bluuebunny/crossref_metadata_2025 huggingface dataset:
A parallel intraphagosomal survival strategy shared by Mycobacterium tuberculosis and Salmonella enterica
Authors: Buchmeier Nancy , Blanc‐Potard Anne , Ehrt Sabine , Piddington Debra , Riley Lee , Groisman Eduardo A.
Published: March 2003

Mycobacterium tuberculosis and Salmonella enterica cause very different diseases and are only distantly related. However, growth within macrophages is crucial for virulence in both of these intracellular pathogens. Here, we demonstrate that in spite of the phylogenetic distance, M. tuberculosis and Salmonella employ a parallel survival strategy for growth within macrophage phagosomes. Previous studies established that the Salmonella mgtC gene is required for growth within macrophages and for virulence in vivo. M. tuberculosis contains an open reading frame exhibiting 38% amino acid identity with the Salmonella MgtC protein. Upon inactivation of mgtC, the resulting M. tuberculosis mutant was attenuated for virulence in cultured human macrophages and impaired for growth in the lungs and spleens of mice. Replication of the mgtC mutant was inhibited in vitro by a combination of low magnesium and mildly acidic pH suggesting that the M. tuberculosis‐containing phagosome has these characteristics. The similar phenotypes displayed by the mgtC mutants of M. tuberculosis and Salmonella suggest that the ability to acquire magnesium is essential for virulence in intracellular pathogens that proliferate within macrophage phagosomes.
Rad6p represses yeast‐hypha morphogenesis in the human fungal pathogen Candida albicans
Authors: Leng P. , Sudbery P. E. , Brown A. J. P.
Published: March 2003

Rad6p plays important roles in post‐replication DNA repair, chromatin organization, gene silencing and meiosis. In this study, we show that Rad6p also regulates yeast‐hypha morphogenesis in the human pathogen Candida albicans. CaRAD6 gene and cDNAs were isolated and characterized revealing that the gene carries two 5′‐proximal introns. CaRad6p shows a high degree of sequence similarity to Rad6 proteins from fungi to man (60–83% identity), and it suppresses the UV sensitivity and lack of induced mutagenesis displayed by a Saccharomyces cerevisiae rad6 mutant. In C. albicans, CaRAD6 expression is induced in response to UV, and CaRad6p depletion confers UV sensitivity, confirming that Rad6p serves a role in protecting this fungus against UV damage. CaRAD6 overexpression inhibits hyphal development, whereas CaRad6p depletion enhances hyphal growth. Also, CaRAD6 mRNA levels decrease during the yeast‐hypha transition. These effects are dependent on Efg1p, but not Cph1p, indicating that CaRad6p acts specifically through the Efg1p morphogenetic signalling pathway to repress yeast‐hypha morphogenesis.
Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters
Authors: Drazek E. Susan , Hammack Craig A. ,   Sr, Schmitt Michael P.
Published: March 2003

Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron‐depleted medium. C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants. The complementing plasmids shared an ≈ 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon. The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram‐negative bacteria and are proposed to be part of an ABC (ATP‐binding cassette) transport system. The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae. HmuT binds in vitro to haemin‐ and haemoglobin‐agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae. This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram‐positive bacterium.
Membrane‐to‐cytosol redistribution of ECF sigma factor AlgU and conversion to mucoidy in Pseudomonas aeruginosa isolates from cystic fibrosis patients
Authors: Rowen D. W. , Deretic V.
Published: March 2003

The conversion to mucoid phenotype in Pseudomonas aeruginosa during chronic infections in cystic fibrosis (CF) is due to mutations in the algU mucABCD gene cluster. This cluster encodes an extreme stress response system conserved in Gram‐negative bacteria. The system includes an ECF sigma factor, AlgU (σE), an inner membrane protein, MucA, which inhibits AlgU activity, and MucB, a periplasmic protein that negatively controls AlgU. In this work, we investigated whether and how these factor interact to transduce signals between different cellular compartments. The mutation mucAΔG440, which renders a large fraction of P. aeruginosa CF isolates mucoid, did not abrogate AlgU–MucA interactions, although it eliminated MucA–MucB interactions in the yeast two‐hybrid system. The mucAΔG440 truncation of the periplasmic C‐terminal tail of MucA destabilized the molecule resulting in low or undetectable steady‐state levels in P. aeruginosa. Somewhat reduced levels of MucA were also seen in cells with inactivated mucB or with the mucACF53 allele carrying the missense P184S mutation, which mildly affected interactions with MucB. The events downstream from MucA destabilization were also investigated. AlgU was found to associate with inner membranes in mucA+ cells. In mutants destabilizing MucA, a limited redistribution of AlgU from the membrane to the cytosol was observed. The redistribution was spontaneous in mucAΔG440 cells, while in mucB and mucACF53 mutants it required additional signals. Despite a large reduction in MucA levels in mucAΔG440 cells, only a small fraction of AlgU was redistributed to the cytosol and a significant portion of this σ factor remained membrane bound and behaved as a peripheral inner membrane protein. The fraction of AlgU that depended on MucA for association with the membrane also brought RNA polymerase into this compartment. These results are consistent with a model in which MucB–MucA–AlgU–RNA polymerase interactions at the membrane allow transduction of potentially lethal stress signals with both rapid reaction times of the preassembled complexes and efficient resupply at the membrane from the prebound components.
Aspergillus SteA (Sterile12‐like) is a homeodomain‐C2/H2‐Zn+2 finger transcription factor required for sexual reproduction
Authors: Vallim Marcelo A. , Miller Karen Y. , Miller Bruce L.
Published: March 2003

Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell‐specific or morphogenesis‐specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63–75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12αp. SteAp and Ste12αp lack the pheromone induction domain found in budding yeast Ste12p, but have C‐terminal C2/H2‐Zn+2 finger domains not present in the other Ste12 proteins. A ΔsteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle‐specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH‐like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.
Control of bioluminescence in Vibrio fischeri by the LuxO signal response regulator
Authors: Miyamoto Carol M. , Lin Yi Hsing , Meighen Edward A.
Published: March 2003

Bioluminescence in the marine bacterium Vibrio fischeri is controlled by the excretion of a N‐acyl homoserine lactone (HSL) autoinducer which interacts with a regulator, LuxR, and activates transcription of the lux operon at high‐cell density. This system has become the prototype for quorum sensing in many bacteria. Although light emission in Vibrio harveyi is also regulated by a N‐acyl‐HSL inducer, in sharp contrast, a completely different and more complex system is involved in quorum sensing which is mediated via LuxO, the response regulator of a phosphorelay signal transduction system. In the present work, luxO and the overlapping luxU gene, also involved in the phosphorelay system in V. harveyi, have been discovered in V. fischeri. By gene replacement technology, a V. fischeri luxO– mutant was generated whose phenotype was similar to that of V. harveyi luxO– showing that LuxO is involved in control of luminescence in V. fischeri. This mutant could be complemented with luxO from either V. fischeri or V. harveyi resulting in the restoration of the dependence of luminescence intensity on cell density. In contrast to V. harveyi luxO–, light emission of V. fischeri luxO– was stimulated by the N‐acyl‐HSL autoinducer indicating that luxO is part of a second signal transduction system controlling luminescence in this species. The presence of a luxO‐based phosphorelay regulatory system as well as the luxR‐based system in V. fischeri suggests that the former system, originally discovered in V. harveyi, may be a general regulatory mechanism in luminescent bacteria.
Analysis of the DNA‐binding domain of Escherichia coli DnaA protein
Authors: Blaesing Franca , Weigel Christoph , Welzeck Michaela , Messer Walter
Published: March 2003

The DNA‐binding domain of the Escherichia coli DnaA protein is represented by the 94 C‐terminal amino acids (domain 4, aa 374–467). The isolated DNA‐binding domain acts as a functional repressor in vivo, as monitored with a mioC::lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site‐directed and random PCR mutagenesis were performed, using the mioC::lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA‐binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N‐terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N‐terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non‐specifically.
ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells
Authors: Thomas Jeffrey G. , Baneyx François
Published: March 2003

DnaK–DnaJ–GrpE and GroEL–GroES are the best‐characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat‐shock conditions. We show that the absence of either ClpB or HtpG at 42°C leads to increased aggregation of preS2‐β‐galactosidase, a fusion protein whose folding depends on DnaK–DnaJ–GrpE, but not GroEL–GroES. However, only the ΔclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL–GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation‐prone protein preS2‐S′‐β‐galactosidase in wild‐type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK–DnaJ, but not GroEL–GroES, restore efficient folding in ΔclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK–DnaJ–GrpE team to interact with newly synthesized polypeptides.
c‐Jun and RACK1 homologues regulate a control point for sexual development inAspergillus nidulans
Authors: Hoffmann Bernd , Wanke Christoph , LaPaglia S. Kirsten , Braus Gerhard H.
Published: March 2003

Amino acid limitation results in impaired sexual fruit body formation in filamentous fungi such asAspergillus nidulans. The starvation signal is perceived by the cross‐pathway regulatory network controlling the biosynthesis of translational precursors and results in increased expression of a transcriptional activator encoded by ac‐Junhomologue. In the presence of amino acids, the gene product of the mammalianRACK1homologuecpcBis required to repress the network. Growth under amino acid starvation conditions permits the initiation of the sexual developmental programme of the fungus, but blocks fruit body formation before completion of meiosis. Accordingly, arrest at this defined control point results in microcleistothecia filled with hyphae. Addition of amino acids results in release of the block and completion of development to mature ascospores. The same developmental block is induced by either overexpression ofc‐Junhomologues or deletion of theRACK1homologuecpcBofA. nidulansin the presence of amino acids. Therefore, the amino acid starvation signal regulates sexual development through the network that also controls the amino acid biosynthetic genes. Expression of theRACK1gene suppresses the block in development caused by a deletion ofcpcB. These data illuminate a connection between metabolism and sexual development in filamentous fungi.
Unravelling the mysteries of virulence gene regulation in Salmonella typhimurium
Authors: Lucas Robin L. , Lee Catherine A.
Published: March 2003

Salmonella typhimurium, which causes gastroenteritis in calves and humans as well as a typhoid‐like disease in mice, uses numerous virulence factors to infect its hosts. Genes encoding these factors are regulated by many environmental conditions and regulatory pathways in vitro. Many virulence genes are specifically induced at particular sites during infection or in cultured host cells. The complex regulation of virulence genes observed in vitro may be necessary to restrict their expression to specific locations within the host. In vitro and in vivo studies provide clues about how virulence genes might be regulated in vivo. Future studies must assess the actual environmental signals and regulators that modulate each virulence gene in vivo and determine how multiple regulatory pathways are integrated to co‐ordinate the appropriate expression of virulence factors at specific sites in vivo.
Different alleles of the response regulator gene bldM arrest Streptomyces coelicolor development at distinct stages
Authors: Molle Virginie , Buttner Mark J.
Published: March 2003

whiK was one of five new whi loci identified in a recent screen of NTG‐induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C‐terminal helix–turn–helix DNA‐binding domain and an apparently typical N‐terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG‐induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G‐62) with aspartate. The more severe mutant, R655, carried a C‐7Y substitution adjacent to the highly conserved DD motif at positions 8–9. However, although BldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate‐54, the putative site of phosphorylation, is not required for BldM function. Constructed mutant alleles carrying either D‐54N or D‐54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D‐54N or the D‐54A substitution at the native chromosomal bldM locus sporulated normally. BldM was not phosphorylated in vitro with either of the small‐molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.
Escherichia coli translocase: the unravelling of a molecular machine
Authors: Manting Erik H. , Driessen Arnold J. M.
Published: March 2003

Protein translocation across the bacterial cytoplasmic membrane has been studied extensively in Escherichia coli. The identification of the components involved and subsequent reconstitution of the purified translocation reaction have defined the minimal constituents that allowed extensive biochemical characterization of the so‐called translocase. This functional enzyme complex consists of the SecYEG integral membrane protein complex and a peripherally bound ATPase, SecA. Under translocation conditions, four SecYEG heterotrimers assemble into one large protein complex, forming a putative protein‐conducting channel. This tetrameric arrangement of SecYEG complexes and the highly dynamic SecA dimer together form a proton‐motive force‐ and ATP‐driven molecular machine that drives the stepwise translocation of targeted polypeptides across the cytoplasmic membrane. Recent findings concerning the translocase structure and mechanism of protein translocation are discussed and shine new light on controversies in the field.
The cis requirements for transcriptional activation by HilA, a virulence determinant encoded on SPI‐1
Authors: Lostroh C. Phoebe , Bajaj Vivek , Lee Catherine A.
Published: March 2003

In several models of pathogenesis, Salmonella requires genes encoded on Salmonella pathogenicity island 1 (SPI‐1) for virulence. In Salmonella enterica serovar Typhimurium (S. typhimurium), most SPI‐1 genes are arranged in operons and are co‐ordinately regulated in response to environmental signals via the SPI‐1‐encoded protein HilA. In order to understand how HilA controls the transcription of SPI‐1 genes, we have analysed the invF and prgH promoters. We have reconstituted HilA‐dependent activation of both promoters in Escherichia coli by supplying hilA on a plasmid, strongly suggesting that HilA acts directly on the promoters. By analysing the HilA‐dependent activity of deletions and mutations in PinvF, we identified cis elements necessary for HilA‐dependent activation. Through biochemical studies, we have defined a probable HilA‐binding sequence in the invF promoter. This ‘HilA box’ is intact in the minimal promoter identified through deletion analysis, and it is disrupted in one class of PinvF mutants that has reduced activation by HilA. The prgH promoter also contains a HilA box in the same position relative to the +1 of transcription. This work is the first to connect HilA‐dependent environmental regulation with a specific sequence in a SPI‐1 virulence gene promoter.
Repeat‐associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58
Authors: Saunders Nigel J. , Jeffries Alex C. , Peden John F. , Hood Derek W. , Tettelin Herve , Rappuoli Rino , Moxon E. Richard
Published: March 2003

Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin‐related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium–host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence‐specific mutational biases that favour the instability of sequences associated with phase variation.
Regulation by external pH and stationary growth phase of the acetolactate synthase from Synechocystis PCC6803
Authors: Maestri O. , Joset F.
Published: March 2003

Several characteristics identify the protein encoded by the alsS gene [sll1981 in Cyanobase (http://www.kazusa.or.jp/cyano/cyano.html)] of Synechocystis PCC6803 as an acetolactate synthase. The AlsS protein is about 60% homologous to the AlsS from Bacillus subtilis or other bacteria. These enzymes condense two pyruvates to form acetolactate, implicated in pH homeostasis via the acetoin−2,3‐butanediol pathway or in valine biosynthesis. Transcriptional fusions revealed that alsS was induced at the onset of stationary phase, as in B. subtilis, a situation leading to an increase in the pHout to above 11 in Synechocystis. This is the first cyanobacterial gene showing a dependence on pH for its expression. Induction was also obtained by the presence of > 100 mM Na+, the effect being prevented by amiloride, in agreement with Na+/H+ exchange in the pH homeostasis process. Homology of the Synechocystis AlsS protein to the close family of acetohydroxy acid synthases (including one in Synechocystis) is around 30%. These enzymes are involved in the parallel routes for valine/leucine and isoleucine biosynthesis. No phenotype of auxotrophy for any of these amino acids was associated with a null mutation in the Synechocystis alsS gene. The AlsS enzyme did not complement the isoleucine deficiency of an acetohydroxy acid synthase‐deficient Escherichia coli mutant.
Restriction–modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer
Authors: Ando Takafumi , Xu Qing , Torres Melaine , Kusugami Kazuo , Israel Dawn A. , Blaser Martin J.
Published: March 2003

Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli–H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction–modification (R–M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild‐type JP26, transformed MboI R−M+ JP26 and heterologous MboI R−M+ wild‐type H. pylori strains. Parallel studies with pHP1 from dam+ and dam−E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.
Spo0A directly controls the switch from acid to solvent production in solvent‐forming clostridia
Authors: Ravagnani Adriana , Jennert Katrin C. B. , Steiner Elisabeth , Grünberg Raik , Jefferies James R. , Wilkinson Shane R. , Young Danielle I. , Tidswell Edward C. , Brown David P. , Youngman Philip , Morris J. Gareth , Young Michael
Published: March 2003

The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C‐terminal DNA‐binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12‐amino‐acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A‐binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1.4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild‐type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post‐exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.
Virulent aggregates of Streptococcus pyogenes are generated by homophilic protein–protein interactions
Authors: Frick Inga‐Maria , Mörgelin Matthias , Björck Lars
Published: March 2003

Many strains of the important human pathogen Streptococcus pyogenes form aggregates when grown in vitro in liquid medium. The present studies demonstrate that this property is crucial for the adherence, the resistance to phagocytosis and the virulence of S. pyogenes. A conserved sequence of 19 amino acid residues (designated AHP) was identified in surface proteins of common S. pyogenes serotypes. This sequence was found to promote bacterial aggregation through homophilic protein–protein interactions between AHP‐containing surface proteins of neighbouring bacteria. A synthetic AHP peptide inhibited S. pyogenes aggregation, reduced the survival of S. pyogenes in human blood and attenuated its virulence in mice. In contrast, mutant bacteria devoid of surface proteins containing AHP‐related sequences did not aggregate or adhere to epithelial cells. These bacteria are also rapidly killed in human blood and show reduced virulence in mice, underlining the pathogenic significance of the AHP sequence and S. pyogenes aggregation.
Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms–aroE intragenic region
Authors: Guy Rebecca L. , Gonias Lauren A. , Stein Murry A.
Published: March 2003

Salmonella‐induced aggregation of host endosomal compartments into tubules, termed lgp‐tubules, requires sifA and ompR. Lgp‐tubules result from Salmonella‐directed alteration of the endocytic system and typify the unique intracellular locale where Salmonella replicate. A high‐throughput method devised to screen 11 520 MudJ mutants for loss of lgp‐tubule formation identified one auxotrophic and nine prototrophic mutants. Molecular characterization identified four new loci required to alter epithelial endocytic structure. Salmonella pathogenicity island 2 (SPI2) is the locus central to the phenotype. A subset of SPI2 effectors is essential: SpiC and SseFG are required, but not SseE. A subset of apparatus proteins is also implicated: SsaJ, L, M, V and P are required. SPI2 was implicated further, as SifA shows similarity with known SPI2 translocation targets, and OmpR regulates SPI2. Another locus lies within the smf–aroE intragenic region. Lgp‐tubule formation also involves a locus on the virulence plasmid pSLT. The pSLT‐encoded SpvR negatively regulates an unknown repressor of the phenotype located on pSLT. Finally, disruption of carB leads to multiple auxotrophy that prevents lgp‐tubule formation. This study demonstrates that lgp‐tubule formation is a virulence mechanism that underlies the selective disruption of host endocytic trafficking and is associated with the formation of a replication‐permissive locale.
The evolution of secondary metabolism – a unifying model
Authors: Firn Richard D. , Jones Clive G.
Published: March 2003

Why do microbes make secondary products? That question has been the subject of intense debate for many decades. There are two extreme opinions. Some argue that most secondary metabolites play no role in increasing the fitness of an organism. The opposite view, now widely held, is that every secondary metabolite is made because it possesses (or did possess at some stage in evolution) a biological activity that endows the producer with increased fitness. These opposing views can be reconciled by recognizing that, because of the principles governing molecular interactions, potent biological activity is a rare property for any molecule to possess. Consequently, in order for an organism to evolve the rare potent, biologically active molecule, a great many chemical structures have to be generated, most of which will possess no useful biological activity. Thus, the two sides of the debate about the role and evolution of secondary metabolism can be accommodated within the view that the possession of secondary metabolism can enhance fitness, but that many products of secondary metabolism will not enhance the fitness of the producer. It is proposed that secondary metabolism will have evolved such that traits that optimize the production and retention of chemical diversity at minimum cost will have been selected. Evidence exists for some of these predicted traits. Opportunities now exist to exploit these unique properties of secondary metabolism to enhance secondary product diversity and to devise new strategies for biotransformation and bioremediation.
Oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon: roles of multiple promoters and the Fnr‐1 and Fnr‐2 binding sites
Authors: Govantes Fernando , Albrecht Jeffrey A. , Gunsalus Robert P.
Published: March 2003

The Escherichia coli cydAB operon encodes the high‐affinity terminal oxidase of the oxygen respiratory chain, cytochrome d oxidase. The sensor–regulator pair, ArcB–ArcA, is responsible for the microaerobic activation of the cydAB operon, whereas the anaerobic regulator Fnr represses its expression in the absence of oxygen. Fnr binds in vitro at two sites within the cydAB promoter element. To discern whether these two regions have an in vivo function in the anaerobic regulation of cydAB, the Fnr‐binding motifs were mutagenized individually and in combination. The effects of these mutations on in vivo gene expression were determined by lac fusion and primer extension analysis. Our results show that the Fnr‐2 site is critical for Fnr‐mediated anaerobic repression of the two main cydAB promoters, P1 and P2. In contrast, the Fnr‐1 site has an auxiliary role in the anaerobic repression of P1, but not of P2. Transcription from P1 did not affect ArcA‐mediated activation or Fnr‐mediated repression of P2, indicating that oxygen regulation is exerted on both promoters in an independent fashion. In addition, three new promoters were identified in the cydAB control region, and the 5′ ends of the corresponding transcripts were mapped. Two of these promoters, designated P3 and P4, are co‐ordinately regulated with P1 and P2 in response to oxygen, ArcA and Fnr. The P5 promoter is not Fnr regulated and is only weakly activated by ArcA. The contribution of these three additional promoters to the overall cydAB expression is most relevant under aerobic conditions. Our results suggest a unique repression model, in which one Fnr dimer bound to one single site (Fnr‐2) is sufficient to downregulate transcription from four cydAB promoters. In conclusion, transcription of the cydAB operon is driven by a complex regulatory element containing at least five promoters that act in unison to provide adequate oxygen control of gene expression.
A study of the YopD–LcrH interaction fromYersinia pseudotuberculosisreveals a role for hydrophobic residues within the amphipathic domain of YopD
Authors: Francis Matthew S. , Aili Margareta , Wiklund Magda‐Lena , Wolf‐Watz Hans
Published: March 2003

The enteropathogenYersinia pseudotuberculosisis a model system used to study the molecular mechanisms by which Gram‐negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced byY. pseudotuberculosisthat act in concert to establish an infection, YopD (Yersiniaouterprotein D) is a crucial component essential foryopregulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two‐hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non‐secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N‐terminal domain and a C‐terminal amphipathic α‐helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic α‐helix abolished the YopD–LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild‐type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during aYersiniainfection.
The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans
Authors: Schweizer Anja , Rupp Steffen , Taylor Brad N. , Röllinghoff Martin , Schröppel Klaus
Published: March 2003

The temporal and spatial expression of stage‐specific genes during morphological development of fungi and higher eukaryotes is controlled by transcription factors. In this study, we report the cloning and functional analysis of the Candida albicans TEC1 (CaTEC1) gene, a new member of the TEA/ATTS family of transcription factors that regulates C. albicans virulence. The promoters of the type 4, 5 and 6 proteinase isogenes (SAP4–6) contain repetitive TEA/ATTS consensus sequence motifs. This finding suggests a possible role for a homologue of Saccharomyces cerevisiae TEC1 during the activation of proteinase gene expression in C. albicans. CaTEC1 is predominantly expressed in the hyphal form of C. albicans. In vitro, serum‐induced hyphal formation as well as evasion from MΦ after phagocytosis is suppressed in catec1/catec1 mutant cells. Furthermore, expression of the proteinase isogenes SAP4–6 is no longer inducible in these mutant cells. The deletion of the CaTEC1 gene attenuates virulence of C. albicans in a systemic model of murine candidiasis, although both mutant and revertant cells that were prepared from infected tissues or the vaginal mucosa grew in a hyphal morphology in vivo. CaTEC1 complements the pseudohyphal and invasive growth defect of haploid and diploid S. cerevisiae tec1/tec1 mutant cells and strongly activates the promoter of FLO11, a gene required for pseudohyphal growth. This study provides the first evidence pointing to an essential role for a member of the TEA/ATTS transcription factor family that had so far only been ascribed to function during development as a virulence regulator in microbial pathogenesis.
Control of directionality in the site‐specific recombination system of the Streptomyces phage φC31
Authors: Thorpe Helena M. , Wilson Stuart E. , Smith Margaret C. M.
Published: March 2003

The genome of the Streptomyces temperate phage φC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, φC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non‐permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non‐permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.
Early lateral transfer of genes encoding malic enzyme, acetyl‐CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica
Authors: Field Jessica , Rosenthal Benjamin , Samuelson John
Published: March 2003

The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl‐CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl‐CoA synthetase, which converts acetyl‐CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl‐CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram‐positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT‐rich codon usage of the parasite.
A new cytotoxin from Bacillus cereus that may cause necrotic enteritis
Authors: Lund Terje , De Buyser Marie‐Laure , Granum Per Einar
Published: March 2003

A cytotoxin (CytK) has been isolated from a Bacillus cereus strain that caused a severe food poisoning outbreak killing three people. A protein of 34 kDa was highly cytotoxic, and the addition of other secreted proteins gave no synergistic effect. CytK was also necrotic and haemolytic. No known B. cereus enterotoxins were produced by this strain. A DNA sequence from 1.8 kb upstream to 0.2 kb downstream of the toxin gene was sequenced. The deduced amino acid sequence of the toxin showed similarity to Staphylococcus aureus leucocidins, γ‐haemolysin and α‐haemolysin, Clostridium perfringensβ‐toxin and B. cereus haemolysin II, all belonging to a family of β‐barrel channel‐forming toxins. There was no sequence similarity between CytK and enterotoxins of B. cereus. The upstream sequence contained a partial sequence of a putative histidine kinase gene. A recognition site for PlcR, which regulates the transcription of enterotoxins HBL and Nhe of B. cereus, was found in the promoter region of the toxin. This new cytotoxin may be responsible for a disease that is similar to, although not as severe as, the necrotic enteritis caused by the β‐toxin of C. perfringens type C.
A 76‐residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12‐dependent Escherichia coli 113/3 cells
Authors: Penfold Christopher N. , Garinot‐Schneider Carole , Hemmings Andrew M. , Moore Geoffrey R. , Kleanthous Colin , James Richard
Published: March 2003

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N‐ and C‐terminal deletions of the N‐terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor‐binding activity consisted of residues 343–418. The results of the in vivo receptor‐binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1–497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor‐binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76‐residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase‐type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor‐binding domain polypeptide inhibited the growth of the vitamin B12‐dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.
SdiA, an Escherichia coli homologue of quorum‐sensing regulators, controls the expression of virulence factors in enterohaemorrhagic Escherichia coli O157:H7
Authors: Kanamaru Kyoko , Kanamaru Kengo , Tatsuno Ichiro , Tobe Toru , Sasakawa Chihiro
Published: March 2003

The quorum‐sensing system in bacteria is a well‐known regulatory system that controls gene expression in a cell density‐dependent manner. A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue) and autoinducer (acyl homoserine lactone) are indispensable for this system in most Gram‐negative bacteria. In this study, we found that SdiA, an Escherichia coli LuxR homologue, is a negative regulator of the expression of virulence factors EspD and intimin in enterohaemorrhagic E. coli (EHEC) O157:H7. The expression of EspD and intimin was inhibited at the RNA level upon SdiA overexpression. SdiA has a DNA‐binding motif in its C‐terminal part and can bind to the promoter regions of the esp and eae genes in vitro. Extracellular factors, which accumulate in culture supernatants of O157:H7 at the stationary phase of growth and inhibit EspD and intimin synthesis, bind to the N‐terminal part of SdiA in vivo and in vitro. O157:H7 overproducing the N‐terminal part of SdiA exhibited hypertranscription of EspD and intimin, suggesting that the overproduced N‐terminal part had inhibited the activity of intact SdiA through titration of the extracellular factors. These results indicate that a quorum‐sensing system including the SdiA protein controls colonization by O157:H7.