bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host
Authors: Tooley Aaron J. , Glazer Alexander N. 
Published: October 2002

ABSTRACT

The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in
Escherichia coli.
Cyanobacterial genes encoding enzymes required for the conversion of heme to 3
Z
-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3
Z-
phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid
trp-lac
(
trc
) promoter. Genes for the apo-phycoerythrocyanin α subunit (
pecA
) and the heterodimeric lyase/isomerase (
pecE
and
pecF
), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the
trc
promoter on a second plasmid. Upon induction
,
recombinant
E. coli
used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered
E. coli
strain that lacks
pecE
and
pecF.
By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
SalmonellaType III Secretion-Associated Protein InvE Controls Translocation of Effector Proteins into Host Cells
Authors: Kubori Tomoko , Galán Jorge E. 
Published: October 2002

ABSTRACTSalmonella entericaencodes a type III secretion system (TTSS) within a pathogenicity island located at centisome 63 (SPI-1), which is essential for its pathogenicity. This system mediates the transfer of a battery of bacterial proteins into the host cell with the capacity to modulate cellular functions. The transfer process is dependent on the function of protein translocases SipB, SipC, and SipD. We report here thatSalmonellaprotein InvE, which is also encoded within SPI-1, is essential for the translocation of bacterial proteins into host cells. AnS. entericaserovar Typhimurium mutant carrying a loss-of-function mutation ininvEshows reduced secretion of SipB, SipC, and SipD while exhibiting increased secretion of other TTSS effector proteins. We also demonstrate that InvE interacts with a protein complex formed by SipB, SipC, and their cognate chaperone, SicA. We propose that InvE controls protein translocation by regulating the function of the Sip protein translocases.
Palindromic Unit-Independent Transposition of IS1397inYersinia pestis
Authors: Wilde Caroline , Bachellier Sophie , Hofnung Maurice , Carniel Elisabeth , Clément Jean-Marie 
Published: October 2002

ABSTRACTPalindromic units (PUs) are intergenic repeated sequences scattered over the chromosomes ofEscherichia coliand several other enterobacteria. In the latter, IS1397, anE. coliinsertion sequence specific to PUs, transposes into PUs with sequences close to theE. coliconsensus. Reasons for this insertion specificity can relate to either a direct recognition of the target (by its sequence or its structure) by the transposase or an interaction between a specific host protein and the PU target DNA sequence. In this study, we show that forYersinia pestis, a species deprived of PUs, IS1397can transpose onto its chromosome, with transpositional hot spots. Our results are in favor of a direct recognition of target DNA by IS1397transposase.
Stochastic Assembly of Two-Component Staphylococcal γ-Hemolysin into Heteroheptameric Transmembrane Pores with Alternate Subunit Arrangements in Ratios of 3:4 and 4:3
Authors: Sugawara-Tomita Noriko , Tomita Toshio , Kamio Yoshiyuki 
Published: October 2002

ABSTRACT

Self-assembling, pore-forming toxins from
Staphylococcus aureus
are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione
S
-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively.
Site-Specific Recombination System Encoded by Toluene Catabolic Transposon Tn4651
Authors: Genka Hiroyuki , Nagata Yuji , Tsuda Masataka 
Published: October 2002

ABSTRACTThe 56-kb class II toluene catabolic transposon Tn4651fromPseudomonas putidaplasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kbtnpT-res-tnpSregion is 48 kb apart from thetnpAgene (M. Tsuda, K.-I. Minegishi, and T. Iino, J. Bacteriol. 171:1386-1393, 1989). Detailed analysis of the 2.4-kb region revealed that thetnpSandtnpTgenes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bpressite. The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS. In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination. Analysis of site-specific recombination under theEscherichia coli recAcells indicated that (i) the site-specific resolution between the two copies of theressite on a single molecule was catalyzed by TnpS, (ii) the functionalressite was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution. It was also found that TnpS catalyzed the site-specific recombination between theressites located at two different molecules to form a cointegrate molecule. Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.
Membrane Protein Degradation by FtsH Can Be Initiated from Either End
Authors: Chiba Shinobu , Akiyama Yoshinori , Ito Koreaki 
Published: October 2002

ABSTRACTFtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins inEscherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.
Factors Affecting Start Site Selection at the
Escherichia coli fis
Promoter
Authors: Walker Kimberly A. , Osuna Robert 
Published: October 2002

ABSTRACT

Transcription initiation with CTP is an uncommon feature among
Escherichia coli
σ
70
promoters. The
fis
promoter (
fis
P), which is subject to growth phase-dependent regulation, is among the few that predominantly initiate transcription with CTP. Mutations in this promoter that cause a switch from utilization of CTP to either ATP or GTP as the initiation nucleotide drastically alter its growth phase regulation pattern, suggesting that the choice of the primary initiating nucleotide can significantly affect its regulation. To better understand what factors influence this choice in
fis
P, we made use of a series of promoter mutations that altered the nucleotide or position used for initiation. Examination of these promoters indicates that start site selection is determined by a combination of factors that include preference for a nucleotide distance from the −10 region (8 > 7 > 9 ≫ 6 ≫ 10 > 11), initiation nucleotide preference (A = G ≫ CTP ≥ UTP), the DNA sequence surrounding the initiation region, the position of the −35 region, and changes in the intracellular nucleoside triphosphate pools. We describe the effects that each of these factors has on start site selection in the
fis
P and discuss the interplay between position and nucleotide preference in this important process.
Structure and Function of Hib Pili fromHaemophilus influenzaeType b
Authors: Mu Xiang-Qi , Egelman Edward H. , Bullitt Esther 
Published: October 2002

ABSTRACTPathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding ofHaemophilus influenzaetype b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 Å in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 Å along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenicEscherichia coliwere used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence ofH. influenzaetype b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.
Imbroglios of Viral Taxonomy: Genetic Exchange and Failings of Phenetic Approaches
Authors: Lawrence Jeffrey G. , Hatfull Graham F. , Hendrix Roger W. 
Published: October 2002

ABSTRACTThe practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linnaeus. The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification. Whereas species have long been established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task and has proven exceedingly difficult for describing viruses and bacteriophages. Moreover, the assembly of viral “species” into higher-order taxonomic groupings has been even more tenuous, since these groupings were based initially on limited numbers of morphological features and more recently on overall genomic similarities. The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms necessitates a reevaluation of the concept of viral species, genera, families, and higher levels of classification. Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes. For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnaean paradigm that necessitate a new view of viral evolution, classification, and taxonomy.
Membrane Association and Kinase-Like Motifs of the RamC Protein of
Streptomyces coelicolor
Authors: Hudson Michael E. , Zhang Dachuan , Nodwell Justin R. 
Published: October 2002

ABSTRACT

The protein RamC is required for the production of the spore-forming cells called aerial hyphae by the filamentous bacterium
Streptomyces coelicolor.
We showed that RamC, which contains several weakly predicted membrane-spanning sequences, is located exclusively in the
S. coelicolor
membrane. By constructing site-directed mutants in the cloned
ramC
gene and complementing a
ramC
null mutant, we showed that protein kinase-like sequence motifs in the amino-terminal half of the protein are required for function in vivo
.
These data suggest that RamC is a membrane-associated receptor kinase.
Comparative Proteome Analysis ofBrucella melitensisVaccine Strain Rev 1 and a Virulent Strain, 16M
Authors: Eschenbrenner Michel , Wagner Mary Ann , Horn Troy A. , Kraycer Jo Ann , Mujer Cesar V. , Hagius Sue , Elzer Philip , DelVecchio Vito G. 
Published: October 2002

ABSTRACTThe genusBrucellaconsists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the differentBrucellaspecies,Brucella melitensisis considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence inB. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the twoB. melitensisproteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.
Purification and Polar Localization of Pneumococcal LytB, a Putative Endo-β-
N
-Acetylglucosaminidase: the Chain-Dispersing Murein Hydrolase
Authors: De Las Rivas Blanca , García José L. , López Rubens , García Pedro 
Published: October 2002

ABSTRACT

The DNA region encoding the mature form of a pneumococcal murein hydrolase (LytB) was cloned and expressed in
Escherichia coli
. LytB was purified by affinity chromatography, and its activity was suggested to be the first identified endo-β-
N
-acetylglucosaminidase of
Streptococcus pneumoniae
. LytB can remove a maximum of only 25% of the radioactivity from [
3
H]choline-labeled pneumococcal cell walls in in vitro assays. Inactivation of the
lytB
gene of wild-type strain R6 (R6B mutant) led to the formation of long chains but did not affect either total cell wall hydrolytic activity at the stationary phase of growth or development of genetic competence. Longer chains were formed when the
lytB
mutation was introduced into the M31 strain (M31B mutant), which harbors a complete deletion of
lytA
, which codes for the major autolysin. Furthermore, the use of this mutant revealed that LytB is the first nonautolytic murein hydrolase of pneumococcus. Purified LytB added to pneumococcal cultures of R6B or M31B was capable of dispersing, in a dose-dependent manner, the long chains characteristic of these mutants into diplococci or short chains, the typical morphology of R6 and M31 strains, respectively. In vitro acetylation of purified pneumococcal cell walls did not affect the activity of LytB, whereas that of the LytA amidase was drastically reduced. On the other hand, the use of a translational fusion between the gene (
gfp
) coding for the green fluorescent protein (GFP) and
lytB
supports the notion that LytB accumulates in the cell poles of either the wild-type R6,
lytB
mutants, or ethanolamine-containing cells (EA cells). The GFP-LytB fusion protein was also able to unchain the
lytB
mutants but not the EA cells. In contrast, translational fusion protein GFP-LytA preferentially bound to the equatorial regions of choline-containing cells but did not affect their average chain length. These observations suggest the existence of specific receptors for LytB that are positioned at the polar region on the pneumococcal surface, allowing localized peptidoglycan hydrolysis and separation of the daughter cells.
Mycobacterium smegmatisl-Alanine Dehydrogenase (Ald) Is Required for Proficient Utilization of Alanine as a Sole Nitrogen Source and Sustained Anaerobic Growth
Authors: Feng Zhengyu , Cáceres Nancy E. , Sarath Gautam , Barletta Raúl G. 
Published: October 2002

ABSTRACTNAD(H)-dependentl-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination ofl-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald inMycobacterium smegmatis, we cloned thealdgene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence ofl- ord-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore,M. smegmatis aldnull mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. Thealdmutants grew poorly in minimal medium withl-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by theM. smegmatis aldmutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.
Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems ofEscherichia coli
Authors: Suzuki Kazushi , Wang Xin , Weilbacher Thomas , Pernestig Anna-Karin , Melefors Öjar , Georgellis Dimitris , Babitzke Paul , Romeo Tony 
Published: October 2002

ABSTRACTThe global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation inEscherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activatescsrBtranscription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreasedcsrBtranscription but did not affectcsrA′-′lacZexpression. TheuvrYdefect was severalfold more severe than that ofbarA. BothcsrAanduvrYwere required for optimalbarAexpression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression ofuvrYsuppressed thecsrB-lacZexpression defects caused byuvrY,csrA, orbarAmutations;csrAsuppressedcsrAorbarAdefects; andbarAcomplemented only thebarAmutation. Purified UvrY protein stimulatedcsrB-lacZexpression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY oncsrBtranscription. Disruption ofsdiA, which encodes a LuxR homologue, decreased the expression ofuvrY′-′lacZandcsrB-lacZfusions but did not affectcsrA′-′lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression ofuvrYstimulated biofilm formation by acsrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate thatuvrYresides downstream fromcsrAin a signaling pathway forcsrBand that CsrA stimulates UvrY-dependent activation ofcsrBexpression by BarA-dependent and -independent mechanisms.
Conidial Viability Assay for Rapid Susceptibility Testing of
Aspergillus
Species
Authors: Balajee S. Arunmozhi , Marr Kieren A. 
Published: October 2002

ABSTRACT

Antifungal susceptibility testing of filamentous fungi has become more important given the recognition of drug-resistant organisms and the availability of therapies other than amphotericin B (AMB). As current microdilution and E-test methods are limited by a 2 to 3 day incubation time required to obtain results, a more rapid method for susceptibility testing of fungi is needed. We report here a flow cytometric assay that relies on conidial metabolism of the viability dye FUN-1. Conidia are incubated in media containing increasing concentrations of AMB for 3 h, exposed to FUN-1, and then analyzed by flow cytometry. Relative susceptibility to AMB can be measured both by forward and side scatter characteristics of the conidial population and by mean fluorescence intensity (MFI) of the dye. MIC, calculated as the concentration of AMB to yield 90% reduction in MFI relative to growth controls, was determined for 27 clinical isolates
Aspergillus
species and correlated well with the standard (i.e., NCCLS) method. The results of these studies illustrate a method by which AMB susceptibility can be rapidly and reproducibly determined by measuring conidial viability.
Multiple
Mycobacterium tuberculosis
Strains in Early Cultures from Patients in a High-Incidence Community Setting
Authors: Richardson Madalene , Carroll Nora M. , Engelke Erica , van der Spuy Gian D. , Salker Faeeza , Munch Zahn , Gie Robert P. , Warren Robin M. , Beyers Nulda , van Helden Paul D. 
Published: October 2002

ABSTRACT

In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled.
Mycobacterium tuberculosis
strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple
M. tuberculosis
strains in the context of the molecular epidemiology of the study setting.