bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts
Authors: Chung Y.‐H. , Chang E.‐J. , Kim S.‐J. , Kim H.‐H. , Kim H.‐M. , Lee S.‐B. , Ko J. S.
Published: May 2006

Background and Objective:  Lipopolysaccharide is thought to be a major virulence factor of pathogens associated with periodontal diseases and is believed to stimulate bone resorption in vivo. Although Prevotella nigrescens has been implicated in periodontitis, its role in osteoclastogenesis has not been reported. In this study, we investigated the effects of lipopolysaccharide from P. nigrescens on the formation of osteoclasts and the production of cytokines related to osteoclast differentiation.

Material and Methods:  Mouse bone marrow mononuclear cells were cultured in the presence of macrophage colony‐stimulating factor (M‐CSF) and receptor activator of nuclear factor κB ligand (RANKL), with or without lipopolysaccharide. Bone marrow mononuclear cells were also cocultured with calvarial osteoblastic cells in the presence or absence of lipopolysaccharide. Osteoclast formation was determined by tartrate‐resistant acid phosphatase cytochemistry. The production of osteoprotegerin (OPG), M‐CSF, tumor necrosis factor alpha (TNF‐α), transforming growth factor‐beta (TGF‐β) and prostaglandin E2 (PGE2) was determined by enzyme‐linked immunosorbent assay (ELISA).

Results:  P. nigrescens lipopolysaccharide inhibited osteoclast differentiation from bone marrow mononuclear cells cultured in the presence of M‐CSF and RANKL. However, in the coculture system, P. nigrescens lipopolysaccharide stimulated osteoclastogenesis. Notably, P. nigrescens lipopolysaccharide decreased OPG production but increased TGF‐β secretion. In addition, treatment with P. nigrescens lipopolysaccharide increased PGE2 production during the late stage of the culture period. There was no difference in M‐CSF and TNF‐α production.

Conclusion:  These results demonstrate that P. nigrescens lipopolysaccharide stimulates osteoclastogenesis in the coculture system by decreasing the production of OPG and increasing the production of TGF‐β and PGE2. Through the mechanisms involving these factors, P. nigrescens lipopolysaccharide may cause alveolar bone resorption in periodontal diseases.
Hormone therapy is associated with smaller Achilles tendon diameter in active post‐menopausal women
Authors: Cook J. L. , Bass S. L. , Black J. E.
Published: May 2006

The effects of hormonal status and activity levels on Achilles tendon structure were examined in asymptomatic post‐menopausal women. It was hypothesized that women using hormone replacement therapy (HRT) would have better tendon structure than those not using HRT and that active women would have poorer tendon structure than inactive women.

Eighty‐five women including 53 active women (regular golf players) and 32 controls (healthy but inactive women) recorded their HRT and menopausal history and underwent basic anthropometric measurements. Women were divided into two groups based on their hormonal status: those currently using HRT; and those who had never used HRT or ceased using HRT at least 12 months prior to the study. Achilles tendons were examined with ultrasound and categorized as normal or abnormal, and the diameter of each tendon (mm) was recorded.

Active women had a greater prevalence of tendon abnormality (P=0.10) and thicker Achilles tendons than inactive women (P<0.05). Active women on HRT had less tendon abnormality (P=0.056) than active women not on HRT and significantly less tendon thickness (P<0.05).

This study indicates that Achilles tendon diameter is greater in active post‐menopausal women. Hormone replacement therapy appeared to ameliorate this effect in active women. A similar effect from HRT on the Achilles tendons of inactive women was not apparent.
Myocardial and peripheral vascular functional adaptation to exercise training
Authors: Hannukainen J. C. , Janatuinen T. , Toikka J. O. , Järvisalo M. J. , Heinonen O. J. , Kapanen J. , Någren K. , Nuutila P. , Kujala U. M. , Kaprio J. , Knuuti J. , Kalliokoski K. K.
Published: May 2006

Exercise training seems to restore impaired vascular function in both peripheral and myocardial vessels in patients with coronary artery and peripheral vascular disease or in patients with risk factors for these diseases. However, the results on the effects of exercise training on vascular function in apparently healthy subjects are controversial. We studied the effects of long‐term volitionally increased physical activity on peripheral and myocardial vascular function in nine young healthy male monozygotic twin pairs discordant for physical activity and fitness. The brothers were divided into more (MAG) and less active groups according to physical activity and fitness. The difference between groups in VO2max was 18±10% (P<0.001). Myocardial perfusion at rest, during adenosine‐induced vasodilatation and during cold‐pressor test and myocardial oxygen consumption were measured with positron emission tomography. In addition, endothelial function was measured using ultrasound in brachial and left anterior descending coronary arteries, and standard echocardiographic measures were taken. No differences were observed in myocardial perfusion measurements between groups. MAG tended to have a lower oxygen extraction fraction (P=0.06), but oxygen consumption was similar between the groups. No differences were found in coronary artery, myocardial resistance vessel or peripheral endothelial function between groups. These results suggest that when the effects of heredity are controlled, myocardial perfusion reserve and endothelial function, both in peripheral arteries and myocardial vessels, are not enhanced by increased physical activity and fitness in young healthy adult men.
Circulating Endothelial Progenitor Cells During Normal Pregnancy and Pre‐Eclampsia
Authors: Matsubara Keiichi , Abe Emiko , Matsubara Yuko , Kameda Kenji , Ito Masaharu
Published: May 2006

Problem

Endothelial progenitor cell (EPC), which mediates neovascularization of uterine endometrium may be involved in the neovascularization in the utero‐placental circulation. We evaluated whether EPC proliferation in pre‐eclampsia (PE) differed from that in normal pregnancy.

Method of study

EPC number in peripheral blood (20 non‐pregnancy, 36 normal pregnancy, 10 PE) was measured using flow cytometry. Peripheral blood mononuclear cell was cultured for 7 days and EPC proliferation was assessed based on detection of the uptake of acetylated low‐density lipoprotein and lectin. Furthermore, the proliferative activity induced by angiotensin II (Ang II) and tumor necrosis factor‐α (TNF‐α) was measured by BrdU assay.

Results

EPC number in peripheral blood did not differ significantly between PE and normal pregnancy; however, EPC proliferation was significantly increased in PE. Furthermore, Ang II and TNF‐α induced the proliferation of EPC derived from patients with PE.

Conclusions

In PE, some factors including Ang II and TNF‐α stimulated EPC proliferation; however, the impairment of EPC mobilization into systemic circulation by serum factors may contribute to insufficient regeneration of EC in disturbed utero‐placental circulation of PE.
Laughing Falcon (Herpetotheres cachinnans) Predation on Coral Snakes (Micrurus nigrocinctus)1
Authors: DuVal Emily H. , Greene Harry W. , Manno Katharina L.
Published: May 2006

Laughing falcon (Herpetotheres cachinnans) predation on coral snakes (Micrurus nigrocinctus) was recorded in two incidents that illustrate previously unreported variation in predatory behavior. In the first, the falcon held a live coral snake by the posterior end for an extended period of time, rather than decapitating it immediately. In the second, the falcon left a decapitated coral snake in a tree for more than 2 h before returning to recover its prey. A variety of behavioral adaptations may protect laughing falcons from coral snake venom.
Feeding Behavior of Purple‐throated Fruitcrow (Querula purpurata: Cotingidae) in the Colombian Amazon and Its Implications for Seed Dispersal1
Authors: Parrado‐Rosselli Angela , Amaya‐Espinel Juan David
Published: May 2006

We studied some feeding behaviors of the purple‐throated fruitcrow (Querula purpurata) in two Colombian Amazonian forests, which affect the primary seed dispersal of the plants on which it feeds. Visit length, number of fruits removed and dispersed, feeding rates, and fruit‐handling times were compared to those obtained for two other cotingas feeding on the same fruiting trees. Querula purpurata exhibited shorter visits (98 sec) and fruit‐handling times (4 sec), and higher mean feeding rates (1.6 fruits/min) than Phoenicircus nigricollis and Cotinga cayana. In contrast, P. nigricollis dispersed the highest number of seeds of four of the five tree species studied. Although Q. purpurata and P. nigricollis exhibited feeding behaviors that increase seed dispersal, Q. purpurata may be more important in the transport of seeds between habitats, while P. nigricollis may be a major seed disperser within the primary forest.
Sesiid Moths Reduce Germination, Seedling Growth, and Survivorship in Pentaclethra macroloba (Mimosoideae), a Locally Dominant Lowland Neotropical Tree1
Authors: McKenna Duane D. , McKenna Katherine M.
Published: May 2006

Monodominant forests are a widespread feature of the humid and wet lowland tropics, but little is known about their origins or factors mediating their persistence. Nonetheless, escape from significant vertebrate and invertebrate seed predation plays a prominent role in most hypotheses. The seeds of Pentaclethra macroloba (Fabaceae: Mimosoideae) have long been thought to be virtually immune to predation, contributing to its local dominance in the canopy of some Mesoamerican forests. Here, we describe herbivory by the larvae of Carmenta surinamensis (Lepidoptera: Sesiidae) on the seeds of P. macroloba, and report the results of studies designed to clarify how this interaction influences germination, seedling growth, and mortality. To this end, we collected P. macroloba seeds at 30‐d intervals for 5 mo along a rain forest transect in Costa Rica. The seeds were monitored in a shade house for 30 d. Adult moths were reared from 43.6 percent of seeds, and significantly affected germination and mortality, and all measures of growth (number of leaves, seedling height, seed and seedling mass at 30 d, and 30‐d change in seed and seedling mass). Based on these observations, we conclude that seed boring by C. surinamensis is a potentially important factor influencing population dynamics in P. macroloba, and warrants further investigation for its prospective role in regulating local abundance in this locally dominant and ecologically significant tree.
Edaphic and Floristic Variation within a 1‐ha Plot of Lowland Amazonian Rain Forest1
Authors: Poulsen Axel Dalberg , Tuomisto Hanna , Balslev Henrik
Published: May 2006

Several studies in lowland tropical rain forests have documented effects of local‐scale topographic variation on plant species distribution and abundance patterns. Few studies have compared the distribution patterns of more than one plant group, however, and even fewer have related these to measured physical and chemical soil characteristics. Here, we document such soil characteristics within a square 1‐ha plot in Amazonian Ecuador, and compare them to the distribution patterns of terrestrial pteridophytes, angiosperm ground herbs, and palms. Substantial variation in soil properties was found within the plot. The three plant groups showed highly correlated floristic patterns within the 1‐ha plot even after the effect of geographical distances had been taken into account. Mantel tests yielded significant correlations between edaphic patterns, as measured by distances in various soil and topographic characteristics, and floristic patterns. For all three plant groups, differences in elevation within the plot were highly correlated with floristic distances, and for terrestrial pteridophytes and palms, distances in soil calcium content and sand content were also important. Our results resembled those obtained at wider spatial scales with the same plant groups, which indicates that soil factors may play an important role for distribution and beta diversity of plants, even at the local scale.
Eucharitid Parasitism of the Neotropical Ant Ectatomma tuberculatum: Parasitoid Co‐occurrence, Seasonal Variation, and Multiparasitism1
Authors: Pérez‐Lachaud Gabriela , López‐Méndez José Antonio , Lachaud Jean‐Paul
Published: May 2006

Multiparasitism is reported for the first time in Eucharitidae (Hymenoptera: Chalcidoidea). Dilocantha lachaudii, Isomerala coronata, and Kapala sp. were found simultaneously attacking the same population of Ectatomma tuberculatum (Olivier) (Hymenoptera: Formicidae: Ectatomminae) in southern Mexico. Adults of D. lachaudii and I. coronata completed development on the same individual host on three occasions. The genus Kapala is reported for the first time as a parasitoid of E. tuberculatum, and I. coronata is reported in Mexico for the first time.
Abundance of Gall Midges on Poulsenia armata (Moraceae): Importance of Host Plant Size and Light Environment in Tropical Rain Forests1
Authors: Castellanos I. , Cuevas‐Reyes P. , Ríos‐Casanova L. , Oyama K. , Quesada M.
Published: May 2006

We studied the effects of contrasting light environments on the relationship between the host plant size of Poulsenia armata and the abundance of two gall midges in a tropical rain forest in Veracruz, Mexico. The number and density of two gall morphs (i.e., laminar and vein‐petiole galls) were positively correlated with plant size only in trees found in the forest but not in gaps. The availability of foliar area of P. armata trees was greater in forest gaps than in the forest. The foliar area was positively correlated with the abundance of laminar galls in trees in forest sites, but not with vein‐petiole galls. We concluded that the abundance of two morphs of gall midges on P. armata was associated with host plant size only in the forest trees. Larger plants had more galls than small plants, although this relationship was affected by local light environments.
Pilot testing of manganese oxidation in a high‐rate sedimentation process for the reprovisioning of the Sha Tin water treatment works, Hong Kong Special Administrative Region
Authors: Ko William C. G. , Mak Bevis W. L. , Pun Suki S. K.
Published: May 2006

The Sha Tin water treatment works (STWTW), Hong Kong Special Adminstrative Region, was commissioned in 1964 with an initial treatment capacity of 364 million litres per day (MLD). Since then it has undergone three stages of expansion to reach the current nominal capacity of 1227 MLD. The plant is in need of an in situ reprovisioning. The challenge is to complete the reprovisioning while maintaining a minimum output of 900 MLD, with any shutdown to be limited to 8 h generally. With the site already fully utilised, the key to success is to first replace the conventional low‐rate sedimentation process with high‐rate robust processes to free up the much needed space. Among other requirements, the chosen process must also be capable of treating water with variable turbidity and removing spikes of manganese effectively. The project team conducted a 4‐month pilot testing programme with an Actiflo® high‐rate pilot plant and the key results are described.
Gene Involved in Transcriptional Activation of the
hrp
Regulatory Gene
hrpG
in
Xanthomonas oryzae
pv. oryzae
Authors: Tsuge Seiji , Nakayama Takeshi , Terashima Shinsaku , Ochiai Hirokazu , Furutani Ayako , Oku Takashi , Tsuno Kazunori , Kubo Yasuyuki , Kaku Hisatoshi
Published: May 2006

A novel regulatory gene,
trh
, which is involved in
hrp
gene expression, is identified in the plant pathogen
Xanthomonas oryzae
pv. oryzae. In the
trh
mutant, expression of HrpG, which is a key regulator for
hrp
gene expression, is reduced both under the in vitro
hrp
-inducing condition and in planta.
Anatomy of a Lactococcal Phage Tail
Authors: Mc Grath Stephen , Neve Horst , Seegers Jos F. M. L. , Eijlander Robyn , Vegge Christina S. , Brøndsted Lone , Heller Knut J. , Fitzgerald Gerald F. , Vogensen Finn K. , van Sinderen Douwe
Published: May 2006

Bacteriophages of the
Siphoviridae
family utilize a long noncontractile tail to recognize, adsorb to, and inject DNA into their bacterial host. The tail anatomy of the archetypal
Siphoviridae
λ has been well studied, in contrast to phages infecting gram-positive bacteria. This report outlines a detailed anatomical description of a typical member of the
Siphoviridae
infecting a gram-positive bacterium. The tail superstructure of the lactococcal phage Tuc2009 was investigated using N-terminal protein sequencing, Western blotting, and immunogold transmission electron microscopy, allowing a tangible path to be followed from gene sequence through encoded protein to specific architectural structures on the Tuc2009 virion. This phage displays a striking parity with λ with respect to tail structure, which reenforced a model proposed for Tuc2009 tail architecture. Furthermore, comparisons with λ and other lactococcal phages allowed the specification of a number of genetic submodules likely to encode specific tail structures.
Crl Activates Transcription Initiation of RpoS-Regulated Genes Involved in the Multicellular Behavior ofSalmonella entericaSerovar Typhimurium
Authors: Robbe-Saule Véronique , Jaumouillé Valentin , Prévost Marie-Christine , Guadagnini Stéphanie , Talhouarne Christelle , Mathout Hayette , Kolb Annie , Norel Françoise
Published: May 2006

InSalmonella entericaserovar Typhimurium, the stationary-phase sigma factor σS(RpoS) is required for virulence, stress resistance, biofilm formation, and development of the rdar morphotype. This morphotype is a multicellular behavior characterized by expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The Crl protein ofEscherichia coliinteracts with σSand activates expression of σS-regulated genes, such as thecsgBACoperon encoding the subunit of the curli proteins, by an unknown mechanism. Here, we showed using in vivo and in vitro experiments that the Crl protein ofSalmonellaserovar Typhimurium is required for development of a typical rdar morphotype and for maximal expression of thecsgD,csgB,adrA, andbcsAgenes, which are involved in curli and cellulose biosynthesis. In vitro transcription assays and potassium permanganate reactivity experiments with purified His6-Crl showed that Crl directly activated σS-dependent transcription initiation at thecsgDandadrApromoters. We observed no effect of Crl on σ70-dependent transcription. Crl protein levels increased during the late exponential and stationary growth phases in Luria-Beratani medium without NaCl at 28°C. We obtained complementation of thecrlmutation by increasing σSlevels. This suggests that Crl has a major physiological impact at low concentrations of σS.
Inhibition of ATP Hydrolysis by Thermoalkaliphilic F
1
F
o
-ATP Synthase Is Controlled by the C Terminus of the ε Subunit
Authors: Keis Stefanie , Stocker Achim , Dimroth Peter , Cook Gregory M.
Published: May 2006

The F
1
F
o
-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile
Bacillus
sp. strain TA2.A1, this activity is intrinsic to the F
1
moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in
Escherichia coli
to produce TA2F
1
complexes from this thermoalkaliphile. Like the native F
1
F
o
-ATP synthase, the recombinant TA2F
1
was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the ε subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F
1
mutant with either a truncated ε subunit [i.e., TA2F
1
(ε
ΔC
)] or substitution of basic residues in the second α-helix of ε with nonpolar alanines [i.e., TA2F
1
(ε
6A
)] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F
1
F
o
-ATP synthase and TA2F
1
(ε
WT
) complex with proteases revealed that the ε subunit was resistant to proteolytic digestion. In contrast, the ε subunit of TA2F
1
(ε
6A
) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that ε was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the ε subunit in the entire holoenzyme, we reconstituted recombinant TA2F
1
complexes with F
1
-stripped native membranes of strain TA2.A1. The reconstituted TA2F
o
F
1
(ε
WT
) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F
1
F
o
-ATP synthase in native membranes. Reconstituted TA2F
o
F
1
(ε
6A
) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the ε subunit also inhibits ATPase activity of TA2F
o
F
1
.
Contribution of the PhoP-PhoQ and PmrA-PmrB Two-Component Regulatory Systems to Mg2+-Induced Gene Regulation inPseudomonas aeruginosa
Authors: McPhee Joseph B. , Bains Manjeet , Winsor Geoff , Lewenza Shawn , Kwasnicka Agnieszka , Brazas Michelle D. , Brinkman Fiona S. L. , Hancock R. E. W.
Published: May 2006

When grown in divalent cation-limited medium,Pseudomonas aeruginosabecomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg2+regulation inP. aeruginosa, microarray transcriptional profiling was conducted to compare wild-typeP. aeruginosagrown under Mg2+-limited and Mg2+-replete conditions to isogenicphoPandpmrAmutants grown under Mg2+-limited conditions. Under Mg2+-limited conditions (0.02 mM Mg2+), approximately 3% of theP. aeruginosagenes were differentially expressed compared to the expression in bacteria grown under Mg2+-replete conditions (2 mM Mg2+). Only a modest subset of the Mg2+-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously knownoprH-phoP-phoQoperon and thepmrHFIJKLM-ugdoperon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg2+in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, andfeoABoperons, in addition to the previously known PA4773-PA4775-pmrABandpmrHFIJKLM-ugdoperons.
Proteomic Insights into Metabolic Adaptations inAlcanivorax borkumensisInduced by Alkane Utilization
Authors: Sabirova Julia S. , Ferrer Manuel , Regenhardt Daniela , Timmis Kenneth N. , Golyshin Peter N.
Published: May 2006

Alcanivorax borkumensisis a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This “hydrocarbonoclastic” bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-knownalkB1gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating β-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded byA. borkumensisand its ecological success in oil-contaminated marine habitats.
6S RNA Regulation of
pspF
Transcription Leads to Altered Cell Survival at High pH
Authors: Trotochaud Amy E. , Wassarman Karen M.
Published: May 2006

6S RNA is a highly abundant small RNA that regulates transcription through direct interaction with RNA polymerase. Here we show that 6S RNA directly inhibits transcription of
pspF
, which subsequently leads to inhibition of
pspABCDE
and
pspG
expression. Cells without 6S RNA are able to survive at elevated pH better than wild-type cells due to loss of 6S RNA-regulation of
pspF
. This 6S RNA-dependent phenotype is eliminated in
pspF
-null cells, indicating that 6S RNA effects are conferred through PspF. Similar growth phenotypes are seen when PspF levels are increased in a 6S RNA-independent manner, signifying that changes to
pspF
expression are sufficient. Changes in survival at elevated pH most likely result from altered expression of
pspABCDE
and/or
pspG
, both of which require PspF for transcription and are indirectly regulated by 6S RNA. 6S RNA provides another layer of regulation in response to high pH during stationary phase. We propose that the normal role of 6S RNA at elevated pH is to limit the extent of the
psp
response under conditions of nutrient deprivation, perhaps facilitating appropriate allocation of diminishing resources.
Functional Genomics of Stress Response in
Pseudomonas putida
KT2440
Authors: Reva Oleg N. , Weinel Christian , Weinel Miryam , Böhm Kerstin , Stjepandic Diana , Hoheisel Jörg D. , Tümmler Burkhard
Published: May 2006

The metabolically versatile soil bacterium
Pseudomonas putida
has to cope with numerous abiotic stresses in its habitats. The stress responses of
P. putida
KT2440 to 4°C, pH 4.5, 0.8 M urea, and 45 mM sodium benzoate were analyzed by determining the global mRNA expression profiles and screening for stress-intolerant nonauxotrophic Tn
5
transposon mutants. In 392 regulated genes or operons, 36 gene regions were differentially expressed by more than 2.5-fold, and 32 genes in 23 operons were found to be indispensable for growth during exposure to one of the abiotic stresses. The transcriptomes of the responses to urea, benzoate, and 4°C correlated positively with each other but negatively with the transcriptome of the mineral acid response. The CbrAB sensor kinase, the cysteine synthase CysM, PcnB and VacB, which control mRNA stability, and BipA, which exerts transcript-specific translational control, were essential to cope with cold stress. The
cyo
operon was required to cope with acid stress. A functional PhoP, PtsP, RelA/SpoT modulon, and adhesion protein LapA were necessary for growth in the presence of urea, and the outer membrane proteins OmlA and FepA and the phosphate transporter PstBACS were indispensable for growth in the presence of benzoate. A lipid A acyltransferase (PP0063) was a mandatory component of the stress responses to cold, mineral acid, and benzoate. Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of
P. putida
to abiotic stresses.
Differential Activation of
Escherichia coli
Chemoreceptors by Blue-Light Stimuli
Authors: Wright Stuart , Walia Bharat , Parkinson John S. , Khan Shahid
Published: May 2006

Enteric bacteria tumble, swim slowly, and are then paralyzed upon exposure to 390- to 530-nm light. Here, we analyze this complex response in
Escherichia coli
using standard fluorescence microscope optics for excitation at 440 ± 5 nm. The slow swimming and paralysis occurred only in dye-containing growth media or buffers. Excitation elicited complete paralysis within a second in 1 μM proflavine dye, implying specific motor damage, but prolonged tumbling in buffer alone. The tumbling half-response times were subsecond for onset but more than a minute for recovery. The response required the chemotaxis signal protein CheY and receptor-dependent activation of its kinase CheA. The study of deletion mutants revealed a specific requirement for either the aerotaxis receptor Aer or the chemoreceptor Tar but not the Tar homolog Tsr. The action spectrum of the wild-type response was consistent with a flavin, but the chromophores remain to be identified. The motile response processed via Aer was sustained, with recovery to either step-up or -down taking more than a minute. The response processed via Tar was transient, recovering on second time scales comparable to chemotactic responses. The response duration and amplitude were dependent on relative expression of Aer, Tar, and Tsr. The main response features were reproduced when each receptor was expressed singly from a plasmid in a receptorless host strain. However, time-resolved motion analysis revealed subtle kinetic differences that reflect the role of receptor cluster interactions in kinase activation-deactivation dynamics.
Utilization of the Methoxymalonyl-Acyl Carrier Protein Biosynthesis Locus for Cloning of the Tautomycin Biosynthetic Gene Cluster from
Streptomyces spiroverticillatus
Authors: Li Wenli , Ju Jianhua , Osada Hiroyuki , Shen Ben
Published: May 2006

Tautomycin (TTM), a potent protein phosphatase inhibitor, consists of a polyketide chain containing a spiroketal moiety and an acyl chain bearing a dialkylmaleic anhydride structure. PCR using degenerate primers was used to clone genes from
Streptomyces spiroverticillatus
for formation of the methoxymalonyl-acyl carrier protein. This locus was found to contain five genes (
ttmC
,
ttmA
,
ttmD
,
ttmB
, and
ttmE
), one of which was used as a probe to clone the 110-kb TTM biosynthetic gene cluster. The involvement of the
ttmA
gene in TTM biosynthesis was confirmed by gene inactivation and mutation complementation experiments.
Bundle-Forming Pili and EspA Are Involved in Biofilm Formation by EnteropathogenicEscherichia coli
Authors: Moreira Cristiano G. , Palmer Kelli , Whiteley Marvin , Sircili Marcelo P. , Trabulsi Luiz R. , Castro Antonio F. P. , Sperandio Vanessa
Published: May 2006

Microcolony formation is one of the initial steps in biofilm development, and in enteropathogenicEscherichia coli(EPEC) it is mediated by several adhesins, including the bundle-forming pilus (BFP) and the EspA filament. Here we report that EPEC forms biofilms on plastic under static conditions and a flowthrough continuous culture system. The abilities of several EPEC isogenic mutants to form biofilms were assessed. Adhesins such as BFP and EspA, important in microcolony formation on epithelial cells, are also involved in bacterial aggregation during biofilm formation on abiotic surfaces. Mutants that do not express BFP or EspA form more-diffuse biofilms than does the wild type. We also determined, usinggfptranscriptional fusions, that, consistent with the role of these adhesins in biofilms, the genes encoding BFP and EspA are expressed during biofilm formation. Finally, expression ofespAis controlled by a quorum-sensing (QS) regulatory mechanism, and the EPECqseAQS mutant also forms altered biofilms, suggesting that this signaling mechanism plays an important role in EPEC biofilm development. Taken together, these studies allowed us to propose a model of EPEC biofilm formation.
Identification of Methylation Sites in
Thermotoga maritima
Chemotaxis Receptors
Authors: Perez Eduardo , Zheng Haiyan , Stock Ann M.
Published: May 2006

Adaptation in bacterial chemotaxis involves reversible methylation of specific glutamate residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins. The specific sites of methylation in
Salmonella enterica
and
Escherichia coli
chemoreceptors, identified 2 decades ago, established a consensus sequence for methylation by methyltransferase CheR. Here we report the in vitro methylation of chemoreceptors from
Thermotoga maritima
, a hyperthermophile that has served as a useful source of chemotaxis proteins for structural analysis. Sites of methylation have been identified by liquid chromatography-mass spectrometry/mass spectrometry. Fifteen sites of methylation were identified within the cytoplasmic domains of four different
T. maritima
chemoreceptors. The results establish a consensus sequence for chemoreceptor methylation sites in
T. maritima
that is distinct from the previously identified consensus sequence for
E. coli
and
S. enterica
. These findings suggest that consensus sequences for posttranslational modifications in one organism may not be directly extrapolated to analogous modifications in other bacteria.
Genomic and Functional Analysis of the IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Its Transposon Tn
4655
Suggests Catabolic Gene Spread by a Tyrosine Recombinase
Authors: Sota Masahiro , Yano Hirokazu , Ono Akira , Miyazaki Ryo , Ishii Hidenori , Genka Hiroyuki , Top Eva M. , Tsuda Masataka
Published: May 2006

The naphthalene-catabolic (
nah
) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from
Pseudomonas putida
G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified
nah
genes are located on a class II transposon, Tn
4655
. Our analysis of the Tn
4655
-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the
attI
site, (ii) the functional
attI
site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-
attI
recombination system in the establishment of these catabolic plasmids.
Role of Dipicolinic Acid in Resistance and Stability of Spores of
Bacillus subtilis
with or without DNA-Protective α/β-Type Small Acid-Soluble Proteins
Authors: Setlow Barbara , Atluri Swaroopa , Kitchel Ryan , Koziol-Dube Kasia , Setlow Peter
Published: May 2006

Dipicolinic acid (DPA) comprises ∼10% of the dry weight of spores of
Bacillus
species. Although DPA has long been implicated in spore resistance to wet heat and spore stability, definitive evidence on the role of this abundant molecule in spore properties has generally been lacking.
Bacillus subtilis
strain FB122 (
sleB spoVF
) produced very stable spores that lacked DPA, and sporulation of this strain with DPA yielded spores with nearly normal DPA levels. DPA-replete and DPA-less FB122 spores had similar levels of the DNA protective α/β-type small acid-soluble spore proteins (SASP), but the DPA-less spores lacked SASP-γ. The DPA-less FB122 spores exhibited similar UV resistance to the DPA-replete spores but had lower resistance to wet heat, dry heat, hydrogen peroxide, and desiccation. Neither wet heat nor hydrogen peroxide killed the DPA-less spores by DNA damage, but desiccation did. The inability to synthesize both DPA and most α/β-type SASP in strain PS3664 (
sspA sspB sleB spoVF
) resulted in spores that lost viability during sporulation, at least in part due to DNA damage. DPA-less PS3664 spores were more sensitive to wet heat than either DPA-less FB122 spores or DPA-replete PS3664 spores, and the latter also retained viability during sporulation. These and previous results indicate that, in addition to α/β-type SASP, DPA also is extremely important in spore resistance and stability and, further, that DPA has some specific role(s) in protecting spore DNA from damage. Specific roles for DPA in protecting spore DNA against damage may well have been a major driving force for the spore's accumulation of the high levels of this small molecule.
Maltose and Maltodextrin Utilization by
Bacillus subtilis
Authors: Schönert Stefan , Seitz Sabine , Krafft Holger , Feuerbaum Eva-Anne , Andernach Iris , Witz Gabriele , Dahl Michael K.
Published: May 2006

Bacillus subtilis
can utilize maltose and maltodextrins that are derived from polysaccharides, like starch or glycogen. In this work, we show that maltose is taken up by a member of the phosphoenolpyruvate-dependent phosphotransferase system and maltodextrins are taken up by a maltodextrin-specific ABC transporter. Uptake of maltose by the phosphoenolpyruvate-dependent phosphotransferase system is mediated by maltose-specific enzyme IICB (MalP; synonym, GlvC), with an apparent

K
m

of 5 μM and a
V
max
of 91 nmol · min
−1
· (10
10
CFU)
−1
. The maltodextrin-specific ABC transporter is composed of the maltodextrin binding protein MdxE (formerly YvdG), with affinities in the low micromolar range for maltodextrins, and the membrane-spanning components MdxF and MdxG (formerly YvdH and YvdI, respectively), as well as the energizing ATPase MsmX. Maltotriose transport occurs with an apparent

K
m

of 1.4 μM and a
V
max
of 4.7 nmol · min
−1
 · (10
10
CFU)
−1
.
Metabolism of Isovanillate, Vanillate, and Veratrate by
Comamonas testosteroni
Strain BR6020
Authors: Providenti Miguel A. , O'Brien Jason M. , Ruff Jürgen , Cook Alasdair M. , Lambert Iain B.
Published: May 2006

In
Comamonas testosteroni
strain BR6020, metabolism of isovanillate (iVan; 3-hydroxy-4-methoxybenzoate), vanillate (Van; 4-hydroxy-3-methoxybenzoate), and veratrate (Ver; 3,4-dimethoxybenzoate) proceeds via protocatechuate (Pca; 3,4-dihydroxybenzoate). A 13.4-kb locus coding for the catabolic enzymes that channel the three substrates to Pca was cloned. O demethylation is mediated by the phthalate family oxygenases IvaA (converts iVan to Pca and Ver to Van) and VanA (converts Van to Pca and Ver to iVan). Reducing equivalents from NAD(P)H are transferred to the oxygenases by the class IA oxidoreductase IvaB. Studies using whole cells, cell extracts, and reverse transcriptase PCR showed that degradative activity and expression of
vanA
,
ivaA
, and
ivaB
are inducible. In succinate- and Pca-grown cells, there is negligible degradative activity towards Van, Ver, and iVan and little to no expression of
vanA
,
ivaA
, and
ivaB
. Growth on Van or Ver results in production of oxygenases with activity towards Van, Ver, and iVan and expression of
vanA
,
ivaA
, and
ivaB
. With iVan-grown cultures,
ivaA
and
ivaB
are expressed, and in assays with whole cells, production of the iVan oxygenase is observed, but there is little activity towards Van or Ver. In cell extracts, though, Ver metabolism is observed, which suggests that the system mediating iVan uptake in whole cells does not mediate Ver uptake.
The Genome of the Obligately Intracellular Bacterium
Ehrlichia canis
Reveals Themes of Complex Membrane Structure and Immune Evasion Strategies
Authors: Mavromatis K. , Doyle C. Kuyler , Lykidis A. , Ivanova N. , Francino M. P. , Chain P. , Shin M. , Malfatti S. , Larimer F. , Copeland A. , Detter J. C. , Land M. , Richardson P. M. , Yu X. J. , Walker D. H. , McBride J. W. , Kyrpides N. C.
Published: May 2006

Ehrlichia canis
, a small obligately intracellular, tick-transmitted, gram-negative, α-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the
E. canis
genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (
n
= 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (
n
= 7).
Functional Analysis of Glucan Binding Protein B from
Streptococcus mutans
Authors: Mattos-Graner Renata O. , Porter Kristen A. , Smith Daniel J. , Hosogi Yumiko , Duncan Margaret J.
Published: May 2006

Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these,
Streptococcus mutans
glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from
S. agalactiae
and
S. pneumoniae
, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true
gbpB
mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable
gbpB
null mutants supports the notion that
gbpB
is an essential gene. Northern blot and real-time PCR analyses suggested that induction of
gbpB
expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division.
The X-Ray Structure of the Haloalcohol Dehalogenase HheA from
Arthrobacter
sp. Strain AD2: Insight into Enantioselectivity and Halide Binding in the Haloalcohol Dehalogenase Family
Authors: de Jong René M. , Kalk Kor H. , Tang Lixia , Janssen Dick B. , Dijkstra Bauke W.
Published: May 2006

Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheA
AD2
from
Arthrobacter
sp. strain AD2 at 2.0-Å resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from
Agrobacterium radiobacter
AD1 shows that HheA
AD2
has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheA
AD2
can bind both enantiomers of the haloalcohol substrate 1-
p
-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheA
AD2
. Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheA
AD2
, in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.
Active Transcription of rRNA Operons Is a Driving Force for the Distribution of RNA Polymerase in Bacteria: Effect of Extrachromosomal Copies of
rrnB
on the In Vivo Localization of RNA Polymerase
Authors: Cabrera Julio E. , Jin Ding J.
Published: May 2006

In contrast to eukaryotes, bacteria such as
Escherichia coli
contain only one form of RNA polymerase (RNAP), which is responsible for all cellular transcription. Using an RNAP-green fluorescent protein fusion protein, we showed previously that
E. coli
RNAP is partitioned exclusively in the nucleoid and that stable RNA synthesis, particularly rRNA transcription, is critical for concentrating a significant fraction of RNAP in transcription foci during exponential growth. The extent of focus formation varies under different physiological conditions, supporting the proposition that RNAP redistribution is an important element for global gene regulation. Here we show that extra, plasmid-borne copies of an rRNA operon recruit RNAP from the nucleoid into the cytoplasmic space and that this is accompanied by a reduction in the growth rate. Transcription of an intact rRNA operon is not necessary, although a minimal transcript length is required for this phenotype. Replacement of the ribosomal promoters with another strong promoter, P
tac
, abolished the effect. These results demonstrate that active synthesis from rRNA promoters is a major driving force for the distribution of RNAP in bacteria. The implications of our results for the regulation of rRNA synthesis and cell growth are discussed.