bluuebunny/crossref_metadata_2025 huggingface dataset:
Conjugative Plasmid Transfer and Adhesion Dynamics in an
Escherichia coli
Biofilm
Authors: Ong Cheryl-Lynn Y. , Beatson Scott A. , McEwan Alastair G. , Schembri Mark A.
Published: August 2009

A conjugative plasmid from the catheter-associated urinary tract infection strain
Escherichia coli
MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in
E. coli
, played an important role in biofilm formation. Biofilm formation by
E. coli
MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the
Enterobacteriaceae
that cause device-related infections in nosocomial settings.
Proteogenomic Monitoring of
Geobacter
Physiology during Stimulated Uranium Bioremediation
Authors: Wilkins Michael J. , VerBerkmoes Nathan C. , Williams Kenneth H. , Callister Stephen J. , Mouser Paula J. , Elifantz Hila , N′Guessan A. Lucie , Thomas Brian C. , Nicora Carrie D. , Shah Manesh B. , Abraham Paul , Lipton Mary S. , Lovley Derek R. , Hettich Robert L. , Long Philip E. , Banfield Jillian F.
Published: August 2009

Implementation of uranium bioremediation requires methods for monitoring the membership and activities of the subsurface microbial communities that are responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a proteomics-based approach for simultaneously documenting the strain membership and microbial physiology of the dominant
Geobacter
community members during in situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three planktonic
Geobacter
-dominated samples were obtained from two wells down-gradient of acetate addition. Over 2,500 proteins from each of these samples were identified by matching liquid chromatography-tandem mass spectrometry spectra to peptides predicted from seven isolate
Geobacter
genomes. Genome-specific peptides indicate early proliferation of multiple M21 and
Geobacter bemidjiensis
-like strains and later possible emergence of M21 and
G. bemidjiensis
-like strains more closely related to
Geobacter lovleyi
. Throughout biostimulation, the proteome is dominated by enzymes that convert acetate to acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides matching tricarboxylic acid cycle proteins and ATP synthase subunits were also detected, indicating the importance of energy generation during the period of rapid growth following the start of biostimulation. Evolving
Geobacter
strain composition may be linked to changes in protein abundance over the course of biostimulation and may reflect changes in metabolic functioning. Thus, metagenomics-independent community proteogenomics can be used to diagnose the status of the subsurface consortia upon which remediation biotechnology relies.
Temperature-Dependent Expression of
phzM
and Its Regulatory Genes
lasI
and
ptsP
in Rhizosphere Isolate
Pseudomonas
sp. Strain M18
Authors: Huang Jiaofang , Xu Yuquan , Zhang Hongyan , Li Yaqian , Huang Xianqing , Ren Bin , Zhang Xuehong
Published: August 2009

Pseudomonas
sp. strain M18, an effective biological control agent isolated from the melon rhizosphere, has a genetic background similar to that of the opportunistic human pathogen
Pseudomonas aeruginosa
PAO1. However, the predominant phenazine produced by strain M18 is phenazine-1-carboxylic acid (PCA) rather than pyocyanin (PYO); the quantitative ratio of PCA to PYO is 105 to 1 at 28°C in strain M18, while the ratio is 1 to 2 at 37°C in strain PAO1. We first provided evidence that the differential production of the two phenazines in strains M18 and PAO1 is related to the temperature-dependent and strain-specific expression patterns of
phzM
, a gene involved in the conversion of PCA to PYO. Transcriptional levels of
phzM
were measured by quantitative real-time PCR, and the activities of both transcriptional and translational
phzM
′-′
lacZ
fusions were determined in strains M18 and PAO1, respectively. Using
lasI
::Gm and
ptsP
::Gm inactivation M18 mutants, we further show that expression of the
phzM
gene is positively regulated by the quorum-sensing protein LasI and negatively regulated by the phosphoenolpyruvate phosphotransferase protein PtsP. Surprisingly, the
lasI
and
ptsP
regulatory genes were also expressed in a temperature-dependent and strain-specific manner. The differential production of the phenazines PCA and PYO by strains M18 and PAO1 may be a consequence of selective pressure imposed on
P. aeruginosa
PAO1 and its relative M18 in the two different niches over a long evolutionary process.
Small-Colony Variant Selection as a Survival Strategy for
Staphylococcus aureus
in the Presence of
Pseudomonas aeruginosa
Authors: Biswas Lalitha , Biswas Raja , Schlag Martin , Bertram Ralph , Götz Friedrich
Published: August 2009

Previously it has been demonstrated that
Staphylococcus aureus
is sensitive toward
Pseudomonas
-secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of
S. aureus
survives these respiratory toxins of
P
seudomonas aeruginosa
by selection of the small-colony variant (SCV) phenotype. Purified pyocyanin alone causes the same effect. A
hem
B
mutant of
S. aureus
survives cocultivation with
P. aeruginosa
without a decrease in CFU.
Protozoan-Induced Regulation of Cyclic Lipopeptide Biosynthesis Is an Effective Predation Defense Mechanism forPseudomonas fluorescens
Authors: Mazzola Mark , de Bruijn Irene , Cohen Michael F. , Raaijmakers Jos M.
Published: August 2009

Environmental bacteria are exposed to a myriad of biotic interactions that influence their function and survival. The grazing activity of protozoan predators significantly impacts the dynamics, diversification, and evolution of bacterial communities in soil ecosystems. To evade protozoan predation, bacteria employ various defense strategies. Soil-dwellingPseudomonas fluorescensstrains SS101 and SBW25 produce the cyclic lipopeptide surfactants (CLPs) massetolide and viscosin, respectively, in a quorum-sensing-independent manner. In this study, CLP production was shown to protect these bacteria from protozoan predation as, compared to CLP-deficient mutants, strains SS101 and SBW25 exhibited resistance to grazing byNaegleria americanain vitro and superior persistence in soil in the presence of this bacterial predator. In the wheat rhizosphere, CLP-producing strains had a direct deleterious impact on the survival ofN. americana. In vitro assays further showed thatN. americanawas three times more sensitive to viscosin than to massetolide and that exposure of strain SS101 or SBW25 to this protozoan resulted in upregulation of CLP biosynthesis genes. Enhanced expression of themassABCandviscABCgenes did not require physical contact between the two organisms as gene expression levels were up to threefold higher in bacterial cells harvested 1 cm from feeding protozoans than in cells collected 4 cm from feeding protozoans. These findings document a new natural function of CLPs and highlight that bacterium-protozoan interactions can result in activation of an antipredator response in prey populations.
The Bacterially Produced Metabolite Violacein Is Associated with Survival of Amphibians Infected with a Lethal Fungus
Authors: Becker Matthew H. , Brucker Robert M. , Schwantes Christian R. , Harris Reid N. , Minbiole Kevin P. C.
Published: August 2009

The disease chytridiomycosis, which is caused by the chytrid fungus
Batrachochytrium dendrobatidis
, is associated with recent declines in amphibian populations. Susceptibility to this disease varies among amphibian populations and species, and resistance appears to be attributable in part to the presence of antifungal microbial species associated with the skin of amphibians. The betaproteobacterium
Janthinobacterium lividum
has been isolated from the skins of several amphibian species and produces the antifungal metabolite violacein, which inhibits
B. dendrobatidis
. In this study, we added
J. lividum
to red-backed salamanders (
Plethodon cinereus
) to obtain an increased range of violacein concentrations on the skin. Adding
J. lividum
to the skin of the salamander increased the concentration of violacein on the skin, which was strongly associated with survival after experimental exposure to
B. dendrobatidis
. As expected from previous work, some individuals that did not receive
J. lividum
and were exposed to
B. dendrobatidis
survived. These individuals had concentrations of bacterially produced violacein on their skins that were predicted to kill
B. dendrobatidis
. Our study suggests that a threshold violacein concentration of about 18 μM on a salamander's skin prevents mortality and morbidity caused by
B. dendrobatidis
. In addition, we show that over one-half of individuals in nature support antifungal bacteria that produce violacein, which suggests that there is a mutualism between violacein-producing bacteria and
P. cinereus
and that adding
J. lividum
is effective for protecting individuals that lack violacein-producing skin bacteria.
Novel Features of the Polysaccharide-Digesting Gliding Bacterium
Flavobacterium johnsoniae
as Revealed by Genome Sequence Analysis
Authors: McBride Mark J. , Xie Gary , Martens Eric C. , Lapidus Alla , Henrissat Bernard , Rhodes Ryan G. , Goltsman Eugene , Wang Wei , Xu Jian , Hunnicutt David W. , Staroscik Andrew M. , Hoover Timothy R. , Cheng Yi-Qiang , Stein Jennifer L.
Published: August 2009

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium
Flavobacterium johnsoniae
(phylum
Bacteroidetes
) is presented.
F. johnsoniae
is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of
F. johnsoniae
gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the “motor” and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to
sprB
that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the
gld
and
spr
genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system.
F. johnsoniae
digests proteins, and 125 predicted peptidases were identified.
F. johnsoniae
also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of
F. johnsoniae
to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to
Bacteroides thetaiotaomicron
SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete
F. johnsoniae
.
Goa1p of
Candida albicans
Localizes to the Mitochondria during Stress and Is Required for Mitochondrial Function and Virulence
Authors: Bambach Adrienne , Fernandes Mariana P. , Ghosh Anup , Kruppa Michael , Alex Deepu , Li Dongmei , Fonzi William A. , Chauhan Neeraj , Sun Nuo , Agrellos Orlando A. , Vercesi Anibal E. , Rolfes Ronda J. , Calderone Richard
Published: August 2009

Using a Tn
7
transposon library of
Candida albicans
, we have identified a mutant that exhibited sensitivity in drop plate assays to oxidants such as menadione and hydrogen peroxide. To verify the role of the mutated gene in stress adaptation, null mutants were constructed and phenotypically characterized. Because of its apparent functions in growth and oxidant adaptation, we have named the gene
GOA1
. Goa1p appears to be unique to the CTG subclade of the
Saccharomycotina
, including
C. albicans
. Mutants of
C. albicans
lacking
goa1
(strain GOA31) were more sensitive to 6 mM H
2
O
2
and 0.125 mM menadione than the wild type (wt) or a gene-reconstituted (GOA32) strain. The sensitivity to oxidants correlated with reduced survival of the GOA31 mutant in human neutrophils and avirulence compared to control strains. Other phenotypes of GOA31 include reduced growth and filamentation in 10% serum, Spider, and SLAD agar media and an inability to form chlamydospores. Since Goa1p has an N-terminal mitochondrion localization site, we also show that green fluorescent protein-tagged Goa1p shows a mitochondrionlike distribution during oxidant or osmotic stress. Further, the inability of GOA31 to grow in medium containing lactate, ethanol, or glycerol as the sole carbon source indicates that the mitochondria are defective in the mutant. To determine how Goa1p contributes to mitochondrial function, we compared the wt, GOA32, and GOA31 strains for mitochondrial electrical membrane potential, respiration, and oxidative phosphorylation. We now show that GOA31, but not the wt or GOA32, had decreased respiration and mitochondrial membrane potential such that mutant cells are unable to drive oxidative phosphorylation. This is the first report in
C. albicans
of a respiratory defect caused by a loss of mitochondrial membrane potential.
Streptococcus mutansCompetence-Stimulating Peptide InhibitsCandida albicansHypha Formation
Authors: Jarosz Lucja M. , Deng Dong Mei , van der Mei Henny C. , Crielaard Wim , Krom Bastiaan P.
Published: August 2009

The oral cavity is colonized by microorganisms growing in biofilms in which interspecies interactions take place.Streptococcus mutansgrows in biofilms on enamel surfaces and is considered one of the main etiological agents of human dental caries.Candida albicansis also commonly found in the human oral cavity, where it interacts withS. mutans. C. albicansis a polymorphic fungus, and the yeast-to-hypha transition is involved in virulence and biofilm formation. The aim of this study was to investigate interkingdom communication betweenC. albicansandS. mutansbased on the production of secreted molecules.S. mutansUA159 inhibitedC. albicansgerm tube (GT) formation in cocultures even when physically separated fromC. albicans. OnlyS. mutansspent medium collected in the early exponential phase (4-h-old cultures) inhibited the GT formation ofC. albicans. During this phase,S. mutansUA159 produces a quorum-sensing molecule, competence-stimulating peptide (CSP). The role of CSP in inhibiting GT formation was confirmed by using synthetic CSP and acomCdeletion strain ofS. mutansUA159, which lacks the ability to produce CSP. OtherS. mutansstrains and otherStreptococcusspp. also inhibited GT formation but to different extents, possibly reflecting differences in CSP amino acid sequences amongStreptococcusspp. or differences in CSP accumulation in the media. In conclusion, CSP, anS. mutansquorum-sensing molecule secreted during the early stages of growth, inhibits theC. albicansmorphological switch.
Replacement of α-Tocopherol by β-Tocopherol Enhances Resistance to Photooxidative Stress in a Xanthophyll-Deficient Strain of
Chlamydomonas reinhardtii
Authors: Sirikhachornkit Anchalee , Shin Jai W. , Baroli Irene , Niyogi Krishna K.
Published: August 2009

Tocopherols (vitamin E) comprise a class of lipid-soluble antioxidants synthesized only in plants, algae, and some cyanobacteria. The majority of tocopherols in photosynthetic cells is in the α form, which has the highest vitamin E activity in humans, whereas the β, γ, and δ forms normally account for a small percentage of total tocopherols. The antioxidant activities of these forms of tocopherol differ depending on the experimental system, and their relative activities in vivo are unclear. In a screen for suppressors of the xanthophyll-deficient
npq1 lor1
double mutant of
Chlamydomonas reinhardtii
, we isolated a
vte3
mutant lacking α-tocopherol but instead accumulating β-tocopherol. The
vte3
mutant contains a mutation in the homolog of a 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase gene found in plants. The
vte3 npq1 lor1
triple mutant with β-tocopherol survived better under photooxidative stress than did the
npq1 lor1
mutant, but the
vte3
mutant on its own did not have an obvious phenotype. Following transfer from low light to high light, the triple mutant showed a higher efficiency of photosystem II, a higher level of cell viability, and a lower level of lipid peroxide, a marker for oxidative stress, than did the
npq1 lor1
mutant. After high-light transfer, the level of the photosystem II reaction center protein, D1, was also higher in the
vte3 npq1 lor1
mutant, but the rate of D1 photodamage was not significantly different from that of the
npq1 lor1
mutant. Taken together, these results suggest that the replacement of α-tocopherol by β-tocopherol in a xanthophyll-deficient strain of
Chlamydomonas reinhardtii
contributes to better survival under conditions of photooxidative stress.
The Monothiol Single-Domain Glutaredoxin Is Conserved in the Highly Reduced Mitochondria of
Giardia intestinalis
Authors: Rada Petr , Šmíd Ondřej , Sutak Robert , Doležal Pavel , Pyrih Jan , Žárský Vojtěch , Montagne Jean-Jacques , Hrdý Ivan , Camadro Jean-Michel , Tachezy Jan
Published: August 2009

The highly reduced mitochondria (mitosomes) of
Giardia intestinalis
are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of
G. intestinalis
thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.
Our Paths Might Cross: the Role of the Fungal Cell Wall Integrity Pathway in Stress Response and Cross Talk with Other Stress Response Pathways
Authors: Fuchs Beth Burgwyn , Mylonakis Eleftherios
Published: August 2009

Fungi occupy diverse environments and are subjected to many extreme conditions. Among the stressful conditions faced by fungi are pH changes, osmotic changes, thermal changes, oxide radicals, nutrient deprivation, and exposure to chemicals. These adversities can be found either in the environment or in animal and human hosts. The cell wall integrity (CWI) pathway provides a means to fortify and repair damages to the cell wall in order to withstand stressful environments. The CWI pathway in comprised of cell wall stress sensors that lead to activation of a mitogen-activated protein kinase (MAPK) cascade. Signaling through the MAPK cascade leads to expression of transcription factors that facilitate biosynthesis of cell wall components and actin organization. Given the relatively limited number of components of the CWI pathway and the very diverse stimuli, there must be a means of expanding the pathway. To manage the diverse stress conditions, the CWI pathway cross talks with other pathways or proteins, and these cross talk events enhance the signaling capabilities of the CWI pathway. Lateral influences that facilitate maintaining the cell wall under stress conditions are TOR signaling, calcineurin signaling, the high-osmolarity glycerol pathway, the cyclic AMP-protein kinase A pathway, and additional proteins. In this article, we highlight several of the cross talk events that have been described for
Saccharomyces cerevisiae
and several other fungi.
Neurofibromin Homologs Ira1 and Ira2 Affect Glycerophosphoinositol Production and Transport in
Saccharomyces cerevisiae
Authors: Bishop Andrew C. , Surlow Beth A. , Anand Puneet , Hofer Katherine , Henkel Matthew , Patton-Vogt Jana
Published: August 2009

Saccharomyces cerevisiae
produces extracellular glycerophosphoinositol through phospholipase-mediated turnover of phosphatidylinositol and transports glycerophosphoinositol into the cell upon nutrient limitation. A screening identified the RAS GTPase-activating proteins Ira1 and Ira2 as required for utilization of glycerophosphoinositol as the sole phosphate source, but the RAS/cyclic AMP pathway does not appear to be involved in the growth phenotype. Ira1 and Ira2 affect both the production and transport of glycerophosphoinositol.
Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from
Methanosarcina acetivorans
Authors: Chen Yi-Hsing , Lin Yuyen , Yoshinaga Aya , Chhotani Benazir , Lorenzini Jenna L. , Crofts Alexander A. , Mei Shou , Mackie Roderick I. , Ishino Yoshizumi , Cann Isaac K. O.
Published: August 2009

Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the
Methanosarcina acetivorans
clamp loader or replication factor C (RFC) homolog.
M. acetivorans
RFC (RFC

Ma

), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal
Escherichia coli
clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFC

Ma

complex.
Mycobacterium tuberculosis
Is Able To Accumulate and Utilize Cholesterol
Authors: Brzostek Anna , Pawelczyk Jakub , Rumijowska-Galewicz Anna , Dziadek Bozena , Dziadek Jaroslaw
Published: August 2009

It is expected that the obligatory human pathogen
Mycobacterium tuberculosis
must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that
M. tuberculosis
grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore,
M. tuberculosis
was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (
kstD
) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that
M. tuberculosis
is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.
Investigation of the Three-Dimensional Architecture of the Collagen Adhesin EmaA ofAggregatibacter actinomycetemcomitansby Electron Tomography
Authors: Yu Chunxiao , Mintz Keith P. , Ruiz Teresa
Published: August 2009

The periodontal pathogenAggregatibacter actinomycetemcomitansdisplays on the bacterial surface a nonfimbrial adhesin, EmaA, which is required for collagen binding. In this study, electron tomography was used to characterize the three-dimensional (3D) architecture of this adhesin. The antenna-like surface appendages, corresponding to EmaA, were found to be composed of an ellipsoidal domain capping a rod-like domain that adopts either a straight or a bent conformation at various positions along the length. The most common flexible point along the length of the EmaA appendage was localized 29.4 nm away from the distal end. One-fifth of the appendages were straight and the remaining showed angles distributed between 140° and 170° at this location. Deletion analysis mapped this bend to amino acids 611 to 640 of the protein sequence. The 3D structure of the collagen binding domain of EmaA was generated by alignment and averaging of 9 subvolumes of the adhesin extracted from tomograms. The structure contains three subdomains: a globular structure with a diameter of ∼5 nm and a cylindrical domain (∼4.4 nm by 5.8 nm) separated by a linker region with a diameter of ∼3 nm, followed by a cylindrical domain (∼4.6 nm by 6.6 nm). This is the first 3D structure of a trimeric autotransporter protein ofA. actinomycetemcomitans.
A Novel Glucosyltransferase Involved in O-Antigen Modification of
Shigella flexneri
Serotype 1c
Authors: Stagg Robert M. , Tang Swee-Seong , Carlin Nils I. A. , Talukder Kaisar A. , Cam Phung D. , Verma Naresh K.
Published: August 2009

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized
gtrI
cluster, which is found within a cryptic prophage at the
proA
locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated
gtrIC
) was present as part of a three-gene cluster, similar to other
S. flexneri
glucosyltransferase genes. Relative to the other
S. flexneri gtr
clusters, the
gtrIC
cluster is more distantly related and appears to have arrived in
S. flexneri
from outside the species. Analysis of surrounding sequence suggests that the
gtrIC
cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.
Crystal Structure of theSalmonella entericaSerovar Typhimurium Virulence Factor SrfJ, a Glycoside Hydrolase Family Enzyme
Authors: Kim Yeon-Gil , Kim Jin-Hong , Kim Kyung-Jin
Published: August 2009

To cause infection,Salmonella entericaserovar Typhimurium uses type III secretion systems, which are encoded on twoSalmonellapathogenicity islands, SPI-1 and SPI-2, the latter of which is thought to play a crucial role in bacterial proliferation inSalmonella-containing vacuoles (SCVs) after invading cells.S.Typhimurium SrfJ, located outside SPI-2, is also known to be involved inSalmonellapathogenicity and has high amino acid sequence homology with human lysosomal glucosylceramidase (GlcCerase). We present the first crystal structure of SrfJ at a resolution of 1.8 Å. The overall fold of SrfJ shares high structure similarities with that of human GlcCerase, comprising two distinctive domains: a (β/α)8-barrel catalytic domain and a β-sandwich domain. As in human GlcCerase, the pocket-shaped active site of SrfJ is located on the C-terminal side of the barrel, and two conserved glutamic acid residues are used for the enzyme catalysis. Moreover, a glycerol-bound form of SrfJ reveals that the glucose ring moiety of the substrate might similarly bind to the enzyme as to human GlcCerase, suggesting that SrfJ might function as a glycoside hydrolase. Although some structural differences are observed between SrfJ and human GlcCerase in the substrate entrance of the active site, we speculate that, based on the high structural similarities to human GlcCerase in the overall fold and the active-site environment, SrfJ might have a GlcCerase activity and use the activity to enhanceSalmonellavirulence by modifying SCV membrane lipids.
Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization
Authors: Kumar C. M. Santosh , Khare Garima , Srikanth C. V. , Tyagi Anil K. , Sardesai Abhijit A. , Mande Shekhar C.
Published: August 2009

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of
Mycobacterium tuberculosis
are unable to act as effective molecular chaperones when expressed in
Escherichia coli
. We demonstrate here that the inability of
M. tuberculosis
GroEL1 to act as a functional chaperone in
E. coli
can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding
M. tuberculosis
GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in
E. coli
might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated
M. tuberculosis-E. coli
chimeras bearing mutually exchanged equatorial domains, which revealed that
E. coli
GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for
M. tuberculosis
GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of
M. tuberculosis
revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native
M. tuberculosis
GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.
Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD
Authors: Ng Sandy Y. M. , VanDyke David J. , Chaban Bonnie , Wu John , Nosaka Yoshika , Aizawa Shin-Ichi , Jarrell Ken F.
Published: August 2009

In
Archaea
, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in
Methanococcus voltae
and
Methanococcus maripaludis
) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study.
M. voltae
preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing
M. voltae
membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of
Sulfolobus solfataricus
, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an
M. maripaludis
Δ
flaK
mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.
Structure of Chlorosomes from the Green Filamentous Bacterium
Chloroflexus aurantiacus
Authors: Pšenčík Jakub , Collins Aaron M. , Liljeroos Lassi , Torkkeli Mika , Laurinmäki Pasi , Ansink Hermanus M. , Ikonen Teemu P. , Serimaa Ritva E. , Blankenship Robert E. , Tuma Roman , Butcher Sarah J.
Published: August 2009

The green filamentous bacterium
Chloroflexus aurantiacus
employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from
C. aurantiacus
was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of
Chlorobi
species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in
Chlorobi
species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols.
C. aurantiacus
chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.
An Extracytoplasmic Function Sigma Factor Controls β-Lactamase Gene Expression inBacillus anthracisand OtherBacillus cereusGroup Species
Authors: Ross Cana L. , Thomason Kerrie S. , Koehler Theresa M.
Published: August 2009

The susceptibility of mostBacillus anthracisstrains to β-lactam antibiotics is intriguing considering that the closely related speciesBacillus cereusandBacillus thuringiensistypically produce β-lactamases and theB. anthracisgenome harbors two β-lactamase genes,bla1andbla2. We show that β-lactamase activity associated withB. anthracisis affected by two genes,sigP(BA2502) andrsiP(BA2503), predicted to encode an extracytoplasmic function sigma factor and an anti-sigma factor, respectively. Deletion of thesigP-rsiPlocus abolished β-lactamase activity in a naturally occurring penicillin-resistant strain and had no effect on β-lactamase activity in a prototypical penicillin-susceptible strain. Complementation withsigPandrsiPfrom the penicillin-resistant strain, but not withsigPandrsiPfrom the penicillin-susceptible strain, conferred constitutive β-lactamase activity in both mutants. These results are attributed to a nucleotide deletion near the 5′ end ofrsiPin the penicillin-resistant strain that is predicted to result in a nonfunctional protein.B. cereusandB. thuringiensis sigPandrsiPhomologues are required for inducible penicillin resistance in these species. Expression of theB. cereusorB. thuringiensis sigPandrsiPgenes in aB. anthracis sigP-rsiP-null mutant confers inducible production of β-lactamase activity, suggesting that whileB. anthraciscontains the genes necessary for sensing β-lactam antibiotics, theB. anthracis sigPandrsiPgene products are not sufficient forblainduction.
One of the Two Genes Encoding Nucleoid-Associated HU Proteins inStreptomyces coelicolorIs Developmentally Regulated and Specifically Involved in Spore Maturation
Authors: Salerno Paola , Larsson Jessica , Bucca Giselda , Laing Emma , Smith Colin P. , Flärdh Klas
Published: August 2009

Streptomycesgenomes encode two homologs of the nucleoid-associated HU proteins. One of them, here designated HupA, is of a conventional type similar toE. coliHUα and HUβ, while the other, HupS, is a two-domain protein. In addition to the N-terminal part that is similar to that of HU proteins, it has a C-terminal domain that is similar to the alanine- and lysine-rich C termini of eukaryotic linker histones. Such two-domain HU proteins are found only amongActinobacteria. In this phylum some organisms have only a single HU protein of the type with a C-terminal histone H1-like domain (e.g., Hlp inMycobacterium smegmatis), while others have only a single conventional HU. Yet others, including the streptomycetes, produce both types of HU proteins. We show here that the two HU genes inStreptomyces coelicolorare differentially regulated and thathupSis specifically expressed during sporulation, whilehupAis expressed in vegetative hyphae. The developmental upregulation ofhupSoccurred in sporogenic aerial hyphal compartments and was dependent on the developmental regulatorswhiA,whiG, andwhiI. HupS was found to be nucleoid associated in spores, and ahupSdeletion mutant had an average nucleoid size in spores larger than that in the parent strain. The mutant spores were also defective in heat resistance and spore pigmentation, although they possessed apparently normal spore walls and displayed no increased sensitivity to detergents. Overall, the results show that HupS is specifically involved in sporulation and may affect nucleoid architecture and protection in spores ofS. coelicolor.
Cell Wall Hydrolases Affect Germination, Vegetative Growth, and Sporulation in
Streptomyces coelicolor
Authors: Haiser Henry J. , Yousef Mary R. , Elliot Marie A.
Published: August 2009

Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium
Streptomyces coelicolor
has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the
S. coelicolor
genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5′ untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave
S. coelicolor
cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of
S. coelicolor
, highlighting both the lack of redundancy in hydrolase activity and the importance of cell wall remodeling in the
S. coelicolor
life cycle.
The ArsR Repressor Mediates Arsenite-Dependent Regulation of Arsenate Respiration and Detoxification Operons of
Shewanella
sp. Strain ANA-3
Authors: Murphy Julie N. , Saltikov Chad W.
Published: August 2009

Microbial arsenate reduction affects the fate and transport of arsenic in the environment. Arsenate respiratory (
arr
) and detoxifying (
ars
) reduction pathways in
Shewanella
sp. strain ANA-3 are induced by arsenite and under anaerobic conditions. Here it is shown that an ArsR family protein, called ArsR2, regulates the arsenate respiratory reduction pathway in response to elevated arsenite under anaerobic conditions. Strains lacking
arsR2
grew faster in the presence of high levels of arsenite (3 mM). Moreover, expression of
arrA
and
arsC
(arsenate reductase-encoding genes) in the Δ
arsR2
mutant of ANA-3 were increased in cells grown under anaerobic conditions and in the absence of arsenic. Mutations in putative arsenic binding amino acid residues in ArsR2 (substitutions of Cys-30 and Cys-32 with Ser) resulted in ANA-3 strains that exhibited anaerobic growth deficiencies with high levels of arsenite and arsenate. DNA binding studies with purified ArsR2 showed that ArsR2 binding to the
arr
promoter region was impaired by trivalent arsenicals such as arsenite and phenylarsine oxide. However, ArsR2 binding occurred in the presence of arsenate. A second known regulator of the
arr
operon, cyclic AMP (cAMP)-cAMP receptor protein (CRP), could bind simultaneously with ArsR2 within the
arr
promoter region. It is concluded that ArsR2 is most likely the major arsenite-dependent regulator of
arr
and
ars
operons in
Shewanella
sp. strain ANA-3. However, anaerobic growth on arsenate will require coregulation with global regulators such as cAMP-CRP.
Dam Methylation Controls O-Antigen Chain Length in
Salmonella enterica
Serovar Enteritidis by Regulating the Expression of Wzz Protein
Authors: Sarnacki Sebastián H. , Marolda Cristina L. , Noto Llana Mariángeles , Giacomodonato Mónica N. , Valvano Miguel A. , Cerquetti María Cristina
Published: August 2009

We reported previously that a
Salmonella enterica
serovar Enteritidis
dam
mutant expressing a truncated Dam protein does not agglutinate in the presence of specific antibodies against O9 polysaccharide. Here we investigate the participation of Dam in lipopolysaccharide (LPS) synthesis in
Salmonella
. The LPS O-antigen profiles of a
dam
null mutant (SEΔ
dam
) and the
Salmonella
serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SEΔ
dam
produced LPS with shorter O-antigen polysaccharide chains. Since Wzz is responsible for the chain length distribution of the O antigen, we investigated whether Dam methylation is involved in regulating
wzz
expression. Densitometry analysis showed that the amount of Wzz produced by SEΔ
dam
is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the
wzz
promoter in SEΔ
dam
was reduced nearly 50% in logarithmic phase and 25% in stationary phase. These results were further confirmed by reverse transcription-PCR showing that
wzz
gene expression was threefold lower in the
dam
mutant than in the parental strain. Our results demonstrate that
wzz
gene expression is downregulated in a
dam
mutant, indicating that Dam methylation activates expression of this gene. This work indicates that
wzz
is a new target regulated by Dam methylation and demonstrates that DNA methylation not only affects the production of bacterial surface proteins but also the production of surface polysaccharides.
Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes
Authors: Kasai Daisuke , Fujinami Toshihiro , Abe Tomokuni , Mase Kohei , Katayama Yoshihiro , Fukuda Masao , Masai Eiji
Published: August 2009

Paenibacillus
sp. (formerly
Bacillus macerans
) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of
praR
and
praABEGFDCHI
. Based on the enzyme activities of cell extracts of
Escherichia coli
carrying
praI
,
praA
,
praH
,
praB
,
praC
, and
praD
, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The
praE
,
praF
, and
praG
gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of
praR
showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that
praABEGFDCHI
constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA.
praR-praABEGFDCHI
conferred the ability to grow on 4HB to
E
.
coli
, suggesting that
praEGF
were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that
praR
encodes a repressor of the
pra
operon.
Bacillus subtilis
SpoIIIJ and YqjG Function in Membrane Protein Biogenesis
Authors: Saller Manfred J. , Fusetti Fabrizia , Driessen Arnold J. M.
Published: August 2009

In all domains of life Oxa1p-like proteins are involved in membrane protein biogenesis.
Bacillus subtilis
, a model organism for gram-positive bacteria, contains two Oxa1p homologs: SpoIIIJ and YqjG. These molecules appear to be mutually exchangeable, although SpoIIIJ is specifically required for spore formation. SpoIIIJ and YqjG have been implicated in a posttranslocational stage of protein secretion. Here we show that the expression of either
spoIIIJ
or
yqjG
functionally compensates for the defects in membrane insertion due to YidC depletion in
Escherichia coli
. Both SpoIIIJ and YqjG complement the function of YidC in SecYEG-dependent and -independent membrane insertion of subunits of the cytochrome
o
oxidase and F
1
F
o
ATP synthase complexes. Furthermore, SpoIIIJ and YqjG facilitate membrane insertion of F
1
F
o
ATP synthase subunit c from both
E. coli
and
B. subtilis
into inner membrane vesicles of
E. coli
. When isolated from
B. subtilis
cells, SpoIIIJ and YqjG were found to be associated with the entire F
1
F
o
ATP synthase complex, suggesting that they have a role late in the membrane assembly process. These data demonstrate that the
Bacillus
Oxa1p homologs have a role in membrane protein biogenesis rather than in protein secretion.