bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Lytic Enzyme Open Reading Frame 9 (ORF9) Derived from
Enterococcus faecalis
Bacteriophage φEF24C
Authors: Uchiyama Jumpei , Takemura Iyo , Hayashi Ikue , Matsuzaki Shigenobu , Satoh Miho , Ujihara Takako , Murakami Masanao , Imajoh Masayuki , Sugai Motoyuki , Daibata Masanori
Published: November 2010

In bacteriophage (phage) therapy against Gram-positive bacteria, such as
Staphylococcus aureus
,
Listeria monocytogenes
, and
Enterococcus faecalis
, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting
E. faecalis
, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (
orf9
) of
E. faecalis
phage φEF24C was analyzed
in silico
, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is
N
-acetylmuramoyl-
l
-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against
E. faecalis
were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn
2+
ions for its activity, contrary to the bioinformatics prediction of a Zn
2+
ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of
E. faecalis
strains to 60% (6 out of 10 strains) of
Enterococcus faecium
strains. Fifth,
N
-acetylmuramoyl-
l
-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.
Application of a Depositional
F
acies
Model to an Acid Mine Drainage Site
Authors: Brown Juliana F. , Jones Daniel S. , Mills Daniel B. , Macalady Jennifer L. , Burgos William D.
Published: November 2010

Lower Red Eyes is an acid mine drainage site in Pennsylvania where low-pH Fe(II) oxidation has created a large, terraced iron mound downstream of an anoxic, acidic, metal-rich spring. Aqueous chemistry, mineral precipitates, microbial communities, and laboratory-based Fe(II) oxidation rates for this site were analyzed in the context of a depositional
facies
model. Depositional
facies
were defined as pools, terraces, or microterracettes based on cm-scale sediment morphology, irrespective of the distance downstream from the spring. The sediments were composed entirely of Fe precipitates and cemented organic matter. The Fe precipitates were identified as schwertmannite at all locations, regardless of
facies
. Microbial composition was studied with fluorescence
in situ
hybridization (FISH) and transitioned from a microaerophilic,
Euglena
-dominated community at the spring, to a
Betaproteobacteria
(primarily
Ferrovum
spp.)-dominated community at the upstream end of the iron mound, to a
Gammaproteobacteria
(primarily
Acidithiobacillus
)-dominated community at the downstream end of the iron mound. Microbial community structure was more strongly correlated with pH and geochemical conditions than depositional
facies
. Intact pieces of terrace and pool sediments from upstream and downstream locations were used in flowthrough laboratory reactors to measure the rate and extent of low-pH Fe(II) oxidation. No change in Fe(II) concentration was observed with
60
Co-irradiated sediments or with no-sediment controls, indicating that abiotic Fe(II) oxidation was negligible. Upstream sediments attained lower effluent Fe(II) concentrations compared to downstream sediments, regardless of depositional
facies
.
The Type II Secretion System Is Essential for Erythrocyte Lysis and Gut Colonization by the Leech Digestive Tract Symbiont
Aeromonas veronii
Authors: Maltz Michele , Graf Joerg
Published: November 2010

Hemolysin and the type II secretion system (T2SS) have been shown to be important for virulence in many pathogens, but very few studies have shown their importance in beneficial microbes. Here, we investigated the importance of the type II secretion pathway in the beneficial digestive-tract association of
Aeromonas veronii
and the medicinal leech
Hirudo verbana
and revealed a critical role for the hemolysis of erythrocytes. A mutant with a miniTn
5
insertion in
exeM
, which is involved in forming the inner membrane platform in the T2SS, was isolated by screening mutants for loss of hemolysis on blood agar plates. A hemolysis assay was used to quantify the mutant's deficiency in lysing sheep erythrocytes and revealed a 99.9% decrease compared to the parent strain. The importance of the T2SS in the colonization of the symbiotic host was assessed. Colonization assays revealed that the T2SS is critical for initial colonization of the leech gut. The defect was tied to the loss of hemolysin production by performing a colonization assay with blood containing lysed erythrocytes. This restored the colonization defect in the mutant. Complementation of the mutant using the promoter region and
exeMN
revealed that the T2SS is responsible for secreting hemolysin into the extracellular space and that both the T2SS and hemolysin export by the T2SS are critical for initial establishment of
A. veronii
in the leech gut.
Characterization of Glycoconjugates of Extracellular Polymeric Substances in Tufa-Associated Biofilms by Using Fluorescence Lectin-Binding Analysis
Authors: Zippel B. , Neu T. R.
Published: November 2010

Freshwater tufa deposits are the result of calcification associated with biofilms dominated by cyanobacteria. Recent investigations highlighted the fact that the formation of microbial calcium carbonates is mainly dependent on the saturation index, which is determined by pH, the ion activity of Ca
2+
and CO
3
2−
, and the occurrence of extracellular polymeric substances (EPS) produced by microorganisms. EPS, which contain carboxyl and/or hydroxyl groups, can strongly bind cations. This may result in inhibition of CaCO
3
precipitation. In contrast, the formation of templates for crystal nucleation was reported by many previous investigations. The purposes of this study were (i) to characterize the
in situ
distribution of EPS glycoconjugates in tufa-associated biofilms of two German hard-water creeks by employing fluorescence lectin-binding analysis (FLBA), (ii) to verify the specific lectin-binding pattern by competitive-inhibition assays, and (iii) to assess whether carbonates are associated with structural EPS domains. Three major
in situ
EPS domains (cyanobacterial, network-like, and cloud-like structures) were detected by FLBA in combination with laser scanning microscopy (LSM). Based on lectin specificity, the EPS glycoconjugates produced by cyanobacteria contained mainly fucose, amino sugars (
N
-acetyl-glucosamine and
N
-acetyl-galactosamine), and sialic acid. Tufa deposits were irregularly covered by network-like EPS structures, which may originate from cyanobacterial EPS secretions. Cloud-like EPS glycoconjugates were dominated by sialic acid, amino sugars, and galactose. In some cases calcium carbonate crystals were associated with cyanobacterial EPS glycoconjugates. The detection of amino sugars and calcium carbonate in close association with decaying sheath material indicated that microbially mediated processes might be important for calcium carbonate precipitation in freshwater tufa systems.
Mechanisms of Contact-Mediated Killing of Yeast Cells on Dry Metallic Copper Surfaces
Authors: Quaranta Davide , Krans Travis , Santo Christophe Espírito , Elowsky Christian G. , Domaille Dylan W. , Chang Christopher J. , Grass Gregor
Published: November 2010

Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate
Candida albicans
and
Saccharomyces cerevisiae
within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive
S. cerevisiae
Ctr1p (ScCtr1p) copper uptake transporter in
Saccharomyces
resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the
C. albicans
Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of
Candida
mutant cells than of wild-type cells.
Candida
and
Saccharomyces
took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with
Saccharomyces
. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC
2
(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.
Prevalence ofHelicobacterpullorumin Conventional, Organic, and Free-Range Broilers and Typing of Isolates
Authors: Manfreda Gerardo , Parisi Antonio , Lucchi Alex , Zanoni Renato Giulio , De Cesare Alessandra
Published: November 2010

Helicobacterpullorumrepresents a potential food-borne pathogen, and avian species appear to be a relevant reservoir of this organism. In this study, the prevalence ofH.pullorumwas investigated at 30 conventional farms where 169 ceca from 34 flocks were tested, at eight organic farms where 39 ceca from eight flocks were tested, and at seven free-range farms where 40 ceca from eight flocks were tested. All of the ceca were obtained from healthy broiler chickens. Moreover, amplified fragment length polymorphism, pulsed-field gel electrophoresis, and automated ribotyping were employed to estimate the levels of genetic variability ofH.pullorumbroiler isolates within and between flocks. Overall, Gram-negative, slender, curved rods, identified asH.pullorumby PCR, were isolated at 93.3% of the farms tested. The percentage of positive free-range farms (54.2%) was significantly lower than that of conventional (100%) or organic (100%) farms (P< 0.001). The level of within-flock genetic variability, calculated as the number of flocks colonized by isolates genetically different by all of the typing methods, was 34.9%. Isolates showing identical profiles by each typing method were observed in 11.6% of the flocks, but they were never detected between flocks. However, groups of isolates clustered together with an overall similarity level of ≥85%. Our results suggest that even though a high level of genetic variability is attributable toH.pullorumbroiler isolates, their hierarchical genotyping produces data useful for epidemiological investigations.
Characterization of Three Lactic Acid Bacteria and Their Isogenic
ldh
Deletion Mutants Shows Optimization for
Y
ATP
(Cell Mass Produced per Mole of ATP) at Their Physiological pHs
Authors: Fiedler Tomas , Bekker Martijn , Jonsson Maria , Mehmeti Ibrahim , Pritzschke Anja , Siemens Nikolai , Nes Ingolf , Hugenholtz Jeroen , Kreikemeyer Bernd
Published: November 2010

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria
Lactococcus lactis
,
Enterococcus faecalis
, and
Streptococcus pyogenes
. Of note, deletion of the
ldh
genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of
ldh
affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for
S. pyogenes
and on the pH for
E. faecalis
. In
S. pyogenes
and
L. lactis
, a change in pH resulted in a clear change in
Y
ATP
(cell mass produced per mole of ATP). The pH that showed the highest
Y
ATP
corresponded to the pH of the natural habitat of the organisms.
Anaerobic Obligatory Xylitol Production inEscherichia coliStrains Devoid of Native Fermentation Pathways
Authors: Akinterinwa Olubolaji , Cirino Patrick C.
Published: November 2010

Anaerobic glucose oxidation was coupled to xylose reduction in a nonfermentativeEscherichia colistrain expressing NADPH-dependent xylose reductase. Xylitol production serves as the primary means of NAD(P)+regeneration, as glucose is converted primarily to acetate and CO2. The membrane-bound transhydrogenase PntAB is required to achieve the maximum theoretical yield of four moles of xylitol per mole of glucose consumed.
Metatranscriptome Analysis for Insight into Whole-Ecosystem Gene Expression during Spontaneous Wheat and Spelt Sourdough Fermentations
Authors: Weckx Stefan , Allemeersch Joke , Van der Meulen Roel , Vrancken Gino , Huys Geert , Vandamme Peter , Van Hummelen Paul , De Vuyst Luc
Published: November 2010

Lactic acid bacteria (LAB) are of industrial importance in the production of fermented foods, including sourdough-derived products. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as extended shelf-life, microbial safety, improved texture, and enhanced organoleptic properties. Triggered by the considerable amount of LAB genomic information that became available during the last decade, transcriptome and, by extension, metatranscriptome studies have become one of the most appropriate research approaches to study whole-ecosystem gene expression in more detail. In this study, microarray analyses were performed using RNA sampled during four 10-day spontaneous sourdough fermentations carried out in the laboratory with an in-house-developed LAB functional gene microarray. For data analysis, a new algorithm was developed to calculate a net expression profile for each of the represented genes, allowing use of the microarray analysis beyond the species level. In addition, metabolite target analyses were performed on the sourdough samples to relate gene expression with metabolite production. The results revealed the activation of different key metabolic pathways, the ability to use carbohydrates other than glucose (e.g., starch and maltose), and the conversion of amino acids as a contribution to redox equilibrium and flavor compound generation in LAB during sourdough fermentation.
Proteolytic Cleavage of Covalently Linked Cell Wall Proteins by Candida albicans Sap9 and Sap10
Authors: Schild Lydia , Heyken Antje , de Groot Piet W. J. , Hiller Ekkehard , Mock Marlen , de Koster Chris , Horn Uwe , Rupp Steffen , Hube Bernhard
Published: November 2010

The cell wall of the human-pathogenic fungus
Candida albicans
is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in
C. albicans
cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as
in vitro
protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the
SAP9
and
SAP10
genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of
CHT2
, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by
SAP9
and
SAP10
deletion had no major impact on the phagocytosis and killing of
C. albicans
by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.
Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis
Authors: Piña Francisco J. , O'Donnell Allyson F. , Pagant Silvere , Piao Hai Lan , Miller John P. , Fields Stanley , Miller Elizabeth A. , Cyert Martha S.
Published: November 2010

Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for
Saccharomyces cerevisiae
survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63.
hph1
Δ
hph2
Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity.
sec71
Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in
hph1
Δ
hph2
Δ and
hph1
Δ
hph2
Δ
sec71
Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates
hph1
Δ
hph2
Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.
The Rag4 Glucose Sensor Is Involved in the Hypoxic Induction of
KlPDC1
Gene Expression in the Yeast Kluyveromyces lactis
Authors: Micolonghi C. , Wésolowski-Louvel M. , Bianchi M. M.
Published: November 2010

Kluyveromyces lactis
is a yeast which cannot grow under strict anaerobiosis. To date, no factors responsible for oxygen sensing and oxygen-dependent regulation of metabolism have been identified. In this paper we present the identification of the glucose sensor Rag4 as a factor essential for oxygen-dependent regulation of the fermentative pathway.
Loss of Heterozygosity at an Unlinked Genomic Locus Is Responsible for the Phenotype of a Candida albicans
sap4
Δ
sap5
Δ
sap6
Δ Mutant
Authors: Dunkel Nico , Morschhäuser Joachim
Published: November 2010

The diploid genome of the pathogenic yeast
Candida albicans
exhibits a high degree of heterozygosity. Genomic alterations that result in a loss of heterozygosity at specific loci may affect phenotypes and confer a selective advantage under certain conditions. Such genomic rearrangements can also occur during the construction of
C. albicans
mutants and remain undetected. The
SAP2
gene on chromosome R encodes a secreted aspartic protease that is induced and required for growth of
C. albicans
when proteins are the only available nitrogen source. In strain SC5314, the two
SAP2
alleles are functionally divergent because of differences in their regulation. Basal expression of the
SAP2
-
2
allele, but not the
SAP2
-
1
allele, provides the proteolytic degradation products that serve as inducers for full
SAP2
induction. A triple mutant lacking the
SAP4
,
SAP5
, and
SAP6
genes, which are located on chromosome 6, has previously been reported to have a growth defect on proteins, suggesting that one of the encoded proteases is required for
SAP2
expression. Here we show that this
sap4
Δ
sap5
Δ
sap6
Δ mutant has become homozygous for chromosome R and lost the
SAP2
-
2
allele. Replacement of one of the
SAP2
-
1
copies in this strain by
SAP2
-
2
and its regulatory region restored the ability of the
sap4
Δ
sap5
Δ
sap6
Δ mutant to utilize proteins as the sole nitrogen source. This is an illustrative example of how loss of heterozygosity at a different genomic locus can cause the mutant phenotype attributed to targeted deletion of a specific gene in
C. albicans
.
The Peptidoglycan of
Mycobacterium abscessus
Is Predominantly Cross-Linked by
l
,
d
-Transpeptidases
Authors: Lavollay Marie , Fourgeaud Martine , Herrmann Jean-Louis , Dubost Lionel , Marie Arul , Gutmann Laurent , Arthur Michel , Mainardi Jean-Luc
Published: November 2010

Few therapeutic alternatives remain for the treatment of infections due to multiresistant
Mycobacterium abscessus
. Here we show that the peptidoglycans of the “rough” and “smooth” morphotypes contain predominantly 3→3 cross-links generated by
l
,
d
-transpeptidases, indicating that these enzymes are attractive targets for the development of efficient drugs.
Isolation and Characterization of P1 Adhesin, a Leg Protein of the Gliding Bacterium
Mycoplasma pneumoniae
Authors: Nakane Daisuke , Adan-Kubo Jun , Kenri Tsuyoshi , Miyata Makoto
Published: November 2010

Mycoplasma pneumoniae
, a pathogen causing human pneumonia, binds to solid surfaces at its membrane protrusion and glides by a unique mechanism. In this study, P1 adhesin, which functions as a “leg” in gliding, was isolated from mycoplasma culture and characterized. Using gel filtration, blue-native polyacrylamide gel electrophoresis (BN-PAGE), and chemical cross-linking, the isolated P1 adhesin was shown to form a complex with an accessory protein named P90. The complex included two molecules each of P1 adhesin and P90 (protein B), had a molecular mass of about 480 kDa, and was observed by electron microscopy to form 20-nm-diameter spheres. Partial digestion of isolated P1 adhesin by trypsin showed that the P1 adhesin molecule can be divided into three domains, consistent with the results from trypsin treatment of the cell surface. Sequence analysis of P1 adhesin and its orthologs showed that domain I is well conserved and that a transmembrane segment exists near the link between domains II and III.
Identification of Nocobactin NA Biosynthetic Gene Clusters inNocardia farcinica
Authors: Hoshino Yasutaka , Chiba Kazuhiro , Ishino Keiko , Fukai Toshio , Igarashi Yasuhiro , Yazawa Katsukiyo , Mikami Yuzuru , Ishikawa Jun
Published: November 2010

We identified the biosynthetic gene clusters of the siderophore nocobactin NA. Thenbtclusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing ofNocardia farcinicaIFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of thenbtclusters and the predicted functions of thenbtgenes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of thenbtAandnbtEgenes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of thenbtSgene revealed that this gene encoded a salicylate synthase. These results indicate that thenbtclusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of thenbtA, -G, -H, -S, and -Tgenes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest thatnbtgenes are regulated coordinately by IdeR protein in an iron-dependent manner. The ΔnbtEmutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence ofN. farcinica.
Structural and Functional Insights into
Aeropyrum pernix
OppA, a Member of a Novel Archaeal OppA Subfamily
Authors: Balestrieri M. , Gogliettino M. , Fiume I. , Pocsfalvi G. , Catara G. , Rossi M. , Palmieri G.
Published: November 2010

In this study we gain insight into the structural and functional characterization of the
Aeropyrum pernix
oligopeptide-binding protein (OppA
Ap
) previously identified from the extracellular medium of an
Aeropyrum pernix
cell culture at late stationary phase. OppA
Ap
showed an N-terminal Q32 in a pyroglutamate form and C-terminal processing at the level of a threonine-rich region probably involved in protein membrane anchoring. Moreover, the OppA
Ap
protein released into the medium was identified as a “nicked” form composed of two tightly associated fragments detachable only under strong denaturing conditions. The cleavage site E569-G570 seems be located on an exposed surface loop that is highly conserved in several three-dimensional (3D) structures of dipeptide/oligopeptide-binding proteins from different sources. Structural and biochemical properties of the nicked protein were virtually indistinguishable from those of the intact form. Indeed, studies of the entire bacterially expressed OppA
Ap
protein owning the same N and C termini of the nicked form supported these findings. Moreover, in the middle exponential growth phase, OppA
Ap
was found as an intact cell membrane-associated protein. Interestingly, the native exoprotein OppA
Ap
was copurified with a hexapeptide (EKFKIV) showing both lysines methylated and possibly originating from an
A. pernix
endogenous stress-induced lipoprotein. Therefore, the involvement of OppA
Ap
in the recycling of endogenous proteins was suggested to be a potential physiological function. Finally, a new OppA from
Sulfolobus solfataricus
, SSO1288, was purified and preliminarily characterized, allowing the identification of a common structural/genetic organization shared by all “true” archaeal OppA proteins of the dipeptide/oligopeptide class.
Genome Copy Numbers and Gene Conversion in Methanogenic Archaea
Authors: Hildenbrand Catherina , Stock Tilmann , Lange Christian , Rother Michael , Soppa Jörg
Published: November 2010

Previous studies revealed that one species of methanogenic archaea,
Methanocaldococcus jannaschii
, is polyploid, while a second species,
Methanothermobacter thermoautotrophicus
, is diploid. To further investigate the distribution of ploidy in methanogenic archaea, species of two additional genera—
Methanosarcina acetivorans
and
Methanococcus maripaludis
—were investigated
. M. acetivorans
was found to be polyploid during fast growth (
t
D
= 6 h; 17 genome copies) and oligoploid during slow growth (doubling time = 49 h; 3 genome copies).
M. maripaludis
has the highest ploidy level found for any archaeal species, with up to 55 genome copies in exponential phase and ca. 30 in stationary phase. A compilation of archaeal species with quantified ploidy levels reveals a clear dichotomy between Euryarchaeota and Crenarchaeota: none of seven euryarchaeal species of six genera is monoploid (haploid), while, in contrast, all six crenarchaeal species of four genera are monoploid, indicating significant genetic differences between these two kingdoms. Polyploidy in asexual species should lead to accumulation of inactivating mutations until the number of intact chromosomes per cell drops to zero (called “Muller's ratchet”). A mechanism to equalize the genome copies, such as gene conversion, would counteract this phenomenon. Making use of a previously constructed heterozygous mutant strain of the polyploid
M. maripaludis
we could show that in the absence of selection very fast equalization of genomes in
M. maripaludis
took place probably via a gene conversion mechanism. In addition, it was shown that the velocity of this phenomenon is inversely correlated to the strength of selection.
Role of the F1 Region in theEscherichia coliAerotaxis Receptor Aer
Authors: Campbell Asharie J. , Watts Kylie J. , Johnson Mark S. , Taylor Barry L.
Published: November 2010

InEscherichiacoli, the aerotaxis receptor Aer is an atypical receptor because it senses intracellular redox potential. The Aer sensor is a cytoplasmic, N-terminal PAS domain that is tethered to the membrane by a 47-residue F1 linker. Here we investigated the function, topology, and orientation of F1 by employing random mutagenesis, cysteine scanning, and disulfide cross-linking. No native residue was obligatory for function, most deleterious substitutions had radically different side chain properties, and all F1 mutants but one were functionally rescued by the chemoreceptor Tar. Cross-linking studies were consistent with the predicted α-helical structure in the N-terminal F1 region and demonstrated trigonal interactions among the F1 linkers from three Aer monomers, presumably within trimer-of-dimer units, as well as binary interactions between subunits. Using heterodimer analyses, we also demonstrated the importance of arginine residues near the membrane interface, which may properly anchor the Aer protein in the membrane. By incorporating these data into a homology model of Aer, we developed a model for the orientation of the Aer F1 and PAS regions in an Aer lattice that is compatible with the known dimensions of the chemoreceptor lattice. We propose that the F1 region facilitates the orientation of PAS and HAMP domains during folding and thereby promotes the stability of the PAS and HAMP domains in Aer.
Acyl-Homoserine Lactone Binding to and Stability of the Orphan
Pseudomonas aeruginosa
Quorum-Sensing Signal Receptor QscR
Authors: Oinuma Ken-Ichi , Greenberg E. Peter
Published: November 2010

The
Pseudomonas aeruginosa
transcription factor QscR responds to a variety of fatty acyl-homoserine lactones (HSLs), including
N
-3-oxododecanoyl-HSL (3OC12-HSL), which is produced and detected by the
P. aeruginosa
quorum-sensing circuit LasI and LasR. As is true for LasR and many other acyl-HSL-dependent transcription factors, production of soluble QscR in sufficient amounts for purification requires growth of recombinant bacteria in the presence of an appropriate acyl-HSL. QscR is thought to bind 3OC12-HSL relatively weakly compared to LasR, and unlike LasR, binding of purified QscR to target DNA was shown to strongly depend on exogenously added 3OC12-HSL. We show that purified QscR is dimeric at sufficiently high concentrations and monomeric at lower concentrations. Furthermore, QscR bound 3OC12-HSL more tightly than previously believed. Purified QscR retained 3OC12-HSL, and at sufficiently high concentrations, it bound target DNA in the absence of added 3OC12-HSL. We also obtained soluble QscR from recombinant
Escherichia coli
grown in the presence of
N
-3-oxohexanoyl-HSL (3OC6-HSL) instead of 3OC12-HSL, and because 3OC6-HSL bound much more loosely to QscR than other acyl-HSLs tested, we were able to exchange 3OC6-HSL with other acyl-HSLs
in vitro
and then estimate binding affinities of QscR for different acyl-HSLs and for target DNA. Our data support a model whereby QscR polypeptides fold properly in the absence of an acyl-HSL, but soluble, acyl-HSL-free QscR does not accumulate because it is subject to rapid aggregation or proteolysis.
Fur and the Novel Regulator YqjI Control Transcription of the Ferric Reductase Gene
yqjH
in
Escherichia coli
Authors: Wang Suning , Wu Yun , Outten F. Wayne
Published: November 2010

Iron acquisition in aerobic habitats is complicated by the low solubility of ferric hydroxides. Siderophores that bind ferric iron with high affinity are used to mobilize iron. The reduction of ferric iron to the ferrous form can be coupled to the release of iron from siderophores. Iron is also stored intracellularly as a ferric mineral in proteins, such as ferritin, and must be reduced during release. In
Escherichia coli
, the
yqjH
gene encodes a putative ferric siderophore reductase that is also part of the Fur regulon. Here we show that YqjH has ferric reductase activity and is required for iron homeostasis in
E. coli
. Divergently transcribed from
yqjH
is the
yqjI
gene, which encodes a novel member of the winged-helix family of transcriptional regulators and also contains an N-terminal extension similar to the Ni
2+
-binding C-terminal tail of SlyD. Deletion of
yqjI
leads to constitutive high-level activity of the
yqjH
and
yqjI
promoters. Purified YqjI binds inverted repeat target sequences within the
yqjH
and
yqjI
promoters. We also observed that YqjI-dependent transcriptional repression is reduced when cells are exposed to elevated nickel levels, resulting in increased expression of
yqjH
and
yqjI
. YqjI binding to nickel or iron reduces YqjI DNA-binding activity
in vitro
. Furthermore, we found that elevated nickel stress levels disrupt iron homeostasis in
E. coli
and that deletion of
yqjH
increases nickel toxicity. Our results suggest that the YqjI protein controls expression of
yqjH
to help maintain iron homeostasis under conditions (such as elevated cellular nickel levels) that disrupt iron metabolism.
Differences in MinC/MinD Sensitivity between Polar and Internal Z Rings in
Escherichia coli
Authors: Shen Bang , Lutkenhaus Joe
Published: November 2010

In
Escherichia coli
the Z ring has the potential to assemble anywhere along the cell length but is restricted to midcell by the action of negative regulatory systems, including Min. In the current model for the Min system, the MinC/MinD division inhibitory complex is evenly distributed on the membrane and can disrupt Z rings anywhere in the cell; however, MinE spatially regulates MinC/MinD by restricting it to the cell poles, thus allowing Z ring formation at midcell. This model assumes that Z rings formed at different cellular locations have equal sensitivity to MinC/MinD in the absence of MinE. However, here we report evidence that differences in MinC/MinD sensitivity between polar and nonpolar Z rings exists even when there is no MinE. MinC/MinD at proper levels is able to block minicell production in
Δmin
strains without increasing the cell length, indicating that polar Z rings are preferentially blocked. In the FtsZ-I374V strain (which is resistant to MinC
C
/MinD), wild-type morphology can be easily achieved with MinC/MinD in the absence of MinE. We also show that MinC/MinD at proper levels can rescue the lethal phenotype of a
min slmA
double deletion mutant, which we think is due to the elimination of polar Z rings (or FtsZ structures), which frees up FtsZ molecules for assembly of Z rings at internal sites to rescue division and growth. Taken together, these data indicate that polar Z rings are more susceptible to MinC/MinD than internal Z rings, even when MinE is absent.
Membrane Topology and DNA-Binding Ability of the Streptococcal CpsA Protein
Authors: Hanson Brett R. , Lowe Beth A. , Neely Melody N.
Published: November 2010

Many streptococcal pathogens require a polysaccharide capsule for survival in the host during systemic infection. The highly conserved CpsA protein is proposed to be a transcriptional regulator of capsule production in streptococci, although the regulatory mechanism is unknown. Hydropathy plots of CpsA predict an integral membrane protein with 3 transmembrane domains and only 27 cytoplasmic residues, whereas other members of the LytR_cpsA_psr protein family are predicted to have a single transmembrane domain. This unique topology, with the short cytoplasmic domain, membrane localization, and large extracellular domain, suggests a novel mechanism of transcriptional regulation. Therefore, to determine the actual membrane topology of CpsA, specific protein domains were fused to beta-galactosidase or alkaline phosphatase. Enzymatic assays confirmed that the predicted membrane topology for CpsA is correct. To investigate how this integral membrane protein may be functioning in regulation of capsule transcription, purified full-length and truncated forms of CpsA were used in electrophoretic mobility shift assays to characterize the ability to bind the capsule operon promoter. Assays revealed that full-length, purified CpsA protein binds specifically to DNA containing the capsule promoter region. Furthermore, the large extracellular domain is not required for DNA binding, but all cytoplasmic regions of CpsA are necessary and sufficient for specific binding to the capsule operon promoter. This is the first demonstration of a member of this protein family interacting with its target DNA. Taken together, CpsA, as well as other members of the LytR_cpsA_psr protein family, appears to utilize a unique mechanism of transcriptional regulation.
Genome Annotation and Intraviral Interactome for the
Streptococcus pneumoniae
Virulent Phage Dp-1
Authors: Sabri Mourad , Häuser Roman , Ouellette Marc , Liu Jing , Dehbi Mohammed , Moeck Greg , García Ernesto , Titz Björn , Uetz Peter , Moineau Sylvain
Published: November 2010

Streptococcus pneumoniae
causes several diseases, including pneumonia, septicemia, and meningitis. Phage Dp-1 is one of the very few isolated virulent
S. pneumoniae
bacteriophages, but only a partial characterization is currently available. Here, we confirmed that Dp-1 belongs to the family
Siphoviridae
. Then, we determined its complete genomic sequence of 56,506 bp. It encodes 72 open reading frames, of which 44 have been assigned a function. We have identified putative promoters, Rho-independent terminators, and several genomic clusters. We provide evidence that Dp-1 may be using a novel DNA replication system as well as redirecting host protein synthesis through queuosine-containing tRNAs. Liquid chromatography-mass spectrometry analysis of purified phage Dp-1 particles identified at least eight structural proteins. Finally, using comprehensive yeast two-hybrid screens, we identified 156 phage protein interactions, and this intraviral interactome was used to propose a structural model of Dp-1.
Contributions of Multiple Binding Sites and Effector-Independent Binding to CodY-Mediated Regulation inBacillus subtilis
Authors: Belitsky Boris R. , Sonenshein Abraham L.
Published: November 2010

CodY is a branched-chain amino acid-responsive transcriptional regulator that controls, directly or indirectly, the expression of more than 100 genes and operons inBacillus subtilis. Using DNase I footprinting and gel-shift experiments, we identified two CodY-binding regions upstream of aB. subtilisgene (bcaP, previously known asyhdG) that encodes a transporter of branched-chain amino acids. Mutational analysis revealed that both CodY-binding regions contribute to repressionin vivoand do so independently of each other. Thus, a single CodY-binding site is apparently sufficient for substantial CodY-dependent regulation. By analyzing affinities of wild-type and mutant CodY-binding sites for CodY and their regulation by wild-type CodY and forms of CodY with various levels of activation by branched-chain amino acids, we concluded that unliganded CodY cannot repress transcriptionin vivoand that the level of endogenously produced effectors is sufficient for CodY-mediated regulation of promoters with stronger sites. Because the sites with higher affinity apparently respond to lower concentrations of CodY effectors and saturate faster as the concentrations of effectors increase, having two sites of binding with different affinities for CodY permits a promoter to respond to a wider range of intracellular concentrations of effectors.
Rok Regulates
yuaB
Expression during Architecturally Complex Colony Development of
Bacillus subtilis
168
Authors: Kovács Ákos T. , Kuipers Oscar P.
Published: November 2010

Transcriptome analysis of a
Bacillus subtilis rok
strain that showed reduced complex colony structure formation revealed significant downregulation of the
yuaB
gene. Overexpression of
yuaB
restored structure formation in the
rok
strain. We show that transcription of
yuaB
is indirectly regulated by Rok, independently from its previously described AbrB-dependent regulation.
Chemotaxis to the Quorum-Sensing Signal AI-2 Requires the Tsr Chemoreceptor and the Periplasmic LsrB AI-2-Binding Protein
Authors: Hegde Manjunath , Englert Derek L. , Schrock Shanna , Cohn William B. , Vogt Christian , Wood Thomas K. , Manson Michael D. , Jayaraman Arul
Published: November 2010

AI-2 is an autoinducer made by many bacteria. LsrB binds AI-2 in the periplasm, and Tsr is thel-serine chemoreceptor. We show that AI-2 strongly attractsEscherichia coli. Both LsrB and Tsr are necessary for sensing AI-2, but AI-2 uptake is not, suggesting that LsrB and Tsr interact directly in the periplasm.
Complete Genome Sequence of
Bifidobacterium
longum
subsp.
longum
BBMN68, a New Strain from a Healthy Chinese Centenarian
Authors: Hao Yanling , Huang Dawei , Guo Huiyuan , Xiao Man , An Haoran , Zhao Liang , Zuo Fanglei , Zhang Bing , Hu Songnian , Song Shuhui , Chen Shangwu , Ren Fazheng
Published: November 2010

Bifidobacterium
longum
subsp.
longum
BBMN68 was isolated from the feces of a healthy centenarian living in an area of BaMa, Guangxi, China, known for longevity. Here we report the main genome features of
B
.
longum
strain BBMN68 and the identification of several predicted proteins associated with the ecological niche of longevity.
Primosomal Proteins DnaD and DnaB Are Recruited to Chromosomal Regions Bound by DnaA inBacillus subtilis
Authors: Smits Wiep Klaas , Merrikh Houra , Bonilla Carla Yaneth , Grossman Alan D.
Published: November 2010

The initiation of DNA replication requires the binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication,oriC. DnaA also binds to many sites around the chromosome, outsideoriC, and acts as a transcription factor at several of these. In low-G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase atoriC, and this depends on DnaA. DnaD and DnaB also are required to load the replicative helicase outsideoriCduring replication restart, independently of DnaA. Using chromatin immunoprecipitation, we found that DnaD and DnaB, but not the replicative helicase, are associated with many of the chromosomal regions bound by DnaA inBacillus subtilis. This association was dependent on DnaA, and the order of recruitment was the same as that atoriC, but it was independent of a functionaloriCand suggests that DnaD and DnaB do not require open complex formation for the stable association with DNA. These secondary binding regions for DnaA could be serving as a reservoir for excess DnaA, DnaD, and DnaB to help properly regulate replication initiation and perhaps are analogous to the proposed function of thedatAlocus inEscherichia coli. Alternatively, DnaD and DnaB might modulate the activity of DnaA at the secondary binding regions. All three of these proteins are widely conserved and likely have similar functions in a range of organisms.
A LytM Domain Dictates the Localization of Proteins to the Mother Cell-Forespore Interface during Bacterial Endospore Formation
Authors: Meisner Jeffrey , Moran Charles P.
Published: November 2010

A large number of proteins are known to reside at specific subcellular locations in bacterial cells. However, the molecular mechanisms by which many of these proteins are anchored at these locations remains unclear. During endospore formation in
Bacillus subtilis
, several integral membrane proteins are located specifically at the interface of the two adjacent cells of the developing sporangium, the mother cell and forespore. The mother cell membrane protein SpoIIIAH recognizes the cell-cell interface through an interaction with the forespore membrane protein SpoIIQ, and then the other proteins are positioned there by the SpoIIIAH-SpoIIQ complex. In this study, we investigated the molecular mechanisms underlying the formation of the SpoIIIAH-SpoIIQ complex. Using gel filtration chromatography and isothermal titration calorimetry, we measured the binding parameters that characterize the SpoIIIAH-SpoIIQ interaction
in vitro
. We also demonstrated that the interaction of SpoIIIAH and SpoIIQ is governed by their YscJ and degenerate LytM domains, respectively. Therefore, the LytM domain of SpoIIQ provides the positional cue that dictates the localization of mother cell membrane proteins to the mother cell-forespore interface.
Complete Genome Sequences of Three
Erwinia amylovora
Phages Isolated in North America and a Bacteriophage Induced from an
Erwinia tasmaniensis
Strain
Authors: Müller I. , Kube M. , Reinhardt R. , Jelkmann W. , Geider K.
Published: November 2010

Fire blight, a plant disease of economic importance caused by
Erwinia amylovora
, may be controlled by the application of bacteriophages. Here, we provide the complete genome sequences and the annotation of three
E. amylovora
-specific phages isolated in North America and genomic information about a bacteriophage induced by mitomycin C treatment of an
Erwinia tasmaniensis
strain that is antagonistic for
E. amylovora
. The American phages resemble two already-described viral genomes, whereas the
E. tasmaniensis
phage displays a singular genomic sequence in BLAST searches.
Complete Genome Sequence of the Haloaromatic Acid-Degrading Bacterium
Achromobacter xylosoxidans
A8
Authors: Strnad Hynek , Ridl Jakub , Paces Jan , Kolar Michal , Vlcek Cestmir , Paces Vaclav
Published: November 2010

Achromobacter xylosoxidans
strain A8 was isolated from soil contaminated with polychlorinated biphenyls. It can use 2-chlorobenzoate and 2,5-dichlorobenzoate as sole sources of carbon and energy. This property makes it a good starting microorganism for further development toward a bioremediation tool. The genome of
A. xylosoxidans
consists of a 7-Mb chromosome and two large plasmids (98 kb and 248 kb). Besides genes for the utilization of xenobiotic organic substrates, it contains genes associated with pathogenesis, toxin production, and resistance. Here, we report the complete genome sequence.
Genome Sequence ofWeissella cibariaKACC 11862
Authors: Kim Dae-Soo , Choi Sang-Haeng , Kim Dong-Wook , Nam Seong-Hyeuk , Kim Ryong Nam , Kang Aram , Kim Aeri , Park Hong-Seog
Published: November 2010

Weissella cibariaKACC 11862 is a Gram-positive, heterofermentative,Leuconostoc-like lactic acid bacterium that is widely distributed in Korean traditional foods such as kimchi. Here we report the draft genome sequence of the type strain,W. cibariaKACC 11862 (1,599 known genes, 80 RNA genes), which consists of 72 large contigs (>100 bp in size).