromaticum
an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes
dapE
,
ddlA
,
glpQ
,
ilvE
,
pyc
,
pyrE
, and
leuS
was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products.
Differing Populations of Endemic Bacteriophages in Cattle Shedding High and Low Numbers of Escherichia coli O157:H7 Bacteria in Feces
Authors: Hallewell J. , Niu Y. D. , Munns K. , McAllister T. A. , Johnson R. P. , Ackermann H.-W. , Thomas J. E. , Stanford K.
Published: April 2014

The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to
Escherichia coli
O157:H7 from cattle shedding high and low numbers of naturally occurring
E. coli
O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ≥10
4
CFU · g
−1
of
E. coli
O157:H7 in feces at a single time point were identified as supershedders (SS;
n
= 6), and cattle excreting <10
4
CFU · g
−1
of feces were identified as low shedders (LS;
n
= 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks.
E. coli
O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of
E. coli
O157:H7 isolated from feces was 14.4% and did not differ between LS and SS (
P
= 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%;
P
= 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of
Myoviridae
and T1-like phage of
Siphoviridae
were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting
E. coli
O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of
E. coli
O157:H7 in cattle.
Reconstruction of
mreB
Expression in Staphylococcus aureus via a Collection of New Integrative Plasmids
Authors: Yepes Ana , Koch Gudrun , Waldvogel Andrea , Garcia-Betancur Juan-Carlos , Lopez Daniel
Published: April 2014

Protein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial models
Escherichia coli
and
Bacillus subtilis
have been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacterium
Staphylococcus aureus
. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of the
S. aureus
chromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression of
mreB
in
S. aureus
, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that in
S. aureus
, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the use
S. aureus
as a model system in exploring diverse aspects of cellular microbiology.
In Situ
Evaluation of Paenibacillus alvei in Reducing Carriage of Salmonella enterica Serovar Newport on Whole Tomato Plants
Authors: Allard Sarah , Enurah Alexander , Strain Errol , Millner Patricia , Rideout Steven L. , Brown Eric W. , Zheng Jie
Published: April 2014

Recently, tomatoes have been implicated as a primary vehicle in food-borne outbreaks of
Salmonella enterica
serovar Newport and other
Salmonella
serovars. Long-term intervention measures to reduce
Salmonella
prevalence on tomatoes remain elusive for growing and postharvest environments. A naturally occurring bacterium identified by 16S rRNA gene sequencing as
Paenibacillus alvei
was isolated epiphytically from plants native to the Virginia Eastern Shore tomato-growing region. After initial antimicrobial activity screening against
Salmonella
and 10 other bacterial pathogens associated with the human food supply, strain TS-15 was further used to challenge an attenuated strain of
S
. Newport on inoculated fruits, leaves, and blossoms of tomato plants in an insect-screened high tunnel with a split-plot design. Survival of
Salmonella
after inoculation was measured for groups with and those without the antagonist at days 0, 1, 2, and 3 and either day 5 for blossoms or day 6 for fruits and leaves. Strain TS-15 exhibited broad-range antimicrobial activity against both major food-borne pathogens and major bacterial phytopathogens of tomato. After
P. alvei
strain TS-15 was applied onto the fruits, leaves, and blossoms of tomato plants, the concentration of
S
. Newport declined significantly (
P
≤ 0.05) compared with controls. Astonishingly, >90% of the plants had no detectable levels of
Salmonella
by day 5 for blossoms. The naturally occurring antagonist strain TS-15 is highly effective in reducing the carriage of
Salmonella
Newport on whole tomato plants. The application of
P. alvei
strain TS-15 is a promising approach for reducing the risk of
Salmonella
contamination during tomato production.
Triggering Respirofermentative Metabolism in the Crabtree-Negative Yeast Pichia guilliermondii by Disrupting the
CAT8
Gene
Authors: Qi Kai , Zhong Jian-Jiang , Xia Xiao-Xia
Published: April 2014

Pichia guilliermondii
is a Crabtree-negative yeast that does not normally exhibit respirofermentative metabolism under aerobic conditions, and methods to trigger this metabolism may have applications for physiological study and industrial applications. In the present study,
CAT8
, which encodes a putative global transcriptional activator, was disrupted in
P. guilliermondii
. This yeast's ethanol titer increased by >20-fold compared to the wild type (WT) during aerobic fermentation using glucose. A comparative transcriptional analysis indicated that the expression of genes in the tricarboxylic acid cycle and respiratory chain was repressed in the
CAT8
-disrupted (Δ
CAT8
) strain, while the fermentative pathway genes were significantly upregulated. The respiratory activities in the Δ
CAT8
strain, indicated by the specific oxygen uptake rate and respiratory state value, decreased to one-half and one-third of the WT values, respectively. In addition, the expression of
HAP4
, a transcriptional respiratory activator, was significantly repressed in the Δ
CAT8
strain. Through disruption of
HAP4
, the ethanol production of
P. guilliermondii
was also increased, but the yield and titer were lower than that in the Δ
CAT8
strain. A further transcriptional comparison between Δ
CAT8
and Δ
HAP4
strains suggested a more comprehensive reprogramming function of
Cat8
in the central metabolic pathways. These results indicated the important role of
CAT8
in regulating the glucose metabolism of
P. guilliermondii
and that the regulation was partially mediated by repressing
HAP4
. The strategy proposed here might be applicable to improve the aerobic fermentation capacity of other Crabtree-negative yeasts.
Cryptococcus neoformans Dual GDP-Mannose Transporters and Their Role in Biology and Virulence
Authors: Wang Zhuo A. , Griffith Cara L. , Skowyra Michael L. , Salinas Nichole , Williams Matthew , Maier Ezekiel J. , Gish Stacey R. , Liu Hong , Brent Michael R. , Doering Tamara L.
Published: April 2014

Cryptococcus neoformans
is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in
Saccharomyces cerevisiae
showed that both are functional, with similar kinetics and substrate specificities
in vitro
. We have now examined these proteins
in vivo
and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore,
gmt1 gmt2
double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.
Trypanosoma cruzi Bromodomain Factor 3 Binds Acetylated α-Tubulin and Concentrates in the Flagellum during Metacyclogenesis
Authors: Alonso Victoria Lucia , Villanova Gabriela Vanina , Ritagliati Carla , Machado Motta María Cristina , Cribb Pamela , Serra Esteban Carlos
Published: April 2014

Bromodomains are highly conserved acetyl-lysine binding domains found mainly in proteins associated with chromatin and nuclear acetyltransferases. The
Trypanosoma cruzi
genome encodes at least four bromodomain factors (
Tc
BDFs). We describe here bromodomain factor 3 (
Tc
BDF3), a bromodomain-containing protein localized in the cytoplasm.
Tc
BDF3 cytolocalization was determined, using purified antibodies, by Western blot and immunofluorescence analyses in all life cycle stages of
T. cruzi
. In epimastigotes and amastigotes, it was detected in the cytoplasm, the flagellum, and the flagellar pocket, and in trypomastigotes only in the flagellum. Subcellular localization of
Tc
BDF3 was also determined by digitonin extraction, ultrastructural immunocytochemistry, and expression of
Tc
BDF3 fused to cyan fluorescent protein (CFP). Tubulin can acquire different posttranslational modifications, which modulate microtubule functions. Acetylated α-tubulin has been found in the axonemes of flagella and cilia, as well as in the subpellicular microtubules of trypanosomatids.
Tc
BDF3 and acetylated α-tubulin partially colocalized in isolated cytoskeletons and flagella from
T. cruzi
epimastigotes and trypomastigotes. Interaction between the two proteins was confirmed by coimmunoprecipitation and far-Western blot assays with synthetic acetylated α-tubulin peptides and recombinant
Tc
BDF3.
Construction and Characterization of a Borrelia burgdorferi Strain with Conditional Expression of the Essential Telomere Resolvase, ResT
Authors: Bandy Nicholas J. , Salman-Dilgimen Aydan , Chaconas George
Published: April 2014

Borrelia
species are unique in the bacterial world in possessing segmented genomes which sometimes contain over 20 genetic elements. Most elements are linear and contain covalently closed hairpin ends requiring a specialized process, telomere resolution, for their generation. Hairpin telomere resolution is mediated by the telomere resolvase, ResT. Although the process has been studied extensively
in vitro
, the essential nature of the
resT
gene has precluded biological studies to further probe the role of ResT. In this work, we have generated a
B. burgdorferi
strain that carries an isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible
resT
gene controlled by a tightly regulated promoter. ResT is expressed in this strain at ∼14,000 monomers per cell, similar to the ∼15,000 monomers observed for the parental strain. We demonstrate ResT depletion with a half-life of 16 h upon IPTG washout. ResT depletion resulted in arrested growth 48 h after washout. Interestingly, not all spirochetes died after ResT washout, and at least 15% remained quiescent and could be resuscitated even at 2 weeks postwashout. Significant levels of DNA synthesis were not observed upon growth arrest, suggesting that ResT might interact directly or indirectly with factors controlling the initiation or elongation of DNA synthesis. Analysis of the linear plasmids lp17 and lp28-2 showed that the linear forms of these plasmids began to disappear and be replaced by higher-molecular-weight forms by 24 h post-IPTG washout. Treatment of DNA from the ResT-depleted strain with ResT
in vitro
revealed the presence of replicated telomeres expected in replication intermediates.
Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery
Authors: Liao Sumei , Klein Marlise I. , Heim Kyle P. , Fan Yuwei , Bitoun Jacob P. , Ahn San-Joon , Burne Robert A. , Koo Hyun , Brady L. Jeannine , Wen Zezhang T.
Published: April 2014

Streptococcus mutans
, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in
S. mutans
biofilms. This study demonstrates that
S. mutans
produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an
in vitro
adherence assay, added chromosomal DNA alone had a limited effect on
S. mutans
adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.
Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1
Authors: Xing Xuanxuan , Zhang Likui , Guo Li , She Qunxin , Huang Li
Published: April 2014

Replication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from the hyperthermophilic archaea of the genus
Sulfolobus
physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3′-5′ exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that
Sulfolobus
RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication.
Txc, a New Type II Secretion System of Pseudomonas aeruginosa Strain PA7, Is Regulated by the TtsS/TtsR Two-Component System and Directs Specific Secretion of the CbpE Chitin-Binding Protein
Authors: Cadoret Frédéric , Ball Geneviève , Douzi Badreddine , Voulhoux Romé
Published: April 2014

We present here the functional characterization of a third complete type II secretion system (T2SS) found in newly sequenced
Pseudomonas aeruginosa
strain PA7. We call this system Txc (third Xcp homolog). This system is encoded by the RGP69 region of genome plasticity found uniquely in strain PA7. In addition to the 11
txc
genes, RGP69 contains two additional genes encoding a possible T2SS substrate and a predicted unorthodox sensor protein, TtsS (type II secretion sensor). We also identified a gene encoding a two-component response regulator called TtsR (type II secretion regulator), which is located upstream of the
ttsS
gene and just outside RGP69. We show that TtsS and TtsR constitute a new and functional two-component system that controls the production and secretion of the RGP69-encoded T2SS substrate in a Txc-dependent manner. Finally, we demonstrate that this Txc-secreted substrate binds chitin, and we therefore name it CbpE (chitin-binding protein E).
Rod-to-Hook Transition for Extracellular Flagellum Assembly Is Catalyzed by the L-Ring-Dependent Rod Scaffold Removal
Authors: Cohen Eli J. , Hughes Kelly T.
Published: April 2014

In
Salmonella
, the rod substructure of the flagellum is a periplasmic driveshaft that couples the torque generated by the basal body motor to the extracellular hook and filament. The rod subunits self-assemble, spanning the periplasmic space and stopping at the outer membrane when a mature length of ∼22 nm is reached. Assembly of the extracellular hook and filament follow rod completion. Hook initiation requires that a pore forms in the outer membrane and that the rod-capping protein, FlgJ, dislodges from the tip of the distal rod and is replaced with the hook-capping protein, FlgD. Approximately 26 FlgH subunits form the L-ring around the distal rod that creates the pore through which the growing flagellum will elongate from the cell body. The function of the L-ring in the mature flagellum is also thought to act as a bushing for the rotating rod. Work presented here demonstrates that, in addition to outer membrane pore formation, L-ring formation catalyzes the removal of the FlgJ rod cap. Rod cap removal allows the hook cap to assemble at the rod tip and results in the transition from rod completion in the periplasm to extracellular hook polymerization. By coupling the rod-to-hook switch to outer membrane penetration, FlgH ensures that hook and filament polymerization is initiated at the appropriate spatial and temporal point in flagellar biosynthesis.
Hypermotility in Clostridium perfringens Strain SM101 Is Due to Spontaneous Mutations in Genes Linked to Cell Division
Authors: Liu Hualan , McCord Kristin D. , Howarth Jonathon , Popham David L. , Jensen Roderick V. , Melville Stephen B.
Published: April 2014

Clostridium perfringens
is a Gram-positive anaerobic pathogen of humans and animals. Although they lack flagella,
C. perfringens
bacteria can still migrate across surfaces using a type of gliding motility that involves the formation of filaments of bacteria lined up in an end-to-end conformation. In strain SM101, hypermotile variants are often found arising from the edges of colonies on agar plates. Hypermotile cells are longer than wild-type cells, and video microscopy of their gliding motility suggests that they form long, thin filaments that move rapidly away from a colony, analogously to swarmer cells in bacteria with flagella. To identify the cause(s) of the hypermotility phenotype, the genome sequences of normal strains and their direct hypermotile derivatives were determined and compared. Strains SM124 and SM127, hypermotile derivatives of strains SM101 and SM102, respectively, contained 10 and 6 single nucleotide polymorphisms (SNPs) relative to their parent strains. While SNPs were located in different genes in the two sets of strains, one feature in common was mutations in cell division genes, an
ftsI
homolog in strain SM124 (
CPR
_
1831
) and a
minE
homolog in strain SM127 (
CPR
_
2104
). Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model explaining the principles underlying the hypermotility phenotype is presented.