bluuebunny/crossref_metadata_2025 huggingface dataset:
Gross pathology and its relationship with histopathology of amoebic gill disease (AGD) in farmed Atlantic salmon, Salmo salar L.
Authors: Adams M B , Ellard K , Nowak B F
Published: March 2004

Gross pathological assessment of amoebic gill disease (AGD) is the only non‐destructive, financially viable method for rapid and broad‐scale disease management of farmed Atlantic salmon, Salmo salar L., in Tasmania. However, given the presumptive nature of this diagnosis, the technique has been considered questionable. This study investigated the degree of conformity between clinical signs and histological lesions observed in a commercial setting. Three groups of Atlantic salmon (n = 42, 100 and 100, respectively) were collected from various farm sites in southern Tasmania between December 2001 and April 2003. Micro‐stereoscopic analysis showed that grossly affected tissue regions correspond to areas of hyperplastic lamellar fusion, generally in association with attached Neoparamoeba sp. Agreement between gross signs of AGD and histopathological diagnosis was compared. Kappa analysis indicated moderate to good agreement between methods (κ = 0.52–0.74). Individual cases of disagreement were further scrutinized and several factors were found to influence the level of agreement between the two methods. Stage of disease development, lesions derived from other pathogens, assessor interpretation/experience, sampling methods, histological technique and/or experience were potential confounding factors. It was concluded that clinical diagnosis is acceptable as a farm‐monitoring tool only. Removal of grossly affected tissue and subsequent histological examination is recommended to improve diagnostic accuracy.
Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, Salmo salar L., and farmed rainbow trout, Oncorhynchus mykiss (Walbaum)
Authors: Jewhurst V A , Todd D , Rowley H M , Walker I W , Weston J H , McLoughlin M F , Graham D A
Published: March 2004

A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96‐well tissue‐culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE‐214) cells. After incubation for 3 days at 15 °C, plates are fixed and stained using a monoclonal antibody (mAb)‐based immunoperoxidase (IPX) detection system, and virus‐infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX‐based virus neutralization (IPX‐VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates.
Sequential pathology after initial freshwater bath treatment for amoebic gill disease in cultured Atlantic salmon, Salmo salar L.
Authors: Adams M B , Nowak B F
Published: March 2004

Freshwater bathing is essential for control of amoebic gill disease (AGD) during the marine phase of the Tasmanian Atlantic salmon production cycle, a practice that is costly, production limiting and increasing in frequency. Although the pathogenesis of gill infection with Neoparamoeba sp. in naïve Atlantic salmon, Salmo salar, is now understood, the progression of re‐infection (post‐treatment) required elucidation. Here, we describe the weekly histopathological progression of AGD from first to second freshwater bath. Halocline cessation and increased water temperature appeared to drive the rapid onset of initial infection prior to bathing. Freshwater bathing cleared lesions of attached trophozoites and associated cellular debris. Subsequent gill re‐infection with Neoparamoeba sp. was evident at 2 weeks post‐bath and had significantly increased (P < 0.001), in severity by 4 weeks post‐bath. No significant difference in gross pathology was observed until 4 weeks post‐bath (P < 0.05). The re‐infective progression of AGD was characterized by localized host tissue responses juxtaposed to adhered trophozoites (epithelial oedema, hypertrophy and hyperplasia), non‐specific inflammatory cell infiltration (macrophages, neutrophils and eosinophilic granule cells) and finally advanced hyperplasia with epithelial fortification. During the post‐bath period, non‐AGD lesions including haemorrhage, necrosis and regenerative hyperplasia were occasionally observed, although no evidence of secondary colonization of these lesions by Neoparamoeba sp. was noted. We conclude that pathogenesis during the inter‐bath period was identical to initial infection although the source of re‐infection remains to be established.
Is there a relationship between risk assessment and observation level?
Authors: KETTLES A. M. , MOIR E. , WOODS P. , PORTER S. , SUTHERLAND E.
Published: March 2004

The literature is divided into two sections, one on assessment and one on observation, with only one study that tries to make any connection between them. This study, conducted in both acute and forensic areas, attempts to show that there are some direct relationships between assessment criteria and observation levels in both these areas. An instrument was designed and implemented prospectively to collect data about both assessment criteria and the resulting decisions about observation level. Logistic Regression Analysis, descriptive, and inferential statistics, such as Chi‐square and cross‐tabulations, were used to analyse the data. The results showed that there is a relationship between the risk assessment and the enhanced observation level that patients are placed on at the time of admission. The nature of the relationship was limited to seven variables from the 12 in the risk assessment used. This implies that the variables may be different in risk assessment content and extent in each clinical area and we can speculate that there may be individual profiles of the assessment–observation level relationship in each clinical area.
Immunological and functional characterization of Mycobacterium leprae protein antigens: an overview
Authors: Thole Jelle E.R. , Wieles Brigitte , Clark‐Curtiss Josephine E. , Ottenhoff Tom H.M. , Rinke de Wit Tobias F.
Published: March 2004

A major focus of leprosy research in the last 10 years has been the identification and characterization of antigens of Mycobacterium leprae that interact with antibodies and T cells of the host's immune response. Through the combined efforts of many different laboratories, a substantial number of protein antigens have been identified and characterized. In this MicroReview we present an updated list of M. leprae protein antigens, and, with emphasis on recent developments, summarize what is known regarding their functional and immunological features.
Modulation of DNA topology by flaR, a new gene from Listeria monocytogenes
Authors: Sanchez‐Campillo María , Dramsi Shaynoor , Gómez‐Gómez Jose M. , Michel Eric , Dehoux Pierre , Cossart Pascale , Baquero Fernando , Pérez‐Díaz J. Claudio
Published: March 2004

We report the identification of a previously unknown Listeria monocytogenes gene, flaR, which modulates DNA topology. Through the analysis of a Tn917 nonmotile mutant, LOSC1, in which production of flagellin was abolished, we have identified a bacterial component involved in gene regulation. The transposon had inserted in flaR, an open reading frame of 531 bp, followed by a second open reading frame of 1252 bp in reverse orientation. On the L. monocytogenes physical map, flaR was located in a different region from that of the flaA gene encoding flagellin. Transcriptional analysis showed that the flaR gene product affects the flaA expression and negatively regulates its own expression. When expressed in Escherichia coli, flaR encodes a protein of 18 kDa (FlaR) whose transcription is osmoregulated. In addition, FlaR also influences the expression of reporter genes containing supercoiling‐sensitive promoters such as proU or ompC. The data presented here suggest that FlaR is a histone‐like bacterial protein which acts at specific sites to influence DNA topology and, therefore, transcription. flaR is the first gene of this class to be described in Gram‐positive pathogenic bacteria.
Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins
Authors: Katayama Tsutomu , Crooke Elliott , Sekimizu Kazuhisa
Published: March 2004

Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30°C. Whereas purified wild‐type DnaA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild‐type protein. In a system reconstituted with purified proteins at 30°C, the mutant protein initiated replication of single‐stranded DNA that contains a DnaA‐binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA‐binding sequence and functions in the loading of DnaB helicase onto single‐stranded DNA. Thermal stabilities of wild‐type DnaA and DnaAcos activities were comparable. Unlike wild‐type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP‐bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.
Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi
Authors: Palmer Katherine L. , Munson Robert S.
Published: March 2004

We previously identified a heat‐ and protease‐labile haemolytic activity expressed by Haemophilus ducreyi. In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram‐negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore‐forming family of haemolysins. Tn916 mutagenesis was employed to isolate haemolysin‐deficient mutants. Approximately 5000 Tn916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000‐1, was further characterized. Sequences flanking the Tn916 element in strain 35 000‐1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000‐1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB, were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens.
A TnphoA insertion within the Bradyrhizobium japonicum sipS gene, homologous to prokaryotic signal peptidases, results in extensive changes in the expression of PBM‐specific nodulins of infected soybean (Glycine max) cells
Authors: Müller P. , Ahrens K. , Keller T. , Klaucke A.
Published: March 2004

Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA mutagenesis of strain 110spc4. A 6.5 kb BamHI kanamycin‐resistance‐coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild‐type fragment. DNA sequence analysis of a 3213 bp BamHI—ClaI fragment revealed that three open reading frames (ORFs) were encoded in the same orientation. Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase). The TnphoA insertion in mutant 132 was found to be in frame near the 3′ end of sipS. The resulting SipS—PhoA hybrid polypeptide was shown to be expressed in free‐living B. japonicum and in Escherichia coli cultures. An immunoblot analysis with a polyclonal antibody directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B. Japonicum and in E. coli, in agreement with the calculated size of the hybrid polypeptide. A much stronger 52 kDa band was also detected. Mutant 132 was specifically disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads. The bacteroids were not stably maintained within the symbiosome. Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells. Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroid membrane indicated a lower expression of these proteins. The mutant phenotype was genetically complemented by 4.4 kb BamHI fragment including sipS. Subfragments thereof also complemented a temperature‐sensitive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lepts in E. coli. Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region. Inactivation of the gene downstream of sipS did not result in a detectable phenotype.
Mode of promoter recognition by the Escherichia coli RNA polymerase holoenzyme containing the σs subunit: identification of the recognition sequence of the fic promoter
Authors: Hiratsu Keiichiro , Shinagawa Hideo , Makino Kozo
Published: March 2004

Transcription of a number of genes during stationary phase in Escherichia coli requires the alternative sigma factor σs, encoded by the rpoS gene. No consensus sequence for the promoters recognized by the RNA polymerase holoenzyme containing the σs subunit (Eσs) is known. To identify the Eσs recognition sequence, we analysed the promoter region of the fic gene, whose expression depends on Eσs both in vivo and in vitro. By random mutagenesis, we isolated a large number of the fic promoter mutants that had defective promoter activities in an rpoS,+ strain. All such mutants contained alterations within the −10 region, TATACT, whereas no mutant was isolated with alterations in the −35 region. Based on further analyses including scanning mutagenesis and DNase 1 footprinting assays of the fic promoter region, and in vitro transcription assays, we conclude that the −10 hexamer is essential to transcription initiation from the fie promoter by Eσs.
Role of σs in transcription from the positively controlled Pm promoter of the TOL plasmid of Pseudomonas putida
Authors: Marqués Silvia , Gallegos M. Trinidad , Ramos Juan L.
Published: March 2004

Transcription from the TOL plasmid Pm promoter is dependent on the XylS regulator activated by benzoate effectors. We analysed transcription from Pm in several backgrounds with differing Escherichia coli alpha and sigma subunits of RNA polymerase. In different RpoA backgrounds, transcription from Pm was as high as in the wild‐type background throughout the growth curve. In the σs‐deficient background provided by E. coli RH90, high levels of transcription from Pm (XylS/3‐methylbenzoate dependent) were observed in the early logarithmic growth phase but not in the late logarithmic phase or early stationary phase. This contrasted with the results obtained in the isogenic σs‐proficient background, in which high levels of transcription were observed throughout the growth curve. XylS/3‐methylbenzoate‐dependent transcription from Pm in the late logarithmic growth phase in the RH90 background was restored by cloned rpoS. The transcription initiation point of Pm was the same regardless of the growth phase and the σs background. The requirement of (σs for stimulation of transcription from Pm in the late logarithmic and early stationary phase was overcome by using certain mutant Pm promoters, e.g. Pm5 (C‐47 → G, A‐44 → G), and the mutant regulator XylSG44S. It is suggested that the transcription from Pm involves the use of two sigma factors: σ70 during the early logarithmic phase and σs thereafter.
yst gene expression in Yersinia enterocolitica is positively regulated by a chromosomal region that is highly homologous to Escherichia coli host factor 1 gene (hfq)
Authors: Nakao Hiroshi , Watanabe Haruo , Nakayama Shu‐ichi , Takeda Tae
Published: March 2004

Yersinia enterocolitica produces heat‐stable enterotoxin (Y‐ST) as one of its virulence factors. The yst gene, however, frequently and spontaneously becomes inactive (silent) during storage, which is accompanied by concurrent changes in some biological properties such as colony morphology, growth rate, carbon fermentation and ornithine decarboxylase activity. Northern blot analysis revealed that the level of mRNA for yst was repressed. To investigate the regulatory region, we transformed a yst‐silent strain with a chromosomal gene library of Y‐ST producing an isogenic counterpart. Out of 3604 clones, one clone resumed the Y‐ST production and concurrently other biological properties. An open reading frame in this clone was designated as yrp, yersinia regulator for pleiotropic phenotype. Deduced from the nucleotide sequence, Yrp was a small protein of 101 amino acids with no similarity with any regulatory factor described, but showed high homology with an Escherichia coli host factor 1 required for Qβ‐replicase, and with an Azorhizobium caulinodans NrfA required for the expression of nifA. The yrp gene mutation caused decreased negative supercoiling of plasmids, as did hfq. The yrp gene could similarly complement Y‐ST production in two other silent strains of Y. enterocolitica. In all three silent strains examined, we found various mutations in the yrp region.
Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL‐8 in gastric epithelial cells
Authors: Tummuru Murali K.R. , Sharma Smita A. , Blaser Martin J.
Published: March 2004

Approximately 60% of Helicobacter pylori strains are cagA+ and this genotype is more frequently associated with duodenal ulcer disease. Although most wild‐type cagA+ strains are both cytotoxigenic and induce enhanced Interleukin‐8 (IL‐8) secretion in gastric epithelial cells, isogenic cagA− mutants retain full activity in these assays; thus, cagA appears to be a marker of enhanced virulence. Delineation of the nucleotide sequence of a 4 kb region upstream of cagA allowed the identification of 966 bp (picA) and 2655 bp (picB) open reading frames encoding 36 kDa and 101 kDa polypeptides, respectively. picA and picB constitute an operon in opposite orientation to cagA. The deduced picB product showed significant homology (26% identity and 50% similarity) with the Bordetella pertussis toxin secretion protein (PtlC). Of 55 H. pylori clinical isolates, the picA and picB segment was conserved exclusively in cagA+ strains and present in all isolates from patients with duodenal ulceration, versus 59% of isolates from patients with gastritis alone (P=0.01). Using gene‐replacement techniques, we constructed picA and picB mutant H. pylori strains and demonstrated that the picB gene product is involved in the induction of IL‐8 expression in gastric epithelial cells. Further, Northern blot hybridization and RT‐PCR data showed that picA and picB are co‐transcribed and an insertional mutation in picA ablates picB expression. These studies indicate a role of picA and picB in the induction of an inflammatory response in gastric epithelial cells either directly or by enabling secretion of an unidentified product, and suggest a mechanism for the overrepresentation of strains possessing these genes in patients with peptic ulceration.
The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa
Authors: McIver Kevin S. , Kessler Efrat , Olson Joan C. , Ohman Dennis E.
Published: March 2004

Several proteases are secreted by Pseudomonas aeruginosa including elastase, an abundantly secreted neutral zinc‐metalloprotease. Elastase (encoded by lasB) is first synthesized with a relatively large propeptide (18 kDa) domain. Here, we present evidence that this propeptide functions as an intramolecular chaperone (IMC) essential for proper maturation of elastase into a hydrolytically active enzyme. An altered elastase allele (lasB6) that encoded an elastase precursor with a precise propeptide deletion was expressed in Escherichia coli, and disrupted cells contained only inactive elastase. However, co‐expression of an allele (lasB7) expressing the propeptide as an independent, non‐covalently linked protein rescued about one‐third of the hydrolytic activity when compared with that obtained with wild‐type lasB. Thus, the propeptide was essential for elastase activity and so defined elastase as an IMC‐containing protease. We examined the possibility that the propeptide of elastase also plays a role in the localization of the mature protein past the outer bacterial membrane. Expression of lasB6 in P. aeruginosa (lasBΔ) in the absence of the propeptide resulted in production of inactive elastase that accumulated within the cell and was not secreted to the culture medium. When lasB7 co‐expressed the non‐covalently linked propeptide in the same cell with lasB6, efficient secretion was restored and active elastase was then found in the supernatant. Thus, the propeptide was needed for secretion of the mature protein as well as enzymatic activity. This chaperone‐like activity of the propeptide appears to involve a direct interaction between the mature and propeptide sequences, and evidence for this was obtained by demonstrating that the non‐covalently attached 18 kDa propeptide was co‐precipitated with elastase using elastase antibodies. These results are consistent with a hypothesis that the propeptide domain acts as an IMC to control both enzymatic activity and competence for secretion.
Identification of Salmonella typhi promoters activated by invasion of eukaryotic cells
Authors: Staendner Lothar H. , Rohde Manfred , Timmis Kenneth N. , Guzmán Carlos A.
Published: March 2004

The interaction of pathogenic microorganisms with host tissues, and the underlying genetic events which regulate these interactions, are difficult to analyse where no suitable animal model exists. The approach described here, for obtaining information on the genes involved in these interactions, employs an infection system based on the invasion of Henle cells by Salmonella typhi to select promoter‐containing DNA sequences able to activate gene expression inside eukaryotic cells. Several DNA fragments exhibiting different promoter strengths and extent of selective activation within eukaryotic cells were identified. Three were selected and characterized according to the expression level of the reporter gene, the polynucleotide sequence, the transcription start, and the dependence upon regulatory proteins. All fragments gave much stronger expression of the reporter gene when the recombinant S. typhi carrier strains invaded cells compared with the expression measured in growth medium. One promoter‐containing region exhibited sequence homology to σ54‐dependent promoters, whereas another appears to be dependent on the stationary‐phase RNA polymerase subunit σs. S. typhi containing the S1 subunit gene of pertussis toxin cloned under the control of these promoters, selectively expressed the S1 subunit following infection of different phagocytic and non‐phagocytic cell lines of human or murine origin. Deletion and point mutant derivatives of two promoters enabled the identification of the main motif required for promoter activity. This method may be helpful for the analysis of pathogenesis in organisms previously difficult to study because of the lack of a convenient animal model, and could provide insights into the chronology and topology of gene expression during infection, including a possible genetic basis for tissue tropism.
Control of P1 plasmid replication by iterons
Authors: Abeles Ann L. , Reaves Lucretia D. , Youngren‐Grimes Brenda , Austin Stuart J.
Published: March 2004

The incA locus of plasmid P1 controls plasmid copy number by inhibiting the replication origin, oriR. Both loci contain repeat sequences (iterons) that bind the P1 RepA protein. Regulation appears to occur by contact of incA and oriR loci of daughter plasmids mediated by RepA‐bound iterons. Synthetic incA iteron arrays were constructed with altered numbers, sequences or spacing of iterons. Using these in in vitro and in vivo assays, we examined two models: (i) that the origin and incA loci form a stable 1:1 complex in which multiple iterons of each locus are paired with those of the other, and (ii) that individual incA iterons act as freely diffusing nucleoprotein units that contact origin iterons in a random and dynamic fashion. The data presented here strongly favour the latter case. The origin, with its five iterons, acts as a target but not as an effector of regulation. We present a model for replication control based on random, dynamic contacts between incA iterons and the origin. This system would display randomness with respect to choice of templates and timing of initiation if multiple replicon copies were present, but would tend to act in a machine‐like fashion in concert with the cell cycle if just two copies were present in a dividing cell.
Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNA II
Authors: Del Solar Gloria , Acebo Paloma , Espinosa Manuel
Published: March 2004

Two elements, the products of genes copG and rnall, are involved in the copy‐number control of plasmid pLS1. RNA II is synthesized in a dosage‐dependent manner. Mutations in both components have been characterized. To determine the regulatory role of the two genes, we have cloned copG, rnall or both elements at various gene dosages into pLS1‐compatible plasmids. Assays of incompatibility towards wild‐type or mutant pLS1 plasmids showed that: (i) the rnall gene product, rather than the DNA sequence encoding it, is responsible for the incompatibility, and (ii) CopG and RNA II act in trans and are able to correct up fluctuations in pLS1 copy number. A correlation between the gene dosage at which the regulatory elements were supplied and the incompatibility effect on the resident plasmid was observed. The entire copG‐rnall circuit has a synergistic effect when compared with any of its components in the correction of pLS1 copy‐number fluctuations, indicating that, in the homoplasmid steady‐state situation, the control of pLS1 replication is exerted by the co‐ordinate action of CopG and RNA II.
Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea
Authors: Van De Vossenberg Jack L.C.M. , Ubbink‐Kok Trees , Elferink Marieke G.L. , Driessen Arnold J.M. , Konings Wil N.
Published: March 2004

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy‐coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton‐ and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.
Inducible transposition in Streptomyces lividans of insertion sequence IS6100 from Mycobacterium fortuitum
Authors: Smith Barry , Dyson Paul
Published: March 2004

Transposition of IS6100, originally isolated as part of the compound transposon Tn610 from Mycobacterium fortuitum, was tested in the related actinomycete Streptomyces lividans. Cointegrate formation was observed, as expected for this IS6‐related element, and involved apparent random integration of the temperature‐sensitive vector carrying IS6100 and concomitant duplication of the insertion sequence. This establishes that a single copy of the insertion sequence can promote transposition and is a precedent for the functioning of a heterologous transposable element in Streptomyces. Transposition could be induced 100‐fold by external transcription emanating from a copy of the thiostrepton‐inducible promoter ptipA located outwith the insertion sequence and resulting in overexpression of the transposase gene. Thus, in constrast to other prokaryotic transposable elements, IS6100 appears to have no effective means of protecting itself from external activation.
A missense mutation in rpoD results in an A‐signalling defect in Myxococcus xanthus
Authors: Davis John M. , Mayor Jocelyne , Plamann Lynda
Published: March 2004

The Myxococcus xanthus asg genes (asgA, asgB, and asgC) are necessary for production of extracellular A‐signal, which is thought to function as a cell‐density signal. Previous analyses of the asgA and asgB genes suggest that they perform regulatory functions. In this work, we localized asgC to a region that contains genes homologous to rpsU, dnaG, and rpoD of the Escherichia coli macromolecular synthesis (MMS) operon. Surprisingly, asgC767 was found to be a mutant allele of rpoD, the gene encoding the major sigma factor of M. xanthus. The mutation in asgC767 results in a glutamate to lysine substitution at amino acid 598, which lies within conserved region 3.1 of the major sigma factors. Previous studies have shown that the asg mutants share a number of growth and developmental phenotypes. We found that A‐signal restores developmental expression of an A‐signal‐dependent gene (Ω4521) in the asgC767 (rpoDEK598) mutant background in a manner similar to that seen in the asgA and asgB mutants. Because the asg mutants have very similar phenotypes and the asg genes encode proteins that appear to have regulatory functions, we hypothesize that the asg gene products function together in a regulatory pathway that is required for extracellular A‐signal production.
Phosphotransfer circuitry of the putative multi‐signal transducer, ArcB, of Escherichia coli: in vitro studies with mutants
Authors: Tsuzuki Masakatsu , Ishige Kazuya , Mizuno Takeshi
Published: March 2004

Recently we demonstrated the occurrence of a novel device of signal transducers in Escherichia coli. This class of bacterial sensory kinases, typified by ArcB and BarA, possesses two phospho‐donor (His) sites, together with a phospho‐accepting (Asp) site. These multi‐phosphorylation sites were suggested to make a phosphotransfer circuit. To clarify this complex circuitry, we carried out a series of in vitro assays involving a set of ArcB mutant proteins which have an amino acid substitution at each putative phosphorylation site (His‐292, Asp‐576 and His‐717). By these in vitro phosphorylation and/or phosphotransfer assays, the followings were assessed: (i) ArcB autophosphorylation; (ii) ArcB‐mediated phosphorylation of the cognate response regulator, ArcA; (iii) ArcB‐mediated phosphorylation of its truncated form (ArcBc) encompassing only the C‐terminal phosphorylation site (His‐717); (iv) phosphotransfer from ArcBc to ArcA; and (v) phosphotransfer from ArcBc to ArcB. On the basis of these in vitro results, a complex circuitry was revealed for the signal transducer ArcB. This evidence obtained in vitro supports the view that ArcB can serve as a powerful device for not only propagating multi‐signals, but also making up signalling networks, in ways more sophisticated than previously thought.
Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol‐3‐phosphate dehydrogenase, one of which is responsible for osmotolerance for growth
Authors: Ohmiya Ryusuke , Yamada Hisami , Nakashima Kyoko , Aiba Hirofumi , Mizuno Takeshi
Published: March 2004

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osms) on agar plates containing high concentrations of either non‐ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH‐dependent glycerol‐3‐phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1+ and gpd2+, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1+ was regulated at the mRNA level in response to osmotic upshift, (ii) It was demonstrated that wild‐type cells markedly accumulated internal glycerol under high‐osmolarity growth conditions. (iii) Δgpd1 mutants, however, failed to do so even in a high‐osmolarity medium, and thus exhibited an Osms phenotype. On the other hand, the gpd2+ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.
The product of the pilQ gene is essential for the biogenesis of type IV pili in Neisseria gonorrhoeae
Authors: Drake Sandra L. , Koomey Michael
Published: March 2004

The product of the Neisseria gonorrhoeae omc gene possesses regions homologous to those found in members of a protein superfamily that are associated with the translocation of proteins and DNA‐protein complexes across the outer membrane. Amongst its protein homologues, Omc has higher overall homology to PilQ, which is required for type IV pilus expression in Pseudomonas aeruginosa, and OrfE, which is required for sequence‐specific DNA uptake by Haemophilus influenzae. The function of Omc, however, is unknown and gonococcal omc mutants have not been described. We constructed gonococcal mutants expressing truncated forms of the protein, and found that these mutants are severely defective for both pilus expression and competence for natural transformation. To be consistent with pre‐existing pilus gene nomenclature, we have redesignated the gene pilQ instead of omc, and its product, PilQ instead of Omc. The MS11 gene was sequenced and found to differ from the DNA sequence reported for that of another gonococcal strain; these differences were associated with a repeated DNA element, suggesting a genetic basis for structural variation in PilQ. The results also show that PilQ− mutants are distinct from previously described gonococcal pilus‐assembly mutants and P. aeruginosa PilQ− mutants by virtue of their expression of rare pilus filaments. Taking these data into account, PilQ is proposed to function in the terminal steps of organelle biogenesis by acting as a pilus channel or pore.
Gene regulation of siderophore‐mediated iron acquisition in Pseudomonas: not only the Fur repressor
Authors: Venturi Vittorio , Weisbeek Peter , Koster Margot
Published: March 2004

Pseudomonads have several siderophore‐mediated iron‐acquisition systems. These can be classified into two groups: (i) the biosynthesis of a siderophore (iron‐transport agent) followed by its uptake; and (ii) uptake of heterologous ferric‐siderophores in which the siderophore is produced by other microbial species. The regulation of these mechanisms employs both positive and negative elements ensuring expression of the relevant genes only when they are absolutely required. Siderophore biosynthesis is induced in response to iron limitation. In contrast, activation of the heterologous transport systems is not only regulated by iron availability but also requires the presence of their cognate ferric‐siderophores. The investigation of these regulation systems in three different Pseudomonas species gave similar results consisting of regulatory elements new to the field of iron regulation. These elements are superimposed upon the regulation by the Fur repressor, which in other bacteria directly regulate the expression of the iron‐assimilation genes in response to iron availability.
Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide
Authors: Petit Chantal , Rigg Gordon P. , Pazzani Carlo , Smith Annabel , Sieberth Veit , Stevens Mark , Boulnois Graham , Jann Klaus , Roberts Ian S.
Published: March 2004

The nucleotide sequence of region 2 of the Escherichia coli K5 capsule gene cluster has been determined. This region, essential for the biosynthesis of the K5 poly‐saccharide, contained four genes, termed kfiA‐D. The G+C ratio was 33.4%, which was lower than the typical G+C ratio for E. coli and that of the flanking regions 1 and 3 in the K5 capsule gene cluster. Three major RNA transcripts were detected within region 2 by Northern blotting and three promoters located by transcript mapping. Promoter activity was confirmed by promoter‐probe analysis. The predicted amino acid sequence of KfiC had homology to a number of glycosyl transferase enzymes and overexpression of the kfiC gene resulted in increased K5 transferase activity. The predicted amino acid sequence of KfiD had homology to a number of NAD‐dependent dehydrogenase enzymes and was demonstrated to be a UDP‐glucose dehydrogenase that catalyses the formation of UDP‐glucuronic acid from UDP‐glucose.
The endogenous Bacillus subtilis (natto) plasmids pTA1015 and pTA1040 contain signal peptidase‐encoding genes: identification of a new structural module on cryptic plasmids
Authors: Meijer Wilfried J.J. , De Jong Anne , Bea G. , Wisman A. , Tjalsma Harold , Venema Gerard , Bron Sierd , Van Dijl Jan Maarten
Published: March 2004

Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling‐circle mechanism. Like plasmids from other Gram‐positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis plasmids pTA1015 and pTA1040. It is composed of two genes: one specifies an unidentified protein with a putative signal peptide; and the other (sipP) specifies a functional type I signal peptidase (SPase). The homologous, but non‐identical, sipP genes of the two plasmids are the first identified plasmid‐specific SPase‐encoding genes. With respect to structure and activity, the corresponding enzymes (denoted SipP) are highly similar to the chromosomally encoded SPase, SipS, of B. subtilis and several newly identified SPases of other bacilli. Our findings suggest that plasmid‐encoded SPases have evolved because, under certain conditions, SPase can be a limiting factor for protein secretion in B. subtilis.
NusG overexpression inhibits Rho‐dependent termination in Escherichia coli
Authors: Burova Elena , Gottesman Max E.
Published: March 2004

The Escherichia coli NusG protein binds Rho in vitro and is required for efficient Rho‐dependent transcription termination in vivo. In this work we examine transcription termination in cells that overexpress NusG. Termination at the Rho‐dependent λtL1 and λtR1 sites was significantly inhibited by excess NusG, whereas the Rho‐independent λtl site was fully functional. Although Western analysis showed no reduction in the levels of soluble Rho, termination was restored when Rho was also overexpressed. Our data indicate that the ratio of NusG and Rho proteins affects the efficiency of transcription termination.
Molecular analysis of the two‐component genes, ompR and envZ, in the symbiotic bacterium Xenorhabdus nematophilus
Authors: Tabatabai Niloofar , Forst Steven
Published: March 2004

In Escherichia coli the histidine kinase sensor protein, EnvZ, undergoes autophosphorylation and subsequently phosphorylates the regulatory protein, OmpR. Modulation of the levels of OmpR‐phosphate controls the differential expression of ompF and ompC. While the phosphotransfer reaction between EnvZ and OmpR has been extensively studied, the domains involved in the sensing function of EnvZ are not well understood. We have used a comparative approach to study the sensing function of EnvZ. During our search of numerous bacteria we found that the symbiotic/pathogenic bacterium Xenorhabdus nematophilus contained the operon encoding both ompR and envZ. Nucleotide sequence analysis revealed that EnvZ of X. nematophilus (EnvZX.n.) is composed of 342 amino acid residues, which is 108 residues shorter than EnvZ of E. coli (EnvZE.c.). Amino acid sequence comparison showed that the cytoplasmic domains of the EnvZ moleculsshared 57% sequence identity. In contrast, the large hydrophilic periplasmic domain of EnvZE.c. was absent in EnvZX.n., and was replaced by a shorter hydrophobic region. Although the periplasmic domains had diverged extensively, envZX.n. was able to complement a ΔenvZ strain of E. coli. OmpF and OmpC were differentially produced in response to changes in medium osmolarity in this strain. Further genetic analysis established that heterologous phosphorylation between EnvZX.n. and OmpR of E. coli (OmpRE.c.) accounted for the complementation of the ΔenvZ strain. In addition we show that the OmpR molecules of X. nematophilus and E. coli share 78% amino acid sequence identity. These results indicate that the EnvZ protein of X. nematophilus was able to sense changes in the osmolarity of the growth environment and properly regulate the levels of OmpR‐phosphate in E. coli.
A genomic locus in Saccharomyces cerevisiae with four genes up‐regulated by osmotic stress
Authors: Miralles Vicente J. , Serrano Ramón
Published: March 2004

A locus within chromosome XIII of Saccharomyces cerevisiae containing four genes upregulated by osmotic stress has been characterized. Two of the genes, but not their osmotic induction, were already described: the DNA damage‐inducible gene DDR48 and the protease inhibitor gene PAI3. The two novel genes encode a cytoplasmic aldehyde dehydrogenase (ALD2) and a peptide of unknown function (SIP18). These genes form a cluster of two pairs of divergent promoters regulated by osmotic stress. The regulation of the divergent ALD2 and DDR48 genes, however, occurs by different mechanisms. ALD2 exhibits maximum induction with 0.3 M NaCl, negative regulation by protein kinase A and dependence on PBS2 and HOG1 protein kinases for osmotic induction. DDR48 requires 1 M NaCl for maximum induction and its expression is independent of PBS2 and HOG1 protein kinases and less sensitive to protein kinase A. PAI3 and SIP18 are as dependent on the above protein kinases as ALD2. Deletion analysis indicates that most of the regulation of the ALD2 promoter is mediated by a negative element counteracted by osmotic stress.
The dnaK operon of Streptomyces coelicolor encodes a novel heat‐shock protein which binds to the promoter region of the operon
Authors: Bucca Giselda , Ferina Giuseppa , Puglia Anna Maria , Smith Colin P.
Published: March 2004

Transcriptional studies have demonstrated that the dnaK gene of Streptomyces coelicolor A3(2) is contained within a 4.3 kb operon. The operon is transcribed from a single (transiently) heat‐inducible promoter, dnaKp, that resembles the typical vegetative (σ70‐recognized) eubacterial consensus promoter sequence. dnaK transcription was found to be heat‐inducible at all stages of development in surface‐grown cultures. In addition, at the normal growth temperature of 30°C, dnaK transcript levels were shown to vary at different stages of development, being more abundant in young germinating cultures and in mycelium undergoing sporogenesis. The nucleotide sequence of the dnaK operon has been completed, revealing the gene organization 5′‐dnaK‐grpE‐dnaJ‐orfX. orfX represents a novel heat‐shock gene. Its predicted product displays high similarity to the GlnR repressor proteins of Bacillus spp. and to the MerR family of eubacterial transcriptional regulators. The S. coelicolor OrfX protein has been over‐produced in Escherichia coli, and DNA‐binding experiments indicate that it interacts specifically with the dnaKp region, binding to three partially related inverted repeat sequences; they are centred at −75, −49 and +4, respectively, relative to the transcription start site of the operon. These results suggest that OrfX plays a direct role in the regulation of the dnaK operon.
A third periplasmic transport system for l‐arginine in Escherichia coli: molecular characterization of the artPIQMJ genes, arginine binding and transport
Authors: Wissenbach U. , Six S. , Bongaerts J. , Ternes D. , Steinwachs S. , Unden G.
Published: March 2004

A new binding‐protein‐dependent transport system of Escherichia coli specific for l‐arginine was characterized by genetic and biochemical means. The system is encoded by five adjacent genes, artPIQMJ (art standing for arginine transport), which are organized in two transcriptional units (artPIQM and artJ). The artl and artJ gene products (Artl and ArtJ) are periplasmic binding proteins with sequence similarity to binding proteins for polar (basic) amino acids. The artQ, artM and artP products are similar to the transmembraneous proteins and the ATPase of binding‐protein‐dependent carriers. The mature Artl and J proteins were localized in the periplasm and lacked signal peptides of 19 amino acid residues. Artl and ArtJ were isolated from overproducing strains. ArtJ specifically binds l‐arginine with high affinity and overproduction of ArtJ stimulated l‐arginine uptake by the bacteria. The substrate for Artl is not known, and isolated Artl did not bind common amino acids, various basic uncommon amino acids or amines. It is concluded that the artPIQM artJ genes encode a third arginine‐uptake system in addition to the known argT hisJQMP system of Salmonella typhimurium and E. coli and the arginine (‐ornithine) carrier (aps) of E. coli
In Rhizobium meliloti, the operon associated with the nod box n5 comprises nodL, noeA and noeB, three host‐range genes specifically required for the nodulation of particular Medicago species
Authors: Ardourel Maryvonne , Lortet Gilles , Maillet Fabienne , Roche Philippe , Truchet Georges , Promé Jean‐Claude , Rosenberg Charles
Published: March 2004

In Rhizobium meliloti, the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes. In this paper, we have characterized three host‐specific nodulation genes, which form a flavonoid‐inducible operon down‐stream of the nod box n5. The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R. meliloti strain AK631. The product of the second gene, NoeA, presents some homology with a methyl transferase. nodL mutants synthesize Nod factors lacking the O‐acetate substituent. In contrast, in strains carrying a mutation in either noeA or noeB, no modification in Nod‐factor structure or production could be detected. On particular hosts, such as Medicago littoralis, mutants of the n5 operon showed a very weak nodule‐forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected. Thus, nodL, noeA and noeB are host‐specific nodulation genes. By using a gain‐of‐function approach, we showed that the presence of nodL, and hence of O‐acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici.