bluuebunny/crossref_metadata_2025 huggingface dataset:
Type III secretion genes identify a putative virulence locus of Chlamydia
Authors: Hsia Ru‐ching , Pannekoek Yvonne , Ingerowski Eric , Bavoil Patrik M.
Published: November 2003

Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact‐dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence‐associated type III secretion loci. The existence of contact‐dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact‐dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.
PspF and IHF bind co‐operatively in the psp promoter‐regulatory region of Escherichia coli
Authors: Jovanovic Goran , Model Peter
Published: November 2003

PspF bound to the psp enhancer activates Eσ54 holoenzyme‐dependent transcription of the Escherichia coli phage‐shock protein (psp ) operon and autogenously represses its own σ70‐dependent transcription, thereby keeping its concentration at a low level. It has been demonstrated previously that integration host factor (IHF) bound to a DNA site located between the psp core promoter and the PspF binding sites stimulates psp expression. We show here that wild‐type IHF strongly retards DNA containing the psp promoter region. In vitro, PspF binding to the psp enhancer facilitates IHF binding, while IHF binding to the pspF–pspA‐E promoter‐regulatory region increases the efficacy of PspF binding to the upstream activating sequences (UASs). This is the first demonstration of co‐operative binding of an activator and IHF in a σ54‐dependent system. In the absence of IHF, in vivo autoregulation of pspF transcription is lifted and, consequently, PspF production is increased, indicating that IHF enhances PspF binding to the psp enhancer in vivo.
Copper‐dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium
Authors: Nielsen Allan K. , Gerdes Kenn , Murrell J. Colin
Published: November 2003

The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b convert methane to methanol using the enzyme, methane monooxygenase (MMO). These bacteria are able to express two distinct MMOs: a cytoplasmic or soluble form (sMMO) and a membrane‐bound or particulate form (pMMO). Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper–biomass ratios, whereas pMMO is expressed at high copper–biomass ratios. In both methanotrophs, transcription of the sMMO gene cluster is negatively regulated by copper ions. Data suggest that transcription of the M. trichosporium OB3b sMMO gene cluster is directed from a σ54‐like and a σ70‐like promoter. The pMMO (pmo ) genes of M. capsulatus (Bath) are transcribed into a polycistronic mRNA of 3.3 kb. The synthesis of this mRNA was activated by copper ions. Activation of pmo transcription by copper ions was concomitant with repression of sMMO gene transcription in both methanotrophs. This suggests that a common regulatory pathway may be involved in the transcriptional switch between sMMO and pMMO gene expression.
Signalling pathways leading to transcriptional regulation of genes involved in the activation of glycolysis in yeast
Authors: Gonçalves Paula M. , Griffioen Gerard , Paul Bebelman Jan , Planta Rudi J.
Published: November 2003

Addition of glucose to yeast cells growing on less preferred carbon sources triggers profound changes in the expression levels of several genes. This paper focuses on the signal transduction pathways leading to transcriptional activation of the glycolysis in Saccharomyces cerevisiae during the transition from respiratory to fermentative growth conditions. To this end, we studied the transcriptional regulation of glycolytic genes (PFK1, PYK1 and PDC ), one gluconeogenic gene (FBP1) and the two genes encoding the 6‐phosphofructo‐2‐kinase isoenzymes (PFK26 and PFK27 ) during this transition. The results of experiments using glycolysis mutants, different fermentable carbon sources and 2‐deoxyglucose indicate that proper transcriptional regulation of these genes is dependent on the ability to form glucose 6‐phosphate by any one of the three hexose kinases. In addition, we conclude that signalling via the Ras–adenylate cyclase pathway is not necessary for the proper transcriptional response of glycolytic and gluconeogenic genes to glucose, because the transcription of these genes is not significantly affected in mutants having either high or low activities of this pathway. In contrast, the transcriptional regulation of the PFK26 and PFK27 genes is significantly altered in several of the Ras–adenylate cyclase pathway mutants studied, indicating that protein kinase A plays an important role in the transcriptional regulation of these genes.
Analysis of cis‐acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene
Authors: Rollins Sean M. , Grundy Frank J. , Henkin Tina M.
Published: November 2003

The Bacillus subtilis tyrS gene belongs to the T box family of aminoacyl‐tRNA synthetase and amino acid biosynthesis genes, which are regulated by a common mechanism of transcriptional antitermination. Each gene is induced by specific amino acid limitation; the uncharged cognate tRNA is the effector inducing transcription of the full‐length message. The leader regions of the genes in this family share a number of conserved primary sequence and secondary structural elements, the functions of which are unknown. In this study, we examine these regions and report the effects of mutations in several of these elements. In addition, two alternative basepairings in the F box region were found to be necessary for tyrS antitermination.
IS911‐mediated intramolecular transposition is naturally temperature sensitive
Authors: Haren Laurence , Bétermier Mireille , Polard Patrice , Chandler Michael
Published: November 2003

It is shown here that the bacterial insertion sequence IS911 exhibits a temperature‐sensitive transposition phenotype. Previous results have demonstrated that elevated levels of the IS911 transposase OrfAB generate significant quantities of a figure‐eight form, created by cleavage and circularization of one of the transposon strands, and of an excised circular form, in which both transposon strands have been circularized. We show here that the level of both types of molecule observed in vivo was greatly reduced at 42°C compared with 37°C. On the other hand, reducing the temperature to 30°C resulted in a significant increase in production. Transposition activity at this temperature was sufficiently high to permit detection in vivo of an excised circular form of a defective single IS911 chromosomal copy when OrfAB is supplied in trans. A similar temperature–activity profile is observed for a cell‐free reaction that uses partially purified OrfAB and generates the figure‐eight form uniquely. Moreover, two point mutants of OrfAB were obtained, which render the reactions partially temperature resistant both in vivo and in vitro. These results suggest that some property of transposase itself is sensitive to elevated temperatures.
Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K‐12 depends on DNA binding site arrangement
Authors: Darwin Andrew J. , Tyson Kerry L. , Busby Stephen J. W. , Stewart Valley
Published: November 2003

The NarL and NarP proteins are homologous response regulators of Escherichia coli that control the expression of several operons in response to nitrate and nitrite. A consensus heptameric NarL DNA‐binding sequence has been identified, and previous observations suggest that the NarP protein has a similar sequence specificity. However, some operons are regulated by NarL alone, whereas others are controlled by both NarL and NarP. In this study, DNase I footprinting experiments with the fdnG, nirB and nrfA control regions revealed that NarP only binds to heptamer sequences organized as an inverted repeat with a 2 bp spacing (7–2–7 sites). The NarL protein also binds to these 7–2–7 sites but, unlike NarP, also recognizes heptamers in other arrangements. These results provide an explanation for the regulation of some operons by NarL alone and for the different effects of NarL and NarP at common target operons, such as fdnG and nrfA. To investigate this differential DNA binding further, derivatives of the nrfA control region were constructed in which the spacing of the 7–2–7 heptamers was increased (7–n–7 constructs). Increasing the spacing to four or more basepairs abolished NarP binding and significantly reduced NarL binding. The NarL protein also had a reduced binding affinity for heptamers adjacent to the 7–n–7 heptamer pair, suggesting a decrease in cooperative interactions. In conclusion, we propose that 7–2–7 sites are preferred by both NarL and NarP. NarL can also recognize other binding site arrangements, an ability that appears to be lacking in NarP.
Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p
Authors: Wemmie John A. , Moye‐Rowley W. Scott
Published: November 2003

Ycf1p is a member of the ATP‐binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single‐copy gene levels, co‐fractionates with the vacuolar membrane and that this co‐fractionation is independent of vps15, vps34 or end3 gene function. Several cystic fibrosis‐associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino‐terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild‐type protein. These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP‐binding cassette transporter proteins, such as the CFTR and MRP.
The lytic enzyme of the pneumococcal phage Dp‐1: a chimeric lysin of intergeneric origin
Authors: Sheehan Michelle M. , García José L. , López Rubens , García Pedro
Published: November 2003

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp‐1. The lytic enzyme of this phage (Pal), previously identified as an N‐acetyl‐muramoyl‐L‐alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram‐positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N‐terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5‐T that infects Lactococcus lactis, whereas the C‐terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C‐terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.
Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases
Authors: Dintilhac Agnès , Alloing Geneviève , Granadel Chantal , Claverys Jean‐Pierre
Published: November 2003

The adcCBA putative operon of Streptococcus pneumoniae, an important human pathogen, was identified in a search for transformation‐deficient mutants. It was found to exhibit homology to ATP‐binding cassette (ABC) transport operons encoding streptococcal adhesins such as FimA of Streptococcus parasanguis and PsaA of S. pneumoniae. The latter was recently shown to be essential for virulence as judged by intranasal or intraperitoneal challenge of mice. We suggested previously that AdcA, together with a set of 14 proteins, including PsaA and homologous adhesins, defines a new family of external solute‐binding proteins specific for metals. In this work, Northern analysis revealed the existence of two adcB–adcA specific transcripts originating within adcC or further upstream, consistent with the hypothesis that adc is an operon. Investigation of growth of adc and psaA mutants in synthetic medium revealed that the addition of Zn improved the growth rate of the former, whereas the latter exhibited an absolute requirement for added Mn. A psaA–adc double mutant turned out to be essentially non‐viable unless both metals were added in the appropriate ratio. Taken together, these results suggest a previously undocumented requirement of S. pneumoniae for Zn and Mn. The addition of Zn also restored near‐normal spontaneous transformation of adc mutant cells in standard transformation medium. Zn was found to be specifically required soon after contact of cells with the competence‐stimulating peptide, revealing an unsuspected need for Zn in transformation of S. pneumoniae. The removal of Mn from standard transformation medium also resulted in transformation deficiency of psaA mutant cells. Taken together, these results lead us to propose that Adc is an ABC‐type Zn permease, the first such protein complex identified in any organism, and that Psa is an ABC‐type Mn permease complex.
A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures
Authors: Graumann Peter , Wendrich Thomas M. , Weber Michael H.W. , Schröder Katja , Marahiel Mohamed A.
Published: November 2003

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two‐dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down‐regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B. subtilis. After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild‐type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co‐operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.
Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans
Authors: Suelmann Rüdiger , Sievers Nicole , Fischer Reinhard
Published: November 2003

Nuclear migration and nuclear positioning are fundamental processes in all eukaryotic cells. They are easily monitored during hyphal growth of filamentous fungi. We expressed the green fluorescent protein (GFP) as a fusion protein with the putative nuclear localization domain of the transcriptional activator stuA in nuclei of Aspergillus nidulans and visualized these organelles in living cells. Nuclear staining was observed in interphase nuclei but not during mitosis. Nuclear division, nuclear migration, septum formation and branching were analysed with time‐lapse video microscopy during hyphal extension. Hyphae elongated at 0.1–1.2 μm min−1 and nuclei moved with similar speeds towards the hyphal tip until they had reached a defined position. An individual regulation of nuclear mobility in a given hyphal compartment was observed. Some representative movies are available on the Internet (http://www.blackwell‐science.com/products/journals/molextra.htm). Nuclear positioning was also studied at the molecular level. The ApsA protein, which regulates nuclear migration, was localized at the cytoplasmic membrane in germlings and hyphae by immunofluorescence and GFP tagging. A model of nuclear migration, nuclear positioning and the role of ApsA is presented.
Characterization of an exported protease from Shiga toxin‐producing Escherichia coli
Authors: Djafari Soudabeh , Ebel Frank , Deibel Christina , Krämer Sylvia , Hudel Martina , Chakraborty Trinad
Published: November 2003

The gene for a novel, high molecular weight protein secreted by Shiga toxin‐producing Escherichia coli (STEC) has been cloned, sequenced and characterized with respect to its activity. This gene, designated pssA, is localized on the large plasmid that also harbours the STEC haemolysin operon. Sequencing of a region comprising 10 630 nt revealed that the sequences flanking the pssA gene are composed of several remnants of different insertion elements. The PssA protein is produced as a 142 kDa precursor molecule that, after N‐ and C‐terminal processing, is released into the culture supernatant as a mature polypeptide of approximately 104 kDa. The primary sequence of PssA is highly related to a family of autonomously transported putative virulence factors from different Gram‐negative pathogens, which includes the Tsh protein of an avian‐pathogenic E. coli strain, the SepA protein from Shigella flexneri and the EspC protein from enteropathogenic E. coli. A common motif present in all four proteins is reminiscent of the catalytic centre of certain serine proteases. PssA (protease secreted by STEC) indeed shows serine protease activity in a casein‐based assay and is moreover cytotoxic for Vero cells. This activity of PssA and probably of other proteins of the Tsh family may be of functional importance during infection of the mucosal cell layer by the bacterial pathogen.
Differential regulation of fasA and fasH expression of Escherichia coli 987P fimbriae by environmental cues
Authors: Edwards Robert A. , Schifferli Dieter M.
Published: November 2003

An early process in the pathogenesis of enteric bacteria is colonization of the intestinal epithelium leading to local multiplication, pathophysiological interactions with the host and further spreading. Attachment is typically mediated by bacterial fimbriae, which are selectively expressed during growth in the intestine. Here we report an analysis of the regulation of 987P fimbrial expression of enterotoxigenic Escherichia coli (ETEC). Expression of both fasH, the transcriptional activator of the 987P fimbrial genes, and fasA, the major fimbrial subunit, is regulated in response to a variety of environmental stimuli. We have found that expression of fasH is regulated in response to the carbon status of the growth medium by the cAMP–CRP complex. Moreover, fasH is regulated in response to both the nitrogen status of the growth medium and the external pH. Expression of fasA is activated by FasH, and is also selectively regulated in response to growth temperature by HNS. Regulation of fimbrial expression by carbon and/or nitrogen gradients is proposed to provide a mechanism that allows preferential colonization of different segments of the intestine by various enteropathogens, such as ETEC, enteropathogenic E. coli and Vibrio cholerae.
Nicking by transesterification: the reaction catalysed by a relaxase
Authors: Byrd Devon R. , Matson Steven W.
Published: November 2003

DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site‐ and strand‐specific manner by catalysing a transesterification. The product of the reaction is a nicked double‐stranded DNA molecule with a sequestered 3′‐OH and the relaxase covalently bound to the 5′ end of the cleaved strand via a phosphotyrosyl linkage. The relaxase‐catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long‐lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase‐catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.
Energy‐dependent changes in the gonococcal transferrin receptor
Authors: Cornelissen Cynthia N. , Anderson James E. , Sparling P. Frederick
Published: November 2003

The pathogenic Neisseria spp. are capable of iron utilization from host iron‐binding proteins including transferrin and lactoferrin. Transferrin iron utilization is an energy‐dependent, receptor‐mediated event in which two identified transferrin‐binding proteins participate. One of these proteins, TbpA, is homologous to the TonB‐dependent family of outer membrane receptors that are required for high‐affinity uptake of vitamin B12 and ferric siderophores. The ‘TonB box’ is a conserved domain near the amino‐terminus of these proteins that has been implicated in interaction with TonB. Interaction between a periplasmic domain of TonB and the TonB box allows energy transduction to occur from the cytoplasmic membrane to the energy‐dependent receptor in the outer membrane. We created a TonB box mutant of gonococcal TbpA and demonstrated that its binding and protease accessibility characteristics were indistinguishable from those of gonococcal Ton system mutants. The protease exposure of the second transferrin‐binding protein, TbpB, was affected by the energization of TbpA, consistent with an interaction between these proteins. TbpB expressed by the de‐energized mutants was readily accessible to protease, similar to TbpB expressed in the absence of TbpA. The de‐energized mutants exhibited a marked decrease in transferrin diffusion rate, suggesting that receptor energization was necessary for ligand release. We propose a model to explain the observed Ton‐dependent changes in the binding parameters and exposures of TbpA and TbpB.
SpsA, a novel pneumococcal surface protein with specific binding to secretory Immunoglobulin A and secretory component
Authors: Hammerschmidt Sven , Talay Susanne R. , Brandtzaeg Per , Chhatwal Gursharan S.
Published: November 2003

The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10−9 M. Free secretory component (SC) also binds to S. pneumoniae, whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA‐ or SC‐binding domain is located in the N‐terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA‐binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S. pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S. pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.
Analysis of the DNA sequence, gene expression, origin of replication and modular structure of the Lactococcus lactis lytic bacteriophage sk1
Authors: Chandry P. Scott , Moore Sean C. , Boyce John D. , Davidson Barrie E. , Hillier Alan J.
Published: November 2003

Bacteriophage sk1 is a small isometric‐headed lytic phage belonging to the 936 species. It infects Lactococcus lactis, a commonly used dairy starter organism. Nucleotide sequence data analysis indicated that the sk1 genome is 28 451 nucleotides long and contains 54 open reading frames (ORFs) of 30 or more codons, interspersed with three large intergenic regions. The nucleotide sequence of several of the sk1 ORFs demonstrated significant levels of identity to genes (many encoding proteins of unknown function) in other lactococcal phages of both small isometric‐headed and prolate‐headed morphotype. Based on this identity and predicted peptide structures, sk1 genes for the terminase, major structural protein and DNA polymerase have been putatively identified. Genes encoding holin and lysin were also identified, subcloned into an Escherichia coli expression vector, and their function demonstrated in vivo. The sk1 origin of replication was located by identifying sk1 DNA fragments able to support the maintenance in L. lactis of a plasmid lacking a functional Gram‐positive ori. The minimal fragment conferring replication origin function contained a number of direct repeats and 179 codons of ORF47. Although no similarity between phage sk1 and coliphage λ at the nucleotide or amino acid sequence level was observed, an alignment of the sk1 late region ORFs with the λ structural and packaging genes revealed a striking correspondence in both ORF length and isoelectric point of the ORF product. It is proposed that this correspondence is indicative of a strong conservation in gene order within these otherwise unrelated isometric‐headed phages that can be used to predict the functions of the sk1 gene products.
The hsd loci of Mycoplasma pulmonis: organization, rearrangements and expression of genes
Authors: Sitaraman Ramakrishnan , Dybvig Kevin
Published: November 2003

Two paralogous, site‐specific invertible loci, designated hsd1 and hsd2 (host specificity determinant), have been identified in the Mycoplasma pulmonis genome. They encode putative type I restriction and modification (R–M) systems with maximum sequence homology to the type IC family, which includes EcoR124II and EcoDXXI. Each locus encodes an endonuclease subunit (HsdR), a methylase subunit (HsdM) and two DNA specificity subunits (HsdS). The gene organization at each locus is such that hsdR and hsdM are flanked by two hsdS genes. Within each locus, one of the hsdS genes, hsdR and hsdM, is encoded in tandem by the same DNA strand, while the second hsdS gene is encoded by the complementary strand but without overlap with the other three hsd genes. The hsdR and hsdM sequences of one locus are almost identical to their counterparts in the other. The four hsdS genes (two per locus) are highly homologous at their 5′ ends and also share sequence similarities in the 3′ ends of their corresponding coding regions. Owing to the disposition of and sequence similarities among the hsdS genes, they form inverted repeats at each locus. Analysis by polymerase chain reaction (PCR) has shown that both loci behave as site‐specific DNA invertible elements with multiple inversion sites, termed ‘vipareetus’, occurring within the hsdS genes. The inversions lead to a reassortment of hsdS sequences, generating an array of recombinant genes that probably encode S subunits possessing alternative DNA‐binding specificities. Sequence information obtained from the analysis of hsd2 transcripts by 5′ RACE (rapid amplification of cDNA ends) indicates that inversion induces the transcription of alternative hsdS genes by the relocation of coding sequences downstream of a promoter and ribosome‐binding site (RBS) situated at one end of each locus.
Transcriptional control of the yeast acetyl‐CoA synthetase gene, ACS1, by the positive regulators CAT8 and ADR1 and the pleiotropic repressor UME6
Authors: Kratzer Steffen , Schüller Hans‐Joachim
Published: November 2003

The ACS1 gene, encoding one out of two acetyl‐CoA synthetase isoenzymes of Saccharomyces cerevisiae, is strictly regulated at the transcriptional level by the carbon source of the medium. While ACS1 is poorly expressed in the presence of a high glucose concentration, a several hundred‐fold derepression occurs with ethanol as the sole carbon source or under conditions of sugar limitation. The molecular mechanism responsible for the carbon source control of ACS1 turned out to be highly complex. A carbon source‐responsive element (CSRE), previously identified upstream of gluconeogenic structural genes, and a binding site of the alcohol dehydrogenase regulator, Adr1p, together mediate about 80% of the derepressed gene activity. Binding of Adr1p synthesized by Escherichia coli to the ACS1 control region was shown by an electrophoretic mobility shift assay. In addition to these activating elements, two URS1 motifs confer negative control on the ACS1 promoter. The URS1 element was found to be a constitutive repression site, which is most effective from a downstream position with respect to an upstream activation site (UAS). In a mutant lacking the URS1‐binding factor, Ume6p, ACS1 expression was partially glucose insensitive. Ume6p must counteract transcription factors that are constitutively active. Site‐directed mutagenesis of Abf1p binding sites in the ACS1 promoter significantly reduced gene expression in the ume6 mutant, grown under repressing conditions. Thus, a functional balance of the pleiotropic positive factor Abf1p and the negative factor Ume6p is in part responsible for glucose repression of ACS1. The combined influence of the regulated UAS elements, CSRE and Adr1p binding site, mediates a strong increase in ACS1 expression under derepressing conditions.
A temperature upshift induces initiation of replication at oriC on the Escherichia coli chromosome
Authors: Botello Emilia , Jiménez‐Sánchez Alfonso
Published: November 2003

A temperature upshift of 10 or more degrees in the growth temperature of a bacterial culture causes induction of extra rounds of chromosome replication. This heat‐induced replication (HIR) initiates at oriC, is transitory, requires RNase H1 and RecA proteins and requires neither RNA polymerase activity nor de novo protein synthesis. The number of origins activated by heat is growth rate and temperature differential dependent. An origin activation higher than 20% increases the DNA:mass ratio around twofold, and this value is kept constant for the subsequent generations of growth at 41°C. We have also shown that HIR is neither related to SDR nor induced by the heat shock response. We suggest that a thermodynamic alteration of oriC structure or of membrane fluidity could explain the observed HIR.
Molecular and functional analysis of genes required for expression of group IB K antigens in Escherichia coli : characterization of the his‐region containing gene clusters for multiple cell‐surface polysaccharides
Authors: Amor Paul A. , Whitfield Chris
Published: November 2003

Escherichia coli group I capsular K antigens are found in two forms on the cell surface. The KLPS form is linked to lipopolysaccharide lipid A core, whereas the high‐molecular‐weight capsular form is assembled independently of lipid A core. Subgroup IB K antigens are generally co‐expressed with either the O8 or O9 antigen and, under the appropriate conditions, with the exopolysaccharide, colanic acid. To examine the relationships between the genetic loci and the synthetic pathways for these various cell‐surface polymers, the gene cluster responsible for expression of a prototype group IB K antigen (serotype K40) was cloned and the flanking chromosomal regions characterized. Analysis of the six orfs within the cluster indicates features typical of Wzy (Rfc)‐dependent O antigens. Synthesis of group IB K antigens is initiated by WecA (Rfe), a UDP‐GlcNAc::undecaprenylphosphate GlcNAc‐1‐phosphate transferase, and the chain length of K40LPS is determined by the wzz gene product. The his‐region of the E. coli O8:K40 prototype is almost exclusively devoted to the expression of three different surface polysaccharides. The rfbK40 cluster is located adjacent to the cps (colanic acid synthesis) and rfbO8 (O8 antigen synthesis) loci in the gene order: his‐rfbO8/O9–wzz–ugd–gnd–rfbK40–galF–cps. Thus, rfbK40 is in the location occupied by other Wzy‐dependent rfb gene clusters, and rfbO8/O9 represents an additional locus.
Aspergillus fumigatus arp1 modulates conidial pigmentation and complement deposition
Authors: Tsai Huei‐Fung , Washburn Ronald G. , Chang Yun C. , Kwon‐Chung K. J.
Published: November 2003

Aspergillus fumigatus is an important pathogen causing invasive pulmonary aspergillosis in immunocompromised patients. The fungus propagates by conidia, which are the infectious structures inhaled by the human host. Opsonophagocytosis is thought to contribute to clearance of the inhaled conidia, a process that is facilitated by complement deposition on conidial surfaces. We now show that conidial colour mutants exhibit significant increases in C3 binding capacity compared with wild type. A reddish‐pink mutation that led to enhanced C3 binding was complemented by a cosmid clone. A 3.3 kb DNA fragment from the subsequently rescued cosmid was sufficient to restore the bluish‐green conidial pigment. The bluish‐green transformant exhibited a level of C3 binding similar to that of the parental strain. A gene, designated arp1, was responsible for the complementation. Comparison of the genomic and cDNA sequences of arp1 revealed that it has two introns and encodes a putative protein of 168 amino acids. Arp1 is very similar to scytalone dehydratase, an enzyme involved in 1,8‐dihydroxynaphthalene‐melanin synthesis in Colletotrichum lagenarium and Magnaporthe grisea. Northern hybridization analysis revealed that arp1 is developmentally regulated, being expressed during conidiation. Disruption of arp1 resulted in reddish‐pink conidia and increased C3 binding. Our studies suggest that arp1 modulates the bluish‐green pigmentation of conidia as well as complement deposition.
SOS induction in mycobacteria: analysis of the DNA‐binding activity of a LexA‐like repressor and its role in DNA damage induction of the recA gene from Mycobacterium smegmatis
Authors: Durbach Steven I. , Andersen Susan J. , Mizrahi Valerie
Published: November 2003

The protein encoded by the lexA gene from Mycobacterium leprae was overproduced in Escherichia coli. The recombinant protein bound to the promoter regions of the M. leprae lexA, M. leprae recA and M. smegmatis recA genes at sites with the sequences 5′‐GAACACATGTTT and 5′‐GAACAGGTGTTC, which belong to the ‘Cheo box’ family of binding sites recognized by the SOS repressor from Bacillus subtilis. Gel mobility shift assays were used to confirm that proteins with the same site specificity of DNA binding are also present in Mycobacterium tuberculosis and M. smegmatis. Complex formation was impaired by mutagenic disruption of the dyad symmetry of the M. smegmatis recA Cheo box. LexA binding was also inhibited by preincubation of the M. smegmatis and M. tuberculosis extracts with anti‐M. leprae LexA antibodies, suggesting that the mycobacterial LexA proteins are functionally conserved at the level of DNA binding. Finally, exposure of M. smegmatis to DNA‐damaging agents resulted in induction of the M. smegmatis recA promoter with concomitant loss of DNA binding of LexA to its Cheo box, confirming that this organism possesses the key regulatory elements of a functional SOS induction system.
Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA
Authors: Söderbom Fredrik , Binnie Uta , Masters Millicent , Wagner E. Gerhart H.
Published: November 2003

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate‐limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E‐generated 3′ cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3′ CopA stem‐loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.
Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein
Authors: Hendrixson David R. , De La Morena Maria L. , Stathopoulos Christos , St Geme III Joseph W.
Published: November 2003

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full‐length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N‐terminal portion of mature Hap (designated Haps). In the present study, we examined the mechanism of processing and secretion of Hap. Site‐directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N‐terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C‐terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hapβ) predicted formation of a β‐barrel with an N‐terminal transmembrane α‐helix followed by 14 transmembrane β‐strands. Additional analysis revealed that the final β‐strand contains an amino acid motif common to other β‐barrel outer membrane proteins. Upon deletion of this entire C‐terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Haps was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C‐terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Haps. These results provide insight into a growing family of Gram‐negative bacterial exoproteins that are secreted by an IgA1 protease‐like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of β‐barrel proteins to the bacterial outer membrane.
CedA is a novel Escherichia coli protein that activates the cell division inhibited by chromosomal DNA over‐replication
Authors: Katayama Tsutomu , Takata Makoto , Sekimizu Kazuhisa
Published: November 2003

We isolated and characterized a new gene related to the control of cell division regulation in Escherichia coli. At 30°C, the dnaAcos mutant causes over‐replication of the chromosome, and colony formation is inhibited. We found that, at this temperature, the dnaAcos cells form filaments; therefore, septum formation is inhibited. This inhibition was independent of SfiA, an inhibitor of the septum‐forming protein, FtsZ. To identify factors involved in this pathway of inhibition, we isolated seven multicopy suppressors for the cold‐sensitive phenotype of the dnaAcos mutant. One of these proved to be a previously unknown gene, which we named cedA. This gene encoded a 12 kDa protein and resided at 38.9 min on the E. coli genome map. A multicopy supply of the cedA gene to the dnaAcos cells did not repress over‐replication of the chromosome but did stimulate cell division of the host, the result being growth of cells with an abnormally elevated chromosomal copy number. Therefore, the expression level of the cedA gene seems to be important for inhibiting cell division of the dnaAcos mutant at 30°C. We propose that over‐replication of the chromosome activates a pathway for inhibiting cell division and that the cedA gene modulates this division control. In the dnaA+ background, cedA also seems to affect cell division.
FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli
Authors: Schneider Robert , Travers Andrew , Muskhelishvili Georgi
Published: November 2003

The Escherichia coli DNA‐binding protein FIS serves as a DNA architectural factor in two unrelated enzymatic reactions, the site‐specific inversion of DNA and transcriptional activation of stable RNA promoters. In both these processes, FIS facilitates the assembly and dynamic transitions of two structurally distinct nucleoprotein complexes. We have proposed previously that, in these systems, FIS stabilizes writhed DNA microloops by binding at multiple helically phased sites in DNA. However, FIS also binds and bends DNA at many non‐specific sites and, at its maximum levels in the early exponential phase, FIS could potentially occupy a considerable part of the E. coli chromosome. Here, we show that fis affects growth phase‐specific alterations in the supercoiling level of DNA. Expression of fis accelerates the accumulation of moderately supercoiled plasmids in stationary phase, which are stabilized by FIS after nutritional shift‐up. In accordance with such a function, FIS modulates the relaxing and supercoiling activities of topoisomerases in vitro in a way that keeps DNA in a moderately supercoiled state. Our results suggest that the primary role of FIS is to modulate chromosomal dynamics during bacterial growth.
Oscillin, an extracellular, Ca2+‐binding glycoprotein essential for the gliding motility of cyanobacteria
Authors: Hoiczyk Egbert , Baumeister Wolfgang
Published: November 2003

Electron microscopic studies have demonstrated that various gliding filamentous cyanobacteria have trichome surfaces with a common structural organization. They contain an S‐layer attached to the outer membrane and an array of parallel fibrils on top of the S‐layer. In all species studied, the helical arrangement of these fibrils corresponds to the sense of rotation of the organism during the gliding movement. We have investigated the surface fibrils of Phormidium uncinatum using electron microscopic, spectroscopic and biochemical techniques. The fibrils consist of a single rod‐shaped protein, which we refer to as oscillin. Oscillin is a 646 amino acid residue protein (Mr 65 807; pI 3.63) and appears to be glycosylated. Sequence analysis reveals a two‐domain structure: a 554 residue domain contains 46 repeats of a Ca2+‐binding motif; it is followed by a 92 residue C‐terminal domain, which might mediate its export. Filaments that do not express oscillin lose their ability to move. Homology studies suggest that similar proteins play comparable roles in other motile cyanobacteria. The structure of oscillin appears to favour a passive role in gliding.
Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel β‐lactam resistance mechanism
Authors: McGowan S. J. , Sebaihia M. , O'Leary S. , Hardie K. R. , Williams P. , Stewart G. S. A. B. , Bycroft B. W. , Salmond G. P. C.
Published: November 2003

Members of two genera of Gram‐negative bacteria, Serratia and Erwinia, produce a β‐lactam antibiotic, 1‐carbapen‐2‐em‐3‐carboxylic acid. We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora. These genes are organized as an operon, carA–H, and are controlled by a LuxR‐type transcriptional activator, encoded by the linked carR gene. We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes. We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule. Three of these, carABC, are absolutely required. In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the carF and carG genes. Both products of these overlapping and potentially translationally coupled genes have functional, N‐terminal signal peptides. Removal of these genes from the Erwinia chromosome results in a carbapenem‐sensitive phenotype. We assume that these novel β‐lactam resistance genes have evolved in concert with the biosynthetic genes to ensure ‘self‐resistance’ in the Erwinia carbapenem producer.