bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine‐scaled geographical population structuring in a highly mobile marine species: the Atlantic cod
Authors: Knutsen H. , Jorde P. E. , André C. , Stenseth N. CHR.
Published: March 2003

Compared with many terrestrial and freshwater environments, dispersal and interbreeding is generally much less restricted in the marine environment. We studied the tendency for a marine species, the Atlantic cod, to be sub‐structured into genetically differentiated populations on a fine geographical scale. We selected a coastal area free of any obvious physical barriers and restricted sampling to a 300‐km region, well within the dispersal ability of this species. Screening 10 polymorphic microsatellite loci in 6 samples we detected a weak, but consistent, differentiation at all 10 loci. The average FST over loci was small (0.0023) but highly significant statistically, demonstrating that genetically differentiated populations can arise and persist in the absence of physical barriers or great distance. We found no geographical pattern in the genetic differentiation and there was no apparent trend of isolation by distance along the coastline. These findings lend support to the notion that low levels of differentiation are due to passive transport of eggs or larvae by the ocean currents rather than to adult dispersal, the latter being strongly dependent on distance.
Lizards in ‘nuclear families’: a novel reptilian social system in Egernia saxatilis (Scincidae)
Authors: O'Connor D. , Shine R.
Published: March 2003

Recent research has revealed unsuspected complexity in social organization among squamate reptiles. In particular, large Australian scincid lizards of the genus Egernia have been reported to occur in large aggregations of closely related individuals. However, the ‘nuclear family’ structure found in many other ‘social’ organisms (especially birds) has not been reported from reptiles. Our field studies on black rock skinks (Egernia saxatilis) in southeastern Australia document exactly this pattern. We quantified group composition using behavioural observations at regular intervals over three field seasons, and took tissue samples for parentage analysis. On the focal rock outcrop 72% of lizards were typically found as part of a stable social grouping, with individuals physically associated with other group members in a third of observations. Eighty‐five per cent of juveniles lived in social groups, 65% in family groups with at least one of their parents (including 39% with both parents as revealed by parentage analysis of five microsatellite loci). Broader sampling in surrounding areas revealed similar patterns of group size, composition and relatedness. Overall, of the groups that contained more than one adult, 83% contained a single adult pair. Long‐term monogamy and group stability were evident from our genetic data, with up to three annual cohorts of full‐sib offspring living with their biological parents. Our data expand the range of social systems known for reptiles, and reveal strong convergence towards ‘nuclear family’ systems in distantly related vertebrates.
Streptolysin O and adherence synergistically modulate proinflammatory responses of keratinocytes to group A streptococci
Authors: Ruiz Natividad , Wang Bei , Pentland Alice , Caparon Michael
Published: March 2003

In contrast to a mutant adhesin‐deficient Streptococcus pyogenes (group A streptococcus), its isogenic parental strain binds to human keratinocytes and promotes a vigorous proinflammatory response, characterized by enhanced expression of several cytokines, a more rapid release of prostaglandin E2 (PGE2) and damage to keratinocyte membranes. However, adherence alone is not sufficient to induce these responses. In this study, we have begun to examine the contribution of other streptococcal products in interactions with keratinocytes by the construction and evaluation of mutants deficient in expression of the secreted pore‐forming haemolysin, streptolysin O (SLO). Inactivation of SLO did not prevent the streptococci from adhering to cultured HaCaT keratinocytes or from expressing an unrelated second streptococcal haemolysin, streptolysin S, during infection of keratinocytes. As measured by a quantitative reverse transcriptase polymerase chain reaction (PCR) assay, inactivation of SLO also did not have a marked effect on the expression of interleukin 1α (IL‐1α) during infection. However, the lack of the ability to produce SLO was associated with a considerable reduction in expression of IL‐1β, IL‐6 and IL‐8 by infected keratinocytes. Measurement of the release of PGE2 by an enzyme‐linked immunosorbent assay demonstrated that the SLO‐deficient mutants were also not capable of promoting the rapid high level of PGE2 release characteristic of the adherent SLO‐producing parental strain. Finally, analyses using the fluorescent probe ethidium homodimer‐1 and measurements of release of keratinocyte lactate dehydrogenase indicated that the failure of the SLO‐deficient mutants to induce responses was associated with the failure of these mutants to damage the integrity of the keratinocyte membrane. These data implicate SLO as a factor that acts synergistically with an adhesin to modulate the signalling responses of keratinocytes during infection.
An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His‐Asp phosphorelay
Authors: Ogino Tomoaki , Matsubara Masahiro , Kato Naoki , Nakamura Yoshihiro , Mizuno Takeshi
Published: March 2003

The Escherichia coli sensory kinase, ArcB, possesses a histidine‐containing phosphotransfer (HPt) domain, which is implicated in the His‐Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His‐Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine–histidine–glycine (RHG) signature, at its N‐terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose‐2,6‐bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose‐1‐phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His‐Asp phosphorelay through regulating the phosphorylation state of the HPt domain.
Mutational analysis of the NH2‐terminal arms of the trp repressor indicates a multifunctional domain
Authors: Mackintosh Samuel G. , McDermott Patrick F. , Hurlburt Barry K.
Published: March 2003

The NH2‐terminal arms of the Escherichia coli trp repressor have been implicated in three functions: formation of repressor–operator complexes via association with non‐operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA. To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutants in vivo using reporter gene fusions. Deletion of any part of the arms resulted in a significant decrease in repressor activity at both the trp and the trpR operons. Positions 4, 5 and 6 were the most sensitive to missense changes. Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild‐type trp repressor. A large percentage of the missense mutants were more active than the wild‐type repressor in medium containing tryptophan and less active in medium without tryptophan. This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor. Also, nine super‐repressor mutants, resulting from substitutions clustered at both ends of the arms, were found. Our results support the hypothesis that the NH2‐terminal arm of the trp repressor is a multifunctional domain and reveal structural components likely to be involved in the various functions.
Xanthine dehydrogenase from the phototrophic purple bacterium Rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes
Authors: Leimkühler Silke , Kern Monika , Solomon Peter S. , McEwan Alastair G. , Schwarz Günter , Mendel Ralf R. , Klipp Werner
Published: March 2003

Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis. Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases. DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase. The deduced amino acid sequence of XDHA contains binding sites for two [2Fe–2S] clusters and FAD, whereas XDHB is predicted to contain the molybdopterin cofactor. In contrast to R. capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases. The amino acid sequence of xanthine dehydrogenase from R. capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase‐related aldehyde oxidoreductase from Desulphovibrio gigas. The expression of an xdhA–lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source. Mutations in nifR1 (ntrC) and nifR4 (rpoN, encoding σ54) had no influence on xdh gene expression. A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine. The transcriptional start site of xdhA was mapped by primer extension analysis. Comparison with known promoter elements revealed no significant homology. Xanthine dehydrogenase from R. capsulatus was purified to homogeneity. The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively. N‐terminal amino acid sequencing of both subunits confirmed the predicted start codons. The molecular mass of the native enzyme was determined to be 275 kDa, indicating an α2β2‐subunit structure. Analysis of the molybdenum cofactor of xanthine dehydrogenase from R. capsulatus revealed that it contains the molybdopterin cofactor and not a molybdopterin dinucleotide derivative.
Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine–lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli
Authors: Eaves D. J. , Grove J. , Staudenmann W. , James P. , Poole R. K. , White S. A. , Griffiths L. , Cole J. A.
Published: March 2003

Cytochrome c552 is the terminal component of the formate‐dependent nitrite reduction pathway of Escherichia coli. In addition to four ‘typical’ haem‐binding motifs, CXXCH‐, characteristic of c‐type cytochromes, the N‐terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild‐type bacteria with cyanogen bromide followed by trypsin were analysed by on‐line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haem‐containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate‐dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low mid‐point potential redox centres. The best fit to the experimental data is for three n = 1 components with mid‐point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), −90 mV (36% of the total) and −210 mV (43% of the total). Plasmids in which the lysine codon of the cysteine–lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth ‘typical’ haem c‐binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf− deletion mutant to Nrf+ or to restore formate‐dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c‐type cytochrome was confirmed by staining proteins separated by SDS–PAGE for covalently bound haem, but the methyl‐viologen‐dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine–lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.
Escherichia coli genes expressed preferentially in an aquatic environment
Authors: Espinosa‐Urgel Manuel , Kolter Roberto
Published: March 2003

Enteric bacteria are frequently found in aquatic environments, where they may pose a risk to human health. Although bacterial survival and persistence in such habitats has been studied extensively, there is almost no information about bacterial adaptation to these conditions at the level of changes in gene expression. As a first exploration of this field, we have carried out a screen designed to identify Escherichia coli genes that show increased expression in an aquatic environment. The screen was performed by subtractive hybridization on a genomic library and led to the identification of several RNA species more abundant in cells inoculated in this medium than in stationary‐ phase cultures after growth in rich medium. The genes identified include specific tRNA operons and a gene of unknown function, gapC, with similarities to glyceraldehyde‐3‐phosphate dehydrogenases. E. coli K‐12 strains appear to have accumulated mutations in gapC, which may impede its translation, whereas natural isolates have an intact gapC gene. Sequence comparison of gapC with related genes suggests its acquisition by horizontal gene transfer from Gram‐positive bacteria.
Involvement of two A‐factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis
Authors: Onaka Hiroyasu , Nakagawa Taishiro , Horinouchi Sueharu
Published: March 2003

Nucleotide sequences homologous to arpA encoding the A‐factor receptor protein (ArpA) of Streptomyces griseus are distributed in a wide variety of streptomycetes. Two genes, cprA and cprB, each encoding an ArpA‐like protein were found and cloned from Streptomyces coelicolor A3(2). CprA and CprB shared 90.7% identity in amino acid sequence and both showed about 35% identity to ArpA. Disruption of cprA by use of an M13 phage‐derived single‐stranded vector resulted in severe reduction of actinorhodin and undecylprodigiosin production. In addition, the timing of sporulation in the cprA disruptants was delayed by 1 day. The cprA gene thus appeared to act as a positive regulator or an accelerator for secondary metabolite formation and sporulation. Consistent with this idea, introduction of cprA on a low‐copy‐number plasmid into the parental strain led to overproduction of these secondary metabolites and accelerated the timing of sporulation. On the other hand, cprB disruption resulted in precocious and overproduction of actinorhodin. However, almost no effect on undecylprodigiosin was detected in the cprB disruptants. Sporulation of the cprB disruptant began 1 day earlier than the parental strain. The cprB gene thus behaved as a negative regulator on actinorhodin production and sporulation. Consistent with this, extra copies of cprB in the parental strain caused reduced production of actinorhodin and delay in sporulation. It is thus concluded that both cprA and cprB play regulatory roles in both secondary metabolism and morphogenesis in S. coelicolor A3(2), just as the arpA/A‐factor system in Streptomyces griseus.
Intramolecular synergism in an engineered exo‐endo‐1,4‐β‐glucanase fusion protein
Authors: Riedel Kathrin , Bronnenmeier Karin
Published: March 2003

Exoglucanase CelY and endoglucanase CelZ from the cellulolytic thermophile Clostridium stercorarium act in synergism to hydrolyse cellulosic substrates. To increase the efficiency of the hydrolytic degradation process, an artificial multienzyme carrying both enzymatic activities on one polypeptide chain was constructed by gene fusion. A segment of CelZ, CelZΔBB′C (designated CelZC′), comprising the catalytic domain and the adjacent domain C′ homologous to the cellulose‐binding domain family IIIc, was fused to the C‐terminus of CelY, yielding the fusion protein CelY–CelZC′, designated CelYZ. The large fusion protein (170 kDa) could be isolated from a recombinant Escherichia coli strain in its intact form retaining the pronounced thermostability of the fusion partners. As a true multienzmye, CelYZ exhibited both exoglucanase and endoglucanase activities. The cellulolytic activity of the fusion protein was three‐ to fourfold higher than the sum of the individual activities. Dilution experiments showed that the enhanced cellulolytic activity of the multienzyme resulted from intramolecular synergism of the fusion partners. The product profiles and the kinetic constants of cellulose hydrolysis support a new mechanistic model proposed for explaining the co‐operativity of the two catalytic domains within the multienzmye.
The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica
Authors: Yuk Ming Huam , Harvill Eric T. , Miller Jeff F.
Published: March 2003

The BvgAS signal transduction system in Bordetella spp. mediates a transition between infectious (Bvg+) and non‐infectious (Bvg−) phases by sensing environmental conditions and regulating gene expression. Using differential display, arbitrary‐primed polymerase chain reaction (PCR), we identified a gene expressed in the Bvg+ phase of Bordetella bronchiseptica that shows a high degree of sequence similarity to a locus involved in providing energy for type III secretion in pathogenic Gram‐negative bacteria (yscN in Yersinia spp.). We determined that the expression of this homologue in B. bronchiseptica (designated bscN ) is regulated by bvg. Several open reading frames surrounding the bscN locus also show sequence similarity to loci encoding type III secretion apparatus components in other bacteria. An in‐frame deletion of bscN in B. bronchiseptica leads to decreased secretion of several proteins, decreased cytotoxicity towards cultured cell lines and a defect in causing tyrosine dephosphorylation of specific proteins in infected cells in vitro. The deletion strain also revealed that bscN‐mediated secretion is required for persistent colonization of the trachea in a rat infection model. Loci encoding type III secretion homologues were identified in four strains of B. pertussis and two strains of B. parapertussis. B. pertussis strain 18323 and an ovine isolate of B. parapertussis show significant transcription of the genes in vitro.
A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: implications for co‐ordination of chromosome segregation and cell division
Authors: Sharpe Michaela E. , Errington Jeffery
Published: March 2003

The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome. Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites. Soon after duplication, sister oriC/Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 μm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle. The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication. It could also explain how segregation can proceed accurately in the absence of cell division. The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.
In vivo relations between pAMβ1‐encoded type I topoisomerase and plasmid replication
Authors: Bidnenko V. , Ehrlich S. D. , Jannière L.
Published: March 2003

A number of large extrachromosomal elements encode prokaryotic type I topoisomerases of unknown functions. Here, we analysed the topoisomerase Topβ encoded by the Gram‐positive broad‐host‐range plasmid pAMβ1. We show that this enzyme possesses the DNA relaxation activity of type I topoisomerases. Interestingly, it is active only on plasmids that use DNA polymerase I to initiate replication, such as pAMβ1, and depends on the activity of this polymerase. This is the first example, to our knowledge, of prokaryotic type I topoisomerase that is specific for a given type of replicon. During pAMβ1 replication in Bacillus subtilis cells, Topβ promotes premature arrest of DNA polymerase I, ≈190 bp downstream of the replication initiation point. We propose that Topβ acts on the early replication intermediates of pAMβ1, which contain D‐loops formed by DNA polymerase I‐mediated strand displacement. The possible role of the resulting DNA Pol I arrest in plasmid replication is discussed.
The extracytoplasmic function sigma factors: role and regulation
Authors: Missiakas Dominique , Raina Satish
Published: March 2003

Alternative sigma factors provide a means of regulating gene expression in response to various extracellular changes. One such class of sigma factors appears to control a variety of functions, including expression of heat‐shock genes in Escherichia coli, biosynthesis of alginates and carotenoids in Pseudomonas aeruginosa and Myxococcus xanthus, respectively, iron uptake in E. coli and Pseudomonas spp., nickel and cobalt efflux in Alcaligenes europhus, plant pathogenicity in Pseudomonas syringae and synthesis of outer membrane proteins in Photobacterium sp. strain SS9. Most of these activities deal with extracytoplasmic functions, and such sigmas have been designated as ECF sigma factors. They have also been characterized in Mycobacteria as well as Gram‐positive bacteria such as Streptomyces coelicolor and Bacillus subtilus and the archaea Sulpholobus acidocaldarius. ECF factors belong to a subfamily of the sigma 70 class, based on their sequence conservation and function across bacterial species. The promoter consensus sequences recognized by the ECF factors are also highly conserved. In most of the cases, the activity of these factors is modulated by a cognate inner membrane protein that has been shown, both in E. coli and in P. aeruginosa, to act as an anti‐sigma activity. This inner membrane protein is presumed to serve as a sensor and signalling molecule, allowing an adaptive response to specific environmental change. Presumably, an on‐and‐off switch of the anti‐sigma activity leads to the release of the sigma factor and thereby to the co‐ordinate transcription of the specific regulon it governs.
The group A streptococcal dipeptide permease (Dpp) is involved in the uptake of essential amino acids and affects the expression of cysteine protease
Authors: Podbielski Andreas , Leonard Bettina A. B.
Published: March 2003

The majority of characterized bacterial dipeptide permeases (Dpp) are membrane‐associated complexes of five proteins belonging to the ABC‐transporter family. They have been found to be involved in the uptake of essential amino acids, haem production, chemotaxis and sporulation. A 5.8 kb genomic DNA fragment of the serotype M49 group A streptococcal (GAS) strain CS101 was sequenced and found to contain five putative GAS Dpp genes (dppA to dppE ). Deduced amino acid sequences exhibited 17–54% similarity to corresponding ABC‐transporter sequences. The operon organization of the five genes was confirmed by transcriptional analysis, and a shorter, more abundant, dppA‐only transcript was detected similar to that found in the GAS oligopeptide permease (Opp) system. Insertional inactivation was used to create serotype M2 and M49 strains that did not express the dppD and dppE ATPase genes or nearly the entire operon. In feeding experiments with di‐ to hexapeptides, the wild‐type strain grew with each peptide tested. The dpp mutants were unable to grow on dipeptides, whereas hexapeptides did not sustain the growth of opp mutants. Expression of the dpp operon was induced approximately fourfold in late exponential growth phase. In addition, a striking increase in the dppA to dppA–E ratio from 5:1 to more than 20:1 occurred during late exponential growth phase in complex medium. Growth in chemically defined medium (CDM) supplemented with various dipeptides specifically induced the expression of dpp and reduced both the dppA to dppA–E and oppA to oppA–F mRNA ratios. Expression of the virulence factor SpeB (major cysteine protease) was reduced eightfold in dpp mutants, whereas dpp expression was decreased about fourfold in a Mga virulence regulator mutant. Taken together, these data indicate a correlation between levels of intracellular essential amino acids and the regulation of virulence factor expression.
Apolar growth of Neurospora crassa leads to increased secretion of extracellular proteins
Authors: Lee In Hyung , Walline Rodney G. , Plamann Michael
Published: March 2003

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount of growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature‐sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive temperature. Incubation of the mcb mutant at restrictive temperature results in a three‐ to fivefold increase in the level of extracellular protein and a 20‐fold increase in carboxymethyl cellulase activity relative to a wild‐type strain. A mutation in the cr‐1 gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb;cr‐1 double mutant was reduced to that of the wild‐type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced after a shift of the mcb mutant to restrictive temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.
The ATP‐dependent Clp protease is essential for acclimation to UV‐B and low temperature in the cyanobacterium Synechococcus
Authors: Porankiewicz Joanna , Schelin Jenny , Clarke Adrian K.
Published: March 2003

ClpP is the proteolytic subunit of the ATP‐dependent Clp protease in eubacteria, mammals and plant chloroplasts. Cyanobacterial ClpP protein is encoded by a multigene family, producing up to four distinct isozymes. We have examined the importance of the first ClpP protein (ClpP1) isolated from the cyanobacterium Synechococcus sp. PCC 7942 for acclimation to ecologically relevant UV‐B and low‐temperature regimens. When the growth light of 50 μmol photons m−2 s−1 was supplemented with 0.5 W m−2 UV‐B for 8 h, the constitutive level of ClpP1 rose eightfold after an initial lag of 1 h. Wild‐type cells readily acclimated to this UV‐B level, recovering after the initial stress to almost the same growth rate as that before UV‐B exposure. Growth of a clpP1 null mutant (ΔclpP1), however, was severely inhibited by UV‐B, being eight times slower than the wild type after 8 h. In comparison, ClpP1 content increased 15‐fold in wild‐type cultures shifted from 37°C to 25°C for 24 h. Wild‐type cultures readily acclimated to 25°C after 24 h, whereas the ΔclpP1 strain did not and eventually lost viability with prolonged cold treatment. During acclimation to either UV‐B or cold, photosynthesis in the wild type was initially inhibited upon the shift but then recovered. Photosynthesis in ΔclpP1 cultures, however, was more severely inhibited by the stress treatment and failed to recover. Acclimation was also monitored by examining the exchange of photosystem II reaction centre D1 proteins that occurs in wild‐type Synechococcus during conditions of excitation stress. During both cold and UV‐B shifts, wild‐type cultures replaced the acclimative form of D1 (D1:1) with the alternative D1 form 2 (D1:2) within the first hours. Once acclimated to either 25°C or 0.5 W m−2 UV‐B, D1:2 was exchanged back for D1:1. In ΔclpP1 cultures, this second exchange between D1 forms did not occur, with D1:2 remaining the predominant D1 form. Our results demonstrate that the ATP‐dependent Clp protease is an essential component of the cold and UV‐B acclimation processes of Synechococcus.
Expression and transcriptional control of the Salmonella typhimurium lpf fimbrial operon by phase variation
Authors: Norris Tracy L. , Kingsley Robert A. , Bäumler Andreas J.
Published: March 2003

The lpf operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches. To investigate expression of this virulence factor, a transcriptional fusion between the lpf operon and the genes lacZYA of Escherichia coli was constructed and introduced into the S. typhimurium chromosome. The resulting strain yielded both Lac+ and Lac− colony phenotypes. Alternation between Lac+ (phase ON) and Lac− (phase OFF) phenotypes occurred by a heritable phase variation mechanism, as inoculation of broth cultures with bacteria picked from a Lac+ colony gave rise to a considerably higher proportion of Lac+ colonies than inoculation with bacteria picked from a Lac− colony. During growth in vitro, phase transition from ON to OFF and from OFF to ON occurred at rates of 6.8 × 10−3 and 2.4 × 10−4 events per cell per generation respectively. In a murine intestinal organ culture model, selection for the ON expression state occurred when attached bacteria were recovered from Peyer's patches, suggesting that Lac phase variation correlated with expression of lpf mediated adherence. Selection for either the ON or the OFF expression state of the lpf operon in vivo was studied in mice immunized with either GST or GST–LpfA fusion protein. A strong selection against phase ON cells occurred only in animals immunized with GST–LpfA. The ability of animals immunized with GST–LpfA to distinguish between phase ON and phase OFF bacteria provided evidence for the presence of LpfA fimbrial protein in phase ON cells and for its exposure to the immune system.
Site‐directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare
Authors: Marklund Britt‐Inger , Mahenthiralingam Eshwar , Stokes Richard W.
Published: March 2003

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare, like many other mycobacteria, produces two proteins with catalase activity: a heat‐stable catalase (KatE) and an inducible, heat‐labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site‐directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H2O2 and had lost peroxidase and heat‐sensitive catalase activity but retained heat‐stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H2O2 and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.
Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis
Authors: Chung Y. S. , Breidt F. , Dubnau D.
Published: March 2003

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N‐terminal sequence motifs typical of type 4 prepilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre‐ComGG differs from pre‐ComGC and pre‐ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide‐cross‐linked homodimer in vivo. ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.
Deletion analysis of MotA and MotB, components of the force‐generating unit in the flagellar motor of Salmonella
Authors: Muramoto Kazumasa , Macnab Robert M.
Published: March 2003

MotA and MotB are cytoplasmic membrane proteins that form the force‐generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow‐motile phenotype) or no rotation at all (non‐motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10‐amino‐acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C‐terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C‐terminus. We also found that deletions in the MotB cytoplasmic region at the N‐terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C‐terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N‐terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C‐terminus are also unimportant.
Transcription‐driven DNA replication of plasmid pAMβ1 in Bacillus subtilis
Authors: Bruand Claude , Ehrlich S. Dusko
Published: March 2003

pAMβ1 is a plasmid isolated from Enterococcus faecalis which replicates in Bacillus subtilis by a unidirectional theta mechanism. It has been shown previously that initiation of pAMβ1 replication requires a plasmid‐encoded protein (RepE) and a short origin and is carried out by the host DNA polymerase I. It is not known which primer is used by this polymerase for initiating replication. Here, we report that a transcription fork passing through the origin is a limiting factor for plasmid replication. Transcription that activates the origin is initiated at the repE promoter and is thus regulated by the plasmid copy‐number control system. Two lines of evidence suggest that the transcription generates the primer for the DNA polymerase I. First, the transcription must start upstream from the origin and progress in the direction of replication to be effective. Second, 3′ ends of RNA transcripts initiated upstream of the origin map within the origin, provided that the Rep protein and an intact origin are present. This is the first report for simultaneous requirement of a transcription fork, a replication protein and the DNA polymerase I in initiation of DNA replication.
Temperature sensing in bacterial gene regulation — what it all boils down to
Authors: Hurme Reini , Rhen Mikael
Published: March 2003

Many bacterial gene regulatory circuits are controlled by temperature. Temperature‐mediated regulation occurs at the level of transcription and translation. Supercoiling, changes in mRNA conformation and protein conformation are all implicated in thermosensing. Bacterial virulence functions are often temperature regulated and thus many an example of thermoregulation comes from pathogenic organisms. H‐NS is at the crossroads of regulation in many such systems. mRNA melting has also been shown to act as a thermosensing mechanism in various contexts. Proteins can also act as temperature sensors as exemplified by the gene regulator TlpA in Salmonella typhimurium.
An experimental chain of infection reveals that distinct Borrelia burgdorferi populations are selected in arthropod and mammalian hosts
Authors: Ryan Jeffrey R. , Levine Jay F. , Apperson Charles S. , Lubke Lori , Wirtz Robert A. , Spears Patricia A. , Orndorff Paul E.
Published: March 2003

The prokaryotic, spirochaetal microorganism Borrelia burgdorferi is the causative agent of Lyme disease, an arthropod‐borne disease of a variety of vertebrates and the most prevalent arthropod‐borne disease of humans in the United States. In order to understand better the normal life cycle of B. burgdorferi, an experimental chain of infection was devised that involved multiple sequential arthropod and mammalian passages. By examining populations of B. burgdorferi emerging from different points in this infectious chain, we demonstrate that selection of B. burgdorferi populations peculiar to arthropod or vertebrate hosts is a property of at least one of the two ecologically distinct strains we examined. Distinct B. burgdorferi populations were identified using an antigenic profile, defined by a set of monoclonal antibodies to eight B. burgdorferi antigens, and a plasmid profile, defined by the naturally occurring plasmids in the starting clonal populations. These two profiles constituted the phenotypical signature of the population. In the strain exhibiting selection in the different hosts, transition from one host to another produced a striking series of alternating phenotypical signatures down the chain of infection. At the molecular level, the alternating signatures were manifested as a reciprocal relationship between the expression of certain antigenic forms of outer surface protein (Osp) B and OspC. In the case of OspC, the antigenic changes could be correlated to the presence of one of two distinctly different alleles of the ospC gene in a full‐length and presumably transcriptionally active state. In the case of OspB, two alleles were again identified. However, their differences were minor and their relationship to OspB antigenic variation more complicated. In addition to the reciprocating changes in the antigenic profile, a reciprocating change in the size (probably the multimeric state) of a 9.0 kbp supercoiled plasmid was also noted. Selection of distinct populations in the tick may be responsible for the microorganism's ability to infect a wide range of vertebrate hosts efficiently, in that the tick might provide selective pressure for the elimination of the population selected in the previous host.
Bilin deletions and subunit stability in cyanobacterial light‐harvesting proteins
Authors: Toole Colleen M. , Plank Tracey L. , Grossman Arthur R. , Anderson Lamont K.
Published: March 2003

Light‐harvesting in cyanobacteria and red algae is a function of the biliproteins, which have covalently bound bilin chromophores. The biliproteins are assembled with linker proteins into the phycobilisome, a large complex that resides on the surface of the photosynthetic membranes. Early steps in the phycobilisome assembly pathway include the folding of biliprotein α‐ and β‐subunits, covalent modification of subunits by bilin attachment and formation of the primary assembly unit, the αβ heterodimer. The potential role of bilins in subunit structure and assembly is examined in this study by site mutagenesis of biliprotein genes. Phycocyanin subunits from Synechocystis sp. 6701 that were unable to bind chromophores at specific sites were generated by changing the codons for bilin‐binding cysteines to alanine residues. The altered genes were then expressed in a phycocyanin‐minus mutant of the transformable Synechocystis sp. strain 6803. Single and multiple chromophore deletions cause specific and reproducible variations in phycobilisome‐associated phycocyanin that do not correlate with transcript levels. Sedimentation equilibrium studies with purified proteins showed that bilin absence reduces the strength of αβ interaction in the heterodimer. These results suggest that phycocyanin instability in bilin‐deletion mutants is a consequence of diversion of unassembled α‐ and β‐subunits to a degradation pathway. Attachment of the central bilin, which is common to all biliprotein subunits, may facilitate αβ interaction by completing the final stage of subunit folding and stabilizing the contact domains of binding partners in the heterodimer.
Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo
Authors: Tatsuta Takashi , Tomoyasu Toshifumi , Bukau Bernd , Kitagawa Masanari , Mori Hirotada , Karata Kiyonobu , Ogura Teru
Published: March 2003

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ32, an alternative sigma factor for heat shock promoters. FtsH, a membrane‐bound AAA‐type metalloprotease, degrades σ32 and has a central role in the control of the σ32 level. The ftsH null mutant was isolated, and establishment of the ΔftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of σ32 (≈20‐fold of the wild type), leading to the impaired downregulation of the level of σ32. Surprisingly, however, ΔftsH cells express heat shock proteins only two‐ to threefold higher than wild‐type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ32 when it is accumulated. Overproduction of DnaK/J reduces the activity of σ32 in ΔftsH cells without any detectable changes in the level of σ32, indicating that the DnaK chaperone system is responsible for the activity control of σ32in vivo. In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ32.
Increased nuclear traffic chaos in hyphae of Aspergillus nidulans: molecular characterization of apsB and in vivo observation of nuclear behaviour
Authors: Suelmann Rüdiger , Sievers Nicole , Galetzka Danuta , Robertson Laura , Timberlake William E. , Fischer Reinhard
Published: March 2003

Filamentous fungi are model microorganisms for studying nuclear migration in eukaryotic cells. Two genes, apsA and apsB (= anucleate primary sterigmata), were identified in Aspergillus nidulans that affect nuclear distribution in hyphae and specifically block conidiophore development at the metula stage when mutant. Here we describe the cloning, sequencing and molecular analysis of apsB. The gene encodes a 121 kDa coiled‐coil, hydrophilic protein that was localized in the cytoplasm. No protein–protein interaction was detected between ApsB and ApsA, a membrane‐associated, previously identified protein. An apsB null mutant was characterized by video epifluorescence microscopy using strains that express green fluorescent protein (GFP) in nuclei. With this novel approach, we have discovered a new mutant phenotype and have found that nuclei display an increased chaotic movement in older hyphal compartments that results in clustering and an uneven distribution of these organelles. These results suggest a regulatory role of ApsB in nuclear migration.
Promoter element spacing controls basal expression and light inducibility of the cyanobacterial secA gene
Authors: Mazouni K. , Bulteau S. , Cassier‐Chauvat C. , Chauvat F.
Published: March 2003

The Synechocystis PCC6803 secA gene was found to be essential for cell viability and to be transcriptionally controlled by the redox state of the cells. The basic promoter (BP, −71 to +47 relative to the transcription start site) is controlled by three cis‐acting elements, which together mediate the fourfold light induction of BP activity. The positively acting element (PE1, −361 to −71) upstream of BP exerts a twofold stimulation of BP; the negative element (NE, +47 to +104) downstream of BP decreases BP strength about sixfold. The PE2 element (+104 to +175) lying in the coding sequence overcomes NE‐dependent downregulation of BP. BP harbours Escherichia coli
σ70‐like promoter elements −35 (5′‐TTGAat‐3′) and −10 (5′‐TAagAT‐3′). The −10 motif, which has the features of an ‘extended −10’ box, is absolutely essential to promoter activity. The −35 hexamer is critical to the enhancement of promoter strength above BP level and to light inducibility, both features involving regulatory elements flanking BP. Most interestingly, reducing the length of the 30 bp spacing between the −35 and −10 boxes down to 17 bp was found to increase promoter activity and to confer light inducibility to BP. This demonstrates that promoter element spacing controls basal expression and light inducibility of the secA gene.