bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulations in lipid A and phospholipid biosynthesis pathways influence outer membrane protein assembly in Escherichia coli K‐12
Authors: Kloser Andrew , Laird Mike , Deng Ming , Misra Rajeev
Published: March 2003

The assembly defect of a mutant outer membrane protein, OmpF315, can be corrected by suppressor mutations that lower lipopolysaccharide (LPS) levels and indirectly elevate phospholipid levels. One such assembly suppressor mutation, asmB1, is an allele of lpxC (envA) whose product catalyses the first rate‐limiting step in the lipid A (LPS) biosynthesis pathway. Besides reducing LPS levels, asmB1 confers sensitivity to MacConkey medium. A mutation, sabA1, that reverses the MacConkey sensitivity phenotype of asmB1 maps within fabZ (whose product is needed for phospholipid synthesis from a precursor) is also required for lipid A synthesis. In addition to reversing MacConkey sensitivity, the sabA1 mutation reverses the OmpF315 assembly suppression phenotype of asmB1. These results show that OmpF315 assembly suppression by asmB1, which is achieved by lowering LPS levels, can be averted by a subsequent aberration in phospholipid synthesis at a point where the biosynthetic pathways for these two lipid molecules split. OmpF315 assembly suppression can also be achieved in an asmB+ background where FabZ expression is increased. The data obtained in this study provide genetic evidence that elevated phospholipid levels and/or phospholipid to LPS ratios are necessary for assembly suppression.
The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages
Authors: Rouquette Corinne , De Chastellier Chantal , Nair Shamila , Berche Patrick
Published: March 2003

Under stress conditions, the facultative intracellular pathogen Listeria monocytogenes produces a ClpC ATPase, which is a general stress protein encoded by clpC and belonging to the HSP‐100/Clp family. A ClpC‐deficient mutant was obtained by gene disruption in strain LO28, which became highly susceptible to stress conditions in vitro. Intracellular growth of this mutant was restricted within macrophages, one of the major target cells of L. monocytogenes, during the infectious process. A quantitative electron microscope study showed that, contrary to wild‐type bacteria that rapidly gain access to the cytoplasm of macrophages, mutant bacteria remained confined to membrane‐bound phagosomes. Only a few mutant bacteria disrupted the phagosome membrane after 4 h of incubation, then polymerized actin filaments and multiplied within the cytoplasm. The ClpC ATPase, therefore, promotes early bacterial escape from the phagosome of macrophages, thus enhancing intracellular survival. The ClpC ATPase was produced in vivo during experimental infection by wild‐type bacteria. The virulence of the ClpC‐deficient mutant was severely attenuated in mice, with a three‐log decrease in its 50% lethal dose compared with wild‐type bacteria. Bacterial growth of mutant bacteria was strongly restricted in organs, presumably because of an impairment of intracellular survival in host tissues. Our results provide evidence that a general stress protein is required for the virulence of L. monocytogenes, which behaves as a virulence factor promoting intracellular survival of this pathogen.
Combinational variation of restriction modification specificities in Lactococcus lactis
Authors: Schouler Catherine , Gautier Michel , Ehrlich S. Dusko , Chopin Marie‐Christine
Published: March 2003

Three genes coding for a type I R‐M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403. In addition, plasmids were found that encode only the HsdS subunit that directs R‐M specificity. The presence of these plasmids in IL1403 conferred a new R‐M phenotype on the host, indicating that the plasmid‐encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits. Such combinational variation of type I R‐M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.
The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures
Authors: Huang Chih‐Hung , Lin Yi‐Shing , Yang Ya‐Ling , Huang Shu‐wen , Chen Carton W.
Published: March 2003

The chromosomes of the Gram‐positive soil bacteria Streptomyces are linear DNA molecules, usually of about 8 Mb, containing a centrally located origin of replication and covalently bound terminal proteins (which are presumably involved in the completion of replication of the telomeres). The ends of the chromosomes contain inverted repeats of variable lengths. The terminal segments of five Streptomyces chromosomes and plasmids were cloned and sequenced. The sequences showed a high degree of conservation in the first 166–168 bp. Beyond the terminal homology, the sequences diverged and did not generally cross‐hybridize. The homologous regions contained seven palindromes with a few nucleotide differences. Many of these differences occur in complementary pairs, such that the palindromicity is preserved. Energy‐optimized modelling predicted that the 3′ strand of the terminal palindromes can form extensive hairpin structures that are similar to the 3′ ends of autonomous parvovirus genomes. Most of the putative hairpins have a GCGCAGC sequence at the loop, with the potential to form a stable single C‐residue loop closed by a sheared G:A pairing. The similarity between the terminal structures of the Streptomyces replicons and the autonomous parvoviral genomes suggests that they may share some structural and/or replication features.
Listeriolysin O: cholesterol inhibits cytolysis but not binding to cellular membranes
Authors: Jacobs Thomas , Darji Ayub , Frahm Nicole , Rohde Manfred , Wehland Jürgen , Chakraborty Trinad , Weiss Siegfried
Published: March 2003

Listeriolysin O (LLO) binds to cholesterol‐containing membranes in which it oligomerizes to form pores. Preincubation of the toxin with cholesterol is known to inhibit haemolysis, whereas the oxidized form of cholesterol has no inhibitory effect. Using immunoblot analyses and flow cytometry we demonstrate that preincubation with cholesterol does not influence binding of the listeriolysin–cholesterol complex to red blood cells, eukaryotic cells or artificial membranes. Lytic activity of membrane‐bound LLO inactivated by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. To determine the step at which cholesterol inhibits lytic activity, we looked for pore formation using electron microscopy. Pores formed by purified listeriolysin could be directly visualized using erythrocyte ghosts. This property was lost upon incubation of the toxin with cholesterol. Quantitative analysis strongly suggest that inhibition of lysis by cholesterol is not due to decreased binding of listeriolysin to target membranes, but rather to an interference with a subsequent step leading to polymerization of the toxin.
PhoP~P and RNA polymerase σA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP~P activator sites within the coding region stimulate transcription in vitro
Authors: Qi Ying , Hulett F. Marion
Published: March 2003

The Bacillus subtilis pstS operon and phoA gene are members of the Pho regulon that is controlled by PhoR, a histidine kinase, and PhoP, a response regulator. Footprinting analysis showed that phosphorylated PhoP extended the PhoP protected region in pstS and phoA promoters, and also bound to a separate site within the coding region of each gene. Our previous in vivo studies have shown that, in contrast to other Pho regulon promoters that are not expressed in either phoP or phoR mutants, a low‐level induction from the pstS promoter (25% of parent strain) can be detected in a phoR mutant. In this study, by using an in vitro transcription system, we demonstrate that (i) only phosphorylated PhoP is a transcriptional activator of the pstS operon and of the phoA gene; (ii) phosphorylated PhoP and RNA polymerase σA holoenzyme are sufficient for in vitro transcription of the pstS promoter and the phoA promoter; (iii) the activation of the pstS promoter requires lower concentrations of phosphorylated PhoP than does the phoA promoter for transcription; and (iv) PhoP binding sites in both the pstS promoter core binding region and in the 5′ coding region of the gene, which have been identified by footprinting analysis, are important for the transcription of the pstS promoter in vitro.
Isolation and characterization of an extracellular haem‐binding protein from Pseudomonas aeruginosa that shares function and sequence similarities with the Serratia marcescens HasA haemophore
Authors: Létoffé Sylvie , Redeker Virginie , Wandersman Cécile
Published: March 2003

The major mechanism by which bacteria acquire free or haemoglobin‐bound haem involves direct binding to specific outer membrane receptors. Serratia marcescens also secretes a haem‐binding protein, HasA, which functions as a haemophore that catches haem and shuttles it to a cell surface specific outer membrane receptor, HasR. We report the isolation and characterization of hasAp, a gene from Pseudomonas aeruginosa. HasAp is an iron‐regulated extracellular haem‐binding protein that shares about 50% identity with HasA. HasAp is required for P. aeruginosa utilization of haemoglobin iron. It can replace HasA for HasR‐dependent haemoblobin acquisition in a system reconstituted in Escherichia coli. HasAp, like HasA, lacks a signal peptide and is secreted by an ABC transporter. These findings show that haemophore‐dependent haem acquisition is not unique to S. marcescens.
The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms
Authors: Dorman Charles J. , Porter Megan E.
Published: March 2003

Shigella flexneri is the causative agent of bacillary dysentery and is a facultative intracellular pathogen. Its virulence regulon is subject to tight control by several mechanisms involving the products of over 20 genes and an array of environmental signals. The regulon is carried on a plasmid that is prone to instability and to integration into the chromosome, with associated silencing of the virulence genes. Closely related regulons are found in other species of Shigella and in enteroinvasive Escherichia coli. A wealth of detailed information is now available on the Shigella virulence gene control circuits, and it is becoming clear that these share many features with regulatory systems found in other bacterial pathogens. All of this makes the S. flexneri virulence gene control system a very attractive topic for those interested in the nature of gene regulatory networks in bacteria.
Assembly of the FtsZ ring at the central division site in the absence of the chromosome
Authors: Sun Qin , Yu Xuan‐Chuan , Margolin William
Published: March 2003

The FtsZ ring assembles between segregated daughter chromosomes in prokaryotic cells and is essential for cell division. To understand better how the FtsZ ring is influenced by chromosome positioning and structure in Escherichia coli, we investigated its localization in parC and mukB mutants that are defective for chromosome segregation. Cells of both mutants at non‐permissive temperatures were either filamentous with unsegregated nucleoids or short and anucleate. In parC filaments, FtsZ rings tended to localize only to either side of the central unsegregated nucleoid and rarely to the cell midpoint; however, medial rings reappeared soon after switching back to the permissive temperature. Filamentous mukB cells were usually longer and lacked many potential rings. At temperatures permissive for mukB viability, medial FtsZ rings assembled despite the presence of apparently unsegregated nucleoids. However, a significant proportion of these FtsZ rings were mislocalized or structurally abnormal. The most surprising result of this study was revealed upon further examination of FtsZ ring positioning in anucleate cells generated by the parC and mukB mutants: many of these cells, despite having no chromosome, possessed FtsZ rings at their midpoints. This discovery strongly suggests that the chromosome itself is not required for the proper positioning and development of the medial division site.
ClpC regulates the fate of a sporulation initiation sigma factor, σH protein, in Bacillus subtilis at elevated temperatures
Authors: Nanamiya Hideaki , Ohashi Yoshiaki , Asai Kei , Moriya Shigeki , Ogasawara Naotake , Fujita Masaya , Sadaie Yoshito , Kawamura Fujio
Published: March 2003

Using a strain carrying a clpC–bgaB transcriptional fusion at the amyE locus, we found that the expression of a clpC operon was induced at the end of exponential growth in a σB‐independent manner and ceased around T3.5 in the wild type but not in a spo0H mutant. This suggests that some gene product(s) whose expression is dependent on σH function is required for the turn‐off of clpC transcription during an early stage of sporulation. A clpC deletion mutant showed a temperature‐sensitive sporulation phenotype and exhibited an abnormally large accumulation of σH in the cell at 45°C after T2, at which time the σH level in the wild type had begun to decrease. These results, together with the fact that spo0H transcription in the clpC deletion mutant was similar to that of the wild type, suggested that ClpC may be responsible for the degradation of σH after the accomplishment of its role in sporulation. Moreover, as expected from these results, overproduction of Spo0A was also observed after the initiation of sporulation in the clpC deletion mutant at 45°C.
TolB protein ofEscherichia coliK‐12 interacts with the outer membrane peptidoglycan‐associated proteins Pal, Lpp and OmpA
Authors: Clavel Thierry , Germon Pierre , Vianney Anne , Portalier Raymond , Lazzaroni Jean Claude
Published: March 2003

The Tol–Pal proteins ofEscherichia coliare involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interactin vitrowith the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross‐linkedin vivowith Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation ofpalandtolBmutants disrupting some interactions between these proteins represents a first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol–Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.
Limits to inducer exclusion: inhibition of the bacterial phosphotransferase system by glycerol kinase
Authors: Rohwer Johann M. , Bader Rechien , Westerhoff Hans V. , Postma Pieter W.
Published: March 2003

The uptake of methyl α‐D‐glucopyranoside by the phosphoenolpyruvate‐dependent phosphotransferase system of Salmonella typhimurium could be inhibited by prior incubation of the cells with glycerol. Inhibition was only observed for glycerol preincubation times longer than 45 s and required the preinduction of both the glucose and the glycerol‐catabolizing systems. Larger extents of inhibition by glycerol correlated with higher intracellular levels of glycerol kinase when the glp regulon had been induced to different extents. Preincubation with lactate did not inhibit methyl α‐D‐glucopyranoside uptake significantly, although both lactate and glycerol were oxidized by the cells. The cellular free‐energy state of the cells (intracellular [ATP]/[ADP] ratio) was virtually identical for lactate and glycerol preincubation, suggesting that the inhibition of phosphotransferase‐mediated uptake was not a metabolic effect. In vitro, phosphotransferase activity was inhibited to a maximal extent of 32% upon titrating cell‐free extracts with high concentrations of commercial glycerol kinase. The results show that uptake systems that have hitherto been regarded merely as targets of the phosphotransferase system component IIAGlc also have the capacity themselves to retroinhibit the phosphotransferase system flux, presumably by sequestration of the available IIAGlc, provided that these systems are induced to appropriate levels.
Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties
Authors: Hell Wolfgang , Meyer Heinz‐Georg W. , Gatermann Sören G.
Published: March 2003

A gene encoding a novel cell wall‐associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N‐acetyl‐muramyl‐L‐alanine amidase and for an endo‐β‐N‐acetyl‐D‐glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N‐terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram‐positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.
Probing the active site of mitogillin, a fungal ribotoxin
Authors: Kao R. , Shea J. E. , Davies J. , Holden D. W.
Published: March 2003

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α‐sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14‐base sequence (the α‐sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met‐mature mitogillin (mitogillin with a Met codon at the N‐terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non‐toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His‐49Tyr, Glu‐95Lys, Arg‐120Lys and His‐136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.
A novel mechanism for upregulation of the Escherichia coli K‐12 hmp (flavohaemoglobin) gene by the ‘NO releaser’, S‐nitrosoglutathione: nitrosation of homocysteine and modulation of MetR binding to the glyA‐hmp intergenic region
Authors: Membrillo‐Hernández Jorge , Coopamah Malini D. , Channa Asif , Hughes Martin N. , Poole Robert K.
Published: March 2003

The flavohaemoglobin gene, hmp, of Escherichia coli is upregulated by nitric oxide (NO) in a SoxRS‐independent manner. We now show that hmp expression is also upregulated by S‐nitrosoglutathione (GSNO, widely used as an NO releaser) and sodium nitroprusside (SNP, which is a NO+ donor). Elevated homocysteine (Hcy) levels, achieved either by adding Hcy extracellularly or using metE mutants, decreased hmp expression. Conversely, metC mutants (defective in Hcy synthesis) had higher levels of hmp expression. Mutations in metR abolished hmp induction by GSNO and SNP, and hmp expression became insensitive to Hcy. We propose that the previously documented modulation by Hcy of MetR binding to the glyA‐hmp intergenic regulatory region regulates hmp transcription. Although two MetR binding sites are present in this region, only the higher affinity site proximal to hmp is required for hmp induction by GSNO and SNP. GSNO and SNP react with Hcy in vitro under physiologically relevant conditions of pH and temperature generating S‐nitrosohomocysteine, although in the latter case this would be co‐ordinated to the Fe in SNP as a stable species. The free S‐nitrosocysteine generated in the reaction with GSNO breaks down to release NO more readily than via homolysis of GSNO. As GSNO and SNP upregulate hmp similarly, the NO released in the former case on reaction with homocysteine cannot be involved in hmp regulation.
A model for early events in the assembly pathway of cyanobacterial phycobilisomes
Authors: Anderson Lamont K. , Toole Colleen M.
Published: March 2003

Biological self‐assembly is remarkable in its fidelity and in the efficient production of intricate molecular machines and functional materials from a heterogeneous mixture of macromolecules. The phycobilisome, a light‐harvesting structure of cyanobacteria, presents the opportunity to study an in vivo assembly process in detail. The phycobilisome molecular architecture is defined, and crystal structures are available for all major proteins, as are a large sequence database (including a genome sequence) and effective genetic systems exist for some cyanobacteria. Recent studies on subunit interaction, covalent modification, and protein stability suggest a model for the earliest events in the phycobilisome assembly pathway. Partitioning of phycobilisome proteins between degradation and assembly is proposed to be controlled by the interaction equilibria between phycobilisome assembly partners, processing enzymes and chaperones. The model provides plausible explanations for existing observations and makes predictions that are amenable to direct experimental investigation.
A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures
Authors: Tillotson R. D. , Wösten H. A. B. , Richter M. , Willey J. M.
Published: March 2003

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m−2 to 30 mJ m−2 at a concentration of 50 μg ml−1. We also report that SapB, like the surface‐active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.
Transient transcriptional activation of the IncI1 plasmid anti‐restriction gene (ardA) and SOS inhibition gene (psiB) early in conjugating recipient bacteria
Authors: Althorpe Nicola J. , Chilley Paul M. , Thomas Angela T. , Brammar William J. , Wilkins Brian M.
Published: March 2003

The ardA gene of the enterobacterial plasmid ColIbP‐9 acts to alleviate restriction of DNA by type I systems, while psiB inhibits induction of the bacterial SOS response. Both genes are transferred early in a round of bacterial conjugation as part of the plasmid leading region. We report here that ardA and psiB are transcribed transiently after their conjugative transport into the recipient cell. Transcript levels, monitored by competitive reverse transcription–polymerase chain reaction (RT–PCR) amplification of RNA templates, started to increase about 5 min after the initiation of conjugation in a cell population and probably before the first round of plasmid transfer was completed. Genetic evidence is given that the expression of ardA and psiB is activated when the genes enter the recipient cell on the transferring plasmid strand. It is proposed that these and other leading region genes function to promote the establishment of the immigrant plasmid in the new host and are expressed by transcription from promoters active only in single‐stranded DNA.
Molecular and functional analysis indicates a mosaic structure of Salmonella pathogenicity island 2
Authors: Hensel Michael , Nikolaus Thomas , Egelseer Christine
Published: March 2003

Two large virulence loci encoding type III secretion systems are present on the chromosome of Salmonella typhimurium. Salmonella pathogenicity island 2 (SPI2) is important for the survival of S. typhimurium in host organs and forms an insertion of about 40 kb at the tRNAVal gene. However, several indications suggested that SPI2 was not the result of a single event of horizontal gene transfer. We characterized the portion of SPI2 towards the 30 cs boundary and performed mutational analysis to investigate the contribution of this region to S. enterica virulence. This analysis indicates that SPI2 may be composed of at least two different genetic elements. About 15 kb of the 40 kb of SPI2 contain genes without a significant contribution to systemic infections in the model of murine salmonellosis. Our study allowed us to define genes in SPI2 important for virulence further and indicated that this locus has a complex mosaic structure.
A ‘Gram‐negative‐type’ DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2)
Authors: Flett Fiona , De Mello Jungmann‐Campello Diana , Mersinias Vassilios , Koh Susie L.‐M. , Godden Robert , Smith Colin P.
Published: March 2003

The Streptomyces coelicolor dnaE gene, encoding the catalytic α‐subunit of DNA polymerase III (pol III) was isolated by genetic complementation of a temperature‐sensitive DNA replication mutant, S. coelicolor ts‐38. The deduced protein sequence (1179 residues) is highly similar to the Escherichia coli‐type pol III α‐subunit, rather than to the PolC‐type α‐subunit that is known to be essential for replication in the ‘low G + C’ Gram‐positive bacteria such as Bacillus subtilisThe dnaE gene is able to restore replication to a ‘slow stop’ mutant (ts‐38) and a ‘fast stop’ mutant (ts‐114); the dnaE gene of ts‐38 carries a single amino acid substitution (Glu‐802 to Lys), and the mutation in ts‐114 has been mapped between codons 697 and 1062 of dnaE. Mutant ts‐38 is considered to be defective in assembly of the multisubunit pol III holoenzyme and, hence, in initiation of replication, whereas ts‐114 is defective in chain elongation. This study provides the first evidence that a DnaE‐type pol III is essential for replication in a Gram‐positive bacterium. In addition, the complementation studies suggest that the C‐terminal 117 residues are not essential for DnaE function in S. coelicolor. When integrated at a distant site on the chromosome, a fragment containing the 3′ half of dnaE (codons 697–1179) is capable of rescuing ts‐38 (but not ts‐114) at the restrictive temperature; it was demonstrated that homogenotization was responsible for this phenomenon.
Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation
Authors: Park Youn‐Il , Sandström Stefan , Gustafsson Petter , Öquist Gunnar
Published: March 2003

Iron deficiency is known to suppress primary productivity in both marine and freshwater ecosystems. In response to iron deficiency, certain cyanobacteria induce a chlorophyll (Chl)–protein complex, CP43′, which is encoded by the isiA gene. The deduced amino‐acid sequence of CP43′ predicts some structural similarity to the CP43 polypeptide of photosystem II, but the function of CP43′ remains uncertain. In order to assess its physiological role, the isiA gene of a cyanobacterium, Synechococcus sp. PCC7942, was inactivated by insertion mutagenesis (giving isiA− cells). Compared with isiA− cells, under iron deprivation, wild‐type cells showed both lower rates of photosystem II‐mediated O2 evolution at limiting light irradiances and decreased yields of room temperature Chl fluorescence at various irradiances. These observations strongly suggest that the decreased photosystem II activity in wild‐type cells with CP43′ is attributable to increased non‐radiative dissipation of light energy. In agreement with this hypothesis, isiA− cells were more susceptible to photoinhibition of photosynthesis than wild‐type cells, resulting in much slower growth rates under iron limitation. Based on these results, we suggest that CP43′ functions as a non‐radiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron‐deficient conditions.
The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post‐transcriptional level
Authors: Strauss Joseph , Horvath Henny K. , Abdallah Basem M. , Kindermann Johanna , Mach Robert L. , Kubicek Christian P.
Published: March 2003

The creA gene of A. nidulans encodes a wide‐domain regulatory protein mediating carbon catabolite repression. Northern blot analysis of creA mRNA revealed a complex expression profile: the addition of monosaccharides to a carbon‐starved culture of A. nidulans provoked a strong transient stimulation of creA transcript formation within a few minutes. In the case of repressing carbon sources, creA mRNA levels were subsequently downregulated, whereas the high creA mRNA levels were maintained in a creA mutant strain and in the presence of derepressing monosaccharides. A high creA transcript level is essential to achieve carbon catabolite repression and is dependent on glucose transport and, at least partially, on the creB gene product. Subsequent downregulation of creA mRNA levels, on the other hand, is typical of carbon catabolite repression and requires a functional CreA recognition site in the creA promoter (and thus involves autoregulation) and formation of glucose‐6‐phosphate. Despite the presence of continuing high transcript levels of creA in the presence of derepressing carbohydrates, EMSA demonstrated the presence of only low levels of a CreA–DNA complex in respective cell‐free extracts. Upon transfer of carbon catabolite derepressed mycelia to catabolite‐repressing conditions, a CreA–DNA complex is formed, and this process is dependent on de novo protein synthesis.
A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256
Authors: Ziebuhr Wilma , Krimmer Vanessa , Rachid Shwan , Lößner Isabel , Götz Friedrich , Hacker Jörg
Published: March 2003

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell‐to‐cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm‐forming Staphylococcus epidermidis strains and their PIA‐negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm‐negative variants by Southern hybridization with ica‐specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in ≈30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA‐negative insertional mutants, the biofilm‐forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.
Introns are necessary for mRNA accumulation in Schizophyllum commune
Authors: Lugones Luis G. , Scholtmeijer Karin , Klootwijk Riko , Wessels Joseph G. H.
Published: March 2003

The cDNA coding sequence of the Agaricus bisporus hydrophobin gene ABH1 under the regulation sequences of the Schizophyllum commune SC3 hydrophobin gene gave no expression in S. commune. In contrast, the genomic coding sequence (containing three introns) produced high levels of ABH1 mRNA when transformed to S. commune in the same configuration. Apparently, introns were needed for the accumulation of mRNAs from the ABH1 gene. When the effect of intron deletion on expression of the homologous genes SC3 and SC6 was examined, it was observed that only the genomic coding sequences were expressed in S. commune. Run‐on analysis with nuclei harbouring intron‐containing and intronless SC6 showed that this effect did not occur at the level of transcription initiation: genomic and cDNA sequences were equally active in this respect. When a 50 bp artificial intron containing the consensus splice and branch sites of S. commune introns, in addition to random‐generated sequences, was introduced in the right orientation into the intronless SC3 transcriptional unit, accumulation of SC3 mRNA was restored. By polymerase chain reaction amplification, no unspliced SC3 mRNA species could be detected. Furthermore, the addition of an intron into the transcriptional unit of the gene for green fluorescent protein (GFP) effected clear fluorescence of the transgenic hyphae. Apparently, splicing is required for the normal processing of primary transcripts in S. commune.
A hydrogenosome with pyruvate formate‐lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism
Authors: Akhmanova Anna , Voncken Frank G. J. , Hosea Ken M. , Harhangi Harry , Keltjens Jan T. , Op Den Camp Huub J. M. , Vogels Godfried D. , Hackstein Johannes H. P.
Published: March 2003

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate‐lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.
Characterization of the SarA virulence gene regulator of Staphylococcus aureus
Authors: Rechtin Tammy M. , Gillaspy Allison F. , Schumacher Maria A. , Brennan Richard G. , Smeltzer Mark S. , Hurlburt Barry K.
Published: March 2003

Staphylococcus aureus is a potent human pathogen that expresses a large number of virulence factors in a temporally regulated fashion. Two pleiotropically acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum‐sensing system from the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA‐binding protein that activates the expression of both agr operons. We have cloned the sarA gene, expressed SarA in Escherichia coli and purified the recombinant protein to apparent homogeneity. The purified protein was found to be dimeric in the presence and absence of DNA and to consist mostly of α‐helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high‐affinity binding sites composed of two half‐sites each. Quantitative electrophoretic mobility shift assays (EMSAs) were used to derive equilibrium binding constants (KD) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragment including all three binding sites. Our data indicate that SarA regulation of the agr operons involves binding to multiple half‐sites and may involve other sites located downstream of the promoters.
A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose‐ and acidification‐induced cAMP signalling and not the basal cAMP level
Authors: Vanhalewyn Mieke , Dumortier Françoise , Debast Gilda , Colombo Sonia , Ma Pingsheng , Winderickx Joris , Van Dijck Patrick , Thevelein Johan M.
Published: March 2003

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related ‘wild‐type’ laboratory yeast strains (e.g. ENY.cat80‐7A, CEN.PK2‐1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303‐1A wild‐type strain largely eliminated glucose‐ and acidification‐induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+‐dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild‐type strain, but GTP/Mg2+‐dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose‐induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose‐induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.
The plasmid prophage N15: a linear DNA with covalently closed ends
Authors: Rybchin Valentin N. , Svarchevsky Alexander N.
Published: March 2003

Coliphage N15 is a temperate bacteriophage whose prophage is a linear plasmid molecule with covalently closed ends (telomeres). The N15 prophage provided the first example of such DNA in prokaryotes and, up to now, it is the only known example of a linear plasmid in Escherichia coli. The linear N15 mature phage DNA has single‐stranded cohesive ends. The phage and plasmid prophage DNAs are circularly permuted. The nucleotide structure of the telomere‐forming site tel RL in phage DNA corresponds to the structures of the terminal hairpin loops. It suggests a unique mechanism for conversion of the circular phage DNA to the linear plasmid form, which is performed by the prokaryotic telomerase (protelomerase). The results of a comparison of the protelomerase with integrases lead us to suggest that these proteins may have evolved from a common ancestor. The mechanism of plasmid N15 replication is unknown. We propose that the protelomerase participates in linear plasmid replication, acting as a resolvase of replicative intermediates that are tail‐to‐tail linear dimers. The sequence analysis of the N15 DNA showed that it represents an evolutionary ‘link’ between plasmids F, P1, P4 and lambdoid bacteriophages.
Involvement of the σN DNA‐binding domain in open complex formation
Authors: Oguiza José A. , Gallegos María‐Trinidad , Chaney Matthew K. , Cannon Wendy V. , Buck Martin
Published: March 2003

σN (σ54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniaeσN mutants, altered in the carboxy DNA‐binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator‐dependent transcription. The mutant holoenzymes have altered activator‐dependent interactions with promoter sequences that normally become melted. Activator‐dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator‐independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I σN, suggesting different defects. The DNA‐binding domain substitutions are within σN sequences previously shown to be buried upon formation of the wild‐type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the σN–core and σN–DNA interfaces has occurred to change holoenzyme activity. Core‐binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of σN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator‐dependent transcription, formed a stable activator‐independent holoenzyme promoter complex on premelted DNA templates.
Characterization of a morphological checkpoint coupling cell‐specific transcription to septation in Bacillus subtilis
Authors: Feucht Andrea , Daniel Richard A. , Errington Jeffery
Published: March 2003

Early in the process of spore formation in Bacillus subtilis, asymmetric cell division produces a large mother cell and a much smaller prespore. Differentiation of the prespore is initiated by activation of an RNA polymerase sigma factor, σF, specifically in that cell. σF is controlled by a regulatory cascade involving an anti‐sigma factor, SpoIIAB, an anti‐anti‐sigma factor, SpoIIAA, and a membrane‐bound phosphatase, SpoIIE, which converts the inactive, phosphorylated form of SpoIIAA back to the active form. SpoIIE is required for proper asymmetric division and much of the protein is sequestered into the prespore during septation. Importantly, activation of σF is dependent on formation of the asymmetric septum. We have now characterized this morphological checkpoint in detail, using strains affected in cell division and/or spoIIE function. Surprisingly, we found that significant dephosphorylation of SpoIIAA occurred even in the absence of septation. This shows that the SpoIIE phosphatase is at least partially active independent of the morphological event and also that cells can tolerate significant levels of unphosphorylated SpoIIAA without activating σF. We also describe a spoIIE mutant in which the checkpoint is bypassed, probably by an increase in the dephosphorylation of SpoIIAA. Taken together, the results support the idea that sequestration of SpoIIE protein into the prespore plays an important role in the control of σF activation and in coupling this activation to septation.
Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae
Authors: Rahner Antje , Hiesinger Margit , Schüller Hans‐Joachim
Published: March 2003

In the yeast Saccharomyces cerevisiae, growth with a non‐fermentable carbon source requires co‐ordinate transcriptional activation of gluconeogenic structural genes by an upstream activation site (UAS) element, designated CSRE (carbon source‐responsive element). The zinc cluster protein encoded by CAT8 is necessary for transcriptional derepression mediated by a CSRE. Expression of CAT8 as well as transcriptional activation by Cat8p is regulated by the carbon source, requiring a functional Cat1p (= Snf1p) protein kinase. The importance of both regulatory levels was investigated by construction of CAT8 variants with a constitutive transcriptional activation domain (INO2TAD) and/or a carbon source‐independent promoter (MET25 ). Whereas a reporter gene driven by a CSRE‐dependent synthetic minimal promoter showed a 40‐fold derepression with wild‐type CAT8, an almost constitutive expression was found with a MET25–CAT8–INO2TAD fusion construct due to a dramatically increased gene activation under conditions of glucose repression. Similar results were obtained with the mRNA of the isocitrate lyase gene ICL1 and at the level of ICL enzyme activity. Taking advantage of a Cat8p size variant, we demonstrate its binding to the CSRE. Our data show that carbon source‐dependent transcriptional activation by Cat8p is the most important mechanism affecting the regulated expression of gluconeogenic structural genes.