bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeny, taxonomy, genetics and global heritage ranks of an imperilled, freshwater snail genus Lithasia (Pleuroceridae)
Authors: Minton Russell L. , Lydeard Charles
Published: March 2003

Numerous aquatic species are threatened with extinction from habitat elimination or modification. One particularly imperilled group is the freshwater gastropod family Pleuroceridae. Pleurocerids reach their greatest diversity in the southeastern United States, and many species are currently considered extinct, endangered or threatened. One issue hindering efforts to implement conservation management plans for imperilled pleurocerid species is that the taxonomy is in an abysmal state. The taxonomy of pleurocerids is currently based on late 19th‐ and early 20th‐century studies, which used a typological or morphospecies concept. Most biologists today doubt the validity of many of the currently recognized species; however, this does not stop them from assigning conservation ranks in an attempt to determine which species are imperilled or currently stable. We conducted a phylogenetic analysis of the pleurocerid genus Lithasia using morphological and mitochondrial DNA sequence (mtDNA) data in an attempt to delimit species boundaries and test previous taxonomic schemes. We found that the current taxonomy of Lithasia does not reflect species diversity adequately within the genus, with two new undescribed species being discovered. The conservation status ranks of the new, undescribed species are imperilled and would have been overlooked had we relied on the conventional taxonomy. Additionally, the undescribed species’ conservation ranks that were previously apparently secure became vulnerable due to being inappropriately assigned as members of formerly widely distributed species instead of the imperilled status they warrant and vice versa, as some taxa that were considered imperilled are now thought to be modestly stable. Our study suggests that conservation ranks should be considered suspect at best in taxonomically poorly known groups until the taxa are reviewed using modern systematic methods.
Lack of genetic and plumage differentiation in the red‐billed quelea Quelea quelea across a migratory divide in southern Africa
Authors: Dallimer M. , Jones P. J. , Pemberton J. M. , Cheke R. A.
Published: March 2003

A migratory divide usually signals the presence of a geographical region over which other traits, such as morphology and genotypes, also undergo rapid change. A migratory divide has been hypothesized in central southern Africa for the abundant migratory weaver, the red‐billed quelea Quelea quelea. The positioning of the divide in the region is based on the patterns of rainfall in the region that stimulate the annual migrations of queleas. Evidence indicates that premigratory queleas near the divide show two distinct preferred directions for migration. We used eight polymorphic microsatellite loci and a range of plumage characters to determine whether there was population structure among red‐billed queleas in southern Africa, and specifically whether this structure coincided with the location of the migratory divide. There was no evidence of population genetic structure. An amova revealed no significant differences between samples taken either side of the migratory divide. Similarly, there was no geographical variation in plumage patterns across southern Africa. For both microsatellites and plumage characteristics, the variation that does exist occurs within each sampled site, with little differentiation between sites. We were therefore unable to find any evidence that either plumage or microsatellite genotypes varied in a similar way to migratory direction preference in red‐billed queleas in southern Africa. This is perhaps because the migratory divide does not act to separate individuals into populations within which genetic and plumage differentiation can be maintained.
Genetic analysis of zwittermicin A resistance in Escherichia coli: effects on membrane potential and RNA polymerase
Authors: Stabb Eric V. , Handelsman Jo
Published: March 2003

Zwittermicin A is a novel aminopolyol antibiotic that represents a new structural class of antibiotic and has diverse biological activities, including the suppression of plant disease and the ability to inhibit prokaryotic and eukaryotic cells. To enhance our fundamental understanding and applications of zwittermicin A, we elucidated mechanisms of zwittermicin A resistance in Escherichia coli. Two classes of zwittermicin A‐resistant mutants of E. coli were selected and characterized. One class included mutants altered in hemA, hemB, hemL, ubi, cydAB or atp, which were defective in generating a proton motive force (PMF) and resistant to aminoglycosides. The mutant analysis, coupled with physiological data, indicated an association between the electrical membrane potential (ΔΨ) component of PMF and zwittermicin A sensitivity. A second class of zwittermicin A‐resistant mutants was aminoglycoside sensitive and was affected in rpoB and rpoC, genes that encode subunits of RNA polymerase. The rpoB and rpoC mutants suggested that zwittermicin A might inhibit transcription, DNA replication, DNA gyrase or topoisomerase I; however, we found no further evidence to support any of these as the target for zwittermicin A. This study elucidated the genetic mechanisms of zwittermicin A resistance in E. coli. The results suggest that ΔΨ drives zwittermicin A uptake, and that, unlike other antibiotics for which resistance maps in rpoB or rpoC, zwittermicin A does not cause the rapid cessation of DNA or RNA synthesis, suggesting a unique mechanism of antibiosis.
Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc
Authors: Decker Katja , Plumbridge Jacqueline , Boos Winfried
Published: March 2003

The maltose regulon consists of 10 genes encoding a multicomponent and binding protein‐dependent ABC transporter for maltose and maltodextrins as well as enzymes necessary for the degradation of these sugars. MalT, the transcriptional activator of the system, is necessary for the transcription of all mal genes. MalK, the energy‐transducing subunit of the transport system, acts phenotypically as repressor, particularly when overproduced. We isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK. The affected gene was sequenced and identified as mlc, a known gene encoding a protein of unknown function with homology to the Escherichia coli NagC protein. The loss of Mlc function led to a threefold increase in malT expression, and the presence of mlc on a multicopy plasmid reduced malT expression. By DNaseI protection assay, we found that Mlc protected a DNA region comprising positions + 1 to + 23 of the malT transcriptional start point. Using a mlc–lacZ fusion in a mlc and mlc+ background, we found that Mlc represses its own expression. As Mlc also regulates another operon (manXYZ, see pages 369–379 of this issue), it may very well constitute a new global regulator of carbohydrate utilization.
Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria
Authors: Wyckoff Elizabeth E. , Duncan Donald , Torres Alfredo G. , Mills Melody , Maase Kamiel , Payne Shelley M.
Published: March 2003

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli. We found that many of these haem‐utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA. These shuA‐positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem‐utilizing pathogenic E. coli strains did not hybridize with shuA, so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae. The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA. The haem transport locus is not present in E. coli K‐12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K‐12. The junctions and flanking sequences in the shuA‐positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae, indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae, and it is located in the same relative position on the chromosome.
Functional and genetic analysis of regulatory regions of coliphage H‐19B: location of shiga‐like toxin and lysis genes suggest a role for phage functions in toxin release
Authors: Neely Melody N. , Friedman David I.
Published: March 2003

Analysis of the DNA sequence of a 17 kb region of the coli lambdoid phage H‐19B genome located the genes encoding shiga‐like toxin I (Stx‐I) downstream of the gene encoding the analogue of the phage λ Q transcription activator with its site of action, qut at the associated pR′ late promoter, and upstream of the analogues of λ genes encoding lysis functions. Functional studies, including measurement of the effect of H‐19B Q action on levels of Stx expressed from an H‐19B prophage, show that the H‐19B Q acts as a transcription activator with its associated pR′(qut) by promoting readthrough of transcription terminators. Another toxin‐producing phage, 933W, has the identical Q gene and pR′(qut) upstream of the stx‐II genes. The H‐19B Q also activates Stx‐II expression from a 933W prophage. An ORF in H‐19B corresponding to the holin lysis genes of other lambdoid phages differs by having only one instead of the usual two closely spaced translation initiation signals that are thought to contribute to the time of lysis. These observations suggest that stx‐I expression can be enhanced by transcription from pR′ as well as a model for toxin release through cell lysis mediated by action of phage‐encoded lysis functions. Functional studies show that open reading frames (ORFs) and sites in H‐19B that resemble components of the N transcription antitermination systems controlling early operons of other lambdoid phages similarly promote antitermination. However, this N‐like system differs significantly from those of other lambdoid phages.
tRNATrp as a key element of antitermination in the Lactococcus lactis trp operon
Authors: Van De Guchte Maarten , Ehrlich S. Dusko , Chopin Alain
Published: March 2003

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon–anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA‐mediated antitermination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.
sigA is an essential gene in Mycobacterium smegmatis
Authors: Gomez Manuel , Doukhan Laurence , Nair Gopalan , Smith Issar
Published: March 2003

sigA encodes a σ factor of the σ70 family, σA, that is found in all mycobacterial species. As σA shows high similarity to the primary σ factor in Streptomyces coelicolor, it was postulated that σA has the same role in mycobacteria. However, a point mutation in sigA, resulting in the replacement of arginine 522 by histidine, was found responsible for the attenuated virulence of the Mycobacterium bovis strain ATCC 35721. This raised the possibility that σA was an alternative σ factor specifically required for virulence gene expression. In this work, we show that sigA can not be disrupted in Mycobacterium smegmatis unless an extra copy of the gene is provided at another chromosomal site, which demonstrates that sigA is essential. To characterize the pattern of sigA expression during exponential and stationary phase in M. smegmatis, we measured the β‐galactosidase activity in a strain carrying a sigA–lacZ transcriptional fusion and monitored σA levels using Western blotting. Our results indicate that sigA is expressed throughout the growth of the culture. The essential character of sigA and its pattern of expression corroborate the hypothesis that sigA codes for the primary σ factor in M. smegmatis and, most likely, in all mycobacteria.
YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells: evidence of a structural domain necessary for translocation
Authors: Francis Matthew S. , Wolf‐Watz Hans
Published: March 2003

Yersinia pseudotuberculosis YopB and YopD proteins are essential for translocation of Yop effector proteins into the target cell cytosol. YopB is suggested to mediate pore formation in the target cell plasma membrane, allowing translocation of Yop effector proteins, although the function of YopD is unclear. To investigate the role in translocation for YopD, a mutant strain in Y. pseudotuberculosis was constructed containing an in frame deletion of essentially the entire yopD gene. As shown recently for the Y. pestis YopD protein, we found that the in vitro low calcium response controlling virulence gene expression was negatively regulated by YopD. This yopD null mutant (YPIII/pIB621) was also non‐cytotoxic towards HeLa cell monolayers, supporting the role for YopD in the translocation process. Although other constituents of the Yersinia translocase apparatus (YopB, YopK and YopN) are not translocated into the host cell cytosol, fractionation of infected HeLa cells allowed us to identify the cytosolic localization of YopD by the wild‐type strain (YPIII/pIB102), but not by strains defective in either YopD or YopB. YopD was also identified by immunofluorescence in the cytoplasm of HeLa cell monolayers infected with a multiple yop mutant strain (YPIII/pIB29MEKA). These results demonstrate a dual function for YopD in negative regulation of Yop production and Yop effector translocation, including the YopD protein itself. To investigate whether an amphipathic domain near the C‐terminus of YopD is involved in the translocation process, a mutant strain (YPIII/pIB155ΔD278–292) was constructed that is devoid of this region. Phenotypically, this small in frame ΔyopD278–292 deletion mutant was indistinguishable from the yopD null mutant. The truncated YopD protein and Yop effectors were not translocated into the cytosol of HeLa cell monolayers infected with this mutant. The comparable regulatory and translocation phenotypes displayed by the small in frame ΔyopD278–292 deletion and ΔyopD null mutants suggest that regulation of Yop synthesis and Yop translocation are intimately coupled. We present an intriguing scenario to the Yersinia infection process that highlights the need for polarized translocation of YopD to specifically establish translocation of Yop effectors. These observations are contrary to previous suggestions that members of the translocase apparatus were not translocated into the host cell cytosol.
Codon usages in different gene classes of the Escherichia coli genome
Authors: Karlin Samuel , Mrázek Jan , Campbell Allan M.
Published: March 2003

A new measure for assessing codon bias of one group of genes with respect to a second group of genes is introduced. In this formulation, codon bias correlations for Escherichia coli genes are evaluated for level of expression, for contrasts along genes, for genes in different 200 kb (or longer) contigs around the genome, for effects of gene size, for variation over different function classes, for codon bias in relation to possible lateral transfer and for dicodon bias for some gene classes. Among the function classes, codon biases of ribosomal proteins are the most deviant from the codon frequencies of the average E. coli gene. Other classes of ‘highly expressed genes’ (e.g. amino acyl tRNA synthetases, chaperonins, modification genes essential to translation activities) show less extreme codon biases. Consistently for genes with experimentally determined expression rates in the exponential growth phase, those of highest molar abundances are more deviant from the average gene codon frequencies and are more similar in codon frequencies to the average ribosomal protein gene. Independent of gene size, the codon biases in the 5′ third of genes deviate by more than a factor of two from those in the middle and 3′ thirds. In this context, there appear to be conflicting selection pressures imposed by the constraints of ribosomal binding, or more generally the early phase of protein synthesis (about the first 50 codons) may be more biased than the complete nascent polypeptide. In partitioning the E. coli genome into 10 equal lengths, pronounced differences in codon site 3 G+C frequencies accumulate. Genes near to oriC have 5% greater codon site 3 G+C frequencies than do genes from the ter region. This difference also is observed between small (100–300 codons) and large (>800 codons) genes. This result contrasts with that for eukaryotic genomes (including human, Caenorhabditis elegans and yeast) where long genes tend to have site 3 more AT rich than short genes. Many of the above results are special for E. coli genes and do not apply to genes of most bacterial genomes. A gene is defined as alien (possibly horizontally transferred) if its codon bias relative to the average gene exceeds a high threshold and the codon bias relative to ribosomal proteins is also appropriately high. These are identified, including four clusters (operons). The bulk of these genes have no known function.
Gyrase and Topo IV modulate chromosome domain size in vivo
Authors: Staczek Pawel , Higgins N. Patrick
Published: March 2003

In bacteria, DNA supercoil movement is restricted to subchromosomal regions or ‘domains.’ To elucidate the nature of domain boundaries, we analysed reaction kinetics for γδ site‐specific resolution in six chromosomal intervals ranging in size from 14 to 90 kb. In stationary cultures of Salmonella typhimurium, resolution kinetics were rapid for both short and long intervals, suggesting that random stationary barriers occur with a 30% probability at approximately 80 kb intervals along DNA. To test the biochemical nature of domain barriers, a genetic screen was used to look for mutants with small domains. Rare temperature‐sensitive alleles of DNA gyrase and Topo IV (the two essential type II topoisomerases) had more supercoil barriers than wild‐type strains in all growth states. The most severe gyrase mutants were found to have twice as many barriers in growing cells as wild type throughout a 90 kb interval of the chromosome. We propose that knots and tangles in duplex DNA restrain supercoil diffusion in living bacteria.
Clumping factor B (ClfB), a new surface‐located fibrinogen‐binding adhesin of Staphylococcus aureus
Authors: Ní Eidhin Déirdre , Perkins Samuel , Francois Patrice , Vaudaux Pierre , Höök Magnus , Foster Timothy J.
Published: March 2003

The surface‐located fibrinogen‐binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall‐anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen‐binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the α‐ and β‐chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the γ‐chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential‐phase cells in a solution of fibrinogen, (ii) adherence of exponential‐phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial‐related infections. However, in wild‐type exponential‐phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary‐phase cultures with fibrinogen. ClfB‐dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca2+ and Mn2+, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low‐affinity inhibitory cation binding site.
Identification of a co‐repressor binding site in catabolite control protein CcpA
Authors: Kraus Alexandra , Küster Elke , Wagner Andrea , Hoffmann Karin , Hillen Wolfgang
Published: March 2003

The catabolite control protein CcpA is the central regulator of carbon catabolite repression in Bacilli and other Gram‐positive bacteria. A comparison of 12 CcpA‐like sequences with regulators from the LacI/GalR family defines a CcpA subfamily based on extensive similarities found among CcpAs and not in 32 other members of the family. These amino acids are clustered in three blocks in the CcpA sequence. Their interpretation, assuming a PurR‐like fold, reveals that almost all of them are surface exposed and form a continuous patch on the N‐terminal subdomain of the protein core extending into the DNA reading head. We introduced nine single amino acid exchanges in the subfamily specific residues of CcpA from Bacillus megaterium. Six mutants, namely CcpA47RS, 79AE, 89YE, 295YR, 299YE and 303RD, are inactive or severely impaired in catabolite repression, underlining their relevance for CcpA function. They are negatively transdominant over wild‐type CcpA demonstrating their ability to correctly fold for dimerization. Five of them are unable or impaired in binding HPr‐Ser‐46‐P in vitro, establishing a correlation between catabolite repression efficiency and HPr‐Ser‐46‐P binding. These results support the hypothesis that the conserved region in CcpA is the HPr‐Ser‐46‐P binding site.
Involvement of the FtsH (HflB) protease in the activity of σ54 promoters
Authors: Carmona Manuel , De Lorenzo Víctor
Published: March 2003

The effect of FtsH, an essential inner membrane‐bound protease, in the regulation of the σ54‐dependent Pu promoter has been examined in vivo. Escherichia coli cells lacking FtsH failed to activate a Pu–lacZ fusion in response to the cognate enhancer‐binding protein XylR. However, the intracellular concentrations of XylR and σ54, as well as their apparent physical integrity were the same regardless of the presence or absence of the protease. The loss of Pu activity in FtsH‐minus cells was not due to the imbalance between sigma factors caused by the lack of the protease. ftsH mutants could not grow in media with glutamine as the only nitrogen source and failed also to induce the σ54 promoters PnifH by NifA and PpspA by PspF. These lesions were fully complemented by a ftsH + plasmid. Therefore, part of the pleiotropic phenotype of FtsH‐less cells corresponded to the lack of σ54 activity. Overproduction of σ54, however, restored both transcriptional activity of Pu and growth in glutamine of a ftsH strain. These observations suggested that the activity of σ54 is checked in vivo by an interplay of factors that ultimately determine the performance of cognate promoters under given physiological conditions.
Control of σ factor activity during Bacillus subtilis sporulation
Authors: Kroos Lee , Zhang Bin , Ichikawa Hiroshi , Yu Yuen‐Tsu Nicco
Published: March 2003

When starved, Bacillus subtilis undergoes asymmetric division to produce two cell types with different fates. The larger mother cell engulfs the smaller forespore, then nurtures it and, eventually, lyses to release a dormant, environmentally resistant spore. Driving these changes is a programme of transcriptional gene regulation. At the heart of the programme are σ factors, which become active at different times, some only in one cell type or the other, and each directing RNA polymerase to transcribe a different set of genes. The activity of each σ factor in the cascade is carefully regulated by multiple mechanisms. In some cases, novel proteins control both σ factor activity and morphogenesis, co‐ordinating the programme of gene expression with morphological change. These bifunctional proteins, as well as other proteins involved in σ factor activation, and even precursors of σ factors themselves, are targeted to critical locations, allowing the mother cell and forespore to communicate with each other and to co‐ordinate their programmes of gene expression. This signalling can result in proteolytic σ factor activation. Other mechanisms, such as an anti‐σ factor and, perhaps, proteolytic degradation, prevent σ factors from becoming active in the wrong cell type. Accessory transcription factors modulate RNA polymerase activity at specific promoters. Negative feedback loops limit σ factor production and facilitate the transition from one σ factor to the next. Together, the mechanisms controlling σ factor activity ensure that genes are expressed at the proper time and level in each cell type.
Salmonella SirA is a global regulator of genes mediating enteropathogenesis
Authors: Ahmer Brian M. M. , Van Reeuwijk Jeroen , Watson Patricia R. , Wallis Tim S. , Heffron Fred
Published: March 2003

SirA of Salmonella typhimurium is known to regulate the hilA and prgH genes within Salmonella pathogenicity island 1 (SPI1). To identify more members of the SirA regulon, we screened 10 000 random lacZY fusions (chromosomal MudJ insertions) for regulation by SirA and identified 10 positively regulated fusions. Three fusions were within the SPI1 genes hilA (an SPI1 transcriptional regulator), spaS (a component of the SPI1 type III export apparatus) and sipB (a substrate of the SPI1 export apparatus). Two fusions were within the sopB gene (also known as sigD). sopB is located within SPI5, but encodes a protein that is exported via the SPI1 export apparatus. In addition, five fusions were within genes of unknown function that are located in SPI4. As spaS and sipB were likely to be hilA dependent, we tested all of the fusions (except hilA) for hilA dependence. Surprisingly, we found that all of the fusions require hilA for expression and that plasmid‐encoded SirA cannot bypass this requirement. Therefore, SirA regulates hilA, the product of which regulates genes within SPI1, SPI4 and SPI5. Both sirA and hilA mutants are dramatically attenuated in a bovine model of gastroenteritis, but have little or no effect in the mouse model of typhoid fever. This study establishes the SirA/HilA regulatory cascade as the primary regulon controlling enteropathogenic virulence functions in S. typhimurium. Because S. typhimurium causes gastroenteritis in both cattle and humans, we believe that this information may be directly applicable to the human disease.
Characterization of the GlnK protein of Escherichia coli
Authors: Atkinson Mariette R. , Ninfa Alexander J.
Published: March 2003

The GlnK and PII signal transduction proteins are paralogues that play distinct roles in nitrogen regulation. Although cells lacking GlnK appear to have normal nitrogen regulation, in the absence of PII, the GlnK protein controls nitrogen assimilation by regulating the activities of the PII receptors glutamine synthetase adenylyltransferase (ATase) and the kinase/phosphatase nitrogen regulator II (NRII or NtrB), which controls transcription from nitrogen‐regulated promoters. Here, the wild‐type GlnK protein and two mutant forms of GlnK were purified, and their activities were compared with those of PII using purified components. GlnK and PII were observed to have unique properties. Both PII and GlnK were potent activators of the phosphatase activity of NRII, although PII was slightly more active. In contrast, PII was approximately 40‐fold more potent than GlnK in the activation of the adenylylation of glutamine synthetase by ATase. While both GlnK and PII were readily uridylylated by the uridylyltransferase activity of the signal‐transducing uridylyltransferase/uridylyl‐removing enzyme (UTase/UR), only PII∼UMP was effectively deuridylylated by the UR activity of the UTase/UR. Finally, there were subtle differences in the regulation of GlnK activity by the small molecule effector 2‐ketoglutarate compared with the regulation of PII activity by this effector. Altogether, these results suggest that GlnK is unlikely to play a significant role in the regulation of ATase in wild‐type cells, and that the main role of GlnK may be to contribute to the regulation of NRII and perhaps additional, unknown receptors in nitrogen‐starved cells. Also, the slow deuridylylation of GlnK∼UMP by the UTase/UR suggests that rapid interconversion of GlnK between uridylylated and unmodified forms is not necessary for GlnK function. One mutant form of GlnK, containing the alteration R47W, was observed to lack specifically the ability to activate the NRII phosphatase in vitro ; it was able to be uridylylated by the UTase/UR and to activate the adenylylation activity of ATase. Another mutant form of GlnK, containing the Y51N alteration at the site of uridylylation, was not uridylylated by the UTase/UR and was defective in the activation of both the NRII phosphatase activity and the ATase adenylylation activity.
In budding yeast, reactive oxygen species induce both RAS‐dependent and RAS‐independent cell cycle‐specific arrest
Authors: Wanke Valeria , Accorsi Katia , Porro Danilo , Esposito Franca , Russo Tommaso , Vanoni Marco
Published: March 2003

The role of mild oxidative stresses elicited by diethylmaleate (DEM)‐induced glutathione depletion in the progression of the yeast cell cycle has been investigated. We found that different wild‐type strains are sensitive to oxidative stresses induced by similar DEM doses: ≈ 1 mM on YPD plates, 5–10 mM in shaken flasks. At lower doses, DEM caused a transient decrease in growth rate, largely because of a decreased G1‐to‐S transition. Treatment with higher DEM doses leads to complete growth arrest, with most cells found in the unbudded G1 phase of the cell cycle. DEM treatment resulted in transcriptional induction of stress‐responsive element (STRE)‐controlled genes and was relieved by treatment with the antioxidant N‐acetyl cysteine. Reciprocal shift experiments with cdc25 and cdc28 mutants showed that the major cell cycle arrest point was located in the Start area, at or near the CDC25‐mediated step, before the step mediated by the CDC28 cyclin‐dependent kinase. The DEM‐induced G1 arrest requires a properly regulated RAS pathway and can be bypassed by overexpressing the G1‐specific cyclin CLN2. However, cells with either a deregulated RAS pathway or overexpressing CLN2 failed to grow and arrested as budded cells, indicating that a second DEM‐sensitive cell cycle step exists.
Mutational analysis of ComS: evidence for the interaction of ComS and MecA in the regulation of competence development in Bacillus subtilis
Authors: Ogura Mitsuo , Liu Li , LaCelle Michael , Nakano Michiko M. , Zuber Peter
Published: March 2003

The development of Bacillus subtilis genetic competence is a highly regulated adaptive response to stationary‐phase stress. A key step in competence development is the activation of the transcriptional regulator ComK, which is required for the expression of genes encoding the products that function in DNA uptake. In log‐phase cultures, ComK is trapped in a complex composed of MecA and ClpC, in which it is rendered inactive. The comS gene, contained within the srf operon, is induced in response to high culture cell density and nutritional stress. Its product functions to release active ComK from the complex, allowing ComK to stimulate the transcription initiation of its own gene as well as that of the late competence operons. Western analysis showed that ComS accumulates to maximal levels between T3 and T4, mirroring the pattern of competence cell development and late competence gene expression. Experiments to examine the target of ComS activity in vitro showed that ComS binds to MecA. This is further supported by coimmunoprecipitation using anti‐MecA antiserum. To clarify the role of ComS in competence regulation, a system for evaluating the effect of comS and mutant derivatives on the expression of comG, one of the late competence operons, was constructed. comS mutations, created by alanine‐scanning mutagenesis, that significantly reduced comG–lacZ expression were clustered within two regions, one at the N‐terminus and the other at the C‐terminus of ComS. ComSI13 → A and ComSW43 → A were selected for further analysis as representative mutants for both regions required for ComS activity. We observed that ComSI13 → A showed significantly reduced affinity for MecA, whereas ComSW43 → A showed near normal binding affinity for MecA. The results show that binding to MecA is critical for ComS function, but do not rule out the possibility that ComS possesses other activities.
The expression of the trpD, trpC and trpBA genes of Streptomyces coelicolor A3(2) is regulated by growth rate and growth phase but not by feedback repression
Authors: Hu D. S.‐J. , Hood D. W. , Heidstra R. , Hodgson D. A.
Published: March 2003

Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD‐coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end‐points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase‐dependent and growth rate‐dependent regulation is the first reported in streptomycete primary metabolism.
Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium
Authors: Piper Kevin R. , Beck von Bodman Susanne , Hwang Ingyu , Farrand Stephen K.
Published: March 2003

Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum‐sensing system. Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl‐homoserine lactone signal molecule Agrobacterium autoinducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR. Complementation analysis of mutations in the genes 5′ to traR showed that the other members of the arc operon are not required for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that AccR‐regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon. Reverse transcriptase–polymerase chain reaction (RT–PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine‐responsive transcriptional regulator.
DNA nicks inflicted by restriction endonucleases are repaired by a RecA‐ and RecB‐dependent pathway in Escherichia coli
Authors: Heitman Joseph , Ivanenko Tatyana , Kiss Antal
Published: March 2003

Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non‐cognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.
Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein
Authors: Saunders Neil F. W. , Houben Edith N. G. , Koefoed Sarah , De Weert Sandra , Reijnders Willem N. M. , Westerhoff Hans V. , De Boer Anthonius P. N. , Van Spanning Rob J. M.
Published: March 2003

The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane‐bound protein with six transmembrane helices, a large periplasmic domain and cysteine‐rich cytoplasmic domains that resemble the binding sites of [4Fe‐4S] clusters in many ferredoxin‐like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI‐deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N‐oxides. The NirX‐deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR‐like transcriptional activators. An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position −41.5 relative to the transcription start sites of both genes. Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad‐host‐range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI‐deficient mutant via homologous recombination in such a way that the wild‐type nirI gene was present directly upstream of the nir operon.
FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly
Authors: Minamino Tohru , González‐Pedrajo Bertha , Yamaguchi Kenta , Aizawa Shin‐Ichi , Macnab Robert M.
Published: March 2003

In wild‐type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at ≈ 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild‐type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook‐filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N‐terminal region of FliK, but not by C‐terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook‐length control and the switching of export substrate specificity.
Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spatial relationship to the core promoter
Authors: Martin Robert G. , Gillette William K. , Rhee Sangkhee , Rosner Judah L.
Published: March 2003

The promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely defined because of its degeneracy and asymmetry and seemingly variable location with respect to the −10 and −35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of promoters. This has yielded a more defined marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the −35 hexamer are oriented opposite those closer to the −35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP.
Dual control by regulatory gene fdsR of the fds operon encoding the NAD+‐linked formate dehydrogenase of Ralstonia eutropha
Authors: Oh Jeong‐Il , Bowien Botho
Published: March 2003

The transcriptional regulator gene fdsR was identified 150 bp upstream of the divergently oriented fdsGBACD operon encoding the soluble, NAD+‐linked formate dehydrogenase in the chemoautotrophic bacterium Ralstonia eutropha H16. Its deduced product, FdsR, displays a basal sequence similarity to the regulatory proteins of the LysR family. The carboxy‐terminal domain of FdsR contains a short region that is conserved in formate dehydrogenases. Deletion of fdsR revealed a dual regulatory effect of FdsR on the fds operon by acting as transcriptional activator in the presence of formate or as repressor in the absence of formate. Studies with fdsR transcriptional fusions also suggested a negative autoregulation of the gene. A promoter structure resembling σ70‐dependent promoters from Escherichia coli was identified upstream of the fdsR transcriptional start site. FdsR purified to homogeneity after overexpression of fdsR in E. coli is a 130 kDa homotetramer binding to the fds control region located between the fdsR and fdsG genes. Formate significantly increased the binding affinity of FdsR for this region. Two FdsR binding sites characterized by the inverted‐repeat structure ATANG‐N10‐CNTAT were identified. The regulatory pattern found in R. eutropha was also observed in the heterologous host E. coli and results from a novel mode of control of formate dehydrogenase genes.