bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel gene whose expression is regulated by the response‐regulator, SphR, in response to phosphate limitation in Synechococcus species PCC7942
Authors: Aiba Hirofumi , Mizuno Takeshi
Published: October 2006

In Synechococcus species PCC7942, the production of a subset of proteins is induced when it is grown in a phosphate‐limited medium. We previously suggested that a pair of cyanobacterial two‐component regulatory proteins, SphS (sensory‐kinase) and SphR (response‐regulator), may be involved in this particular response to phosphate limitation. Here it was found that a protein with an apparent molecular mass of 33 kDa became particularly abundant when the total cellular proteins from cells grown in a phosphate‐limited medium were analysed by SDS‐PAGE. A deletion mutant lacking both the sphS and the sphR genes failed to produce this 33 kDa protein in response to phosphate limitation. Thus it was reasonable to assume that this protein is a member of the group of proteins involved in the Synechococcus phosphate regulatory circuit (hence, it was named SphX). The SphX protein was purified to near homogeneity, and the corresponding structural gene was cloned. The determined nucleotide sequence revealed that the sphX gene encodes a novel protein with a calculated molecular mass of 36 374 Da, which was demonstrated to be located in the cytoplasmic membrane. Structural features of the SphX promoter were then clarified by determining its transcription start site, from which transcription was triggered in response to phosphate limitation. Furthermore, the putative response‐regulator, SphR, was demonstrated to bind to the upstream region of the SphX promoter by means of in vitro DNase I footprinting. From these results, we conclude that the sphX gene is a member of the Synechococcus phosphate regulatory circuit, in which the two signal‐transduction components, SphS and SphR, are crucially involved as transcriptional regulators. The SphX protein may play a role in phosphate assimilation in Synechococcus.
Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K‐12
Authors: Tsui Ho‐Ching Tiffany , Leung Hon‐Chiu Eastwood , Winkler Malcolm E.
Published: October 2006

The region immediately downstream from the miaA tRNA modification gene at 94.8 min contains the hfq gene and the hflA region, which are important in the bacteriophage Qβ and lambda life cycles. The roles of these genes in bacteria remain largely unknown. We report here the characterization of two chromosomal hfq insertion mutations. An omega (ω) cassette insertion near the end of hfq resulted in phenotypes only slightly different from the parent, although transcript mapping demonstrated that the insertion was completely polar on hfq expression. In contrast, an equally polar omega cassette insertion near the beginning of hfq caused pronounced pleiotropic phenotypes, including decreased growth rates and yields, decreased negative supercoiling of piasmids in stationary phase, increased cell size, osmosensitivity, increased oxidation of carbon sources, increased sensitivity to ultraviolet light, and suppression of bgl activation by hns mutations, hfq::ω mutant phenotypes were distinct from those caused by omega insertions early in the miaA tRNA modification gene. On the other hand, both hfq insertions interfered with lambda phage plaque formation, probably by means of polarity at the hflA region. Together, these results show that hfq function plays a fundamental role in Escherichia coli physiology and that hfq and the hflA region are in the amiB‐mutL‐miaA‐hfq‐hflX superoperon.
Bacteriophage‐like particle of Rochalimaea henselae
Authors: Anderson Burt , Goldsmith Cynthia , Johnson Angela , Padmalayam Indira , Baumstark Barbara
Published: October 2006

Sumnnary

An extracellular particle approximately 40 nM in diameter was detected in culture supernatant from the fastidious bacterium Rochalimaea henselae. This particle has at least three associated proteins and contains 14kbp linear DNA segments that are heterogeneous in sequence. The 14kbp DNA was also present in R. henselae cells as an extrachromosomal element for all 14 strains tested. Despite attempts to induce lysis of R henselae, plaque formation was not observed. A similar particle, also containing 14 kbp DNA, was observed in Bartonella bacilliformis, and may be analogous to a bacteriophage that has been described elsewhere for B. bacilliformis.
A conserved DNA sequence is required for efficient gonococcal pilin antigenic variation
Authors: Wainwright Leslie Ann , Pritchard Kristen Hoikka , Selfert H. Steven
Published: October 2006

Antigenic variation of the Neisseria gonorrhoeae pilus occurs when a variant pilin sequence from a silent locus recombines into the expression locus by predominantly unidirectional, homologous recombination. At the 3’end of all pilin loci lies a conserved ONA sequence, called the Sma/Cla repeat, which has sequence similarity to several recombinase‐binding sites, and therefore may be involved in pilin recombination. We have developed a novel reverse transcriptase/polymerase chain reaction (RT‐PCR) assay for direct monitoring of pilin recombination, and both RT‐PCR and phase variation were used to examine pilin recombination in a gonococcal strain that had had the pilE Sma/Cla repeat removed. Results from these experiments showed a decrease in pilin recombination when the Sma/Cla sequence was deleted from the expression locus, showing that a specialized site (Sma/Cla) is involved in efficient pilin recombination.
A non‐directed, hydroxylamine‐generated suppressor mutation in the P3 pairing region of the bacteriophage T4 td intron partially restores self‐splicing capability
Authors: Brown Michael D. , DeYoung Katherine L. , Hair Dwight H.
Published: October 2006

Hydroxylamine (HA) mutagenesis of an HA‐induced splicing‐defective bacteriophage T4 td intron mutant with a mutation in the intron P3 RNA pairing region was used to generate pseudorevertants. Because HA can only cause GC to AT transitions, the original mutant (H104A) could not undergo true reversion, yet the compensatory mutation on the opposite side of the P3 helix, which was complementary to the original H104A mutation, could occur. A pseudorevertant was isolated that contained both the original H104A mutation and the compensatory mutation HS9. By phenotypic and molecular genetic criteria, this double mutant (H104A‐HS9) was shown to be able to undergo significant RNA splicing, thus confirming the existence and functional importance of the long‐range P3 pairing region in this phage intron. The second‐site suppressor mutation (HS9) was isolated by phage cross and also exhibited some self‐splicing ability. A correlation exists between the strength of P3 helix Watson‐Crick base pairing and the apparent level of splicing when wild‐type, H104A, HS9, and H104A‐HS9 are compared. This suggests that the primary role of the P3 RNA pairing region in the T4 td intron is structural in contributing to the critical RNA secondary structure.
Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis
Authors: Agaisse Hervé , Lereclus Didier
Published: October 2006

The promoter region of the cryIIIA toxin gene of Bacilius thuringiensis is composed of at least three domains: an upstream region extending from nucleotide positions ‐635 to ‐553 (with reference to the translational start codon of cryIIIA), an internal region extending from nucleotide positions ‐553 to ‐367, and a downstream region extending from nucleotide position 367 to +18. Deletion analysis and transcriptional fusions to the lacZ gene indicate that full expression of cryIIIA requires the association of the upstream and the downstream region. Primer extension experiments reveal a major cryIIIA transcript (designated T‐129) starting at nucleotide position ‐129 and another transcript (designated T‐558) starting at nucleotide position ‐558. Mutation in the ‐35 region of the promoter responsible for the initiation of T‐558 indicates that the upstream promoter is essential for full expression of cryIIIA, although not sufficient. Deletion of the DNA region carrying the previously described cryIIIA promoter does not affect full expression of cryIIIA and does not modify the 5 end of T‐129. Taken together, these results indicate that the 5 end of T‐129 is not a transcriptional start site. Therefore, we propose that T‐129 results from the processing of the mRNA initiated at the upstream promoter (T‐558), generating a stable mRNA with a 5’extremity at nucleotide position ‐129. From primer extension analysis and transcriptional fusions to lacZ, it appears that the upstream promoter is weakly but significantly expressed during the vegetative phase of growth, is activated at the onset of sporula‐tion and remains active at least until t5. However, unlike the promoters of other cry genes, this promoter is similar to σa‐dependent promoters rather than sporulation‐specific promoters. This promoter may therefore be transcribed by the EσA form of RNA polymerase. Activation at the onset of sporulation could result from the disappearance of a repressor, or the appearance of a stationary‐phase‐specific activator.
Cloning and sequencing of Vibrio cholerae mannose‐sensitive haemagglutinin pilin gene: localization of mshA within a cluster of type 4 pilin genes
Authors: Jonson Gunhild , Lebens Mikael , Holmgren Jan
Published: October 2006

The mannose‐sensitive haemagglutinin (MSHA) pilus that is associated with Vibrio cholerae strains of EI Tor biotype has been shown to be a potential colonization factor and protective antigen. The gene encoding the structural subunit of MSHA pili was cloned from size‐fractionated Sacl‐cleaved chromosomal DNA in the expression phage vector lambda ZAPII. Positive clones carried a c. 5.3 kb Sacl fragment and were identified on the basis of MSHA expression and hybridization with a synthetic oligonucleotide probe based upon the N‐terminus of MshA, the structural subunit of MSHA. The mshA gene vitas localized to a 2.6kb Sall‐EcoRI fragment, which was subcloned and shown to express MshA from Its own promoter in Escherichia coli. Nucleotide sequencing of the entire fragment revealed six open reading frames (ORFs) of which four were complete. The mshA gene encodes an 18094 Da prepilin protein, which in its mature form has a size of 17 436 Da. MshA is a type 4 (N‐MePhe) pilin protein that is more homologous to pilins produced by Pseudomonas aeruginosa and Neisseria gonorrhoeae than to TcpA, the structural subunit of the toxin‐coregulated pilus of V. cholerae. The protein seems to be directly involved in receptor binding, as an in‐frame mutation in the mshA gene was found to abolish both d‐mannose‐dependent haemagglutination and binding of V. cholerae bacteria to d‐mannose‐containing agarose beads. Three additional ORFs, all in the same transcriptional orientation as mshA, were found to encode type 4 pilin‐like proteins. A potential promoter with a sequence homologous to that of cAMP‐CRP‐activated promoters in E. coli was identified upstream of ORF3, the gene preceding mshA.
The HAP2,3,4 transcriptional activator is required for derepression of the yeast citrate synthase gene, CIT1
Authors: Rosenkrantz Mark , Keil Christine S. , Pennell Elizabeth A. , Devenish Louise J.
Published: October 2006

The yeast nuclear gene CIT1 encodes mitochondrial citrate synthase, which catalyses the first and rate‐limiting step of the tricarboxylic acid (TCA) cycle. Transcription of CIT1 is subject to glucose repression. Mutations in HAP2, HAP3 or HAP4 block derepression of a CIT1‐lacZ gene fusion. The HAP2,3,4 transcriptional activator also activates nuclear genes encoding components of the mitochondrial electron transport chain, and thus it co‐ordinates derepression of two major mitochondrial functions. Two DNA sequences resembling the consensus HAP2,3,4‐binding site (ACCAATNA) are located at approximately‐ 310 and ‐290, upstream of the CIT1 coding sequence. Deletion and mutation analysis indicates that the 290 element is critical for activation by HAP2,3,4. Glucose‐repressed expression of CIT1 is largely independent of HAP2,3,4, is repressed by glutamate, and requires a DNA sequence between ‐367 and ‐348. Evidence is presented for a second HAP2,3,4‐independent activation element located just upstream and overlapping the ‐290 HAP2,3,4 element.
Role of hns in the virulence phenotype of pathogenic salmonellae
Authors: Harrison Julia A. , Pickard Derek , Higgins Christopher F. , Khan Anjam , Chatfield Stephen N. , Ali Tahir , Dorman Charles J. , Hormaeche Carlos E. , Dougan Gordon
Published: October 2006

A TnphoA‐generated mutant C5060, attenuated for virulence, was derived from the mouse‐virulent Salmonella typhimurium strain C5. This mutation, designated hns‐112::TnphoA, harbours the transposon in the 3 end of hns, with the alkaline phosphatase open reading frame in the opposite orientation to that of hns. Bacterial strains harbouring hns‐112::TnphoA were mucoid and had altered levels of DNA supercoiling, as monitored using pUC18 as a reporter plasmid. Transduction of hns‐112::TnphoA into mouse virulent strains, including S. typhimurium SL1344 and Salmonella enteritidis Se795, resulted in attenuation. When an independent hns mutation, harbouring a kanamycin‐resistance cassette inserted into the Kpnl site at base pair 237 of the hns gene, was introduced into S. typhimurium C5, the isolates were also attenuated. S. typhimurium C5 isolates harbouring the multicopy plasmid pGB651, which encodes the Escherichia coli hns gene, were partially attenuated in mice. Transductional analysis, using Tn10 insertions located close to the hns gene, showed that virulence could be restored In genetic crosses that eliminated the resident hns mutations. However, some hns+ transductants were stilt attenuated, suggesting that secondary attenuating lesions can accumulate in hns‐deficient strains. These studies show that the hns locus plays a role in Salmonella virulence.
The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp‐Fnr family of transcription regulators
Authors: Lampidis Robert , Gross Roy , Sokolovic Zeljka , Goebel Werner , Kreft Jürgen
Published: October 2006

The two pathogenic Listeria species, L. ivanovii and L monocytogenes, can be differentiated biochemically and show different host ranges. Virulence of L. monocytogenes is dependent on the integrity of prfA which positively and coordinately regulates transcription of several virulence genes. Until now, a prfA homologue had not been identified in L. ivanovii. We have now cloned a chromosomal region from L. ivanovii comprising two genes with high homology to the picA and prfA genes from L. monocytogenes. Distal from prfA, an open reading frame highly homologous to a phosphorlbosyl pyrophosphate synthetase gene (prs) was newly identified, defining the border of the virulence gene cluster. Transcription of the gene for ivanolysin O and expression of other genes of the virulence gene cluster in L. ivanovii were dependent on PrfA. The pattern of PrfA‐dependent proteins (PdPs) expressed in L ivanovii was similar, but not identical to that of L. monocytogenes. The PrfA proteins, as predicted from nucleotide sequences of both pathogenic Listeria species, are very similar and show significant homology to the Crp‐Fnr family of global transcription regulators.
The human gastric pathogen Helicobacter pylori has a gene encoding an enzyme first classified as a mucinase in Vibrio cholerae
Authors: Smith Andrew W. , Chahal Bhajan , French Gary L.
Published: October 2006

The human bacterial pathogen Helicobacter pylori has been suggested to be the causative agent of the most common chronic infection of man. Since its first isolation in 1982, H. pylori has been associated with gastric and duodenal ulcer disease, and more recently, gastric cancer. The proteolytic digestion of gastric mucus by this microorganism has been suggested as an important mechanism by which its pathogenicity is at least partly exerted. Here we report the detection of protease activity in H. pylori total‐cell and supernatant extracts. On the basis that zinc metalloproteases are common microbial pathogenicity factors, we identified a single protein in H. pylori protein extracts with antibodies to the Pseudomonas aeruginosa elastase (a secreted zinc metallo‐protease). This same protein was identified by pooled serum from patients infected with H. pylori. We used the functional and immunological relationship between the P. aeruginosa elastase and the Vibrio cholerae haemagglutinin/protease (HAP) to clone the H. pylori hap gene, which was over 99% similar to the V. cholerae hap gene in the coding region. A 4 kb DNA fragment containing the entire cloned gene was highly unstable in Escherichia coli and Bacillus subtilis cloning vectors. We also demonstrated that a hap‐like gene sequence is present in ail nine Helicobacter species so far discovered. The V. cholerae HAP was first classified on the basis of its mucinase activity.
Nod factors of Rhizobium are a key to the legume door
Authors: Relić B. , Perret X. , Estrada‐García M. T. , Kopcinska J. , Golinowski W. , Krishnan H. B. , Pueppke S. G. , Broughton W. J.
Published: October 2006

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo‐oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis‐specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR‐factors to signal‐production‐deficient mutants of the broad‐host‐range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 −7 M and 10−6 M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non‐host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild‐type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.
Membrane association and role in DNA uptake of the Bacillus subtilis PriA anaiogue ComF1
Authors: Londoño‐Vallejo J. A. , Dubnau D.
Published: October 2006

The late competence protein ComF1 is required for genetic transformation in Bacillus subtilis. Because of the sequence similarities of ComF1 to known ATP‐dependent DNA helicases and translocases, we have hypothesized that this protein either unwinds bound double‐stranded DNA or helps in the translocation of the transforming single‐stranded DNA across the cell membrane. Two important implications of this hypothesis (the association of ComF1 with the membrane and its specific requirement for DNA uptake) have been tested in this report. Using cell fractionation techniques and Western blotting analysis, we show that ComF1 is located almost exclusively on the cell membrane and that it is membrane‐targeted independently of other competence proteins. Moreover, ComF1 behaves like an integral membrane protein in extractability and detergent partition assays. We also show that this protein is required for the DNA‐uptake step during transformation but not for DNA binding to the ceil surface. DNA uptake is blocked in strains with null mutations or in‐frame deletions in comF1 but also in strains that overproduce the ComF1 protein under competence conditions. This last observation suggests that ComF1 expression must be balanced with that of other competence proteins, with which it may interact to form a multisubunit complex for DNA uptake.
Variable expression of the Opc outer membrane protein in Neisseria meningitidis is caused by size variation of a promoter containing poly‐cytidine
Authors: Sarkari Jasmine , Pandit Niketan , Moxon E. Richard , Achtman Mark
Published: October 2006

Opa proteins of Neisseria meningitidis exhibit translational phase variation via addition or deletion of repetitive coding repeat units within the DNA encoding the protein leader sequence. In contrast, Opc phase variation is the result of transcriptional regulation. Transcription starts 13 nucleotides after the ‐10 region of an unusual promoter sequence containing a variable number of contiguous cytidine residues and lacking a ‐ 35 region. Efficient expression of Opc occurred in strains with 12 to 13 cytidine residues, intermediate expression in strains with 11 or 14 residues and no expression with ≤ 10 or ≥ 15 residues. This unusual regulation may have evolved because the Opc protein enables meningococcal invasion and is immunogenic.
Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress conditions
Authors: Kwaik Yousef Abu , Engleberg N. Cary
Published: October 2006

The synthesis of a global stress protein (GspA) of Legionella pneumophila is induced in the intracellular environment of the phagocytic ceil and by various in vitro stress stimuli. We used techniques of reverse genetics to isolate the gspA gene from a genomic library of L. pneumophila. Sequence analysis of approximately 1700 bp of a representative clone (pBSP1) showed the presence of two open reading frames (ORFs). 0RF1 encoded for a polypeptide with an inferred molecular mass of 19kDa and an iso‐electric point of 6.1. These predictions correlated with the migration of the GspA protein on two‐dimensional SDS‐polyacrylamide gels. The predicted amino acid sequence of the GspA protein was identicai to 22/23 residues of the N‐terminal amino acid sequence derived by Edman degradation of the purified protein. The GspA protein was 41.3% and 36.5% identical to the 16 kDa ibpA and IbpB heat‐shock proteins, respectively, of Escherichia coli. Primer extension from mRNA isolated from L. pneumophila showed that transcription of the gspA gene was controlled by two overlapping promoters. One of the promoters was a σ70 promoter, while the other was a heat‐shock promoter and was regulated by the σ32 transcription factor in E. coli. Northern biol analysis showed that the level of gspA mRNA was elevated 3.4‐, 5.0‐ and 6.7‐foid after exposure of L. pneumophila to heat shock, oxidative stress and osmotic shock, respectiveiy. The gspA gene was conserved among 13 serogroups of L pneumophila. Our data showed that the gspA gene of L. pneumophila, which is induced by intracellular infection and by various stress stimuli, is controlled transcriptionally by two overlapping and separately regulated promoters.
The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function
Authors: Rakin Alexander , Saken Elisabeth , Harmsen Dag , Heesemann Jürgen
Published: October 2006

The iron‐repressible outer membrane protein FyuA of Yersinia enterocolitica operates as a receptor with dual function: (i) as a receptor for the Y. pestis bacterlocin pesticin, and (ii) as a receptor for yersiniabactin, a siderophore that is produced by mouse‐viruient Y. enterocolitica strains of biogroup IB. Cloning of the FyuA‐encoding gene was achieved by mobilization of a genomic cosmid library of the pesticin‐sensitive and mouse‐virulent Y. enterocolitica O:8 strain WA into the pesticin‐reslstant WA fyuA mutant and subsequent in vivo selection of transconjugants for the ability to survive and multiply in mice (phenotype mouse viruience). The reisolated transconjugants which survived in mice for 3d harboured a unique cosmid and phenotypicaity were pesticin sensitive. From this cosmid a 2650 bp SalI‐PstI fragment conferring pesticin sensitivity was subcioned. Sequencing of this DNA fragment revealed a single open reading frame of 2022 bp, which encodes a deduced polypeptide of 673 amino acids with a predicted molecular mass of 73 677 Da. Cleavage of a putative signal sequence composed of 22 amino acids should lead to a mature protein of 651 amino acids with a molecular mass of 71 368 Da. The open reading frame is preceded by a sequence which shares homoiogy with the postulated consensus Fur iron‐repressor protein‐binding site. FyuA shows homology to other iron‐regulated TonB‐dependent outer membrane proteins with receptor functions (e.g. BtuB, CirA, FepA, lutA, FhuA, FoxA, FcuA). On the basis of multiple alignment of amino acid sequences of FyuA and other TonB‐dependent receptors, a phylogenetic tree was constructed, demonstrating that FyuA probably belongs to the citrate subfamily or represents a new subfamily of TonB‐dependent receptors. Moreover, by complementation of the WA fyuA mutant by the cioned fyuA gene.
The dps promoter is activated by OxyR during growth and by IHF and σs in stationary phase
Authors: Altuvia Shoshy , Almiron Marta , Huisman Gjalt , Kolter Roberto , Storz Gisela
Published: October 2006

Dps is a non‐specific DNA‐binding protein abundant In starved Escherichia coli cells and is important for the defence against hydrogen peroxide. We found that dps mRNA levels are controlled by rpoS‐encoded σs, the transcriptional activator OxyR and the histone‐like IHF protein, in exponentially growing cells, dps is induced by treatment with hydrogen peroxide in an OxyR‐dependent manner. This OxyR‐dependent induction occurs only during log phase, although the OxyR protein is present in stationary phase, in the stationary phase cells, dps is expressed in a σs ‐ and IHF‐dependent manner. The purified OxyR and IHF proteins are also shown to bind upstream of the dps promoter. Our results suggest that the dps promoter is recognized by both σ70‐holoenzyme and σs‐holoenzyme, since OxyR acts through σ70 and the starts of the OxyR‐ and σs‐dependent transcripts are identical.
Activation of a vmp pseudogene in Borrella hermsii: an alternate mechanism of antigenic variation during relapsing fever
Authors: Restrepo B. I. , Carter C. J. , Barbour A. G.
Published: October 2006

The relapsing fever agent, Borrella hermsii, undergoes multiphasic antigenic variation to evade its host's immune response. A frequently observed switch is serotype 7 to 26. Unlike silent vmp genes previously characterized, the transcriptionally silent vmp26 sequence was a pseudogene in lacking a start codon. In serotype 7 the location of the silent vmp26 sequence just downstream of vmp7 on the expression plasmid, as well as on the silent plasmid, was also unique. The demonstration of a predicted circular recombination product in serotype 7 but not serotype 21 populations indicates that the pseudogene was activated by an intramolecular recombination producing a deletion of DNA between 20‐nucleotide direct repeats in vmp7 and Ψvmp26.
Cloning, sequencing, and characterization of multicopy suppressors of a mukB mutation in Escherichia coli
Authors: Yamanaka Kunitoshl , Mitani Tadao , Ogura Teru , Niki Hironorl , Hiraga Sota
Published: October 2006

The mukB gene codes for a 177kDa protein, which might be a candidate for a force‐generating enzyme in chromosome positioning in Escherichia coli. The mukB106 mutant produces normal‐sized, anucleate cells and shows a temperature‐sensitive colony formation. To Identify proteins interacting with the MukB protein, we isolated three multicopy suppressors (msmA, msmB, and msmC) to the temperature‐sensitive colony formation of the mukB106 mutation. The msmA gene, which could not suppress the production of anucleate cells, was found to be identical to the dksA gene. The msmB and msmC genes suppressed the production of anucleate cells as well as the temperature‐sensitive colony formation. However, none of them couid suppress both phenotypes in a mukB null mutation. DNA sequencing revealed that the msmB gene was identicai to the cspC gene and that the msmC gene had not been described before. A homology search revealed that the amino acid sequences of both MsmB and MsmC possessed high similarity to proteins containing the cold‐shock domain, such as CspA of E. coliand the Y‐box binding proteins of eukaryotes; this suggests that MsmB and MsmC might be DNA‐binding proteins that recognize the CCAAT sequence. Hence, the msmB and msmC genes were renamed cspC and cspE, respectively. Possible mechanisms for suppression of the mukB106 mutation are discussed.
The purified SoxABCD quinol oxidase complex of Sulfolobus acidocaldarius contains a novel haem
Authors: Lübben Mathias , Warne Antony , Albracht Simon P. J. , Saraste Matti
Published: October 2006

A respiratory quinol oxidase complex that is encoded by the soxABCD operon has been purified from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The enzyme was solubilized with dodecyl maltoside and purified in the presence of this detergent and ethylene glycol. The complex is hydro‐dynamically homogeneous and contains at least five different polypeptides. In addition to the major subunits SoxA, SoxB and SoxC, it has two small polypeptides. One of these is the translation product of a short open reading frame (now called the soxD gene) at the end of the operon. The optical and electron paramagnetic resonance spectra of the SoxABCD compiex have been characterized. It probably contains four A‐type haems which are bound to SoxB and SoxC. The structure of these haems is not identical to haem A. The novel haem Aa has a 2‐hydroxyethyl geranylgeranyl in position 2 of the porphyrin ring whereas haem A has the related farnesyl‐containing side‐chain.
Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins
Authors: Proft Thomas , Herrmann Richard
Published: October 2006

Eleven hitherto unknown Mycoplasma pneumoniae proteins were identified and characterized with respect to their size and subcellular location. This was carried out through the construction of in vitro gene fusions between a modified mouse dehydrofolate reductase (dhfr) gene and selected regions (cosmid clones) of the M. pneumoniae genome and expressing them in Escherichia coli. Positive clones were identified using antibodies against specific fractions of M. pneumoniae. The deduced protein sequences of 11 out of 30 clones did not show significant homologies to known proteins in protein databank searches. Monospecific antibodies against these 11 fusion proteins were used to determine the size and cellular location of the corresponding M. pneumoniae proteins by immunoscreening Western blots of SDS‐acrylamide gels from M. pneumoniae cell extracts.
Multiplicity of genes encoding secreted aspartic proteinases in Candida species
Authors: Monod Michel , Togni Giuseppe , Hube Bernhard , Sanglard Dominique
Published: October 2006

The secreted aspartic proteinases (SAP) of Candida sp. are presumed to be potential virulence factors. In the opportunistic pathogen Candida albicans the proteinase genes identified to date, SAP1, SAP2, SAP3 and SAP4, constitute a multigene family. Before addressing the possible role of each proteinase in virulence, we sought to isolate all the members of this multigene family by screening a genomic library with a SAP1 probe for additional C. albicans SAP genes using low‐stringency hybridization conditions. Three putative new members, SAP5, SAP6 and SAP7 were isolated and sequenced. The N‐terminal segments of the deduced amino acid sequences of SAP5 and SAP6 contained secretion signal sequences similar to those of other Candida SAPs. Upon comparison and alignment with the other reported SAP amino acid sequences, SAP7 is not only the most divergent protein but also exhibits a much longer putative pro‐sequence with a single Lys‐Lys putative processing site. Using SAP1 to SAP7 as probes, the overall number of SAP genes in C. albicans was tentatively estimated by low‐stringency hybridization to EcoRI‐digested genomic DNA. While each isolated SAP gene could be assigned to distinct EcoRI bands, the existence of two additional genes not isolated after screening of the C. albicans gene library was inferred. Furthermore, evidence was obtained for the existence of SAP muttigene families in other Candida species such as C. tropicalis, C. parapsilosis and C. guiller‐mondii.
MicroCorrespondence
Authors: Mot Rene De , Vanderleyden Jos
Published: October 2006

A conserved surface‐exposed domain in major outer membrane proteins of pathogenic Pseudomonas and Branhamella species shares sequence homology with the calcium‐binding repeats of the eukaryotic extracellular matrix protein thrombospondin
Micro Review tRNA‐dircted transcription antitermination
Authors: Henkin Tina M.
Published: October 2006

Summary

At least 18 aminoacyl‐tRNA synthetase and amino acid biosynthesis genes in several Gram‐positive genera appear to be regulated by a common transcription anti‐termination mechanism. Each gene is induced by limitation for the appropriate amino acid, and not by general amino acid limitation. The mRNA leader regions of these genes exhibit extensive structural conservation. haracterization of the Bacillus subtilis tyrS gene revealed that uncharged tyrosyl‐tRNA promotes readthrough of a leader‐region terminator; a conformational switch in the leader mRNA between a terminator structure and an antiterminator structure is postulated to mediate antitermination. Two sites of interaction between the tRNA and the leader have been identified by genetic analysis: the tRNA anti‐codon interacts with a single codon displayed at a precise position in the leader‐region structure, and the acceptor end of the tRNA interacts with a side‐bulge on the antiterminator.
Bees aren't the only ones: swarming in Gram‐negative bacteria
Authors: Harshey Rasika M.
Published: October 2006

Summary

Swarming is a form of active surface motility that is widespread among flagellated. Gram–negative bacteria. In the laboratory, growth of the bacteria on certain agar surfaces leads to induction of the differentiated swarmer‐cell state. Swarmer cells are generally long and multinucleate, always hyperflagellated, and can move rapidly over the agar surface in a coordinated manner. Some swarm colonies exude large amounts of ‘slime’, which could be essential for promoting intimate cell–cell contacts during swarming. There is evidence that the differentiated swarmer‐cell stage facilitates pathogenic associations with host tissue. Almost nothing is known about the molecular signalling mechanism of surface sensing. Increased viscosity appears to be sensed by several bacteria, but other environmental cues, specific to each bacterium, are also important. In organisms in which swarming motility has been studied in some detail, the chemotaxis system has been shown to play an important rote. The recent discovery of swarming motility in two genetically well‐characterized organisms –Escherichia coli and Salmonella typhimurium– should lead to rapid progress in understanding this process.
The actin‐based motility of the facultative intracellular pathogen Listeria monocytogenes
Authors: Cossart Pascale , Kocks Christine
Published: October 2006

Summary

The Gram‐positive bacterium Listeria monocytogenes is a facultative intracellular parasite that invades and multiplies within diverse eukaryotic cell types. An essential pathogenicity determinant is its ability to move in the host cell cytoplasm and to spread within tissues by directly passing from one cell to another. The propulsive force for intracellular movement is thought to be generated by continuous actin assembly at the rear end of the bacterium. Moving bacteria that reach the plasma membrane induce the formation of long membranous protrusions that are internalized by neighbouring cells, thus mediating the spread of infection. The unrelated pathogens Shigella and Rickettsia use a similar process of actin‐based motility to disseminate in infected tissues. This review focuses on the bacterial and cellular factors involved in the actin‐based motility of L monocytogenes.
Sequence changes in the pilus subunit lead to tropism variation of Neisseria gonorrhoeae to human tissue
Authors: Jonsson Ann‐Beth , Ilver Dag , Falk Per , Pepose Jay , Normark Staffan
Published: October 2006

Summary

Pili of Neisseria gonorrhoeae are correlated with Increased bacterial attachment to epithelial cells and undergo both phase and antigenic variation. Phase variation of gonococcal pili can be brought about by recombination events in the pilin structural gene, pilE, or by the on/off switch in expression of PilC, a pilus biogenesis protein for which two loci exist. We have studied the binding to epithelial cell lines and to fixed tissue sections of N. gonorrhoeae MS11 derivatives and mutants carrying structurally defined PilE and PilC proteins, in situ binding studies of N. gonorrhoeae to formalin‐fixed tissue sections resulted in a binding pattern similar to that obtained using viable epithelial cell lines of different origin. Piliated gonococcal clones, containing different pilE sequences, varied dramatically from one another in their efficiencies at binding to corneal and conjunctival tissue, but bound equally well to cervical and endometrial tissues. Further, the binding data suggested that PJIC expression by itself, i.e. without pili, cannot confer bacterial binding and that expression of either PilC1 or PiiC2 does not confer different binding properties to the bacterial cells. Possible receptors for piliated gonococci were expressed in human tissues, such as cervix, endometrium, cornea, intestine, stomach, mid‐brain and meninges, but not in human kidney. Pretreatment of the target tissues with Proteinase K decreased the gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta‐periodate, which cleave carbon‐carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect attachment of gonococci. These data argue that pilus‐dependent attachment of N. gonorrhoeae to human tissue may be mediated by a eukaryotic receptor having protein characteristics, and that the pilus subunit sequence may play an important role in the interaction with human cornea.
The rfb locus from Pseudomonas aeruginosa strain PA103 promotes the expression of O antigen by both LPS‐rough and LPS‐smooth isolates from cystic fibrosis patients
Authors: Evans David J. , Pier Gerald B. , Coyne Michael J. , Goldberg Joanna B.
Published: October 2006

Summary

Strains of Pseudomonas aeruginosa initially isolated from patients with cystic fibrosis (CF) often express a smooth lipopolysaccharide (LPS) containing many long O side‐chain antigens, but once a chronic infection is established, strains recovered from these patients express little or no LPS O antigen. The genetic basis for this loss of O antigen expression by P. aeruginosa CF isolates is unknown. We report here that 20 CF isoiates of P. aeruginosa, 13 of which are LPS‐rough, were each capable of expressing serogroup 011 antigen when provided with the rfb iocus from P. aeruginosa serogroup 011 strain PA103 on the recombinant plasmid pLPS2. Eight of the thirteen LPS‐rough isolates co‐expressed another, presumably endogenous, O antigen when they contained pLPS2. Different subcloned regions of pLPS2 complemented distinct strains to restore endogenous O antigen expression. These data suggest that the loss of O antigen expression by P. aeruginosa CF isolates results from alterations specific to the rfb region, and is not due to mutations involving other loci or ancillary LPS genes.
Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling
Authors: Tokishita Shin‐ichi , Mizuno Takeshi
Published: October 2006

Summary

The Escherichia coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane‐located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane‐spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signalling, a set of EnvZ mutants, each having amino acid substitutions within the membrane‐spanning regions, was characterized in terms of both their in vivo phenotype and in vitro catalytic activities. One of them, characterized further, has an amino acid change (Pro‐41 to Ser or Leu) In TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF−/OmpC‐constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibiting a wild‐type phenotype of OmpF+/OmpC+, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. Furthermore, they exhibited restored phosphatase activity (i.e., both kinase+ and phosphatase+ activities). It was further demonstrated that one of the suppressors, EnvZ(Arg‐180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and the in vitro catalytic activities caused by EnvZ(P41S), through intermolecular complementation. These results are best interpreted as meaning that an intimate intermolecular interaction between the membrane–spanning segments of EnvZ is crucial for transmembrane signalling per se in response to an external osmotic stimulus.
The protein HU can displace the LexA repressor from its DNA‐binding sites
Authors: Preobrajenskaya Olga , Boullard Annie , Boubrik Fatima , Schnarr Manfred , Rouvière‐Yaniv Josette
Published: October 2006

The major bacterial histone‐like protein HU is a small, basic, dimeric protein composed of two closely related subunits. HU is involved in several processes in the bacterial cell such as the initiation of replication, transposition, gene inversion and cell division. It has been suggested that HU could introduce structural changes to the DNA which would facilitate or inhibit the binding of regulatory proteins to their specific sites. In this study we investigated the effect of HU on the binding of LexA protein, the regulator of SOS functions, to three of its specific binding sites. We show that HU can displace LexA from its binding sites on the operators of the lexA, recA and sfiA genes. The lexA operator was the most sensitive while the higher affinity sfiA operator was the least sensitive. Since HU, like its homologue IHF, probably binds DNA in the minor groove we tested the effect of distamycin, a drug which binds to the minor groove, on LexA binding. Like HU, this drug disrupted LexA–operator complexes. These results suggest that distortion of the minor groove of the lexA operators excludes the binding of the repressor to the major groove.
Neither absence nor excess of λ O initiator‐digesting ClpXP protease affects λ plasmid or phage replication in Escherichia coli
Authors: Szalewska Agnieszka , Wȩgrzyn Grzegorz , Taylor Karol
Published: October 2006

Owing to rapid proteolysis of the coliphage λ‐coded initiator protein, λ O, this protein is considered to carry a rate–limiting step in λ DNA replication. The discovery of ClpXP protease responsible for λ O protein turnover allowed an opportunity to verify this hypothesis. However, neither absence nor excess of this protease significantly affected the transformation efficiency and copy number of λ plasmid, or the Kinetics of the λ phage growth. These results are also incompatible with the hypothesis that the stabilization of λ O plays a role in the switch from early (circle‐to‐circle) to late (rolling‐circle) λ phage DNA replication. Tran‐scriptional activation of oriλ probably assisted by the Escherichia coli DnaA function, remains as the possible rate‐limiting step in λ DNA replication.
An analogue of the DnaJ molecular chaperone whose expression is controlled by σS during the stationary phase and phosphate starvation in Escherichia coli
Authors: Yamashino Takafumi , Kakeda Minoru , Ueguchi Chiharu , Mizuno Takeshi
Published: October 2006

The Escherichia coli CbpA protein appears to be an analogue of the molecular chaperone, DnaJ, as judged from not only its structure but also its possible function. The expression of cbpA, however, was not significantly affected by up‐shift of the growth temperature. Remarkably, it was found that the expression of cbpA was induced under certain growth conditions, such as the entry of cells into stationary phase, or growth in a phosphate‐limited medium. Such conditional expression of cbpA was regulated at the transcriptional level in a σS ‐dependent manner. The structure of this σS ‐dependent cbpA promoter was clarified by determining its transcription start site. The cbpA promoter region was found to contain an unusual DNA structure (i.e. DNA curvature). From these results, it was suggested that, in contrast to DnaJ, CbpA may function as a molecular chaperone in an adaptive response to environmental stresses other than heat shock.
Analysis of membrane protein interaction: ToxR can dimerize the amino terminus of phage lambda repressor
Authors: Dziejman Michelle , Mekalanos John J.
Published: October 2006

The ToxR protein of Vibrio cholerae is an integral membrane protein that co‐ordinately regulates virulence determinant expression. ToxR directiy activates the cholera toxin operon, but maximal activation is achieved in the presence of ToxS, an integral membrane protein thought to interact with ToxR periplasmic sequences. Studies that substitute alkaline phosphatase sequences for the periplasmic domain of ToxR have led to a model for ToxR activation based on dimerization and ToxS interaction. We constructed λ‐ToxR chimeric proteins using the DNA‐binding domain of the phage λ repressor, which cannot effectively dimerize by itself, to assess the ability of ToxR to form dimers in Escherichia coli The results suggest that ToxR sequences can propagate dimerization, and that ToxS can influence the ability to dimerize.
Regulation of xylose utilization in Bacillus licheniformis: Xyl repressor‐xyl‐operator interaction studied by DNA modification protection and interference
Authors: Scheler Andrea , Hillen Wolfgang
Published: October 2006

Xylose utilization in Bacillus licheniformis is inducible by xylose. We establish here that the Xyl repressor recognizes and binds an xyl operator sequence located 12 nucleotides downstream from the transcription start site of the xyl operon. DNA‐retardation experiments employing xyl regulatory DNA and soluble protein extracts indicate complex formation in the presence of Xyl repressor. Two repressor–operator complexes are distinguished by different gel mobilities. They yield the same in situ copper‐phenanthroline footprint. This result suggests that a single xyl operator may be bound by different oligomers of Xyl repressor. Methylation and hydroxyl radical cleavage protection of the xyl operator by Xyl repressor binding and ethylation interference of Xyl repressor binding to the xyl operator reveals symmetrical interaction of the repressor with two half sites of the operator, which show palindromic symmetry and are located on the same side of the B‐form DNA structure.