bluuebunny/crossref_metadata_2025_split huggingface dataset:
The first committed step in the biosynthesis of sialic acid by Escherichia coli K1 does not involve a phosphorylated N‐acetylmannosamine intermediate
Authors: Ringenberg Michael A. , Steenbergen Susan M. , Vimr Eric R. 
Published: October 2003

SummaryA variety of pathogens or commensals use at least one of four distinct mechanisms for decorating their surfaces with sialic acid as a strategy to avoid, subvert or inhibit host innate immunity. The metabolism of sialic acid thus is central to a range of host–pathogen interactions. The first committed step in this process, the production of free N‐acetylmannosamine (ManNAc), has not been defined. Here we show that ManNAc‐6‐phosphate (ManNAc‐6‐P) is not an obligate sialate precursor in Escherichia coli K1. This conclusion was supported by 31P NMR spectroscopy of E. coli K1 derivatives engineered with different combinations of mutations in nanA (sialate aldolase or lyase), nanK (ManNAc kinase), nanE (ManNAc‐6‐P 2‐epimerase), neuS (polysialyltransferase) and neuB (sialate synthase). The product specificities for purified NanK and NanE were determined by chromatographic analyses. Direct biochemical analysis showed that ManNAc‐6‐P was stable in a nanE mutant extract. The combined results indicate that neither ManNAc‐6‐P nor specific or non‐specific phosphatase are necessary to generate the requisite ManNAc for sialate biosynthesis. Our results imply that the neuC gene product encodes an UDP‐N‐acetylglucosamine 2‐epimerase that generates ManNAc directly from the dinucleotide‐sugar precursor despite detection of only this enzyme's UDP‐GlcNAc hydrolase activity. This study describes the first use of NMR for analysing intermediate flux within the sialate biosynthetic pathway.
Upstream short sequence repeats regulate expression of the alpha C protein of group B Streptococcus
Authors: Puopolo Karen M. , Madoff Lawrence C. 
Published: October 2003

SummaryGroup B streptococci (GBS) express a family of repeat‐containing surface proteins, the prototype of which is the alpha C protein expressed in type Ia/C strain A909. We have isolated a series of mutant GBS strains by mouse‐passage of A909 that do not produce normal levels of the alpha C protein. Polymerase chain reaction amplification and sequencing of the gene encoding the alpha C protein, bca, from four mutant strains revealed the presence of a full‐length gene in each strain. However, Northern and RT‐PCR analysis revealed greatly reduced levels of RNA encoding the alpha C protein. Sequence analysis of the mutant genes found the coding region unchanged from the wild‐type gene in each case, but variation was observed in a specific locus located 110 bp upstream of the start codon. The presence of a 5‐nucleotide repeat, AGATT, and a string of adenine residues mark this locus. Both deletion and expansion of the AGATT motif were associated with the complete null phenotype. Deletions in the string of adenine residues were associated with both a decreased‐production phenotype and a complete null phenotype. Cloning of this upstream region into a green‐fluorescent protein (GFP) reporter system in GBS demonstrated promoter activity that was completely abolished by changes in the pentanucleotide repeat or adenine string. Primer extension studies of the wild‐type strain revealed one dominant and two minor transcription start sites. Primer extension studies of the null and low‐expression mutant strains revealed that the dominant transcript is completely absent in each mutant. The short sequence repeat locus is located at position − 55 to − 78 relative to the start site of the dominant transcript. We have demonstrated in vitro phase variation in expression of the alpha C protein associated with variation at the pentanucleotide repeat locus. We conclude that this short sequence repeat motif is located upstream of the dominant promoter for the alpha C protein and represents a regulatory site for alpha C protein expression. This is the first evidence of transcriptional regulation by short‐sequence repeats in a Gram‐positive organism.
Segregation of the Escherichia coli chromosome terminus
Authors: Li Yongfang , Youngren Brenda , Sergueev Kirill , Austin Stuart 
Published: October 2003

SummaryWe studied the segregation of the replication terminus of the Escherichia coli chromosome by time‐lapse and still photomicroscopy. The replicated termini lie together at the cell centre. They rapidly segregate away from each other immediately before cell division. At fast growth rate, the copies move progressively and quickly toward the centres of the new‐born cells. At slow growth rate, the termini usually remain near the inner cell pole and migrate to the cell centre in the middle of the cell cycle. A terminus domain of about 160kb, roughly centred on the dif recombination site, segregated as a unit at cell division. Sequences outside this domain segregated before division, giving two separate foci in predivision cells. Resolution of chromosome dimers via the terminus dif site requires the XerC recombinase and an activity of the FtsK protein that is thought to align the dif sequences at the cell centre. We found that anchoring of the termini at the cell centre and proper segregation at cell division occurred normally in the absence of recombination via the XerC recombinase. Anchoring and proper segregation were, however, frequently disrupted when the C‐terminal domain of FtsK was truncated.
Nitrogen metabolism and virulence of Candida albicans require the GATA‐type transcriptional activator encoded by GAT1
Authors: Limjindaporn Thawornchai , Khalaf Roy A. , Fonzi William A. 
Published: October 2003

SummaryNitrogen acquisition and metabolism is central to microbial growth. A conserved family of zinc‐finger containing transcriptional regulators known as GATA‐factors ensures efficient utilization of available nitrogen sources by fungi. GATA factors activate expression of nitrogen catabolic pathways when preferred nitrogen sources are absent or limiting, a phenomenon known as nitrogen catabolite repression. GAT1 of Candida albicans encodes a GATA‐factor homologous to the AREA protein of Aspergillus nidulans and related transcription factors involved in nitrogen regulation. Two observations implicated GAT1 in nitrogen regulation. The growth of mutants lacking GAT1 was reduced when isoleucine, tyrosine or tryptophan were the sole source of nitrogen. Secondly, when cultured on a secondary nitrogen source, gat1Δ mutants were unable to activate expression of GAP1, UGA4 or DAL5, which were shown to be nitrogen regulated in C. albicans. This regulatory defect did not prevent filamentation of gat1Δ mutants in nitrogen repressing or non‐repressing conditions, demonstrating that nitrogen catabolite repression does not influence dimorphism. The mutants were, however, highly attenuated in a murine model of disseminated candidiasis. Attenuation was not associated with any diminution of growth in serum or ability to utilize serum amino acids. The results indicate an important role for nitrogen regulation in the virulence of C. albicans.
Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors
Authors: De Jonge Marien I. , Hamstra Hendrik Jan , Van Alphen Loek , Dankert Jacob , Van Der Ley Peter 
Published: October 2003

SummaryThe opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs). Several studies have identified the Opa‐binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM‐receptors. The high degree of sequence variation in the surface‐exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV‐1 and HV‐2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV‐1 and HV‐2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV‐1 and HV‐2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV‐1 and HV‐2 regions.
The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA
Authors: Rand Lucinda , Hinds Jason , Springer Burkhard , Sander Peter , Buxton Roger S. , Davis Elaine O. 
Published: October 2003

SummaryIn many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild‐type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.
Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin‐binding proteins during the cell cycle
Authors: Morlot Cécile , Zapun André , Dideberg Otto , Vernet Thierry 
Published: October 2003

SummaryThe bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin‐binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP‐deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ‐ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP‐defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.
The NblAI protein from the filamentous cyanobacterium Tolypothrix PCC 7601: regulation of its expression and interactions with phycobilisome components
Authors: Luque Ignacio , Ochoa de Alda Jesús A. G. , Richaud Catherine , Zabulon Gérald , Thomas Jean‐Claude , Houmard Jean 
Published: October 2003

SummaryCyanobacteria respond to changes in light or nutrient availability by modifications in their photosynthetic light harvesting antenna. In unicellular cyanobacteria a small polypeptide (NblA) is required for phycobilisome degradation following environmental stresses. In the filamentous strain Tolypothrix sp. PCC 7601 the nblAI gene, encoding a NblA homologue, is located upstream of the operon coding for phycoerythrin (cpeBA). The nblAI transcripts all originate from a single transcription start point; their intracellular levels vary according to nitrogen regimes but not with light spectral quality. Using recombinant His‐tagged NblAI protein, we found that in vitro NblAI has affinity for both phycocyanin and phycoerythrin subunits from Tolypothrix sp. PCC 7601, but not for allophycocyanin from this cyanobacterium or for phycobiliproteins from other cyanobacterial species. We also observed that although nblAI is mainly expressed under nitrogen starvation, NblAI polypeptides are always present in the cell; a significant portion of them co‐purify with phycobilisome preparations but only if cells were grown under red light. Our data indicate that NblAI attaches to the phycobilisomes even under non‐inducing conditions and suggest a preferential affinity of NblAI for phycocyanin.
Internalization of OspA in rsCD14 complex and aggregated forms
Authors: Welty Diane M. , Snyder D. Scott 
Published: October 2003

SummaryAlthough the spirochetal protein OspA is capable of stimulating immune cells in a CD14‐ and TLR2‐dependent manner, little is known about how TLR2 receptor complex ligands, such as OspA, are handled by the cell once delivered. We examine here the internalization of the fluorescently derivatized forms of both the full length OspA lipoprotein delivered as a recombinant soluble CD14 (rsCD14) complex and the corresponding lipohexapeptide given to the cells as an aggregate. Both forms of OspA are internalized in a similar manner to acetylated low density lipoprotein (AcLDL), a scavenger receptor ligand. Acetylated low density lipoprotein is capable of competing for internalization with OspA even when OspA is delivered as a rsCD14 complex. We observe co‐localization of OspA with lysosomes but not with the Golgi complex. These phenomena are similar between RAW264.7 macrophages and endothelial cells but change drastically when the cells are deprived of serum. Upon serum starvation, OspA shows some localization to the Golgi apparatus whereas the lipohexapeptide remains on the cell surface. Inhibition of internalization of OspA via treatment with cytochalasin D or of the lipohexapeptide via serum starvation does not interfere with TNF induction activity, consistent with signalling from the cell surface.
NarE: a novel ADP‐ribosyltransferase from Neisseria meningitidis
Authors: Masignani Vega , Balducci Enrico , Di Marcello Federica , Savino Silvana , Serruto Davide , Veggi Daniele , Bambini Stefania , Scarselli Maria , Aricò Beatrice , Comanducci Maurizio , Adu‐Bobie Jeannette , Giuliani Marzia M. , Rappuoli Rino , Pizza Mariagrazia 
Published: October 2003

SummaryMono ADP‐ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile‐based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP‐ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat‐labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP‐ribosylating and NAD‐glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP‐ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET‐5 and Lineage 3 but absent in ET‐37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co‐transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed.
A six amino acid deletion, partially overlapping the VanSB G2 ATP‐binding motif, leads to constitutive glycopeptide resistance in VanB‐type Enterococcus faecium
Authors: Depardieu Florence , Courvalin Patrice , Msadek Tarek 
Published: October 2003

Summary Enterococcus faecium
clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal
vanB
cluster, including the
vanS
B
/
vanR
B
two‐component system regulatory genes.
Enterococcus faecium
strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The
ddl
gene of BM4525 had a 2 bp insertion leading to an impaired
d
‐alanine:
d
‐alanine ligase. Sequencing of the
vanB
operon in BM4525 also revealed an 18 bp deletion in the
vanS

B

gene designated
vanS

BΔ

. The resulting six amino acid deletion partially overlapped the G2 ATP‐binding domain of the VanS
BΔ
histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base‐pairs. The VanS
B
, VanS
BΔ
and VanR
B
proteins were overproduced in
Escherichia coli
and purified.
In vitro
autophosphorylation of the VanS
B
and VanS
BΔ
histidine kinases and phosphotransfer to the VanR
B
response regulator did not differ significantly. However, VanS
BΔ
was deficient in VanR
B
phosphatase activity leading to accumulation of phosphorylated VanR
B
. Increased glycopeptide resistance in
E. faecium
BM4525 was therefore a result of the lack of production of
d
‐alanyl‐
d
‐alanine ending pentapeptide and to constitutive synthesis of
d
‐alanyl‐
d
‐lactate terminating peptidoglycan precursors, following loss of
d
‐alanine:
d
‐alanine ligase and of VanS
B
phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in
vanS

B
may favour the appearance of high level constitutively expressed vancomycin resistance through a ‘slippage’ type of genetic rearrangement in VanB‐type strains.
A Neisserial autotransporter NalP modulating the processing of other autotransporters
Authors: Van Ulsen Peter , Van Alphen Loek , Ten Hove Jan , Fransen Floris , Van Der Ley Peter , Tommassen Jan 
Published: October 2003

SummaryAutotransporters constitute a relatively simple secretion system in Gram‐negative bacteria, depending for their translocation across the outer membrane only on a C‐terminal translocator domain. We have studied a novel autotransporter serine protease, designated NalP, from Neisseria meningitidis strain H44/76, featuring a lipoprotein motif at the signal sequence cleavage site. Indeed, lipidation of NalP could be demonstrated, but the secreted 70 kDa domain of NalP lacked the lipid‐moiety as a result of additional N‐terminal processing. A nalP mutant showed a drastically altered profile of secreted proteins. Mass‐spectrometric analysis of tryptic fragments identified the autotransporters IgA protease and App, a homologue of the adhesin Hap of Haemophilus influenzae, as the major secreted proteins. Two forms of both of these proteins were found in the culture supernatant of the wild‐type strain, whereas only the lower molecular‐weight forms predominated in the culture supernatant of the nalP mutant. The serine‐protease active site of NalP was required for the modulation of the processing of these autotransporters. We propose that, apart from the autoproteolytic processing, NalP can process App and IgA protease and hypothesize that this function of NalP could contribute to the virulence of the organism.
Lateral gene transfer: when will adolescence end?
Authors: Lawrence Jeffrey G. , Hendrickson Heather 
Published: October 2003

SummaryThe scope and impact of horizontal gene transfer (HGT) in Bacteria and Archaea has grown from a topic largely ignored by the microbiological community to a hot‐button issue gaining staunch supporters (on particular points of view) at a seemingly ever‐increasing rate. Opinions range from HGT being a phenomenon with minor impact on overall microbial evolution and diversification to HGT being so rampant as to obfuscate any opportunities for elucidating microbial evolution – especially organismal phylogeny – from sequence comparisons. This contentious issue has been fuelled by the influx of complete genome sequences, which has allowed for a more detailed examination of this question than previously afforded. We propose that the lack of common ground upon which to formulate consensus viewpoints probably stems from the absence of answers to four critical questions. If addressed, they could clarify concepts, reject tenuous speculation and solidify a robust foundation for the integration of HGT into a framework for long‐term microbial evolution, regardless of the intellectual camp in which you reside. Here, we examine these issues, why their answers shape the outcome of this debate and the progress being made to address them.
Death's toolbox: examining the molecular components of bacterial programmed cell death
Authors: Rice Kelly C. , Bayles Kenneth W. 
Published: October 2003

SummaryProgrammed cell death (PCD) is a genetically determined process of cellular suicide that is activated in response to cellular stress or damage, as well as in response to the developmental signals in multicellular organisms. Although historically studied in eukaryotes, it has been proposed that PCD also functions in prokaryotes, either during the developmental life cycle of certain bacteria or to remove damaged cells from a population in response to a wide variety of stresses. This review will examine several putative examples of bacterial PCD and summarize what is known about the molecular components of these systems.
Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long‐distance transport of iron and differentially regulated by iron
Authors: Inoue Haruhiko , Higuchi Kyoko , Takahashi Michiko , Nakanishi Hiromi , Mori Satoshi , Nishizawa Naoko K. 
Published: October 2003

SummaryNicotianamine (NA), a chelator of metals, is ubiquitously present in higher plants. In graminaceous plants, NA is a biosynthetic precursor of phytosiderophores and is thus a crucial component for iron (Fe) acquisition. Here, we show that three rice NA synthase (NAS) genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long‐distance transport of Fe and that the three genes are differentially regulated by Fe. OsNAS1 and OsNAS2 transcripts were detected in Fe‐sufficient roots but not in leaves, and levels of both increased markedly in both roots and leaves in response to Fe deficiency. In contrast, the OsNAS3 transcript was present in leaves but was very low in roots of Fe‐sufficient plants. Further, OsNAS3 expression was induced in roots but was suppressed in leaves in response to Fe deficiency. Promoter–GUS analysis revealed that OsNAS1 and OsNAS2 were expressed in Fe‐sufficient roots in companion cells and pericycle cells adjacent to the protoxylem. With Fe deficiency, OsNAS1 and OsNAS2 expression extended to all root cells along with an increase in phytosiderophore secretion. In Fe‐deficient plants, OsNAS1 and OsNAS2 were expressed in the vascular bundles of green leaves and in all cells of leaves showing severe chlorosis. OsNAS3 expression was restricted to the pericycle and companion cells of the roots, and in companion cells of leaves irrespective of Fe status. These results strongly suggested that NAS and NA play an important role in long‐distance transport of Fe in rice plants, in addition to their roles in phytosiderophore secretion from roots.