bluuebunny/crossref_metadata_2025 huggingface dataset:
Internal transcribed spacer repeat‐specific primers and the analysis of hybridization in the Glycine tomentella (Leguminosae) polyploid complex
Authors: Rauscher Jason T. , Doyle Jeff J. , Brown A. H. D.
Published: March 2003

Polyploid and diploid hybridization is a ubiquitous and evolutionarily important phenomenon in the plant world. Determining the parental species of a hybrid, however, is difficult. Molecular markers such as the nuclear ribosomal DNA gene complex, particularly its internal transcribed spacer (ITS) region, have proved powerful in determining hybrid parentage. In some cases, population and genomic phenomena, such as genetic drift and concerted evolution, result in the loss of all or many of the tandemly repeated copies derived from one parental species, making the recovery of hybrid history difficult or impossible. Methods such as direct sequencing and cloning are typically used to find ITS sequences contributed from parental species, but are limited in their ability to detect rare repeat types. Here we report that repeat‐specific polymerase chain reaction primers can recover rare parental ITS sequences in the Glycine tomentella polyploid complex. In three allopolyploid lineages of this complex, repeat‐specific primers reliably detected rare repeats that both direct sequencing and the screening of many cloned sequences failed to detect. Other strategies, such as the use of exclusion primers, may detect rare parental repeat types in hybrids when previous hypotheses regarding the second parental species are lacking.
Sperm length influences fertilization success during sperm competition in the snail Viviparus ater
Authors: Oppliger A. , Naciri‐Graven Y. , Ribi G. , Hosken D. J.
Published: March 2003

Sperm form and size is tremendously variable within and across species. However, a general explanation for this variation is lacking. It has been suggested that sperm size may influence sperm competition, and there is evidence for this in some taxa but not others. In addition to normal fertilizing sperm, a number of molluscs and insects produce nonfertile sperm that are also extremely morphologically variable, and distinct from fertilizing forms. There is evidence that nonfertile sperm play an indirect role in sperm competition by decreasing female remating propensity in Lepidopterans, but in most taxa the function of parasperm is unknown. We investigated the role of nonfertile (oligopyrene) sperm during sperm competition in the fresh water snail Viviparus ater. Previous studies found that the proportion of oligopyrene sperm increased with the risk of sperm competition, and hence it seems likely that these sperm influence fertilization success during competitive matings. In mating experiments in which females were sequentially housed with males, we examined a range of male characteristics which potentially influence fertilization success. We found that the size of oligopyrene sperm was the best predictor of fertilization success, with males having the longer sperm siring the highest proportion of offspring. Furthermore, we found a positive shell size and sperm concentration effect on paternity, and females with multiply sired families produced more offspring than females mating with only one male. This result suggests polyandry is beneficial for female snails.
Cell division is required for resolution of dimer chromosomes at the dif locus of Escherichia coli
Authors: Steiner Walter W. , Kuempel Peter L.
Published: March 2003

The dif locus is a RecA‐independent resolvase site in the terminus region of the chromosome of Escherichia coli. The locus reduces dimer chromosomes, which result from sister chromatid exchange, to monomers. A density label assay demonstrates that recombination occurs at dif, and that it requires XerC and XerD. The frequency of this recombination is ≈14% per site per generation, which is doubled in polA12 mutants. We have determined that recombination occurs late in the cell cycle, and that resolution is blocked if cell division is inhibited with cephalexin or by a ftsZts mutation. Fluorescence microscopy has demonstrated that abnormal nucleoids are present in cells incubated in cephalexin, and this is increased in polA12 mutants.
Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase‐encoding genes and the haemagglutinin‐encoding gene of Porphyromonas gingivalis
Authors: Nakayama Koji , Ratnayake Dinath B. , Tsukuba Takayuki , Kadowaki Tomoko , Yamamoto Kenji , Fujimura Setsuo
Published: March 2003

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black‐pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N‐terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine‐specific cysteine proteinase (Arg‐gingipain, RGP)‐encoding gene (rgp1) and was also present in genes for lysine‐specific cysteine proteinases (prtP and kgp) and a haemagglutinin (hagA) of P. gingivalis. The HGP15 domain protein was purified from an HGP15‐overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH‐dependent manner. The anti‐HGP15 antiserum reacted with the 19 kDa haemoglobin‐binding protein in the envelope of P. gingivalis. P. gingivalis wild‐type strain showed pH‐dependent haemoglobin adsorption, whereas its non‐pigmented mutants that produced no HGP15‐related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis.
Activity of the quorum‐sensing regulator TraR of Agrobacterium tumefaciens is inhibited by a truncated, dominant defective TraR‐like protein
Authors: Zhu Jun , Winans Stephen C.
Published: March 2003

Horizontal transfer of Agrobacterium tumefaciens tumour‐inducing plasmids requires opines, which are released from plant tumours as nutrients for the bacteria. The opine octopine causes synthesis of the quorum‐sensing TraR protein, which activates several tra promoters in the presence of a pheromone called Agrobacterium autoinducer (AAI). A gene, traS, was previously found on the same Ti plasmid in an operon that directs the uptake of mannopine, another opine. TraS strongly resembles TraR but lacks a DNA‐binding module. TraS did not activate a TraR‐dependent promoter and blocked TraR function, probably by forming inactive heteromultimers. Expression of traS was induced by mannopine, although this induction was strongly inhibited by the favoured catabolites succinate, glutamine and tryptone. Mannopine inhibited conjugation in a TraS‐dependent fashion, and artificial overexpression of TraS also inhibited conjugation. Favoured catabolites restored tra gene expression in wild‐type strains but not in strains that overexpress TraS. Downstream of traS is a gene encoding a truncated, defective chemoreceptor whose expression abolished chemotaxis.
Heparin interferes with translocation of Yop proteins into HeLa cells and binds to LcrG, a regulatory component of the Yersinia Yop apparatus
Authors: Boyd Aoife P. , Sory Marie‐Paule , Iriarte Maite , Cornelis Guy R.
Published: March 2003

Yersiniae are equipped with the Yop virulon, an apparatus that allows extracellular bacteria to deliver toxic Yop proteins inside the host cell cytosol in order to sabotage the communication networks of the host cell or even to cause cell death. LcrG is a component of the Yop virulon involved in the regulation of secretion of the Yops. In this paper, we show that LcrG can bind HeLa cells, and we analyse the role of proteoglycans in this phenomenon. Treatment of the HeLa cells with heparinase I, but not chondroitinase ABC, led to inhibition of binding. Competition assays indicated that heparin and dextran sulphate strongly inhibited binding, but that other glycosaminoglycans did not. This demonstrated that binding of HeLa cells to purified LcrG is caused by heparan sulphate proteoglycans. LcrG could bind directly to heparin‐agarose beads and, in agreement with these results, analysis of the protein sequence of Yersinia enterocolitica LcrG revealed heparin‐binding motifs. In vitro production and secretion by Y. enterocolitica of the Yops was unaffected by the addition of heparin. However, the addition of exogenous heparin decreased the level of YopE–Cya translocation into HeLa cells. A similar decrease was seen with dextran sulphate, whereas the other glycosaminoglycans tested had no significant effect. Translocation was also decreased by treatment of HeLa cells with heparinitase, but not with chondroitinase. Thus, heparan sulphate proteoglycans have an important role to play in translocation. The interaction between LcrG and heparan sulphate anchored at the surface of HeLa cells could be a signal triggering deployment of the Yop translocation machinery. This is the first report of a eukaryotic receptor interacting with the type III secretion and associated translocation machinery of Yersinia or of other bacteria.
Use of signature‐tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization
Authors: Chiang Su L. , Mekalanos John J.
Published: March 2003

The pathogenesis of cholera begins with colonization of the host intestine by Vibrio cholerae. The toxin co‐regulated pilus (TCP), a fimbrial structure produced by V. cholerae, is absolutely required for colonization (i.e. the persistence, survival and growth of V. cholerae in the upper intestinal milieu), but many other aspects of the colonization process are not well understood. In this study, we use signature‐tagged transposon mutagenesis (STM) to conduct a screen for random insertion mutations that affect colonization in the suckling mouse model for cholera. Of approximately 1100 mutants screened, five mutants (approximately 0.5%) with transposon insertions in TCP biogenesis genes were isolated, validating the use of STM to identify attenuated mutants. Insertions in lipopolysaccharide, biotin and purine biosynthetic genes were also found to cause colonization defects. Similar results were observed for mutations in homologues of pta and ptfA, two genes involved in phosphate transfer. Finally, our screen identified several novel genes, disruption of which also caused colonization defects in the mouse model. These results demonstrate that STM is a powerful method for isolating colonization‐defective mutants of V. cholerae.
ClpP ofBacillus subtilisis required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation
Authors: Msadek Tarek , Dartois Véronique , Kunst Frank , Herbaud Marie‐Laure , Denizot François , Rapoport Georges
Published: March 2003

The nucleotide sequence of theBacillus subtilis clpPgene was determined. The predicted protein shows very high similarity to members of the ClpP family of proteolytic subunits (68% amino acid sequence identity with that ofEscherichia coli ). We show that ClpP plays an essential role in stationary phase adaptive responses. Indeed, a ΔclpPmutant was constructed and shown to display a pleiotropic phenotype, including a deficiency in both sporulation initiation and competence for DNA uptake. The ΔclpPmutant has a highly filamentous morphology and appears to be non‐motile, as judged by swarm plate assays. Expression ofclpPis strongly induced under heat shock conditions, and ClpP is shown to be essential for growth ofB. subtilisat high temperature. The role of ClpP in the sporulation and competence regulatory pathways was investigated. ClpP is required for expression of thespoIIAandspoIIGoperons, encoding the σFandσEsporulation‐specific sigma factors. ClpP is also necessary for the expression of thecomKgene, encoding a positive transcriptional regulator of competence genes. ComK‐dependent transcription ofsacB, encoding the exocellular degradative enzyme levansucrase, was found to be abolished in the ΔclpPmutant. MecA has been characterized previously as a negative regulator ofcomKexpression, whose overproduction inhibits both sporulation and competence development. Expression of amecA′–′lacZtranslational fusion is shown to be increased in the ΔclpPmutant. We suggest that ClpP is involved in controlling MecA levels in the cell through proteolysis. Increased levels of MecA in the absence of ClpP are at least partly responsible for the observed pleiotropic phenotype of the ΔclpPmutant.
Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter
Authors: Römling Ute , Sierralta Walter D. , Eriksson Kristina , Normark Staffan
Published: March 2003

A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR− and rpoS− derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37°C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37°C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.
Regulation of type 1 fimbrial expression in uropathogenic Escherichia coli : heterogeneity of expression through sequence changes in the fim switch region
Authors: Leathart J. B. S. , Gally D. L.
Published: March 2003

Over 80% of uropathogenic Escherichia coli express type 1 fimbriae. Expression is phase variable, and regulation of phase switching can differ between isolates. Previously, this was explained by differences in the expression of the fim recombinases, FimB and FimE. Our study of 50 uropathogenic E. coli isolates confirms variation in the regulation of type 1 fimbriae but, in many cases, the variation could be accounted for by sequence changes within and adjacent to the fim switch, rather than by differences in recombinase expression. This was demonstrated by moving the switch from the isolates into an isogenic background and comparing the switching behaviour with that of the original isolate. Isolates could be arranged into groups based on fim switch regulation and sequence similarity. In certain cases, the altered regulation was located to specific basepair changes within the fim switch. Sequence changes were found that had a marked effect on the activity of either FimB or FimE switching, while others affected FimB switching in only one direction. These results emphasize the value of using naturally selected sequence variation to further the understanding of gene regulation.
Regulation of compatible solute accumulation in bacteria
Authors: Poolman Bert , Glaasker Erwin
Published: March 2003

In their natural habitats, microorganisms are often exposed to osmolality changes in the environment. The osmotic stress must be sensed and converted into an activity change of specific enzymes and transport proteins and/or it must trigger their synthesis such that the osmotic imbalance can be rapidly restored. On the basis of the available literature, we conclude that representative Gram‐negative and Gram‐positive bacteria use different strategies to respond to osmotic stress. The main focus of this paper is on the initial response of bacteria to hyper‐ and hypo‐osmotic conditions, and in particular the osmosensing devices that allow the cell to rapidly activate and/or to synthesize the transport systems necessary for uptake and excretion of compatible solutes. The experimental data allow us to discriminate the transport systems by the physicochemical parameter that is sensed, which can be a change in external osmotic pressure, turgor pressure, membrane strain, internal osmolality and/or concentration of specific signal mmicule. We also evaluate the mmicular basis for osmosensing by reviewing the unique structural features of known osmoregulated transport systems.
Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends
Authors: Pandza Suada , Biuković Goran , Paravić Andrea , Dadbin Ali , Cullum John , Hranueli Daslav
Published: March 2003

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6‐501 in mutant MV25 was shown to be due to a single cross‐over at a 4 bp common sequence. pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level. This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region. The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross‐over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c. 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster. The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb. Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme.
Generation of Escherichia coli intimin derivatives with differing biological activities using site‐directed mutagenesis of the intimin C‐terminus domain
Authors: Frankel Gad , Philips Alan D. , Novakova Michaela , Batchelor Miranda , Hicks Susan , Dougan Gordon
Published: March 2003

Intimins, encoded by eae genes, are outer membrane proteins involved in attaching–effacing (A/E) lesion formation and host cell invasion by pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium. A series of intimins, harbouring specific mutations close to the C‐terminus, were constructed using pCVD438, which encodes the eae gene from EPEC strain E2348/69. These mutant plasmids were introduced into EPEC strain CVD206 and C. rodentium strain DBS255, which both contain deletion mutations in their eae genes. CVD206, CVD206(pCVD438) and CVD206(pCVD438) derivatives were assessed for their ability to promote A/E lesion formation or invasion of HEp‐2 cells and to induce A/E lesions on fresh human intestinal in vitro organ cultures (IVOC). The pathogenicity of C. rodentium DBS255 harbouring these plasmid derivatives was also studied in mice. Here, we report that intimin‐mediated A/E lesion formation can be segregated from intimin‐mediated HEp‐2 cell invasion. Moreover, adherence to IVOC, EPEC‐induced microvillus elongation and colonization of the murine intestine by C. rodentium were also modulated by the modified intimins.
DNA polymerase template switching at specific sites on the φ29 genome causes the in vivo accumulation of subgenomic φ29 DNA molecules
Authors: Murthy Vanishree , Meijer Wilfried J. J. , Blanco Luis , Salas Margarita
Published: March 2003

The accumulation of subgenomic phage φ29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage φ29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single‐stranded self‐complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double‐stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo. Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5′‐TGTTtCAC‐GTGgAACA‐3′ being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the φ29 genome. In addition, the almost identical sequence 5′‐TgGTTTCAC‐GTGGAAtCA‐3′ was found once. These five DSEs are all located in the right half of the φ29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic φ29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.
What are archaebacteria: life's third domain or monoderm prokaryotes related to Gram‐positive bacteria? A new proposal for the classification of prokaryotic organisms
Authors: Gupta Radhey S.
Published: March 2003

The evolutionary relationship within prokaryotes is examined based on signature sequences (defined as conserved inserts or deletions shared by specific taxa) and phylogenies derived from different proteins. Archaebacteria are indicated as being monophyletic by a number of proteins related to the information transfer processes. In contrast, for several other highly conserved proteins, common signature sequences are present in archaebacteria and Gram‐positive bacteria, whereas Gram‐negative bacteria are indicated as being distinct. For these proteins, archaebacteria do not form a phylogenetically distinct clade but show polyphyletic branching within Gram‐positive bacteria. A closer relationship of archaebacteria to Gram‐positive bacteria in comparison with Gram‐negative bacteria is generally seen for the majority of the available gene/protein sequences. To account for these results and the fact that both archaebacteria and Gram‐positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between monoderm prokaryotes (surrounded by a single membrane) and diderm prokaryotes (i.e. all true Gram‐negative bacteria containing both an inner cytoplasmic membrane and an outer membrane). This proposal is consistent with both cell morphology and signature sequences in different proteins. The monophyletic nature of archaebacteria for some genes, and their polyphyletic branching within Gram‐positive bacteria as suggested by others, is critically examined, and several explanations, including derivation of archaebacteria from Gram‐positive bacteria in response to antibiotic selection pressure, are proposed. Signature sequences in proteins also indicate that the low‐G + C Gram‐positive bacteria are phylogenetically distinct from the high‐G + C Gram‐positive group and that the diderm prokaryotes (i.e. Gram‐negative bacteria) appear to have evolved from the latter group. Protein phylogenies and signature sequences also show that all eukaryotic cells have received significant gene contributions from both an archaebacterium and a Gram‐negative eubacterium. Thus, the hypothesis that archaebacteria and eukaryotes shared a common ancestor exclusive of eubacteria is not supported. These observations provide evidence for an alternate view of the evolutionary relationship among living organisms that is different from the currently popular three‐domain proposal.
Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus
Authors: Levin James C. , Wessels Michael R.
Published: March 2003

The hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M‐type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two‐component regulatory systems. We constructed an in‐frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement‐mediated opsonophagocytic killing in vitro and with a 500‐fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a two‐component regulatory system that influences capsule expression and virulence.
Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili
Authors: Pratt Leslie A. , Kolter Roberto
Published: March 2003

We have used Escherichia coli as a model system to investigate the initiation of biofilm formation. Here, we demonstrate that E. coli forms biofilms on multiple abiotic surfaces in a nutrient‐dependent fashion. In addition, we have isolated insertion mutations that render this organism defective in biofilm formation. One‐half of these mutations was found to perturb normal flagellar function. Using defined fli, flh, mot and che alleles, we show that motility, but not chemotaxis, is critical for normal biofilm formation. Microscopic analyses of these mutants suggest that motility is important for both initial interaction with the surface and for movement along the surface. In addition, we present evidence that type I pili (harbouring the mannose‐specific adhesin, FimH) are required for initial surface attachment and that mannose inhibits normal attachment. In light of the observations presented here, a working model is discussed that describes the roles of both motility and type I pili in biofilm development.
The ATP‐binding cassette subunit of the maltose transporter MalK antagonizes MalT, the activator of the Escherichia coli mal regulon
Authors: Panagiotidis Cynthia H. , Boos Winfried , Shuman Howard A.
Published: March 2003

Transcription of the mal regulon of Escherichia coli K‐12 is regulated by the positive activator, MalT. In the presence of ATP and maltotriose, MalT binds to decanucleotide MalT boxes that are found upstream of mal promoters and activates transcription at these sites. The earliest studies of the mal regulon, however, suggested a negative role for the MalK protein, the ATP‐binding cassette subunit of the maltose transporter, in regulating mal gene expression. More recently, it was found that overexpression of the MalK protein resulted in very low levels of mal gene transcription. In this report we describe the use of tagged versions of MalT to provide evidence that it physically interacts with the MalK protein both in vitro and in vivo. In addition, we show that a novel malK mutation, malK941, results in an increased ability of MalK to down‐modulate MalT activity in vivo. The fact that the MalK941 protein binds but does not hydrolyse ATP suggests that the MalK941 mutant protein mimics the inactive, ATP‐bound form of the normal MalK protein. In contrast, cells with high levels of MalK ATPase show a reduced ability to down‐modulate MalT and express several mal genes constitutively. These results are consistent with a model in which the inactive form of MalK down‐modulates MalT and decreases transcription, whereas the active form of MalK does not. This model suggests that bacteria may be able to couple information about extracellular substrate availability to the transcriptional apparatus via the levels of ATP hydrolysis associated with transport.
Translational control of mRNA processing in the F1845 fimbrial operon of Escherichia coli
Authors: Loomis Wendy P. , Moseley Steve L.
Published: March 2003

Endoribonucleolytic processing followed by differential decay of the cleavage products is an increasingly recognized mechanism for achieving co‐ordinate regulation of functionally related proteins encoded by bacterial polycistronic transcripts. Unlike most examples when RNases E or III initiate decay, the daa transcript encoding F1845 fimbriae, a member of the Dr family of adhesins in Escherichia coli, is processed by an as yet unidentified endoribonuclease using a unique recognition mechanism. An open reading frame (ORF) predicted to encode a 57‐amino‐acid polypeptide was identified flanking the daa processing site. To determine whether this ORF is involved in processing, site‐directed mutagenesis was used to generate mutants with altered translational efficiencies. A mutation in the putative ribosome binding site preceding the ORF significantly inhibited processing while the introduction of a premature stop codon abolished processing. Site‐directed mutagenesis was used to introduce a limited number of mutations into the ORF, designated daaP, to alter the reading frame such that a different polypeptide of a similar size was encoded. Despite the presumed presence of trafficking ribosomes, this mutant failed to be processed, suggesting that the sequence of the DaaP peptide is important. However, the failure of a wild‐type copy of the daaP gene to complement these mutations in trans suggested that the presence of wild‐type daaP gene product was not sufficient to promote processing. Although active translation has been found to inhibit processing by RNases E and III, our data suggest that translation of the daaP gene is required in cis to promote processing by the endonuclease, perhaps due to an interaction of the nascent peptide with the ribosome or the daaP mRNA.
Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae
Authors: Gagiano Marco , Van Dyk Dewald , Bauer Florian F. , Lambrechts Marius G. , Pretorius Isak S.
Published: March 2003

In Saccharomyces cerevisiae, a network of signal transduction pathways governs the switch from yeast‐type growth to pseudohyphal and invasive growth that occurs in response to nutrient limitation. Important elements of this network have been identified, including nutrient signal receptors, GTP‐binding proteins, components of the pheromone‐dependent MAP kinase cascade and several transcription factors. However, the structural and functional mapping of these pathways is far from complete. Here, we present data regarding three genes, MSN1/MSS10, MSS11 and MUC1/FLO11which form an essential part of the signal transduction network establishing invasive growth. Both MSN1 and MSS11 are involved in the co‐regulation of starch degradation and invasive growth. Msn1p and Mss11p act downstream of Mep2p and Ras2p and regulate the transcription of both STA2 and MUC1. We show that MUC1 mediates the effect of Msn1p and Mss11p on invasive growth. In addition, our results suggest that the activity of Msn1p is independent of the invasive growth MAP kinase cascade, but that Mss11p is required for the activation of pseudohyphal and invasive growth by Ste12p. We also show that starch metabolism in S. cerevisiae is subject to regulation by components of the MAP kinase cascade.
Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli
Authors: Ogura Teru , Inoue Koichi , Tatsuta Takashi , Suzaki Toshinobu , Karata Kiyonobu , Young Katherine , Su Lin‐Hui , Fierke Carol A. , Jackman Jane E. , Raetz Christian R. H. , Coleman Jack , Tomoyasu Toshifumi , Matsuzawa Hiroshi
Published: March 2003

The suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R‐3‐hydroxyacyl‐ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42°C. This was accompanied by a dramatic increase in the amount of UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase [the lpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse‐chase experiments and in vitro assays with purified components showed that FtsH, the AAA‐type membrane‐bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl‐ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R‐3‐hydroxyacyl‐ACP pool, is regulated by FtsH.
Mta, a global MerR‐type regulator of the Bacillus subtilis multidrug‐efflux transporters
Authors: Baranova Natalya N. , Danchin Antoine , Neyfakh Alexander A.
Published: March 2003

Little is known about the natural functions of multidrug‐efflux transporters expressed by bacteria. Although identified as membrane proteins actively extruding exogenous toxins from the cell, they may actually be involved in the transport of as yet unidentified specific natural substrates. The expression of two highly similar multidrug transporters of Bacillus subtilis, Bmr and Blt, is regulated by specific transcriptional activators, BmrR and BltR, respectively, which respond to different inducer molecules, thus suggesting distinct functions for the two transporters. Here, we describe an alternative mechanism of regulation, which involves a global transcriptional activator, Mta, a member of the MerR family of bacterial regulatory proteins. The individually expressed N‐terminal DNA‐binding domain of Mta interacts directly with the promoters of bmr and blt and induces transcription of these genes. Additionally, this domain stimulates the expression of the mta gene itself and at least one more gene, ydfK, which encodes a hypothetical membrane protein. These results and the similarity of Mta to the thiostrepton‐induced protein TipA of Streptomyces lividans strongly suggest that Mta is an autogenously controlled global transcriptional regulator, whose activity is stimulated by an as yet unidentified inducer. This stimulation is mimicked by the removal of the C‐terminal inducer‐binding domain. The fact that both Bmr and Blt are controlled by this regulator demonstrates that some of their functions are either identical or, at least, related. Further analysis of Mta‐mediated regulation may reveal the natural function of the system of multidrug transporters in B. subtilis and serve as a paradigm for similar systems in other bacteria.
Site‐specific integration of the conjugal Vibrio cholerae SXT element into prfC
Authors: Hochhut Bianca , Waldor Matthew K.
Published: March 2003

Vibrio cholerae O139, the first non‐O1 serogroup of V. cholerae to give rise to epidemic cholera, is characteristically resistant to the antibiotics sulphamethoxazole, trimethoprim, chloramphenicol and streptomycin. Resistances to these antibiotics are encoded by a 62 kb self‐transmissible, conjugative, chromosomally integrating element designated the ‘SXT element’. We found that the SXT element integrates site specifically into both V. cholerae and Escherichia coli K‐12 into the 5′ end of prfC, the gene encoding peptide chain release factor 3. Integration of the SXT element interrupts the chromosomal prfC gene, but the element encodes a new 5′ end of prfC that restores the reading frame of this gene. The recombinant prfC allele created upon element integration is functional. The integration and excision mechanism of the SXT element shares many features with site‐specific recombination found in lambdoid phages. First, like λ, the SXT element forms a circular extrachromosomal intermediate through specific recombination of the left and right ends of the integrated element. Second, chromosomal integration of the element occurs via site‐specific recombination in a 17 bp sequence found in the circular form of the SXT element and a similar 17 bp sequence in prfC. Third, both chromosomal integration and excision of the SXT element were found to require an element‐encoded int gene with strong similarities to the λ integrase family. Based on the properties of the SXT element, we propose to classify this element as a CONSTIN, an acronym for a conjugative, self‐transmissible, integrating element.
High‐resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234
Authors: Perret Xavier , Freiberg Christoph , Rosenthal André , Broughton William J. , Fellay Rémy
Published: March 2003

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad‐host‐range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA‐σ54 regulatory sequences, which are thought to co‐ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)‐like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.
Interactions of pathogenic Neisseria with host cells. Is it possible to assemble the puzzle?
Authors: Nassif Xavier , Pujol Céline , Morand Philippe , Eugène Emmanuel
Published: March 2003

Neisseria meningitidis and Neisseria gonorrhoeae are human pathogens that have to interact with mucosa and/or cellular barriers for their life cycles to progress. Even though they both give rise to dramatically different diseases, the use of in vitro models has shown that most of the mechanisms mediating cellular interactions are common to N. meningitidis and N. gonorrhoeae. This suggests that bacterial cell interactions may be essential not only for pathogenesis but also for other aspects of the bacterial life cycle that are common to both N. meningitidis and N. gonorrhoeae. This manuscript will review the most recent developments concerning the mechanisms mediating cellular interaction of pathogenic Neisseria and will then try to put them into the perspective of pathogenesis and bacterial life cycle.
Utilization of exogenous folate in the human malaria parasite Plasmodium falciparum and its critical role in antifolate drug synergy
Authors: Wang Ping , Brobey Reynolds K. B. , Horii Toshihiro , Sims Paul F. G. , Hyde John E.
Published: March 2003

The antifolate combination pyrimethamine/sulphadoxine (PYR/SDX; Fansidar) is frequently used to combat chloroquine‐resistant malaria. Its success depends upon pronounced synergy between the two components, which target dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folate pathway. This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained. Plasmodium falciparum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these conditions. However, we have measured parasite inhibition by SDX/PYR combinations in assays in which folate levels are strictly controlled. In parasites that use exogenous folate efficiently, SDX inhibition can be restored by levels of PYR significantly lower than those required to inhibit DHFR. Isobolograms show that the degree of synergy between PYR and SDX is highly dependent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was observed at physiological drug levels when PYR/SDX acted on purified DHFR, whether wild type or mutant. We conclude that the primary basis for antifolate synergy in these organisms arises from PYR targeting a site (or sites) in addition to DHFR, which restores DHPS as a relevant target for SDX.
Host‐dependent requirement for specific DnaA boxes for plasmid RK2 replication
Authors: Doran Kelly S. , Helinski Donald R. , Konieczny Igor
Published: March 2003

The replication origin of the broad‐host‐range plasmid RK2, oriV, contains four DnaA boxes, which bind the DnaA protein isolated from Escherichia coli. Using a transformation assay, mutational analysis of these boxes showed a differential requirement for replication in different Gram‐negative bacteria. DnaA boxes 3 and 4 were required in E. coli and Pseudomonas putidabut not as strictly in Azotobacter vinelandii and not at all in P. aeruginosa. In vitro replication results using an extract prepared from E. coli demonstrated that the activity of origin derivatives containing mutations in boxes 3 or 4 or a deletion of all four DnaA boxes could be restored by the addition of increasing amounts of purified DnaA protein. High levels of DnaA protein in the presence of the TrfA protein also resulted in the stimulation of open complex formation and DnaB helicase loading on oriV, even in the absence of the four DnaA boxes. These observations at least raise the possibility that an alternative mechanism of initiation of oriV is being used in the absence of the four DnaA boxes and that this mechanism may be similar to that used in P. aeruginosa, which does not require these four DnaA boxes for replication.
pH‐dependent secretion of SseB, a product of the SPI‐2 type III secretion system of Salmonella typhimurium
Authors: Beuzón Carmen R. , Banks Geoff , Deiwick Jörg , Hensel Michael , Holden David W.
Published: March 2003

The type III secretion system of Salmonella pathogenicity island 2 (SPI‐2) is required for bacterial replication inside macrophages. SseB has been considered a putative target of the secretion system on the basis of its similarity with EspA, a protein secreted by the type III secretion system of enteropathogenic Escherichia coli (EPEC). EspA forms a filamentous structure on the bacterial cell surface and is involved in translocation of proteins into the eukaryotic cytosol. In this paper, we show that SseB is a secreted protein that associates with the surface of the bacterial cell and might, therefore, also be required for delivery of SPI‐2 effector proteins to the eukaryotic cell cytosol. SseB begins to accumulate inside the bacterial cell when the culture enters early stationary phase. However, SseB is only secreted if the bacteria are grown at low pH or if the pH is shifted after growth from 7.0 to below pH 5.0. The secretion occurs within minutes of acidification and is totally dependent on a functional SPI‐2 type III secretion system. As the pH of the Salmonella‐containing vacuole inside host cells has been shown to acidify to between pH 4.0 and 5.0, and as SPI‐2 gene expression occurs inside host cells, low pH might be a physiological stimulus for SPI‐2‐mediated secretion in vivo.
Sequence‐selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli
Authors: Phadtare Sangita , Inouye Masayori
Published: March 2003

The CspA family of Escherichia coli comprises nine homologous proteins, CspA to CspI. CspA, the major cold shock protein, binds RNA with low sequence specificity and low binding affinity. This is considered to be important for its proposed function as an RNA chaperone to prevent the formation of secondary structures in RNA molecules, thus facilitating translation at low temperature. The cellular functions of other Csp proteins are yet to be fully elucidated, and their sequence specific binding capabilities have not been identified. As a step towards identification of the target genes of Csp proteins, we investigated the RNA binding specificities of CspB, CspC and CspE by an in vitro selection approach (SELEX). In the present study, we show that these proteins are able to bind preferentially to specific RNA/single‐stranded DNA sequences. The consensus sequences for CspB, CspC and CspE are U/T stretches, AGGGAGGGA and AU/AT‐rich regions, especially AAAUUU, respectively. CspE and CspB have Kd values in the range 0.23–0.9 × 10−6 M, while CspC has 10‐fold lower binding affinity. Consistent with our recent findings of transcriptional regulation of cspA by CspE, we have identified a motif identical to the CspE consensus. This motif is the putative CspE‐mediated transcription pause recognition site in a 5′‐untranslated region of the cspA mRNA.
The sigma 54 DNA‐binding domain includes a determinant of enhancer responsiveness
Authors: Chaney Matthew , Buck Martin
Published: March 2003

The bacterial σ54 protein associates with core RNA polymerase to form a holoenzyme that functions in enhancer‐dependent transcription. Isomerization of the σ54 polymerase and its engagement with melted DNA in open promoter complexes requires nucleotide hydrolysis by an enhancer‐binding activator. We show that a single amino acid substitution, RA336, in the Klebsiella pneumoniaeσ54 C‐terminal DNA‐binding domain allows the holoenzyme to isomerize, engage with stably melted DNA and to transcribe from transiently melting DNA without an activator. Activator responsiveness for the formation of stable open complexes remained intact. The activator‐independent transcription phenotype of RA336 is shared with mutants in amino‐terminal Region I sequences. Thus, in σ54, two distinct domains function for enhancer responsiveness. A σ54‐DNA contact mediated by R336 appears to be part of a network of interactions necessary for maintaining the transcriptionally inactive state of the holoenzyme. We suggest activator functions to change these interactions and facilitate open complex formation through promoting polymerase isomerization.