bluuebunny/crossref_metadata_2025 huggingface dataset:
Biotransformations of 2-Methylisoborneol by Camphor-Degrading Bacteria
Authors: Eaton Richard W. , Sandusky Peter
Published: December 2008

Many camphor-degrading bacteria that are able to transform 2-methylisoborneol (2-MIB) have been identified. Three of these strains have been examined in detail.Rhodococcus ruberT1 metabolizes camphor through 6-hydroxycamphor but converts 2-MIB to 3-hydroxy-2-MIB.Pseudomonas putidaG1, which metabolizes camphor through 5-hydroxycamphor, converts MIB primarily to 6-hydroxy-2-MIB.Rhodococcus wratislaviensisDLC-cam converts 2-MIB through 5-hydroxy-2-MIB to 5-keto-2-MIB. Together, these three strains produce metabolites resulting from hydroxylation at all of the three available secondary carbons on the six-member ring of 2-MIB.
Bioproduction of
p
-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium
Pseudomonas putida
S12 in a Two-Phase Water-Decanol Fermentation
Authors: Verhoef Suzanne , Wierckx Nick , Westerhof R. G. Maaike , de Winde Johannes H. , Ruijssenaars Harald J.
Published: December 2008

Two solvent-tolerant
Pseudomonas putida
S12 strains, originally designed for phenol and
p
-coumarate production, were engineered for efficient production of
p
-hydroxystyrene from glucose. This was established by introduction of the genes
pal
and
pdc
encoding
l
-phenylalanine/
l
-tyrosine ammonia lyase and
p
-coumaric acid decarboxylase, respectively. These enzymes allow the conversion of the central metabolite
l
-tyrosine into
p
-hydroxystyrene, via
p
-coumarate. Degradation of the
p
-coumarate intermediate was prevented by inactivating the
fcs
gene encoding feruloyl-coenzyme A synthetase. The best-performing strain was selected and cultivated in the fed-batch mode, resulting in the formation of 4.5 mM
p
-hydroxystyrene at a yield of 6.7% (C-mol of
p
-hydroxystyrene per C-mol of glucose) and a maximum volumetric productivity of 0.4 mM h
−1
. At this concentration, growth and production were completely halted due to the toxicity of
p
-hydroxystyrene. Product toxicity was overcome by the application of a second phase of 1-decanol to extract
p
-hydroxystyrene during fed-batch cultivation. This resulted in a twofold increase of the maximum volumetric productivity (0.75 mM h
−1
) and a final total
p
-hydroxystyrene concentration of 21 mM, which is a fourfold improvement compared to the single-phase fed-batch cultivation. The final concentration of
p
-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM (17.6 g liter
−1
) was obtained in the 1-decanol phase. Thus, a
P. putida
S12 strain producing the low-value compound phenol was successfully altered for the production of the toxic value-added compound
p
-hydroxystyrene.
Energy Metabolism Response to Low-Temperature and Frozen Conditions in
Psychrobacter cryohalolentis
Authors: Amato Pierre , Christner Brent C.
Published: December 2008

Studies of cold-active enzymes have provided basic information on the molecular and biochemical properties of psychrophiles; however, the physiological strategies that compensate for low-temperature metabolism remain poorly understood. We investigated the cellular pools of ATP and ADP in
Psychrobacter cryohalolentis
K5 incubated at eight temperatures between 22°C and −80°C. Cellular ATP and ADP concentrations increased with decreasing temperature, and the most significant increases were observed in cells that were incubated as frozen suspensions (<−5°C). Respiratory uncoupling significantly decreased this temperature-dependent response, indicating that the proton motive force was required for energy adaptation to frozen conditions. Since ATP and ADP are key substrates in metabolic and energy conservation reactions, increasing their concentrations may provide a strategy for offsetting the kinetic temperature effect, thereby maintaining reaction rates at low temperature. The adenylate levels increased significantly <1 h after freezing and also when the cells were osmotically shocked to simulate the elevated solute concentrations encountered in the liquid fraction of the ice. Together, these data demonstrate that a substantial change in cellular energy metabolism is required for the cell to adapt to the low temperature and water activity conditions encountered during freezing. This physiological response may represent a critical biochemical compensation mechanism at low temperature, have relevance to cellular survival during freezing, and be important for the persistence of microorganisms in icy environments.
Genome Survey and Characterization of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and Development of Poplar Trees
Authors: Taghavi Safiyh , Garafola Craig , Monchy Sébastien , Newman Lee , Hoffman Adam , Weyens Nele , Barac Tanja , Vangronsveld Jaco , van der Lelie Daniel
Published: December 2008

The association of endophytic bacteria with their plant hosts has a beneficial effect for many different plant species. Our goal is to identify endophytic bacteria that improve the biomass production and the carbon sequestration potential of poplar trees (
Populus
spp.) when grown in marginal soil and to gain an insight in the mechanisms underlying plant growth promotion. Members of the
Gammaproteobacteria
dominated a collection of 78 bacterial endophytes isolated from poplar and willow trees. As representatives for the dominant genera of endophytic gammaproteobacteria, we selected
Enterobacter
sp. strain 638,
Stenotrophomonas maltophilia
R551-3,
Pseudomonas putida
W619, and
Serratia proteamaculans
568 for genome sequencing and analysis of their plant growth-promoting effects, including root development. Derivatives of these endophytes, labeled with
gfp
, were also used to study the colonization of their poplar hosts. In greenhouse studies, poplar cuttings (
Populus deltoides
×
Populus nigra
DN-34) inoculated with
Enterobacter
sp. strain 638 repeatedly showed the highest increase in biomass production compared to cuttings of noninoculated control plants. Sequence data combined with the analysis of their metabolic properties resulted in the identification of many putative mechanisms, including carbon source utilization, that help these endophytes to thrive within a plant environment and to potentially affect the growth and development of their plant hosts. Understanding the interactions between endophytic bacteria and their host plants should ultimately result in the design of strategies for improved poplar biomass production on marginal soils as a feedstock for biofuels.
Novel Nicotine Oxidoreductase-Encoding Gene Involved in Nicotine Degradation by
Pseudomonas putida
Strain S16
Authors: Tang Hongzhi , Wang Lijuan , Meng Xiangzhou , Ma Lanying , Wang Shuning , He Xiaofei , Wu Geng , Xu Ping
Published: December 2008

There are quite a few ongoing biochemical investigations of nicotine degradation in different organisms. In this work, we identified and sequenced a gene (designated
nicA
) involved in nicotine degradation by
Pseudomonas putida
strain S16. The gene product, NicA, was heterologously expressed and characterized as a nicotine oxidoreductase catalyzing the initial steps of nicotine metabolism. Biochemical analyses using resting cells and the purified enzyme suggested that
nicA
encodes an oxidoreductase, which converts nicotine to 3-succinoylpyridine through pseudooxynicotine. Based on enzymatic reactions and direct evidence obtained using H
2
18
O labeling, the process may consist of enzyme-catalyzed dehydrogenation, followed by spontaneous hydrolysis and then repetition of the dehydrogenation and hydrolysis steps. Sequence comparisons revealed that the gene showed 40% similarity to genes encoding NADH dehydrogenase subunit I and cytochrome
c
oxidase subunit I in eukaryotes. Our findings demonstrate that the molecular mechanism for nicotine degradation in strain S16 involves the pyrrolidine pathway and is similar to the mechanism in mammals, in which pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen, is produced.
Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in
Saccharomyces cerevisiae
Authors: Sullivan David P. , Georgiev Alexander , Menon Anant K.
Published: December 2008

Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated
Saccharomyces cerevisiae
mutants with defects in sterol transport. We used
Upc2-1
cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [
3
H]cholesterol in the form of [
3
H]cholesteryl ester requires an intact PM-ER sterol transport pathway.
Upc2-1
cells were mutagenized using a transposon library, incubated with [
3
H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [
3
H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as
DET1
(
d
ecreased
e
rgosterol
t
ransport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in
det1
Δ cells. These data indicate that the cytoplasmic protein encoded by
DET1
is involved in intracellular sterol transport.
Mutational Analysis of the
Candida albicans
Ammonium Permease Mep2p Reveals Residues Required for Ammonium Transport and Signaling
Authors: Dabas Neelam , Schneider Sabrina , Morschhäuser Joachim
Published: December 2008

The ammonium permease Mep2p mediates ammonium uptake and also induces filamentous growth in the human-pathogenic yeast
Candida albicans
in response to nitrogen limitation. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is not required for ammonium transport but is essential for Mep2p-dependent morphogenesis. Progressive C-terminal truncations showed Y433 to be the last amino acid that is essential for the induction of filamentous growth, thereby delimiting the Mep2p signaling domain. To understand in more detail how the signaling activity of Mep2p is regulated by ammonium availability and transport, we mutated conserved amino acid residues that have been implicated in ammonium binding or uptake. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is thought to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filament formation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filament formation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filament formation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. These results provide important insights into ammonium transport and control of morphogenesis by Mep2p in
C. albicans
.
Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads andAgrobacterium tumefaciensStrain C58
Authors: Yoon Sang-Hwal , Moon Tae Seok , Iranpour Pooya , Lanza Amanda M. , Prather Kristala Jones
Published: December 2008

Uronate dehydrogenase has been cloned fromPseudomonas syringaepv. tomato strain DC3000,Pseudomonas putidaKT2440, andAgrobacterium tumefaciensstrain C58. The genes were identified by using a novel complementation assay employing anEscherichia colimutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library ofP. syringaewas screened in the mutantE. coliby growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to beudh. Homologous enzymes inP. putidaandA. tumefacienswere identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed inE. coliwere purified and characterized. For all three enzymes, the turnover number (kcat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (Km) for galacturonate was lower than that for glucuronate. TheA. tumefaciensenzyme was found to have the highest rate constant (kcat= 1.9 × 102s−1on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.
Delineation of the Species
Haemophilus influenzae
by Phenotype, Multilocus Sequence Phylogeny, and Detection of Marker Genes
Authors: Nørskov-Lauritsen Niels , Overballe Merete D. , Kilian Mogens
Published: December 2008

To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of
Haemophilus influenzae
by comparative analysis of
H. influenzae
reference strains with closely related bacteria including strains assigned to
Haemophilus haemolyticus
, cryptic genospecies biotype IV, and the never formally validated species “
Haemophilus intermedius”
. Multilocus sequence phylogeny based on six housekeeping genes separated a cluster encompassing the type and the reference strains of
H. influenzae
from 31 more distantly related strains. Comparison of 16S rRNA gene sequences supported this delineation but was obscured by a conspicuously high number of polymorphic sites in many of the strains that did not belong to the core group of
H. influenzae
strains. The division was corroborated by the differential presence of genes encoding
H. influenzae
adhesion and penetration protein, fuculokinase, and Cu,Zn-superoxide dismutase, whereas immunoglobulin A1 protease activity or the presence of the
iga
gene was of limited discriminatory value. The existence of porphyrin-synthesizing strains (“
H. intermedius
”) closely related to
H. influenzae
was confirmed. Several chromosomally encoded hemin biosynthesis genes were identified, and sequence analysis showed these genes to represent an ancestral genotype rather than recent transfers from, e.g.,
Haemophilus parainfluenzae
. Strains previously assigned to
H. haemolyticus
formed several separate lineages within a distinct but deeply branching cluster, intermingled with strains of “
H. intermedius
” and cryptic genospecies biotype IV. Although
H. influenzae
is phenotypically more homogenous than some other
Haemophilus
species, the genetic diversity and multicluster structure of strains traditionally associated with
H. influenzae
make it difficult to define the natural borders of that species.
The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in
Neisseria meningitidis
Authors: Metruccio Matteo M. E. , Fantappiè Laura , Serruto Davide , Muzzi Alessandro , Roncarati Davide , Donati Claudio , Scarlato Vincenzo , Delany Isabel
Published: December 2008

Previous microarray studies have suggested that an indirect mechanism of Fur regulation may be present in meningococcus at the posttranscriptional level through a small regulatory RNA (sRNA) system analogous to that of
Escherichia coli
and
Pseudomonas aeruginosa
. Recently, a Fur-regulated sRNA, NrrF, was identified that is involved in the iron regulation of the
sdhA
and
sdhC
succinate dehydrogenase genes. Here we report a detailed transcriptional analysis of the
nrrF
gene and show that NrrF is a Hfq-dependent sRNA. The Hfq protein mediates
nrrF
downregulation and Fur-dependent upregulation of the
sdhCDAB
operon, the major in vivo NrrF-regulated operon. NrrF forms a duplex in vitro with a region of complementarity overlapping the
sdhDA
mRNA junction. Furthermore, Hfq binds to NrrF in vitro and considerably enhances the efficiency of the interaction of the sRNA with the identified target. Our data suggest that Hfq-meditated binding of NrrF to the in vivo target in the
sdhCDAB
mRNA may cause the rapid degradation of the transcript, resulting in Fur-dependent positive regulation of succinate dehydrogenase. In addition, while the upregulation of
sodB
and
fumB
by Fur is dependent on the Hfq protein, it is unaffected in the
nrrF
knockout, which suggests that there is more than one sRNA regulator involved in iron homeostasis in meningococcus.
Catabolite Repression Control ofnapF(Periplasmic Nitrate Reductase) Operon Expression inEscherichia coliK-12
Authors: Stewart Valley , Bledsoe Peggy J. , Chen Li-Ling , Cai Amie
Published: December 2008

Escherichia coli, a facultative aerobe, expresses two distinct respiratory nitrate reductases. The periplasmic NapABC enzyme likely functions during growth in nitrate-limited environments, whereas the membrane-bound NarGHI enzyme functions during growth in nitrate-rich environments. Maximal expression of thenapFDAGHBCoperon encoding periplasmic nitrate reductase results from synergistic transcription activation by the Fnr and phospho-NarP proteins, acting in response to anaerobiosis and nitrate or nitrite, respectively. Here, we report that, during anaerobic growth with no added nitrate, less-preferred carbon sources stimulatednapFoperon expression by as much as fourfold relative to glucose. Deletion analysis identified a cyclic AMP receptor protein (Crp) binding site upstream of the NarP and Fnr sites as being required for this stimulation. ThenapDandnrfAoperon control regions fromShewanellaspp. also have apparent Crp and Fnr sites, and expression from theShewanella oneidensis nrfAcontrol region cloned inE. coliwas subject to catabolite repression. In contrast, the carbon source had relatively little effect on expression of thenarGHJIoperon encoding membrane-bound nitrate reductase under any growth condition tested. Carbon source oxidation state had no influence on synthesis of either nitrate reductase. The results suggest that the Fnr and Crp proteins may act synergistically to enhance NapABC synthesis during growth with poor carbon sources to help obtain energy from low levels of nitrate.
HicA of
Escherichia coli
Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea
Authors: Jørgensen Mikkel G. , Pandey Deo P. , Jaskolska Milena , Gerdes Kenn
Published: December 2008

Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the
hicAB
loci constitute a novel TA gene family. Using the
hicAB
locus of
Escherichia coli
K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong
hicAB
transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that
hicAB
loci are abundant in bacteria and archaea.
Conjugative Interaction Induces Transposition of IS
Pst9
in
Pseudomonas stutzeri
AN10
Authors: Christie-Oleza J. A. , Lanfranconi M. P. , Nogales B. , Lalucat J. , Bosch R.
Published: December 2008

IS
Pst9
is an IS
L3
-like insertion sequence (IS) that was recently described in the naphthalene-degrading organism
Pseudomonas stutzeri
strain AN10. In this paper we describe a novel strong IS regulation stimulus; transposition of IS
Pst9
is induced in all
P. stutzeri
AN10 cells after conjugative interaction with
Escherichia coli
. Thus, we observed that in all
P. stutzeri
AN10 cells that received genetic material by conjugation the IS
Pst9
genomic dose and/or distribution was changed. Furthermore, IS
Pst9
transposition was also observed when
P. stutzeri
AN10 cells were put in contact with the plasmidless conjugative strain
E. coli
S17-1λ

pir

, but not when they were put in contact with
E. coli
DH5α (a nonconjugative strain). The mechanism of IS
Pst9
transposition was analyzed, and transposition was shown to proceed by excision from the donor DNA using a conservative mechanism, which generated 3- to 10-bp deletions of the flanking DNA. Our results indicate that IS
Pst9
transposes, forming double-stranded DNA circular intermediates consisting of the IS and a 5-bp intervening DNA sequence probably derived from the IS
Pst9
flanking regions. The kinetics of IS circle formation are also described.
The Global Virulence Regulator PhcA Negatively Controls the
Ralstonia solanacearum hrp
Regulatory Cascade by Repressing Expression of the PrhIR Signaling Proteins
Authors: Yoshimochi Takeshi , Hikichi Yasufumi , Kiba Akinori , Ohnishi Kouhei
Published: December 2008

PhcA positively and negatively regulates many genes responsible for pathogenicity of
Ralstonia solanacearum
. The type III secretion system-encoding
hrp
regulon is one of the negatively controlled operons. PhcA bound to the promoter region of
prhIR
and repressed its expression, demonstrating that PhcA shuts down the most upstream component of a signal transfer system for
hrpB
activation.
Trigger Factor from the Psychrophilic Bacterium
Psychrobacter frigidicola
Is a Monomeric Chaperone
Authors: Robin Sylvain , Togashi Denisio M. , Ryder Alan G. , Wall J. Gerard
Published: December 2008

In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from
Psychrobacter frigidicola
(TF

Pf

) was cloned, produced in
Escherichia coli
, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric
E. coli
TF (TF

Ec

). Moreover, TF

Pf

did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its
E. coli
counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the
E. coli
chaperone. TF

Pf

was also significantly more effective than TF

Ec

at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF

Pf

differs from TF

Ec

in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.
The Pentapeptide Repeat Proteins MfpA
Mt
and QnrB4 Exhibit Opposite Effects on DNA Gyrase Catalytic Reactions and on the Ternary Gyrase-DNA-Quinolone Complex
Authors: Mérens Audrey , Matrat Stéphanie , Aubry Alexandra , Lascols Christine , Jarlier Vincent , Soussy Claude-James , Cavallo Jean-Didier , Cambau Emmanuelle
Published: December 2008

MfpA
Mt
and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The
mfpA
Mt
gene is chromosome borne in
Mycobacterium tuberculosis
, while
qnrB4
is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA
Mt
on
M. tuberculosis
gyrase and the effect of QnrB4 on
Escherichia coli
gyrase. Whereas QnrB4 inhibited
E. coli
gyrase activity only at concentrations higher than 30 μM, MfpA
Mt
inhibited all catalytic reactions of the
M. tuberculosis
gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 μM. We showed that the D87 residue in GyrA has a major role in the MfpA
Mt
-gyrase interaction, as D87H and D87G substitutions abolished MfpA
Mt
inhibition of
M. tuberculosis
gyrase catalytic reactions, while A83S modification did not. Since MfpA
Mt
and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of
E. coli
gyrase at 0.1 μM as described for other Qnr proteins, but MfpA
Mt
did not modify
M. tuberculosis
gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA
Mt
also inhibited
E. coli
gyrase function, while QnrB4 did not reverse quinolone inhibition of
M. tuberculosis
gyrase. In conclusion, our in vitro experiments showed that MfpA
Mt
and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.
Chenodeoxycholate Is an Inhibitor of
Clostridium difficile
Spore Germination
Authors: Sorg Joseph A. , Sonenshein Abraham L.
Published: December 2008

Some cholate derivatives that are normal components of bile can act with glycine to induce the germination of
Clostridium difficile
spores, but at least one bile component, chenodeoxycholate, does not induce germination. Here we show that chenodeoxycholate inhibits the germination of
C. difficile
spores in response to cholate and taurocholate.
Expression of
yeeK
during
Bacillus subtilis
Sporulation and Localization of YeeK to the Inner Spore Coat using Fluorescence Microscopy
Authors: Takamatsu Hiromu , Imamura Daisuke , Kuwana Ritsuko , Watabe Kazuhito
Published: December 2008

The
yeeK
gene of
Bacillus subtilis
is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in
yabG
mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the
yeeK
gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of
yeeK
did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in
yabG
mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.
Purine Utilization byKlebsiella oxytocaM5al: Genes for Ring-Oxidizing and -Opening Enzymes
Authors: Pope Scott D. , Chen Li-Ling , Stewart Valley
Published: December 2008

The enterobacteriumKlebsiella oxytocauses a variety of inorganic and organic nitrogen sources, including purines, nitrogen-rich compounds that are widespread in the biosphere. We have identified a 23-gene cluster that encodes the enzymes for utilizing purines as the sole nitrogen source. Growth and complementation tests with insertion mutants, combined with sequence comparisons, reveal functions for the products of these genes. Here, we report our characterization of 12 genes, one encoding guanine deaminase and the others encoding enzymes for converting (hypo)xanthine to allantoate. Conventionally, xanthine dehydrogenase, a broadly distributed molybdoflavoenzyme, catalyzes sequential hydroxylation reactions to convert hypoxanthine via xanthine to urate. Our results show that these reactions inK. oxytocaare catalyzed by a two-component oxygenase (HpxE-HpxD enzyme) homologous to Rieske nonheme iron aromatic-ring-hydroxylating systems, such as phthalate dioxygenase. Our results also reveal previously undescribed enzymes involved in urate oxidation to allantoin, catalyzed by a flavoprotein monooxygenase (HpxO enzyme), and in allantoin conversion to allantoate, which involves allantoin racemase (HpxA enzyme). The pathway also includes the recently described PuuE allantoinase (HpxB enzyme). The HpxE-HpxD and HpxO enzymes were discovered independently by de la Riva et al. (L. de la Riva, J. Badia, J. Aguilar, R. A. Bender, and L. Baldoma, J. Bacteriol. 190:7892-7903, 2008). Thus, several enzymes in thisK. oxytocapurine utilization pathway differ from those in other microorganisms. Isofunctional homologs of these enzymes apparently are encoded by other species, includingAcinetobacter,Burkholderia,Pseudomonas,Saccharomyces, andXanthomonas.
Comparison of Responses to Double-Strand Breaks betweenEscherichia coliandBacillus subtilisReveals Different Requirements for SOS Induction
Authors: Simmons Lyle A. , Goranov Alexi I. , Kobayashi Hajime , Davies Bryan W. , Yuan Daniel S. , Grossman Alan D. , Walker Graham C.
Published: December 2008

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in bothEscherichia coliandBacillus subtilis. InE. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell.E. colistrains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that inB. subtilisboth ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction inE. colibut not inB. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in bothE. coliandB. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction inB. subtilis. Furthermore, we found thatB. subtiliscells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover,B. subtilisRecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially betweenE. coliandB. subtilis.
Functional Roles of
arcA
,
etrA
, Cyclic AMP (cAMP)-cAMP Receptor Protein, and
cya
in the Arsenate Respiration Pathway in
Shewanella
sp. Strain ANA-3
Authors: Murphy Julie N. , Durbin K. James , Saltikov Chad W.
Published: December 2008

Microbial arsenate respiration can enhance arsenic release from arsenic-bearing minerals—a process that can cause arsenic contamination of water. In
Shewanella
sp. strain ANA-3, the arsenate respiration genes (
arrAB
) are induced under anaerobic conditions with arsenate and arsenite. Here we report how genes that encode anaerobic regulator (
arcA
and
etrA
[
fnr
homolog]) and carbon catabolite repression (
crp
and
cya
) proteins affect arsenate respiration in ANA-3. Transcription of
arcA
,
etrA
, and
crp
in ANA-3 was similar in cells grown on arsenate and cells grown under aerobic conditions. ANA-3 strains lacking
arcA
and
etrA
showed minor to moderate growth defects, respectively, with arsenate. However,
crp
was essential for growth on arsenate. In contrast to the wild-type strain,
arrA
was not induced in the
crp
mutant in cultures shifted from aerobic to anaerobic conditions containing arsenate. This indicated that cyclic AMP (cAMP)-cyclic AMP receptor (CRP) activates
arr
operon transcription. Computation analysis for genome-wide CRP binding motifs identified a putative binding motif within the
arr
promoter region. This was verified by electrophoretic mobility shift assays with cAMP-CRP and several DNA probes. Lastly, four putative adenylate cyclase (
cya
) genes were identified in the genome. One particular
cya
-like gene was differentially expressed under aerobic versus arsenate respiration conditions. Moreover, a double mutant lacking two of the
cya
-like genes could not grow with arsenate as a terminal electron acceptor; exogenous cAMP could complement growth of the double
cya
mutant. It is concluded that the components of the carbon catabolite repression system are essential to regulating arsenate respiratory reduction in
Shewanella
sp. strain ANA-3.
Streptomyces
Telomeres Contain a Promoter
Authors: Lin Yuh-ru , Hahn Mi-Young , Roe Jung-Hye , Huang Tzu-Wen , Tsai Hsiu-Hui , Lin Yung-Feng , Su Tsung-Sheng , Chan Yu-Jiun , Chen Carton W.
Published: December 2008

Bidirectional replication of the linear chromosomes and plasmids of
Streptomyces
spp. results in single-strand overhangs at their 3′ ends, which contain extensive complex palindromic sequences. The overhangs are believed to be patched by DNA synthesis primed by a terminal protein that remains covalently bound to the 5′ ends of the telomeres. We discovered that in vitro a conserved 167-bp telomere DNA binds strongly to RNA polymerase holoenzyme and exhibits promoter activities stronger than those of an rRNA operon. In vivo, the telomere DNA exhibited promoter activity in both orientations on a circular plasmid in
Streptomyces
. The telomere promoter is also active on a linear plasmid during exponential growth. Such promoter activity in a telomere has not hitherto been observed in eukaryotic or prokaryotic replicons.
Streptomyces
telomere promoters may be involved in priming the terminal Okazaki fragment (during replication) replicative transfer (during conjugation), or expression of downstream genes (including a conserved
ttrA
helicase-like gene involved in conjugal transfer). Interestingly, the
Streptomyces
telomeres also function as a promoter in
Escherichia coli
and as a transcription enhancer in yeast.
Control of Gluconate Utilization in
Sinorhizobium meliloti
Authors: Steele Tyler T. , Fowler Christopher W. , Griffitts Joel S.
Published: December 2008

The
Sinorhizobium meliloti
megaplasmid pSymA has previously been implicated in gluconate utilization. We report a locus on pSymA encoding a putative tripartite ATP-independent periplasmic (TRAP) transporter that is required for gluconate utilization. The expression of this locus is negatively regulated by a GntR family regulator encoded adjacent to the transporter operon.
Mycobacterium aviumGenes MAV_5138 and MAV_3679 Are Transcriptional Regulators That Play a Role in Invasion of Epithelial Cells, in Part by Their Regulation of CipA, a Putative Surface Protein Interacting with Host Cell Signaling Pathways
Authors: Harriff Melanie J. , Danelishvili Lia , Wu Martin , Wilder Cara , McNamara Michael , Kent Michael L. , Bermudez Luiz E.
Published: December 2008

TheMycobacterium aviumcomplex (MAC) is an important group of opportunistic pathogens for birds, cattle, swine, and immunosuppressed humans. Although invasion of epithelial cells lining the intestine is the chief point of entry for these organisms, little is known about the mechanisms by which members of the MAC are taken up by these cells. Studies withM. aviumhave shown that cytoskeletal rearrangement via activation of the small G-protein Cdc42 is involved and that this activation is regulated in part by theM. avium fadD2gene. ThefadD2gene indirectly regulates a number of genes upon exposure to HEp-2 cells, including transcriptional regulators, membrane proteins, and secreted proteins. Overexpression of two fadD2-associated regulators (MAV_5138 and MAV_3679) led to increased invasion of HEp-2 cells, as well as altered expression of other genes. The protein product of one of the regulated genes, named CipA, has domains that resemble the PXXP motif of human Piccolo proteins, which bind SH3 domains in proteins involved in the scaffold complex formed during cytoskeletal rearrangement. Although CipA was not detected in the cytoplasm of HEp-2 cells exposed toM. avium, the recombinant protein was shown to be potentially expressed on the surface ofMycobacterium smegmatisincubated with HEp-2 cells and, possibly, to interact with human Cdc42. The interaction was then confirmed by showing that CipA activates Cdc42. These results suggest that members of theM. aviumcomplex have a novel mechanism for activating cytoskeletal rearrangement, prompting uptake by host epithelial cells, and that this mechanism is regulated in part byfadD2, MAV_5138, and MAV_3679.
Swimming Motility Mutants of Marine
Synechococcus
Affected in Production and Localization of the S-Layer Protein SwmA
Authors: McCarren J. , Brahamsha B.
Published: December 2008

The S-layer protein SwmA is required for nonflagellar swimming in marine
Synechococcus
. An analysis of mutations in seven genes at two loci in the
Synechococcus
sp. strain WH8102 genome indicates that a multicomponent transporter and glycosyltransferases are required for the production and proper localization of SwmA.
Y4lO of
Rhizobium
sp. Strain NGR234 Is a Symbiotic Determinant Required for Symbiosome Differentiation
Authors: Yang Feng-Juan , Cheng Li-Li , Zhang Ling , Dai Wei-Jun , Liu Zhe , Yao Nan , Xie Zhi-Ping , Staehelin Christian
Published: December 2008

Type 3 (T3) effector proteins, secreted by nitrogen-fixing rhizobia with a bacterial T3 secretion system, affect the nodulation of certain host legumes. The open reading frame y4lO of
Rhizobium
sp. strain NGR234 encodes a protein with sequence similarities to T3 effectors from pathogenic bacteria (the YopJ effector family). Transcription studies showed that the promoter activity of y4lO depended on the transcriptional activator TtsI. Recombinant Y4lO protein expressed in
Escherichia coli
did not acetylate two representative mitogen-activated protein kinase kinases (human MKK6 and MKK1 from
Medicago truncatula
), indicating that YopJ-like proteins differ with respect to their substrate specificities. The y4lO gene was mutated in NGR234 (strain NGRΩ
y4lO
) and in NGRΩ
nopL
, a mutant that does not produce the T3 effector NopL (strain NGRΩ
nopL
Ω
y4lO
). When used as inoculants, the symbiotic properties of the mutants differed.
Tephrosia vogelii, Phaseolus vulgaris
cv. Yudou No. 1, and
Vigna unguiculata
cv. Sui Qing Dou Jiao formed pink effective nodules with NGR234 and NGRΩ
nopL
Ω
y4lO
. Nodules induced by NGRΩ
y4lO
were first pink but rapidly turned greenish (ineffective nodules), indicating premature senescence. An ultrastructural analysis of the nodules induced by NGRΩ
y4lO
revealed abnormal formation of enlarged infection droplets in ineffective nodules, whereas symbiosomes harboring a single bacteroid were frequently observed in effective nodules induced by NGR234 or NGRΩ
nopL
Ω
y4lO
. It is concluded that Y4lO is a symbiotic determinant involved in the differentiation of symbiosomes. Y4lO mitigated senescence-inducing effects caused by the T3 effector NopL, suggesting synergistic effects for Y4lO and NopL in nitrogen-fixing nodules.
Covalently Linked Trimer of the AcrB Multidrug Efflux Pump Provides Support for the Functional Rotating Mechanism
Authors: Takatsuka Yumiko , Nikaido Hiroshi
Published: December 2008

Escherichia coli
AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three
acrB
sequences were connected together through short linker sequences. The “linked-trimer” AcrB was expressed well in the inner membrane fraction of Δ
acrB
Δ
recA
strains, as a large protein of ∼300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.
Pseudomonas aeruginosa
PAO1 Pyocin Production Affects Population Dynamics within Mixed-Culture Biofilms
Authors: Waite Richard D. , Curtis Michael A.
Published: December 2008

Transcriptomic and phenotypic studies showed that pyocins are produced in
Pseudomonas aeruginosa
PAO1 aerobic and anaerobic biofilms. Pyocin activity was found to be high in slow-growing anaerobic biofilms but transient in aerobic biofilms. Biofilm coculture of strain PAO1 and a pyocin-sensitive isolate showed that pyocin production had a significant impact on bacterial population dynamics, particularly under anaerobic conditions.
Toxin-Antitoxin Systems in
Escherichia coli
Influence Biofilm Formation through YjgK (TabA) and Fimbriae
Authors: Kim Younghoon , Wang Xiaoxue , Ma Qun , Zhang Xue-Song , Wood Thomas K.
Published: December 2008

The roles of toxin-antitoxin (TA) systems in bacteria have been debated. Here, the role of five TA systems in regard to biofilm development was investigated (listed as toxin/antitoxin: MazF/MazE, RelE/RelB, ChpB, YoeB/YefM, and YafQ/DinJ). Although these multiple TA systems were reported previously to not impact bacterial fitness, we found that deletion of the five TA systems decreased biofilm formation initially (8 h) on three different surfaces and then increased biofilm formation (24 h) by decreasing biofilm dispersal. Whole-transcriptome profiling revealed that the deletion of the five TA systems induced expression of a single gene,
yjgK
, which encodes an uncharacterized protein; quantitative real-time PCR (qRT-PCR) confirmed consistent induction of this gene (at 8, 15, and 24 h). Corroborating the complex phenotype seen upon deleting the TA systems, overexpression of YjgK decreased biofilm formation at 8 h and increased biofilm formation at 24 h; deletion of
yjgK
also affected biofilm formation in the expected manner by increasing biofilm formation after 8 h and decreasing biofilm formation after 24 h. In addition, YjgK significantly reduced biofilm dispersal. Whole-transcriptome profiling revealed YjgK represses fimbria genes at 8 h (corroborated by qRT-PCR and a yeast agglutination assay), which agrees with the decrease in biofilm formation upon deleting the five TA systems at 8 h, as well as that seen upon overexpressing YjgK. Sand column assays confirmed that deleting the five TA systems reduced cell attachment. Furthermore, deletion of each of the five toxins increased biofilm formation at 8 h, and overexpression of the five toxins repressed biofilm formation at 8 h, a result that is opposite that of deleting all five TA systems; this suggests that complex regulation occurs involving the antitoxins. Also, the ability of the global regulator Hha to reduce biofilm formation was dependent on the presence of these TA systems. Hence, we suggest that one role of TA systems is to influence biofilm formation.
Complete Genome Sequence of the Extremophilic
Bacillus cereus
Strain Q1 with Industrial Applications
Authors: Xiong Zhaohui , Jiang Yan , Qi Danhua , Lu Huaibao , Yang Fan , Yang Jian , Chen Lihong , Sun Lilian , Xu Xingye , Xue Ying , Zhu Yafang , Jin Qi
Published: December 2008

Bacillus cereus
strain Q1 was isolated from a deep-subsurface oil reservoir in the Daqing oil field in northeastern China. This strain is able to produce biosurfactants and to survive in extreme environments. Here we report the finished and annotated genome sequence of this organism.
Upregulation of Human T-Cell Leukemia Virus Type 1 Antisense Transcription by the Viral Tax Protein
Authors: Landry Sébastien , Halin Marilène , Vargas Amandine , Lemasson Isabelle , Mesnard Jean-Michel , Barbeau Benoit
Published: December 2008

Several studies have recently demonstrated the existence of human T-cell leukemia virus type 1 (HTLV-1) antisense transcripts, which allow the synthesis of the newly described HBZ protein. Although previous reports have been aimed at understanding the potential role of the HBZ protein in HTLV-1 pathogenesis, little is known as to how this viral gene is regulated. Here, using our K30-3′asLuc reporter construct, we show that the viral Tax protein upregulates antisense transcription through its action on the TRE sequences located in the 3′ long terminal repeat. Generation of stable clones in 293T cells demonstrated that Tax-induced HBZ expression is importantly influenced by the integration site in the host genome. The cellular DNA context could thus affect the level of HBZ mRNA expression in infected cells.