bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium: characterization of the dipeptide permease (Dpp) and the dipeptide‐binding protein
Authors: Abouhamad W. N. , Manson M. , Gibson M. M. , Higgins C. F.
Published: October 2006

The dipeptide permease (Dpp) is one of three genetically distinct peptide‐transport systems in enteric bacteria. Dpp also plays a role in chemotaxis towards peptides. We have devised three selections for dpp mutations based on resistance to toxic peptides (bacilysin, valine‐containing peptides, and biala‐phos). All dpp mutations mapped to a single chromosomal locus between 77 and 78 min in Salmonella typhimurium and at 79.2 min in Escherichia coli. Expression of dpp was constitutive in both species but the absolute level of expression varied widely between strains. At least in part this difference in expression levels is determined by c/s‐acting sequences. The dpp locus of E. coli was cloned. The first gene in the operon, dppA, encodes a periplasmic dipeptide‐binding protein (DBP) required for dipeptide transport and chemotaxis. Downstream of dppA are other genes required for transport but not for chemotaxis. The dipeptide‐binding protein was found to share 26.5% sequence identity with the periplasmic oligopeptide‐binding protein OppA.
DNA sequence and analysis of the bet genes encoding the osmoregulatory choline—glycine betaine pathway of Escherichia coli
Authors: Lamark T. , Kaasen I. , Eshoo M. W. , Falkenberg P. , McDougall J. , Strøm A. R.
Published: October 2006

The sequence was determined of 6493 nucleotides encompassing the bet genes of Escherichia coli which encode the osmoregulatory choline—glycine betaine pathway. Four open reading frames were identified: betA encoding choline dehydrogenase, a flavoprotein of 61.9kDa; betB encoding betaine aldehyde dehydrogenase (52.6kDa); betT encoding a proton‐motive‐force‐driven, high‐affinity transport system for choline (75.8kDa); and betl, capable of encoding a protein of 21.8 kDa, implicated as a repressor involved in choline regulation of the bet genes. Identification of the genes was supported by subcloning, physical mapping of λplac Mu53 insertions, amino acid sequence similarity, or N‐terminal amino acid sequencing. The bet genes are tightly spaced, with bet T located upstream of, and transcribed divergently to, the tandemly linked betlBA genes.
Binding of the immunity protein inactivates colicin M
Authors: Ölschläger T. , Turba A. , Braun V.
Published: October 2006

Colicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma‐producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N‐terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.
Common and variable domains of the flagellin gene, flaA, in Campylobacter jejuni
Authors: Fischer S. H. , Nachamkin I.
Published: October 2006

The organization of the flagellin gene locus in Campylobacter jejuni strain IN1 (Lior 7) was determined using the polymerase chain (PCR) reaction and a series of oligonucleotide primers. Two tandemly arranged flagellin genes of approximately 1.7 kb were found to be joined by an intervening segment of c. 0.2 kb, similar to that reported for Campylobacter coli. The 5′ flagellin gene, flaA, was generated by PCR and both strands sequenced. Comparison of the deduced amino acid sequence for C. jejuni FlaA with the published sequence for C. coli FlaA showed 77% identical amino acids between the proteins. Two common regions, C1 and C2, comprising the N‐terminal 170 amino acids and C‐terminal 100 amino acids, exhibit amino acids 94% and 96% identical to those of C. coli, respectively. The variable region, V1, comprising the middle of the protein, shows 61% identical residues with C. coli. Comparison of these regions with other bacterial flagellins reveals a similar pattern but with much less identity. Several areas within the V1 region correspond to predicted surface‐exposed regions and may represent areas in which surface epitopes are located.
Light and oxygen effects share a common regulatory DNA sequence in Rhodobacter capsulatus
Authors: Klug G. , Gad'on N. , Jock S. , Narro M. L.
Published: October 2006

External factors regulate the formation of pigment protein complexes in facultatively photosynthetic bacteria. The puf operon of Rhodobacter capsulatus encodes the pigment binding proteins of the reaction centre and light‐harvesting I complex. Here we demonstrate that a single base‐pair exchange within a sequence of dyad symmetry upstream of the puf promoter affects both the oxygen regulation and the light regulation of the formation of reaction‐centre and light‐harvesting I complexes in Rhodobacter capsulatus. Our resuts imply that effects of oxygen or light ultimately act on the same regulatory DNA sequence, although it is still unknown how these environmental signals are sensed and transmitted to a transcriptional regulator.
The role of the OOP antisense RNA in coliphage λ development
Authors: Krinke L. , Mahoney M. , Wulff D. L.
Published: October 2006

We have made a derivative of bacteriophage λ that makes no OOP antisense RNA. The mutant phage carries a point mutation that inactivates the OOP promoter, po. The phages λ+ and λp o‐ have identical plaque morphologies, one‐step growth curves, and frequencies of lysogenization of a sensitive host. OOP RNA synthesis is weakly repressed by the Escherichia coli LexA protein. Consonant with this inducibility of OOP RNA synthesis by ultraviolet light, we find a two‐fold greater phage burst following ultraviolet induction of a λ+ than of a λp o− prophage. In λ+ infections, OOP RNA causes two cleavage events in dl mRNA: one is in the 3′ ‐end of the coding region, and the second is in the intercistronic region between the dl and O genes. The dl gene fragments are subject to additional hydrolytic events, and dl mRNA levels are several‐fold lower in λ+ than in λp o‐ infections late in the infection cycle. However, O mRNA levels are almost unaffected by the P o‐ mutation.
Mucoid Pseudomonas aeruginosa in cystic fibrosis: signal transduction and histone‐like elements in the regulation of bacterial virulence
Authors: Deretic V. , Mohr C. D. , Martin D. W.
Published: October 2006

The profuse production of the exopolysaccharide alginate results in mucoidy, a critical virulence factor expressed by Pseudomonas aeruginosa during chronic respiratory tract infections in cystic fibrosis. Studies of the regulation of this pathogenic determinant have unravelled at least two levels of control, including bacterial signal transduction systems and histone‐like elements. Although only in its initial phase, an understanding of the dual control of mucoidy may help to illuminate adaptive processes that depend on the combination of these regulatory factors. Integration of specific signals transduced by the two‐component systems with inputs generated by the general state of bacterial nucleoids may govern the expression of certain virulence determinants and provide a framework facilitating selection of pheno‐types successful under particular environmental conditions and selective pressures.
The gef gene from Escherichia coli is regulated at the level of translation
Authors: Poulsen L. K. , Refn A. , Molin S. , Andersson P.
Published: October 2006

We describe post‐transcriptional regulation of the chromosomal gene, gef, from Escherichia coli. The gef gene is a member of a gene family consisting of the chromosomal gef and relF genes from Escherichia coli and the hok, flmA, smB, and pndA genes, which are situated on conjugative plasmids. All the genes encode small, toxic proteins of approximately 50 amino acids which are functionally and structurally homologous. Furthermore, the gene family shares post‐transcriptional regulation of expression, albeit by different mechanisms. We demonstrate here that translation of gef is coupled to an upstream open reading frame which, in turn, is regulated by a transacting factor, probably an antisense RNA.
In vitro activation of Escherichia coli prohaemolysin to the mature membrane‐targeted toxin requires HlyC and a low molecular‐weight cytosolic polypeptide
Authors: Hardie K. R. , Issartel J.‐P. , Koronakis E. , Hughes C. , Koronakis V.
Published: October 2006

The c. 110kDa haemolysin toxin secreted by Escherichia coli and other pathogenic Gram‐negative bacteria is synthesized as the non‐toxic precursor, prohaemolysin (proHlyA), which is unable to target mammalian cell membranes until activated intracellularly by an unknown mechanism dependent upon the coexpressed c. 20kDa protein, HlyC. We have established in vitro post‐translational activation of proHlyA in membrane‐depleted cell extract fractions from E. coli recombinant strains containing (separately) the proHlyA and HlyC proteins. In vitro activation was calcium‐independent and effective over a pH range of 6 to 9 and at temperatures from 42°C to 4°C. HlyC cell extract was also able to activate proHlyA which had been secreted out of cells containing the export proteins HlyB and HlyD. Fractionation of HlyC cell extracts by sucrose gradient centrifugation and molecular weight chromatography revealed activating fractions as having a molecular mass of 40kDa, suggesting that the HlyC activator is present physiologically in a multimeric form. Cell extracts containing activation‐competent HlyC and proHlyA were inactive following dialysis, but activity was restored by complementation with a cell extract lacking both proteins. HlyC and proHlyA proteins which were overproduced separately from recombinant expression plasmids were inactive following purification, but activity could again be restored with a Hly‐negative cell extract. These experiments demonstrated that HlyC is not sufficient for activation; an additional cellular factor is required. The cellular factor was found in enterobacteria but not other bacteria or eukaryotic cells. It was cytosolic, protease‐sensitive, and behaved as a c. 10kDa polypeptide in a number of assays including dialysis, sucrose gradient centrifugation, and gel filtration chromatography. Thus activation was possible in a defined in vitro reaction containing only purified proHlyA, HlyC, and the cellular factor. Ki stic studies in which the relative concentrations of the three components of proHlyA activation were varied suggested that neither HlyC nor the cellular factor acts as a conventional enzyme, with each participating in a finite number of activation events.
Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation
Authors: Pla J. , Sanchez M. , Patacios P. , Vicente M. , Aldea M.
Published: October 2006

An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild‐type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non‐viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.
The three‐dimensional structure of the regular surface protein of Comamonas acidovorans derived from native outer membranes and reconstituted two‐dimensional crystals
Authors: Engelhardt H. , Gerbt‐Rleger S. , Santarius U. , Baumelster W.
Published: October 2006

The three‐dimensional structure of the regular surface protein (p4 symmetry, lattice constant a=b=10.5 nm) of Comamonas acidovorans has been determined to a resolution of about 1.5 nm by means of electron microscopy and image processing. Three‐dimensional reconstructions were performed using native outer membranes and artificial two‐dimensional crystals of the surface protein, which was selectively solubilized by deoxycholate and recrystallized on carbon films. The two‐fold symmetric morphological complex is composed of two identical monomers which are in tight contact with the outer membrane and presumably anchored to it by a small hydrophobic domain.
Purification and mutant analysis of Citrobacter freundii AmpR, the regulator for chromosomal AmpC β‐lactamase
Authors: Bartowsky E. , Normark S.
Published: October 2006

AmpR, the transcriptional regulator for the Citrobacter freundii ampC β‐lactamase gene, was purified. The purified AmpR had DNA‐binding activity, the same molecular mass (32 kDa) on sodium dodecyl sulphate polyacrylamide gel electrophoresis as previously described, and N‐terminal sequencing of the first 15 amino acids was in agreement with that predicted from the nucleotide sequence. Two mutants were isolated that abolish DNA‐binding and β‐lactamase induction and which map in the amino‐ and carboxyl‐terminal ends of AmpR, respectively. The mutation in the amino terminus (S35F) was located in a helix‐turn‐helix region showing high homology to other members of the LysR regulator family. Therefore this mutation may directly abolish the contact between AmpR and its operator sequence. It is suggested that the C‐terminal mutation (Y264N) affects subunit interactions in AmpR. One constitutive mutant was isolated which mapped in the centre of the ampR gene. This G102E mutant leads to constitutive β‐lactamase expression in the absence of both β‐lactam inducer and ampG, a gene essential for induction in wild‐type enterobacteria. Another mutant protein, D135Y, showed wild‐type properties In an ampG* and an ampG::kan background, but could, unlike wild‐type AmpR, activate the ampC gene in an ampG1 mutant background. It is thought that ampG1 is a missense mutant. These two types of ampR mutants suggest that activation of ampC transcription is dependent on the conversion of AmpR into a transcriptional activator and that this activation may normally involve interactions with AmpG.
Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans
Authors: Rousset M. , Dermoun Z. , Chippaux M. , Bélaich J. P.
Published: October 2006

A strain of Desulfovibrio fructosovorans deleted from the hydN [NiFe]hydrogenase structural gene was constructed. A plasmid carrying a 7 kb DNA fragment on which the hydN gene had been replaced by the npt reporter gene (kanamycin‐resistant, KnR) was introduced into D. fructosovorans by electroporation. Southern analysis of one KnR clone demonstrated that the hydN gene had been eliminated by marker exchange. This mutant, which was devoid of the [NiFe]hydrogenase gene, still showed a 10% residual hydrogenase activity. Its ability to grow efficiently with H2 as sole energy source is discussed. This is the first report, in a member of the sulphate‐reducing bacteria, of a successful transformation and concomitant homologous recombination leading to a fully controlled genotype.
Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE‐dependent endonucleolytic processing
Authors: Nilsson P. , Uhtin B. E.
Published: October 2006

Differential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities. As the result of a post‐transcriptional processing event, a papA gene‐specific mRNA product (mRNA‐A) accumulates in large excess relative to the primary mRNA‐BA transcript. Our results show that the processed product, mRNA‐A, is a translationally active molecule and that it is generated from the mRNA‐BA precursor by an RNaseE‐dependent mechanism. The processing did not occur under non‐permissive conditions in an E. coli me mutant strain with a temperature‐sensitive RNaseE. The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA. A comparison of nucleotide sequences of mRNA‐BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites. Mutations abolishing the activity of either of two other endoribonucleases, RNaselll and RNaseP, did not affect the pap mRNA processing event. However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E. coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency. Our findings are consistent with the idea that ams is related/alletic to rne. Absence of the processing event in the RNaseE mutant (me‐3071) strain led to a four‐fold stabilization of the mRNA‐BA primary transcript. We conclude that the RNaseE‐dependent processing event is the rate‐limiting step in the decay of the papB‐coding part of the primary transcript and in the production of the stable mRNA‐A product. The present results with the papBA transcripts and previous analyses of rne mutants clearly suggest an important role for RNaseE in turnover of several E. coli mRNAs. Furthermore, the novel processing event exemplified by the pap system demonstrates that RNaseE can have a crucial role at the post‐transcriptional stage in the production of stable and efficiently translated mRNA molecules from E. coli operons.
The role of the NAC protein in the nitrogen regulation of Klebsiella aerogenes
Authors: Bender R. A.
Published: October 2006

The NAC (nitrogen assimilation control) protein from Klebsiella aerogenes is a LysR‐like regulator for transcription of several operons involved in nitrogen metabolism, and couples the transcription of these σ70‐dependent operons to regulation by the σ54‐dependent NTR system. NAC activates expression of operons (e.g. histidine utilization, hut), allowing use of poor nitrogen sources, and represses expression of operons (e.g. glutamate dehydrogenase, gdh) allowing assimilation of the preferred nitrogen source, ammonium. NAC is both necessary and sufficient to activate transcription, but the expression of the nac gene is totally dependent on the central nitrogen regulatory system (NTR) and RNA polymerase carrying the σ54 sigma factor (RNAPσ54). Nitrogen starvation signals the NTR system to transcribe nac, and NAC activates the transcription of hut, put (pro‐line utilization), and urease. NAC does not affect the transcription of RNAP σ54‐dependent operons like gInA or nifLA, which respond directly to the NTR system, but activates transcription of RNAPσ70‐dependent operons. Thus NAC acts as a bridge between RNAPσ70‐dependent operons like hut and the RNAPσ54‐dependent NTR system. The activation of operons like hut by NAC in response to nitrogen starvation is at least superficially similar to their activation by CAP‐cAMP in response to carbon and energy starvation.
Pseudomonad replication origins: a paradigm for bacterial origins?
Authors: Smith D. W. , Yee T. W. , Baird C. , Krishnapiilai V.
Published: October 2006

Structural features of three analysed bacterial DNA replication origin classes (six enteric origins, three pseudomonad origins, and the Bacillus subtilis origin region) are compared in order to deduce characteristics common to all bacterial origins and characteristics that distinguish the three origin classes. The two Pseudomonas aeruginosa origins are shown to map within 10 kb of each other, and correlations are drawn with four potential origin regions in B. subtilis. The enteric origin class is further distinguished from the other two classes by its genetic organization, the presence of GATC sites, and the role of Dam methylation in enteric initiation. The pseudomonad origin class has the most features that are common to all of these bacterial origins, and hence may be the paradigm bacterial origin class.
Structure and function of DnaA and the DnaA‐box in eubacteria: evolutionary relationships of bacterial replication origins
Authors: Yoshikawa H. , Ogasawara N.
Published: October 2006

DnaA protein (a trans‐acting element) and its binding sequence, DnaA‐box: (a cis‐acting element) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other enteric bacteria. Recently these two elements have been found to be conserved in three Gram‐positive bacteria (Bacillus subtilis, Micrococcus luteus and Mycoplasma capricolum) as well as in Gram‐negative pseudomonads. DnaA protein was also found to be essential in the initiation of the replication of the B. subtilis chromosome, and regions containing multiple repeats of DnaA‐box (DnaA‐box region) are found to be active as autonomously replicating elements both in B. subtilis and pseudomonads. In this MicroReview we compare first the structures of these DnaA‐box regions and their locations on the chromosome and then functional aspects of DnaA protein and DnaA‐box regions in the initiation and regulation of chromosomal replication. From these observations we propose evolutionary relationships between replication origins of eubacteria.
The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes
Authors: Inoue C. , Sugawara K. , Kusano T.
Published: October 2006

Two distinct merR genes, which regulate expression of the mercuric ion resistance gene (mer), of Thiobacillus ferrooxidans strain E‐15 have been cloned, sequenced and termed merR1, and merR2. As a result of gene walking around two merR genes, it was found that these two genes were quite close in distance. The nucleotide sequence of the region (5001 base pairs; PstI‐EcoRI fragment) containing the merR genes was determined. Between the two merR genes, there were five potential open reading frames (ORFs). Two of these were identified as merC genes, and the other three as ORFs 1 to 3. ORFs 1 to 3 show significant homology to merA, tnsA from transposon Tn7, and merA, respectively. Both merR genes consist of a 408 bp ORF coding for 135 amino acids. Their gene products, MerRl and MerR2, differed at three amino acid positions, and shared 56‐57% and 32‐38% identity with the MerRs from other Gram‐negative and Gram‐positive bacteria, respectively. Competitive primer extension analysis revealed that both regulatory genes were expressed in the host cells. These merR genes were located more than 6 kb from either end of the mer structural genes (merC‐merA). This is the first example of merR being separated from the mer structural genes. The two merC genes, each of which coded for a 140‐amino‐acid protein, appeared to be functionally active because Escherichia coli cells carrying these merC geneson plasmid vectors showed hypersensitivity to HgCl2. However, ORFs 1 and 3, which were homologous to merA, seemed to be inactive both structurally and enzymatically. The gene arrangement in this region took on a mirror image, with the truncated tnsA as the symmetrical centre. It is suggested that the Tn7‐like factor may have participated in gene duplication events of the mer region, and in its chromosomal integration.
Investigation of the specificity of the interaction between colicin E9 and its immunity protein by site‐directed mutagenesis
Authors: Curtis M. D. , James R.
Published: October 2006

Comparison of the amino acid sequences of the C‐terminal domain of three DNAase type E colicins has identified six candidate specificity determinants for the interaction of these E colicins with their homologous immunity proteins. We have changed these candidate specificity determinants of colicin E9, using site‐directed mutagenesis, to the corresponding amino‐acids of colicin E8. A‘mutant’colicin E9, in which four of the six candidate specificity determininants have been changed, demonstrated colicin activity against Escherichia coli indicator strains which carried either the E8imm or the E9imm genes, indicative of a‘novel’E colicin. After changing all six of the candidate specificity determinants, the resulting colicin E9‘mutant’exhibited a phenotype very similar to that of colicin E8.
Skp is a periplasmic Escherichia coli protein requiring SecA and SecY for export
Authors: Thome B. M. , Müller M.
Published: October 2006

Skp of Escherichia coli (OmpH of Salmonella typhimurium) is a protein whose precise function has been obscured by its ubiquity in a wide range of sub‐cellular fractions such as those containing DNA, ribosomes, and outer membranes. Combining in vitro and in vivo techniques we show that Skp is synthesized as a larger precursor that is processed upon translocation across the plasma membrane. Translocation is dependent on the H+‐gradient, ATP, SecA, and SecY. Upon cellular subfractionation (avoiding non‐specific electrostatic interactions) Skp partitions with β‐lactamase into the fraction of soluble, periplasmic proteins. In the context of the export factor properties of Skp previously demonstrated in vitro it is conceivable that this protein is involved in the later steps of protein translocation across the plasma membrane and/or sorting to the outer membrane.
A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants
Authors: Torres L. , Martín H. , García‐Saez M. I. , Arroyo J. , Molina M. , Sánchez M. , Nombela C.
Published: October 2006

By genetic analysis of a thermosensitive autolytic mutant whose phenotype was complemented by osmotic stabilization with sorbitol, we identified gene LYT2 of Saccharomyces cerevisiae, which is probably involved in cell wall formation. A yeast gene complementing lyt2 strains was cloned and shown to carry an open reading frame coding for a 484‐amino‐acid protein exhibiting all the characteristic domains of serine/threonine protein kinases and highly homologous to other yeast protein kinases involved in control of the mitotic cycle. Mutants disrupted in the cloned gene also displayed an autolytic phenotype complemented by osmotic stabilization with sorbitol. However, genetic comparison of lyt2 mutants and disruptants of the protein kinase gene revealed that the cloned gene is not the structural gene LVT2 but a suppressor of the lytic phenotype, named gene SLT2, that was mapped to chromosome V. The product of gene SLT2 is the first protein kinase to be described in relation to the yeast cell‐wall functions.
Yeast genes involved in DNA‐repair processes: new looks on old faces
Authors: Friedberg E. C.
Published: October 2006

Multiple genes (many of which are designated RAD (for radiation resistance)) are required for cellular responses to DNA damage in the yeast Saccharomyces cerevisiae. In recent years a number of these genes have been cloned and sequenced and in some cases their polypeptide products have been purified and characterized biochemically. These studies are beginning to yield clues about the possible nature of the multiple biochemical pathways for DNA‐damage processing in yeast.
Yeast dsRNA viruses: replication and killer phenotypes
Authors: Tipper D. J. , Schmitt M. J.
Published: October 2006

The cytoplasmic L‐A dsRNA virus of Saccharomyces cerevisiae consists of a 4.5kb dsRNA and the two gene products it encodes: the capsid (cap) and at least one copy of the capsid–polymerase (cap–pol) fusion protein. Virion cap–pol catalyses transcription of the plus (sense)‐strand; this is extruded from the virus and serves as messenger for synthesis of cap and cap–pol. Nascent cap–pol binds to a specific domain in the plus strand to initiate encapsidation and then catalyses minus‐strand synthesis to complete the replication cycle. Products of at least three host genes are required for replication, and virus copy number is kept at tolerable levels by the SKI antivirus system. S. cerevisiae killer viruses are satellite dsRNAs that use a similar encapsidation domain to parasitize the L‐A replication machinery. They encode precursors of secreted polypeptide toxins and immunity (specific resistance) determinants and are self‐selecting. Three unique killer types, K1, K2 and K28, are currently recognized. They are distinguished by an absence of cross‐immunity and by toxin properties and lethal mechanisms; while K1 and K2 toxins bind to cell‐wall glucan and disrupt membrane functions, K28 toxin binds to mannoprotein and causes inhibition of DNA synthesis.
Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae
Authors: Latterich M. , Watson M. D.
Published: October 2006

The yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation. Evidence suggests that it is also involved in osmohomeostasis of the cell. We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium. Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and α‐mannosidase. The mutants fall into at least 17 complementation groups, termed ssv for salt‐sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others. We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.
Characterization, localization and transmembrane organization of the three proteins PrtD, PrtE and PrtF necessary for protease secretion by the Gram‐negative bacterium Erwinia chrysanthemi
Authors: Delepelaire P. , Wandersman C.
Published: October 2006

Erwinia chrysanthemi, a Gram‐negative phythopathogenic bacterium, secretes two related extracellular metalloproteases, B and C, which do not have N‐terminal signal sequences. The specific pathway by which they are secreted, which has been reconstituted in Escherichia coli, comprises three proteins — PrtD, PrtE and PrtF. Hybrid proteins containing segments of these proteins fused to the C‐terminus of protease B were purified and used to immunize rabbits. The antisera thus obtained were used to study the location and membrane topology of the three proteins. PrtD and PrtE were found to cofractionate almost exclusively with the cytoplasmic membrane, whereas PrtF was found to co‐fractionate mostly with the outer membrane. Proteinase K accessibility experiments as well as sequence data lead us to propose that PrtF has one or both ends exposed to the periplasm, that PrtE has one transmembrane segment with its amino‐terminus facing the cytoplasm and its C‐terminal hydrophilic domain exposed to the periplasm, and that PrtD has six transmembrane segments with its N‐terminus and its C‐terminal hydrophilic domain in the cytoplasm.
Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG‐like family of proteins
Authors: Venkatesan M. M. , Buysse J. M. , Hartman A. B.
Published: October 2006

Oligonucleotide primers derived from the ipaH7.8 sequence have been used to determine the boundaries of DNA sequence homology among five lpaH genes on the invasion plasmid (pWR100) of Shigella flexneri 5, strain M9OT‐W. The primary structure of lpaH4.5 has been established from DNA sequence analysis. The first 197 amino acids in lpaH7.8 were replaced in lpaH4.5 by a unique set of 251 amino acids, generating two related proteins with variable and conserved sequences. The amino‐terminal region of lpaH4.5 displayed an internal repeat structure, also seen in lpaH7.8, characteristic of members of the leucine‐rich glycoprotein (LRG) family. The DNA sequences of ipaH2.5 and ipaH1.4 indicate that these genes are truncated versions of lpaH7.8. Western blot analysis of a λgt11 ipaH recombinant (W7) subclone demonstrated that the antigenicity of lpaH7.8 resides outside the leucine‐rich repetitive region.
Physical and genetic map of the Neisseria gonorrhoeae strain MS11‐N198 chromosome
Authors: Bihlmater A. , Römling U. , Meyer T. F. , Tümmler B. , Gibbs C. P.
Published: October 2006

A macro‐restriction map of the Neisseria gonorrhoeae chromosome was constructed using the enzymes Nhel and Spel. Combinations of one‐and two‐dimensional electrophoresis of completely or partially digested chromosomal DNA were performed to align the restriction fragments. The chromosome is circular, with an estimated size of 2.33Mb±35kb. A genetic map was derived from the physical map; positions of over 60 defined loci were determined by Southern hybridization.
Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β‐galactosidase fused to the Hly C‐terminal signal domain
Authors: Kenny B. , Haigh R. , Holland I. B.
Published: October 2006

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post‐translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C‐terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids.

In this paper, we have sought to confirm the presence of a C‐terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C‐terminal fragments of HlyA to heterologous polypeptides.

A C‐terminal fragment (23 kDa) of HlyA, when fused at the C‐terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloram‐phenicol acetyltransferase (CAT) and 98 per cent of the β‐galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences.

Analysis of the nature of the C‐terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.
Molecular aspects of microbial ice nucleation
Authors: Warren G. , Wolber P.
Published: October 2006

Certain organisms nucleate the crystallization of ice. This requires a small volume of water to be induced, probably by lattice‐matching with a solid template, to form an‘ice embryo’—a region sharing at least some of the characteristics of macroscopic ice. It is of particular interest to understand the structure and function of biological structures capable of lattice‐matching (or otherwise inducing a quasi‐crystalline state). Some strains of the Gram‐negative eubacterial genera Erwinia, Pseudomonas, and Xanthomonas, and the mycobionts of certain lichens, display ice‐nucleating activity. In bacteria, the activity is conferred by a protein that contains three nested periodicities of repetition, which probably reflects a hierarchy of three motifs of structural repetition. Thus the tertiary structure of the ice‐nucleation protein is likely to be regular, consistent with the expectation of its forming a template for lattice‐matching. Even within a clonal culture, the nucleating sites formed by bacteria and lichens vary considerably in the threshold temperatures at which they display activity; this indicates wide variations in either the size of the template, or its structural regularity, or both. However, ice‐nucleating sites of lichen and bacterial origin are clearly differentiated by their sensitivities to experimental treatments.
The stringent response in Streptomyces coelicolor A3(2)
Authors: Strauch E. , Takano E. , Baylts H. A. , Bibb M. J.
Published: October 2006

The stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of act III occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis atone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).
Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b
Authors: Cardy D. L. N. , Laidler V. , Salmond G. P. C. , Murrell J. C.
Published: October 2006

The oxidation of methane to methanol in methano‐trophic bacteria is catalysed by the enzyme methane monooxygenase (MMO). This multicomponent enzyme catalyses a range of oxidations including that of aliphatic and aromatic compounds and therefore has potential for commercial exploitation. This study details the molecular characterization of the soluble MMO (sMMO) genes from the Type II methanotroph Methylosinus trichosporium OB3b. The structural genes encoding the α, β and γ subunits of sMMO protein A and the structural gene encoding component B have been isolated and sequenced. These genes have been expressed and their products identified using an in vitro system. A comparative analysis of sMMO predicted sequences of M. trichosporium OB3b and the taxonomically related M. capsulatus (Bath) is also presented.
The general protein‐export pathway is directly required for extracellular pullulanase secretion in Escherichia coli k12
Authors: Pugsley A. P. , Kornacker M. G. , Poquet I.
Published: October 2006

Pullulanase is an extracellular, cell surface‐anchored lipoprotein produced by Gram‐negative bacteria belonging to the genus Klebsiella. Its correct localization in recombinant Escherichia coli requires the products of 14 genes that are linked to the enzyme structural gene in the Klebsiella chromosome. In addition, we show here that six sec genes (secA, secB, secD, secE, secF, and secY) are all required for processing of the prepulluianase signal peptide to occur. This implies that pullulanase crosses the cytoplamic membrane via the general export pathway of which the sec gene products are essential components. Removal or drastic alteration of the prepullulanase signal peptide cause the enzyme to remain cytoplasmic. We propose that pullulanase secretion occurs in two steps, the first of which is common to all signal peptide‐bearing precursors of exported and secreted proteins, whereas the second is specifically involved in translocating pullulanase to the cell surface.
Promoter sequence requirements for Fnr‐dependent activation of transcription of the narGHJI operon
Authors: Walker M. S. , DeMoss J. A.
Published: October 2006

The sequence requirements for Fnr‐dependent transcription of the narGHJI operon of Escherichia coli were studied in plasmids carrying a narG::lacZ protein fusion with the 5′ end of the promoter deleted so that expression was controlled exclusively by Fnr. These plasmids were subjected to in vitro mutagenesis, and β‐galactostdase activities were determined in transformed strains after aerobic and anaerobic growth. A single base‐pair change in the Fnr box, a sequence which is highly conserved in all Fnr‐dependent promoters, essentially abolished anaerobic induction of expression. Primer extension analysis located the transcription start site 57 bp upstream from the narG translation start site and placed the Fnr box at a position centred between –41 and –42 bases from the transcription start site. The position of the Fnr box relative to the transcription start site was critical for anaerobic induction of expression. The deletion of 2bp or addition of 4, 6, 10, 14, 20, 22, 28, 30, and 40 bp immediately downstream from the Fnr box abolished anaerobic induction. Sequence changes between the Fnr box and the transcription start site had different effects, depending upon the region mutagenized. Base changes immediately downstream from the Fnr box, including bases –20 to –29, did not lead to any decrease in anaerobic induction of expression, but in most instances resulted in increased expression. Base changes further downstream prevented anaerobic induction of expression and suggested the requirement for a –10 hexamer which is partially homologous to the –10 consensus sequence for σ70‐specific promoters of E. coli. In addition, specific bases in the region immediately 5′ to the –10 hexamer were found to be required for full anaerobic induction. The critical location of the Fnr box and the absence of sequence specificity for the region which separates the Fnr box from the –10 region suggests that the interaction of Fnr with the Fnr box may replace the requirement for a –35 sequence in the activation of transcription from the narGHJI promoter.
Detection of a gene encoding a phosphatidylinositol‐specific phospholipase C that is co‐ordinately expressed with listeriolysin in Listeria monocytogenes
Authors: Leimeister‐Wächter M. , Domann E. , Chakraborty T.
Published: October 2006

A phosphatidylinositol‐specific phospholipase C (PI‐PLC) that is unique to the pathogenic Listeria species L. monocytogenes and L. ivanovii has been detected. Deletion analysis performed with Escherichia coli recombinants expressing PI‐PLC activity together with maxicell analysis showed that a 34kDa polypeptide was responsible for this activity. Nucleotide sequencing revealed that the gene encoding this polypeptide comprises 317 amino acid residues with a 22‐amino‐acid signal peptide. This gene, designated pic for phosphatidylInositol‐specific phospholipase C, is located back to back with the listeriolysin gene on the chromosome of L. monocytogenes where these genes are transcribed by divergent non‐overlapping promoters. Expression of the pic gene is dependent on the product of the prfA gene, which also regulates expression of the listeriolysin gene in L. monocytogenes.
Nucleotide sequence of the Escherichia coli thymidine kinase gene provides evidence for conservation of functional domains and quaternary structure
Authors: Black M. E. , Hruby D. E.
Published: October 2006

Using λ bacteriophage clones from the Kohara Escherichia coli library spanning minutes 25.5 to 26.5 on the E. coli chromosome (strain W3110), two overlapping DNA fragments were identified which were able to confer thymidine kinase (TK) enzyme activity to a TK‐ strain of E. coli (KY895). This genetic complementation assay was used in concert with subcloning procedures to identify the minimal region (a 900bp EcoRI‐SalI fragment) which contained the E. coli thymidine kinase gene (tdk.) The nucleotide sequence of the Eco RI–SalI fragment and a small portion of the adjoining downstream fragment was determined. Computer analysis of the derived sequence indicated the presence of a rightward‐reading open reading frame of 615bp which was capable of encoding a 205‐amino‐acid polypeptide with a predicted Mr, of 23458 daltons. The in vivo transcriptional activity of this locus was confirmed by Northern blot hybridization analysis of RNA isolated from E. coli JM101 or KY895 which detected a 650‐nucleotide RNA transcribed from this region. This places the tdk gene at approximately minute 27.35 on the E. coli W3110 chromosome, about 15 kb downstream from the narG locus and approximately 25 kb upstream of the trp operon. Although the predicted Mr of the E. coli TK protein was 23.5 kDa, gel‐filtration analyses suggested that, like eukaryotic thymidine kinases, the active form of this enzyme is a multimeric complex. Furthermore, comparisons between the predicted amino acid sequences of the TK proteins encoded by E. coli, bacteriophage T4, and a mammalian poxvirus (vaccinia virus) revealed several regions of amino acid identity, one of which appears to be the ATP‐binding site, thereby suggesting that the essential functional domains of this enzyme have been well‐conserved throughout evolution.
Functioning of the stable signal peptide of the pCloDF13‐encoded bacteriocin release protein
Authors: Luirink J. , Duim B. , de Gier J. W. L. , Oudega B.
Published: October 2006

The pCloDF13‐encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino‐terminal signal peptide that appears to be stable after cleavage. The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, ‘lysis’ and leakage of periplasmic proteins. The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp). The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells. Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas ‘lysis’ and the release of periplasmic enzymes were unaffected. These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.
The role of a stress‐response protein in Salmonella typhimurium virulence
Authors: Johnson K. , Charles I. , Dougan G. , Pickard D. , O'Gaora P. , Costa G. , Ali T. , Miller I. , Hormaeche C.
Published: October 2006

We recently described the use of selective transposon mutagenesis to generate a series of a virulent mutants of a pathogenic strain of Salmonella typhimurium. Cloning and sequencing of the insertion sites from two of these mutants reveals that both have identical locations within an open reading frame that is highly homologous to a gene, htrA, encoding a heat‐shock protein in Escherichia coli. DNA sequence analysis of S. typhimurium htrA reveals the presence of a gene capable of encoding a protein with a calculated Mr of 49316 that has 88.7% protein:protein homology with its E. coli counterpart. In E. coli, lesions in this gene, also known as degP, reduce proteolytic degradation of aberrant periplasmic proteins. Characteristics of the S. typhimurium htrA mutants, 046 and 014, in vivo and in vitro suggested that they are avirulent because of impaired ability to survive and/or replicate in host tissues. In vitro, the S. typhimurium htrA mutants 046 and 014 are not temperature‐sensitive but were found to be more susceptible to oxidative stress than the parent, suggesting that they may be less able to withstand oxidative killing within macrophages.