bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Ferredoxin-Dependent Glutamate Synthase from the Hydrogen-Oxidizing Chemoautotrophic BacteriumHydrogenobacter thermophilusTK-6
Authors: Kameya Masafumi , Ikeda Takeshi , Nakamura Miyuki , Arai Hiroyuki , Ishii Masaharu , Igarashi Yasuo 
Published: January 2007

ABSTRACTGlutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPH-dependent glutamate synthases. In this study,Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme fromH. thermophiluswas shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, theH. thermophilusglutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fd1 and Fd2 from this bacterium) containing [4Fe-4S] clusters. Interestingly, theH. thermophilusglutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.
Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading
Pseudomonas
Strain C4
Authors: Swetha Vandana P. , Basu Aditya , Phale Prashant S. 
Published: January 2007

ABSTRACT

Pseudomonas
sp. strain C4 metabolizes carbaryl (1-naphthyl-
N
-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from
Pseudomonas
sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants

K
m

and
V
max
for 1-naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min
−1
mg
−1
, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The

K
i

for 1-naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.
Precise Excision and Self-Integration of a Composite Transposon as a Model for Spontaneous Large-Scale Chromosome Inversion/Deletion of the
Staphylococcus haemolyticus
Clinical Strain JCSC1435
Authors: Watanabe Shinya , Ito Teruyo , Morimoto Yuh , Takeuchi Fumihiko , Hiramatsu Keiichi 
Published: January 2007

ABSTRACT

Large-scale chromosomal inversions (455 to 535 kbp) or deletions (266 to 320 kbp) were found to accompany spontaneous loss of beta-lactam resistance during drug-free passage of the multiresistant
Staphylococcus haemolyticus
clinical strain JCSC1435. Identification and sequencing of the rearranged chromosomal loci revealed that ISSha
1
of
S. haemolyticus
is responsible for the chromosome rearrangements.
Cysteine Residues in the Transmembrane Regions of M13 Procoat Protein Suggest that Oligomeric Coat Proteins Assemble onto Phage Progeny
Authors: Nagler Christof , Nagler Gisela , Kuhn Andreas 
Published: January 2007

ABSTRACT

The M13 phage assembles in the inner membrane of
Escherichia coli
. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the −6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.
Quorum-Sensing Regulation of Adhesion in
Serratia marcescens
MG1 Is Surface Dependent
Authors: Labbate Maurizio , Zhu Hua , Thung Leena , Bandara Rani , Larsen Martin R. , Willcox Mark D. P. , Givskov Michael , Rice Scott A. , Kjelleberg Staffan 
Published: January 2007

ABSTRACT

Serratia marcescens
is an opportunistic pathogen and a major cause of ocular infections. In previous studies of
S. marcescens
MG1, we showed that biofilm maturation and sloughing were regulated by
N
-acyl homoserine lactone (AHL)-based quorum sensing (QS). Because of the importance of adhesion in initiating biofilm formation and infection, the primary goal of this study was to determine whether QS is important in adhesion to both abiotic and biotic surfaces, as assessed by determining the degree of attachment to hydrophilic tissue culture plates and human corneal epithelial (HCE) cells. Our results demonstrate that while adhesion to the abiotic surface was AHL regulated, adhesion to the HCE cell biotic surface was not. Type I fimbriae were identified as the critical adhesin for non-QS-mediated attachment to the biotic HCE cell surface but played no role in adhesion to the abiotic surface. While we were not able to identify a single QS-regulated adhesin essential for attachment to the abiotic surface, four AHL-regulated genes involved in adhesion to the abiotic surface were identified. Interestingly, two of these genes,
bsmA
and
bsmB
, were also shown to be involved in adhesion to the biotic surface in a non-QS-controlled fashion. Therefore, the expression of these two genes appears to be cocontrolled by regulators other than the QS system for mediation of attachment to HCE cells. We also found that QS in
S. marcescens
regulates other potential cell surface adhesins, including exopolysaccharide and the outer membrane protein OmpX. We concluded that
S. marcescens
MG1 utilizes different regulatory systems and adhesins in attachment to biotic and abiotic surfaces and that QS is a main regulatory pathway in adhesion to an abiotic surface but not in adhesion to a biotic surface.
Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in
Geobacillus stearothermophilus
NRS 2004/3a
Authors: Steiner Kerstin , Novotny René , Patel Kinnari , Vinogradov Evgenij , Whitfield Chris , Valvano Miguel A. , Messner Paul , Schäffer Christina 
Published: January 2007

ABSTRACT

The glycan chain of the S-layer glycoprotein of
Geobacillus stearothermophilus
NRS 2004/3a is composed of repeating units [→2)-α-
l
-Rha
p-
(1→3)-β-
l
-Rha
p
-(1→2)-α-
l
-Rha
p
-(1→], with a 2-
O
-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-
l
-rhamnose residues, and a β-
d
-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of
Geobacillus stearothermophilus
NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from
Salmonella enterica
. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of
Escherichia coli
and
Salmonella enterica
serovar Typhimurium.
DRS Is Far Less Divergent than Streptococcal Inhibitor of Complement of Group A Streptococcus
Authors: Sagar Vivek , Kumar Rajesh , Ganguly Nirmal K. , Menon Thangam , Chakraborti Anuradha 
Published: January 2007

ABSTRACT

When 100 group A streptococcus isolates were screened,
drs
, a variant of
sic
, was identified in
emm12
and
emm55
isolates. Molecular characterization showed that the
drs
gene sequence is highly conserved, unlike the
sic
gene sequence. However, the variation in gene size observed was due to the presence of extra internal repeat sequences.
The Essential GTPase YqeH Is Required for Proper Ribosome Assembly in
Bacillus subtilis
Authors: Uicker William C. , Schaefer Laura , Koenigsknecht Mark , Britton Robert A. 
Published: January 2007

ABSTRACT

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in
Bacillus subtilis
.
The W-Beijing Lineage of
Mycobacterium tuberculosis
Overproduces Triglycerides and Has the DosR Dormancy Regulon Constitutively Upregulated
Authors: Reed Michael B. , Gagneux Sebastien , DeRiemer Kathryn , Small Peter M. , Barry Clifton E. 
Published: January 2007

ABSTRACT

The Beijing family of
Mycobacterium tuberculosis
strains has been associated with epidemic spread and an increased likelihood of developing drug resistance. The characteristics that predispose this family to such clinical outcomes have not been identified, although one potential candidate, the phenolic glycolipid PGL-tb, has been shown to mediate a fulminant lethal disease in mice and rabbits due to lipid-mediated immunosuppression. However, PGL-tb is not uniformly expressed throughout the Beijing lineage and may not be the only unique virulence trait associated with this family. In an attempt to define phenotypes common to all Beijing strains, we interrogated a carefully selected set of isolates representing the five extant lineages of the Beijing family. Comparison of lipid production in this set revealed that all Beijing strains accumulated large quantities of triacylglycerides in in vitro aerobic culture. This accumulation was found to be coincident with upregulation of Rv3130c, whose product was previously characterized as a triacylglyceride synthase. Rv3130c is a member of the DosR-controlled regulon of
M. tuberculosis
, and further examination revealed that several members of this regulon were upregulated throughout this strain family. The upregulation of the DosR regulon may confer an adaptive advantage for growth in microaerophilic or anaerobic environments encountered by the bacillus during infection and thus may be related to the epidemiological phenomena associated with this important strain lineage.
Regulatory Role of PopN and Its Interacting Partners in Type III Secretion ofPseudomonas aeruginosa
Authors: Yang Hongjing , Shan Zhiying , Kim Jaewha , Wu Weihui , Lian Wei , Zeng Lin , Xing Laijun , Jin Shouguang 
Published: January 2007

ABSTRACTThe type III secretion system (T3SS) ofPseudomonas aeruginosaplays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and bothpopNandpcr1mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of thepopNoperon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. UnlikepopNandpcr1mutants,pcr3andpcr4mutants are totally defective in type III secretion, while apcr2mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.
Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38
Authors: Aoki Kazuteru , Shinohara Miki , Itoh Tateo 
Published: January 2007

ABSTRACT
The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, α and β, in the origins as specificity determinants and showed that when each component of the region A-site α pair and the region B-site β pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site α. By using an electrophoretic mobility shift assay, we demonstrated that region B and site β play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site α were involved in the efficient unwinding of several nucleotide residues around site α, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.
Controlled Expression of
nif
and
isc
Iron-Sulfur Protein Maturation Components Reveals Target Specificity and Limited Functional Replacement between the Two Systems
Authors: Dos Santos Patricia C. , Johnson Deborah C. , Ragle Brook E. , Unciuleac Mihaela-Carmen , Dean Dennis R. 
Published: January 2007

ABSTRACT

The nitrogen-fixing organism
Azotobacter vinelandii
contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by
nif
genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by
isc
genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory
iscR
gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.
Regulators of Expression of the Oligopeptide Permease A Proteins of
Borrelia burgdorferi
Authors: Medrano Melisa S. , Ding Yanpeng , Wang Xing-Guo , Lu Peng , Coburn Jenifer , Hu Linden T. 
Published: January 2007

ABSTRACT

Borrelia burgdorferi
undergoes an infectious cycle that requires adaptation to different hosts and marked differences in environment.
B. burgdorferi
copes with its different environments by regulating the expression of proteins required for survival in specific settings. The
B. burgdorferi
oligopeptide permease (Opp) is one of only a few transporters encoded by the
B. burgdorferi
genome. Opp proteins in other bacteria serve multiple environmental adaptation functions.
B. burgdorferi
appears to broaden the usage of this transporter by utilizing five different substrate binding proteins (OppA proteins) that interact with the integral membrane components of the transporter. Expression of the OppA proteins is individually regulated and may play different roles in adaptation to host environments. Very little is known about the mechanisms used by
B. burgdorferi
to regulate the expression of different OppA proteins. Here we show that the alternative sigma factors, RpoS and RpoN, regulate the expression of
oppA5
but not that of other
oppA
genes. Using a reporter assay with
Escherichia coli
and gel shift binding assays, we also show that the
B. burgdorferi
BosR/Fur homologue interacts with the
oppA4
promoter and that another candidate transcription factor, EbfC, interacts with the
oppA5
promoter. Binding to the promoters was confirmed by gel shift assays. Expression of BosR/Fur in its different hosts does appear to parallel the expression of
oppA4
. A better understanding of the factors involved in gene regulation in
B. burgdorferi
will help to identify coregulated proteins that may cooperate to allow the organism to survive in a specific environment.
Positive Regulation of
fur
Gene Expression via Direct Interaction of Fur in a Pathogenic Bacterium,
Vibrio vulnificus
Authors: Lee Hyun-Jung , Bang So Hyun , Lee Kyu-Ho , Park Soon-Jung 
Published: January 2007

ABSTRACT

In pathogenic bacteria, the ability to acquire iron, which is mainly regulated by the ferric uptake regulator (Fur), is essential to maintain growth as well as its virulence. In
Vibrio vulnificus
, a human pathogen causing gastroenteritis and septicemia,
fur
gene expression is positively regulated by Fur when the iron concentration is limited (H.-J. Lee et al., J. Bacteriol. 185:5891-5896, 2003). Footprinting analysis revealed that an upstream region of the
fur
gene was protected by the Fur protein from DNase I under iron-depleted conditions. The protected region, from −142 to −106 relative to the transcription start site of the
fur
gene, contains distinct AT-rich repeats. Mutagenesis of this repeated sequence resulted in abolishment of binding by Fur. To confirm the role of this
cis
-acting element in Fur-mediated control of its own gene in vivo,
fur
expression was monitored in
V. vulnificus
strains using a transcriptional fusion containing the mutagenized Fur-binding site (
fur
mt
::
luxAB
). Expression of
fur
mt
::
luxAB
showed that it was not regulated by Fur and was not influenced by iron concentration. Therefore, this study demonstrates that
V. vulnificus
Fur acts as a positive regulator under iron-limited conditions by direct interaction with the
fur
upstream region.
Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in
Staphylococcus aureus
Authors: Zheng Li , Yu Chuanxin , Bayles Kenneth , Lasa Iñigo , Ji Yinduo 
Published: January 2007

ABSTRACT

Our previous studies demonstrated that a putative
Staphylococcus aureus
glycoprotease (Gcp) is essential for bacterial survival, indicating that Gcp may be a novel target for developing antibacterial agents. However, the biological function of Gcp is unclear. In order to elucidate the reason that Gcp is required for growth, we examined the role of Gcp in bacterial autolysis, which is an important biological process for bacterial growth. Using both a
spacp
-regulated
gcp
expression strain and a TetR-regulated
gcp
antisense expression strain, we found that the down-regulation of
gcp
expression can effectively inhibit Triton X-100-induced lysis, eliminate penicillin- and vancomycin-caused cell lysis, and dramatically increase tolerance to hydrolases. Moreover, we determined whether resistance to lysis is due to a defect in murein hydrolase activity by using a zymogram analysis. The results showed that the cell lysate of a down-regulated
gcp
expression mutant displayed several bands of decreased murein hydrolytic activity. Furthermore, we explored the potential mechanism of Gcp's involvement in autolysis and demonstrated that Gcp may function independently from several key autolysins (Atl, LytM, and LytN) and regulators (ArlRS, Mgr/Rat, and CidA). Taken together, the above results indicate that the essential Gcp is involved in the modification of substrates of murein hydrolases as well as in the regulation of expression and/or activity of some murein hydrolases, which, in turn, may play important roles in bacterial viability.