bluuebunny/crossref_metadata_2025 huggingface dataset:
Negative control of fae (K88) expression by the‘global’regulator Lrp is modulated by the‘local’regulator FaeA and affected by DNA methylation
Authors: Huisman Tako T. , de Graaf Frits K.
Published: October 2006

Expression of the K88 (fae) operon is negatively controlled by the co‐operative binding of Lrp and FaeA to the fae regulatory region and is dependent on the methylation status of three GATC sites present in this region. In this paper, we describe the binding of Lrp to a T‐rich DNA helix between GATC site I and site II. FaeA stabilized and modified the Lrp binding, thereby extending the Lrp footprint over GATC site I and site III. Methylation of GATC site I prevented the binding of Lrp/FaeA at this site and appeared to be essential for the cells, since mutation of this site into GTTC resulted in a lethal overproduction of K88 fimbriae. Methylation of GATC site II and site III reduced the stability of Lrp/FaeA binding. Moreover, methylation of GATC site III stimulated faeB promoter activity. The plasmid population in cells harbouring multiple copies of a K88 plasmid consisted of two differentially methylated forms. Form A plasmids with a methylated GATC site I and site III and a non‐methylated site II (+,‐,+) represented 20% of the population and were responsible for high‐level expression. Form B plasmids with a methylated GATC site I and a non‐methylated site II and site III (+,‐,‐) represented 80% of the population and were responsible for low‐level expression. Apparently, K88 fimbriae expression in vivo is balanced at its maximal possible level by modulation of the methylation status of GATC site III. The ratio (1:4) between these populations is stabilized by a constitutive synthesis of FaeA resulting from the presence of an IS1 insertion upstream of faeA. This IS1 insertion separates the faeA promoter from the FaeB‐binding sites, thereby neutralizing the control by FaeB activity on expression of FaeA. Instead, faeA transcription is stimulated by binding of FaeA to the faeA promoter region.
Bacillus licheniformis bacitracin‐resistance ABC transporter: relationship to mammalian multidrug resistance
Authors: Podlesek Zdravko , Comino Aleksandra , Herzog‐Velikonja Blagajana , Žgur‐Bertok Darja , Komel Radovan , Grabnar Miklavž
Published: October 2006

The nucleotide sequence of the Bacillus licheniformis bacitracin‐resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP‐binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro‐phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram‐negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide‐binding sites, BcrA protein and mammalian multidrug transporter or P‐glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide‐binding protein to determine the Importance of various regions in transport.
rpsL+: a dominant selectable marker for gene replacement in mycobacteria
Authors: Sander Peter , Meier Albrecht , Böttger Erik C.
Published: October 2006

Molecular genetic manipulations in mycobacteria would benefit from procedures which efficiently select for double‐crossover events by homologous recombination. Here we describe a vector‐host system for gene replacement in mycobacteria, the utility of which was investigated using functional inactivation of the pyrF gene in Mycobacterium smegmatis as a model. This system is based on the expression of the wild‐type rpsL gene coding for ribosomal protein S12 in a streptomycin‐resistant host. Owing to the absence of a mycobacterial origin the plasmids are unable to replicate autonomously in mycobacteria. The first selection for maintenance of cloned sequences is conferred by the kanamycin‐resistance gene. The second simultaneous selection by streptomycin is against maintenance of cloned sequences which contain the gene encoding the streptomycin‐sensitive allele of the rpsL gene. By placing the gene for positive selection and that used for negative selection within and outside the target gene of interest, respectively, gene replacement is obtained. A one‐step double selection procedure provides a means to distinguish strictly between gene replacement by double crossover versus homologous recombination by single crossover events. The system should have considerable potential for genera or species where single‐crossover events or even illegitimate recombination are the predominant recombination mechanisms. It should also be of wide use for the construction of mutants without a selectable phenotype.
Molecular dissection of PapD interaction with PapG reveals two chaperone‐binding sites
Authors: Xu Zheng , Jones C. Hal , Haslam David , Pinkner Jerome S. , Dodson Karen , Kihlberg Jan , Hultgren Scott J.
Published: October 2006

P pili are composite adhesive fibres that allow uropathogenic Escherichia coli to gain a foothold in the host by binding to receptors present on the uroepithalium via the adhesin PapG. The assembly of P pili requires a periplasmic chaperone, PapD, that has an immunoglobulin‐like three‐dimensional structure. PapD‐subunit complex formation involves a conserved anchoring mechanism in the chaperone cleft and a‘molecular zippering’to the extreme C‐terminus of pilus subunits. A chaperone‐binding assay was developed using fusions of the C‐terminus of PapG to maltose‐binding protein (MBP/G fusions) to investigate whether chaperone‐subunit complex formation requires additional interactions. PapD bound strongly to an MBP/G fusion containing the C‐terminal 140 amino acids of PapG (MBP/G175‐314) but only weakly to the MBP/G234‐314 fusion containing 81 C‐terminal residues, arguing that the region between residues 175‐234 contains additional information that is required for strong PapD‐PapG interactions. PapD was shown to interact with a PapG C‐terminal truncate containing residues 1‐198 but not a truncate containing residues 1‐145, suggesting the presence of a second, independent PapD interactive site. Four peptides overlapping the second site region were tested for binding to PapD in vitro to further delineate this motif. Only one of the peptides synthesized was recognized by PapD. The MBP/G fusion containing both binding sites formed a tight complex with PapD in vivo and inhibited pilus assembly by preventing chaperone‐subunit complex formation.
Treponema pallidum and the quest for outer membrane proteins
Authors: Radolf Justin D.
Published: October 2006

Treponema pallidum, the syphilis spirochaete, has a remarkable ability to evade the humoral and cellular responses it elicits in infected hosts. Although formerly attributed to the presence of an outer coat comprised of serum proteins and/or mucopolysaccharides, current evidence indicates that the immuno‐evasiveness of this bacterium is largely the result of its unusual molecular architecture. Based upon a combination of molecular, biochemical, and ultrastructural data, it is now believed that the T. pallidum outer membrane (OM) contains a paucity of poorly immunogenic transmembrane proteins (‘rare outer membrane proteins’) and that its highly immunogentc proteins are lipoproteins anchored predominantly to the periplasmic leaflet of the cytoplasmic membrane. The presence in the T. pallidum OM of a limited number of transmembrane proteins has profound implications for understanding syphilis pathogenesis as well as treponemal physiology. Two major strategies for molecular characterization of rare outer membrane proteins have evolved. The first involves the identification of candidate OM proteins as fusions with Escherichia coli alkaline phosphatase. The second involves the characterization of candidate OM proteins identified in outer membranes isolated from virulent T. pallidum. Criteria to define candidate OM proteins and for definitive identification of rare OM proteins are proposed as a guide for future studies.
The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli
Authors: Ramseier Tom M. , Bledig Stefan , Michotey Valerie , Feghali Rita , Jr Saier Milton H.
Published: October 2006

The Escherichia coli fructose repressor, FruR, is known to regulate expression of several genes concerned with carbon utilization. Using a previously derived consensus sequence for FruR binding, additional potential operators were identified and tested for FruR binding in DNA band migration retardation assays. Operators in the control regions of operons concerned with carbon metabolism bound FruR, while those in operons not concerned with carbon metabolism did not. In vivo assays with transcriptional lacZ fusions showed that FruR controls the expression of FruR operator‐containing genes encoding key enzymes of virtually every major pathway of carbon metabolism. Moreover, a fruR null mutation altered the rates of utilization of at least 36 carbon sources. In general, oxidation rates for glycolytic substances were enhanced while those for gluconeogenic substances were depressed. Alignment of FruR operators revealed that the consensus sequence for FruR binding is the same for operons that are activated and repressed by FruR and permitted formulation of a revised FruR‐binding consensus sequence. The reported observations indicate that FruR modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with oxidative and gluconeogenic carbon flow and by repression of those concerned with fermentative carbon flow.
The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence
Authors: Dai Zhihao , Sirard Jean‐Claude , Mock Michèle , Koehler Theresa M.
Published: October 2006

Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen). Expression of the toxin genes by B. anthracis is enhanced during growth under elevated levels of CO2. This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a trans‐activator of anthrax toxin synthesis. Here we show that transcription of atx A does not appear to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new efficient method for gene replacement in B. anthracis, we constructed an atx A‐null mutant in which the atx A‐coding sequence on pXO1 is replaced with an ωkm‐2 cassette. Transcription of all three toxin genes is decreased in the absence of atx A. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5′ ends mapping to P1 are decreased in the atx A‐null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the Pi site is sufficient for atx A‐mediated activation of this transcript. The cya and lef genes each have one apparent start site for transcription. Transcripts with 5′ ends mapping to these sites are not detected in the afxA‐null mutant. The atx A‐null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atx A‐null mutant‐infected mice. These data suggest that the atx A gene product also regulates toxin gene expression during infection.
Response of a yeast glycogen synthase gene to stress
Authors: Ni Hsiao‐Tzu , LaPorte David C.
Published: October 2006

In the yeast Saccharomyces cerevisiae, glycogen synthase is encoded by two genes: GSY1 and GSY2. The activity of the enzymes increases as cultures enter the stationary phase of growth. Using a GSy2::lacZ fusion gene, we have demonstrated that the increase in glycogen synthase activity resulted, at least in part, from an increase in the level of the protein rather than simply from a change in its phosphorylation state. Northern blot analysis showed a parallel increase in the level of the GSY2 mRNA, which is consistent with transcriptional activation of GSY2. Deletion analysis identified three regions upstream of GSY2 which are involved in GSy2 expression: regions A (‐390 to ‐347 relative to the start of translation), B (‐252 to ‐209) and C (‐209 to ‐167). Region A or C independently activated expression of GSY2. In contrast, region B alone yielded only modest expression. Expression of GSY2 is induced by growth to stationary phase, heat shock or nitrogen starvation. Response to these stressors is mediated by elements within regions A and C. These elements appear to be related to the stress‐response elements found in other stress‐responsive genes.
Role of the conserved C‐repeat region of the M protein of Streptococcus pyogenes
Authors: Perez‐Casal José , Okada Nobuhiko , Caparon Michael G. , Scott June R.
Published: October 2006

The surface‐located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C‐repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M‐protein‐mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C‐repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C‐repeat domain, a region of the M‐protein molecule distinct from the C‐repeat domain confers upon S. pyogenes its ability to resist phagocytosis.
Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora
Authors: Bugert Peter , Geider Klaus
Published: October 2006

A16 kb transcript of the ams region, which is essential for biosynthesis of amylovoran, the acidic exopolysaccharide of Erwinia amylovora, was detected by Northern hybridization analysis. The positive regulator RcsA enhanced transcription of the targe mRNA from the ams operon. The nucleotide sequence of this area revealed 12 open reading frames (ORFs), which are all transcribed in the same direction. Five ORFs corresponded to the previously mapped genes amsA to amsE, Sequence analysis of the insertion sites of several Tn5 mutations confirmed these data. Tn5 or site‐directed mutagenesis of the ORFs 477, 377, 144, and 743 reveated an amytovoran‐deficient phenotype, and the newly identified genes were named amsG, amsH, amsI, and amsF, respectivety. The predicted amino acid sequence of AmsG is highly homologous to gatactosyl‐1 ‐phosphate undecaprenyl‐phosphate transferases. AmsB and AmsD are similar to other glycosyl transferases, and AmsH may be related to BexD. A significant homology to mammalian phosphatases was observed for Amsl. AmsA shows characteristic motifs for membrane association and ATP binding. AmsF carries a secretory signal sequence in the N‐terminus and could be involved in periplasmic processing of the repeating units. Complementation experiments located a promoter region required for gene expression as far as 500 bp upstream of amsG. It is preceded by a typical transcriptional termination sequence. A mutation upstream of the terminator did not affect amylovoran synthesis. Partial nucleotide sequences further upstream of the ams region showed homology to genes mapped at 45min on the Escherichia coli chromosome. A termination sequence was also found downstream of the ams operon at a distance of 16 kb from the promoter. Between amsF and this terminator, three additional ORFs were detected.
Sequence‐specific DNA binding by NIT4, the pathway‐specific regulatory protein that mediates nitrate induction in Neurospora
Authors: Fu Y. H. , Feng B. , Evans S. , Marzluf G. A.
Published: October 2006

The expression of the structural genes nit‐3 and nit‐6, which encode the nitrate assimilatory enzymes nitrate reductase and nitrite reductase, respectively, is highly regulated by the global‐acting NIT2 regulatory protein. These structural genes are also controlled by nitrogen catabolite repression and by specific induction via nitrate. A pathway‐specific regulatory protein, NIT4, appears to mediate nitrate induction of nit‐3 and of nit‐6. The NIT4 protein, composed of 1090 amino acids, contains a putative GAL4‐like Cys‐6 zinc cluster DNA‐binding motif, which is joined by a short segment to a stretch of amino acids that appear to constitute a coiled‐coil dimerization domain. Chemical crosslinking studies demonstrated that a truncated form of NIT4 forms homodimers. Mobility‐shift and DNA‐footprinting experiments have identified two NIT4‐binding sites of significantly different strengths in the promoter region of the nit‐3 gene. The stronger binding site contains a symmetrical octameric sequence, TCCGCGGA, whereas the weaker site has a related sequence. Sequences related to this palindromic element can be found upstream of the nit‐6 gene.
Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells
Authors: Rockey Daniel D. , Heinzen Robert A. , Hackstadt T.
Published: October 2006

Chlamydiae are obligate intracellular bacteria which occupy a non‐acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin‐killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post‐translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.
Structural identification of the iipo‐chitin oligosaccharide nodulation signals of Rhizobium loti
Authors: López‐Lara Isabel M. , van den Berg Jorrit D. J. , Thomas‐Oates Jane E. , Glushka John , Lugtenberg Ben J. J. , Spaink Herman P.
Published: October 2006

Rhizobium loti is a fast‐growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus. Nodulation studies show that Lotus plants are nodulated by R loti, but not by most other Rhizobium strains, indicating that R. loti produces specific lipo‐chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five different Rhizobium ioti strains have been purified and were shown to be N‐acetylglucosamine pentasaccharides of which the non‐reducing residue is N‐methylated and N‐acylated with c/s‐vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In one R. loti strain, NZP2037, an additional carbamoyl group is present on the non‐reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4‐O‐acetylfucose. Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia.
The bacteriophage T4 middle promoter PuvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription
Authors: March‐Amegadzie Roslyn , Hinton Deborah M.
Published: October 2006

Bacteriophage T4 middle promoters, which are transcribed using phage‐modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host σ70 consensus sequence at – 10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at – 30. While the T4 middle promoter Puvsx has these – 10 and –30 motifs, it also has matches to the MotA box at –35, –51, –70, and –87. We show that MotA binds to Puvsx DNA, footprinting a region that includes the MotA boxes at –30, –35, and –51. Very high levels of MotA are required for footprinting and gel‐shift experiments, and protein‐DNA complexes formed in the presence of both phage‐modified polymerase and MotA are more resistant to Hindlll cleavage than those formed with either protein alone. These results suggest that MotA‐DNA interactions may be stabilized by phage‐modified polymerase. Sequences between –18 and –38 are absolutely required for MotA activation of transcription, but sequences upstream of –38 are stimulatory, particularly when chloride instead of glutamate is the major anion. Our results dissect Puvsx into a core promoter, downstream of ‐38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions less favourable for protein‐DNA interactions.
Analysis of Vibrio cholierae ToxR function by construction of novel fusion proteins
Authors: Ottemann Karen M. , Mekalanos John J.
Published: October 2006

The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR‐PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), β‐lactamase (monomeric, ToxR–Bla), or the leucine zipper of GCN4 (dimeric, ToxR‐GCN4‐M) could substitute functionally for the ToxR periplasmic domain. ToxR‐GCN4 fusion proteins, in which the ToxR trans‐membrane domain was eliminated (ToxR‐GCN4‐C), were inactive, but an additional fusion protein that contained a heterologous membrane‐spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR‐GCN4‐M and ToxR‐Bla responded like wild‐type strains to in vitro growth conditions. In the infant‐mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild‐type ToxR, and strains containing monomeric ToxR‐Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.
Requirement for FlhA in flagella assembly and swarm‐cell differentiation by Proteus mirabilis
Authors: Gygi Daniel , Bailey Marc J. , Allison Clive , Hughes Colin
Published: October 2006

Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical rods into long, aseptate swarm cells that overexpress flagella and virulence factors, particularly haemolysin. A non‐swarming Tn5phoA mutant was unable to synthesize flagella, to fully elongate or to induce high levels of the toxin. The mutation lay within a 2091 bp gene encoding a homologue of the Escherichia coli FlhA belonging to a family of proteins that are required for assembly of flagella or virulence proteins and that are suggested to act either directly in membrane trans‐location and/or in regulating synthesis of the export apparatus. In trans expression of multicopy flhA restored cell elongation and migration and generated differentiation‐specific hyperexpression of flagellin and toxin genes to levels above those seen in the wild‐type strain. Transcription of flhA was strongly induced during differentiation, from its own putative σ28 promoter. The results suggest a mechanistic coupling of flagella assembly and swarm‐cell differentiation.
Complementation studies with the gas vesicle‐encoding p‐vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p‐gvpDE genes
Authors: Offner Sonja , Pfeifer Felicitas
Published: October 2006

Gas‐vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p‐vac region consisting of 14 different gvp genes that are arranged in two clusters: p‐gvpACNO and, oppositely oriented, p‐gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p‐gvpDE and p‐gvpF–M. The 5′‐terminus of the p‐gvpF–M mRMA was located 169 nucleotides upstream of p‐gvpF within p‐gvpE. The p‐gvpG and p‐gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas‐vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p‐gvp gene expression for gas‐vesicle synthesis was determined by transformation experiments using the Vac− species Haloferax volcanii as recipient. Construct ΔA containing all p‐gvp genes except for p‐gvpA, encoding the major gas‐vesicle structural protein, produced Vac− transformants, but the addition of p‐gvpA on a second vector restored gas‐vesicle synthesis to wild‐type level (Vac++). Similarly, double transformants containing p‐gvpD–M plus p‐gvpACNO, or p‐gvpG–M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p‐gvpFED–ACNO were Vac++. Transformants containing the p‐vac region either lacking gvpA, gvpF, or gvpGHI were Vac−, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas‐vesicle formation. Double transformants containing the constructs p‐gvpF–M plus p‐gvpACNO (ΔDE) accumulated gas vesicles (Vac+) but synthesized fewer than the wild type, showing that the p‐gvpDE genes are not necessary for gas‐vesicle assembly. A repressor function affecting the synthesis of the p‐gvpF–M mRNA could be suggested for p‐gvpD and the 5′‐ region of its mRNA.
dewA encodes a fungal hydrophobin component of the Aspergillus spore wall
Authors: Stringer Mary A. , Timberlake William E.
Published: October 2006

An anonymous cDNA clone, pCAN4, was shown previously to correspond to an mRNA that accumulates preferentially during asexual sporulation of the filamentous fungus Aspergillus nidulans. The peptide encoded by pCAN4 is a fungal hydrophobin, a group of small, hydrophobic cell wall proteins. When the CAN4 gene was disrupted, conidia and conidiophores appeared to be normal, but sporulating colonies wetted more rapidly with detergent solutions than did the wild type. We renamed CAN4 dewA for the detergent wettable phenotype and mapped it to chromosome V, 24 map units from cysC. The A. nidulans rodA gene also encodes a sporulation‐specific fungal hydrophobin. Spores of a dewA rodA double mutant were less hydrophobic than those of either mutant alone, showing that dewA and rodA contribute independently to spore‐wall hydrophobicity, Immuno‐localization of DewA by epitope tagging demonstrated that DewA is present in the spore wall, but not in the walls of germ tubes, hyphae or cells of the spore‐producing conidiophore. We conclude that dewA encodes a new fungal hydrophobin component of the conidial wall.
Novel colicin 10: assignment of four domains to TonB‐and TolC dependent uptake via the Tsx receptor and to pore formation
Authors: Pilsl Holger , Braun Volkmar
Published: October 2006

Uptake of a new colicin, colicin 10(Col10), into cells of Escherichia coli required TonB, ExbBD (Ton system), but its cognate receptor, Tsx, functioned independently of Ton and TolQRAB (Tol system). Uptake of Col10 also required TolC which is unique for a Ton‐coupled translocation through the outer membrane. A 2470bp DNA fragment from the natural plasmid pCol10 encoding the Col10 activity (cta), immunity (cti) and lysis (ctl) genes was sequenced. The Cta, Cti and Ctl proteins, as deduced from the nucleotide sequences, consisted of 490 (Mr 53 342), 96 (Mr 11586) and 43 (Mr 4484) amino acid residues, respectively. Col10 (Cta) was highly homologous to colicin E1 in two regions which determined the common TolC requirement for uptake and the pore‐forming activity. Col10 and E1 differed entirely in the regions which are predicted to determine the Ton dependence of Col10 and the Ton dependence of E1, and binding to the receptors Tsx and BtuB, respectively. The region responsible for the Ton‐dependent uptake of Col10 was localized in the sequence ranging from residues 1 to 43 (Ton region), and the region responsible for the Tol‐dependent uptake of colicin E1 extended from residues 1 to 34 (Tol region). Each Tol‐dependent colicin contained a pentapeptide homologous to the sequence DGSGS in the Tol region of E1 which is proposed to be implicated in Tol‐dependent uptake (TolA box). After the exchange of the Ton and the Tol regions between Col10 and E1, the Col10‐E1 fusion protein was carried into cells via the Ton system and BtuB, whereas the E1–Col10 fusion protein was imported via the Tol system and Tsx. Although the immunity proteins of Col10 and E1 displayed a low homology, Cti conferred full immunity to E1, in contrast to the immunity protein of E1 which did not protect cells against Col10. It is proposed that Col10 belongs to the colicin E1, Ia, Ib group as opposed to the colicin A, B, N group of pore‐forming colicins. Col10 consists of 4 domains of which two are very similar and two are very different to E1, supporting our previous proposal that colicins evolved by recombination of DNA fragments which encode uptake and activity domains.
Helicobacter pylori nickel‐transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions
Authors: Mobley Harry L. T. , Garner Rachel M. , Bauerfeind Peter
Published: October 2006

Urease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastritis and peptic ulceration. This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea. When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structural subunits are overexpressed, exogenous NiCl2 is added, and the host strain is grown in medium that does not chelate free Ni2+. As wild‐type H. pylori does not require such conditions for very high levels of urease expression, we reasoned that additional genes were required to accumulate the metal ion. To isolate such genes, E. coll SE5000 (pHP808), which carries the H. pylori urease gene cluster, was complemented with a λ ZAP‐derived plasmid library of the H. pylori chromosome. One of 1000 ampicillin‐resistant clones, plated onto urea segregation agar, produced detectable urease. Urease activity of this co‐transformant, grown in Luria broth containing 1 μM NiCl2, was 36μmol NH3min−1 mg−1 protein. Urease‐enhancing activity, which is not directly linked to the urease gene cluster, was localized by subcloning and nucleotide sequencing. The largest open reading frame, designated nixA, predicted a polypeptide of 34317 Da that displayed characteristics of an integral membrane protein. In vitro transcription‐translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size. An in‐frame Bal31 deletion within nixA abolished urease‐enhancing activity. At 50 nM NiCl2, E. coli containing the nixA clone transported 1250±460 pmol Ni2+min−110−8 cells, whereas the vector control transported only 140±85pmol Ni+2 min−1 108 cells, i.e. significantly less (P=0.01). We conclude that NixA confers upon E. coli a high‐affinity nickel‐transport system (KT=11.3±2.4nM; Vmax=1750±220 pmol Ni2+ min−110−8 cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.
UreR activates transcription at multiple promoters within the plasmid‐encoded urease locus of the Enterobacteriaceae
Authors: D'Orazio Sarah E. F. , Collins Carleen M.
Published: October 2006

Urease activity is produced by members of the family Enterobacteriaceae that contain the plasmid‐encoded urease locus only when urea is present in the growth medium. The plasmid‐encoded urease locus contains seven tandem urease structural and accessory genes (ureDABCEFG). Previously we showed that transcription of the first gene in this cluster. ureD, is initiated at a urea‐dependent promoter (ureD p). Expression from ureD p requires the product of ure R, which is transcribed divergently from the plasmid‐encoded ureDABCEFG. From DNA sequence analysis, UreR is predicted to be a 34kDa protein with identity to the AraC family of transcriptional activators. In this report we demonstrate that there are two additional urea and UreR‐dependent promoters within the plasmid‐encoded urease locus: ureR p and ureG p. A low‐level constitutive promoter was also identified upstream of ureE (ureE p). Three major mRNA transcripts were induced when urea was present in the growth medium: a transcript containing ureDABCEF, a transcript corresponding to ureG, and a transcript corresponding to ureR. These results indicate that expression of each of the plasmid‐encoded urease genes is transcriptionally regulated in response to urea and suggest that there is autogenous regulation of ureR. Therefore UreR is one of three AraC family members described thus far that are positively auto‐regulated.
Beta‐lactamases and bacterial resistance to antibiotics
Authors: Frère Jean‐Marie
Published: October 2006

The efficiency of β‐lactam antibiotics, which are among our most useful chemotherapeutic weapons, is continuously challenged by the emergence of resistant bacterial strains. This is most often due to the production of β‐lactamases by the resistant cells. These enzymes inactivate the antibiotics by hydrolysing the β‐lactam amide bond. The elucidation of the structures of some β‐lactamases by X‐ray crystallography has provided precious insights into their catalytic mechanisms and revealed unsuspected similarities with the DD‐transpeptidases, the bacterial enzymes which constitute the lethal targets of β‐lactams. Despite numerous kinetic, structural and site‐directed mutagenesis studies, we have not completely succeeded in explaining the diversity of the specificity profiles of β‐lactamases and their surprising catalytic power. The solutions to these problems represent the cornerstones on which better antibiotics can be designed, hopefully on a rational basis.
The role of anti‐sigma factors in gene regulation
Authors: Brown Kit L. , Hughes Kelly T.
Published: October 2006

Despite the isolation of an anti‐sigma factor over 20 years ago, it is only recently that the concept of an anti‐sigma factor emerged as a general mechanism of transcriptional regulation in prokaryotic systems. Anti‐sigma factors bind to sigma factors and inhibit their transcriptional activity. Studies on the mechanism of action of anti‐sigma factors has shed new light on the regulation of gene expression in bacteria, as the anti‐sigma factors add another layer to transcriptional control via negative regulation. Their cellular roles are as diverse as FlgM of Salmonella typhimurium, which can be exported to sense the structural state of the flagellar organelle, to SpollAB of Bacillus subtilis participating in the switch from one cell type to another during the process of sporulation. Additionally, the bacteriophage T4 uses an anti‐sigma factor to sabotage the Escherichia coli E·σ70 RNA polymerase in order to direct exclusive transcription of its own genes. Cross‐linking., co‐immuno‐precipitations, and co‐purification indicate that the anti‐sigma factors directly interact with their corresponding sigma factor to negatively regulate transcription. in B. subtilis, anti anti‐sigma factors regulate anti‐sigma factors by preventing an anti‐sigma factor from interacting with its cognate sigma factor, thereby allowing transcription to occur.
IHF‐ and RpoN‐dependent regulation c hydrogenase expression in Bradyrhizobium japonicum
Authors: Black Lori K. , Maier Robert J.
Published: October 2006

Sequence analysis of the Bradyrhizobium japonicum hydrogenase promoter regulatory region indicated the presence of a –24/–12 type promoter, which is recognized by RpoN, and a potential integration host factor (IHF)‐binding site. B. japonicum rpoN−/rpoN2 double mutants were deficient in hydrogen‐uptake activity. Using plasmid‐borne hup‐lacZ fusions, it was shown that the rpoN mutants were also deficient in nickel‐dependent transcriptional regulation of hydrogenase. Gel‐shift assays of the hydrogenase promoter regulatory region showed that purified IHF from Escherichia coli binds to a 210bp fragment. DNase footprint analysis revealed a protected region of 31 bp between bases –44 and –75 from the transcription start site. Western analysis with B. japonicum soluble extract and antibodies against E. coli IHF gave two bands equivalent to molecular masses of 12 and 14kDa approximately. When the IHF‐binding area is mutated on a plasmid‐borne hup‐lacZ fusion, nickel‐dependent transcriptional regulation of hydrogenase is still observed, but the transcriptional rates are clearly less than in the parent hup‐lacZ fusion plasmid. Like the results with nickel, regulation of hydrogenase by other transcriptional regulators (hydrogen and oxygen) still occurs, but at a diminished level in the IHF‐binding‐area‐mutated construct.
Organization of tcp, acf, and toxT genes within a ToxT‐dependent operon
Authors: Brown R. Clark , Taylor Ronald K.
Published: October 2006

The toxin coregulated pilus (TCP) is required for Vibrio cholerae to colonize the human intestine. The expression of the pilin gene, tcpA, is dependent upon ToxR and upon ToxT. The toxT gene was recently mapped within the TCP biogenesis gene cluster and shown to be capable of activating a tcpA::TnphoA fusion when cloned in Escherichia coli. In this study, we determined that ToxR/ToxT activation occurs at the level of tcpA transcription. ToxT expressed in E. coli could activate a tcp operon fusion, while ToxR, ToxR with ToxS, or a ToxR‐PhoA fusion failed to activate the tcp operon fusion and we could not demonstrate binding of a ToxR extract to the tcpA promoter region in DNA mobility‐shift assays. The start site for the regulated promoter was shown by primer extension to lie 75 bp upstream of the first codon of tcpA. An 800‐base tcpA message was identified, by Northern analysis, that correlates by size to the distance between the transcriptional start and a hairpin‐loop sequence between tcpA and tcpB. The more‐sensitive assay of RNase protection analysis demonstrated that a regulated transcript probably extends through the rest of the downstream tcp genes, including ToxT and the adjacent accessory colonization factor (acf) genes. An in‐frame tcpA deletion, but not a polar tcpA::TnphoA fusion, could be complemented for pilus surface expression by providing tcpA in trans. This evidence suggests that the tcp genes, including toxT, are organized in an operon directly activated by ToxT in a ToxR‐dependent manner. Most of the ToxT expression under induced conditions requires transcription of the tcpA promoter. Further investigation of how tcp::TnphoA insertions that are polar on ToxT expression retain regulation showed that a low basal level of toxT expression is present in toxR and tcp::TnphoA strains. Overall, these observations support the ToxR/ToxT cascade of regulation for tcp. Once induced, toxT expression becomes autoregulatory via the tcp promoter, linking tcp expression to that of additional colonization factors, exotoxin production, and genes of unknown function in cholera pathogenesis.
Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy
Authors: Lordanescu Serban
Published: October 2006

The replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC lo the PT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild‐type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram‐positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.
Characterization of the pilF—pilD pilus‐assembly locus of Neisseria gonorrhoeae
Authors: Freitag Nancy E. , Seifert H. Steven , Koomey Michael
Published: October 2006

Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre‐pilin peptidase, were unable to process the pre‐pilin subunit into pilin and thus were non‐piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S‐pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide‐binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.
Resistance to mecillinam produced by the co‐operative action of mutations affecting iipopolysaccharide, spoT, and cya or crp genes of Salmonella typhimurium
Authors: Antón Dora N.
Published: October 2006

Lipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium. However, when mutations of two of these types were combined in the same strain, high‐level resistance appeared, and increased even further when all three types of mutations were present. Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees. The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations. This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it. Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of SpoT, galE, and cya or crp mutations. All the strains produced spherical cells in medium with a low concentration (0.05 μg ml−1) of the antibiotic. These results suggest that the antibacterial action of mecillinam on S. typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT. The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K‐12 is probably due to the natural LPS defectiveness of this strain.
The chaperone‐like protein YerA of Yersinia pseudotuberculosis stabilizes YopE in the cytoplasm but is dispensible for targeting to the secretion loci
Authors: Frithz‐Lindsten Elisabet , Rosqvist Roland , Johansson Lenore , Forsberg Åke
Published: October 2006

The virulence plasmid‐encoded YopE cytotoxin of Yersinia pseudotuberculosis is secreted across the bacterial membranes and subsequently translocated into the eukaryotic cell. Translocation of YopE into target cells was recently shown to be polarized and only occurred at the zone of contact between the pathogen and the eukaryotic cell.

Immunogold electron microscopy on cryosectioned Y. pseudotuberculosis revealed that YopE is secreted and deposited on the bacterial cell surface when the bacteria are grown in Ca2+‐depleted media at 37°C. No YopE was detected in the cytoplasm or in the membranes. In yerA mutants which are downregulated for VopE at a post‐transcriptional level, the cytotoxin could only be detected in the cytoplasm. The overall recovery of YopE from the yerA mutant strain was, however, considerably lower than from the wild‐type strain. yerA had no major effect on the translation of YopE, but was found to stabilize YopE in the cytoplasm. YerA was shown to specifically interact with YopE in the cytoplasm in vivo and this binding also correlated with YopE secretion. Targeting of YopE to the secretion loci as well as translocation of YopE into HeLa cells occurred also in the absence of YerA. Based on our findings, we suggest that YerA by binding to YopE stabilizes and maintains the cytotoxin in a secretion‐competent conformation.