bluuebunny/crossref_metadata_2025 huggingface dataset:
Starvation‐independent sporulation in Myxococcus xanthus involves the pathway for β‐lactamase induction and provides a mechanism for competitive cell survival
Authors: O'Connor Kathleen A. , Zusman David R.
Published: November 2003

Myxococcus xanthus is a Gram‐negative, soil‐dwelling bacterium with a complex life cycle which includes fruiting body formation and sporulation in response to starvation. This developmental process is slow, requiring a minimum of 24–48 h, and requires cells to be at high cell density on a solid surface. It is known that, in the absence of starvation, vegetatively growing cell suspensions can form ‘glycerol spores’ when exposed to high levels of glycerol, usually 0.5 M. The cells differentiate from rods to resistant spheres rapidly (2–4 h) and synchronously. We have found that the chromosomally encoded β‐lactamase of M. xanthus can be induced by numerous β‐lactam antibiotics as well as by non‐specific inducers including glycine and many D‐amino acids. In addition, D‐cycloserine, phosphomycin, and hen egg‐white lysozyme also induce β‐lactamase in this bacterium. Unexpectedly, agents which induce β‐lactamase can induce ‘glycerol spores’; all of the agents tested which induce glycerol spores (glycerol, DMSO, ethylene glycol) also induce β‐lactamase. During the induction of sporulation, β‐lactamase activity increases, reaching a peak during the morphological transition from rod‐shaped cells to spherical spores. These spores are viable and resistant to many treatments which disrupt vegetatively growing rods but are not as resistant as fruiting body spores. The concomitant induction of β‐lactamase and starvation‐independent sporulation suggests that these processes share a common signal‐transduction pathway. These results also suggest that starvation‐independent sporulation may be an adaptation of cells in order to resist agents that damage peptidoglycan structure and therefore threaten cell survival.
Methanol production during chemotaxis to amino acids in Bacillus subtilis
Authors: Kirby John R. , Kristich Christopher J. , Feinberg Shelby L. , Ordal George W.
Published: November 2003

The 20 common amino acids act as attractants during chemotaxis by the Gram‐positive organism Bacillus subtilis. In this study, we report that all amino acids induce B. subtilis to produce methanol both upon addition and removal of the chemoeffector. Asparagine‐induced methanol production is specific to the McpB receptor and aspartate‐induced methanol production correlates with receptor occupancy. These findings suggest that addition and removal of all amino acids cause demethylation of specific receptors which results in methanol production. We also demonstrate that certain attractants cause greater production of methanol after multiple stimulations. CheC and CheD, while affecting the levels of receptor methylation, are not absolutely required for either methylation or demethylation. In contrast, CheY is necessary for methanol formation upon removal of attractant but not upon addition of attractant. We conclude that methanol formation due to negative stimuli indicates the existence of a unique adaptational mechanism in B. subtilis involving the response regulator, CheY.
Catabolite repression by glucose 6‐phosphate, gluconate and lactose in Escherichia coli
Authors: Hogema Boris M. , Arents Jos C. , Inada Toshifumi , Aiba Hiroji , Van Dam Karel , Postma Pieter W.
Published: November 2003

While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non‐PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6‐phosphate. In this article it is shown that enzyme IIAGlc of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of β‐galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6‐phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP‐independent CRP* protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6‐phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these non‐PTS carbon sources.
Identification and molecular cloning of a gene encoding a fibronectin‐binding protein (CadF) from Campylobacter jejuni
Authors: Konkel Michael E. , Garvis Steven G. , Tipton Stephanie L. , Anderson, Jr Donald E. , Cieplak, Jr Witold
Published: November 2003

Campylobacter jejuni, a Gram‐negative bacterium, is a common cause of gastrointestinal disease. By analogy with other enteric pathogens such as Salmonella and Shigella, the ability of C. jejuni to bind to host cells is thought to be essential in the pathogenesis of enteritis. Scanning electron microscopy of infected INT407 cells suggested that C. jejuni bound to a component of the extracellular matrix. Binding assays using immobilized extracellular matrix proteins and soluble fibronectin showed specific and saturable binding of fibronectin to C. jejuni. Ligand immunoblot assays using 125I‐labelled fibronectin revealed specific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel‐purified protein, reacted with a 37 kDa protein in all C. jejuni isolates (n = 15) as tested by immunoblot analysis. Antibodies present in convalescent serum from C. jejuni‐infected individuals also recognized a 37 kDa protein. The gene encoding the immunoreactive 37 kDa protein was cloned and sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a calculated molecular mass of 36 872 Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhesin protein from Pseudomonas fluorescens. Isogenic C. jejuni mutants which lack the 37 kDa outer membrane protein, which we have termed CadF, displayed significantly reduced binding to fibronectin. Biotinylated fibronectin bound to a protein with an apparent molecular mass of 37 kDa in the outer membrane protein extracts from wild‐type C. jejuni as judged by ligand‐binding blots. These results indicate that the binding of C. jejuni to fibronectin is mediated by the 37 kDa outer membrane protein which is conserved among C. jejuni isolates.
Evidence for autolysin‐mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface
Authors: Heilmann Christine , Hussain Muzaffar , Peters Georg , Götz Friedrich
Published: November 2003

Biofilm formation on a polymer surface which involves initial attachment and accumulation in multilayered cell clusters (intercellular adhesion) is proposed to be the major pathogenicity factor in Staphylococcus epidermidis foreign‐body‐associated infections. We have characterized two distinct classes of biofilm‐negative Tn917 mutants in S. epidermidis affected in initial attachment (class A) or intercellular adhesion (class B). mut1 (class A mutant) lacks five surface‐associated proteins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be complemented by transformation with a 16.4 kb wild‐type DNA fragment. The complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1. Subcloning experiments revealed that the 60 kDa protein is sufficient for initial attachment. Immunofluorescence microscopy using an antiserum raised against the 60 kDa protein showed that this protein is located at the cell surface. DNA‐sequence analysis of the complementing region revealed a single open reading frame which consists of 4005 nucleotides and encodes a deduced protein of 1335 amino acids with a predicted molecular mass of 148 kDa. The amino acid sequence exhibits a high similarity (61% identical amino acids) to the atl gene product of Staphylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE. By analogy with the S. aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by proteolytic processing. The 120 kDa protein missing from mut1 presumably represents the unprocessed amidase and glucosaminidase domain after proteolytic cleavage of the signal‐ and propeptide. The 45 and 38 kDa proteins are probably the degradation products of the 60 and 52 kDa proteins, respectively. Additionally, AtlE was found to exhibit vitronectin‐binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein‐coated polymer surfaces during later stages of adherence. Our findings provide evidence for a new function of an autolysin (AtlE) in mediating the attachment of bacterial cells to a polymer surface, representing the prerequisite for biofilm formation.
Genes involved in conjugative DNA processing of plasmid R6K
Authors: Núñez Belén , Avila Pilar , De La Cruz Fernando
Published: November 2003

The conjugative transfer region of the IncX plasmid R6K (TRAX) was analysed by transposon mutagenesis and DNA sequencing. Tn5tac1 insertional mutations localized TRAX to a 14.8 kb segment containing the α origin of transfer (oriTα), genes involved in conjugative DNA‐processing (DtrX) and genes involved in pilus synthesis and assembly (MpfX). A second functional oriT, oriTβ, was located at a distance of 5.3 kb from oriTα and was outside TRAX. MpfX occupied a segment of 10 kb, as judged by the location of insertions conferring resistance to infection by the X pilus‐specific phage X‐2. At both sides of MpfX there were insertions that were Tra− but X‐2 sensitive, suggesting that the mutations were in DtrX. This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriTα–taxA–taxC–MpfX–taxB. taxC coded for a oriT‐relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F‐like plasmids and to the Arc‐repressor superfamily. TaxB showed similarity to TraG‐like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA‐transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein π, which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein π for conjugation.
Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell‐binding and catalytic domains
Authors: Lemichez Emmanuel , Flatau Gilles , Bruzzone Marc , Boquet Patrice , Gauthier Michel
Published: November 2003

Cytotoxic necrotizing factor type 1 (CNF1) induces, in epithelial cells, the development of stress fibres via the GTPase Rho pathway. We showed that CNF1 is able to modify Rho both in vitro and in vivo. Recombinant N‐terminal 33 kDa (CNF1Nter) and C‐terminal 14.8–31.5 kDa (CNF1Cter) regions of the CNF1 protein allowed us to demonstrate that the N‐terminal region contains the cell‐binding domain of the toxin and that the C‐terminal region is responsible for its catalytic activity. CNF1Nter lowered the activity of CNF1 when provided to cells before the toxin whereas CNF1Cter had no effect on CNF1 cell toxicity. CNF1Cter was sufficient to induce a typical CNF1 phenotype when microinjected into African green monkey kidney cells (Vero cells), and was able to modify Rho as previously reported for CNF1. The C‐terminal domain lost its catalytic activity when deleted of various subdomains, suggesting a scattered distribution of catalytic‐site amino acids. Elucidation of the CNF1 functional organization and analysis of amino acid homologies between CNFs (CNF1, CNF2), Pasteurella multocida toxin (PMT) and dermonecrotic toxin of Bordetella pertussis (DNT) allowed us to postulate that CNFs and DNT act on Rho via the same enzymatic activity located in their C‐terminus, and that CNFs and PMT probably bind to analogous cell receptors.
A new cytolethal distending toxin (CDT) from Escherichia coli producing CNF2 blocks HeLa cell division in G2/M phase
Authors: Pérès Sylvie Y. , Marchès Olivier , Daigle France , Nougayrède Jean‐Philippe , Hérault Frédéric , Tasca Christian , De Rycke Jean , Oswald Eric
Published: November 2003

Escherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4 h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non‐pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell‐cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell‐cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli, Campylobacter jejuni and Shigella dysenteriae. Southern hybridization experiments demonstrated that the pVir of other CNF2‐producing E. coli strains contained sequences related to cdt. Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.
The Neisseria type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial cells
Authors: Lin Lan , Ayala Patricia , Larson Jason , Mulks Martha , Fukuda Minoru , Carlsson Sven R. , Enns Caroline , So Magdalene
Published: November 2003

Infection of human epithelial cells by Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) increases the rate of degradation of LAMP1, a major integral membrane glycoprotein of late endosomes and lysosomes. Several lines of evidence indicate that the neisserial IgA1 protease is directly responsible for this LAMP1 degradation. LAMP1 contains an IgA1‐like hinge region with potential cleavage sites for the neisserial type 1 and type 2 IgA1 proteases. Neisserial type 2 IgA1 protease cleaves purified LAMP1 in vitro. Unlike its wild‐type isogenic parent, an iga− mutant of N. gonorrhoeae cannot affect LAMP1 turnover and its growth in epithelial cells is dramatically reduced. Thus, IgA1 protease cleavage of LAMP1 promotes intracellular survival of pathogenic Neisseria spp.
mRNA stability is regulated by a coding‐region element and the unique 5′ untranslated leader sequences of the three Synechococcus psbA transcripts
Authors: Kulkarni Resham D. , Golden Susan S.
Published: November 2003

The psbAI and psbAIII transcripts in Synechococcus sp. strain PCC 7942 are subject to accelerated turnover when cells are exposed to high light intensities, but psbAII message stability is unaffected. We used a psbAI‘minigene’ which has a part of the coding sequence removed as a reporter gene in order to identify the cis‐acting elements of the transcript that determine stability. While engineering the minigene to optimally mimic the native gene, we identified a stabilizer element within the open reading frame, corresponding to the coding region for the first membrane span of the D1 protein, the presence of and translation through which was essential for normal psbA mRNA stability. We propose that this stabilizer is a site for ribosome pausing, and that accumulation of ribosomes on the transcript upstream of the pause site increases stability. To identify the elements that regulate the differential responses of the psbA transcripts to high‐light growth, sequences from psbAII and psbAIII were substituted in the psbAI minigene reporter. The chimeric reporter transcripts established that the psbAI and psbAIII untranslated leaders determine the faster turnover of these messages. The untranslated leader regions of the psbA transcripts may regulate mRNA stability by modulating translation and thereby stability, or by recruiting RNA‐binding proteins that affect mRNA turnover more directly.
Proteolysis of the phage λ CII regulatory protein by FtsH (HflB) of Escherichia coli
Authors: Shotland Y. , Koby S. , Teff D. , Mansur N. , Oren D. A. , Tatematsu K. , Tomoyasu T. , Kessel M. , Bukau B. , Ogura T. , Oppenheim A. B.
Published: November 2003

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage λ CII transcriptional activator plays a key role in the lysis‐lysogeny decision by phage λ. Here we demonstrate that the E. coli ATP‐dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat‐shock transcription factor σ32. Proteolysis of σ32 requires, in vivo, the presence of the DnaK‐DnaJ‐GrpE chaperone machine. Neither DnaK‐DnaJ‐GrpE nor GroEL‐GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP‐dependent proteolysis of CII in vitro. The degradation of CII is at least 10‐fold faster than that of σ32. Electron microscopy revealed that purified FtsH forms ring‐shaped structures with a diameter of 6–7 nm.
Roles for motility in bacterial–host interactions
Authors: Ottemann Karen M. , Miller Jeff F.
Published: November 2003

The ability to move in a directed manner may confer distinct advantages upon host‐adapted prokaryotes. Potential benefits of motility include increased efficiency of nutrient acquisition, avoidance of toxic substances, the ability to translocate to preferred hosts and access optimal colonization sites within them, and dispersal in the environment during the course of transmission. The costs of motility also may be significant. These include the metabolic burden of synthesizing flagellar components, the energetic expense of fuelling flagellar motors and the presentation of polymeric and highly antigenic targets to the immune system. It is therefore not surprising that synthesis of the motility apparatus is usually subject to strict control. Studies of a variety of bacterial–host interactions demonstrate roles for motility, and its regulation, at points throughout the infectious cycle.
Adherence of Porphyromonas gingivalis to matrix proteins via a fimbrial cryptic receptor exposed by its own arginine‐specific protease
Authors: Kontani Masanori , Kimura Shigenobu , Nakagawa Ichiro , Hamada Shigeyuki
Published: November 2003

Porphyromonas gingivalis, a Gram‐negative anaerobe, is known to be involved in the pathogenesis of periodontitis. P. gingivalis fimbriae, which are proteinaceous appendages extending from the cell surface, may contribute to the adherence of the organism to the host cell surface. We previously suggested that arginine‐specific protease produced by P. gingivalis enhanced the adherence of purified fimbriae to fibroblasts or matrix proteins. In this study, we have revealed the mechanism of the enhanced binding of fimbriae by the protease in more detail. Arg‐specific protease and fimbriae were obtained from P. gingivalis 381 cells and purified. We then analysed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the real‐time biomolecular interaction analysis (BIAcore) system with an optical biosensor based on the principles of surface plasmon resonance. BIAcore profiles demonstrated an enhanced interaction between fimbriae and protease‐degraded fibronectin. We also showed specific binding of fimbriae to the degraded fibronectin by means of BIAcore analysis. The binding of biotinylated fimbriae to immobilized fibronectin was examined by enzyme‐linked biotin–avidin assay. The purified protease enhanced the fimbrial binding to the immobilized fibronectin. The enhancement was inhibited by the addition of l‐Arg, or oligopeptides containing the Arg residue at the C‐terminus in the fimbrial binding reaction, suggesting that the P. gingivalis fimbriae may potentially have an ability to bind tightly to the Arg residue at C‐terminus. Taken together, these studies indicate that P. gingivalis arginine‐specific protease can expose a cryptitope in the matrix protein molecules, i.e. the C‐terminal Arg residue of the host matrix proteins, so that the organism can adhere to the surface layer in the oral cavity through fimbriae–Arg interaction (a novel host–parasite relationship).
Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937
Authors: Shevchik Vladimir E. , Hugouvieux‐Cotte‐Pattat Nicole
Published: November 2003

Erwinia chrysanthemi causes soft‐rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase‐encoding genes, pelA–pelE–pelD–paeY–pemA. The paeY open reading frame is 1650 bases long and encodes a 551‐residue precursor protein of 60 704 Da, including a 25‐amino‐acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar‐beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de‐esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar‐beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of paeY expression appears to be dependent on the KdgR repressor, which controls all the steps of pectin catabolism, and on the catabolite regulatory protein (CRP), the global activator of sugar catabolism. The contiguous pelD, paeY and pemA genes are transcribed as an operon from a promoter proximal to pelD which allows the regulation by KdgR and CRP. However, transcription can be interrupted at the intra‐operon Rho‐independent terminator situated between pelD and paeY. The paeY mutant inoculated into Saintpaulia plants was less invasive than the wild‐type E. chrysanthemi strain 3937, demonstrating the important role of PaeY in the soft‐rot disease.
Isolation of a dnaE mutation which enhances RecA‐independent homologous recombination in the Escherichia coli chromosome
Authors: Bierne Hélène , Vilette Didier , Ehrlich S. Dusko , Michel Bénédicte
Published: November 2003

The mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA‐independent recombination is increased 15‐fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA‐independent recombination. It is proposed that tandem‐repeat recombination occurs by a replication slippage mechanism. RecA‐independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.
Mutations leading to increased levels of resistance to glycopeptide antibiotics in VanB‐type enterococci
Authors: Baptista Marina , Depardieu Florence , Reynolds Peter , Courvalin Patrice , Arthur Michel
Published: November 2003

The vanB gene cluster mediates glycopeptide resistance by production of peptidoglycan precursors ending in the depsipeptide D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac) instead of D‐Ala‐D‐Ala found in susceptible enterococci. Synthesis of D‐Ala‐D‐Lac and hydrolysis of D‐Ala‐D‐Ala is controlled by the VanRBSB two‐component regulatory system that activates transcription of the resistance genes in response to vancomycin but not to teicoplanin. Two substitutions (A30→G or D168→Y) in the VanSB sensor kinase resulted in induction by teicoplanin, indicating that the N‐terminal domain of the protein was involved in glycopeptide sensing. A substitution (T237→K) located in the vicinity of the putative autophosphorylation site of VanSB (H233) was associated with a constitutive phenotype and affected a conserved residue known to be critical for the phosphatase activity of related kinases. A mutant producing an impaired host D‐Ala:D‐Ala ligase required vancomycin for growth, since D‐Ala‐D‐Lac was only produced under inducing conditions. The ddl and vanSB mutations, alone or in combination, resulted in various resistance phenotypes that were determined by the amount of D‐Ala‐D‐Ala and D‐Ala‐D‐Lac incorporated into peptidoglycan precursors under different inducing conditions.
Spontaneous duplication of a 661 bp element within a two‐component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii, cause of brown blotch disease of mushrooms
Authors: Han Bin , Pain Arnab , Johnstone Keith
Published: November 2003

Spontaneous sectoring of Pseudomonas tolaasii colonies results in a phenotypic switch from the smooth, pathogenic form (designated 1116S) to the rough non‐pathogenic form (designated 1116R). This phenotypic switch can also be induced by mutation of the pheN master regulatory locus, which encodes a 99 kDa protein with homology to the conserved family of sensor regulator proteins. Southern blot analysis of genomic DNA from 1116S and 1116R probed with a 3.4 kb XhoI–BamHI fragment containing the pheN gene has revealed restriction fragment length polymorphisms in the pheN locus of 1116R. In order to characterize the genetic basis of this variation, the pheN locus (designated pheN′ ) was cloned from 1116R and its nucleotide sequence determined. A 661 bp duplication was identified within pheN′ introducing a frameshift mutation in the predicted pheN open reading frame (ORF). A resulting predicted ORF of pheN′ designated ORF2 encodes a polypeptide of 706 amino acid residues, with a predicted molecular weight of 77 kDa, and which lacks part of the PheN sensor domain. Southern blot analysis of genomic DNA using a probe within the duplicated sequence revealed the presence of two bands in 1116R but only one band in the 1116S form. Polymerase chain reaction (PCR) analysis of 25 independently isolated 1116R sectors using primers flanking the duplication site in pheN confirmed the presence of the duplicated 661 bp sequence within this region in all of the sectors and the absence of the duplicated sequence in spontaneous revertants from 1116R to 1116S. Northern blot analysis of RNA from 1116S and 1116R using a pheN probe showed that ORF2 was transcribed in the 1116R form. The presence of a truncated PheN protein in 1116R was verified by Western blot analysis of total cell protein using a LemA antiserum, which revealed the presence of 99 kDa and 77 kDa cross‐reactive bands in 1116S and 1116R respectively. It is concluded that the spontaneous colony‐sectoring event that results in the 1116R phenotypic variant form of P. tolaasii arises owing to a 661 bp DNA duplication within the 5′ end of the pheN gene, which results in loss of the periplasmic sensor domain of PheN and elimination of normal PheN function.
Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5′ leader sequence
Authors: Tedin Karsten , Resch Armin , Bläsi Udo
Published: November 2003

It has previously been proposed that Escherichia coli ribosomal protein S1 is required for the translation of highly structured mRNAs. In this study, we have examined the influence of structural features at or near the start codon of different mRNAs. The requirement for ribosomal protein S1 for translation initiation was determined when (i) the ribosome‐binding site (RBS) was either preceded by a 5′ non‐translated leader sequence; (ii) the RBS was located 5′ proximal to a mRNA start codon; and (iii) the start codon was the 5′ terminal codon as exemplified by leaderless mRNAs. In vitro translation studies revealed that the leaderless lambda cI mRNA is translated with Bacillus stearothermophilus ribosomes, naturally lacking a ribosomal protein S1 homologue, whereas ompA mRNA containing a 5′ leader is not. These studies have been verified by toeprinting with E. coli ribosomes depleted for S1. We have shown that S1 is required for ternary complex formation on ompA mRNA but not for leaderless mRNAs or for mRNAs in which the RBS is close to the 5′ end.
Residence at the expression site is necessary and sufficient for the transcription of surface antigen genes of Pneumocystis carinii
Authors: Sunkin Susan M. , Stringer James R.
Published: November 2003

The major surface glycoprotein (MSG) of P. carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSG isoforms start with the same sequence, called the upstream conserved sequence (UCS), which is encoded by a single locus. The mechanism by which the UCS becomes part of different MSG mRNAs is not obvious because at least 15 loci, which are distributed throughout the genome, encode MSGs. One possibility is that attachment to the UCS locus is required for the transcription of an MSG gene. The alternative to this expression site model is that mRNAs acquire the UCS by RNA splicing. To distinguish between these two models, UCS/MSG junctions in the genome were compared with UCS/MSG junctions in mRNA. The UCS/MSG junctions in the mRNA matched those in the genome, as would be expected if splicing did not contribute to the attachment of the UCS to the 5′ ends of MSG mRNAs. Given that few if any MSG mRNAs lack the UCS, the correspondence between the UCS/MSG junctions in transcripts and those in the genome indicates that attachment to the UCS is both necessary and sufficient for transcription of an MSG gene.
The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification
Authors: Finnie Christine , Hartley Nigel M. , Findlay Kim C. , Downie J. Allan
Published: November 2003

NodO is a secreted protein from Rhizobium leguminosarum bv. viciae with a role in signalling during legume nodulation. A Tn5‐induced mutant was identified that was defective in NodO secretion. As predicted, the secretion defect decreased pea and vetch nodulation but only when the nodE gene was also mutated. This confirms earlier observations that NodO plays a particularly important role in nodulation when Nod factors carrying C18:1 (but not C18:4) acyl groups are the primary signalling molecules. In addition to NodO secretion and nodulation, the secretion mutant had a number of other characteristics. Several additional proteins including at least three Ca2+‐binding proteins were not secreted by the mutant and this is thought to have caused the pleiotropic phenotype. The nodules formed by the secretion mutant were unable to fix nitrogen efficiently; this was not due to a defect in invasion because the nodule structures appeared normal and nodule cells contained many bacteroids. The mutant formed sticky colonies and viscous liquid cultures; analysis of the acidic exopolysaccharide revealed a decrease in the ratio of reducing sugars to total sugar content, indicating a longer chain length. The use of a plate assay showed that the mutant was defective in an extracellular glycanase activity. DNA sequencing identified the prsDE genes, which are homologous to genes encoding protease export systems in Erwinia chrysanthemi and Pseudomonas aeruginosa. An endoglycanase (Egl) from Azorhizobium caulinodans may be secreted from R. leguminosarum bv. viciae in a prsD‐dependent manner. We conclude that the prsDE genes encode a Type I secretion complex that is required for the secretion of NodO, a glycanase and probably a number of other proteins, at least one of which is necessary for symbiotic nitrogen fixation.
Negative autoregulation of the Rhizobium meliloti fixK gene is indirect and requires a newly identified regulator, FixT
Authors: Foussard Marie , Garnerone Anne‐Marie , Ni Fudi , Soupène Eric , Boistard Pierre , Batut Jacques
Published: November 2003

fixK genes are crp/fnr homologues that have been discovered in diverse Rhizobium spp., in which they are usually essential for symbiotic nitrogen fixation. One recurrent function of fixK genes in rhizobia is to activate the transcription of operons required for respiration in the microoxic environment of the nodule. In a similar manner to its Escherichia coli crp and fnr homologues, R. meliloti fixK regulates its own expression negatively. However, we demonstrate here that fixK negative autoregulation is not direct and, instead, involves a newly identified gene, fixT, the expression of which depends on fixK. Inactivation of fixT resulted in derepression of fixK expression under free‐living microoxic conditions. Furthermore, constitutively expressed fixT strongly repressed fixK–lacZ expression in the absence of a functional fixK gene. Several lines of evidence indicate that fixT is active via its protein product FixT. FixT does not resemble any protein present in databases so far. Nodules induced by a fixT mutant were Fix+, thus demonstrating that fixT is not essential for symbiotic nitrogen fixation.
Arginine boxes and the argR gene in Streptomyces clavuligerus: evidence for a clear regulation of the arginine pathway
Authors: Rodríguez‐García Antonio , Ludovice Madalena , Martín Juan F. , Liras Paloma
Published: November 2003

The argR gene of Streptomyces clavuligerus has been located in the upstream region of argG. It encodes a protein of 160 amino acids with a deduced Mr of 17 117 for the monomer. Transformants containing the amplified argR gene showed lower activity (50%) of the biosynthetic ornithine carbamoyltransferase (OTC) activity and higher levels (380%) of the catabolic ornithine aminotransferase (OAT) activity than control strains. Amplification of an arginine (ARG) box‐containing sequence results in a 2‐ to 2.5‐fold derepression of ornithine acetyltransferase and OTC, suggesting that the repressor is titrated out. Footprinting experiments using the pure homologous arginine repressor (AhrC) of B. subtilis showed a protected 38 nt region (ARG box) in the coding strand upstream of argC. The protected region contained two tandemly repeated imperfect palindromic 18‐nt ARG boxes. The repressor–operator interaction was confirmed by band‐shift experiments of the DNA fragment containing the protected region. By computer analysis of the Streptomyces sequences available in the databases, a consensus ARG box has been deduced for the genus Streptomyces. This is the first example of a clear regulation of an amino acid biosynthetic pathway in Streptomyces species, challenging the belief that actinomycetes do not have a well‐developed regulatory system of these pathways.
The membrane‐bound cell division protein DivIB is localized to the division site in Bacillus subtilis
Authors: Harry E. J. , Wake R. G.
Published: November 2003

The cell division gene divIB of Bacillus subtilis is essential for the normal rate of growth and division. The gene product, DivIB, is a membrane‐bound protein in which the bulk of the protein (at the C‐terminal end) is on the exterior surface of the cell membrane. DivIB is involved in the early stages of septum formation, but its exact role in cell division is unknown. To gain more information about the mode of action of DivIB in septum formation, we determined the location of DivIB within the cell membrane using immunofluorescence. This immunolocalization approach established that DivIB becomes localized to the division site before visible septation and remains localized to this site throughout the division process. Various DivIB immunostaining patterns were observed in immunofluorescence experiments and, together with cell length and nucleoid distance measurements, have allowed us to propose two models to describe DivIB localization during the cell cycle.
Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection
Authors: Clausen Johannes D. , Christiansen Gunna , Holst Henrik U. , Birkelund Svend
Published: November 2003

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia‐containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule‐associated Chlamydia vesicles also collocated with tyrosine‐phosphorylated McCoy cell proteins. After migration, the Chlamydia‐containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule‐dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule‐dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR‐1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR‐1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.
InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association
Authors: Braun Laurence , Dramsi Shaynoor , Dehoux Pierre , Bierne Hélène , Lindahl Gunnar , Cossart Pascale
Published: November 2003

Listeria monocytogenes is an intracellular bacterial pathogen that expresses several surface proteins critical for the infectious process. Such proteins include InlA (internalin) and InlB, involved in bacterial entry into the host cell, and ActA, required for bacterially induced actin‐based motility. Although the molecular mechanisms of attachment of InlA and ActA have been characterized, essentially nothing is known about how InlB is anchored to the bacterial surface. Using a genetic approach, we demonstrate that the last 232 amino acids of InlB are both necessary and sufficient for anchoring this protein to the bacterial surface. An InlB mutant protein deleted for the last 232 amino acids was secreted and not detected at the cell surface. A ‘domain‐swapping’ strategy in which these 232 amino acids were used to replace the normal cell wall‐anchoring domain of InlA resulted in a chimeric protein that was anchored to the cell surface and able to confer entry. Interestingly, surface association of InlB also occurred when InlB was added externally to bacteria, suggesting that association may be able to occur after secretion. This association was productive for invasion, as it conferred bacterial entry into host cells. The C‐terminal anchoring region in InlB contains 80‐amino‐acid repeats beginning with the sequence GW that is also present in a newly identified surface‐associated bacteriolysin of L. monocytogenes, called Ami. Addition of GW repeats to the C‐terminal of InlB improves anchoring of the protein to the cell surface. These and other data suggest that such ‘GW’ repeats may constitute a novel motif for cell‐surface anchoring in Listeria and other Gram‐positive bacteria. This motif may have important consequences for the release of surface proteins involved in interactions with eukaryotic cells.
Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification
Authors: Jayaraman Gayatri C. , Penders Jana E. , Burne Robert A.
Published: November 2003

The dental pathogen Streptococcus mutans persists and causes diseases in highly dynamic environments and gains a selective ecological advantage in environmental conditions that frequently exceed the limits for growth of the organism, particularly with regard to environmental pH. The goal of this study was to begin a molecular genetic analysis of a major stress protein, DnaK/Hsp70, to begin to understand how stress responses are regulated in this lactic acid bacterium and to establish a relationship between dnaK gene expression and exposure to acidic environments. Cloning and nucleotide sequence analysis revealed that the dnaK gene is preceded by, and is in an operon‐like arrangement with, the hrcA and grpE genes, although intergenic spacing was unlike that described in other bacteria. An inverted repeat (a CIRCE element) was identified by sequence analysis and, using primer extensions, a heat shock‐responsive, σA‐type promoter, P1, 5′ to the hrcA gene, and a σB‐type promoter, 5′ to the grpE translational start site, were identified. No promoters were detected between grpE and dnaK. A strain carrying a strongly polar insertion in the hrcA gene had markedly diminished levels of dnaK mRNA, indicating that dnaK was transcribed as part of an operon from P1, and to a lesser extent from P2. Results from physiological manipulation of S. mutans in continuous chemostat culture demonstrated that steady‐state levels of S. mutans dnaK mRNA and DnaK protein were (i) increased in response to acid shock; (ii) elevated in acid ‘adapted’ cells; and (iii) induced in response to alkali shock of acid ‘adapted’ cells. In all cases, increased amounts of dnaK mRNA could be correlated with enhanced transcription from P1. This study provides the first detailed analysis of the expression of a heat shock gene from an oral isolate, and the evidence provided suggests that σB‐like promoters may also be involved in class I heat shock gene expression in some Gram‐positive organisms.
Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin‐based motility
Authors: Shere Kalpana D. , Sallustio Sandra , Manessis Anastasios , D’Aversa Teresa G. , Goldberg Marcia B.
Published: November 2003

Shigella pathogenesis involves bacterial invasion of colonic epithelial cells and movement of bacteria through the cytoplasm and into adjacent cells by means of actin‐based motility. The Shigella protein IcsA (VirG) is unipolar on the bacterial surface and is both necessary and sufficient for actin‐based motility. IcsA is inserted into the outer membrane as a 120‐kDa polypeptide that is subsequently slowly cleaved, thereby releasing the 95‐kDa amino‐terminal portion into the culture supernatant. IcsP, the major Shigella protease that cleaves IcsA, was identified and cloned. It has significant sequence similarity to the E. coli serine proteases, OmpP and OmpT. Disruption of icsP in serotype 2a S. flexneri leads to a marked reduction in IcsA cleavage, increased amounts of IcsA associated with the bacterium and altered distribution of IcsA on the bacterial surface. The icsP mutant displays significantly increased rates of actin‐based motility, with a mean speed 27% faster than the wild‐type strain; moreover, a significantly greater percentage of the icsP mutant moves in the cytoplasm. Yet, plaque formation on epithelial monolayers by the mutant was not altered detectably. These data suggest that IcsA, and not a host protein, is limiting in the rate of actin‐based motility of wild‐type serotype 2a S. flexneri.
The erythrocyte receptor for the channel‐forming toxin aerolysin is a novel glycosylphosphatidylinositol‐anchored protein
Authors: Cowell Simon , Aschauer Wolfgang , Gruber Hermann J. , Nelson Kim L. , Buckley J. Thomas
Published: November 2003

The plasma membrane of rat erythrocytes contains a 47‐kDa glycoprotein that binds the channel‐forming toxin aerolysin with high affinity and accounts for the sensitivity of these cells to the toxin. The receptor was purified so that its N‐terminal sequence could be determined after Western blotting. The sequence did not match any sequences in the databases, indicating that the receptor is a novel erythrocyte surface protein. However, it exhibited considerable homology to the N‐termini of a group of membrane proteins that are thought to be involved in ADP‐ribosyl transfer reactions. A common property of these proteins is that they are attached to plasma membranes by C‐terminal glycosylphosphatidylinositol (GPI) anchors. The aerolysin receptor was shown to be anchored in the same way by treating rat erythrocytes with phosphatidylinositol‐specific phospholipase C. This caused the selective release of the receptor and a reduction in the rodent cells’ sensitivity to aerolysin. Human and bovine erythrocytes were shown to contain an aerolysin‐binding protein with similar properties to the rat erythrocyte receptor. Proteins with GPI anchors are thought to have unusually high lateral mobility, and this may be an advantage for a toxin, such as aerolysin, which must oligomerize after binding to become insertion competent.