bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transferrin binding in Staphylococcus aureus: involvement of a cell wall‐anchored protein
Authors: Taylor John M. , Heinrichs David E. 
Published: March 2003

SummaryThe ability to gain access to iron is pivotal for bacterial pathogens during infection. Although much is known about iron acquisition systems in Gram‐negative bacteria, comparatively little is known about how Gram‐positive pathogens access iron from host iron sources. A previous study showed that, in the Gram‐positive human pathogen Staphylococcus aureus, a cell surface‐associated glyceraldehyde‐3‐phosphate

dehydrogenase (GAPDH) enzyme (Gap, or Tpn) is capable of binding human transferrin,

representing a potential means by which this bacterium is able to access iron in

vivo. We have investigated this property of S. aureus further and shown

that, in S. aureus RN6390, GAPDH is expressed on the S. aureus cell

surface independent of exogenous iron concentrations, and that overexpressed and

purified Gap, although retaining GAPDH activity, has no affinity for human transferrin.

Moreover, although a S. aureus gap mutant was devoid of surface‐associated

and cytoplasmic GAPDH activity, it retained the ability to bind human transferrin,

equivalent to wild type. We concluded from these results that the Gap protein is

not involved in S. aureus binding to human transferrin. We identified the

transferrin‐binding protein as a novel cell wall‐anchored protein, designated StbA

for staphylococcal transferrin‐binding protein A, which shared

no significant similarities with any other bacterial transferrin‐binding proteins.

StbA contained a C‐terminal cell wall‐anchoring motif (LPKTG), and expression of

StbA in the cell wall was strictly controlled by exogenous iron concentrations. The

stbA gene is found within a 7 kb region in the S. aureus chromosome

that contains a total of six iron‐regulated genes. Immediately downstream from stbA

is an iron‐regulated gene whose product was predicted to be another cell wall‐anchored

protein with no significant similarity to proteins with characterized functions.

Transcribed in the opposite direction from stbA is a four‐gene operon whose expression is also regulated by iron. While the deduced products of the first two genes lack similarity to known proteins, the last two genes encode, respectively, putative lipoprotein and permease components of an ABC transporter that shares significant similarities with several iron(III) ABC transporters in a variety of bacteria.
Developmental switch of S‐layer protein synthesis in Bacillus anthracis
Authors: Mignot Tâm , Mesnage Stéphane , Couture‐Tosi Evelyne , Mock Michèle , Fouet Agnès 
Published: March 2003

SummaryAdjustment of the synthesis of abundant protein to the requirements of the cell involves processes critical to the minimization of energy expenditure. The regulation of S‐layer genes might be a good model for such processes because expression must be controlled, such that the encoded proteins exactly cover the surface of the bacterium. Bacillus anthracis has two S‐layer genes, sap and eag, encoding

the S‐layer proteins Sap and EA1 respectively. We report that the production and

surface localization of Sap and EA1 are under developmental control, suggesting that

an exponential phase ‘Sap layer’ is subsequently replaced by a stationary

phase ‘EA1 layer’. This switch is controlled at the transcriptional

level: sap is most certainly transcribed by RNA polymerase containing σA,

whereas eag expression depends on σH. More importantly,

Sap is required for the temporal control of eag, and EA1 is involved in strict

feedback regulation of eag. This control may be direct because both S‐layer

proteins bind, in vitro, the eag promoter, specifically suggesting that they might act as transcriptional repressors.
The repressor for an organic peroxide‐inducible operon is uniquely regulated at multiple levels
Authors: Mongkolsuk Skorn , Panmanee Warunya , Atichartpongkul Sopapan , Vattanaviboon Paiboon , Whangsuk Wirongrong , Fuangthong Mayuree , Eiamphungporn Warawan , Sukchawalit Rojana , Utamapongchai Supa 
Published: March 2003

Summary ohrR encodes a novel organic peroxide‐inducible transcription repressor,
and we have demonstrated that ohrR is regulated at the transcriptional and
the post‐transcriptional levels. Primer extension results show that ohrR transcription
initiates at the A residue of the ATG translation initiation codon for the ohrR
coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1)
has high homology to the consensus sequence for Xanthomonas promoters, which
is reflected in the high in vivo promoter activity of P1. Deletion of a 139
bp fragment containing the P1 promoter showed that the sequences upstream of –35
regions were required for neither the promoter activity nor OhrR autoregulation.
In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting
of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands.
The protected regions include the –35 and –10 regions of P1. We suggest
that OhrR represses gene expression by blocking RNA polymerase binding to the promoter.
There are two steps in the post‐transcriptional regulation of ohrR, namely
differential stability and inefficient translation of the mRNA. The bicistronic ohrR–ohr
mRNA was highly labile and underwent rapid processing in vivo to give only
stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore,
the ohrR mRNA was inefficiently translated. We propose that, in uninduced
cells, the concentration of OhrR is maintained at low levels by the autoregulation
mechanism at the transcriptional levels and by the ohrR mRNA instability coupled
with inefficient translation at the post‐transcriptional level. Upon exposure to
an organic peroxide, the compound probably interacts with OhrR and prevents it from
repressing the P1 promoter, thus allowing high‐level expression of the ohrR–ohr
operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic per‐oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.
Structural mechanisms of multidrug recognition and regulation by bacterial multidrug transcription factors
Authors: Schumacher Maria A. , Brennan Richard G. 
Published: March 2003

SummaryThe increase in bacterial resistance to multiple drugs represents a serious and growing health risk. One component of multidrug resistance (MDR) is a group of multidrug transporters that are often regulated at the transcriptional level by repressors and/or activators. Some of these transcription factors are also multidrug‐binding proteins, frequently recognizing the same array of drugs that are effluxed by the transporters that they regulate. How a single protein can recognize such chemically disparate compounds is an intriguing question from a structural standpoint and an important question in future drug development endeavours. Unlike the multidrug transporters, the cytosolic multidrug‐binding regulatory proteins are more tractable systems for structural analyses. Here, we describe recent crystallographic studies on MarR, BmrR and QacR, three bacterial transcription regulators that are also multidrug‐binding proteins. Although our understanding of multidrug binding and transcriptional regulation by MarR is in its initial stages, the structure of a BmrR–TPP+–DNA complex has revealed important insights into the novel transcription activation mechanism of the MerR family, and the structures of a QacR–DNA complex and QacR bound to six different drugs have revealed not only the mechanism of induction of this repressor but has afforded the first view of any MDR protein bound to multiple drugs.
Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non‐cellulosomal endoglucanase EngD
Authors: Murashima Koichiro , Kosugi Akihiko , Doi Roy H. 
Published: March 2003

SummaryEnhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non‐cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a com‐bination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.
Dynamic distribution of BIMGPP1 in living hyphae of Aspergillus indicates a novel role in septum formation
Authors: Fox H. , Hickey P. C. , Fernández‐Ábalos J. M. , Lunness P. , Read N. D. , Doonan J. H. 
Published: March 2003

SummaryMutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimGPP1 in mitosis and hyphal growth and are highly dynamic. BIMG‐GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG‐GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG‐GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes.
Agrobacterium type IV secretion is a two‐step process in which export substrates associate with the virulence protein VirJ in the periplasm
Authors: Pantoja Mario , Chen Lishan , Chen Yuching , Nester Eugene W. 
Published: March 2003

SummaryType IV secretion systems are virulence determinants in many bacteria and share extensive homology with many conjugal transfer systems. Although type IV systems and their homologues have been studied widely, the mechanism by which substrates are secreted remains unclear. In Agrobacterium, we show that type IV secretion substrates that lack signal peptides form a soluble complex in the periplasm with the virulence protein VirJ. Additionally, these proteins co‐precipitate with constituents of the type IV transporter: the VirB pilus and the VirD4 protein. Our findings suggest that the substrate proteins localized to the periplasm may associate with the pilus in a manner that is mediated by VirJ, and suggest a two‐step process for type IV secretion in Agrobacterium. Our analyses of protein–protein interactions in a variety of mutant backgrounds indicate that substrates are probably secreted independently of one another.
Molecular analysis of tyrosine‐ and phenylalanine‐mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli
Authors: Yang Ji , Camakaris Helen , Pittard James 
Published: March 2003

SummaryThe mechanism of repression of the tyrB promoter by TyrR protein has been studied in vivo and in vitro. In tyrR+ strains, transcription of tyrB is repressed by either tyrosine or phenylalanine. Both of the TyrR binding sites (strong and weak TyrR boxes) lie downstream of the tyrB transcription start site and are required for tyrosine‐ or phenylalanine‐mediated repression. Our results establish that the binding of the TyrR protein to the weak box, induced by cofactor tyrosine or phenylalanine, is critical for repression to occur. Neither the binding of the TyrR protein dimer formed in the presence of phenylalanine, nor the binding of the hexamer formed in the presence of tyrosine, blocks the binding of RNA polymerase to the promoter. Instead, open complex formation is inhibited in the presence of tyrosine whereas a step(s) following open complex formation is inhibited in the presence of phenylalanine. Moving the TyrR boxes 3 bp or more further away from the promoter affects tyrosine‐mediated repression without affecting phenylalanine‐mediated repression which remains unaltered until 6 bp are inserted between the TyrR boxes and the promoter. Analysis of deletion and insertion mutants fails to reveal any face of the helix specificity for either tyrosine‐ or phenylalanine‐mediated ­repression.
A locus of group A streptococcus involved in invasive disease and DNA transfer
Authors: Hidalgo‐Grass Carlos , Ravins Miriam , Dan‐Goor Mary , Jaffe Joseph , Moses Allon E. , Hanski Emanuel 
Published: March 2003

SummaryGroup A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic‐tag‐lengths‐transposon‐mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft‐tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil, streptococcal invasion locus. sil contains at least five genes (silA‐E) and is highly homologous to the quorum‐sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two‐component system whereas silD and silE encode two putative ABC transporters. silC is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of silC was restored when injected together with the avirulent emm14‐deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm14‐deletion mutant and mutants of sil occurred also in vitro, at a frequency of ∼ 10‐8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.
Integration and excision of the Mycobacterium tuberculosis prophage‐like element, φRv1
Authors: Bibb Lori A. , Hatfull Graham F. 
Published: March 2003

SummaryThe genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage‐like elements, φRv1 and φRv2. The φRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases φRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for φRv1. Although φRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv φRv1 element does indeed encode an active site‐specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic φRv1‐encoded protein (Rv1584c) controls the directionality of re‐combination. Integration‐proficient plasmid vectors derived from φRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.
Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket
Authors: Liu Hui , Radhakrishnan Padhma , Magoun Loranne , Prabu Moses , Campellone Kenneth G. , Savage Pamela , He Feng , Schiffer Celia A. , Leong John M. 
Published: March 2003

SummaryAttachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two‐hybrid assay for intimin–Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions. Many of the mutations fell within a 50‐residue region near the C‐terminus of intimin. Alanine‐scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition. Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin–Tir complex of enteropathogenic E. coli predicts that each of these four intimin residues lies at the intimin–Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir. Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.
Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium
Authors: Maisnier‐Patin Sophie , Berg Otto G. , Liljas Lars , Andersson Dan I. 
Published: March 2003

SummaryMost chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 → AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty‐one independent lineages of the low‐fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be ≥ 10−7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin‐sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single‐nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.
Governor of the glnAp2 promoter of Escherichia coli
Authors: Atkinson Mariette R. , Pattaramanon Narinporn , Ninfa Alexander J. 
Published: March 2003

SummaryLow‐affinity sites for the activator NRI∼P (NtrC∼P) that map between the enhancer and the glnAp2 promoter were responsible for limiting promoter activity at high concentrations of NRI∼P in intact cells and in an in vitro transcription system consisting of purified bacterial components. That is, the low‐affinity sites constitute a ‘governor’, limiting the maximum promoter activity. As the governor sites are themselves far from the promoter, they apparently act either by preventing the formation of the activation DNA loop that brings the enhancer‐bound activator and the promoter‐bound polymerase into proximity or by preventing a productive interaction between the enhancer‐bound activator and polymerase. The combination of potent enhancer and governor sites at the glnAp2 promoter provides for efficient activation of the promoter when the activator concentration is low, while limiting the maximum level of promoter activity when the activator concentration is high.
Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae
Authors: Trotter Eleanor W. , Grant Chris M. 
Published: March 2003

SummaryThioredoxins are small, highly conserved oxidoreductases that are required to maintain the redox homeostasis of the cell. They have been best characterized for their role as antioxidants in protection against reactive oxygen species. We show here that thioredoxins (TRX1, TRX2) and thioredoxin reductase (TRR1) are also required for protection against a reductive stress induced by exposure to dithiothreitol (DTT). This sensitivity to reducing conditions is not a general property of mutants affected in redox control, as mutants lacking components of the glutathione/glutaredoxin system are unaffected. Furthermore, TRX2 gene expression is induced in response to DTT treatment, indicating that thioredoxins form part of the cellular response to a reductive challenge. Our data indicate that the sensitivity of thioredoxin mutants to reducing stress appears to be a consequence of elevated glutathione levels, which is present predominantly in the reduced form (GSH). The elevated GSH levels also result in a constitutively high unfolded protein response (UPR), indicative of an accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, there does not appear to be a general defect in ER function in thioredoxin mutants, as oxidative protein folding of the model protein carboxypeptidase Y occurs with similar kinetics to the wild‐type strain, and trx1 trx2 mutants are unaffected in sensitivity to the glycosylation inhibitor tunicamycin. Furthermore, trr1 mutants are resistant to tunicamycin, consistent with their high UPR. The high UPR seen in trr1 mutants can be abrogated by the GSH‐specific reagent 1‐chloro‐2,4‐dinitrobenzene. In summary, thioredoxins are required to maintain redox homeostasis in response to both oxidative and reductive stress conditions.