bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of vascular endothelial growth factor in the formation of the thecal layer and vasculature during follicular development in the ovaries of neonatal female rats
Authors: IIJIMA Koji , TAWARA Yukiko , SHIMIZU Takashi , YOGO Keiichiro , SASADA Hiroshi , SATO Eimei
Published: December 2006

Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the ovary, but the localization of VEGF in the ovary of neonatal animals is poorly understood. A clear understanding of the relationship between the formation of the thecal layer and the cell‐specific expression of the VEGF system during follicular development in the neonatal ovary is still lacking. Immature female Wistar‐Imamichi rats used in this study were killed by decapitation 5, 7, 9 and 11 days after birth, and their ovaries were removed and subjected to histological and immunohistochemical observation. The number of primordial follicles had decreased in the ovaries at day 11 compared with that at day 5. The number of secondary follicles significantly increased with age. In the morphological observation of secondary follicles, we found that the theca layer (70 µm in diameter of follicles) began to form at day 9 and was completely formed at day 11. An endothelial cell marker, CD31, VEGF and Flk‐1 were located in the stromal tissues in the ovaries on each day examined after birth. In particular, in the ovaries at day 9 and day 11, when the secondary follicles appeared, CD31, VEGF and Flk‐1 were expressed in the theca layer. Flt‐1 was expressed in the oocytes of the ovaries at day 5 and day 7, and the sites of its expression changed to stromal and thecal tissues at day 9 and day 11. In conclusion, we provide the first evidence that the theca layer of secondary follicles begin to form at day 9 after birth and that VEGF and Flk‐1 may be able to stimulate the differentiation of stromal‐interstitial cells into thecal cells and the formation of the thecal vasculature in the neonatal rat ovaries, suggesting that the VEGF system may be involved in the formation of the thecal layer and vasculature during folliculogenesis in the neonatal rat.
The effect of exogenous purine supply on the endogenous excretion of purine derivatives in the urine of growing lambs
Authors: FUJIHARA Tsutomu , SHEM Martin N. , MATSUI Tohru
Published: December 2006

An experiment was made to determine the absorption of purine metabolites in dietary nucleic acids through the digestive tract, and also to determine the utilization of nucleic acids absorbed in the body, using growing lambs. Two pairs of 120‐ and 180‐day‐old twin female lambs with a bodyweight of 18.2–19.0 kg were kept in metabolism crates and fed on purine‐free milk replacement (MR) with supplements of exogenous purine (purine base or purine nucleoside) at a level of 0.2 mmol/BW0.75/d for 5 consecutive days, and thereafter they were maintained in the crates for 4 days. The daily amount of exogenous purine supply was calculated based on the urinary excreted purine derivatives (PD) in lambs fed on milk replacement alone. A urine sample was collected daily for 9 consecutive days, and the urinary excretion of PD was determined daily. Urinary PD excretion opened to increase within 24 h after the dose of purine bases, and the level was recovered on 3 days after ceasing the exogenous purine supply. The recovery of PD in the urine was about 70% of the purine supplement. When purine nucleosides were added to the feed, urinary PD excretion was initiated within 24 h after dosing, and the values were recovered after ending the purine nucleoside supply. The recovery rate of PD in the urine was only 30% of the supplemented purine. The plasma allantoin levels were almost similar after feeding purine bases and purine nucleosides, and the values were mostly in the range (40–60 µmol/L). These findings indicate that an exogenous purine can be directly incorporated into the body, and the purine as nucleoside is more effectively utilized for the synthesis of nucleic acids than as a purine base in the body of growing lambs.
The effects of potato pulp and feeding level of supplements on digestibility, in situ forage degradation and ruminal fermentation in beef steers
Authors: SUGIMOTO Masahito , SAITO Waka , OOI Motoki , SATO Yukinobu , SAITO Toshiro , MORI Kiyokazu
Published: December 2006

Six ruminally cannulated Wagyu (Japanese Black) steers (average initial bodyweight (BW) 387 ± 29 kg) were used in a split‐plot design experiment, comprising a 3 × 3 Latin square design (whole plot) and a randomized block design (subplot). The whole plot treatments were three different feeding levels of supplemental diet, fed at 0.2, 0.4 and 0.6% of BW, on a dry matter (DM) basis. Subplot treatments were two different supplemental diets: a potato pulp silage‐based diet (PPS) and a grain‐based diet (GRAIN). Chopped, medium‐quality cool‐season grass hay (predominately Timothy, Phleum pratense L) was fed daily at 0.7% BW (on a DM basis) as the basal diet. Each period consisted of 21 d, which included 11 d of adaptation to the diets and 10 d of the collection period. Chromium oxide was used as an indigestible marker. In situ forage degradation was measured using the nylon bag technique. The dry matter intake increased (linear; P < 0.01) as the feeding level increased and was not affected by the diet. Digestibility was not affected by any treatments. The GRAIN diet tended to decrease the rate of in situ forage degradation as the feeding level increased, but this trend was not found in the steers fed the PPS diet. Steers fed the GRAIN diet had a lower (P < 0.1) ruminal pH compared with steers fed the PPS diet. Ruminal pH was not significantly affected by feeding level; however, it was numerically higher for steers supplemented at 0.2% per BW than that for the steers supplemented above 0.4% per BW due probably to the higher starch intake. The total volatile fatty acids concentration numerically increased as the feeding level increased and was not affected by the diet. Increasing the feeding level decreased (linear, P < 0.01) the proportion of acetate. Neither diet nor the feeding level had any effects on the proportion of ruminal propionate. The results suggested that, for steers fed the PPS diet, there are not adverse effects on forage digestion in the rumen that occur as the feeding level is increased.
Changes in single‐unit activity in the ventromedial hypothalamic nucleus of goats during feeding
Authors: NAGAMINE Itsuki , SUNAGAWA Katsunori , SHINJO Akihisa
Published: December 2006

In this study we used a single‐unit activity (SUA) recording electrode to investigate the existence of appetite‐regulating neurons in the ventromedial hypothalamic nucleus (VMH) in goats. Five male Japanese Saanen goats were used. The animals were fed at 10.00 hours with alfalfa hay cubes, and at 16.00 hours with concentrate feed and hay. The animals were allowed free access to drinking water. The present experiment consisted of two treatments, non‐infusion (NI) and an intra‐ruminal water infusion (RWI). The SUA recordings were carried out continuously over a 130 min period beginning 10 min prior to the commencement of morning feeding. In the RWI treatment, warm water was infused intra‐ruminally before the SUA recording. Eating rates in the NI treatment decreased sharply by the time 40 min had elapsed and then remained at low levels. In the RWI treatment, eating rates decreased by the time 50 min had elapsed and then remained at low levels. This study recorded 19 units, of which four units increased their firing rates when the animals became satiated (i.e. when eating rates declined to low levels) in both treatments. The four units were located in the VMH. The results suggest that cells located in the VMH of goats are active in the control of hay intake.
Treatment of cow‐waste slurry by a microbial fuel cell and the properties of the treated slurry as a liquid manure
Authors: YOKOYAMA Hiroshi , OHMORI Hideyuki , ISHIDA Mitsuyoshi , WAKI Miyoko , TANAKA Yasuo
Published: December 2006

Resource recycling and the proper treatment of animal waste to reduce its environmental impact are currently important issues for the livestock industry. A microbial fuel cell (MFC), a new type of bioreactor, is expected to play roles in both waste‐water purification and energy recovery. However, the generation of electricity from cow waste has not yet been examined. In this study, using an MFC, we examined the possibility of generating electricity from dairy‐cow waste slurry, and analyzed the properties of the treated slurry as liquid manure for resource recycling. The MFC treatment of the slurry generated electricity in a dose‐dependent manner, and the maximum power output by the MFC from a 1 g of chemical oxygen demand/L slurry was 0.34 mW/m2. After the MFC treatment, 84% of the biological oxygen demand in the slurry was removed and three essential fertilizer elements (nitrogen, phosphorus, and potassium) were retained at 84, 70, and 91% levels, respectively. The amount of ammonia nitrogen in the slurry, as an element of fast‐release fertilizer, was increased by 1.9‐fold. Although the treated slurry displayed properties that made it preferable as liquid manure, further studies to improve the electrical power output by the MFC are required for practical use.
Biochemical and Structural Characterization of the Secreted Chorismate Mutase (Rv1885c) from
Mycobacterium tuberculosis
H
37
R
v
: an *AroQ Enzyme Not Regulated by the Aromatic Amino Acids
Authors: Kim Sook-Kyung , Reddy Sathyavelu K. , Nelson Bryant C. , Vasquez Gregory B. , Davis Andrew , Howard Andrew J. , Patterson Sean , Gilliland Gary L. , Ladner Jane E. , Reddy Prasad T.
Published: December 2006

The gene Rv1885c from the genome of
Mycobacterium tuberculosis
H
37
R
v
encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in
Escherichia coli
and the absence of a discrete periplasmic compartment in
M. tuberculosis
, we show here that *MtCM secretes into the culture filtrate of
M. tuberculosis
. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a

K
m

of 0.5 ± 0.05 mM and a
k
cat
of 60 s
−1
per active site (at 37°C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 Å resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg
134
. We provide evidence by site-directed mutagenesis that the residues Arg
49
, Lys
60
, Arg
72
, Thr
105
, Glu
109
, and Arg
134
constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.
A Homologue of the 3-Oxoacyl-(Acyl Carrier Protein) Synthase III Gene Located in the Glycosylation Island of
Pseudomonas syringae
pv. tabaci Regulates Virulence Factors via
N
-Acyl Homoserine Lactone and Fatty Acid Synthesis
Authors: Taguchi Fumiko , Ogawa Yujiro , Takeuchi Kasumi , Suzuki Tomoko , Toyoda Kazuhiro , Shiraishi Tomonori , Ichinose Yuki
Published: December 2006

Pseudomonas syringae
pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames:
orf1
,
orf2
, and
orf3
. Our previous study revealed that
orf1
and
orf2
encode glycosyltransferases; on the other hand,
orf3
has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an
orf3
deletion mutant (Δ
orf3
mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Δ
orf3
mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H
2
O
2
and antibiotics were enhanced in the Δ
orf3
mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Δ
orf3
mutant. The phenotypes of the Δ
orf3
mutant and an AHL synthesis (Δ
psyI
) mutant were similar, although the mutant-specific characteristics were more extreme in the Δ
orf3
mutant. The swarming motility of the Δ
orf3
mutant was greater than that of the Δ
psyI
mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Δ
orf3
mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in
P. syringae
pv. tabaci 6605.
Comparative Genetics of the rdar Morphotype inSalmonella
Authors: White A. P. , Surette M. G.
Published: December 2006

TheSalmonellardar morphotype is a distinct, rough and dry colony morphology formed by the extracellular interaction of thin aggregative fimbriae (Tafi or curli), cellulose, and other polysaccharides. Cells in rdar colonies are more resistant to desiccation and exogenous stresses, which is hypothesized to aid in the passage of pathogenicSalmonellaspp. between hosts. Here we analyzed the genetic and phenotypic conservation of the rdar morphotype throughout the entireSalmonellagenus. The rdar morphotype was conserved in 90% of 80 isolates representing all 7Salmonellagroups; however, the frequency was only 31% in a reference set of 16 strains (Salmonella referencecollection C [SARC]). Comparative gene expression analysis was used to separatecis- andtrans-acting effects on promoter activity for the 16 SARC strains, focusing on the 780-bp intergenic region containing divergent promoters for the master regulator of the rdar morphotype (agfD) and the Tafi structural genes (agfB). Surprisingly, promoter functionality was conserved in most isolates, and loss of the phenotype was due primarily to defects intrans-acting regulatory factors. We hypothesize thattransdifferences have been caused by domestication, whereascisdifferences, detected forSalmonella entericasubsp.arizonaeisolates, may reflect an evolutionary change in lifestyle. Our results demonstrate that the rdar morphotype is conserved throughout the salmonellae, but they also emphasize that regulation is an important source of variability among isolates.
Escherichia coliRNA Polymerase Recognition of a σ70-Dependent Promoter Requiring a −35 DNA Element and an Extended −10 TGn Motif
Authors: Hook-Barnard India , Johnson Xanthia B. , Hinton Deborah M.
Published: December 2006

Escherichia coliσ70-dependent promoters have typically been characterized as either −10/−35 promoters, which have good matches to both the canonical −10 and the −35 sequences or as extended −10 promoters (TGn/−10 promoters), which have the TGn motif and an excellent match to the −10 consensus sequence. We report here an investigation of a promoter, Pminor, that has a nearly perfect match to the −35 sequence and has the TGn motif. However, Pminorcontains an extremely poor σ70−10 element. We demonstrate that Pminoris active both in vivo and in vitro and that mutations in either the −35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the −10 element more canonical, thus converting the −35/TGn promoter to a −35/−10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with Pminor, the DNA is single stranded from positions −11 to +4. We also demonstrate that transcription from Pminorincorporates nontemplated ribonucleoside triphosphates at the 5′ end of the Pminortranscript, which results in an anomalous assignment for the start site when primer extension analysis is used. Pminorrepresents one of the few −35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized −10/−35 and extended −10 promoters used byE. coliRNA polymerase.
Identification of a Gene Negatively Affecting Antibiotic Production and Morphological Differentiation inStreptomyces coelicolorA3(2)
Authors: Li Wencheng , Ying Xin , Guo Yuzheng , Yu Zhen , Zhou Xiufen , Deng Zixin , Kieser Helen , Chater Keith F. , Tao Meifeng
Published: December 2006

SC7A1 is a cosmid with an insert of chromosomal DNA fromStreptomyces coelicolorA3(2). Its insertion into the chromosome ofS. coelicolorstrains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frameSCO5582asnsdA, a genenegatively affectingStreptomycesdifferentiation. The disruption of chromosomalnsdAcaused the overproduction of spores and of three of four knownS. coelicolorantibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying thatnsdAin the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster.nsdAexpression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, aStreptomycesprotein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in someStreptomycesgenomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of thensdA-like geneSCO4114had no obvious phenotypic effects onS. coelicolor. ThensdAorthologueSAV2652inS. avermitiliscould complement theS. coelicolor nsdA-null mutant phenotype.
Identification of a
nisI
Promoter within the
nisABCTIP
Operon That May Enable Establishment of Nisin Immunity Prior to Induction of the Operon via Signal Transduction
Authors: Li Haiping , O'Sullivan Daniel J.
Published: December 2006

Certain strains of
Lactococcus lactis
produce the broad-spectrum bacteriocin nisin, which belongs to the lantibiotic class of antimicrobial peptides. The genes encoding nisin are organized in three contiguous operons:
nisABTCIP
, encoding production and immunity (
nisI
);
nisRK
, encoding regulation; and
nisFEG
, also involved in immunity. Transcription of
nisABTCIP
and
nisFEG
requires autoinduction by external nisin via signal transducing by NisRK. This organization poses the intriguing question of how sufficient immunity (NisI) can be expressed when the nisin cluster enters a new cell, before it encounters external nisin. In this study, Northern analysis in both
Lactococcus
and
Enterococcus
backgrounds revealed that
nisI
mRNA was present under conditions when no
nisA
transcription was occurring, suggesting an internal promoter within the operon. The
nisA
transcript was significantly more stable than
nisI
, further substantiating this. Reverse transcriptase PCR analysis revealed that the transcription initiated just upstream from
nisI
. Fusing this region to a
lacZ
gene in a promoter probe vector demonstrated that a promoter was present. The transcription start site (TSS) of the
nisI
promoter was mapped at bp 123 upstream of the
nisI
translation start codon. Ordered 5′ deletions revealed that transcription activation depended on sequences located up to bp −234 from the TSS. The presence of poly(A) tracts and computerized predictions for this region suggested that a high degree of curvature may be required for transcription initiation. The existence of this
nisI
promoter is likely an evolutionary adaptation of the nisin gene cluster to enable its successful establishment in other cells following horizontal transfer.
Expression of the
pyr
Operon of
Lactobacillus plantarum
Is Regulated by Inorganic Carbon Availability through a Second Regulator, PyrR
2
, Homologous to the Pyrimidine-Dependent Regulator PyrR
1
Authors: Arsène-Ploetze Florence , Kugler Valérie , Martinussen Jan , Bringel Françoise
Published: December 2006

Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of
Lactobacillus plantarum
. At low IC levels, one-third of natural isolated
L. plantarum
strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-CO
2
-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the
pyrR
1
BCAa
1
Ab
1
DFE
operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated
pyr
genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the
L. plantarum
genome. PyrR
1
is an RNA-binding protein that regulates the
pyr
genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of PyrR
2
was investigated by allelic gene replacement. Unlike the
pyrR
1
mutant, the
ΔpyrR
2
strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO
2
was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in
pyrR
1
and
pyrR
2
mutants suggested that only PyrR
2
positively regulates the expression levels of the
pyr
genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of PyrR
1
and PyrR
2
in the
pyr
regulon expression.
Malonyl-Coenzyme A Reductase in the Modified 3-Hydroxypropionate Cycle for Autotrophic Carbon Fixation in Archaeal
Metallosphaera
and
Sulfolobus
spp
Authors: Alber Birgit , Olinger Marc , Rieder Annika , Kockelkorn Daniel , Jobst Björn , Hügler Michael , Fuchs Georg
Published: December 2006

Autotrophic members of the
Sulfolobales
(
Crenarchaeota
) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO
2
fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO
2
into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of
Metallosphaera sedula
contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum
Sulfolobus tokodaii
. The
Sulfolobus
gene was cloned and heterologously expressed in
Escherichia coli
, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H
+
→ malonate-semialdehyde + CoA + NADP
+
. In its native state it is associated with small RNA. Its activity was stimulated by Mg
2+
and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (

K
m

= 25 μM) and malonyl-CoA (

K
m

= 40 μM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from
Chloroflexus aurantiacus.
This shows that the autotrophic pathway in
Chloroflexus
and
Sulfolobaceae
has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.
Mesorhizobium lotiProducesnodPQ-Dependent Sulfated Cell Surface Polysaccharides
Authors: Townsend Guy E. , Forsberg Lennart S. , Keating David H.
Published: December 2006

Leguminous plants and bacteria from the familyRhizobiaceaeform a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms betweenSinorhizobium melilotiand alfalfa requires biosynthesis of Nod factor, a β-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification.S. melilotialso produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants ofS. melilotiwith reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of theS. meliloticarbohydrate sulfotransferase, LpsS, inMesorhizobium loti. M. lotiparticipates in a determinate symbiosis with the legumeLotus japonicus. We showed thatM. lotiproduces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides inM. loti, we identified and disabled anM. lotihomolog of the sulfate-activating genes,nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled anM. loti cysHhomolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that thenodPQmutant, but not thecysHmutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume,Lotus japonicus. Interestingly, thenodPQmutant also exhibited a more rapid growth rate and appeared to outcompete wild-typeM. lotifor nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization inM. loti.
Lysophosphatidylethanolamine Is a Substrate for the Short-Chain Alcohol Dehydrogenase SocA from
Myxococcus xanthus
Authors: Avadhani Madhavi , Geyer Roland , White David C. , Shimkets Lawrence J.
Published: December 2006

Short-chain alcohol dehydrogenases (SCADHs) synthesize a variety of intercellular signals and other chemically diverse products. It is difficult to predict the substrate of a SCADH on the basis of amino acid sequence homology, as the substrates are not known for most SCADHs. In
Myxococcus xanthus
, the SCADH CsgA is responsible for C signaling during fruiting body development, although the mechanism is unclear. Overexpression of the SCADH SocA compensates for the lack of CsgA and restores development and C signaling in
csgA
mutants. The potential of SocA in generating the C signal enzymatically was explored by developing a dehydrogenase assay-based screen to purify the SocA substrate(s). A SocA substrate was extracted from
M. xanthus
cells with acidified ethyl acetate and sequentially purified by solid-phase extraction on silica gel and by reverse-phase high-performance liquid chromatography. The fraction with the highest SocA dehydrogenase activity contained the lysophospholipid 1-acyl 2-hydroxy-
sn
-glycerophosphoethanolamine (lyso-PE) as indicated by the fragment ions and a phosphatidylethanolamine-specific neutral loss scan following liquid chromatography coupled to mass spectrometry. The abundant lysophospholipid with the mass
m/z
450 (molecular ion [M-H]
−
) had a monounsaturated acyl chain with 16 carbons. SocA oxidizes lyso-PE containing either saturated or unsaturated fatty acids but exhibits poor activity on
l
-α-glycerophosphorylethanolamine, suggesting that an acyl chain is important for activity. Of the five different head groups, only ethanolamine showed appreciable activity. The apparent

K
m

and
V
max
for lyso-PE 18:1 were 116 μM and 875 μmol min
−1
mg
−1
, respectively. The catalytic efficiency (
k
cat
/

K
m

) was 1 × 10
8
M
−1
s
−1
. The proposed product, 1-acyloxy-3-(2-aminoethylphosphatyl) acetone was unstable, and the fragmented products were unable to rescue
csgA
mutant development. The active fraction from thin-layer chromatography also contained an unidentified SocA substrate that had morphogenic properties.
Iron Starvation Triggers the Stringent Response and Induces Amino Acid Biosynthesis for Bacillibactin Production in
Bacillus subtilis
Authors: Miethke Marcus , Westers Helga , Blom Evert-Jan , Kuipers Oscar P. , Marahiel Mohamed A.
Published: December 2006

Iron deprivation in bacteria causes the derepression of genes controlled by the ferric uptake regulator (Fur). The present microarray analysis of iron-starved
Bacillus subtilis
cells grown in minimal medium unveils additional physiological effects on a large number of genes linked to stringent-response regulation and to genes involved in amino acid biosynthesis associated with pathways essential for bacillibactin production.
Characterization of a Highly Conserved Island in the Otherwise Divergent
Bordetella holmesii
and
Bordetella pertussis
Genomes
Authors: Diavatopoulos D. A. , Cummings C. A. , van der Heide H. G. J. , van Gent M. , Liew S. , Relman D. A. , Mooi F. R.
Published: December 2006

The recently discovered pathogen
Bordetella holmesii
has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of
B. holmesii
are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions,
B. holmesii
strains were analyzed by comparative genomic hybridization (CGH) to a
Bordetella pertussis
microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between
B. pertussis
and
B. holmesii
. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS
481
elements that may have been laterally transferred from
B. pertussis
to
B. holmesii
. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by
B. holmesii
may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between
B. pertussis
and
B. holmesii
may also explain the unusually high sequence identity of their 16S rRNA genes.
N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant
Authors: Kolkenbrock Stephan , Parschat Katja , Beermann Bernd , Hinz Hans-Jürgen , Fetzner Susanne
Published: December 2006

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation byArthrobacter nitroguajacolicusRü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the α/β-hydrolase-fold superfamily of enzymes; inactivation of (His6-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (kcat/Km= 208 mM−1s−1), while among the aryl-acetylamides,o-carboxy- oro-nitro-substituted analogues were preferred overp-substituted or unsubstituted compounds. Hydrolysis by His6Amq of primary amides, lactams,N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1%N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His6AmqC22A/C63A markedly differed from those of His6Amq. The replacements effected some changes inKms of the enzyme and increasedkcats for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasingkcatfor the formyl analogueN-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in α-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.
Functional Analysis of AtlA, the MajorN-Acetylglucosaminidase ofEnterococcus faecalis
Authors: Eckert Catherine , Lecerf Maxime , Dubost Lionel , Arthur Michel , Mesnage Stéphane
Published: December 2006

The major peptidoglycan hydrolase ofEnterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is anN-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of theatlAgene inEscherichia colior partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms ofE. faecalisextracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from anatlAnull mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA inE. faecalisextracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.
Mycobacterium tuberculosisSigM Positively Regulates Esx Secreted Protein and Nonribosomal Peptide Synthetase Genes and Down Regulates Virulence-Associated Surface Lipid Synthesis
Authors: Raman Sahadevan , Puyang Xiaoling , Cheng Tan-Yun , Young David C. , Moody D. Branch , Husson Robert N.
Published: December 2006

TheMycobacterium tuberculosisgenome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigatedM. tuberculosisSigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in asigMmutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and thekasA-kasBoperon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in asigM-disrupted strain and is undetectable in asigMoverexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions.
Kinase Activity of Overexpressed HipA Is Required for Growth Arrest and Multidrug Tolerance in
Escherichia coli
Authors: Correia Frederick F. , D'Onofrio Anthony , Rejtar Tomas , Li Lingyun , Karger Barry L. , Makarova Kira , Koonin Eugene V. , Lewis Kim
Published: December 2006

Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in
Escherichia coli
. HipA is a “toxin” that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the
hipBA
operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg
2+
-binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation.
Role of RelGsuin Stress Response and Fe(III) Reduction inGeobacter sulfurreducens
Authors: DiDonato Laurie N. , Sullivan Sara A. , Methé Barbara A. , Nevin Kelly P. , England Reg , Lovley Derek R.
Published: December 2006

Geobacterspecies are key members of the microbial community in many subsurface environments in which dissimilatory metal reduction is an important process. The genome ofGeobacter sulfurreducenscontains a gene designatedrelGsu, which encodes a RelA homolog predicted to catalyze both the synthesis and the degradation of guanosine 3′,5′-bispyrophosphate (ppGpp), a regulatory molecule that signals slow growth in response to nutrient limitation in bacteria. To evaluate the physiological role of RelGsuinG. sulfurreducens, arelGsumutant was constructed and characterized, and ppGpp levels were monitored under various conditions in both the wild-type andrelGsumutant strains. In the wild-type strain, ppGpp and ppGp were produced in response to acetate and nitrogen deprivation, whereas exposure to oxygen resulted in an accumulation of ppGpp alone. Neither ppGpp nor ppGp could be detected in therelGsumutant. TherelGsumutant consistently grew to a higher cell density than the wild type in acetate-fumarate medium and was less tolerant of oxidative stress than the wild type. The capacity for Fe(III) reduction was substantially diminished in the mutant. Microarray and quantitative reverse transcription-PCR analyses indicated that during stationary-phase growth, protein synthesis genes were up-regulated in therelGsumutant and genes involved in stress responses and electron transport, including several implicated in Fe(III) reduction, were down-regulated in the mutant. The results are consistent with a role for RelGsuin regulating growth, stress responses, and Fe(III) reduction inG. sulfurreducensunder conditions likely to be prevalent in subsurface environments.
Characterization of XynC from
Bacillus subtilis
subsp.
subtilis
Strain 168 and Analysis of Its Role in Depolymerization of Glucuronoxylan
Authors: St. John Franz J. , Rice John D. , Preston James F.
Published: December 2006

Secretion of xylanase activities by
Bacillus subtilis
168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAX
n
[where n represents the number of xylose residues]) in the hemicellulose component of lignocellulosics to biobased products. In addition to the characterized glycosyl hydrolase family 11 (GH 11) endoxylanase designated XynA,
B. subtilis
168 secretes a second endoxylanase as the translated product of the
ynfF
gene. This sequence shows remarkable homology to the GH 5 endoxylanase secreted by strains of
Erwinia chrysanthemi
. To determine its properties and potential role in the depolymerization of MeGAX
n
, the
ynfF
gene was cloned and overexpressed to provide an endoxylanase, designated XynC, which was characterized with respect to substrate preference, kinetic properties, and product formation. With different sources of MeGAX
n
as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the β-1,4-xylan chain. With MeGAX
n
from sweetgum as a preferred substrate, XynC exhibited a
V
max
of 59.9 units/mg XynC, a

K
m

of 1.63 mg MeGAX
n
/ml, and a
k
cat
of 2,635/minute at pH 6.0 and 37°C. Matrix-assisted laser desorption ionization—time of flight mass spectrometry and
1
H nuclear magnetic resonance data revealed that each hydrolysis product has a single glucuronosyl substitution penultimate to the reducing terminal xylose. This detailed analysis of XynC from
B. subtilis
168 defines the unique depolymerization process catalyzed by the GH 5 endoxylanases. Based upon product analysis,
B. subtilis
168 secretes both XynA and XynC. Expression of
xynA
was subject to MeGAX
n
induction;
xynC
expression was constitutive with growth on different substrates. Translation and secretion of both GH 11 and GH 5 endoxylanases by the fully sequenced and genetically malleable
B. subtilis
168 recommends this bacterium for the introduction of genes required for the complete utilization of products of the enzyme-catalyzed depolymerization of MeGAX
n
.
B. subtilis
may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.
BadM Is a Transcriptional Repressor and One of Three Regulators That Control Benzoyl Coenzyme A Reductase Gene Expression in
Rhodopseudomonas palustris
Authors: Peres Caroline M. , Harwood Caroline S.
Published: December 2006

The rate-limiting enzyme of anaerobic benzoate degradation by
Rhodopseudomonas palustris
, benzoyl coenzyme A (CoA) reductase, is highly sensitive to oxygen, and its synthesis is tightly regulated. We determined that a previously unknown gene in the benzoate gene cluster,
badM
, encodes a transcriptional repressor of benzoyl-CoA reductase gene expression. BadM controls gene expression from the benzoyl-CoA reductase promoter in concert with two previously described transcriptional activators.
Mutational Analysis To Define an Activating Region on the Redox-Sensitive Transcriptional Regulator OxyR
Authors: Wang Xunde , Mukhopadhyay Partha , Wood Matthew J. , Outten F. Wayne , Opdyke Jason A. , Storz Gisela
Published: December 2006

The OxyR transcription factor is a key regulator of the
Escherichia coli
response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of
oxyR
. The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.
Heme Biosynthesis in
Methanosarcina barkeri
via a Pathway Involving Two Methylation Reactions
Authors: Buchenau Bärbel , Kahnt Jörg , Heinemann Ilka U. , Jahn Dieter , Thauer Rudolf K.
Published: December 2006

The methanogenic archaeon
Methanosarcina barkeri
synthesizes protoheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reactions utilizing
S
-adenosyl-
l
-methionine. The existence of this pathway, previously exclusively found in the sulfate-reducing δ-proteobacterium
Desulfovibrio vulgaris
, was demonstrated for
M. barkeri
via the incorporation of two methyl groups from methionine into protoheme.
Membrane Topology Mapping of the Na
+
-Pumping NADH: Quinone Oxidoreductase from
Vibrio cholerae
by PhoA- Green Fluorescent Protein Fusion Analysis
Authors: Duffy Ellen B. , Barquera Blanca
Published: December 2006

The membrane topologies of the six subunits of Na
+
-translocating NADH:quinone oxidoreductase (Na
+
-NQR) from
Vibrio cholerae
were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (
phoA
) or green fluorescent protein (
gfp
) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of
Escherichia coli
homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na
+
-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed.