bluuebunny/crossref_metadata_2025 huggingface dataset:
The alpha subunit of RNA polymerase and transcription antitermination
Authors: Schauer Alan T. , Cheng Sheau‐Wei C. , Zheng Chuanhai , St. Pierre Linda , Alessi Diane , Hidayetoglu Debra L. , Costantino Nina , Court Donald L. , Friedman David I.
Published: November 2003

The N gene product of coliphage λ, with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription‐termination signals. Mutations in host nus genes diminish this N‐mediated antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the α subunit of RNA polymerase, that enhances N‐mediated antitermination. A deletion of the 3′ terminus of rpoA, resulting in the expression of an α subunit missing the CTD, also enhances N‐mediated antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N–Nus complex may be affected through contacts with the CTD of the α subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N–Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N–Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the α subunit, that influences antitermination. Although our results provide evidence that interactions of the α subunit of RNA polymerase must be considered in forming models of transcription antitermination, they do not provide information as to whether the interactions of α that ultimately influence antitermination occur during initiation or during elongation of transcription.
Salmonella spp. are cytotoxic for cultured macrophages
Authors: Chen Li Mei , Kaniga Koné , Galán Jorge E.
Published: November 2003

We have shown by a variety of microscopical and biochemical techniques that Salmonella spp. are cytotoxic for cultured J774A.1 and bone marrow‐derived murine macrophages. The cytotoxicity is initially manifested by inhibition of membrane ruffling and macropinocytosis in infected macrophages, and is followed by cell death. Macrophages killed by Salmonella spp. exhibited features of apoptosis such as condensation and fragmentation of chromatin, membrane blebbing, and the presence of cytoplasmic nucleosomes and apoptotic bodies. Cytotoxicity does not require bacterial internalization as cytochalasin D, a drug that prevents bacterial uptake, did not prevent Salmonella‐induced macrophage cell death. However, the cytotoxic effects are strictly dependent upon the expression of the invasion‐associated Type III protein‐secretion system encoded at centisome 63 of the Salmonella chromosome. Wild‐type Salmonella typhimurium grown under conditions that do not allow optimal expression of this system or strains of Salmonella carrying mutations in genes that encode components of this protein‐secretion system were devoid of macrophage cytotoxicity. In addition, mutations in invJ, spaO, sipB, sipC and sipD, which encode proteins that are secreted via this secretion apparatus and are required for bacterial entry into non‐phagocytic cells, also abolished the toxicity. In contrast, mutations in sipA and sptP, which encode secreted proteins that are not required for bacterial invasion, had no effect on macrophage cytotoxicity. These results indicate a close correlation between the mechanisms of bacterial internalization into non‐phagocytic cells and those that lead to macrophage cytotoxicity. Host‐adapted serotypes of Salmonella such as S. typhi, S. gallinarum and S. dublin were also toxic for murine macrophages, indicating that this virulence property is probably present in most Salmonella spp. and is not associated with the mechanisms responsible for host range.
Very short patch repair: reducing the cost of cytosine methylation
Authors: Lieb Margaret , Bhagwat Ashok S.
Published: November 2003

In Escherichia coli and related bacteria, the product of gene dcm methylates the second cytosine of 5′‐CCWGG sequences (where W is A or T). Deamination of 5‐methylcytosine (5meC) results in C to T mutations. The mutagenic potential of 5meC is reduced by a system called very short patch (VSP) repair, which replaces T with C. T:G and U:G mispairs in the methylatable sequence and in related sequences are recognized by the product of vsr, a gene adjacent to dcm. Vsr creates a nick just 5′ of the mispaired pyrimidine to initiate the repair. Additional products known to be required for VSP repair are DNA polymerase I and DNA ligase. MutS and MutL have a stimulatory role but are not required. The ability of Vsr to recognize T:G mispairs in sequences related to CCWGG is probably responsible for over‐ and under‐representation of certain tetranucleotides in the E. coli genome. Although VSP repair reduces spontaneous mutations at 5meCs in replicating bacteria, mutation hot‐spots persist at these sites. Under conditions that more accurately mimic the natural environment of E. coli, VSP repair appears to be effective in preventing mutation at 5meC.
A newly identified gene cluster in Aspergillus nidulans comprises five novel genes localized in the alc region that are controlled both by the specific transactivator AlcR and the general carbon‐catabolite repressor CreA
Authors: Fillinger Sabine , Felenbok Béatrice
Published: November 2003

Ethanol‐utilization in Aspergillus nidulans is mediated by alcohol dehydrogenase I and aldehyde dehydrogenase encoded by alcA and aldA, respectively. Both genes are under the transcriptional control of the specific activator AlcR and the general carbon catabolite repressor CreA. The alcR and alcA genes are closely linked in chromosome VII; aldA is located in chromosome VIII. We have identified five other transcripts that are expressed from the same genomic region as alcA and alcR. They are inducible by the gratuitous inducer ethyl methyl ketone (EMK), and are carbon catabolite repressed. The corresponding genes, designated alcMalcSalcOalcP, and alcU, are differentially regulated by the specific transcriptional activator AlcR, and they are not all under the direct control by the CreA repressor. Some of the inducible transcripts are very abundant in the cell, whereas others are poorly expressed. Two sets of genes, alcM/alcS and alcR/alcO, are divergently transcribed and probably share a common cis‐acting region, whereas alcP and alcU are individually transcribed from the same strand as alcA and alcR, and have their own promoters. The significance of the alc gene clustering is discussed. At least four of the five novel alc genes in the cluster are not essential for ethanol metabolism.
Identification and sequence of a nifJ‐like gene in Rhodospirillum rubrum: partial characterization of a mutant unaffected in nitrogen fixation
Authors: Lindblad Anders , Jansson Janet , Brostedt Erica , Johansson Magnus , Hellman Ulf , Nordlund Stefan
Published: November 2003

A nifJ‐like gene was identified in the photosynthetic purple non‐sulphur bacterium Rhodospirillum rubrum. A DNA segment hybridizing to Klebsiella pneumoniae nifJ was isolated, the gene was inactivated, and a mutant strain, SNJ‐1, was constructed by allele replacement. The mutation was confirmed by DNA sequencing, Northern blotting and by the lack of pyruvate oxidoreductase activity. This is the first report of a nifJ‐like gene in photosynthetic bacteria. Unexpectedly, SNJ‐1 was capable of nitrogen fixation, and growth was similar to the wild‐type strain under all conditions investigated. Therefore, this is also the first demonstration that a nifJ homologue, when present, is not essential for nitrogen fixation in a diazotroph. The nifJ‐like gene was sequenced and found to have considerable similarity to published nifJ gene sequences from other organisms. By primer extension, the initiation site for transcription was located, and a typical σ54 promoter sequence was identified.
TOL plasmid transcription factor XylS binds specifically to the Pm operator sequence
Authors: Kaldalu Niilo , Mandel Tiiu , Ustav Mart
Published: November 2003

XylS, an AraC family transcription factor, positively regulates transcription of Pseudomonas putida TOL plasmid meta operon from the Pm promoter. A tandem of 15 bp homologous direct repeats, separated by 6 bp and overlapping with the −35 hexamer of the promoter, is required for the activation of Pm by XylS in vivo. In this study we have characterized specific binding of XylS to the Pm operator Om. XylS was overexpressed with an epitope tag in its N‐terminus Tagged XylS (N‐XylS) was immunopurified and was shown to specifically bind to Om. We have used matrix‐bound N‐XylS in DNA footprinting and methylation interference experiments. Binding of N‐XylS protects 44 bp in the Om region on both strands from DNase I digestion and generates hypersensitive sites (within the protected area) which lie on the same face of the DNA helix. Results of hydroxyl radical footprinting and methylation interference assays indicate that XylS binds along one side of the DNA and covers four helical turns. The protein has base‐specific contacts in four adjacent major groove regions on the same helical face. Our data are in accord with the prediction of the presence of two separate DNA‐binding units in an XylS molecule which are involved in base‐specific contacts in two adjacent major‐groove regions of a half‐site. The direct repeat arrangement of the binding site and the mode of DNA binding of XylS are similar to the arrangement of recognition sites and the DNA contact pattern of AraC protein from Escherichia coli
STAB‐SD: a Shine–Dalgarno sequence in the 5′ untranslated region is a determinant of mRNA stability
Authors: Agaisse Hervé , Lereclus Didier
Published: November 2003

Transcription of the Bacillus thuringiensis cryIIIA toxin gene is initiated at nucleotide position −558 (relative to the cryIIIA start codon). However, the major cryIIIA transcript is an mRNA with its 5′ end at nucleotide position −129. Fusions to the lacZ reporter gene revealed that the cryIIIA 5′ untranslated region downstream of nucleotide position −129 acts as a 5′ mRNA stabilizer. Deletion and mutation analysis suggest that the determinant of stability is a Shine–Dalgarno (SD) sequence close to the 5′ end of the stable transcript. This SD sequence, designated STAB‐SD, does not direct translation initiation. However, mutations predicted to weaken the putative interaction between the SD sequence and the 3′ end of 16S rRNA lead to reduced mRNA stability. The binding of a 30S subunit to STAB‐SD may be required for stabilization of cryIIIA mRNA as a transcript with a 5′ end at nucleotide position −129. Similar STAB‐SD sequences are found in the 5′ untranslated regions of other cryIII genes, and in the 5′ untranslated regions of genes from Gram‐positive bacteria other than B. thuringiensis and may therefore be a widespread determinant of mRNA stability.
Analysis of nitrate regulatory protein NarL‐binding sites in the fdnG and narG operon control regions of Escherichia coli K‐12
Authors: Darwin Andrew J. , Li Jing , Stewart Valley
Published: November 2003

During anaerobic growth, expression of the fdnGHI and narGHJI operons of Escherichia coli is induced by the NarL protein in response to nitrate. The fdnG operon control region contains four NarL‐binding sites (termed NarL heptamers) between positions −70 and −130. The two central NarL heptamers of fdnG are arranged as an inverted repeat and are essential for regulation by NarL. We used mutational analysis of these central heptamers to investigate the precise sequence requirements for NarL‐dependent induction. Mutations were examined for their effects on NarL‐dependent expression in vivo. Substitutions at position 1 of either heptamer had the strongest effect whereas substitutions at position 7 had the weakest effect. For some positions, alterations in both heptamers had a stronger effect than either of the single changes. The 2 bp spacing between these NarL heptamers was also important for normal nitrate induction. The narG operon control region has at least eight NarL heptamers arranged in two groups. Previous work has shown that nucleotide substitutions in two of these heptamers, centred at positions −195 and −89, severely reduce nitrate induction of narG operon expression in vivo and significantly interfere with NarL–DNA interactions in vitro. Substitutions in heptamers −185 and −101 affected narG operon induction only when the concentration of phospho‐NarL was low (during growth in the presence of nitrite). Changes in each of the other four NarL heptamers studied had little or no effect on nitrate or nitrite induction of narG operon expression or on NarL–DNA interactions in vitro
The Aspergillus fumigatus chsC and chsG genes encode Class III chitin synthases with different functions
Authors: Mellado Emilia , Aufauvre‐Brown Agnès , Gow Neil A. R. , Holden David W.
Published: November 2003

Two genes, designated chsC and chsG were isolated from DNA libraries of the opportunistic fungal pathogen, Aspergillus fumigatus. The genes were characterized with respect to their nucleotide sequences and mutant phenotypes. The complete deduced amino acid sequences of chsC and chsG show that the products of both genes are Class III zymogen‐type enzymes. A mutant strain constructed by disruption of chsC is phenotypically indistinguishable from the wild‐type strain, but chsG− and chsC− chsG− strains have reduced colony radial growth rate and chitin synthase activity, conidiate poorly and produce highly branched hyphae. Despite these defects, the double‐mutant strain retained the ability to cause pulmonary disease in neutropenic mice. However, in comparison to the wild‐type strain, there was a decrease in mortality and delay in the onset of illness in mice inoculated with the double‐mutant strain, which was associated with smaller and more highly branched fungal colonies in lung tissue.
Modulation of lambda integrase synthesis by rare arginine tRNA
Authors: Zahn Kenneth , Landy Arthur
Published: November 2003

Lambda’s int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda. These are the least frequent codons in genes of E. coli and are recognized by the rarest tRNAs. The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism.
The 19‐residue pro‐peptide of staphylococcal nuclease has a profound secretion‐enhancing ability in Escherichia coli
Authors: Suciu Dominic , Inouye Masayori
Published: November 2003

Staphylococcus aureus secretes two forms of extracellular nuclease, nuclease A and nuclease B. Nuclease A, consisting of 149 residues, is a proteolytic product of nuclease B, which is a processing intermediate that has a 19‐residue N‐terminal pro‐peptide between the signal peptide and nuclease A. It has been shown that nuclease A can be secreted by Escherichia coli by fusing it to the OmpA signal peptide. We now demonstrate that the addition of the pro‐peptide between the OmpA signal peptide and nuclease A leads to a significantly enhanced secretion rate in E. coli. The processing and secretion rates of nuclease B at 37°C were at least 10 times faster than those of nuclease A. Nuclease B was also secreted efficiently under conditions which blocked the secretion of nuclease A, such as secA mutations and the addition of phenethyl alchohol or sodium azide. This enhancing effect of the pro‐peptide was not as striking when it was attached to β‐lactamase, indicating that the pro‐peptide acts as a specific secretion enhancer for nuclease A. Equilibrium circular dichroism on purified nuclease A and nuclease B indicated that the pro‐peptide itself had no significant destabilizing effect on the mature protein. The existence of similar pro‐peptides in Gram‐positive bacterial secretory proteins indicates that they may also serve as secretion enhancers for individual proteins.
Membrane topology of the yeast uracil permease
Authors: Garnier Catherine , Blondel Marie‐Odile , Haguenauer‐Tsapis Rosine
Published: November 2003

The uracil permease of Saccharomyces cerevisiae is a 633 residue polytopic plasma membrane protein. Hydropathy profile analysis indicates that this protein has long hydrophilic N‐and C‐termini and 10–12 potential transmembrane segments. Previous results based on analysis of hybrid proteins allowed identification of the first transmembrane segment of uracil permease, and provided a preliminary indication of the cytoplasmic orientation of its N‐terminus. In this work, other experimental approaches were used to confirm this orientation, and to determine that of the C‐terminus. Epitopes in the N‐and the C‐termini of the protein were protected against trypsin degradation on intact protoplasts, but readily digested on permeabilized protoplasts. Immunofluorescent analysis showed that antibodies to the last 10 amino acids of uracil permease bind to detergent‐treated protoplasts, but not to intact ones. Carboxypeptidase digested the C‐terminus of uracil permease inserted into sealed dog‐pancreas microsomes. These results establish that both N‐ and C‐termini are cytoplasmic, the permease polypeptide spanning the membrane an even number of times. The orientation of several hydrophilic loops with respect to the membrane was investigated by introducing potential glycosylation sites into these regions. We checked whether the resulting mutant proteins were glycosylated when expressed in the presence of dog‐pancreas microsomes. Our data show that two loops of the protein are lumenal. Together with previous results, this work indicates that uracil permease is a 10 membrane‐spanning protein, with rather small external loops and three main cytoplasmic regions (the N‐and C‐termini and a central 60‐residue loop).
A class of gyrase mutants of Salmonella typhimurium show quinolone‐like lethality and require Rec functions for viability
Authors: Garí Eloi , Figueroa‐Bossi Nara , Blanc‐Potard Anne‐Beatrice , Spirito Flavia , Schmid Molly B. , Bossi Lionello
Published: November 2003

We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon. Thus, these mutants mimic the response of wild‐type cells to gyrase inhibitors of the quinolone family. SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product. Introduction of recA or recB null mutations into these strains exacerbates their temperature‐sensitive phenotype and prevents growth at the otherwise permissive temperature of 37°C. Selection of suppressors that concomitantly restore growth at 37°C and SOS induction in a recB− background yielded mutations that relieve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE. Such mutations also restore SOS induction in quinolone‐treated gyr+recB− strains. These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37°C and suggest that recombinational repair intermediates constitute the SOS‐inducing signal in the mutants as well as in quinolone‐treated wild‐type bacteria. Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of ‘cleavable’ gyrase–DNA complexes in plasmid or chromosomal DNA. Yet gyrA208 (the only allele tested) was found to trigger RecB‐mediated reckless degradation of chromosomal DNA in recA− cells at restrictive temperatures. Indirect evidence suggests that double‐stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA‐replication forks. We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone‐treated bacteria.
In vitro characterization of constitutive CysB proteins from Salmonella typhimurium
Authors: Colyer Tracey E. , Kredich Nicholas M.
Published: November 2003

Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR‐type transcriptional activator CysB and by the inducer N‐acetyl‐l‐serine. Sulphide and thiosulphate are anti‐inducers. Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIHcysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N‐acetyl‐l‐serine, and to be insensitive to the effects of anti‐inducers. At 10mM MgCl2, the in vitro transcription activity of CysB(T149M) was maximal without N‐acetyl‐l‐serine, but that of CysB(T149P) was increased by inducer. At 2mM MgCl2, both proteins were fully active without inducer. A third mutant protein, CysB(W166R), was totally inactive at 10mM MgCl2, but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl2. Surprisingly, wild‐type CysB was also constitutive for the cysK promoter at 2mM MgCl2 but not at 10mM MgCl2; it required inducer for cysJIH promoter activation at both concentrations. Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half‐sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter. We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site. In vitro activities of wild‐type and mutant CysB proteins correlated much better with in vivo behaviour at 2mM than at 10mM MgCl2, suggesting that the former is the more physiological concentration.
Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four‐stranded intermediate and the order of strand exchange
Authors: Hakimi Janette M. , Scocca John J.
Published: November 2003

The integrase encoded by the temperate phage HP1 promotes the site‐specific recombination between DNA sites on its genome (the attP site) and on the genome of the host Haemophilus influenzae (the attB site). The protein has been overproduced in Escherichia coli, and purified to apparent homogeneity. HP1 integrase promotes recombination of supercoiled attP‐containing molecules with linear segments with attB sites. Reaction was enhanced by spermidine and by the bacterial DNA‐bending protein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concentration of the supercoiled attP substrate. These relationships probably originate from the need to assemble a multi‐protein complex on the attP DNA. The reaction promoted by HP1 integrase produced a four‐stranded initial reaction product in which one pair of DNA strands had undergone transfer while the other pair remained intact. This four‐stranded component was produced more rapidly than any product, and its steady‐state level was proportional to the overall rate of reaction. This component had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was used to determine that the two strand exchanges required for recombination of the duplex substrates proceed in a defined order.
Protein F2, a novel fibronectin‐binding protein from Streptococcus pyogenes, possesses two binding domains
Authors: Jaffe Joseph , Natanson‐Yaron Shira , Caparon Michael G. , Hanski Emanuel
Published: November 2003

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)‐binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn‐binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene (prtF2) from GAS strain 100076 encoding a novel Fn‐binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high‐affinity Fn binding. prtF2‐related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn‐binding protein in GAS. Protein F2 is highly homologous to Fn‐binding proteins from Streptococcus dysgalactiae and Strep‐tococcus equisimilis, particularly in its carboxy‐terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non‐repeated stretch of approximately 100 amino acids and is located 100 amino acids amino‐terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.
Three, four or more: the translational stop signal at length
Authors: Tate Warren P. , Mannering Sally A.
Published: November 2003

Translational stop signals are defined in the genetic code as UAA, UAG and UGA, although the mechanism of their decoding via protein factors is clearly different from that of the other codons. There are strong biases in the upstream and downstream nucleotides surrounding stop codons. Experimental tests have shown that termination‐signal strength is strongly influenced by the identity of the nucleotide immediately downstream of the codon (+4), with a correlation between the strength of this four‐base signal and its occurrence at termination sites. The +4 nucleotide and other biases downstream of the stop codon may reflect sites of contact between the release factor and the mRNA, whereas upstream biases may be due to coding restrictions, with the release factor perhaps recognizing the final tRNA and the last two amino acids of the polypeptide undergoing synthesis. This means that the translational stop signal is probably larger than the triplet codon, but its exact length will be clearer when it is known which nucleotides are in direct contact with the release factor. Ultimately it will be defined exactly when a crystal structure of the release factor with its recognition substrate becomes available.
Identification of a ClpC ATPase required for stress tolerance and in vivo survival of Listeria monocytogenes
Authors: Rouquette Corinne , Ripio Maria‐Teresa , Pellegrini Elisabeth , Bolla Jean‐Michel , Tascon Ruben Ivan , Vázquez‐Boland José‐Antonio , Berche Patrick
Published: November 2003

We identified a new chromosomal locus involved in the virulence of the facultative intracellular pathogen Listeria monocytogenes. This locus displays the same genetic organization as that of the clpC/mecB locus of Bacillus subtilis. It contains a thermoregulated operon of four genes, whose transcription is upregulated at 42°C. The last gene of this operon is clpC, which encodes a protein of 826 amino acid residues, identified as a ClpC ATPase, sharing a strong peptide sequence identity (78%) with ClpC/MecB of B. subtilis. Tn917 insertions inactivating the entire operon, or only clpC, gave mutants highly susceptible to stress, including iron limitation, elevated temperatures and high osmolarity. The virulence of these mutants was severely impaired in the mouse. A clpC insertional mutant was also restricted in its capacity to grow in bone‐marrow‐derived macrophages. These results demonstrate that the ClpC ATPase of L. monocytogenes is a general stress protein involved in intracellular growth and in vivo survival of this pathogen in host tissues.
Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1
Authors: Bliss Joseph M. , Silver Richard P.
Published: November 2003

Capsules are well‐studied components of the bacterial surface that modulate interactions between the cell and its environment. Generally composed of polysaccharide, they are key virulence determinants in invasive infections in humans and other animals. Genetic determinants involved in capsule expression have been isolated from a number of organisms, but perhaps the best characterized is the kps cluster of Escherichia coli K1. In this review, the current understanding of the functions of the kps gene products is summarized. Further, a proposed mechanistic model for capsule expression is presented and discussed. The model is based on the premise that the numerous components of the kps cluster form a hetero‐oligomeric complex responsible for synthesis and concurrent translocation of the capsular polysialic acid through sites of inner and outer membrane fusion. We view the ATP‐binding cassette (ABC) transporter, KpsMT, to be central to the functioning of the complex, interacting with the biosynthetic apparatus as well as the extracytoplasmic components of the cluster to co‐ordinate synthesis and translocation. The model provides the basis for additional experimentation and reflects emerging similarities among systems responsible for macromolecular export in Gram‐ negative bacteria.
Broad‐host‐range plasmid replication: an open question
Authors: del Solar Gloria , Alonso Juan C. , Espinosa Manuel , Díaz‐Orejas Ramón
Published: November 2003

Many factors can influence the ability of plasmids to colonize different hosts, efficient replication probably being the most critical one. Two major strategies seem to facilitate promiscuous plasmid replication: (i) initiation independent of host initiation factors; and (ii) versatile communication between plasmid and host initiation factors. Appropriate communication between a replicon and the different hosts, which becomes crucial at the initation of plasmid replication, plays a major role in plasmid promiscuity. Fused replicons or mechanisms that rescue collapsed replication forks may increase the efficiency of plasmid propagation. However, their contribution to plasmid promiscuous replication remains to be fully evaluated. Several examples of host‐specific adaptation of promiscuous plasmids point to an enormous flexibility of these replicons.
Molecular characterization of group A streptococcal (GAS) oligopeptide permease (Opp) and its effect on cysteine protease production
Authors: Podbielski Andreas , Pohl Barbara , Woischnik Markus , Körner Christiane , Schmidt Karl‐Hermann , Rozdzinski Eva , Leonard Bettina A. B.
Published: November 2003

Bacterial oligopeptide permeases are membrane‐associated complexes of five proteins belonging to the ABC‐transporter family, which have been found to be involved in obtaining nutrients, cell‐wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10 192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50–84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra‐operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.
Role of the conserved carboxy‐terminal α‐helix of Rad6p in ubiquitination and DNA repair
Authors: Dor Yuval , Raboy Bilha , Kulka Richard G.
Published: November 2003

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin‐conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C‐terminal α‐helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin‐conjugating enzymes (Ubcs). This specific conservation suggests that the C‐terminal a‐helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error‐free DNA repair in vivo. C‐terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C‐terminal u‐helix is also essential for error‐free DNA repair in vivo. Overexpression of Rad18p, a single‐stranded DNA‐binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C‐terminal α‐helix mutants to different degrees. This indicates that the C‐terminal α‐helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild‐type capacity for error‐free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.
The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells
Authors: Davis M. A. , Radnedge L. , Martin K. A. , Hayes F. , Youngren B. , Austin S. J.
Published: November 2003

The P1 ParA protein is an ATPase that recognizes the parA promoter region where it acts to autoregulate the P1 parA–parB operon. The ParB protein is essential for plasmid partition and recognizes the cis‐acting partition site parS. The regulatory role of ParA is also essential because a controlled level of ParB protein is critical for partition. However, we show that this regulatory activity is not the only role for ParA in partition. Efficient partition can be achieved without autoregulation as long as Par protein levels are kept within a range of low values. The properties of ParA mutants in these conditions showed that ParA is essential for some critical step in the partition process that is independent of par operon regulation. The putative nucleotide‐binding site for the ParA ATPase was identified and disrupted by mutation. The resulting mutant was substantially defective for autoregulation and completely inactive for partition in a system in which the need for autoregulation is abolished. Thus, the ParA nucleotide‐binding site appears to be necessary both for the repressor activity of ParA and for some essential step in the partition process itself. We propose that the nucleotide‐bound form of the enzyme adopts a configuration that favours binding to the operator, but that the ATPase activity of ParA is required for some energetic step in partition of the plasmid copies to daughter cells.
Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplifiable element AUD1 of Streptomyces lividans 66
Authors: Volff J.‐N. , Eichenseer C. , Viell P. , Piendl W. , Altenbuchner J.
Published: November 2003

The amplifiable unit of DNA no. 1 (AUD1) of Streptomyces lividans consists of three 1 kb repeats (left direct repeat, LDR; middle direct repeat, MDR; and the slightly different right direct repeat, RDR) and two 4.7 kb repeats alternately arranged in identical orientation to each other. Both 4.7 kb repeats have been sequenced. They are identical and contain one open reading frame (orf4.7 ). The deduced amino acid sequence has a low similarity to chitinases, and two amino acid repeats present high similarities to fibronectin type III modules. Sequencing had previously shown that the ORF corresponding to each 1 kb repeat encodes a putative DNA‐binding protein. Crude extracts of Escherichia coli overexpressing the orfRDR‐encoded protein and of S. lividans Jni1, having a high amplification of AUD1 and therefore orfMDR, were used in gel retardation assays. The orfRDR‐ and probably the orfMDR‐encoded proteins can bind to an imperfect palindromic sequence upstream from MDR and RDR and to another sequence downstream from RDR. An extrachromosomal DNA amplification system was constructed containing different combinations of the sequences composing AUD1. In mutants having a deletion of the chromosomal AUD1, the 4.7 kb repeats could be reduced in size, mutated or replaced by E. coli DNA without altering the ability to amplify when RDR was present. Therefore, the only function of the 4.7 kb repeats in amplification is to provide directly repeated DNA sequences. When RDR was lacking or mutated, no amplification was observed. This strongly suggests that the DNA‐binding protein encoded by orfRDR is required for AUD1 amplification.
Programmed cell death in bacteria: translational repression by mRNA end‐pairing
Authors: Franch Thomas , Gerdes Kenn
Published: November 2003

The hok/sok and pnd systems of plasmids R1 and R483 mediate plasmid maintenance by killing plasmid‐free cells. Translation of the exceptionally stable hok and pnd mRNAs is repressed by unstable antisense RNAs. The different stabilities of the killer mRNAs and their cognate repressors explain the onset of translation in plasmid‐free cells. The full‐length hok and pnd mRNAs are inert with respect to translation and antisense RNA binding. We have previously shown that the mRNAs contain two negative translational control elements. Thus, the mRNAs contain upstream anti‐Shine–Dalgarno elements that repress translation by shielding the Shine–Dalgarno ele‐ments. The mRNAs also contain fold‐back‐inhibition elements (fbi ) at their 3′ ends that are required to maintain the inert mRNA configuration. Using genetic complementation, we show that the 3′fbi elements pair with the very 5′ ends of the mRNAs. This pairing sets the low rate of 3′ exonucleolytical processing, which is required for the accumulation of an activatable pool of mRNA. Unexpectedly, the hok and pnd mRNAs were found to contain translational activators at their 5′ ends (termed tac ). Thus, the fbi elements inhibit translation of the full‐length mRNAs by sequestration of the tac elements. The fbi elements are removed by 3′ exonucleolytical processing. Mutational ana‐lyses indicate that the 3′ processing triggers refolding of the mRNA 5′ ends into translatable configurations in which the 5′tac elements base pair with the anti‐Shine–Dalgarno sequences.
Role of stoichiometry between mRNA, translation factor SeIB and selenocysteyl‐tRNA in selenoprotein synthesis
Authors: Tormay P. , Sawers A. , Böck A.
Published: November 2003

The specialized translation factor SeIB forms a quaternary complex in vitro with selenocysteyl‐tRNASec, the selenoprotein mRNA and guanine nucleotides. To gain information on whether this complex is required for selenocysteine insertion in vivo we have studied the effect of unbalanced ratios of the individual components of the complex on UGA readthrough. It was found that overproduction of SeIB in an otherwise wild‐type genetic background reduced UGA read‐through to less than 1 %. Concomitant overexpression of seIC (the gene for selenocysteine‐specific tRNASec) completely reversed the inhibition. Truncation of SeIB from the C‐terminal end abolished function as a translation factor but the truncated molecules, when overproduced, were still able to suppress UGA read‐through. The inhibition was also reversed by overproduction of tRNASec. The most plausible explanation is that overproduction of SeIB impairs the statistics of formation of the quaternary complex and that the C‐terminally truncated molecules are still able to bind selenocysteyl‐tRNASec and remove it from the pool. The mRNA‐binding capacity, therefore, is physically separated from the selenocysteyl‐tRNA‐binding domain.
SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm
Authors: Van Der Does Chris , Den Blaauwen Tanneke , De Wit Janny G. , Manting Erik H. , Groot Nancy A. , Fekkes Peter , Driessen Arnold J. M.
Published: November 2003

SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide‐modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase,the SecY, SecE and SecG polypeptides, results in an increased level of membrane‐bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane‐associated form of SecA indicates that it exposes a carboxy‐terminal domain to the periplasmic face of the membrane.
A TonB‐like protein and a novel membrane protein containing an ATP‐binding cassette function together in exotoxin secretion
Authors: Howard S. Peter , Meiklejohn Heather G. , Shivak David , Jahagirdar Ravindra
Published: November 2003

Protein secretion by many Gram‐negative bacteria occurs via the type II pathway involving translocation across the cytoplasmic and outer membranes in separate steps. The mechanism by which metabolic energy is supplied to the translocation across the outer membrane is unknown. Here we show that two Aeromonas hydrophila inner membrane proteins, ExeA and ExeB, are required for this process. ExeB bears sequence as well as topological similarity to TonB, a protein which opens gated ports for the inward translocation of ligands across the outer membrane. ExeA is a novel membrane protein which contains a consensus ATP‐binding site. Mutations in this site dramatically decreased the rate of secretion of the toxin aerolysin from the cell. ExeB was stable when overproduced in the presence of ExeA, but was degraded when synthesized in its absence, indicating that the two proteins form a complex. These results suggest that ExeA and ExeB may act together to transduce metabolic energy to the opening of a secretion port in the outer membrane.
Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine‐rich loop of diphtheria toxin
Authors: Chiron Marielle F. , Ogata Masato , FitzGerald David J.
Published: November 2003

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg‐279 and Gly‐280. Cleavage, which is mediated by the cellular protease furin, generates an active C‐terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro, furin‐mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5–10% of cell‐associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine‐rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE‐DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE‐cleavage site were changed to amino acids found at the DT‐cleavage site. Mutant proteins were expressed in Escherichia coli, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell‐mediated cleavage, and cytotoxic activity relative to wild‐type PE. At pH 5.5, the rate of both furin‐mediated cleavage and trypsin‐mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin‐mediated cleavage of PE toxins, which remained at pH 5.0–5.5. When radioactive versions of selected PE‐DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild‐type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE‐DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild‐type PE.
Molecular characterization of mutants affected in the osmoprotectant‐dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1
Authors: Sage Andrew E. , Vasil Adriana I. , Vasil Michael L.
Published: November 2003

Production of the two phospholipases C (PLCs) in Pseudomonas aeruginosa PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine. Tn5 mutagenesis was performed on strain PAO1 to isolate mutants deficient in choline‐dependent induction of PLC. Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate‐replete media supplemented with choline. A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced. The DNA flanking the Tn5T1 insertion contains an open reading frame predicted to encode a peptide that is approx. 60% identical to the N‐terminus of a previously identified protein (P35) of unknown function from Escherichia coli. The P35 gene, which is located in the nusA–infB operon in E. coli, was designated orp (osmoprotectant regulator of PLC). Haemolytic titres, total PlcH protein and β‐galactosidase activity expressed from a chromosomally inserted plcH–lacZ operon fusion werereduced in strain Tn5T1 in comparison with the parental strain (PAO1) carrying the same fusion. However, this mutant expressed several‐fold higher levels of plcH message than strain PAO1 in the presence of choline, while the phosphate‐starvation‐dependent transcript of plcH could not be detected in this mutant. The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO1, that carries the orp gene. The deduced amino acid sequence of the DNA fragment cloned from Tn5G19 exhibits 84% identity with the betB gene product of E. coli that has betaine aldehyde dehydrogenase activity. This enzyme catalyses the conversion of betaine aldehyde to glycine–betaine. Unlike the parental strain, the Tn5G19 mutant could not utilize choline as a sole carbon, nitrogen and energy source, and it was deficient in betaine aldehyde dehydrogenase activity. Also, consistent with a disruption of betB in Tn5G19, choline inhibited growth of this strain in media containing 0.7 M NaCl, while glycine–betaine restores growth to wild‐type levels. The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene. The orp gene is located between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.