bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Helicobacter pylori Bacteriophage KHP30
Authors: Uchiyama Jumpei , Takeuchi Hiroaki , Kato Shin-ichiro , Gamoh Keiji , Takemura-Uchiyama Iyo , Ujihara Takako , Daibata Masanori , Matsuzaki Shigenobu
Published: March 2013

Helicobacter pyloriinhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported inH. pylori. We have isolated anH. pyloriphage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinicalH. pyloriisolates. The latent period was ca. 140 min, shorter than the doubling time ofH. pylori(ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome inH. pyloristrain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage familiesCorticoviridaeandTectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.
Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata
Authors: Ovrutsky Alida R. , Chan Edward D. , Kartalija Marinka , Bai Xiyuan , Jackson Mary , Gibbs Sara , Falkinham Joseph O. , Iseman Michael D. , Reynolds Paul R. , McDonnell Gerald , Thomas Vincent
Published: March 2013

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms.
Acanthamoeba
spp. (genotype T4) were mainly recovered (8/17), followed by
Hartmannella vermiformis
(7/17) as well as one isolate closely related to the genus
Flamella
and one isolate only distantly related to previously described species. Concerning mycobacteria,
Mycobacterium gordonae
was the most frequently found isolate (9/17), followed by
Mycobacterium peregrinum
(4/17),
Mycobacterium chelonae
(2/17),
Mycobacterium mucogenicum
(1/17), and
Mycobacterium avium
(1/17). The propensity of
Mycobacterium avium
hospital isolate H87 and
M. avium
collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in
Acanthamoeba lenticulata
. As
A. lenticulata
was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of
M. avium
in drinking water networks and the epidemiology of disease due to this species.
Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase
Authors: Theisen Joanne , Zylstra Gerben J. , Yee Nathan
Published: March 2013

The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in
Escherichia coli
K-12. The results show that mutants deleted of the
moaA
,
moaB
,
moaE
, or
mog
gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the
modB
or
modC
gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in
E. coli
involves a molybdoenzyme.
Coordination of K
+
Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated
Authors: Rivetta Alberto , Allen Kenneth E. , Slayman Carolyn W. , Slayman Clifford L.
Published: March 2013

Fungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organism
Neurospora crassa
, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the gene
HAK1
to be much more highly regulated than is
TRK1
. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K
+
] and/or the coexpression of
HAK1
; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K
+
and by coexpression of
TRK1
; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ∼500:1,
viz.
, 10- to 50-fold larger than that in K
+
- and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K
+
stress by means of progressively increasing transporter affinity for extracellular K
+
. The underlying observations are (iv) that K
+
influx via Trk1p remains nearly constant at ∼9 mM/h when extracellular K
+
is progressively depleted below 0.05 mM and (v) that K
+
influx via Hak1p remains at ∼3 mM/h when extracellular K
+
is depleted below 0.1 mM.
Does Candida albicans Als5p Amyloid Play a Role in Commensalism in Caenorhabditis elegans?
Authors: Bois Michael , Singh Sean , Samlalsingh Alyssa , Lipke Peter N. , Garcia Melissa C.
Published: March 2013

Candida albicans
, a dimorphic fungus and an opportunistic pathogen, possesses a myriad of adherence factors, including members of the agglutinin-like sequence (Als) family of mannoproteins. The adhesin Als5p mediates adhesion to many substrates and is upregulated during commensal interactions but is downregulated during active
C. albicans
infections. An amyloid-forming core sequence at residues 325 to 331 is important for Als5p function, because a single-amino-acid substitution at position 326 (V326N) greatly reduces Als5p-mediated adherence. We evaluated the role of Als5p in host-microbe interactions by using
Caenorhabditis elegans
nematodes as a host model and feeding them
Saccharomyces cerevisiae
expressing Als5p on the surface. Als5p-expressing yeast had 8.5- and 3.5-fold-increased intestinal accumulation rates compared to Als5p-nonexpressing
S. cerevisiae
or yeast expressing amyloid-deficient Als5p
V326N
, respectively. Surprisingly, this accumulation delayed
S. cerevisiae
-induced killing of
C. elegans.
The median survival time was nearly twice as long as that of nematodes fed nonexpressing or non-amyloid-forming Als5p
V326N
-expressing
S. cerevisiae
. Treatment with the amyloid-inhibiting dye Congo red or repression of Als5p expression abrogated the protective effect of Als5p. Furthermore, Als5p had no effect on oocyte quantity or quality, since nematodes fed either empty vector (EV)- or Als5p
V326N
-expressing
S. cerevisiae
had similar egg-laying and egg-hatching rates. This study is the first, to our knowledge, to show that expression of an amyloid-forming protein can attenuate pathogenicity in
C. elegans
.
Nuclear Pores and Perinuclear Expression Sites ofvarand Ribosomal DNA Genes Correspond to Physically Distinct Regions in Plasmodium falciparum
Authors: Guizetti Julien , Martins Rafael Miyazawa , Guadagnini Stéphanie , Claes Aurélie , Scherf Artur
Published: March 2013

The human malaria parasitePlasmodium falciparummodifies the erythrocyte it infects by exporting variant proteins to the host cell surface. Thevargene family that codes for a large, variant adhesive surface protein calledP. falciparumerythrocyte membrane protein 1 (PfEMP1) plays a particular role in this process, which is linked to pathogenesis and immune evasion. A single member of this gene family is highly transcribed while the other 59 members remain silenced. Importantly,vargene transcription occurs at a spatially restricted, but yet undefined, perinuclear site that is distinct from repressedvargene clusters. To advance our understanding of monoallelic expression, we investigated whether nuclear pores associate with thevargene expression site. To this end, we studied the nuclear pore organization during the asexual blood stage using a specific antibody directed against a subunit of the nuclear pore,P. falciparumNup116 (PfNup116). Ring and schizont stage parasites showed highly polarized nuclear pore foci, whereas in trophozoite stage nuclear pores redistributed over the entire nuclear surface. Colocalization studies ofvartranscripts and anti-PfNup116 antibodies showed clear dissociation between nuclear pores and thevargene expression site in ring stage. Similar results were obtained for another differentially transcribed perinuclear gene family, the ribosomal DNA units. Furthermore, we show that in the poised state, thevargene locus is not physically linked to nuclear pores. Our results indicate thatP. falciparumdoes form compartments of high transcriptional activity at the nuclear periphery which are, unlike the case in yeast, devoid of nuclear pores.
Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea
Authors: Harren Karin , Tudzynski Bettina
Published: March 2013

In the filamentous phytopathogen
Botrytis cinerea
, the Ca
2+
/calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The
cch1
and
mid1
genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca
2+
-chelating agents EGTA and 1,2-bis(
o
-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetic acid (BAPTA). The impact of external Ca
2+
was investigated by supplementing with CaCl
2
and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1–green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in
B. cinerea
.
Comparison of Sterol Import under Aerobic and Anaerobic Conditions in Three Fungal Species, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae
Authors: Zavrel Martin , Hoot Sam J. , White Theodore C.
Published: March 2013

Sterol import has been characterized under various conditions in three distinct fungal species, the model organism
Saccharomyces cerevisiae
and two human fungal pathogens
Candida glabrata
and
Candida albicans
, employing cholesterol, the sterol of higher eukaryotes, as well as its fungal equivalent, ergosterol. Import was confirmed by the detection of esterified cholesterol within the cells. Comparing the three fungal species, we observe sterol import under three different conditions. First, as previously well characterized, we observe sterol import under low oxygen levels in
S. cerevisiae
and
C. glabrata
, which is dependent on the transcription factor Upc2 and/or its orthologs or paralogs. Second, we observe sterol import under aerobic conditions exclusively in the two pathogenic fungi
C. glabrata
and
C. albicans
. Uptake emerges during post-exponential-growth phases, is independent of the characterized Upc2-pathway and is slower compared to the anaerobic uptake in
S. cerevisiae
and
C. glabrata
. Third, we observe under normoxic conditions in
C. glabrata
that Upc2-dependent sterol import can be induced in the presence of fetal bovine serum together with fluconazole. In summary,
C. glabrata
imports sterols both in aerobic and anaerobic conditions, and the limited aerobic uptake can be further stimulated by the presence of serum together with fluconazole.
S. cerevisiae
imports sterols only in anaerobic conditions, demonstrating aerobic sterol exclusion. Finally,
C. albicans
imports sterols exclusively aerobically in post-exponential-growth phases, independent of Upc2. For the first time, we provide direct evidence of sterol import into the human fungal pathogen
C. albicans
, which until now was believed to be incapable of active sterol import.
Agl16, a Thermophilic Glycosyltransferase Mediating the Last Step of
N
-Glycan Biosynthesis in the Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius
Authors: Meyer Benjamin H. , Peyfoon Elham , Dietrich Carsten , Hitchen Paul , Panico Maria , Morris Howard R. , Dell Anne , Albers Sonja-Verena
Published: March 2013

Recently, the S-layer protein of
Sulfolobus acidocaldarius
was shown to be N-linked with a tribranched hexasaccharide, composed of Man
2
Glc
1
GlcNAc
2
and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of
agl16
, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the
N
-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the
agl16
deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed
N
-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the
agl16
deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δ
agl16
mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δ
agl16
deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the
N
-glycan size.
The Vitamin B
6
Biosynthesis Pathway in Streptococcus pneumoniae Is Controlled by Pyridoxal 5′-Phosphate and the Transcription Factor PdxR and Has an Impact on Ear Infection
Authors: El Qaidi Samir , Yang Jun , Zhang Jing-Ren , Metzger Dennis W. , Bai Guangchun
Published: March 2013

Vitamin B
6
is an essential cofactor for a large number of enzymes in both prokaryotes and eukaryotes. In this study, we characterized the pyridoxal 5′-phosphate (PLP) biosynthesis pathway in
Streptococcus pneumoniae
. Our results revealed that
S. pneumoniae
possesses a
de novo
vitamin B
6
biosynthesis pathway encoded by the
pdxST
genes. Purified PdxS functionally displayed as PLP synthase, whereas PdxT exhibited glutaminase activity
in vitro
. Deletion of
pdxS
, but not
pdxT
, resulted in a vitamin B
6
auxotrophic mutant. The defective growth of the Δ
pdxS
mutant in a vitamin B
6
-depleted medium could be chemically restored in the presence of the B
6
vitamers at optimal concentrations. By analyzing PdxS expression levels, we demonstrated that the expression of
pdxS
was repressed by PLP and activated by a transcription factor, PdxR. A pneumococcal Δ
pdxR
mutant also exhibited as a vitamin B
6
auxotroph. In addition, we found that disruption of the vitamin B
6
biosynthesis pathway in
S. pneumoniae
caused a significant attenuation in a chinchilla middle ear infection model and a minor attenuation in a mouse pneumonia model, indicating that the impact of vitamin B
6
synthesis on virulence depends upon the bacterial infection niche.
The Nucleotide Excision Repair System of Borrelia burgdorferi Is the Sole Pathway Involved in Repair of DNA Damage by UV Light
Authors: Hardy Pierre-Olivier , Chaconas George
Published: March 2013

To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized inEscherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes forBorrelia burgdorferisurvival to UV-induced DNA damage was assessed. In contrast toE. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival ofB. burgdorferiexposed to UV light. However, the disruption of theB. burgdorferinucleotide excision repair (NER) pathway (uvrA,uvrB,uvrC, anduvrD) resulted in a 10- to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required forB. burgdorferiresistance to nitrosative damage. Finally, disruption ofuvrA,uvrC, anduvrDhad only a minor effect upon murine infection by increasing the time required for dissemination.
SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism
Authors: Li Qinglian , Wang Lifei , Xie Yunying , Wang Songmei , Chen Ruxian , Hong Bin
Published: March 2013

Sansanmycins, produced by
Streptomyces
sp. strain SS, are uridyl peptide antibiotics with activities against
Pseudomonas aeruginosa
and multidrug-resistant
Mycobacterium tuberculosis
. In this work, the biosynthetic gene cluster of sansanmycins, comprised of 25 open reading frames (ORFs) showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster, was identified. SsaA, the archetype of a novel class of transcriptional regulators, was characterized in the sansanmycin gene cluster, with an N-terminal fork head-associated (FHA) domain and a C-terminal LuxR-type helix-turn-helix (HTH) motif. The disruption of
ssaA
abolished sansanmycin production, as well as the expression of the structural genes for sansanmycin biosynthesis, indicating that SsaA is a pivotal activator for sansanmycin biosynthesis. SsaA was proved to directly bind several putative promoter regions of biosynthetic genes, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA binding sequence, GTMCTGACAN
2
TGTCAGKAC. The DNA binding activity of SsaA was inhibited by sansanmycins A and H in a concentration-dependent manner. Furthermore, sansanmycins A and H were found to directly interact with SsaA. These results indicated that SsaA strictly controls the production of sansanmycins at the transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end products. As the first characterized regulator of uridyl peptide antibiotic biosynthesis, the understanding of this autoregulatory process involved in sansanmycin biosynthesis will likely provide an effective strategy for rational improvements in the yields of these uridyl peptide antibiotics.
Insights into Denitrification in Methylotenera mobilis from Denitrification Pathway and Methanol Metabolism Mutants
Authors: Mustakhimov Ildar , Kalyuzhnaya Marina G. , Lidstrom Mary E. , Chistoserdova Ludmila
Published: March 2013

We investigated phenotypes of mutants of
Methylotenera mobilis
JLW8 with lesions in genes predicted to encode functions of the denitrification pathway, as well as mutants with mutations in methanol dehydrogenase-like structural genes
xoxF1
and
xoxF2
, in order to obtain insights into denitrification and methanol metabolism by this bacterium. By monitoring the accumulation of nitrous oxide, we demonstrate that a periplasmic nitrate reductase, NAD(P)-linked and copper-containing nitrite reductases, and a nitric oxide reductase are involved in the denitrification pathway and that the pathway must be operational in aerobic conditions. However, only the assimilatory branch of the denitrification pathway was essential for growth on methanol in nitrate-supplemented medium. Mutants with mutations in each of the two
xoxF
genes maintained their ability to grow on methanol, but not the double XoxF mutant, suggesting that XoxF proteins act as methanol dehydrogenase enzymes in
M. mobilis
JLW8. Reduced levels of nitrous oxide accumulated by the XoxF mutants compared to the wild type suggest that these enzymes must be capable of donating electrons for denitrification.
Dynamic Localization of a Transcription Factor in Bacillus subtilis: the LicT Antiterminator Relocalizes in Response to Inducer Availability
Authors: Rothe Fabian M. , Wrede Christoph , Lehnik-Habrink Martin , Görke Boris , Stülke Jörg
Published: March 2013

Bacillus subtilis
transports β-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the β-glucoside permease BglP is induced in the presence of the substrate salicin, and this induction requires the binding of the antiterminator protein LicT to a specific RNA target in the 5′ region of the
bglP
mRNA to prevent the formation of a transcription terminator. LicT is composed of an N-terminal RNA-binding domain and two consecutive PTS regulation domains, PRD1 and PRD2. In the absence of salicin, LicT is phosphorylated on PRD1 by BglP and thereby inactivated. In the presence of the inducer, the phosphate group from PRD1 is transferred back to BglP and consequently to the incoming substrate, resulting in the activation of LicT. In this study, we have investigated the intracellular localization of LicT. While the protein was evenly distributed in the cell in the absence of the inducer, we observed a subpolar localization of LicT if salicin was present in the medium. Upon addition or removal of the inducer, LicT rapidly relocalized in the cells. This dynamic relocalization did not depend on the binding of LicT to its RNA target sites, since the localization pattern was not affected by deletion of all LicT binding sites. In contrast, experiments with mutants affected in the PTS components as well as mutations of the LicT phosphorylation sites revealed that phosphorylation of LicT by the PTS components plays a major role in the control of the subcellular localization of this RNA-binding transcription factor.
Characterization of the
phd-doc
and
ccd
Toxin-Antitoxin Cassettes from Vibrio Superintegrons
Authors: Guérout Anne-Marie , Iqbal Naeem , Mine Natacha , Ducos-Galand Magaly , Van Melderen Laurence , Mazel Didier
Published: March 2013

Toxin-antitoxin (TA) systems have been reported in the genomes of most bacterial species, and their role when located on the chromosome is still debated. TA systems are particularly abundant in the massive cassette arrays associated with chromosomal superintegrons (SI). Here, we describe the characterization of two superintegron cassettes encoding putative TA systems. The first is the

phd-doc
SI

system identified in
Vibrio cholerae
N16961. We determined its distribution in 36
V. cholerae
strains and among five
V. metschnikovii
strains. We show that this cassette, which is in position 72 of the
V. cholerae
N16961 cassette array, is functional, carries its own promoter, and is expressed from this location. Interestingly, the

phd-doc
SI

system is unable to control its own expression, most likely due to the absence of any DNA-binding domain on the antitoxin. In addition, this SI system is able to cross talk with the canonical P1 phage system. The second cassette that we characterized is the

ccd
Vfi

cassette found in the
V. fischeri
superintegron. We demonstrate that CcdB
Vfi
targets DNA-gyrase, as the canonical CcB
F
toxin, and that

ccd
Vfi

regulates its expression in a fashion similar to the
ccd
F
operon of the conjugative plasmid F. We also establish that this cassette is functional and expressed in its chromosomal context in
V. fischeri
CIP 103206T. We tested its functional interactions with the

ccdAB
F

system and found that CcdA
Vfi
is specific for its associated CcdB
Vfi
and cannot prevent CcdB
F
toxicity. Based on these results, we discuss the possible biological functions of these TA systems in superintegrons.
Influence of Copper Resistance Determinants on Gold Transformation by Cupriavidus metallidurans Strain CH34
Authors: Wiesemann Nicole , Mohr Juliane , Grosse Cornelia , Herzberg Martin , Hause Gerd , Reith Frank , Nies Dietrich H.
Published: March 2013

Cupriavidus metalliduransis associated with gold grains and may be involved in their formation. Gold(III) complexes influence the transcriptome ofC. metallidurans(F. Reith et al., Proc. Natl. Acad. Sci. U. S. A. 106:17757–17762, 2009), leading to the upregulation of genes involved in the detoxification of reactive oxygen species and metal ions. In a systematic study, the involvement of these systems in gold transformation was investigated. Treatment ofC. metalliduranscells with Au(I) complexes, which occur in this organism's natural environment, led to the upregulation of genes similar to those observed for treatment with Au(III) complexes. The two indigenous plasmids ofC. metallidurans, which harbor several transition metal resistance determinants, were not involved in resistance to Au(I/III) complexes nor in their transformation to metallic nanoparticles. Upregulation of acupA-lacZfusion by the MerR-type regulator CupR with increasing Au(III) concentrations indicated the presence of gold ions in the cytoplasm. A hypothesis stating that the Gig system detoxifies gold complexes by the uptake and reduction of Au(III) to Au(I) or Au(0) reminiscent to detoxification of Hg(II) was disproven. ZupT and other secondary uptake systems for transition metal cations influenced Au(III) resistance but not the upregulation of thecupA-lacZfusion. The two copper-exporting P-type ATPases CupA and CopF were also not essential for gold resistance. ThecopABCDdeterminant on chromosome 2, which encodes periplasmic proteins involved in copper resistance, was required for full gold resistance inC. metallidurans. In conclusion, biomineralization of gold particles via the reduction of mobile Au(I/III) complexes inC. metalliduransappears to primarily occur in the periplasmic space via copper-handling systems.
A Mutant with Aberrant Extracellular LcrV-YscF Interactions Fails To Form Pores and Translocate Yop Effector Proteins but Retains the Ability To Trigger Yop Secretion in Response to Host Cell Contact
Authors: Harmon Dana E. , Murphy Julia L. , Davis Alison J. , Mecsas Joan
Published: March 2013

The plasmid-encoded type three secretion system (TTSS) of
Yersinia
spp. is responsible for the delivery of effector proteins into cells of the innate immune system, where these effectors disrupt the target cells' activity. Successful translocation of effectors into mammalian cells requires
Yersinia
to both insert a translocon into the host cell membrane and sense contact with host cells. To probe the events necessary for translocation, we investigated protein-protein interactions among TTSS components of the needle-translocon complex using a chemical cross-linking-based approach. We detected extracellular protein complexes containing YscF, LcrV, and YopD that were dependent upon needle formation. The formation of these complexes was evaluated in a secretion-competent but translocation-defective mutant, the YscFD28AD46A strain (expressing YscF with the mutations D28A and D46A). We found that one of the YscF and most of the LcrV and YopD cross-linked complexes were nearly absent in this mutant. Furthermore, the YscFD28AD46A strain did not support YopB insertion into mammalian membranes, supporting the idea that the LcrV tip complex is required for YopB insertion and translocon formation. However, the YscFD28AD46A strain did secrete Yops in the presence of host cells, indicating that a translocation-competent tip complex is not required to sense contact with host cells to trigger Yop secretion. In conclusion, in the absence of cross-linkable LcrV-YscF interactions, translocon insertion is abolished, but
Yersinia
still retains the ability to sense cell contact.
Cyclic Di-GMP Modulates the Disease Progression of Erwinia amylovora
Authors: Edmunds Adam C. , Castiblanco Luisa F. , Sundin George W. , Waters Christopher M.
Published: March 2013

The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease
Erwinia amylovora
has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in
E. amylovora
(
edc
genes, for

E
rwinia

d
iguanylate
c
yclase),
edcA
,
edcC
, and
edcE
, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in
E. amylovora
, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of
hrpA
, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in
E. amylovora
and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.
SMC Condensation Centers in Bacillus subtilis Are Dynamic Structures
Authors: Kleine Borgmann Luise A. K. , Hummel Hanna , Ulbrich Maximilian H. , Graumann Peter L.
Published: March 2013

SMC and MukB complexes consist of a central SMC dimer and two essential binding partners, ScpA and ScpB (MukE and MukF), and are crucial for correct chromosome compaction and segregation. The complexes form two bipolar assemblies on the chromosome, one in each cell half. Using fluorescence recovery after photobleaching (FRAP), we provide evidence that the SMC complex has high exchange rates. This depends to a considerable degree on
de novo
protein synthesis, revealing that the bacterial SMC complex has high on and off rates for binding to the chromosome. A mutation in SMC that affects ATPase activity and results in exaggerated DNA binding
in vitro
causes a strong segregation defect
in vivo
and affects the localization of the entire SMC complex, which localizes to many more sites in the cell than under normal conditions. These data indicate that ATP turnover is important for the function of
Bacillus subtilis
SMC. In contrast, the centromere protein Spo0J and DNA gyrase showed much less exchange between distinct binding sites on the chromosome than that seen with SMC. Binding of Spo0J to the origin regions was rather static and remained partially conserved until the next cell cycle. Our experiments reveal that the SMC complex has a high, condensin-like turnover rate and that an alteration of the ATPase cycle affects SMC function
in vivo
, while several nucleoid-associated proteins feature limited or slow exchange between different sites on the nucleoid, which may be the basis for epigenetic-like phenomena observed in bacteria.
PelN Is a New Pectate Lyase of Dickeya dadantii with Unusual Characteristics
Authors: Hassan Susan , Shevchik Vladimir E. , Robert Xavier , Hugouvieux-Cotte-Pattat Nicole
Published: March 2013

The plant-pathogenic bacterium
Dickeya dadantii
produces several pectinolytic enzymes that play a major role in the soft-rot disease. Eight characterized endopectate lyases are secreted in the extracellular medium by the type II secretion system, Out. They cleave internal glycosidic bonds of pectin, leading to plant tissue maceration. The
D. dadantii
pectate lyases belong to different families, namely, PL1, PL2, PL3, and PL9. Analysis of the
D. dadantii
3937 genome revealed a gene encoding a new protein of the PL9 family, which already includes the secreted endopectate lyase PelL and the periplasmic exopectate lyase PelX. We demonstrated that PelN is an additional extracellular protein secreted by the Out system. However, PelN has some unusual characteristics. Although most pectate lyases require a very alkaline pH and Ca
2+
for their activity, the PelN activity is optimal at pH 7.4 and in the presence of Fe
2+
as a cofactor. PelN is only weakly affected by the degree of pectin methyl esterification. The PelN structural model, constructed on the basis of the PelL structure, suggests that the PelL global topology and its catalytic amino acids are conserved in PelN. Notable differences concern the presence of additional loops at the PelN surface, and the replacement of PelL charged residues, involved in substrate binding, by aromatic residues in PelN. The
pelN
expression is affected by different environmental conditions, such as pH, osmolarity, and temperature. It is controlled by the repressors KdgR and PecS and by the activator GacA, three regulators of
D. dadantii
pectinase genes. Since a
pelN
mutant had reduced virulence on chicory leaves, the PelN enzyme plays a role in plant infection, despite its low specific activity and its unusual cofactor requirement.
Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans
Authors: Childress Catherine , Feuerbacher Leigh A. , Phillips Linda , Burgum Alex , Kolodrubetz David
Published: March 2013

Aggregatibacter actinomycetemcomitans
, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the
ltxA
operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases
ltxA
expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible for the anaerobic induction of
ltxA
RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of
ltxA
RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions −69 to −35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which is a repressor in
Escherichia coli
, functions as a direct transcriptional activator in
A. actinomycetemcomitans
; an
mlc
deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the −68 to −40 region of the leukotoxin promoter. Furthermore, we show that CRP activates
ltxA
expression indirectly by increasing the levels of Mlc. Analyses of Δ
mlc
, Δ
ihf
, and Δ
ihf
Δ
mlc
strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses
ltxA
RNA synthesis mainly by blocking Mlc binding. Finally, a Δ
ihf
Δ
mlc
mutant still induces
ltxA
during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented.
Neisseria gonorrhoeae RecQ Helicase HRDC Domains Are Essential for Efficient Binding and Unwinding of the
pilE
Guanine Quartet Structure Required for Pilin Antigenic Variation
Authors: Cahoon Laty A. , Manthei Kelly A. , Rotman Ella , Keck James L. , Seifert H. Steven
Published: March 2013

The strict human pathogen
Neisseria gonorrhoeae
utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (
pilE
) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of
pilE
. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one “helicase and RNase D C-terminal” (HRDC) domain, whereas the
N. gonorrhoeae
RecQ helicase gene encodes three HRDC domains, which are critical for pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of pilin Av comparable to that obtained with a functional knockout. We demonstrate that the
N. gonorrhoeae
RecQ helicase can bind and unwind the
pilE
G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the
pilE
G4 structure.
Gap2 Promotes the Formation of a Stable Protein Complex Required for Mature Fap1 Biogenesis
Authors: Echlin Haley , Zhu Fan , Li Yirong , Peng Zhixiang , Ruiz Teresa , Bedwell Gregory J. , Prevelige Peter E. , Wu Hui
Published: March 2013

Serine-rich repeat glycoproteins (SRRPs) are important bacterial adhesins conserved in streptococci and staphylococci. Fap1, a SRRP identified in
Streptococcus parasanguinis
, is the major constituent of bacterial fimbriae and is required for adhesion and biofilm formation. An 11-gene cluster is required for Fap1 glycosylation and secretion; however, the exact mechanism of Fap1 biogenesis remains a mystery. Two glycosylation-associated proteins within this cluster—Gap1 and Gap3—function together in Fap1 biogenesis. Here we report the role of the third glycosylation-associated protein, Gap2. A
gap2
mutant exhibited the same phenotype as the
gap1
and
gap3
mutants in terms of Fap1 biogenesis, fimbrial assembly, and bacterial adhesion, suggesting that the three proteins interact. Indeed, all three proteins interacted with each other independently and together to form a stable protein complex. Mechanistically, Gap2 protected Gap3 from degradation by ClpP protease, and Gap2 required the presence of Gap1 for expression at the wild-type level. Gap2 augmented the function of Gap1 in stabilizing Gap3; this function was conserved in Gap homologs from
Streptococcus agalactiae
. Our studies demonstrate that the three Gap proteins work in concert in Fap1 biogenesis and reveal a new function of Gap2. This insight will help us elucidate the molecular mechanism of SRRP biogenesis in this bacterium and in pathogenic species.
Involvement of Cyclic AMP Receptor Protein in Regulation of the
rmf
Gene Encoding the Ribosome Modulation Factor in Escherichia coli
Authors: Shimada Tomohiro , Yoshida Hideji , Ishihama Akira
Published: March 2013

The decrease in overall translation in stationary-phase
Escherichia coli
is accompanied with the formation of functionally inactive 100S ribosomes mediated by the ribosome modulation factor (RMF). At present, however, little is known regarding the regulation of stationary-phase-coupled RMF expression. In the course of a systematic screening of regulation targets of DNA-binding transcription factors from
E. coli
, we realized that CRP (cyclic AMP [cAMP] receptor protein), the global regulator for carbon source utilization, participates in regulation of some ribosomal protein genes, including the
rmf
gene. In this study, we carried out detailed analysis of the regulation of the RMF gene by cAMP-CRP. The cAMP-dependent binding of CRP to the
rmf
gene promoter was confirmed by gel shift and DNase I footprinting assays. By using a reporter assay system, the expression level of RMF was found to decrease in the
crp
knockout mutant, indicating the involvement of CRP as an activator of the
rmf
promoter. In good agreement with the reduction of
rmf
promoter activity, we observed decreases in RMF production and 100S ribosome dimerization in the absence of CRP. Taken together, we propose that CRP regulates transcription activation of the
rmf
gene for formation of 100S ribosome dimers. Physiological roles of CRP involvement in RMF production are discussed.
Biochemistry and Physiology of the β Class Carbonic Anhydrase (Cpb) from Clostridium perfringens Strain 13
Authors: Kumar R. Siva Sai , Hendrick William , Correll Jared B. , Patterson Andrew D. , Melville Stephen B. , Ferry James G.
Published: March 2013

The carbonic anhydrase (Cpb) from
Clostridium perfringens
strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the β class, the first β class enzyme investigated from a strictly anaerobic species of the domain
Bacteria
. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with

K
m

and
k
cat
/

K
m

values of 3.1 mM CO
2
and 4.8 × 10
6
s
−1
M
−1
, respectively. Analyses of a
cpb
deletion mutant of
C. perfringens
strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO
2
. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO
2
in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO
2
fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.
Cloning and Analysis of the Planosporicin Lantibiotic Biosynthetic Gene Cluster of Planomonospora alba
Authors: Sherwood Emma J. , Hesketh Andrew R. , Bibb Mervyn J.
Published: March 2013

The increasing prevalence of antibiotic resistance in bacterial pathogens has renewed focus on natural products with antimicrobial properties. Lantibiotics are ribosomally synthesized peptide antibiotics that are posttranslationally modified to introduce (methyl)lanthionine bridges. Actinomycetes are renowned for their ability to produce a large variety of antibiotics, many with clinical applications, but are known to make only a few lantibiotics. One such compound is planosporicin produced by
Planomonospora alba
, which inhibits cell wall biosynthesis in Gram-positive pathogens. Planosporicin is a type AI lantibiotic structurally similar to those which bind lipid II, the immediate precursor for cell wall biosynthesis. The gene cluster responsible for planosporicin biosynthesis was identified by genome mining and subsequently isolated from a
P. alba
cosmid library. A minimal cluster of 15 genes sufficient for planosporicin production was defined by heterologous expression in
Nonomuraea
sp. strain ATCC 39727, while deletion of the gene encoding the precursor peptide from
P. alba
, which abolished planosporicin production, was also used to confirm the identity of the gene cluster. Deletion of genes encoding likely biosynthetic enzymes identified through bioinformatic analysis revealed that they, too, are essential for planosporicin production in the native host. Reverse transcription-PCR (RT-PCR) analysis indicated that the planosporicin gene cluster is transcribed in three operons. Expression of one of these,
pspEF
, which encodes an ABC transporter, in
Streptomyces coelicolor
A3(2) conferred some degree of planosporicin resistance on the heterologous host. The inability to delete these genes from
P. alba
suggests that they play an essential role in immunity in the natural producer.
Subacute combined degeneration of the spinal cord following recreational nitrous oxide use
Authors: Cheng Hui Mei , Park Jae Hong , Hernstadt David
Published: March 2013

We describe a case of a young woman who developed myelopathy and peripheral neuropathy following 1 year of recreational nitrous oxide (N2O) use. She presented with uncomfortable tingling sensation in her feet and poor balance. Physical examination revealed mild weakness and hyper-reflexia in the lower limbs. Proprioception and vibration were absent in the lower limbs. Pinprick and light touch sensations were reduced below the sixth thoracic dermatome. A broad-based gait and a positive Romberg sign were noted. The level of vitamin B12was low (76 pmol/l). T2-weighted MRI scans showed hyperintensity changes at the posterior column from the second to the fifth cervical vertebrae. She made a full recovery following injections of vitamin B12and physiotherapy. This case discusses subacute combined degeneration of the spinal cord on a background of N2O abuse.
Delayed resolution of eyelid swelling in preseptal cellulitis in a child: beware of causing occlusion amblyopia
Authors: Upendran Muralidharan R , McLoone Eibhlin
Published: March 2013

A 7-month-old child presented with a 6-day history of right eyelid swelling and redness. She was being treated with chloramphenicol ointment and oral flucloxacillin by the general practitioner. Clinical examination revealed features of preseptal cellulitis. There was no evidence of orbital involvement and the child was generally well otherwise. Her oral antibiotics were changed and she was adviced to attend for a review in 2 days time. However, the child did not return for a review. Three weeks later following resolution of the eyelid swelling, the child represented with a divergent squint in the right eye. Clinical and orthoptic investigations suggested occlusion amblyopia. The child is currently receiving patching treatment for the amblyopia and is awaiting surgery for squint eye.