bluuebunny/crossref_metadata_2025 huggingface dataset:
VirE1 is a specific molecular chaperone for the exported single‐stranded‐DNA‐binding protein VirE2 in Agrobacterium
Authors: Deng Wanyin , Chen Lishan , Peng Wen‐Tao , Liang Xiaoyou , Sekiguchi Susumu , Gordon Milton P. , Comai Luca , Nester Eugene W.
Published: March 2003

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T‐complex, from the bacterium to the plant cell. The T‐complex consists of a single‐stranded DNA (ssDNA) segment, the T‐DNA, and VirD2, an endonuclease covalently attached to the 5′ end of the T‐DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T‐complex and VirE2, a ssDNA‐binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two‐hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA‐binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self‐interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self‐interaction domain. Incubation of extracts from Escherichia coli overexpressing His‐VirE1 with the extracts of E. coli overexpressing His‐VirE2 increased the yield of His‐VirE2 in the soluble fraction. In a similar purified protein solubility assay, His‐VirE1 increased the amount of His‐VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co‐expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export‐competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.
Evolutionary relationships among photosynthetic prokaryotes (Heliobacterium chlorum, Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidum and proteobacteria): implications regarding the origin of photosynthesis
Authors: Gupta Radhey S. , Mukhtar Tariq , Singh Bhag
Published: March 2003

The presence of shared conserved insertions or deletions in proteins (referred to as signature sequences) provides a powerful means to deduce the evolutionary relationships among prokaryotic organisms. This approach was used in the present work to deduce the branching orders of various eubacterial taxa consisting of photosynthetic organisms. For this purpose, portions of the Hsp60 and Hsp70 genes, covering known signature sequence regions, were PCR‐amplified and sequenced from Heliobacterium chlorum, Chloroflexus aurantiacus and Chlorobium tepidum. This information was integrated with sequence data for several other proteins from numerous species to deduce the branching orders of different photosynthetic taxa. Based on signature sequences that are present in different proteins, it is possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low G+C Gram‐positive (H. chlorum ) → high G+C Gram‐positive → Deinococcus–Thermus → green non‐sulphur bacteria (Cf. aurantiacus ) → cyanobacteria → spirochaetes →Chlamydia–Cytophaga–Aquifex–flavobacteria–green sulphur bacteria (Cb. tepidum ) → proteobacteria (α, δ and ε) and → proteobacteria (β and γ). The members of the Heliobacteriaceae family that contain a Fe–S type of reaction centre (RC‐1) and represent the sole photosynthetic phylum from the Gram‐positive or monoderm group of prokaryotes are indicated to be the most ancestral of the photosynthetic lineages. Among the Gram‐negative bacteria or diderm prokaryotes, green non‐sulphur bacteria such as Cf. aurantiacus, which contains a pheophytin–quinone type of reaction centre (RC‐2), are indicated to have evolved very early. Thus, the organisms containing either RC‐1 or RC‐2 existed before the evolution of cyanobacteria, which contain both these reaction centres to carry out oxygenic photosynthesis. The eubacterial divisions consisting of green sulphur bacteria and proteobacteria are indicated to have diverged after cyanobacteria. Some implications of these results concerning the origin of photosynthesis and the earliest prokaryotic fossils are discussed.
Neurospora crassa ro‐10 and ro‐11 genes encode novel proteins required for nuclear distribution
Authors: Minke Peter F. , Lee In Hyung , Tinsley John H. , Bruno Kenneth S. , Plamann Michael
Published: March 2003

Movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule‐associated motor cytoplasmic dynein. We have isolated hundreds of Neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. Three of the ro genes, ro‐1, ro‐3 and ro‐4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. In this report, we describe the isolation and initial characterization of two additional ro genes, ro‐10 and ro‐11. ro‐10 and ro‐11 are non‐essential genes that encode novel 24 kDa and 75 kDa proteins respectively. Both ro‐10 and ro‐11 mutants retain the ability to generate long cytoplasmic microtubule tracks, suggesting that the nuclear distribution defect is not caused by a gross defect in the microtubule cytoskeleton. RO10, as well as RO4 (actin‐related protein ARP1, the most abundant subunit of dynactin), appears to be required for the stability of RO3 (p150Glued), the largest subunit of dynactin. We propose that ro‐10 mutants lack proper nuclear distribution, because RO10 is either a subunit of dynactin and required for dynactin activity or essential for assembly of the dynactin complex. ro‐11 mutations have no effect on RO1 or RO3 levels and have only a very slight effect on the localization pattern of cytoplasmic dynein and dynactin. The role of RO11 in the movement and distribution of nuclei in N. crassa hyphae remains unknown.
Cloning and allelic exchange mutagenesis of two flagellin genes of Helicobacter felis
Authors: Josenhans Christine , Ferrero Richard L. , Labigne Agnès , Suerbaum Sebastian
Published: March 2003

Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections. Attempts to manipulate H. felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium. In common with other Helicobacter spp., H. felis is a highly motile organism. To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin‐encoding genes, flaA and flaB, from H. felis. These genes encode two flagellin proteins that are expressed simultaneously under the control of putative σ28 and σ54 promoters respectively. Isogenic mutants of H. felis in flaA and flaB were generated by electroporation‐mediated allelic disruption and replacement, showing for the first time that H. felis could be manipulated genetically. Both types of H. felis flagellin mutants exhibited truncated flagella and were poorly motile. H. felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model.
Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases
Authors: Aleksenko Alexei , Liu Wenguang , Gojkovic Zoran , Nielsen Jens , Piskur Jure
Published: March 2003

The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme. Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans. The first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucleotide identity with the Ura2 gene in Saccharomyces cerevisiae. The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD. However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase‐encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae. The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrimidine starvation is noticeably enhanced in pyrE mutants that accumulate dihydroorotic acid. The pyrABCN gene maps to the distal portion of the right arm of the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brlA gene and located to the right of it. Our data on mitotic recombination should help to verify the genetic map of the chromosome VIII. Comparison of amino acid sequences of active dihydroorotases with related enzymes and with their non‐functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway. The ‘silent’ dihydroorotase domains of the multifunctional enzymes from fungi and active DHOase domains of the multifunctional enzymes in higher eukaryotes are more closely related to bacterial dehydroorotases.
AsgD, a new two‐component regulator required for A‐signalling and nutrient sensing during early development of Myxococcus xanthus
Authors: Cho Kyungyun , Zusman David R.
Published: March 2003

Myxococcus xanthus has a complex life cycle that includes fruiting body formation. One of the first stages in development has been called A‐signalling. The asg (A‐signalling) mutants have been proposed to be deficient in producing A‐signal, resulting in development arresting at an early stage. In this paper, we report the identification of a new asg locus asgD. This locus appears to be involved in both environmental sensing and intercellular signalling. Expression of asgD was undetected during vegetative growth, but increased dramatically within 1 h of starvation. The AsgD protein is predicted to contain 773 amino acids and to be part of a two‐component regulatory system because it has a receiver domain located at the N‐terminus and a histidine protein kinase at the C‐terminus. An asgD null mutant was defective in fruiting body formation and sporulation on CF medium. However, the defects of the mutant were complemented extracellularly when cells were mixed with wild‐type strains or with bsgA, csgA, dsgA or esgA mutants, but were not complemented extracellularly by asgA, asgB or asgC mutants. In addition, the mutant was rescued by a subset of A‐factor amino acids. Surprisingly, when the mutant was plated on stringent starvation medium rather than CF, cells were able to form fruiting bodies. Thus, it appears that AsgD is directly or indirectly involved in sensing nutritionally limiting conditions. The discovery of the asgD locus provides an important sensory transduction component of early development in M. xanthus.
PhoP–PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer‐membrane protein OprH and polymyxin B resistance
Authors: Macfarlane Emma L. A. , Kwasnicka Agnieszka , Ochs Martina M. , Hancock Robert E. W.
Published: March 2003

Rapid adaptation to environmental challenge is essential for the survival of many bacterial species, and is often effectively mediated by two‐component regulatory systems. Part of the adaptive response of Pseudomonas aeruginosa to Mg2+ starvation is overexpression of the outer‐membrane protein OprH and increased resistance to the polycationic antibiotic polymyxin B. Two overlapping open reading frames that encoded proteins with high similarities to the PhoP–PhoQ two‐component regulatory system of Salmonella typhimurium were identified downstream of the oprH gene. A P. aeruginosa PhoP‐null mutant, H851, was constructed by means of a phoP::xylE‐GmR transcriptional fusion, and shown to be deficient in OprH expression. In contrast, an analogous PhoQ‐null mutant, H854 (phoQ::xylE‐GmR), exhibited constitutive overexpression of OprH. Normal Mg2+‐regulated OprH expression could be restored in both mutants by complementation with a plasmid carrying the phoP and phoQ genes. Measurement of the catechol‐2,3‐dioxygenase activity, expressed from the xylE transcriptional fusion in strains H851 and H854, indicated that PhoP–PhoQ is involved in the regulation of phoP–phoQ as well as oprH. Reverse transcription polymerase chain reaction experiments and Northern blot analysis revealed linkage of oprH, phoP and phoQ into an operon that was demonstrated to be under the joint control of PhoP–PhoQ and Mg2+ ion concentration. In addition, studies of the polymyxin B resistance of the two mutant strains, H851 and H854, indicated that PhoP–PhoQ is involved in regulating P. aeruginosa polymyxin resistance in response to external Mg2+ concentrations.
The anti‐immunity system of phage‐plasmid N15: identification of the antirepressor gene and its control by a small processed RNA
Authors: Ravin Nikolai V. , Svarchevsky Alexander N. , Dehò Gianni
Published: March 2003

N15 is a temperate virus of Escherichia coli related to lambdoid phages. However, unlike all other known phages, the N15 prophage is maintained as a low copy number linear DNA molecule with covalently closed ends. The primary immunity system at the immB locus is structurally and functionally comparable to that of lambdoid phages, and encodes the immunity repressor CB. We have characterized a second locus, immA, in which clear plaque mutations were mapped, and found that it encodes an anti‐immunity system involved in the choice between the lytic and the lysogenic cycle. Three open reading frames at the immA locus encode an inhibitor of cell division (icd ), an antirepressor (antA) and a gene that may play an ancillary role in anti‐immunity (antB ). These genes may be transcribed from two promoters: the upstream promoter Pa is repressed by the immunity repressor CB, whereas the downstream promoter Pb is constitutive. Full repression of the anti‐immunity system is achieved by premature transcription termination elicited by a small RNA (CA RNA) produced by processing of the leader transcript of the anti‐immunity operon. The N15 anti‐immunity system is structurally and functionally similar to the anti‐immunity system of bacteriophage P1 and to the immunity system of satellite phage P4.
Cross‐talk between ammonium transporters in yeast and interference by the soybean SAT1 protein
Authors: Marini Anne‐Marie , Springael Jean‐Yves , Frommer Wolf B. , André Bruno
Published: March 2003

Ammonium uptake in the yeast Saccharomyces cerevisiae involves three membrane transporters (Mep1, ‐2 and ‐3) belonging to an evolutionarily conserved protein family that also includes the rhesus (Rh) blood group polypeptides of erythrocytes. We show here that, in the 26972c mutant defective in NH4+ transport, the Mep1 protein carrying an amino acid substitution in its cytoplasmic C‐terminus trans‐inhibits the closely related Mep3 protein. The same mutation introduced into Mep3 leads to loss of transport activity and this inactive form also trans‐inhibits native Mep3. Inhibition of Mep3 is post‐translational and can be overcome by overexpression. These results are consistent with a direct interaction between Mep proteins, as is the case for the Rh polypeptides. The soybean GmSAT1 gene, recently cloned for its ability to complement the NH4+ transport defect of strain 26972c, has been described as an NH4+ channel protein involved in the transfer of fixed nitrogen from the bacteroid to the host plant. We show here that GmSAT1 contains a sequence homologous to the DNA‐binding domain of basic helix–loop–helix (bHLH) transcription factors. We also show that GmSAT1 restores NH4+ uptake in the yeast mutant by interfering with the inhibition of Mep3. Our results are not consistent with a direct role of GmSAT1 in ammonium transport.
Genetic and biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for Penner serotype specificity
Authors: Karlyshev Andrey V. , Linton Dennis , Gregson Norman A. , Lastovica Albert J. , Wren Brendan W.
Published: March 2003

Campylobacter jejuni, a Gram‐negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post‐infection neurological complications of the Miller–Fisher and Guillain–Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one‐third of strains also producing high‐molecular‐weight LPS (referred to as O‐antigen). In this report, we demonstrate the presence of the high‐molecular‐weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O‐antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O‐antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.
Characterization of two novel regulatory genes affecting Salmonella invasion gene expression
Authors: Altier Craig , Suyemoto Mitsu , Ruiz Angela I. , Burnham Kara D. , Maurer Russell
Published: March 2003

A Salmonella typhimurium chromosomal deletion removing ≈19 kb of DNA at centisome 65 reduces invasion of cultured epithelial cells as well as the expression of lacZY operon fusions to several genes required for the invasive phenotype. As the deleted region contains no genes previously known to affect Salmonella invasion, we investigated the roles of individual genes in the deleted region using a combination of cloning, complementation and directed mutation. We find that the deletion includes two unrelated regulatory genes. One is the Salmonella homologue of Escherichia coli barA (airS ), which encodes a member of the multistep phosphorelay subgroup of two‐component sensor kinases. The action of BarA is coupled to that of SirA, a member of the phosphorylated response regulator family of proteins, and includes both HilA‐dependent and HilA‐independent components. The other regulatory gene removed by the deletion is the Salmonella homologue of E. coli csrB, which specifies a regulatory RNA implicated in controlling specific message turnover in E. coli. These results identify a protein that is likely to play a key role in the environmental control of Salmonella invasion gene expression, and they also suggest that transcriptional control of invasion genes could be subject to refinement at the level of message turnover.
Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa
Authors: Walsh Andrew G. , Matewish Mauricia J. , Burrows Lori L. , Monteiro Mario A. , Perry Malcolm B. , Lam Joseph S.
Published: March 2003

Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P. aeruginosa lipopolysaccharide. The first gene is a waaP homologue, whereas the other two (wapP and wapQ) are unique to P. aeruginosa. Repeated attempts using a variety of membrane‐stabilizing conditions to generate waaP::Gm (Gm, gentamicin) or wapP::Gm mutants were unsuccessful. We were able to generate a chromosomal waaP mutant that had a wild‐type copy of either waaPPa or waaPEcin trans, but were unable to cure this plasmid‐borne copy of the gene. These results are consistent with the fact that P. aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement of Hep‐linked phosphate for P. aeruginosa viability. A wapQ::Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer‐membrane permeability. Nuclear magnetic resonance and methylation linkage analysis showed that WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaPPa increased the outer‐membrane integrity of these complemented mutants, as evidenced by 35‐fold and 75‐fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutant transformed with both waaP and wapP had over 250‐fold and 1000‐fold increases, respectively, in these MICs. The inner core phosphates of P. aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.
Families of transmembrane sugar transport proteins
Authors: Saier Milton H.
Published: March 2003

We describe here 20 families of secondary (pmf‐driven) carriers which, in addition to nine families within the ATP‐dependent ABC superfamily, and seven families of Gram‐negative bacterial outer membrane porins, largely account for the stereospecific transport of sugars and their derivatives into and out of all living cells on earth. Family characteristics as well as struc‐tural and functional properties of the family constituents are described. By reference to our website (http://www‐biology.ucsd.edu/~msaier/transport/), phylogenetic relationships, detailed substrate specificity information and both primary and secondary references are also available. This review provides a comprehensive guide to the diversity of carriers that mediate the transport of sugar‐containing molecules across cell and organellar membranes.
Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system
Authors: Lacks Sanford A. , Ayalew Sahlu , De La Campa Adela G. , Greenberg Bill
Published: March 2003

The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double‐stranded and the other on single‐stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH‐containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.
cis‐Acting regulatory sequences for antitermination in the transcript of the Bacillus subtilis hut operon and histidine‐dependent binding of HutP to the transcript containing the regulatory sequences
Authors: Oda Masanao , Kobayashi Nobuhiro , Ito Akiko , Kurusu Yasurou , Taira Kazunari
Published: March 2003

The location of the cis‐acting regulatory region for histidine‐dependent antitermination of the Bacillus subtilis hut operon was determined. A secondary structure, whose sequences partially overlap with the downstream terminator, was found in the regulatory region of the hut transcript. Mutational analysis of the regulatory region showed that the secondary structure was required for histidine‐dependent antitermination. An electrophoretic mobility‐shift assay demonstrated that, in response to the presence of histidine and Mg2+, purified HutP bound hut RNA bearing putative secondary structure but not RNA lacking the potential to form putative secondary structure. Native gel electrophoresis showed that HutP existed as a hexamer. A filter‐binding assay revealed that the concentration of histidine required for half‐maximal binding of HutP to RNA was 3.1 mM and that the Kd for binding of HutP to RNA was ≈ 0.56 µM in the presence of histidine. These results suggested that putative secondary structure in the regulatory region of hut mRNA could function as an antiterminator to inhibit the formation of the terminator structure and that HutP causes expression of the hut structural genes by binding to the putative antiterminator structure in response to the presence of histidine.
The carbon metabolism‐controlled Synechocystis gap2 gene harbours a conserved enhancer element and a Gram‐positive‐like −16 promoter box retained in some chloroplast genes
Authors: Figge R. M. , Cassier‐Chauvat C. , Chauvat F. , Cerff R.
Published: March 2003

The two glyceraldehyde‐3‐phosphate dehydrogenase‐encoding genes (gap) of Synechocystis were shown to be expressed as monocistronic transcripts. Whereas gap1 expression is slow and weak, gap2 gene induction is rapid and strong. Transcription of the gap2 gene was shown to depend on functional photosynthetic electron transport and on active carbon metabolism. The basal promoter of gap2 (P, −45 to +34, relative to the transcription start site) is controlled by three cis‐acting elements designated A (−443 to −45), B (+34 to +50, in the untranslated leader region) and C (+50 to +167, in the coding region) that, together, promote a 100‐fold stimulation of P activity. Element B was found to behave as a transcriptional enhancer, in that it was active regardless of its position, orientation and distance relative to P. All three cis‐acting stimulatory elements exhibit a common 5′‐agaTYAACg‐3′ nucleotide motif that appears to be conserved in cyanobacteria and may be the target for a transcriptional enhancer. We also report that gap2 transcription depends on a Gram‐positive‐like −16 promoter box (5′‐TRTG‐3′) that was obviously conserved throughout the evolution of chloroplasts. This is the first report on the occurrence of a −16 promoter element in photoautotrophic organisms.
Genesis of BRO β‐lactamase‐producing Moraxella catarrhalis: evidence for transformation‐mediated horizontal transfer
Authors: Bootsma Hester J. , Van Dijk Hans , Vauterin Paul , Verhoef Jan , Mooi Frits R.
Published: March 2003

The dramatic rise in BRO‐producing M. catarrhalis strains observed in the last decades is without precedence. The aim of this study was to elucidate the events that led to the emergence of BRO‐1 and BRO‐2 β‐lactamases. Previously, we showed bro1 and bro2 to be >99% identical. Data presented here suggested that bro2 was acquired by a fortuitous event and inserted between M. catarrhalis genes orf1 and orf3. Subsequently, bro1 evolved from bro2. Promoter‐up mutations increased fitness of bro2, explaining its present predominance. The highly conserved nature of bro compared with orf1 and orf3 suggested that acquisition has occurred relatively recently. The random distribution of bro among M. catarrhalis fingerprint types indicated that bro has spread by horizontal transfer. Sequence analysis revealed that 80–200 bp is generally cotransferred with bro, serving as regions of homology that target bro to the same chromosomal locus. A region of 160 bases upstream of bro1 lacked polymorphism, indicating it was derived from the original strain that acquired bro2. We observed that bro was readily transferred by transformation between M. catarrhalis strains in vitro, suggesting a mechanism by which bro has disseminated. In conclusion, we have been able to reconstruct the steps that led to the emergence of BRO‐producing M. catarrhalis.
Sequence and expression of a halobacterial β‐galactosidase gene
Authors: Holmes Melissa L. , Dyall‐Smith Michael L.
Published: March 2003

Studies of gene expression in haloarchaea have been greatly hindered by the lack of a convenient reporter gene. In a previous study, a β‐galactosidase from Haloferax alicantei was purified and several peptide sequences determined. The peptide sequences have now been used to clone the entire β‐galactosidase gene (designated bgaH) along with some flanking chromosomal DNA. The deduced amino acid sequence of BgaH was 665 amino acids (74 kDa) and showed greatest amino acid similarity to members of glycosyl hydrolase family 42 [classification of Henrissat, B., and Bairoch, A. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293: 781–788]. Within this family, BgaH was most similar (42–43% aa identity) to enzymes from extremely thermophilic bacteria such as Thermotoga and Thermus. Family 42 enzymes are only distantly related to the Sulfolobus LacS and Escherichia coli LacZ enzymes (families one and two respectively). Three open reading frames (ORFs) upstream of bgaH were readily identified by database searches as glucose–fructose oxidoreductase, 2‐dehydro‐3‐deoxyphosphogluconate aldolase and 2‐keto‐3‐deoxygluconate kinase, enzymes that are also involved in carbohydrate metabolism. Downstream of bgaH there was an ORF which contained a putative fibronectin III motif. The bgaH gene was engineered into a halobacterial plasmid vector and introduced into Haloferax volcanii, a widely used strain that lacks detectable β‐galactosidase activity. Transformants were shown to express the enzyme; colonies turned blue when sprayed with Xgal and enzyme activity could be easily quantitated using a standard ONPG assay. In an accompanying publication, Patenge et al. (2000) have demonstrated the utility of bgaH as a promoter reporter in Halobacterium salinarum.
FtsZ ring formation without subsequent cell division after replication runout in Escherichia coli
Authors: Gullbrand Björn , Nordström Kurt
Published: March 2003

In this report, we have investigated cell division after inhibition of initiation of chromosome replication in Escherichia coli. In a culture grown to the stationary phase, cells containing more than one chromosome were able to divide some time after restart of growth, under conditions not allowing initiation of chromosome replication. This shows that there is no requirement for cell division to take place within a certain time after initiation of chromosome replication. Continued growth without initiation of replication resulted in filamented cells that generally did not have any constrictions. Interestingly, FtsZ rings were formed in a majority of these cells as they reached a certain cell length. These rings appeared and were maintained for some time at the cell quarter positions on both sides of the centrally localized nucleoid. These results confirm previous findings that cell division sites are formed independently of chromosome replication and indicate that FtsZ ring assembly is dependent on cell size rather than on the capacity of the cell to divide. Disruption of the mukB gene caused a significant increase in the region occupied by DNA after the replication runout, consistent with a role of MukB in chromosome condensation. The aberrant nucleoid structure was accompanied by a shift in FtsZ ring positioning, indicating an effect of the nucleoid on the positioning of the FtsZ ring. A narrow cell length interval was found, under and over which primarily central and non‐central FtsZ rings, respectively, were observed. This finding correlates well with the previously observed oscillatory movement of MinC and MinD in short and long cells.
Non‐typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor
Authors: Swords W. Edward , Buscher Benjamin A. , Ver Steeg Ii Kyle , Preston Andrew , Nichols Wade A. , Weiser Jeffrey N. , Gibson Bradford W. , Apicella Michael A.
Published: March 2003

Adherence and invasion are thought to be key events in the pathogenesis of non‐typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS‐coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB::ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB::ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co‐localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti‐ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.
Functional roles of conserved amino acid residues surrounding the phosphorylatable histidine of the yeast phosphorelay protein YPD1
Authors: Janiak‐Spens Fabiola , West Ann H.
Published: March 2003

The histidine‐containing phosphotransfer (HPt) protein YPD1 is an osmoregulatory protein in yeast that facilitates phosphoryl transfer between the two response regulator domains associated with SLN1 and SSK1. Based on the crystal structure of YPD1 and the sequence alignment of YPD1 with other HPt domains, we site‐specifically engineered and purified several YPD1 mutants in order to examine the role of conserved residues surrounding the phosphorylatable histidine (H64). Substitution of the positively charged residues K67 and R90 destabilized the phospho‐imidazole linkage, whereas substitution of G68 apparently reduces accessibility of H64. These findings, together with the effect of other mutations, provide biochemical support of the proposed functional roles of conserved amino acid residues of HPt domains.
‘Intergenic’blr gene in Escherichia coli encodes a 41‐residue membrane protein affecting intrinsic susceptibility to certain inhibitors of peptidoglycan synthesis
Authors: Wong Rebecca S. Y. , McMurry Laura M. , Levy Stuart B.
Published: March 2003

In the annotation of genomic sequences, small open reading frames (ORFs) are often neglected, particularly if they have no homology to other ORFs or proteins. A mini‐TnphoA insertion in a 602 bp ‘intergenic’ region of the Escherichia coli chromosome at genomic nucleotide 1702674 gave rise to a membrane‐bound PhoA fusion protein and a two‐ to fourfold increase in the intrinsic susceptibility to a wide spectrum of beta‐lactam antibiotics without affecting beta‐lactamase activity or susceptibility to tetracycline, chloramphenicol, gentamicin or quinolones. Susceptibility was also increased to cycloserine and bacitracin, but not to fosfomycin or valinomycin; these drugs, like beta‐lactams, inhibit peptidoglycan synthesis, although by different mechanisms. A clone bearing only 358 bp of this ‘blr’ region restored resistance to the parental level. Two amber mutations in the clone prevented such restoration and were counteracted by an amber suppressor, proving that the active species is a protein. The Blr protein has 41 amino acids, with a single predicted transmembrane helix, but no clear homology to any other protein. A transcriptional start exists 39 bp upstream from the translational start. The membrane location of Blr suggests that it may be part of an efflux pump or involved in murein metabolism. The results indicate that genes for other very small functional proteins may lie within ‘intergenic’ regions.
Plasmid copy number control: an ever‐growing story
Authors: Del Solar Gloria , Espinosa Manuel
Published: March 2003

Bacterial plasmids maintain their number of copies by negative regulatory systems that adjust the rate of replication per plasmid copy in response to fluctuations in the copy number. Three general classes of regulatory mechanisms have been studied in depth, namely those that involve directly repeated sequences (iterons), those that use only antisense RNAs and those that use a mechanism involving an antisense RNA in combination with a protein. The first class of control mechanism will not be discussed here. Within the second class (the most ‘classical’ one), exciting insights have been obtained on the molecular basis of the inhibition mechanism that prevents the formation of a long‐range RNA structure (pseudoknot), which is an example of an elegant solution reached by some replicons to control their copy number. Among the third class, it is possible to distinguish between (i) cases in which proteins play an auxiliary role; and (ii) cases in which transcriptional repressor proteins play a real regulatory role. This latter type of regulation is relatively new and seems to be widespread among plasmids from Gram‐positive bacteria, at least for the rolling circle‐replicating plasmids of the pMV158 family and the theta‐replicating plasmids of the Inc18 streptococcal family.
The Escherichia coli aquaporin‐Z water channel
Authors: Calamita Giuseppe
Published: March 2003

The membrane pathway of the rapid fluxes of water by which microorganisms adapt promptly to abrupt changes in environmental osmolality have begun to be understood since the discovery of the Escherichia coli aquaporin‐Z water channel, AqpZ. As in animals and plants, aquaporins are variously represented among microorganisms, in which 31 homologous genes have already been identified in eubacteria, Archaea, fungi and protozoa. The AqpZ channel is selectively permeable to water, although other functions are not excluded. Consistent with a conservation over the course of evolution, AqpZ and AQP1, a human counterpart, share similar structures. The aqpZ gene is growth phase and osmotically regulated. AqpZ has a role in both the short‐ and the long‐term osmoregulatory response and is required by rapidly growing cells. AqpZ‐like proteins seem to be necessary for the virulence expressed by some pathogenic bacteria. Microbial aquaporins are also likely to be involved in spore formation and/or germination. Additional roles may still be unknown. The use of AqpZ as a model system will continue to provide insight into the understanding of the importance of aquaporins.
A temperature‐sensitive Krp1 allows in vivo characterization of kexin activation
Authors: Ladds Graham , Davis Kevin , Powner Dale , Davey John
Published: March 2003

Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes are themselves made as inactive precursors and we have investigated the activation of Krp1, a kexin from the fission yeast Schizosaccharomyces pombe. As Krp1 is essential for cell growth, we have used a krp1ts strain to investigate the role of the prosequence in the activation process. Mutations that reduce either the efficiency with which the prosequence is released or the rate at which the released prosegment is subsequently cleaved at an internal site are less active when assayed in vivo. We also show that prosegments lacking an internal dibasic motif can act as autoinhibitors and prevent activation of the catalytic fragment. Krp1 constructs containing prosequences based on these inhibitors do not become active in vitro. Surprisingly, the same constructs do become active in the intact cell and appear to suggest that alternative activation processes can be used by these enzymes.
Iron availability regulates DNA recombination in Neisseria gonorrhoeae
Authors: Serkin Carla D. , Seifert H. Steven
Published: March 2003

The pilus of Neisseria gonorrhoeae (the gonococcus Gc), the causative agent of gonorrhoea, promotes attachment of the gonococcus to the host epithelium and is essential for the establishment of disease. The ability of N. gonorrhoeae to infect previously exposed individuals is partially due to pilus antigenic variation. In addition, variation of the pilus has been proposed to function in the adaptation of the gonococcus to host environments. Previously, we described the development of a competitive reverse transcriptase (RT)‐PCR assay that quantifies the frequency of pilin antigenic variation within a gonococcal population. Using this assay, the effect of different biologically relevant environmental conditions on the frequency of pilin antigenic variation was tested. Of the environmental conditions examined in vitro, only limited iron affected a significant change in the frequency of antigenic variation. Further investigation revealed that an observed increase in pilin antigenic variation reflected an increase in other DNA recombination and DNA repair processes within iron‐starved cultures. In addition, this low iron‐induced increase was determined to be independent of changes in RecA expression and was observed in a Fur mutant strain. As gonococci encounter conditions of low iron during infection, these data suggest that iron‐limitation signals for increased recombinational events that are important for gonococcal pathogenesis.
The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection
Authors: Scherer Christina A. , Cooper Emily , Miller Samuel I.
Published: March 2003

Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS). The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer. Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection. In addition, a yeast two‐hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified. Three individual carboxyl‐terminal point mutations within SspC that disrupt the yeast two‐hybrid interaction were isolated. Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC. These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions.
Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation
Authors: Howell‐Adams Becky , Seifert H. Steven
Published: March 2003

The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high‐frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high‐frequency gene conversions that mediate pilin Av.
Type IV pilus biogenesis and motility in the cyanobacterium Synechocystis sp. PCC6803
Authors: Bhaya Devaki , Bianco Nicole R. , Bryant Donald , Grossman Arthur
Published: March 2003

We have recently shown that phototactic movement in the unicellular cyanobacterium Synechocystis sp. PCC6803 requires type IV pilins. To elucidate further type IV pilus‐dependent motility, we inactivated key genes implicated in pilus biogenesis and function. Wild‐type Synechocystis sp. PCC6803 cells have two morphologically distinct pilus types (thick and thin pili). Of these, the thick pilus morphotype, absent in a mutant disrupted for the pilin‐encoding pilA1 gene, appears to be required for motility. The thin pilus morphotype does not appear to be altered in the pilA1 mutant, raising the possibility that thin pili have a function distinct from that of motility. Mutants disrupted for pilA2, which encodes a second pilin‐like protein, are still motile and exhibit no difference in morphology or density of cell‐surface pili. In contrast, inactivation of pilD (encoding the leader peptidase) or pilC (encoding a protein required for pilus assembly) abolishes cell motility, and both pilus morphotypes are absent. Thus, the PilA1 polypeptide is required for the biogenesis of the thick pilus morphotype which, in turn, is necessary for motility (hence we refer to them as type IV pili). Furthermore, PilA2 is critical neither for motility nor for pilus biogenesis. Two genes encoding proteins with similarity to PilT, which is considered to be a component of the motor essential for type IV pilus‐dependent movement, were also inactivated. A pilT1 mutant is (i) non‐motile, (ii) hyperpiliated and (iii) accumulates higher than normal levels of the pilA1 transcript. In contrast, pilT2 mutants are motile, but are negatively phototactic under conditions in which wild‐type cells are positively phototactic.